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Aims: The cell-surface display of Cex, which encodes xylanase and exoglucanase from Cellulomonas fimi, was constructed on Escherichia coli using PgsA as the anchor protein. Characterization of the cell-surface display of Cex was performed. Methods and Results: PgsA was fused to the N-terminus of Cex and six histidines were utilized as spacers between the targeting and anchor proteins. Successful cell-surface display of Cex was demonstrated by Western blot and immunofluorescence analyses on E. coli C41 (DE3). According to the time-course analysis, the xylanase activity of Cex was achieved at 49 U g−1 dry cell weight after 12 h culture at 37°C. The optimal temperature and pH ranges of the cell-surface displayed protein with whole-cell were broader than the corresponding ranges of the purified form. Further determination of thermostability indicated that the half-life of cell-surface displayed Cex was 1·6 times longer than that of purified Cex at 60°C. Conclusions: We have successfully developed the cell-surface display of xylanase on E. coli. The cell-surface display can enhance the stability of xylanase against changes in temperature and has the potential of becoming a whole-cell biocatalyst for industrial applications, such as biobleaching of paper and production of renewable energy. Significance and Impact of the Study: The results demonstrated that the cell-surface display of xylanase embedded in the cell membrane is more stable than that of the purified enzyme. Thus, to improve the stability of heterologous proteins production, cell-surface display using the PgsA anchor protein as a tool can be considered in E. coli. [ABSTRACT FROM AUTHOR]

Hydrophobins are amphipathic molecules which form part of fungal cell walls and extracellular matrices and perform a variety of roles in fungal growth and development. The tomato pathogen Cladosporium fulvum has six hydrophobin genes, HCf-1 to -6. We have devised an epitope tagging approach for establishing hydrophobin localization during growth in culture and in plants. In this paper we localize HCf-2, -3, -4 and -5 and compare the data to our previous observations for HCf-1 and -6. In culture, HCf-1, -2, -3 and 4 localize to conidia and also appear on aerial hyphae. HCf-4 is unique in that it appears on submerged hyphae. HCf-5 expression is tightly regulated and appears on aerial hyphae early on during growth. Only HCf-1, -3 and -6 were observed during infection; HCf-3 appears on both conidia and emerging germ tubes. We also show that HCf-6 is secreted and coats surfaces under and around growing hyphae and demonstrate the effect of deleting HCf-6 on the adhesion of germinating C. fulvum conidia to glass slides. [ABSTRACT FROM AUTHOR]

MARCH comprises a recently identified family of transmembrane RING-finger proteins which is implicated in diverse biological functions, such as immune regulation, protein quality control, and membrane trafficking. We previously identified MARCH-II, as a binding partner of syntaxin 6, which plays a role in endosomal protein transport. In this paper, we describe the cloning and characterization of MARCH-III which is the closest homolog of MARCH-II. It is broadly expressed at relatively high levels in spleen, colon, and lung. An immunofluorescence study of HeLa cells demonstrated that MARCH-III is present in peripheral vesicles partially colocalized with transferrin receptor. Overexpression of MARCH-III resulted in the redistribution of TGN46 and strong inhibition of transferrin uptake. Immunoprecipitation studies revealed that MARCH-III is associated with syntaxin 6 and MARCH-II. Mutational analyses revealed that the PDZ-binding motif and RING finger are essential for the subcellular localization of MARCH-III and the inhibitory effect on transferrin uptake. The location, associated molecules, and effects of overexpression suggest that MARCH-III is involved in the regulation of vesicular trafficking in endosomes. [ABSTRACT FROM PUBLISHER]

This paper examines the molecular machinery involved in membrane exchange within the plant endomembrane system. A study has been undertaken on β‐COP‐like proteins in plant cells using M3A5, an antibody raised against the conserved sequence of mammalian β‐COP proteins. In mammalian cells, β‐COP proteins are part of a complex named the coatomer, which probably recruits some specific areas of the endomembrane system. Immunofluorescence analyses by confocal laser scanning microscopy showed that β‐COP‐like proteins marked predominantly the plant Golgi apparatus. Other proteins known to be part of a potential machinery for COPI vesicle formation (γ‐COP, β′‐COP and Arf1 proteins) were immunolocalized on the same membraneous structures as β‐COP. Moreover, β‐COP and other COPI antibodies stained the cell plate in dividing cells. It is further shown that, in maize root cells, and in contrast to observations upon mammalian cells, the drug Brefeldin A (BFA) does not induce the release of β‐COP and Arf1 proteins from the Golgi membrane into the cytosol. These data clearly demonstrate that the antibody M3A5 is a valuable marker for studies on trafficking events in plant cells. They also report for the first time the location of COP components in plant tissue at the light level, especially on a model well known for secretion, i.e. the maize root cells. They also suggest that the membrane recruitment machinery may function in a plant‐specific way. [ABSTRACT FROM PUBLISHER]

Urticaria is known to occur in the pre-icteric phase of acute viral hepatitis. Often such urticaria is a symptom of a serum sickness-like syndrome which can be a prodrome of viral hepatitis. Immune complexes arc thought to be pathogenetic in this condition. In this paper we present two patients with a serum sickness-like syndrome, urticaria and hepatitis B. By immunofluorescence techniques Hepatitis B surface antigen, CIq and C3 could be demonstrated in the blood vessels of the superficial dermis. These findings are consistent with the immune complex hypothesis. [ABSTRACT FROM AUTHOR]

This paper describes the spontaneous occurrence of an autoimmune uveoretinitis in nude (nu/nu) mice reconstituted when 4 weeks old by the grafting of rat embryonic thymus. The uveoretinitis was characterized histologically by progressive loss of the photoreceptor layer, observed in 4.0, 17.6, 42.9% and 71.4% of such mice at 3, 5, 7 and 12 months of age, respectively. Mice with uveoretinitis were shown to have serum IgG antibody reactive by indirect immunofluorescence with retinal photoreceptors, and with interphotoreceptor retinoid-binding protein (IRBP), but not retinal S-antigen, by immunoblotting and ELISA. A uveoretinitis could be adoptively transferred to syngeneic ungrafted nude mice by splenic CD4+ T cells from diseased animals. This is the first experimental model of a (T cell mediated) autoimmune uveoretinitis which develops spontaneously and which is not dependent upon deliberate sensitization with retinal antigens and adjuvants. [ABSTRACT FROM AUTHOR]

Brain-reactive autoantibodies are thought to play an important role in mediating central nervous system (CNS) disorders in systemic lupus erythematosus (SLE), In this paper the developmental occurrence ofthese antibodies in the sera of autoimmune mice, i,e, NZB, NZB/W, MRL/I and BXSB mice were examined. All murine strains tested, whether autoimmune or not, showed some degree of serum reactivity toward brain antigens. Autoimmune mice, however, displayed higher levels of serum brain-reactive antibodies, and at earlier ages, than non-autoimmune mice. Immunofluorescence assays against brain sections and adsorption assays, with both neural and non-neural tissue, indicated a heterogeneity in the specificity of the populations of brain-reactive antibodies present. These studies provide an important step in characterizing the appearance and diversity of brain-reactive autoantibodies, with the goal of better understanding their significance and potential role in mediating CNS dysfunction in SLE. [ABSTRACT FROM AUTHOR]

This paper describes the course of infection and the immunological response in thymus-deprived and normal CBA mice after intradermal inoculation with promastigotes of L. tropica. Infection of normal mice resulted in the development of a cutaneous ulcer healing within 12 weeks. As the infection progressed and the draining lymph nodes increased in weight, changes in the paracortical and follicular regions were accompanied by the development of delayed hypersensitivity and the production of antibodies detectable by immunofluorescence and a parasite agglutination test. Lesions in thymectomized irradiated mice healed more slowly and the draining lymph nodes were smaller than in normal mice. Follicular reactions were feeble and paracortical activity depressed. The most noticeable feature of these lymph nodes was a persistent and intense macrophage infiltration. Delayed hypersensitivity and antibodies detectable by immunofluorescence were correspondingly low; but parasite agglutinating antibody was not depressed. The course of infection and immunological response of a control group of sham thymectomized, irradiated mice resembled that of normal mice. These experiments indicate that thymus-dependent cell populations play an important role in the response of mice to infection with L. tropica. [ABSTRACT FROM AUTHOR]

An antibody against neurones, which has previously been demonstrated by immunofluorescence, is present in the sera of patients with sensory carcinomatous neuropathy.. This antibody was demonstrated by a new technique, namely the use of purified peroxidase-labelled sheep anti-human γ-globulin. In cryostat sections of brain treated with a first Jayer of serum antibody followed by a second layer of peroxidase labelled ant -human γ-globulin, heavy coarse brown deposits in nerve cells only from different parts of the central nervous system indicated a positive reaction. The special technical precautions necessary to obtain, specific staining of neurones are outlined, [ABSTRACT FROM AUTHOR]

Similarly to microbial pathogens, plant-parasitic nematodes secrete into their host plants proteins that are essential to establish a functional interaction. Identifying the destination of nematode secreted proteins within plant cell compartment(s) will provide compelling clues on their molecular functions. Here the fine localization of five nematode secreted proteins was analysed throughout parasitism in Arabidopsis thaliana. An immunocytochemical method was developed that preserves both the host and the pathogen tissues, allowing the localization of nematode secreted proteins within both organisms. One secreted protein from the amphids and three secreted proteins from the subventral oesophageal glands involved in protein degradation and cell wall modification were secreted in the apoplasm during intercellular migration and to a lower extent by early sedentary stages during giant cell formation. Conversely, another protein produced by both subventral and dorsal oesophageal glands in parasitic stages accumulated profusely at the cell wall of young and mature giant cells. In addition, secretion of cell wall-modifying proteins by the vulva of adult females suggested a role in egg laying. The study shows that the plant apoplasm acts as an important destination compartment for proteins secreted during migration and during sedentary stages of the nematode. [ABSTRACT FROM PUBLISHER]

The tripeptide glutathione is a major antioxidant and redox buffer with multiple roles in plant metabolism. Glutathione biosynthesis is restricted to the cytosol and the plastids and the product is distributed to the various organelles by unknown mechanisms. In the present study immunogold cytochemistry based on anti-glutathione antisera and transmission electron microscopy was used to determine the relative concentration of glutathione in different organelles of Arabidopsis thaliana leaf and root cells. Glutathione-specific labelling was detected in all cellular compartments except the apoplast and the vacuole. The highest glutathione content was surprisingly not found in plastids, which have been described before as a major site of glutathione accumulation, but in mitochondria which lack the capacity for glutathione biosynthesis. Mitochondria of both leaf and root cells contained 7-fold and 4-fold, respectively, higher glutathione levels than plastids while the density of glutathione labelling in the cytosol, nuclei, and peroxisomes was intermediate. The accuracy of the glutathione labelling is supported by two observations. First, pre-adsorption of the anti-glutathione antisera with glutathione reduced the density of the gold particles in all organelles to background levels. Second, the overall glutathione-labelling density was reduced by about 90% in leaves of the glutathione-deficient Arabidopsis mutant pad2-1 and increased in transgenic plants with enhanced glutathione accumulation. Hence, there was a strong correlation between immunocytochemical and biochemical data of glutathione accumulation. Interestingly, the glutathione labelling of mitochondria in pad2-1 remained very similar to wild-type plants thus suggesting that the high mitochondrial glutathione content is maintained in a situation of permanent glutathione-deficiency at the expense of other glutathione pools. High and constant levels of glutathione in mitochondria appear to be particularly important in cell survival strategies and it is predicted that mitochondria must have highly competitive mitochondrial glutathione uptake systems. The present results underline the suggestion that subcellular glutathione concentrations are not controlled by a global mechanism but are controlled on an individual basis and it is therefore not possible to conclude from global biochemical glutathione analysis on the status of the various organellar pools. [ABSTRACT FROM PUBLISHER]

Plants are the organisms containing the most complex multigenic family for thioredoxins (TRX). Several types of TRXs are targeted to chloroplasts, which have been classified into four subgroups: m, f, x, and y. Among them, TRXs f and m were the first plastidial TRXs characterized, and their function as redox modulators of enzymes involved in carbon assimilation in the chloroplast has been well-established. Both TRXs, f and m, were named according to their ability to reduce plastidial fructose-1,6-bisphosphatase (FBPase) and malate dehydrogenase (MDH), respectively. Evidence is presented here based on the immunocytochemistry of the localization of f and m-type TRXs from Pisum sativum in non-photosynthetic tissues. Both TRXs showed a different spatial pattern. Whilst PsTRXm was localized to vascular tissues of all the organs analysed (leaves, stems, and roots), PsTRXf was localized to more specific cells next to xylem vessels and vascular cambium. Heterologous complementation analysis of the yeast mutant EMY63, deficient in both yeast TRXs, by the pea plastidial TRXs suggests that PsTRXm, but not PsTRXf, is involved in the mechanism of reactive oxygen species (ROS) detoxification. In agreement with this function, the PsTRXm gene was induced in roots of pea plants in response to hydrogen peroxide. [ABSTRACT FROM PUBLISHER]

Many patients with SARS-CoV-2 infection develop neurological signs and symptoms, though, to date, little evidence exists that primary infection of the brain is a significant contributing factor. We present the clinical, neuropathological, and molecular findings of 41 consecutive patients with SARS-CoV-2 infections who died and underwent autopsy in our medical center. The mean age was 74 years (38-97 years), 27 patients (66%) were male and 34 (83%) were of Hispanic/Latinx ethnicity. Twenty-four patients (59%) were admitted to the intensive care unit (ICU). Hospital-associated complications were common, including 8 (20%) with deep vein thrombosis/pulmonary embolism (DVT/PE), 7 (17%) patients with acute kidney injury requiring dialysis, and 10 (24%) with positive blood cultures during admission. Eight (20%) patients died within 24 hours of hospital admission, while 11 (27%) died more than 4 weeks after hospital admission. Neuropathological examination of 20-30 areas from each brain revealed hypoxic/ischemic changes in all brains, both global and focal; large and small infarcts, many of which appeared hemorrhagic; and microglial activation with microglial nodules accompanied by neuronophagia, most prominently in the brainstem. We observed sparse T lymphocyte accumulation in either perivascular regions or in the brain parenchyma. Many brains contained atherosclerosis of large arteries and arteriolosclerosis, though none had evidence of vasculitis. Eighteen (44%) contained pathologies of neurodegenerative diseases, not unexpected given the age range of our patients. We examined multiple fresh frozen and fixed tissues from 28 brains for the presence of viral RNA and protein, using quantitative reverse-transcriptase PCR (qRT-PCR), RNAscope, and immunocytochemistry with primers, probes, and antibodies directed against the spike and nucleocapsid regions. qRT-PCR revealed low to very low, but detectable, viral RNA levels in the majority of brains, although they were far lower than those in nasal epithelia. RNAscope and immunocytochemistry failed to detect viral RNA or protein in brains. Our findings indicate that the levels of detectable virus in COVID-19 brains are very low and do not correlate with the histopathological alterations. These findings suggest that microglial activation, microglial nodules and neuronophagia, observed in the majority of brains, do not result from direct viral infection of brain parenchyma, but rather likely from systemic inflammation, perhaps with synergistic contribution from hypoxia/ischemia. Further studies are needed to define whether these pathologies, if present in patients who survive COVID-19, might contribute to chronic neurological problems. [ABSTRACT FROM AUTHOR]

Background Extensins are plant cell wall hydroxyproline-rich glycoproteins known to be involved in cell wall reinforcement in higher plants, and in defence against pathogen attacks. The ability of extensins to form intra- and intermolecular cross-links is directly related to their role in cell wall reinforcement. Formation of such cross-links requires appropriate glycosylation and structural conformation of the glycoprotein. Scope Although the role of cell wall components in plant defence has drawn increasing interest over recent years, relatively little focus has been dedicated to extensins. Nevertheless, new insights were recently provided regarding the structure and the role of extensins and their glycosylation in plant–microbe interactions, stimulating an interesting debate from fellow cell wall community experts. We have previously revealed a distinct distribution of extensin epitopes in Arabidopsis thaliana wild-type roots and in mutants impaired in extensin arabinosylation, in response to elicitation with flagellin 22. That study was recently debated in a Commentary by Tan and Mort (Tan L, Mort A. 2020. Extensins at the front line of plant defence. A commentary on: 'Extensin arabinosylation is involved in root response to elicitors and limits oomycete colonization'. Annals of Botany 125: vii–viii) and several points regarding our results were discussed. As a response, we herein clarify the points raised by Tan and Mort, and update the possible epitope structure recognized by the anti-extensin monoclonal antibodies. We also provide additional data showing differential distribution of LM1 extensin epitopes in roots between a mutant defective in PEROXIDASES 33 and 34 and the wild type, similarly to previous observations from the rra2 mutant defective in extensin arabinosylation. We propose these two peroxidases as potential candidates to specifically catalyse the cross-linking of extensins within the cell wall. Conclusions Extensins play a major role within the cell wall to ensure root protection. The cross-linking of extensins, which requires correct glycosylation and specific peroxidases, is most likely to result in modulation of cell wall architecture that allows enhanced protection of root cells against invading pathogens. Study of the relationship between extensin glycosylation and their cross-linking is a very promising approach to further understand how the cell wall influences root immunity. [ABSTRACT FROM AUTHOR]

Study Question: Do human granulosa cells (GCs) ingest and destroy apoptotic oocytes?Summary Answer: Somatic GCs ingest and destroy apoptotic oocytes and other apoptotic substrates through unconventional autophagy-assisted phagocytosis.What Is Known Already: Most (99%) ovarian germ cells undergo apoptosis through follicular atresia. The mode of cleaning of atretic follicles from the ovary is unclear. Ovarian GCs share striking similarities with testicular Sertoli cells with respect to their origin and function. Somatic Sertoli cells are responsible for the elimination of apoptotic spermatogenic cells through unconventional autophagy-assisted phagocytosis.Study Design, Size, Duration: Human GCs were tested for the ability to ingest and destroy the apoptotic oocytes and other apoptotic substrates. A systemic study of the main phagocytosis steps has been performed at different time points after loading of apoptotic substrates into the GC.Participants/materials, Setting, Methods: Primary cultures of GC retrieved following controlled ovarian stimulation of five women for IVF/ICSI and a human granulosa KGN cell line were incubated with different apoptotic substrates: oocytes which underwent spontaneous apoptosis during the cultivation of immature germ cells for IVF/ICSI; apoptotic KGN cells; and apoptotic membranes from rat retinas. Cultured GC were analyzed for the presence of specific molecular markers characteristic of different steps of phagocytic and autophagy machineries by immunocytochemistry, confocal microscopy, transmission electron microscopy and western blotting, before and after loading with apoptotic substrates.Main Results and the Role Of Chance: Incubation of human GC with apoptotic substrates resulted in their translocation in cell cytoplasm, concomitant with activation of the phagocytosis receptor c-mer proto-oncogene tyrosine kinase MERTK (P < 0.001), clumping of motor molecule myosin II, recruitment of autophagy proteins: autophagy-related protein 5 (ATG5), autophagy-related protein 6 (Beclin1) and the rise of a membrane form of microtubule-associated protein 1 light chain 3 (LC3-II) protein. Ingestion of apoptotic substrates was accompanied by increased expression of the lysosomal protease Cathepsin D (P < 0.001), and a rise of lysosomes in the GCs, as assessed by different techniques. The level of autophagy adaptor, sequestosome 1/p62 (p62) protein remained unchanged.Large Scale Data: N/A.Limitations, Reasons For Caution: The number of patients described here is limited. Also the dependence of phagocytosis on reproductive hormone status of patients should be analyzed.Wider Implications Of the Findings: Removal of apoptotic oocytes by surrounding GC seems likely to be a physiological mechanism involved in follicular atresia. Proper functioning of this mechanism may be a new strategy for the treatment of ovarian dysfunctions associated with an imbalance in content of germ cells in the ovaries, such as premature ovarian failure and polycystic ovary syndrome.Study Funding/competing Interest(s): The study was funded by Rennes Metropole (AIS 2015) and Agence de BioMédecine. This work was supported by funding from Université de Rennes1, Institut National de la Santé et de la Recherche Médicale (INSERM) and CHU de Rennes. A.B. is funded in part by the program Actions Concertées Interpasteuriennes (ACIP) and a research grant from the European Society of Pediatric Endocrinology. This work is supported by the Agence Nationale de la Recherche Grants ANR-17-CE14-0038 and ANR-10-LABX-73. The authors declare no competing interests. [ABSTRACT FROM AUTHOR]

This article presents information about the research paper entitled "Preferential activation of CD4 T-lymphocytes in the lamina propria of dermatitis herpetiformis and coeliac disease," presented by C. E. M. Griffiths, I. Barrison, J. N. Leonard, and others at the British Society for Investigative Dermatology Annual Meeting, Cambridge, September 1987. The distribution and activation of T-lymphocyte subsets in the small intestinal mucosa of coeliac disease and dermatitis herpetiformis subjects on a normal diet has been studied and compared with normal controls. Double-labelling immunofluorescence techniques in conjunction with monoclonal antibodies were used on cryostat tissue sections.

Sesuvium sesuvioides (Sesuvioideae, Aizoaceae) is a perennial, salt-tolerant herb distributed in flats, depressions, or disturbed habitats of southern Africa and the Cape Verdes. Based on carbon isotope values, it is considered a C4 species, despite a relatively high ratio of mesophyll to bundle sheath cells (2.7:1) in the portulacelloid leaf anatomy. Using leaf anatomy, immunocytochemistry, gas exchange measurements, and enzyme activity assays, we sought to identify the biochemical subtype of C4 photosynthesis used by S. sesuvioides and to explore the anatomical, physiological, and biochemical traits of young, mature, and senescing leaves, with the aim to elucidate the plasticity and possible limitations of the photosynthetic efficiency in this species. Assays indicated that S. sesuvioides employs the NADP-malic enzyme as the major decarboxylating enzyme. The activity of C4 enzymes, however, declined as leaves aged, and the proportion of water storage tissue increased while air space decreased. These changes suggest a functional shift from photosynthesis to water storage in older leaves. Interestingly, S. sesuvioides demonstrated CO2 compensation points ranging between C4 and C3–C4 intermediate values, and immunocytochemistry revealed labeling of the Rubisco large subunit in mesophyll cells. We hypothesize that S. sesuvioides represents a young C4 lineage with C4-like photosynthesis in which C3 and C4 cycles are running simultaneously in the mesophyll. [ABSTRACT FROM AUTHOR]

Background and Aims Changes in the arrangement of cell wall components determine cell wall properties (integrity, stiffness), thereby affecting the macro-scale properties of fruits, which are important for consumers and industry. Arabinogalactan proteins (AGPs) are ubiquitous components of the plant cell, in which they have various functions. Currently, AGPs are considered to be one of the less well-known, enigmatic proteoglycans, a consequence of their heterogeneous structure and unclear mechanism of activity. Methods An immunocytochemical study was conducted to elucidate the distribution of AGPs and pectic polysaccharides contained in apple (Malus × domestica) fruit during senescence. De-esterified homogalacturonan (LM19), methyl-esterified homogalacturonan (LM20), processed arabinan (LM16) and three AGP epitopes (JIM13, JIM15, MAC207) were identified in the fruit at three stages: fresh fruit, and fruit at 1 and 3 months of post-harvest storage. Key Results Microscopy revealed spatio-temporal changes in the localization of all examined epitopes. Changes of fruit cell wall assembly and its degradation were confirmed by determination of the galacturonic acid content in the WSP (water soluble pectins), CSP (chelator soluble pectins) and DASP (dilute alkali soluble pectins) fractions. Conclusions The results revealed dependencies between AGPs, arabinan and homogalacturonan distribution in apple fruit, which are correlated with changes in microstructure during senescence. We propose that AGPs are involved in establishment of the cell wall – plasma membrane continuum. [ABSTRACT FROM AUTHOR]

The author comments on the article "Early- and late-onset psoriasis: a cross-sectional clinical and immunocytochemical investigation," by E. Theodorakopoulou and colleagues. Topics discussed include subtypes of chronic plaque psoriasis such as type I and type II psoriasis, the link of interleukins with early-onset psoriasis (EOP) and late-onset psoriasis (LOP), and the impact of T-cell infiltration for psoriasis

Study Question: What are the effects of exposure to bisphenol A (BPA) or bisphenol S (BPS) during IVM on bovine oocyte maturation, spindle morphology and chromosome alignment?Summary Answer: Exposure to BPA or BPS during IVM resulted in increased spindle abnormalities and chromosome misalignment, even at very low concentrations.What Is Known Already: BPA is an endocrine disrupting chemical that alters oocyte maturation, spindle morphology and chromosome alignment in a range of species. The use of BPA substitutes, such as BPS, is increasing and these substitutes often display different potencies and mechanisms of action compared with BPA.Study Design, Size, Duration: Bovine cumulus-oocyte complexes (COCs) underwent IVM with BPA or BPS for 24 h, together with vehicle-only controls. Overall, 10 different concentrations of BPA or BPS were used ranging from 1 fM to 50 μM in order to detect low dose or non-monotonic effects. An incomplete block design was utilized for the study, with at least three replicates per block. A total of 939 oocytes (250 of which were controls) were used for the BPA experiments, and 432 (110 controls) for the BPS experiments. Following the IVM period, the oocytes were denuded and fixed for immunocytochemistry.Participants/materials, Setting, Methods: Immunocytochemistry was used to label the chromatin, actin, and microtubules in the fixed oocytes. The meiotic stage was assessed using immunofluorescence, and the metaphase-II (MII) oocytes were further assessed for spindle morphology and chromosome alignment (in all MII oocytes regardless of spindle morphology) using immunofluorescence and confocal microscopy. Significant differences between the treatment and control groups were determined using chi-square and Fisher's exact tests.Main Results and the Role Of Chance: There was no effect of BPA or BPS on the proportion of bovine oocytes that reached MII (P > 0.05). BPA and BPS increased spindle abnormalities in MII oocytes at almost all concentrations tested, including those as low as 1 fM (P = 0.013) or 10 fM (P < 0.0001), respectively, compared to control. Oocytes with flattened spindles with broad poles were observed at a higher frequency at some concentrations of BPA (P = 0.0002 and P = 0.002 for 10 nM and 50 μM, respectively) or BPS (P = 0.01 for 100 nM BPS), while this spindle phenotype was absent in the controls. BPA increased chromosome misalignment at concentrations of 10 fM, 10 nM and 50 μM (P < 0.0001 to P = 0.043 depending on the dose). BPS increased chromosome misalignment at concentrations of 10 fM, 100 pM, 10 nM, 100 nM and 50 μM (P < 0.0001 to P = 0.013 depending on the dose).Limitations Reasons For Caution: Exposures to BPA or BPS were performed during the IVM of COCs to allow for determination of direct effects of these chemicals on oocyte maturation. Whole follicle culture or in vivo studies will confirm whether follicular cell interactions modify the effects of BPA or BPS on oocyte meiotic maturation. Investigation into the effects of BPA or BPS on other oocyte functions will determine whether these chemicals alter oocyte quality via mechanisms independent of the meiotic endpoints characterized here.Wider Implications Of the Findings: The findings of this study show that both BPA and BPS induce spindle abnormalities and chromosome misalignment in bovine in a non-monotonic manner, and at concentrations that are orders of magnitude below those measured in humans. Taken in context with previous studies on the effects of BPA in a range of species, our data support the literature that BPA may reduce oocyte quality and lead to subsequent infertility. Additionally, these results contribute to the burgeoning field of research on BPS and suggest that BPS may indeed be a 'regrettable substitution' for BPA.Study Funding/competing Interest(s): This study was supported by funding from the National Institutes of Health (NIH) (Grant 1R15ES024520-01). The authors declare no conflict of interest. [ABSTRACT FROM AUTHOR]

Superwarfarins are very long-lasting rodenticides effective in warfarin-resistant rodents at extremely low doses. The consequences of chronic superwarfarin levels in tissues, due to biological half-lives on the order of 20 days, have not been examined. We now characterized the neurological effects of brodifacoum(BDF), one of the most widely used superwarfarins, in adult male Sprague Dawley rats. Dosing curves established the acute oral lethal dose for BDF as 221 ± 14 μg/kg. Measurement of tissue BDF levels showed accumulation throughout the body, including the central nervous system, with levels diminishing over several days. Immunocytochemical staining showed that both astrocyte and microglial activation was increased 4 days after BDF administration, as were levels of carbonylated proteins, and neuronal damage assessed by fluorojade B staining. Direct toxic effects of BDF on neurons and glia were observed using enriched cultures of cerebellar neurons and cortical astrocytes. Proteomic analysis of cerebellar lysates revealed that BDF altered expression of 667 proteins in adult rats. Gene ontology and pathway analysis identified changes in several functional pathways including cell metabolism, mitochondria function, and RNA handling with ribosomal proteins comprising the largest group. In vitro studies using primary astrocytes showed that BDF suppressed de novo protein synthesis. These findings demonstrate that superwarfarin accumulation increases indices of neuroinflammation and neuropathology in adult rodents, suggesting that methods which minimize BDF toxicity may not address delayed neurological sequelae. [ABSTRACT FROM AUTHOR]

Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011. [ABSTRACT FROM PUBLISHER]

Thyroid fine-needle aspiration (FNA) cytology is a fast growing field. One of the most developing areas is represented by molecular tests applied to cytological material. Patients that could benefit the most from these tests are those that have been diagnosed as 'indeterminate' on FNA. They could be better stratified in terms of malignancy risk and thus oriented with more confidence to the appropriate management. Taking in to consideration the need to improve and keep high the yield of thyroid FNA, professionals from various fields (i.e. molecular biologists, endocrinologists, nuclear medicine physicians and radiologists) are refining and fine-tuning their diagnostic instruments. In particular, all these developments aim at increasing the negative predictive value of FNA to improve the selection of patients for diagnostic surgery. These advances involve terminology, the application of next-generation sequencing to thyroid FNA, the use of immunocyto- and histo-chemistry, the development of new sampling techniques and the increasing use of nuclear medicine as well as molecular imaging in the management of patients with a thyroid nodule. Herein, we review the recent advances in thyroid FNA cytology that could be of interest to the 'thyroid-care' community, with particular focus on the indeterminate diagnostic category. [ABSTRACT FROM AUTHOR]

Objective Pigment epithelium-derived factor ( PEDF) has proven anti-osteosarcoma activity. However, the mechanism(s) underpinning its ability to reduce primary bone tumour (osteosarcoma) metastasis is unknown. Methods Adult and fetal murine bone were immunostained for PEDF, collagen I (major protein in bone) and its processing proteins, heat shock protein 47 ( HSP47, a chaperone protein for collagen I), membrane type I matrix metalloproteinase ( MT1- MMP, a collagenase), and matrix metalloproteinase 2 ( MMP-2, which is activated by MT1- MMP). Immunoblotting and immunocytochemistry were used to observe levels of the above biomarkers when human osteosarcoma cells were treated with PEDF. Key findings Immunohistochemical staining in adult and fetal bone mirrors collagen I. PEDF localised to ridges of trabecular bone in tibial cortex and to megakaryocytes within bone marrow. Second, we observed that PEDF upregulates collagen I, HSP47 and MT1- MMP, while downregulating MMP-2 in osteosarcoma cells in vitro. Conclusion PEDF is a promising antagonist to osteosarcoma cell metastasis via downregulation of MMP-2, and can induce tumour cells to further adopt differentiative properties, thereby possibly reducing their aggressive growth in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Background and Aims Innovations in vegetative and reproductive characters were key factors in the evolutionary history of land plants and most of these transformations, including dramatic changes in life cycle structure and strategy, necessarily involved cell-wall modifications. To provide more insight into the role of cell walls in effecting changes in plant structure and function, and in particular their role in the generation of vascularization, an antibody-based approach was implemented to compare the presence and distribution of cell-wall glycan epitopes between (free-living) gametophytes and sporophytes of Ceratopteris richardii ‘C-Fern’, a widely used model system for ferns. Methods Microarrays of sequential diamino-cyclohexane-tetraacetic acid (CDTA) and NaOH extractions of gametophytes, spores and different organs of ‘C-Fern’ sporophytes were probed with glycan-directed monoclonal antibodies. The same probes were employed to investigate the tissue- and cell-specific distribution of glycan epitopes. Key Results While monoclonal antibodies against pectic homogalacturonan, mannan and xyloglucan widely labelled gametophytic and sporophytic tissues, xylans were only detected in secondary cell walls of the sporophyte. The LM5 pectic galactan epitope was restricted to sporophytic phloem tissue. Rhizoids and root hairs showed similarities in arabinogalactan protein (AGP) and xyloglucan epitope distribution patterns. Conclusions The differences and similarities in glycan cell-wall composition between ‘C-Fern’ gametophytes and sporophytes indicate that the molecular design of cell walls reflects functional specialization rather than genetic origin. Glycan epitopes that were not detected in gametophytes were associated with cell walls of specialized tissues in the sporophyte. [ABSTRACT FROM PUBLISHER]

Background and Aims Parasitic plants obtain nutrients from their hosts through organs called haustoria. The hyaline body is a specialized parenchymatous tissue occupying the central parts of haustoria in many Orobanchaceae species. The structure and functions of hyaline bodies are poorly understood despite their apparent necessity for the proper functioning of haustoria. Reported here is a cell wall-focused immunohistochemical study of the hyaline bodies of three species from the ecologically important clade of rhinanthoid Orobanchaceae. Methods Haustoria collected from laboratory-grown and field-collected plants of Rhinanthus minor, Odontites vernus and Melampyrum pratense attached to various hosts were immunolabelled for cell wall matrix glycans and glycoproteins using specific monoclonal antibodies (mAbs). Key Results Hyaline body cell wall architecture differed from that of the surrounding parenchyma in all species investigated. Enrichment in arabinogalactan protein (AGP) epitopes labelled with mAbs LM2, JIM8, JIM13, JIM14 and CCRC-M7 was prominent and coincided with reduced labelling of de-esterified homogalacturonan with mAbs JIM5, LM18 and LM19. Furthermore, paramural bodies, intercellular deposits and globular ergastic bodies composed of pectins, xyloglucans, extensins and AGPs were common. In Rhinanthus they were particularly abundant in pairings with legume hosts. Hyaline body cells were not in direct contact with haustorial xylem, which was surrounded by a single layer of paratracheal parenchyma with thickened cell walls abutting the xylem. Conclusions The distinctive anatomy and cell wall architecture indicate hyaline body specialization. Altered proportions of AGPs and pectins may affect the mechanical properties of hyaline body cell walls. This and the association with a transfer-like type of paratracheal parenchyma suggest a role in nutrient translocation. Organelle-rich protoplasts and the presence of exceptionally profuse intra- and intercellular wall materials when attached to a nitrogen-fixing host suggest subsequent processing and transient storage of nutrients. AGPs might therefore be implicated in nutrient transfer and metabolism in haustoria. [ABSTRACT FROM PUBLISHER]

STUDY QUESTION Is SIRT1 involved in the oxidative stress (OS) response in mouse oocytes? SUMMARY ANSWER SIRT1 plays a pivotal role in the adaptive response of mouse germinal vesicle (GV) oocytes to OS and promotes a signalling cascade leading to up-regulation of the MnSod gene. WHAT IS KNOWN ALREADY OS is known to continuously threaten acquisition and maintenance of oocyte developmental potential during in vivo processes and in vitro manipulations. Previous studies in somatic cells have provided strong evidence for the role of SIRT1 as a sensor of the cell redox state and a protector against OS and aging. STUDY DESIGN, SIZE, DURATION GV oocytes obtained from young (4–8 weeks) and reproductively old (48–52 weeks) CD1 mice were blocked in the prophase stage by 0.5 µM cilostamide. Groups of 30 oocytes were exposed to 25 µM H2O2 and processed following different times for the analysis of intracellular localization of SIRT1 and FOXO3A, and evaluation of Sirt1, miRNA-132, FoxO3a and MnSod gene expression. Another set of oocytes was cultured in the presence or absence of the SIRT1-specific inhibitor Ex527, and exposed to H2O2 in order to assess the involvement of SIRT1 in the activation of a FoxO3a-MnSod axis and ROS detoxification. In the last part of this study, GV oocytes were maturated in vitro in the presence of different Ex527 concentrations (0, 2.5, 5, 10, 20 µM) and assessed for maturation rates following 16 h. Effects of Ex527 on spindle morphology and ROS levels were also evaluated. PARTICIPANTS/MATERIALS, SETTING, METHODS SIRT1 and FOXO3A intracellular distribution in response to OS was investigated by immunocytochemistry. Real-time RT–PCR was employed to analyse Sirt1, miR-132, FoxO3a and MnSod gene expression. Reactive oxygen species (ROS) production was evaluated by in vivo measurement of carboxy-H2DCF diacetate labelling. Spindle and chromosomal distribution in in vitro matured oocytes were analysed by immunocytochemistry and DNA fluorescent labelling, respectively. MAIN RESULTS AND THE ROLE OF CHANCE Specific changes in the intracellular localization of SIRT1 and up-regulation of Sirt1 gene were detected in mouse oocytes in response to OS. Moreover, increased intracellular ROS were observed when SIRT1 activity was inhibited by Ex527. In aged oocytes Sirt1 was expressed more than in young oocytes but SIRT1 protein was undetectable. Upon OS, significant changes in miR-132 micro-RNA, a validated Sirt1 modulator, were observed. A negative correlation between Sirt1 mRNA and miR-132 levels was observed when young oocytes exposed to OS were compared with young control oocytes, and when aged oocytes were compared with young control oocytes. FoxO3a and MnSod transcripts were increased upon OS with the same kinetics as Sirt1 transcripts, and up-regulation of MnSod gene was prevented by oocyte treatment with Ex527, indicating that SIRT1 acts upstream to the FoxO3a-MnSod axis. Finally, the results of the in vitro maturation assay suggested that SIRT1 might be involved in oocyte maturation by regulating the redox state and ensuring normal spindle assembly. LIMITATIONS, REASONS FOR CAUTION The main limitation of this study was the absence of direct quantification of SIRT1 enzymatic activity due to the lack of an appropriately sensitive method. WIDER IMPLICATIONS OF THE FINDINGS The present findings may provide a valuable background for studying the regulation of SIRT1 during oogenesis and its relevance as a sensor of oocyte redox state and energy status. The antioxidant response orchestrated by SIRT1 in oocytes seems to decrease with aging. This suggests that SIRT1 could be an excellent pharmacological target for improving oocyte quality and IVF outcome in aging or aging-like diseases. STUDY FUNDING/COMPETING INTEREST(S) The work was ... [ABSTRACT FROM PUBLISHER]

Background Pemphigus typically has a chronic course, although there is great variability in disease duration ( DD) and time taken to disease remission ( DR) between individuals with the disease. The reasons for this are unclear. Objectives To explore the prognostic influence of epidemiological, clinical, immunological and genetic factors on disease course and remission in pemphigus vulgaris ( PV) and pemphigus foliaceus ( PF). Methods This was a retrospective study of patients with PV and PF, recruited from a single UK centre. Direct and indirect immunofluorescence and enzyme-linked immunosorbent assay studies for antidesmoglein ( Dsg) antibodies were used to assess immunological factors. Polymerase chain reaction with sequence specific primers ( PCR- SSP) was used to assess the Class II human leukocyte antigen status of patients. Prognostic endpoints investigated were time to initial first DR and total DD. Results Ninety-five patients were recruited (79 PV and 16 PF). Patients of Indo-Asian origin were significantly associated with longer DD than White-British patients ( P = 0·029). In addition, younger age at onset was associated with a worse prognosis in terms of DD: the mean age at presentation of patients with DD of < 5 years was 49 years ( SEM = 3·4) compared with 40 years ( SEM = 1·9) in those with DD > 5 years ( P = 0·039). A higher initial intercellular antibody titre on normal human skin substrate was associated with a greater time to initial DR ( P = 0·007) and high anti-Dsg 3 levels at baseline were associated with a longer total DD ( P = 0·03). Conclusions Ethnic group, age at presentation, initial intercellular antibody titre and initial Dsg 3 antibody levels all had a significant impact on prognosis of pemphigus. [ABSTRACT FROM AUTHOR]

Skeletal muscle shows dynamic changes in mass that correlate with activity and weight bearing loads. The electrical excitation of the muscle of Procambarus clarkii (Girard, 1952) used in this study is graded which requires refined nerve-muscle matching in synaptic efficacy. We used the anterior levator (a.l.) muscle in crayfish as a model to address matching of the extent of nerve terminal and muscle size. This muscle repetitively becomes loaded and unloaded for various lengths of time due to limb autotomy. When an adult P. clarkii loses a cheliped, by autotomy, the a.l. muscle will atrophy over time. The leg stump still moves suggesting functional innervation. During atrophy, the muscle is drastically reduced in mass as compared to the contralateral control with a functional intact cheliped. The a.l. muscle is innervated by multiple excitatory neurons and at least 1 inhibitory neuron. Since the innervation is less extensive and identifiable for the inhibitory neuron the focus was on the innervation profile based on anti-GABA immunocytochemistry. Preliminary findings based on electron microscopic images of a few samples suggest that terminals on atrophied muscles have fewer synapses than terminals on control muscles. In addition, the extent of terminals on atrophied muscle is much more extensive as compared to muscle per surface area for animals with intact chelipeds. The atrophied muscles appear to be hyperinnervated when considering terminal length per surface area of muscle fiber. [ABSTRACT FROM AUTHOR]

Aims Thimet oligopeptidase (TOP, endopeptidase EC3.4.24.15) is a soluble metallopeptidase known to be expressed within the mammalian vasculature. We examine for the first time the relationship between TOP expression and laminar shear stress, a haemodynamic force associated with endothelium-mediated vascular homeostasis. Methods and results Human and bovine aortic endothelial cells were exposed to physiological levels of laminar shear (0–10 dynes/cm2, 24–48 h) and monitored for TOP expression using promoter activity assay, qRT–PCR, western blotting, and immunocytochemistry. Using a luciferase reporter encoding the full-length rat TOP promoter, initial studies demonstrated shear-dependent promoter activation (∼five-fold). TOP mRNA and protein were also consistently up-regulated by shear, events which could be completely prevented by pre-treatment of cells with either N-acetylcysteine, superoxide dismutase, or catalase, confirming ROS involvement. Consistent with this, targeted inhibition of NADPH oxidase (apocynin, NSC23766, NOX4 siRNA) had a similar blocking effect. Finally, in view of its pivotal role in cellular antigen presentation and major histocompatibility complex (MHC) class-1 regulation, we hypothesized that the shear-dependent induction of TOP may lower MHC1 expression. In this respect, we observed that recombinant TOP over-expression in static HAECs dose-dependently depleted MHC1 (>60%), while siRNA-mediated blockade of TOP induction in sheared HAECs led to substantially elevated MHC1 (∼66%). Conclusion Our findings demonstrate that laminar shear positively regulates endothelial TOP expression. Moreover, a role for ROS production by NADPH oxidase is indicated. Finally, our studies suggest that shear-dependent TOP induction down-regulates MHC1 levels, pointing to a role for TOP in the flow-mediated regulation of endothelial immunogenicity. [ABSTRACT FROM PUBLISHER]

Objective: In the field of fine-needle aspiration cytology, the category of suspicious for malignancy (SM) thyroid lesions, that bears 55-85% risk of malignant histology, is a challenging topic in which morphology alone is not always able to make a correct diagnosis. Recently, immunocytochemistry (ICC) has been referred to as helpful in differentiating low- and high-malignant risk lesions and BRAF activating mutations have been identified in a significant amount of papillary thyroid carcinomas (PTC). The introduction of the liquid-based cytology (LBC) may simplify the application of these techniques to thyroid cytology . Design: Our aim is to evaluate the diagnostic and prognostic role of both ICC and BRAF mutation for the SM category on LBC. Methods: From October 2010 through June 2011, 113 LBC cytological cases (including 37 SM and 76 PTC) underwent surgery . All cases were studied for BRAF mutation and ICC. Results: ICC resulted positive in 26 (86.6%) histologically malignant SM with 15 of which (40.5%) expressing a BRAF mutation. Overall, 63 cases showed a BRAF mutation resulting in PTC. Concerning the prognostic role of BRAF mutation for the two categories, we reported a significant correlation with multifocality, nodal involvement and extra-capsular invasion (P < 0.0001). Conclusions: Special techniques such as ICC and molecular markers might be successfully carried out on LBC-processed material. For both categories, ICC is more sensitive whereas BRAF analysis is an interesting support due to its high specificity adding a prognostic value in both SM and PTCs. [ABSTRACT FROM AUTHOR]

BACKGROUND The role of the plasminogen-plasmin (PLG-PLA) system in fertilization is unknown, although its dysfunction has been associated with subfertility in humans. We have recently detected and quantified plasminogen in the oviductal fluid of two mammals and showed a reduction in sperm penetration during IVF when plasminogen is present. The objective of this study was to describe the mechanism by which PLG-PLA system regulates sperm entry into the oocyte. METHODS AND RESULTS By combining biochemical, functional, electron microscopic, immunocytochemical and live cell imaging methods, we show here that (i) plasminogen is activated into the protease plasmin, by gamete interaction; (ii) urokinase-type and tissue-type plasminogen activators are present in oocytes, but they are not of cortical granule origin; (iii) sperm binding to oocytes triggers the releasing of plasminogen activators and (iv) the generated plasmin causes sperm detachment from the zona pellucida. CONCLUSIONS Our results describe a novel mechanism for the success or failure of fertilization in mammals, by which molecules present in the oviductal environment are activated by molecules originating within the gametes. We anticipate that therapeutic up- or down-regulation of this physiological mechanism may be used to help in conception or as a contraceptive tool. Since components of the PLG-PLA system are already available as drugs for heart attacks or cancer therapies, basic research on this novel function would be rapidly transferable for clinical application. [ABSTRACT FROM AUTHOR]

The article presents a study which investigates the Anethum graveolens flower (AGF) extract which inhibits a lipopolysaccharide (LPS)-induced inflammatory response by inducible nitric oxide synthase (iNOS) expression in macrophages. The study employs western blotting, immunocytochemistry, and reverse-transcriptase polymerase chain reaction (RT-PCR). Results suggest that AGF exerts an anti-inflammatory effect in LPS-stimulated RAW 264.7 cells by inhibiting iNOS expression.

Background and Aims The ultrastructure of the pollen tubes and the unusual multicellular stigmatic hairs of Trithuria, the sole genus of Hydatellaceae, are described in the context of comparative studies of stigmatic and transmitting tissue in other early-divergent angiosperms. Methods Scanning and transmission electron microscopy and immunocytochemistry are used to study the structure and composition of both mature and immature stigmatic hair cells and pollen-tube growth in Trithuria. Key Results Trithuria possesses a dry-type stigma. Pollen tubes grow within the cell walls of the long multicellular stigmatic hairs. Immunocytochemistry results suggest that arabinogalactan proteins are involved in attracting the pollen tubes through the stigmatic cuticle. Most tubes grow along the hair axis towards its base, but some grow towards the hair apex, suggesting that pollen tubes are guided by both physical constraints such as microfibril orientation and the presence of binding factors such as unesterified pectins and adhesive proteins. Conclusions The presence of a dry-type stigma in Trithuria supports the hypothesis that this condition is ancestral in angiosperms. Each multicellular stigmatic hair of Hydatellaceae is morphologically homologous with a stigmatic papilla of other angiosperms, but functions as an independent stigma and style. This unusual combination of factors makes Hydatellaceae a useful model for comparative studies of pollen-tube growth in early angiosperms. [ABSTRACT FROM PUBLISHER]

DESIGN: Fine-needle aspiration biopsy (FNAB) is the most reliable diagnostic tool in the diagnosis of thyroid nodules. A cytologic diagnosis of follicular neoplasm with atypical cells of undetermined significance (FN/AUS) implies that the selection of patients between surgery and follow-up is difficult. In this setting immunocytochemical stainings might be helpful. The efficacy of a panel made up of HBME-1 and Galectin-3 antibodies is evaluated in cases processed by liquid-based cytology (LBC). METHODS: Out of 7091 thyroid FNAB processed by LBC method, 120 cases undergoing surgery successively were selected. These cases were classified as benign lesion (BL, eight cases), FN, including the ACUS category of the Bethesda classification (FN/AUS, 50 cases), suspicious for malignancy (SM, 59 cases), and malignant neoplasm (MN, three cases). Immunostains for HBME-1 and Galectin-3 were carried out on the LBC slides. RESULTS: All MN and BL were histologically confirmed. FN/AUS and SM showed a malignancy risk of 24 and 72.9% respectively. The complete immunocytochemical panel was positive in 83.3% of the cases resulting in malignancy and negative in 87.5% of cases resulting in benign histology. Among the FN/AUS, the complete positive immunocytochemical panel was detected in 76.9% of cases resulting as malignant and the complete negative immunocytochemical panel was observed in 96.8% of cases resulting as benign at histology. CONCLUSIONS: The expression of HBME-1 and Galectin-3 in cases classified as FN/AUS on LBC-processed FNABs can effectively distinguish lesions, which need immediate surgery (high risk or FNH or Thy 3h) from those which can be followed-up (low risk or FNL or Thy 3l). [ABSTRACT FROM AUTHOR]

Manganese (Mn) has been associated with embryo toxicity as it impairs differentiation of neural and skeletogenic cells in vertebrates. Nevertheless, information on the mechanisms operating at the cellular level remains scant. We took advantage of an amenable embryonic model to investigate the effects of Mn in biomineral formation. Sea urchin (Paracentrotus lividus) embryos were exposed to Mn from fertilization, harvested at different developmental stages, and analyzed for their content in calcium (Ca), expression of skeletogenic genes, localization of germ layer markers, and activation of the extracellular signal-regulated kinase (ERK). By optical and immunofluorescence microscopy, we found that Mn exposure produced embryos with no skeleton, by preventing the deposition of the triradiate calcitic spicules usually produced only by specialized mesoderm cells. On the contrary, ectoderm and endoderm differentiation was not impaired. Endogenous Ca content in whole embryos and its localization in Golgi regions of skeletogenic cells was strongly reduced, as measured by atomic absorption spectrometry and in vivo calcein labeling. Spicule-lacking embryos showed persistent ERK activation by immunocytochemistry and immunoblotting, contrary to the physiological oscillations observed in normal embryos. The expression of the skeletogenic genes, Pl-msp130 and Pl-sm30, was also differentially affected if compared with controls. Here, we showed for the first time the ability of Mn to interfere with Ca uptake and internalization into skeletogenic cells and demonstrate that Ca content regulates ERK activation/inactivation during sea urchin embryo morphogenesis. The use of Mn-exposed sea urchin embryos as a new model to study signaling pathways occurring during skeletogenesis will provide new insights into the mechanisms involved in Mn embryo toxicity and underlie the role of calcium in the biomineralization process in vertebrates. [ABSTRACT FROM AUTHOR]

Plant cell vacuoles are diverse and dynamic structures. In particular, during seed germination, the protein storage vacuoles are rapidly replaced by a central lytic vacuole enabling rapid elongation of embryo cells. In this study, we investigate the dynamic remodeling of vacuolar compartments during Arabidopsis seed germination using immunocytochemistry with antibodies against tonoplast intrinsic protein (TIP) isoforms as well as proteins involved in nutrient mobilization and vacuolar acidification. Our results confirm the existence of a lytic compartment embedded in the protein storage vacuole of dry seeds, decorated by γ-TIP, the vacuolar proton pumping pyrophosphatase (V-PPase) and the metal transporter NRAMP4. They further indicate that this compartment disappears after stratification. It is then replaced by a newly formed lytic compartment, labeled by γ-TIP and V-PPase but not AtNRAMP4, which occupies a larger volume as germination progresses. Altogether, our results indicate the successive occurrence of two different lytic compartments in the protein storage vacuoles of germinating Arabidopsis cells. We propose that the first one corresponds to globoids specialized in mineral storage and the second one is at the origin of the central lytic vacuole in these cells. [ABSTRACT FROM PUBLISHER]

BACKGROUND Grafting of isolated follicles represents an approach to prevent the risk of reimplanting malignant cells with cryopreserved ovarian fragments. Optimal conditions and cell types required to sustain human follicular growth need to be identified. To help improve the grafting technique, we investigated whether short-term xenografting of a suspension containing ovarian stromal and endothelial cells without follicles could enhance graft survival and revascularization. METHODS In human ovary, CD34 selectively labels endothelial cells of blood vessels. A CD34-replete ovarian stromal cell group, including stromal and endothelial cells, was obtained after enzymatic digestion of fresh human ovarian cortex. Magnetic-activated cell sorting was used to establish a CD34-depleted ovarian stromal cell group. Proportions of CD34-positive cells were evaluated by flow cytometry and immunocytochemistry. Cell suspensions were embedded in human plasma clots and grafted (n = 10 for each group, 7 days) to the ovarian bursa of nude mice. Angiogenesis was quantified after human/mouse CD34 immunostaining. RESULTS CD34-replete grafts had a well-organized and vascularized stromal structure, containing tubular components staining for human CD34 and corresponding to functional vessels, as evidenced by intraluminal red blood cells. CD34-depleted grafts tended to be smaller than CD34-replete grafts and poorly vascularized with central necrosis. Global microvessel density was higher in the CD34-replete than depleted group (337.9 versus 187.3 vessels/mm2, P < 0.05), with a greater proportion of human vessels (68.02 versus 6.95%, respectively, P < 0.05). CONCLUSIONS We demonstrated the importance of co-transplanting ovarian endothelial and stromal cells to ensure the formation of a well-vascularized and structured ovarian-like stroma after short-term xenografting, for future application in the transplantation of isolated follicles. [ABSTRACT FROM AUTHOR]

Zea mays and Arabidopsis thaliana Brittle 1 (ZmBT1 and AtBT1, respectively) are members of the mitochondrial carrier family. Although they are presumed to be exclusively localized in the envelope membranes of plastids, confocal fluorescence microscopy analyses of potato, Arabidopsis and maize plants stably expressing green fluorescent protein (GFP) fusions of ZmBT1 and AtBT1 revealed that the two proteins have dual localization to plastids and mitochondria. The patterns of GFP fluorescence distribution observed in plants stably expressing GFP fusions of ZmBT1 and AtBT1 N-terminal extensions were fully congruent with that of plants expressing a plastidial marker fused to GFP. Furthermore, the patterns of GFP fluorescence distribution and motility observed in plants expressing the mature proteins fused to GFP were identical to those observed in plants expressing a mitochondrial marker fused to GFP. Electron microscopic immunocytochemical analyses of maize endosperms using anti-ZmBT1 antibodies further confirmed that ZmBT1 occurs in both plastids and mitochondria. The overall data showed that (i) ZmBT1 and AtBT1 are dually targeted to mitochondria and plastids; (ii) AtBT1 and ZmBT1 N-terminal extensions comprise targeting sequences exclusively recognized by the plastidial compartment; and (iii) targeting sequences to mitochondria are localized within the mature part of the BT1 proteins. [ABSTRACT FROM AUTHOR]

Several recent studies have expanded our conception of the role of astrocytes in neurogenesis, proposing that these cells may contribute to this phenomenon not only as a source of trophic substances, but also as stem cells themselves. We recently observed in vitro that human mature astrocytes can be induced to differentiate into cells with a neuronal phenotype. Antidepressant drugs have been shown to increase neurogenesis in the adult rodent hippocampus. In order to better understand the role of astroglia in antidepressant-induced neurogenesis, primary astrocyte cultures were treated with the antidepressant imipramine. Cell morphology was rapidly modified by treatment. In fact, whereas untreated astrocytes showed large, flat morphology, after a few hours of treatment cells exhibited a round-shaped cell body with long, thin processes. The expression of neuronal markers was analysed by immunocytochemistry, Western Blot and RT-PCR at different treatment times. Results showed an increase in neuronal markers such as neurofilament and neuron-specific enolase (NSE), whereas glial fibrillary acidic protein (GFAP) and nestin expression were not significantly modified by treatment. Similar results were obtained with fluoxetine and venlafaxine. Hes1 mRNA significantly increased after 2 h of treatment, suggesting involvement of this transcription factor in this process. These results confirm the role of astrocytes in neurogenesis and suggest that these cells may represent one of the targets of antidepressants. [ABSTRACT FROM AUTHOR]

Heparan sulfate (HS) has been found associated with amyloid deposits, including the toxic amyloid-beta (Aβ) peptide aggregates in cerebral vasculature and neuronal tissues in patients with Alzheimer's disease. However, the pathophysiological significance of the HS-Aβ interaction has remained unclear. In the present study, we applied cell models to gain insight into the roles of HS in relation to Aβ toxicity. Wild-type Chinese hamster ovary (CHO-WT) cells showed loss of viability following exposure to Aβ40, whereas the HS-deficient cell line, pgsD-677, was essentially resistant. Immunocytochemical analysis showed Aβ internalization by CHO-WT, but not pgsD-677 cells. Aβ40 toxicity was also attenuated in human embryonic kidney cells overexpressing heparanase. Finally, addition of heparin to human umbilical vein endothelial cells prevented internalization of added Aβ40 and protected against Aβ toxicity. Taken together, these findings suggest that cell-surface HS mediates Aβ internalization and toxicity. [ABSTRACT FROM PUBLISHER]

Phytomonas serpens, a phytoflagellate trypanosomatid, shares common antigens with Trypanosoma cruzi. In the present work, we compared the hydrolytic capability of cysteine peptidases in both trypanosomatids. Trypanosoma cruzi epimastigotes presented a 10-fold higher efficiency in hydrolyzing the cysteine peptidase substrate Z-Phe-Arg-AMC than P. serpens promastigotes. Moreover, two weak cysteine-type gelatinolytic activities were detected in P. serpens, while a strong 50-kDa cysteine peptidase was observed in T. cruzi. Cysteine peptidase activities were detected at twofold higher levels in the cytoplasmic fraction when compared with the membrane-rich or the content released from P. serpens. The cysteine peptidase secreted by P. serpens cleaved several proteinaceous substrates. Corroborating these findings, the cellular distribution of the cruzipain-like molecules in P. serpens was attested through immunocytochemistry analysis. Gold particles were observed in all cellular compartments, including the cytoplasm, plasma membrane, flagellum, flagellar membrane and flagellar pocket. Interestingly, some gold particles were visualized free in the flagellar pocket, suggesting the release of the cruzipain-like molecule. The antigenic properties of the cruzipain-like molecules of P. serpens were also analyzed. Interestingly, sera from chagasic patients recognized both cellular and extracellular antigens of P. serpens, including the cruzipain-like molecule. These results point to the use of P. serpens antigens, especially the cruzipain-like cysteine-peptidases, as an alternative vaccination approach to T. cruzi infection. [ABSTRACT FROM AUTHOR]

Background: Our knowledge of what determines the mammalian oocyte developmental competence is meagre. By comparing the transcriptional profiles of developmentally competent surrounded nucleolus (SN) and incompetent not surrounded nucleolus (NSN) mouse MII oocytes, we recently demonstrated that Oct-4 and Stella are key factors in the establishment of the oocytes' developmental competence. Methods: Using RT-PCR, microarray and immunocytochemistry assays, we analysed expression of genes and proteins in oocytes isolated throughout folliculogenesis and classified based on their SN- or NSN-type of chromatin organization. Results: We show that: (1) Oct-4 and Stella are expressed concurrently at the beginning of oocytes' growth and only in SN oocytes; (2) Germ Cell Nuclear Factor is a putative regulator of Oct-4 expression in MII oocytes; (3) the function of Oct-4 is directed at the Nanog locus, regulating the expression of Stella and Foxj2. Conclusions: (1) A number of factors that act upstream and downstream of Oct-4 emerge as candidate players in the acquisition of the oocyte's developmental competence; (2) we define molecular markers that identify a specific group of ovarian oocytes (SN) that have a potential to acquire developmental competence; (3) the presence of SN and NSN oocytes in human ovaries extends the interest of these results to the field of human reproduction. [ABSTRACT FROM AUTHOR]

Stem cell-based treatment is a promising frontier for neurodegenerative diseases. We propose a novel protocol for inducing the differentiation of rat mesenchymal stem cells (MSCs) toward neurotrophic factor (NTF)-secreting cells as a possible neuroprotective agent. One of the major caveats of stem cell transplantation is their fate post-transplantation. To test the viability of the cells, we tracked the transplanted cells in vivo by magnetic resonance imaging (MRI) scans and validated the results by histology. MSCs went through a two-step medium-based differentiation protocol, followed by in vitro characterization using immunocytochemistry and immunoblotting analysis of the cell media. We examined the migratory properties of the cells in the quinolinic acid (QA)-induced striatal lesion model for Huntington's disease. The induced cells were labeled and transplanted posterior to the lesion. Rats underwent serial MRI scans to detect cell migration in vivo. On the 19th day, animals were sacrificed, and their brains were removed for immunostaining. Rat MSCs postinduction exhibited both neuronal and astrocyte markers, as well as production and secretion of NTFs. High-resolution two-dimensional and three-dimensional magnetic resonance images revealed that the cells migrated along a distinct route toward the lesion. The in vivo MRI results were validated by the histological study, which demonstrated that phagocytosis had only partially occurred and that MRI could correctly depict the status of the migrating cells. The results show that these cells migrated toward a QA lesion and therefore survived for 19 days post-transplantation. This gives hope for future research harnessing these cells for treating neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

The POU-domain transcription factor OCT4 is associated with the pluripotent state of cells comprising the inner cell mass of pre-implantation embryos and has been known to play a critical role in the maintenance of pluripotency of embryonic stem cells. Reactivation of OCT4 expression is postulated to occur in differentiated cells that have undergone carcinogenesis, or tumor formation. In contrast to earlier studies, recent reports describe OCT4 expression in several human tumor cell lines. To resolve the apparent discrepancy in OCT4 expression between earlier and recent studies, we determined OCT4 expression in the cervical carcinoma cell line HeLa and the breast cancer cell line MCF7 in comparison with the human teratoma cell line nTera by immunofluorescence, Western blot, and RT-PCR analyses. We were unable to detect staining of the OCT4 transcription factor in the nucleus of HeLa and MCF7 cells by immunofluorescence using two different monoclonal antibodies. Faint cytoplasmic staining in HeLa and MCF7 cells was observed; however, no OCT4 signal could be detected by Western blot analysis. In addition, we were unable to detect significant levels of OCT4 mRNA in HeLa and in MCF7 cells by RT-PCR. Furthermore, the OCT4 promoter region is highly methylated in HeLa and MCF7 cells. We argue that recent reports of OCT4 expression in these and other cancer cell lines could actually be attributed to OCT4 pseudogene expression or misinterpretation of background signals in immunofluorescence experiments. In conclusion, we emphasize the need for adequate controls in investigations of OCT4 expression in somatic cell lines by immunofluorescence and RT-PCR. [ABSTRACT FROM AUTHOR]

Membrane-associated guanylate kinase inverted (MAGI)-1 plays a role as a scaffold at cell junctions in non-neuronal cells, while S-SCAM, its neuronal isoform, is involved in the organization of synapses. A search for MAGI-1–interacting proteins by yeast two-hybrid screening of a kidney cDNA library yielded dendrin. As dendrin was originally reported as a brain-specific postsynaptic protein, we tested the interaction between dendrin and S-SCAM and revealed that dendrin binds to the WW domains of S-SCAM. Dendrin is known to be dendritically translated but its function is largely unknown. To gain insights into the physiological meaning of the interaction, we performed a second yeast two-hybrid screening using dendrin as a bait. We identified CIN85, an endocytic scaffold protein, as a putative dendrin-interactor. Immunocytochemistry and subcellular fractionation analysis supported the synaptic localization of CIN85. The first SH3 domain and the C-terminal region of CIN85 bind to the proline-rich region and the N-terminal region of dendrin, respectively. In vitro experiments suggest that dendrin forms a ternary complex with CIN85 and S-SCAM and that this complex formation facilitates the recruitment of dendrin and S-SCAM to vesicle-like structures where CIN85 is accumulated. [ABSTRACT FROM PUBLISHER]

BACKGROUND: Hrb is an HIV-1 Rev-binding/interacting protein and is a cofactor for Rev export pathway. Hrb interacts with Eps15 homology (EH) domain-containing proteins and is a component of EH network and functions in vesicle sorting. Earlier, we reported that Hrb-deficient male mice are infertile and that they show oligozoospermia. Their sperm lack acrosomes and present globozoospermia. The aim of this study was: (i) to investigate the additional defects in spermatogenesis in Hrb-deficient mice; and (ii) to investigate the effect of acrosomelessness on spermatid differentiation in Hrb-deficient mice. METHODS: Hrb/ testes, epididymides, spermatids and sperm were analyzed by histology and electron microscopy. Centrioles were analyzed in spermatids and sperm by indirect immunofluorescence technique. RESULTS: Hrb/ male mice exhibited multiple anomalies during meiosis and spermiogenesis that produced developmentally impaired sperm with unshaped or deformed nuclei, loss in cell polarity, intracellular flagellar coiling, multinucleation, supernumerary centrioles and multiflagellation. A total of 13.0% Hrb/ sperm showed macrocephaly. The Hrb/ sperm exhibited variation in head size and shape, disarranged cellular organelles, nuclear and cytoplasmic vacuolization, mitochondrial loss or scattering and no forward motility. CONCLUSIONS: These aberrations, in Hrb/ mouse spermatids and sperm, are reminiscent of human familial male infertility with oligoasthenoteratozoospermia syndrome. The Hrb-deficient mouse may be useful in understanding familial oligoasthenoteratozoospermia syndrome. [ABSTRACT FROM AUTHOR]

Mutations inCOL17A1, coding for type XVII collagen, cause junctional epidermolysis bullosa with an ultrastructural plane of cleavage through the lamina lucida of the epidermal basement membrane.To identify theCOL17A1mutations in a child with reduced type XVII collagen expression and intraepidermal blister formation.Protein expression and level of tissue separation were studied by immunofluorescence and electron microscopy. The mutations were identified by analysing the patient's DNA and mRNA.Immunofluorescence microscopy performed on nonlesional skin demonstrated absence of the type XVII collagen endodomain and presence, although reduced, of the shed ectodomain. Electron microscopy showed that the plane of cleavage was through the basal cells, not through the lamina lucida. Two heterozygous mutations were identified inCOL17A1: a new 3′-acceptor splice-site mutation in intron 21 (1877–2A→C), and a deletion in exon 48 (3432delT). The splice-site mutation in intron 21 results in alternative transcripts of which two are in-frame, with deletions of the first nine codons of exon 22 and the entire exon 22, respectively. By Western blot analysis, a type XVII collagen molecule was detected that was slightly smaller than normal.Occasionally mutations in theCOL17A1gene may result in split levels suggesting epidermolysis bullosa simplex rather than junctional epidermolysis bullosa. [ABSTRACT FROM AUTHOR]

Pseudopelade of Brocq (PB) is an acquired progressive cicatricial alopecia which is characterized by some distinctive clinical features. It may represent either a distinct entity, i.e. an idiopathic primary scarring alopecia, or the end stage of various forms of scarring alopecia such as discoid lupus erythematosus (DLE) or lichen planopilaris (LPP). The aim of the study was to evaluate a set of patients with a clinically defined PB, to ascertain whether their PB was idiopathic or secondary, and then to study the phenotype of the inflammatory infiltrate and the presence of any fibrogenic and antifibrogenic cytokines to identify idiopathic or secondary forms in more detail. Twelve female patients with PB were studied by means of histology, direct immunofluorescence (DIF) and immunohistochemistry, by using monoclonal antibodies to cell markers (lymphocyte subtypes, Langerhans cells, macrophages, fibroblasts, mastocytes and activation markers) and fibrogenic and antifibrogenic cytokines. Using histology and DIF, we diagnosed two cases as DLE and three cases as LPP. Seven cases had nonspecific histology or DIF appearances and were classified as noncharacterized pseudopelade (NCPB). Two major phenotypic patterns of dermal infiltrate were identified by immunohistochemistry. These were: (i) a conspicuous infiltrate of CD3+ cells with a high CD4+/CD8+ ratio, variable numbers of macrophages, mast cells and fibroblasts always fewer than lymphocytes; (ii) an infiltrate of CD3+ cells with variable CD4+/CD8+ ratio and conspicuous amounts of macrophages, mast cells and fibroblasts, more numerous than infiltrating lymphocytes. The first pattern was typical of DLE and LPP, the second one was typical of NCPB. Fibrogenic cytokines were observed in all cases, but basic fibroblastic growth factor (bFGF) and transforming growth factor (TGF)-β were more strongly expressed in NCPB. Interferon (IFN)-γ was found in LPP. In our PB patients we identified five of 12 secondary PB and seven of 12 idiopathic PB by means of histology and DIF. The phenotypic pattern of infiltration allowed us to further differentiate secondary (richer in lymphocytes) from idiopathic PB (richer in resident cells). The pattern of cytokine expression showed the presence of fibrogenic molecules (interleukins 4 and 6, bFGF and TGF-β) in all cases, suggesting the involvement of mechanisms mediated by T-helper 2 and 3 cytokines in PB. [ABSTRACT FROM AUTHOR]

Ultraviolet (UV) irradiation has been involved in both initiation and promotion of carcinogenesis in melanoma. Alterations of cellular proliferation proteins, such as p73, Nup88, Id1 and p27 have been considered to play critical roles in melanoma development. However, the molecular mechanisms behind melanoma carcinogenesis are still poorly understood. In this study, we used human skin melanocytes as an experimental model system to investigate effects of UV irradiation on protein expression concerning cellular proliferation. The melanocytes prepared from human foreskin were separately exposed to various doses of UVA or UVB and post-cultivated for 24 or 48 h. Total proteins were isolated from the melanocytes, and expression of p73, Nup88, Id1, p27, bcl-2 and proliferating cell nuclear antigen (PCNA) proteins was examined by western blotting and immunocytochemistry. Results showed that expression of p73 and Nup88 was enhanced by UVA irradiation in a dose- and time-dependent manner. However, expression of Id1, p27, bcl-2 and PCNA proteins was not changed upon exposure to the UVA. Id1 and p27 proteins were over-expressed by exposure to UVB, but expression of p73, Nup88, bcl-2 and PCNA proteins was not changed by the UVB irradiation. The data suggested that UVA and UVB irradiation might lead to alterations of the different intracellular proteins. UVA enhanced protein expression concerning cell growth (p73 and Nup88) and UVB might over-express proteins concerning cellular proliferation (Id1 and p27). UVA and UVB may induce initiation of melanoma via separate intracellular pathways. [ABSTRACT FROM AUTHOR]