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Candida albicans is one of the most common clinical pathogenic microorganisms and it is becoming a serious health threat, particularly to immunocompromised populations. Drug resistance of Candida species has also frequently emerged, and combination therapy for fungal infections has attracted considerable attention. In this study, we established the Qinling Mountains myxobacterial secondary metabolites library and a synergic assay in combination with ketoconazole against C. albicans was introduced for metabolites screening. Two active compounds with synergic anticandidal activities were obtained, which were identified as trans-resveratrol and cis-resveratrol. According to our study, resveratrol can reduce the dosage to 1/64 of ketoconazole as well as itraconazole. Furthermore, synergistic anticandidal activity of resveratrol combined with azoles was verified against a panel of clinical C. albicans isolates, and the combination strategy enhanced the azoles susceptibility of three fluconazole-resistant isolates. These findings suggest that resveratrol enhances the efficacy of azoles and provides a promising application in therapy of C. albicans infection., (© 2021 The Society for Applied Microbiology.)

Background: Port-wine stain (PWS) is a vascular malformation characterized by progressive dilatation of postcapillary venules, but the molecular pathogenesis remains obscure., Objectives: To illustrate that PWS endothelial cells (ECs) present a unique molecular phenotype that leads to pathoanatomical PWS vasculatures., Methods: Immunohistochemistry and transmission electron microscopy were used to characterize the ultrastructure and molecular phenotypes of PWS blood vessels. Primary culture of human dermal microvascular endothelial cells and in vitro tube formation assay were used for confirmative functional studies., Results: Multiple clinicopathological features of PWS blood vessels during the development and progression of the disease were shown. There were no normal arterioles and venules observed phenotypically and morphologically in PWS skin; arterioles and venules both showed differentiation impairments, resulting in a reduction of arteriole-like vasculatures and defects in capillary loop formation in PWS lesions. PWS ECs showed stemness properties with expression of endothelial progenitor cell markers CD133 and CD166 in non-nodular lesions. They also expressed dual venous/arterial identities, Eph receptor B1 (EphB1) and ephrin B2 (EfnB2). Co-expression of EphB1 and EfnB2 in normal human dermal microvascular ECs led to the formation of PWS-like vasculatures in vitro, for example larger-diameter and thick-walled capillaries., Conclusions: PWS ECs are differentiation-impaired, late-stage endothelial progenitor cells with a specific phenotype of CD133 + /CD166 + /EphB1 + /EfnB2 + , which form immature venule-like pathoanatomical vasculatures. The disruption of normal EC-EC interactions by coexistence of EphB1 and EfnB2 contributes to progressive dilatation of PWS vasculatures., (© 2017 British Association of Dermatologists.)

Aims: LI-Fs are a family of highly potent cyclic lipodepsipeptide antibiotics with a broad antimicrobial spectrum (Gram-positive bacteria and fungi). In this study, LI-F-type antimicrobial peptides (AMP-jsa9) composing of LI-F03a, LI-F03b, LI-F04a, LI-F04b and LI-F05b were isolated from Paenibacillus polymyxa JSA-9. To better understand the antimicrobial mechanism of AMP-jsa9, the potency and action(s) of AMP-jsa9 against Bacillus cereus were examined., Methods and Results: Flow cytometry, confocal laser microscopy, scanning electron microscopy, transmission electron microscopy (TEM) and atomic force microscopy observation, as well as determination of peptidoglycan and cell wall-associated protein and other methods were used. The results indicate that AMP-jsa9 exhibits strong, broad-spectrum antimicrobial activity. Moreover, AMP-jsa9 targets the cell wall and membrane of B. cereus to impair membrane integrity, increase membrane permeability and enhance cytoplasm leakage (e.g. K + , protein, nucleic acid). This leads to bacterial cells with irregular, withered and coarse surfaces. In addition, AMP-jsa9 is also able to bind to DNA and break down B. cereus biofilms., Conclusions: In this study, the action mechanism of LI-Fs against B. cereus was clarified in details., Significance and Impact of the Study: The results of this study provide a theoretical basis for utilizing AMP-jsa9 or similar analogues as natural and effective preservatives in the food and feed industries. These efforts could also stimulate research activities interested in understanding the specific effects of other antimicrobial agents., (© 2017 The Society for Applied Microbiology.)

This study aims to explore the radiosensitivity of sunitinib on esophageal cancer cell lines. For in vitro studies, human esophageal squamous cell carcinoma (ESCC) cell lines were treated with sunitinib 24 hours before irradiation. ESCC cell lines were treated with sunitinib with or without radiation. Cell proliferation was detected by Cell Counting Kit 8 assay. Radiosensitization was evaluated by clonogenic survival assay. Cell apoptosis and cell cycle analysis were detected by flow cytometry. Deoxyribonucleic acid (DNA) double-strand breaks were performed by immunocytofluorescence analysis. Western blot analysis was used to determine the effect of sunitinib on radiation induced signal transduction. Sunitinib potently sensitized ESCC cells to radiation with a sensitization enhancement ratio of 1.13-1.72. Furthermore, sunitinib increased radiation induced DNA double-strand breaks, promoted the apoptosis of ESCC cells and induced the G2/M arrest. Radiosensitization was accompanied with enhanced apoptosis and regulated by the intrinsic pathway of apoptosis. Sunitinib sensitized ESCC cells to the cytotoxic effects of radiation. This compound is promising for future clinical trials with chemoradiation in esophageal cancer., (© 2015 International Society for Diseases of the Esophagus.)

Aims: Piliated Lactobacillus rhamnosus (pLR) strains have attracted much attention owing to their excellent mucus adhering capacity and immunomodulatory effects. Here, we aimed to develop a rapid, sensitive method for isolating pLR strains in complex ecosystems using immunomagnetic separation (IMS) with colony immunoblotting (CIB)., Methods and Results: Magnetic nanobeads (diameter: 180 nm) conjugated with anti-pLR SpaA pilin antibodies (anti-SpaA) were prepared and used to preconcentrate pLR strains in samples, followed by confirmation with anti-SpaA-based CIB analysis. Under optimized experimental conditions, IMS-CIB selectively recovered pLR strains from 10 7  CFU ml -1 of faecal microbiota samples spiked with 2·9 × 10 1 to 2·4 × 10 6  CFU ml -1 of pLR strains. No positive colonies were detected in samples without addition of pLR strains. The detection limit of IMS-CIB was 29 CFU pLR ml -1 of faecal microbiota, which is much lower than that of CIB without IMS preconcentration (2·0 × 10 4  CFU ml -1 )., Conclusions: IMS-CIB allowed selective preconcentration of pLR strains in highly heterogeneous bacterial suspensions and direct detection of pLR colonies, which remained readily available for subsequent isolation., Significance and Impact of the Study: Our findings established an effective method for selective enrichment and detection of pLR strains., (© 2016 The Society for Applied Microbiology.)

Aims: To reveal the microbial communities from Qinghai-Tibetan Plateau wetland soils that have the potential to be used in the utilization of cellulosic and chitinous biomass at low temperatures (≤25°C)., Methods and Results: Soil samples collected from six wetlands on Qinghai-Tibetan Plateau were supplemented with or without cellulose and chitin flakes, and anaerobically incubated at 25 and 15°C; high-throughput 16S rRNA gene sequencing was used to access the composition and localization (in the slurry and on the surface) of enriched microbial communities; a hypothetical model was constructed to demonstrate the functional roles of involved microbes mainly at genus level. Overall, microbial communities from Qinghai-Tibetan Plateau wetlands showed significant potential to convert both cellulose and chitin to methane at low temperatures; Clostridium III, Clostridium XIVa, Paludibacter, Parcubacteria, Saccharofermentans, Pelotomaculum, Methanosaeta, Methanobrevibacter, Methanoregula, Methanospirillum and Methanosarcina participated in methanogenic degradation of both cellulose and chitin through the roles of hydrolytic, saccharolytic and secondary fermenters and methanogens respectively. Acetotrophic methanogens were mainly enriched in the slurries, while hydrogenotrophic methanogens could be both in the slurries and on the surface., Conclusions: The composition and localization of microbial communities that could effectively convert cellulose and chitin to methane at low temperatures have been revealed by high-throughput 16S rRNA gene sequencing methods, and reviewing the literatures on the microbial pure culture helped to elucidate functional roles of significantly enriched microbes., Significance and Impact of the Study: This study will contribute to the understanding of carbon and nitrogen cycling of cellulose and chitin in cold-area wetlands and provide fundamental information to obtain microbial resources for the utilization of biomass wastes at low temperatures., (© 2016 The Society for Applied Microbiology.)

The granulomatous variant of the pigmented purpuric dermatoses (PPDs) is a rare and infrequently described condition, with a total of 16 cases published to date. We report a case of granulomatous PPD in a 59-year-old white woman who demonstrated involvement of the arms, legs, chest and back with concurrent hyperlipidaemia. Histopathological examination revealed a lymphohistiocytic infiltrate obscuring the dermoepidermal junction, and loose granuloma formation in the superficial dermis, with extravasated erythrocytes. Other conditions within the differential diagnosis such as atypical infection, papular sarcoidosis and generalized granuloma annulare were excluded on clinical and histological grounds. Our patient represents the ninth patient reported to have granulomatous PPD with coexisting hyperlipidaemia, and the fifth patient with granulomatous PPD and a lichenoid infiltrate., (© 2014 British Association of Dermatologists.)

Background: The recurrence of port-wine stain (PWS) blood vessels by pulsed dye laser (PDL)-induced angiogenesis is a critical barrier that must be overcome to achieve a better therapeutic outcome., Objectives: To determine whether PDL-induced angiogenesis can be suppressed by topical axitinib., Methods: The mRNA expression profiles of 86 angiogenic genes and phosphorylation levels of extracellular signal regulated kinases (ERKs), phosphorylated protein kinase B (AKT) and ribosomal protein S6 kinase (p70S6K) in rodent skin were examined with or without topical axitinib administration after PDL exposure., Results: The PDL-induced increased transcriptional levels of angiogenic genes peaked at days 3-7 post-PDL exposure. Topical application of 0·5% axitinib effectively suppressed the PDL-induced increase in mRNA levels of the examined angiogenic genes and activation of AKT, P70S6K and ERK from days 1 to 7 post-PDL exposure. After topical administration, axitinib penetrated into rodent skin to an approximate depth of 929·5 μm., Conclusions: Topical application of 0·5% axitinib can systematically suppress the PDL-induced early stages of angiogenesis via inhibition of the AKT/mammalian target of rapamycin/p70S6K and Src homology 2 domain containing transforming protein-1/mitogen-activated protein kinase kinase/ERK pathway cascades., (© 2014 British Association of Dermatologists.)

Aims: Tilletia controversa is an internationally quarantined pathogenic fungus that causes dwarf bunt of wheat and is similar to Tilletia caries in both teliospore morphology and genetic structure. This study developed a rapid and sensitive immunofluorescence method for differentiating the teliospores of T. controversa from T. caries., Methods and Results: The method utilizes monoclonal antibody D-1 against teliospores of T. controversa as well as a PE-Cy3-conjugated goat anti-mouse antibody (overlapping light excitation of 495 and 555 nm). The orange cycle fluorescent signal was stronger against T. controversa teliospores in the outer spore wall and net ridge, whereas only the green signal was observed for the protoplasm of T. caries teliospores. The detection limit of this method was 2.0 μg ml(-1) of the D-1 monoclonal antibody., Conclusion: This study describes the production and diagnostic application of a novel mouse monoclonal antibody specific to T. controversa teliospores., Significance and Impact of the Study: This method could be used for the on-site identification of T. controversa teliospores in the near future and will help in selecting fungicides to control dwarf bunt of wheat as further technical developments are achieved., (© 2014 The Society for Applied Microbiology.)

Objective: To explore the diffusion tensor imaging (DTI) characteristics of thigh muscles in patients with polymyositis (PM) and dermatomyositis (DM)., Methods: 12 patients with known PM/DM and 10 healthy volunteers were enrolled in this study. Both DTI and conventional MR sequences were performed on both thighs of all subjects. Apparent diffusion coefficient (ADC), fractional anisotropy (FA) and three eigenvalues were compared between the PM/DM group and the healthy group. One-way analysis of variance and Student's t-test were used for statistical analyses with a significance of p < 0.05., Results: In the healthy group, the vastus intermedius muscle showed the highest ADC value and the gracilis (GA) muscle showed the lowest ADC value. These results were statistically significant when compared with other muscles (p < 0.05). The GA, semi-tendinosus and semi-membranosus muscles showed higher FA values than the other three thigh muscles (p < 0.05). The mean ADC value and three eigenvalues of oedematous muscles in the PM/DM group were higher on average and showed a statistically significant difference when compared with unaffected (non-oedematous muscles in patients) and normal muscles (p < 0.05). There was no statistical difference in the mean FA value between oedematous and normal muscles. The mean ADC, FA and three eigenvalues in unaffected muscles (in patients) showed no statistical differences from those in normal muscles (p > 0.05)., Conclusion: DTI can be used to quantitatively evaluate the anisotropic diffusion characteristics of muscles in patients with PM/DM., Advances in Knowledge: A new application of DTI is proposed for inflammatory myopathies. The results show that ADC and the three eigenvalues were significantly different between diseased and normal muscles, a finding of potential value in both diagnosis and treatment monitoring of myopathies.

Aims: Wheat (Triticum aestivum) is one of the most important crop species, but yields are often drastically reduced by rust epidemics. In this report, we describe a rapid and sensitive immunofluorescence method for the detection of urediniospores of the fungi Puccinia striiformis f. sp. tritici, Puccinia triticina and Puccinia graminis f. sp. tritici, which are causal agents of wheat rust., Methods and Results: The method uses monoclonal antibody LPT-2 against the urediniospores of P. triticina and PE-cy3 goat anti-mouse. Urediniospores of P. triticina or those of two species that are difficult to distinguish from P. triticina, P. striiformis f. sp. tritici or P. graminis f. sp. tritici were immobilized on a glass slide, and the sample was then treated with LPT-2. Thereafter, a second antibody, goat anti-mouse conjugated PE-cy3, was added, and the slide was observed in a fluoroscope. The fluorescent signal was strong with P. triticina urediniospores, weak with P. striiformis f. sp. tritici urediniospores and weak-to-intermediate with P. graminis f. sp. tritici urediniospores. The detection limit of this method was 2 ng ml(-1) of the monoclonal antibody LPT-2., Conclusions: In this article, we describe the production and diagnostic application of a novel mouse monoclonal antibody specific to urediniospores of P. triticina., Significance and Impact of the Study: After further technical development, this method may become a tool for on-site identification of P. triticina urediniospores and will therefore help in the selection and timing of fungicide applications for control of wheat rust outbreaks., (© 2013 The Society for Applied Microbiology.)

Objective: The aim of this study was to evaluate the actual dose variability to the targets and organs at risk (OARs) during nasopharyngeal carcinoma (NPC) intensity-modulated radiotherapy (IMRT) and to investigate the significance of replanning., Methods: 11 NPC patients were included in this study. Each patient had both a planning CT and weekly repeated CT. Simulated plans that were generated by using the same beam configurations mapped to the repeated CT represented the actual delivered doses to the target volumes and OARs. An IMRT replanning was performed with the fifth week CT scan. Doses among the initial plan, the simulated plans and replanning were compared., Results: There were no significant dosimetric differences in the gross tumour volume, clinical target volume (CTV) 1 or CTV2 for either the simulated plans or the replanning compared with the initial plan. Dosimetric variability of both parotid glands and the brain stem were unique to each individual, and doses to the spinal cord were always maintained within the limit. Replanning in the fifth week had significantly decreased the doses delivered to both parotids (p-values of the mean dose were 0.015 and 0.026 for the left and right parotid, respectively), whereas it did not reduce the doses to the brain stem and spinal cord. There was no relationship between dose variability and weight loss., Conclusions: There are no significant dose changes for target volumes and spinal cord, and doses to the brain stem and both parotid glands changed individually during NPC IMRT. Replanning helps to spare bilateral parotids.

Background: dementia risk conferred by apolipoprotein-E (APOE) and angiotensin-1-converting enzyme (ACE) polymorphisms have been reported for the MRC Cognitive Function and Ageing Study (CFAS) at 6-year follow-up. We concentrate on incident dementia risk over 10 years., Methods: participants come from MRC CFAS, a multi-centre longitudinal population-based study of ageing in England and Wales. Three follow-up waves of data collection were used: 2, 6 and 10 years. Logistic regressions were undertaken to investigate associations between APOE (n = 955) and ACE (n = 856) alleles/genotypes and incident dementia. Two types of control groups were used: non-demented and highly functioning non-demented. Results were back-weighted., Results: compared to APOE epsilon3, epsilon2 conferred protection of odds ratio (OR) = 0.3 (95% confidence interval, CI = 0.1-0.6) and epsilon4 risk of OR = 2.9 (95% CI = 1.7-4.9) for incident dementia. Compared to epsilon3/epsilon3, the epsilon3/epsilon4 and epsilon4/epsilon4 genotypes conferred risks of OR = 3.6 (95% CI = 1.8-7.3) and OR = 7.9 (95% CI = 1.6-39.2), respectively. The epsilon3/epsilon2 genotype protected against dementia (OR = 0.2, 95% CI = 0.1-0.7), and epsilon2/epsilon2 had a similar protective effect but with wide CIs (OR = 0.3, 95% CI = 0.1-1.7). Restricting the control group accentuated these differentials. The effects of ACE alleles/genotypes on incident dementia risk were small., Conclusions: APOE but not ACE is associated with late-onset incident dementia in the population. Using longer term follow-up with proper adjustment for attrition and incident cases increases estimates of risk.

Aims: Dwarf bunt of wheat, caused by Tilletia controversa Kühn, is a destructive disease on wheat as well as an important international quarantined disease in many countries. The objective of this investigation was to develop a diagnostic molecular marker generated from amplified fragment length polymorphism (AFLP) for rapid identification of T. controversa., Methods and Results: A total of 30 primer combinations were tested by AFLP to detect DNA polymorphisms between T. controversa and related species. The primer combination E08/M02 generated a polymorphic pattern displaying a 451-bp DNA fragment specific for T. controversa. The marker was converted into a sequence-characterized amplified region (SCAR), and specific primers (SC-01(49)/SC-02(415)), designed for use in PCR detection assays, amplified a unique DNA fragment in all isolates of T. controversa, but not in the related pathogens. The detection limit with the primer set SC-01(49)/SC-02(415) was 10 ng of DNA which could be obtained from 11 microg of teliospores in a 25-microl PCR reaction., Conclusions: An approach to distinguish T. controversa from similar pathogenic fungi has been developed based on the use of a SCAR marker., Significance and Impact of the Study: Development of the simple, high throughput assay kit for the rapid diagnosis of dwarf bunt of wheat and detection of T. controversa is anticipated in further studies.

Aim: Spring viraemia of carp virus (SVCV) is the causative agent of SVC disease. The main aim of our study was to develop a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid, sensitive and effective detection of SVCV., Methods and Results: A set of four specific primers, two outer and two inner primers were designed based on the SVCV M gene for RT-LAMP assay. The sensitivity and specificity of RT-LAMP were determined and clinical test was performed under optimized amplification conditions (64 degrees C, 60 min). The results showed that the assay has a high specificity and the detection limit was 80 copies using 10-fold series dilutions of SVCV RNA, 10 times more sensitive than nest reverse transcription-polymerase chain reaction. In the detection of 472 fish samples, this assay showed excellent agreement with the standard virus isolation method (kappa = 0.807)., Conclusions: A sensitive and specific RT-LAMP assay was successfully developed to monitor and detect SVCV., Significance and Impact of the Study: This work provides a robust method for evaluating the risk of SVCV. Given the advantages of LAMP in the detection of SVCV, this method can be applied to diagnose other viruses, which pose serious threats to the aquaculture industry.

Reduced fibrinolytic activity has been described in primary anti-phospholipid syndrome (PAPS), and may be responsible for thrombotic events. Antibodies to tissue type plasminogen activator (t-PA) or plasminogen (PLG) might contribute to the hypofibrinolytic state in autoimmune diseases, but the clinical significance of these antibodies is still unclear in recurrent pregnancy loss (RPL). The aim of this study is to evaluate the prevalence and clinical significance of anti-PLG and anti-t-PA antibodies in 87 patients with a history of RPL: 54 women with well-defined PAPS (mean age 32.5 years; range 26-38) and 33 women with unexplained RPL (mean age 30 years; range 24-39). IgG anti-PLG antibodies were found in 20 and four patients from the group with RPL/PAPS and unexplained RPL, respectively; IgG anti-t-PA antibodies were found in 11 and two patients from the above two groups, respectively. IgG anti-PLG antibodies were associated with the high risk of RPL (OR 7.2, P = 0.004), especially with RPL/PAPS (OR 11.2, P < 0.001) evaluated by Fisher's exact test, while IgG anti-t-PA were associated with RPL/PAPS (OR 10.0, P = 0.01) but not with RPL (OR 6.8, P = 0.06). A significant inhibition of exogenous fibrinolysis was observed by IgG fractions from patients with anti-PLG or anti-t-PA antibodies on microplates and on the human umbilical vein endothelial cells, compared with those from healthy controls. The prevalence of IgG anti-PLG antibodies was high in RPL patients, especially in RPL/PAPS, while the prevalence of IgG anti-t-PA antibodies was high in RPL/PAPS but not in RPL, and some of them might inhibit fibrinolysis in patients.

Mesangial cell proliferation is a common cellular response to a variety of different types of glomerular injury. Complement C5b-9 is a prime candidate to mediate mesangial cell proliferation, especially sublytic C5b-9, which can induce the production of multiple inflammatory factors and cytokines. Transforming growth factor (TGF)-beta1 plays a major role in the accumulation of extracellular matrix (ECM), while thrombospondin (TSP)-1 has been identified as an activator of latent TGF-beta1 in an in vitro system. Using rat glomerular mesangial cells (GMCs) as a model system, we assessed the effect of sublytic C5b-9 on the expression of TSP-1 and TGF-beta1 and explored the relevant pathway of signal transduction. First, we ensured the concentrations of anti-Thy1 antibody and complement, which were regarded as a sublytic C5b-9 dose, and examined whether the sublytic C5b-9 induced expression of TSP-1 in rat GMCs which, in turn, activated latent TGF-beta1 by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Then, we investigated the role of the PI3-k/Akt pathway in sublytic C5b-9-induced TSP-1 production in rat GMCs by Western blot analysis. The addition of sublytic C5b-9 (5% anti-Thy1 antibody and 4% normal serum) to rat GMCs induced activation of latent TGF-beta1 via TSP-1. The addition of sublytic C5b-9 apparently increased the protein of Akt phosphorylation, whereas PI3-k inhibitor LY294002 could clearly reduce the increase of TSP-1 induced by sublytic C5b-9. These results indicate that TSP-1 is an activator of latent TGF-beta1 in sublytic C5b-9-induced rat GMCs; furthermore, the PI3-k/Akt signal transduction pathway may play a key role in sublytic C5b-9-induced TSP-1 production.

Adoptive antigen-specific immunotherapy is an attractive concept for the treatment of cancer because it does not require immunocompetence of patients, and the specificity of transferred lymphocytes can be targeted against tumour-associated antigens that are poorly immunogenic and thus fail to effectively trigger autologous T cell responses. As the isolation and in vitro expansion of antigen-specific lymphocytes is difficult, 'conventional' adoptive T cell therapy can only be carried out in specialized centres in small numbers of patients. However, T cell receptor (TCR) genes isolated from antigen-specific T cells can be exploited as generic therapeutic molecules for 'unconventional' antigen-specific immunotherapy. Retroviral TCR gene transfer into patient T cells can readily produce populations of antigen-specific lymphocytes after a single round of polyclonal T cell stimulation. TCR gene modified lymphocytes are functionally competent in vitro, and can have therapeutic efficacy in murine models in vivo. TCR gene expression is stable and modified lymphocytes can develop into memory T cells. Introduction of TCR genes into CD8(+) and CD4(+) lymphocytes provides an opportunity to use the same TCR specificity to produce antigen-specific killer and helper T lymphocytes. Thus, TCR gene therapy provides an attractive strategy to develop antigen-specific immunotherapy with autologous lymphocytes as a generic treatment option.

Gonococci (strain BS4(agar)), emerging from lag-phase during 1-1.5 h incubation in a medium containing glucose (28 mM) and either 5 microM or 50 microM sodium lactate, show enhanced capacity for their lipopolysaccharide (LPS) to be sialylated by cytidine 5'-monophospho-N-acetyl neuraminic acid. The sialyltransferase content of the lactate-treated gonococci was not greater than that of control organisms and showed no differences in LPS components. However, the total LPS content of the lactate-treated gonococci was 10-20% higher than that of control organisms, so lactate enhancement may be due to more sialyl receptors becoming available due to an overall stimulation of LPS synthesis. The protein and pentose contents of the lactate-treated gonococci were also higher than those of controls, indicating stimulation of protein synthesis and ribosome production. Electron microscopy showed hair-like external appendages on control but not on lactate-treated gonococci. The above growth conditions are unnatural. However, when concentrations of glucose and lactate were adjusted to values akin to those occurring in vivo (glucose 5 mM alone and with either 1 mM or 10 mM lactate), and gonococcal multiplication occurred during the short incubation period (1-1.5 h), lactate again induced greater contents of LPS, protein and pentose. A high content of LPS, which will contribute to pathogenicity, should be a constant feature of gonococci growing in human urogenital tissues, where lactate is ever present with glucose.

Coiled bodies (CBs) are nuclear organelles whose morphological structure and molecular composition have been conserved from plants to animals. Furthermore, CBs are often found to co-localize with specific DNA loci in both mammalian somatic nuclei and amphibian oocytes. Much as rDNA sequences are called nucleolus organizers, we term these coiled body-associated sequences 'coiled body organizers' (CBORs). The only sequences that have been shown to be CBORs in human cells are the U1, U2 and histone gene loci. We wanted to determine whether other snRNA genes might also act as CBORs. In this paper we show that human U3 genes (the RNU3 locus) preferentially associate with CBs in interphase cells. In addition, we have analyzed the genomic organization of the RNU3 locus by constructing a BAC and P1 clone contig. We found that, unlike the RNU1 and RNU2 loci, U3 genes are not tandemly repeated. Rather, U3 genes are clustered on human chromosome 17p11.2, with evidence for large inverted duplications within the cluster. Thus all of the CBORs identified to date are composed of either tandemly repeated or tightly clustered genes. The evolutionary and cell biological consequences of this type of organization are discussed.

Ribonuclease P (RNase P) is the enzyme responsible for endonucleolytically separating the 5'-leader sequence from precursor tRNA molecules. In bacteria, and in the nuclei and mitochondria of all eukaryotes studied so far, RNase P contains an RNA subunit which is necessary for activity in vitro and in vivo. In contrast, we showed earlier that partially-purified RNase P from spinach chloroplasts had physical properties inconsistent with the presence of any RNA. We now report that the properties of the chloroplast enzyme, after 500 to 1500-fold purification, are consistent with enzymatic activity residing in a approximately 70 kDa polypeptide. Gel filtration chromatography on Sephacryl S-200 and S-300 provides a mass for chloroplast RNase P of approximately 70 +/- 5 kDa. A single polypeptide of approximately 70-80 kDa can be crosslinked to iodoUMP-substituted pre-tRNA. The labeling intensity of this polypeptide corresponds closely to the peak of RNase P activity on Sephacryl S-200 chromatography. Unlike the bacterial ribozyme-type RNase P, chloroplast RNase P is not a metalloenzyme. We showed previously that phosphodiester bond cleavage by the E. coli RNA enzyme absolutely requires Mg2+ or Mn2+ coordinated to the pro-Rp oxygen of the scissile phosphodiester phosphate. In contrast, we now find that chloroplast RNase P has no such requirement, and can accurately and efficiently cleave pre-tRNA containing an Rp-thio-substitution at the scissile bond. These data are entirely consistent with the hypothesis that RNase P in plant chloroplasts is not a ribozyme, but a conventional protein enzyme.