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Ethnopharmacological relevance Tougu Xiaotong capsule (TXC) is a Chinese herbal compound that belongs to a range of Chinese herbs functioning as ‘kidney invigorators and liver softeners’ commonly used to treat osteoarthritis (OA) in China. Aims of the study: The aims of the present study are to confirm the therapeutic effects of TXC in an OA cell model and to determine the mechanisms involved in such effects. Materials and methods A tunicamycin (Tm)-exposed OA cell model was employed, and the effects of TXC were confirmed by observing cell viability and apoptosis. The reduced cell viability and increased apoptosis caused by Tm were improved by TXC, confirming the cellular protection of TXC. We then investigated the expression of biomarkers related to the endoplasmic reticulum (ER) stress pathway, including microRNA-211 (miR-211), a regulator in the ER stress pathway. Results Downregulation of X-box binding protein 1 (Xbp-1) and miR-211 expression following Tm administration was reversed by TXC. Moreover, the upregulation by Tm of the expression levels of binding immunoglobulin protein, Xbp-1, activating transcription factor 4, C/EBP-homologous protein, Caspase-9 and Caspase-3 was downregulated by TXC. These results indicated that the ER stress pathway-related mechanism may play a potential role in the therapeutic effects of TXC. Conclusions The present study provides evidence of the therapeutic effects of TXC at the cell level and describes a cellular model for establishing the mechanisms of the effects of TXC used in the treatment of OA.

Tuberculosis (TB) is a major global threat, mostly due to the development of antibiotic-resistant forms of Mycobacterium tuberculosis, the causal agent of the disease. Driven by the pressing need for new anti-mycobacterial agents several natural products (NPs) have been shown to have in vitro activities against M. tuberculosis. The utility of any NP as a drug lead is augmented when the anti-mycobacterial target(s) is unknown. To suggest these, we used a molecular reverse docking approach to predict the interactions of 53 selected anti-mycobacterial NPs against known &ldquo, druggable&rdquo, mycobacterial targets ClpP1P2, DprE1, InhA, KasA, PanK, PknB and Pks13. The docking scores/binding free energies were predicted and calculated using AutoDock Vina along with physicochemical and structural properties of the NPs, using PaDEL descriptors. These were compared to the established inhibitor (control) drugs for each mycobacterial target. The specific interactions of the bisbenzylisoquinoline alkaloids 2-nortiliacorinine, tiliacorine and 13&prime, bromotiliacorinine against the targets PknB and DprE1 (&minus, 11.4, &minus, 10.9 and &minus, 9.8 kcal&middot, mol&minus, 1, &minus, 12.7, &minus, 10.3 kcal&middot, 1, respectively) and the lignan &alpha, cubebin and Pks13 (&minus, 11.0 kcal&middot, 1) had significantly superior docking scores compared to controls. Our approach can be used to suggest predicted targets for the NP to be validated experimentally, but these in silico steps are likely to facilitate drug optimization.

The discovery of antimalarial artemisinin from Artemisia annua L. is an example of how Traditional Chinese Medicine (TCM) may be exploited to meet a recognized need. In this study, we systemically investigated A. annua L. for its antimicrobial activity and assessed it as a source of bioactive natural products for anti-mycobacterial activity.We used a silica gel column to perform antimicrobial activity-guided purification of the A. annua leaf, whose identity was confirmed by rbcL DNA barcoding, and used UHPLC-HRMS and NMR to elucidate the structure of purified active compounds. The antimicrobial activity of crude extracts, isolated compounds and the control artemisinin (Apollo Scientific Ltd) was assessed against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Mycobacterium smegmatis strains by serial micro dilution method (31.25-1000 μg/mL). The isolated compounds were tested for synergistic effects against mycobacterium.Bioactive compounds were purified and identified as deoxyartemisinin and artemisinic acid. Artemisinic acid (MIC 250 μg/mL) was more effective in comparison to deoxyartemisinin (MIC 500 μg/mL) and artemisinin (MIC 1000 μg/mL) against M. smegmatis. We used a molecular docking approach to investigate the interactions between selected anti-mycobacterial compounds and proteins involved in vital physiological functions in M. tuberculosis, namely MtPks13, MtPknB, MtPanK, MtKasA, MtInhA and MtDprE1 and found artemisinic acid showed docking scores superior to the control inhibiters for MtKasA, suggesting it to be a potential nick for further in vitro biological evaluation and anti-TB drug design.

Using an enzyme-linked immunosorbent assay (ELISA) and limited dilution methods to screen and clone antigen-specific hybridoma cells is extremely time-consuming and labor-intensive. This work features a simple and rapid cell surface fluorescence immunosorbent assay (CSFIA), designed for the detection and isolation of antigen-specific hybridoma clones. In this assay, antigens are first anchored to the hybridoma cell surface through a dual-functioning molecular Oleyl-PEG4000-NHS. Specific antibodies secreted from hybridoma cells are then captured by the antigens on the cell surface. Positive hybridoma cells are stained using a fluorescently labeled anti-mouse IgG-Fc antibody. After the addition of a methylcellulose semisolid medium, positive clones are easily picked using a pipet. These positive cell clones can be used to produce monoclonal antibodies after direct expansion. Using this method, positive hybridoma clones against both malachite green and porcine epidemic diarrhea virus are selected with high efficiency. Compared to the ELISA-based method, the CSFIA-based method achieved the capability of isolating2-fold more hybridoma clones in25% of the corresponding processing time. In brief, the CSFIA-based method is highly efficient and inexpensive with a simple and direct operation, which is an excellent candidate method for antigen-specific positive clone isolation in a monoclonal antibody preparation.

Expanded hexanucleotide GGGGCC repeat in a noncoding region of C9ORF72 is the most common cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). However, its molecular pathogenesis remains unclear. In our previous study, the expanded GGGGCC repeats have been shown to be sufficient to cause neurodegeneration. In order to investigate the further role of expanded GGGGCC repeats in the neuron, the normal r(GGGGCC)3 and mutant-type expanded r(GGGGCC)30 expression vectors were transfected into Neuro-2a cells. Cell proliferation, dendrite development, and the proteins’ levels of microtubule-associated protein-2 (MAP2) and cyclin-dependent kinase-5 (CDK5) were used to evaluate the cell toxicity of GGGGCC repeats on Neuro-2a cells. The results were shown that expression of expanded GGGGCC repeats caused neuronal cell toxicity in Neuro-2a cells, enhanced the expression of pMAP2 and pCDK5. Moreover, overexpression of Purα repaired expanded GGGGCC repeat-inducing neuronal toxicity in Neuro-2a cells and reduced the expression of pMAP2 and pCDK5. In all, our findings suggested that the expanded GGGGCC repeats might cause neurodegeneration through destroyed neuron cells. And the GGGGCC repeat-induced neuronal cell toxicity was inhibited by upregulation of Purα. We inferred that Purα inhibits expanded GGGGCC repeat-inducing neurodegeneration, which might reveal a novel mechanism of neurodegenerative diseases ALS and FTD.

Background Aconitum plants have been widely used in China for thousands of years. Recent evidences indicate that aconitine, the main active ingredient of Aconitum, has immunomodulatory properties that might be useful for treating autoimmune diseases, such as rheumatoid arthritis. In this study, we conducted a pilot study to explore the effect and mechanisms of aconitine on the treatment of systemic lupus erythematosus. Methods A pristane-induced murine model was used. The pristane-induced mice were treated with aconitine (25, 75 μg kg −1 d −1 , po ) for 9 weeks. Every three weeks, proteinuria was detected to monitor the kidney damage and blood was collected to measure serum levels of autoantibodies, besides the kidney pathological examination. The major B cell activating factor and major pro-inflammatory mediators, PGE2, IL-17a and IL-6, were also detected. Results We found that aconitine significantly improved the mouse health, decreased the elevated blood leukocyte counts, reduced the serum level of anti-double-stranded DNA (anti-dsDNA) antibody, greatly ameliorated renal histopathologic damage and reduced IgG deposit in glomerular. Furtherly, the levels of PGE2, IL-17a and IL-6, were found to have decreased in aconitine treated mice. Conclusion We have demonstrated that aconitine can inhibit the progression of disease and ameliorate the pathologic lesion of systemic lupus erythematosus.

The upregulation of ELTD1 ([epidermal growth factor (EGF), latrophilin and seven transmembrane domain-containing 1] on chromosome 1) in tumor cells has been reported in several types of cancer and correlates with poor cancer prognosis. However, the role of ELTD1 in glioma progression remains unknown. In this study, we examined ELTD1 expression levels in human glioma cell lines and in sixteen human gliomas of different grades. The molecular effects of ELTD1 in glioma cells were measured using quantitative polymerase chain reaction (qRT-PCR), Western blotting, Cell proliferation assays, Matrigel migration and invasion assays and brain orthotopic xenografts. We found that high expression levels of ELTD1 were positively associated with cancer progression and poor prognosis in human glioma. Mechanistically, ELTD1 activated the JAK/STAT3/HIF-1α signaling axis and p-STAT3 bound with HIF-1α. Taken together, our data provide a plausible mechanism for ELTD1-modulated glioma progression and suggest that ELTD1 may represent a potential therapeutic target in the prevention and therapy of glioma.

Receptor tyrosine kinases are believed to be activated through ligand-induced dimerization. We now demonstrate that in cultured neurons, a substantial amount of endogenous TrkB, the receptor for brain-derived neurotrophic factor (BDNF), exists as an inactive preformed dimer, and the application of BDNF activates the pre-existing dimer. Deletion of the extracellular juxtamembrane motif (EJM) of TrkB increased the amount of preformed dimer, suggesting an inhibitory role of EJM on dimer formation. Further, binding of an agonistic antibody (MM12) specific to human TrkB-EJM activated the full-length TrkB and unexpectedly also truncated TrkB lacking ECD (TrkBdelECD365), suggesting that TrkB is activated by attenuating the inhibitory effect of EJM through MM12 binding-induced conformational changes. Finally, in cells co-expressing rat and human TrkB, MM12 could only activate TrkB human-human dimer but not TrkB human-rat TrkB dimer, indicating that MM12 binding to two TrkB monomers is required for activation. Our results support a model that TrkB preforms as an inactive dimer and BDNF induces TrkB conformation changes leading to its activation.

The temporomandibular joint (TMJ), a unique synovial joint whose development differs from that of other synovial joints, develops from two distinct mesenchymal condensations that grow toward each other and ossify through different mechanisms. The short stature homeobox 2 (Shox2) gene serves an important role in TMJ development and previous studies have demonstrated that Shox2SHOX KI/KI mice display a TMJ defective phenotype, congenital dysplasia and premature eroding of the articular disc, which is clinically defined as a TMJ disorder. In the present study, Shox2SHOX KI/KI mouse models were used to investigate the mechanisms of congenital osteoarthritis (OA)‑like disease during postnatal TMJ growth. Shox2SHOX KI/KI mice were observed to develop a severe muscle wasting syndrome from day 7 postnatal. Histological examination indicated that the condyle and glenoid fossa of Shox2SHOX KI/KI mice was reduced in size in the second week after birth. The condyles of Shox2SHOX KI/KI mice exhibited reduced expression levels of collagen type II and Indian hedgehog, and increased expression of collagen type I. A marked increase in matrix metalloproteinase 9 (MMP9) and MMP13 in the condyles was also observed. These cellular and molecular defects may contribute to the observed (OA)‑like phenotype of Shox2SHOX KI/KI mouse TMJs.

Background Contrast-induced nephropathy (CIN) after coronary angiography is frequently observed in patients with chronic renal insufficiency and no effective measures have been developed for prevention of CIN. There is evidence showing that trimetazidine (TMZ) has renoprotective effect on CIN. This study was to evaluate the role of TMZ in the prevention of CIN in renal dysfunction patients undergoing coronary angiography. Methods A total of 132 patients with renal dysfunction who underwent coronary angiography were enrolled in our study and divided into control group (n = 70) and TMZ group (n = 62). Standard hydration was administered in all the patients. In TMZ group, patients were administered TMZ orally for 48 hours before and 24 hours after coronary angiography. Serum creatinine (SCr) and cystatin (CysC) were detected before and after contrast media injection, and the incidence of CIN was evaluated according to the elevation of SCr. Adverse events were observed in 12 months. Results In both groups, CysC and SCr increased significantly after coronary angiography and peaked at 24 and 48 hours, respectively. CysC and SCr were significantly lower in TMZ group than in control group after coronary angiography. The incidence of CIN and adverse events was reduced in TMZ group when compared with control group ( P = 0.034 and P = 0.043, respectively). Conclusions TMZ in combination with standard hydration is more effective than isotonic saline alone in protecting renal function in patients with renal dysfunction who undergo coronary angiography and can reduce the adverse events within 12 months.

Women are vulnerable to adverse stress events, especially during perimenopause. Substantial evidence has associated the impaired neuronal plasticity with abnormal behaviors under stressful conditions in animals. The Notch signaling pathway is critical for neuronal plasticity in the structure and function of brain areas. In this study, the mid-aged female rats were subjected to chronic restraint stress(CRS) in combination with isolated rearing for 6 weeks. The behavior tests and HPA activity were conducted to evaluate the model. The mRNA and protein levels of Notch1 signaling related genes in the hippocampus(HIP) and prefrontal cortex(PFC) were analyzed by RT-qPCR and western blotting. The promoter methylation levels were measured by bisulfite sequencing PCR analysis. CRS induced depression-like and anxiety-like behaviors in mid-aged stressed females, as shown by decreased locomotor activity, sucrose consumption and increased HPA activity. Moreover, after CRS, the rats exhibited decreased mRNA and protein levels in Jagged1, Notch1 and Hes5 in the HIP and Notch1, Hes1 and Hes5 in the PFC. However, there were no significant promotor methylation changes between the stressed and control female rats. These findings suggest that Notch1 signaling pathway may contribute to the behavioral changes following CRS in mid-aged female rats and the upstream cause of the gene expression changes needs to be further investigated.

While Brain-derived Neurotrophic Factor (BDNF) has long been implicated in treating neurological diseases, recombinant BDNF protein has failed in multiple clinical trials. In addition to its unstable and adhesive nature, BDNF can activate p75NTR, a receptor mediating cellular functions opposite to those of TrkB. We have now identified TrkB agonistic antibodies (TrkB-agoAbs) with several properties superior to BDNF: They exhibit blood half-life of days instead of hours, diffuse centimeters in neural tissues instead millimeters, and bind and activate TrkB, but not p75NTR. In addition, TrkB-agoAbs elicit much longer TrkB activation, reduced TrkB internalization and less intracellular degradation, compared with BDNF. More importantly, some of these TrkB-agoAbs bind TrkB epitopes distinct from that by BDNF, and work cooperatively with endogenous BDNF. Unlike BDNF, the TrkB-agoAbs exhibit a half-life of days/weeks and diffused readily in nerve tissues. We tested one of TrkB-agoAbs further and showed that it enhanced motoneuron survival in the spinal-root avulsion model for motoneuron degeneration in vivo. Thus, TrkB-agoAbs are promising drug candidates for the treatment of neural injury.

Plasma membrane (PM) and mitochondrial membrane (MM) proteins of rat hippocampal neurons were identified and comparatively characterized on the basis of a sequential digestion-centered combinative strategy for sample treatment. A total of 478 membrane proteins were identified, of which 240 had PM localization, 170 had MM localization, and 33 had both of the two subcellular localizations. Compared with the PM proteome, the MM proteome not only was smaller, more basic, and more hydrophobic, but also had a narrower protein molecular mass distribution range and a higher proportion of transmembrane proteins. By functional enrichment analysis, 287 molecular function terms for the PM proteome and 173 for the MM proteome were obtained. The MM proteome had a lower percentage of binding function terms and a higher percentage of catalysis function terms than the PM proteome, suggesting that mitochondrial proteins were more inclined to affect the physiological and biochemical processes by binding various molecules and as enzymes. Biological process enrichment showed that the genes of the PM and MM proteomes were mapped to 1104 and 460 biological processes, respectively. The biological processes with the most mapped genes of the PM proteome included those involved in vesicle recycling, transmitter release, neuronal development, protein and ion transport, etc., whereas those involved in electron transport, ATP synthesis, mitochondrial transport, mitochondrial apoptosis, etc., were the most gene-mapped biological processes for the MM proteome. The present work has deepened our understanding of the structure and function of hippocampal neurons and provided reference methods for research in the related field. Graphical abstract Functional comparison of the plasma membrane and mitochondrial membrane proteomes.

A novel acetolactate synthase (ALS) gene was cloned from Anabaena azotica , which was isolated from China and is regarded as a vital and beneficial photosynthetic microorganism that can contribute to soil fertility and maintain ecosystem stability. The full-length gene consists of an open reading frame (ORF) of 1644 bp and encodes a 59.2 kDa 547 amino acid protein. Sequence alignment using the BLAST similarity search in the GenBank database revealed that the amino acid sequence was similar to ALS derived from Anabaena sp. PCC 7120. The ALS gene was subcloned into the plasmid pET28a (+) and expressed in Escherichia coli BL21 (DE3) by IPTG induction. The recombinant protein was purified and subsequently characterized. The properties of ALS from the E. coli transformant are similar to that of an enzyme from the original A. azotica . Conserved amino acid sequence and the predicted structure of the active site were consistent with those observed for the bacterial enzyme Klebsiella pneumoniae ALS in similar experiments. Our results represent a significant functional demonstration of the existence of an algae ALS subunit and provide a solid basis for further studies on the structure, function and properties of ALS, as well as its response mechanism to inhibitors.

Background: Appropriate expression and regulation of the transcriptome, which mainly comprise of mRNAs and lncRNAs, are important for all biological and cellular processes including the physiological activities of bone microvascular endothelial cells (BMECs). Through an intricate intracellular signaling systems, the transcriptome regulates the pharmacological response of the cells. Although studies have elucidated the impact of glucocorticoids (GCs) cell-specific gene expression signatures, it remains necessary to comprehensively characterize the impact of lncRNAs to transcriptional changes. Methods: BMECs were divided into two groups. One was treated with GCs and the other left untreated as a paired control. Differential expression was analyzed with GeneSpring software V12.0 (Agilent, Santa Clara, CA, USA) and hierarchical clustering was conducted using Cluster 3.0 software. The Gene Ontology (GO) analysis was performed with Molecular Annotation System provided by CapitalBio Corporation. Results: Our results highlight the involvement of genes implicated in development, differentiation and apoptosis following GC stimulation. Elucidation of differential gene expression emphasizes the importance of regulatory gene networks induced by GCs. We identified 73 up-regulated and 166 down-regulated long noncoding RNAs, the expression of 107 of which significantly correlated with 172 mRNAs induced by hydrocortisone. Conclusions: Transcriptome analysis of BMECs from human samples was performed to identify specific gene networks induced by GCs. Our results identified complex RNA crosstalk underlying the pathogenesis of steroid-induced necrosis of femoral head.

Amyloid β-peptide (Aβ) has been implicated as a key molecule in the neurodegenerative cascades of Alzheimer's disease (AD). Humanin (HN) is a secretory peptide that inhibits the neurotoxicity of Aβ. However, the mechanism(s) by which HN exerts its neuroprotection against Aβ-induced AD-like pathological changes and memory deficits are yet to be completely defined. In the present study, we provided evidence that treatment of rats with HN increases the number of dendritic branches and the density of dendritic spines, and upregulates pre- and post-synaptic protein levels; these effects lead to enhanced long-term potentiation and amelioration of the memory deficits induced by Aβ(1-42). HN also attenuated Aβ(1-42)-induced tau hyperphosphorylation, apparently by inhibiting the phosphorylation of Tyr307 on the inhibitory protein phosphatase-2A (PP2A) catalytic subunit and thereby activating PP2A. HN also inhibited apoptosis and reduced the oxidative stress induced by Aβ(1-42). These findings provide novel mechanisms of action for the ability of HN to protect against Aβ(1-42)-induced AD-like pathological changes and memory deficits.

Magnetic manipulation of cell activity offers advantages over optical manipulation but an ideal tool remains elusive. The MagR protein was found through its interaction with cryptochrome (Cry) and the protein in solution appeared to respond to magnetic stimulation (MS). After we initiated an investigation on the specific role of MagR in cellular response to MS, a subsequent study claimed that MagR expression alone could achieve cellular activation by MS. Here we report that despite systematically testing different ways of measuring intracellular calcium and different magnetic stimulation protocols, it was not possible to detect any cellular or neuronal responses to magnetic stimulation in MagR-expressing HEK cells or primary neurons from the dorsal root ganglion and the hippocampus. By contrast, in neurons co-expressing MagR and channelrhodopin, optical but not magnetic stimulation increased calcium influx in hippocampal neurons. Our results indicate that MagR alone is not sufficient to confer cellular magnetic responses.

Tongue inspection is a unique and important method of diagnosis in traditional Chinese medicine (TCM). It is a diagnostic approach which involves observing the changes in the tongue proper and tongue coating in order to understand the physiological functions and pathological changes of the body. However, the biological basis of TCM tongue diagnosis remains to be poorly understood and lacks systematic investigation at the molecular level. In this study, we evaluated the effects of tongue coating microbiome on changes in the tongue texture and coating in patients with post-menopausal osteoporosis (PMO) of Gan‑shen deficiency syndrome type. Our aim was to delineate the mechanisms of tongue coating microbiome-induced changes in the tongue texture and coating by investigating the histomorphological changes and performing a bacterial analysis of the tongue coating. We found that the number of intermediate cells in the red tongue with a thin coating was higher, while the number of superficial cells in the red tongue with a thin coating was lower. The maturation value (MV) of tongue exfoliated cells in the red tongue with a thin coating decreased, compared with that in the pale red tongue with a thin white coating. Furthermore, the total bacterial count, oral streptococcus, Gram‑positive (G+) and Gram‑negative (G-) anaerobic bacteria in the red tongue with a thin coating was significantly decreased compared with the pale red tongue with a thin white coating. The results of ultrastructural examination demonstrated that the number of epithelial cells and bacteria in the red tongue with a thin coating decreased compared with that in the pale red tongue with a thin white coating. These observations indicate that the tongue coating microbiome may be an important factor contributing to changes in the tongue in patients with PMO of Gan‑shen deficiency syndrome type.

The ability of neurons in the adult mammalian central nervous system (CNS) to regenerate after injury is limited by inhibitors in CNS myelin. Nogo-66 is the most important myelin inhibitor but the mechanisms of Nogo-66 inhibition of neurite outgrowth remain poorly understood. Particularly, the relationship between Nogo-66 and microtubule-affinity regulating kinase 2 (MARK2) has not been examined. This study investigated the role of MARK2 in Nogo-66 inhibition and the function of MARK2 in neurite elongation in neurons in vitro. MARK2 and phosphorylated MARK2 at Ser212 (p-Ser212) alterations in Neuro 2a cells were assessed at different Nogo-66 exposure times; the relationships between MARK2 and microtubule-associated proteins (MAPs) were determined via the overexpression or interference of MARK2. Our study reports that Nogo-66 inhibited the expression of total MARK2 but also reduced Ser212 phosphorylation of MARK2, whereas levels of MAP1-b and tau varied depending on MARK2 overexpression or reduced expression. Furthermore, MARK2 increased the proportion of tyrosinated α-tubulin, thereby disrupting the stability of tubulin, most likely affecting axonal growth. In line with these results, overexpression of MARK2 promoted neurite elongation and therefore is able to rescue the inhibitory effect of Nogo-66 on neurite growth. In conclusion, the intracellular PKB/MARK2/MAPs/α-tubulin pathway appears to be essential for neurite elongation in neurons in vitro. These results suggest a critical role for MARK2 in overcoming Nogo-66-induced inhibition of axon outgrowth in neurons. Pharmacological activators of MARK2 may be applicable to promote successful axonal outgrowth following many types of CNS injuries.

Nogo-A, a member of the reticulon family, is one of the most important myelin-associated inhibitors for axonal growth, regeneration, and plasticity in the central nervous system. RhoA has been targeted pharmacologically to promote neurite outgrowth and functional recovery in the brain and spinal cord. However, the underlying mechanism of the inhibition of neurite outgrowth by Nogo-A has not yet been fully defined. Protein kinase B (PKB, also known as Akt) is a protein serine/threonine kinase that plays a key role in intracellular signaling and cellular homeostasis. This study reports the role of PKB signaling on Nogo-A-treated PC12 neuronal cells. An inhibitory fragment of Nogo-A (Nogo-66) activated RhoA and reduced the phosphorylation of PKB at Ser473 in a time-dependent manner. In contrast, pretreatment with Y27632, a specific inhibitor of Rho-A, resulted in an increase of the phosphorylation of PKB. Nogo-66 also inhibited the neurite outgrowth of PC12 cells, whereas pretreatment with LY294002, a specific inhibitor of PKB, ameliorated the neurite outgrowth. These data suggest that PKB is involved in the inhibition of neurite outgrowth by Nogo-A in PC12 cells.

The tropomyosin-related kinase A (TrkA) receptor and its ligand, nerve growth factor (NGF), play crucial roles in the development and function of the nervous system. NGF is believed to activate TrkA by bridging two TrkA monomers, leading to TrkA transphosphorylation. However, here we show that the majority of TrkA receptors exist as preformed, yet inactive, homodimers prior to NGF binding by using three different approaches such as chemical crosslinking and enzyme fragment complementation assay. Furthermore, TrkA homodimers are formed in endoplasmic reticulum before newly synthesized receptors reach the cell surface. These findings shed light on molecular mechanisms underlying transmembrane signaling by TrkA.Structured summaryTrkA physically interacts with TrkA by protein complementation assay (View interaction)TrkA physically interacts with TrkA by bimolecular fluorescence complementation (View interaction)TrkA physically interacts with TrkA by cross-linking study (View interaction)

When developing malaria vaccines, the most crucial step is to elucidate the mechanisms involved in protective immunity against the parasites. We found that CD8(+) T cells contribute to protective immunity against infection with blood-stage parasites of Plasmodium yoelii. Infection of C57BL/6 mice with P. yoelii 17XL was lethal, while all mice infected with a low-virulence strain of the parasite 17XNL acquired complete resistance against re-infection with P. yoelii 17XL. However, the host mice transferred with CD8(+) T cells from mice primed only with P. yoelii 17XNL failed to acquire protective immunity. On the other hand, the irradiated host mice were completely resistant to P. yoelii 17XL infection, showing no grade of parasitemia when adoptively transferred with CD8(+) T cells from immune mice that survived infection with both P. yoelii XNL and, subsequently, P. yoelii 17XL. These protective CD8(+) T cells from immune WT mice had the potential to generate IFN-gamma, perforin (PFN) and granzyme B. When mice deficient in IFN-gamma were used as donor mice for CD8(+) T cells, protective immunity in the host mice was fully abrogated, and the immunity was profoundly attenuated in PFN-deficient mice. Thus, CD8(+) T cells producing IFN-gamma and PFN appear to be involved in protective immunity against infection with blood-stage malaria.

Nitrogen-fixing cyanobacteria are vital photosynthetic microorganisms that contribute to soil fertility by fixing atmospheric nitrogen and are also important for maintaining ecosystem stability. These microorganisms can be very sensitive to herbicides because they possess many characteristics of higher plants. Six days after the application of monosulfuron at 0.03 to 0.3 nmol L 21 under laboratory conditions, growth of the nitrogen-fixing cyanobacteria Anabaena flos-aquae, Anabaena azollae, and Anabaena azotica was stimulated, but at higher concentrations (30 to 300 nmol L 21 ) protein synthesis was inhibited. The production of 16 amino acids in A. flos-aquae was reduced from 7 to 69% with increasing monosulfuron concentration. Application of monosulfuron at 3 to 300 nmol L 21 substantially inhibited in vitro acetolactate synthase (ALS) activity as indicated by 50% inhibition index values of 3.3, 65.2, and 101.3 nmol L 21 for A. flos-aquae, A. azollae, and A. azotica, respectively. In contrast, extractable ALS activity was not affected in these algal species with monosulfuron treatments ranging from 0.03 to 300 nmol L 1 except in A. flos-aquae at higher concentrations (30 to 300 nmol L 21 ). The most sensitive species to monosulfuron was A. flos-aquae, followed by A. azollae and A. azotica. Molecular analyses showed that the genomic DNA of A. azollae and A. azotica differed in only one amino acid. Results from photogenetic analyses revealed a high degree of homology between these algae. In contrast, the genomic DNA of A. flos-aquae differed from that of A. azollae and A. azotica in 44 and 45 amino acids, respectively. Our findings support the view that monosulfuron toxicity in these three nitrogen-fixing cyanobacteria is due to interference with protein metabolism via inhibition of branch-chain amino acid biosynthesis, and particularly ALS activity. Nomenclature: Monosulfuron; Anabaena flos-aquae (Lyngb) Breb; Anabaena azollae Strasb; Anabaena azotica Ley. Nitrogen-fixing cyanobacteria are vital photosynthetic microorganisms that contribute to soil fertility by fixing atmospheric nitrogen (Habte and Alexander 1980; Sinha and Kumar 1992) and maintaining ecosystem stability (Irisarri et al. 2001; Shen and Lu 2005). The agronomic importance of free-living symbiotic cyanobacteria as biofertilizers has been recognized (Habte and Alexander 1980). Some cyanobacterial strains that thrive in fields release small quantities of ammonia, as well as other small nitrogenous polypeptides during active growth. Recent research by Shen and Lu (2005) demonstrated that the application of a mixture of 12 species of nitrogen-fixing cyanobacteria (cultured for 90 d; 1 g dry algae weight per liter) after the sowing of a rice (Oryza sativa L.) crop was more beneficial to rice yield and soil fertility than the application (0.78 g L 21 ) of a urea fertilizer [CO(NH2)2] or a nonfertilizer control treatment. After the addition of the algal mixture to the rice paddies, total nitrogen content of rice stems and leaves increased by 27 and 39%, and rice yields increased by 9 and 12% relative to the urea fertilizer and control treatments, respectively. Three of the most common cyanobacteria in agricultural fields in China are the filamentous nitrogen-fixing species Anabaena azotica Ley (a free-living soil cyanobacterium with a high nitrogen-fixing capability), Anabaena flos-aquae (Lyngb) Breb. (a free-living soil cyanobacterium that may be toxic to some organisms), and Anabaena azollae Strasb (a cyanobacterium that forms a symbiotic relationship with the water fern, Azolla spp.) (Li 1962; Shen and Li 1993; Wu and Zhou 2004).

The effects of monosulfuron on growth and photosynthetic pigments of the nitrogen-fixing cyanobacterium Anabaena flos-aquae grown exposed to 2000-, 3000-, and 4000-lux light intensity were studied. Exposed to three light intensities, the seven concentrations of monosulfuron tested can significantly inhibit algal growth in a dose-dependent manner. The cell numbers and growth rate were decreased with the increase in mono-sulfuron concentration, and A. flos-aquae had different degrees of sensitivity to monosulfuron with the most sensitive light intensity being 4000-lux followed by 3000-lux and 2000-lux. The herbicide monosulfuron appeared to have different effects on the synthesis of photosynthetic pigments. The chlorophyll appeared to tackle monosulfuron concentrations. The caroteniod content of algae treated with 0.008 and 0.08 mg/L monosulfuron exposed to 2000-lux had a different stimulatory effect from that of treatments exposed to 3000-lux and 4000-lux, but an inhibitory effect at concentration above 0.8 mg/L. The effect of monosulfuron on biliprotein in cells of A. flos-aquae exposed three light intensities displayed contrary dose dependence.

Acquired immunity against infection with Trypanosoma cruzi is dependent on CD8 + T cells. Here, to develop a vaccine strategy taking advantage of activated CD8 + T cells, we constructed a DNA vaccine, designated pGFP-TSA1, encoding a fusion protein linking GFP to a single CTL epitope of TSA1, a leading candidate for vaccine against T. cruzi . C57BL/6 mice vaccinated with this plasmid showed suppressed parasitemia and prolonged survival. Vaccination with pGFP-TSA1 enhanced epitope-specific cytotoxicity and IFN-γ secretion by CD8 + T cells. Furthermore, the depletion of CD8 + T cells prior to challenge infection with T. cruzi completely abolished this protection, indicating that CD8 + T cells are the principal effector T cells involved. When mice deficient in the proteasome activator PA28α/β or the immunoproteasome subunits LMP2 and LMP7 were used, the protective immunity against infection was profoundly attenuated. Our findings clearly demonstrate that vaccination with pGFP-TSA1 successfully induces protection dependent on CD8 + T cell activation, in which immunoproteasomes play a crucial role. It is noteworthy to document that physical binding of the epitope and GFP is required for induction of this protection, since mice vaccinated with pTSA1-IRES-GFP failed to acquire resistance, probably because the epitope and GFP are separately expressed in the antigen-presenting cells.

The aim of the present study was to investigate the underlying mechanism of the Kidney-Yang deficiency (KYD) pattern of osteoporosis in postmenopausal women of a certain age range by comparing the effect of serum from postmenopausal women with osteoporosis exhibiting the KYD pattern with that of serum from postmenopausal women without osteoporosis on bone formation in an hFOB 1.19 human osteoblastic cell line. A random selection of 30 female, postmenopausal volunteers aged 60-70 years, including 15 cases without osteoporosis and 15 cases with the KYD pattern of osteoporosis, were enrolled at the Physical Examination Center of the Second Affiliated Hospital of Fujian University of Traditional Chinese Medicine. Venous blood was extracted and the serum was separated. The hFOB 1.19 cells were treated with 10% KYD pattern-serum or control serum from postmenopausal women of the same age range without osteoporosis. It was found that the KYD pattern-serum significantly decreased the cell viability, activity of alkaline phosphatase and number of calcified nodules, as well as downregulated the expression of osteocalcin and osteoprotegerin (OPG) and upregulated that of receptor activator of nuclear factor κB ligand (RANKL) in the hFOB 1.19 cells. In addition, the present results showed that the concentrations of estradiol (E2), OPG and insulin-like factor-1 (IGF-1) in the KYD pattern-serum were lower than those in the control serum. In combination, these findings suggest that the downregulation of E2, OPG and IGF-1 in the KYD pattern-serum inhibits the OPG/RANKL system, leading to a decrease in bone formation in the hFOB 1.19 cells. This indicates that the alterations in E2, OPG and IGF-1 may account for the susceptibility of certain postmenopausal women to the KYD pattern of osteoporosis.

Appropriate gene expression patterns form the basis for bone microvascular endothelial cells' function in femoral head. Although previous studies have elucidated the impact of glucocorticoids on these cells' specific gene expression the exact differential transcriptomes and comprehensive gene expression profiles remain unknown. Using microarray-based platforms we investigated the transcriptome patterns before and after hydrocortisone administration of bone microvascular endothelial cells from human femoral head. Our results highlight the involvement of development differentiation and apoptosis in the bone microvascular endothelial cells. Elucidation of differential gene expression before and after hydrocortisone administration emphasizes the importance of regulatory networks to gene co-expression within biological processes induced by glucocorticoids. With Benjamini–Hochberg characterization we identified 73 up-regulated and 166 down-regulated long noncoding RNAs the expression of 107 of which significantly correlated with 172 mRNAs after administration of hydrocortisone. Transcriptome analysis of bone microvascular endothelial cells from human femoral head samples is highly informative because it is deduced from data comprised of large number of genes expressed above background. The data have been submitted to the repository of Gene Expression Omnibus (Series GSE60332).

MUT1 is an H-2Kb-restricted 8-mer CTL epitope expressed in Lewis lung carcinoma (3LL) tumor cells derived from C57BL/6 (B6) mice. We constructed a chimeric gene encoding ubiquitin-fused MUT1 (pUB-MUT1). By using a gene gun, B6 mice were immunized with the gene prior to challenge with 3LL tumor cells. Tumor growth and lung metastasis were prominently suppressed in mice immunized with pUB-MUT1 but only slightly in those immunized with the MUT1 gene (pMUT) alone. CD8+ T cells were confirmed to be the final effector by in vitro experiments and in vivo removal of the cells with a corresponding antibody. Anti-tumor immunity was profoundly suppressed in mice deficient in an immuno-subunit of proteasome, LMP7. Furthermore, mice deficient in a proteasome regulator, PA28alpha/beta, failed to acquire protective immunity. Thus, application of the ubiquitin-fusion degradation pathway was useful even in immunization with genes encoding a single CTL epitope for induction of specific and active CD8+ T cells.

Laboratory and greenhouse studies were conducted to determine the effect of temperature, soil moisture, light, planting depth, and rhizome water content on shoot emergence and vegetative growth of alligatorweed. Optimum shoot emergence and growth occurred at constant 30 C, and no shoot emergence was found below constant 5 C. A maximum shoot emergence of 93% occurred at constant soil moisture of 30% with temperatures of 10 to 35 C. Shoot emergence and growth decreased as rhizome water content decreased, and shoot emergence did not occur below a rhizome water content of 20%. Shoot emergence and growth decreased with burial depth; shoot emergence was above 90% when rhizomes were buried 0.5 to 1.0 cm deep compared to 16% when they were buried 18 cm deep. Alligatorweed shoot emergence and vegetative growth were not significantly affected by light. In the fields, shoot emergence began in late March and culminated in May and June. These data help explain why this species is most commonly found in crop fields, orchards, roadsides, rivers, lakes, ponds, and irrigation canals. This information may aid in the development of more effective management measures, such as bringing alligatorweed rhizomes to the surface or below 20 cm deep to restrain its emergence and growth at winter or summer plowing.

The chemokine stromal cell-derived factor-1 (SDF-1) has been implicated in homing of bone marrow cells to sites of injury. We investigated the time course of myocardial SDF-1 expression and effects of intravenously administered bone marrow mesenchymal stem cells (MSC) in rats with myocardial infarction (MI).SDF-1 expression was measured by RT-PCR and Western blot in sham operated or infarcted hearts at 1/2, 1, 2, 4, 8 and 16 days post operation. MSCs from donor rats were labeled with BrdU. A total of 5 x 10(6) cells in 2.5 mL of PBS or equal volume PBS alone were injected through the tail vein at above mentioned time points. The number of the labeled MSCs in the infarcted hearts was counted 3 days post injection. Cardiac function and vessel numbers were assessed 28 days post injection.Myocardial SDF-1 expression increased and peaked at the first day and decreased thereafter post MI and remained unchanged in sham operated hearts. The MSCs enrichment and angiogenesis in the host hearts were more abundant in the 1 day transplantation group than in the other groups (P0.01). Cardiac function was only improved in rats received intravenous MSCs injection within 4 days post MI and not affected by PBS injection.Myocardial SDF-1 expression was increased only in the early phase post MI. MSCs intravenous infused at the early phase of MI were recruited to injured heart, enhanced angiogenesis and improved cardiac function.

A new subfamily of the Nudix hydrolases, identified by conserved amino acids upstream and downstream of the Nudix box, has been characterized. The cloned, expressed, and purified orthologous enzymes have major activities on the non-canonical nucleoside triphosphate 5-methyl-UTP (ribo-TTP) and the canonical nucleotide UTP. In addition to their homologous signature sequences and their similar substrate specificities, the members of the subfamily are inhabitants of or are related to the bacterial rhizosphere. We propose the acronym and mnemonic, utp, for the gene designating this unique UTPase.

All 21 of the Nudix hydrolase genes from the radiation-resistant organism Deinococcus radiodurans have been cloned into vectors under the control of T7 promoters and expressed as soluble proteins in Escherichia coli. Their sizes range from 9.8 kDa (91 amino acids) to 59 kDa (548 amino acids). Two novel proteins were identified, each with two Nudix boxes in its primary structure, unique among all other known Nudix hydrolases. Extracts of each of the expressed proteins were assayed by a generalized procedure that measures the hydrolysis of nucleoside diphosphate derivatives, and several enzymatic activities were tentatively identified. In addition to representatives of known Nudix hydrolase subfamilies active on ADP-ribose, NADH, dinucleoside polyphosphates or (deoxy)nucleoside triphosphates, two new enzymes, a UDP-glucose pyrophosphatase and a CoA pyrophosphatase, were identified.

Gallic acid (GA), a natural agent, is widely distri-buted in plants with a range of biological effects and has been of potential interest as anticancer agent. However, its effects on chondrosarcoma cell apoptosis are still undefined. In the present study, the possible mechanisms of GA-induced apoptosis were explored in SW1353 cells, a human chondrosarcoma cell line. Our results showed that GA inhibited cell viability dose- and time-dependently. Morphological examination of GA-treated cells exhibited the typical features of cell death, such as rounding up of the cells and cell shrinkage. Wound-healing assay indicated that GA inhibited the migratory abilities of SW1353 cells. Hoechst 33258 staining assay and Annexin V/PI staining assay exhibited apoptosis induction by GA. To determine the molecular mechanism of GA-induced apoptosis, the expression levels Bcl-2, Bax, caspase-3 and caspase-9 were determined in SW1353 cells treated with GA. We found that GA downregulated the expression of the anti-apoptotic protein Bcl-2, and upregulated the expression of the pro-apoptotic protein Bax, and the activation of caspase-3 and caspase-9. To identify the possible mechanisms, the changes of microRNA expression were tested using the miRCURY™ LNA expression array. It was observed that the miR-518b gene was upregulated in treated cells. Taken together, these data show that GA induces apoptosis and inhibits cell migration by upregulating miR-518b in SW1353 cells.

N-acetylserotonin (NAS) is synthesized from serotonin by arylalkylamine N -acetyltransferase (AANAT), which is predominantly expressed in the pineal gland and retina. NAS activates TrkB in a circadian manner and exhibits antidepressant effects in a TrkB-dependent manner. It also enhances neurogenesis in hippocampus in sleep-deprived mice. Here we report the identification of NAS derivatives that possess much more robust neurotrophic effects with improved pharmacokinetic profiles. The compound N -[2-(5- h ydroxy-1H- i ndol-3-yl)ethyl]-2- o xopiperidine-3- c arboxamide (HIOC) selectively activates TrkB receptor with greater potency than NAS. It potently protects retinas from light-induced retinal degeneration (LIRD), which is tightly coupled with pronounced TrkB activation in retinas. Pharmacokinetic studies demonstrate that this compound is stable in serum and liver microsomes. It can pass the blood–brain barrier and blood–retinal barrier. Hence, HIOC is a good lead compound for further drug development for treating retinal degenerative diseases.

The axons of the adult mammalian brain and spinal cord fail to regenerate after injury, and it has been suggested that Nogo-66 could prevent CNS axon repair. However, the mechanism of Nogo-66 inhibiting neurite outgrowth remains unknown. Our previous results indicated that protein kinase B (PKB) is involved in the inhibition of the neurite outgrowth by Nogo-66. Glycogen synthase kinase-3β (GSK-3β) is implicated in many processes in the nervous system, including differentiation, specification, polarity, plasticity and axon growth. In addition, GSK-3β is one of the most important molecules downstream of PKB. In the present study, we report on the role of GSK-3β signaling on Nogo-66-treated mouse neuroblastoma N2a cells. Nogo-66 reduced the phosphorylation of GSK-3β at Ser9 in N2a cells. In contrast, pretreatment with SB216763, a specific inhibitor of GSK-3β, resulted in an amelioration of neurite outgrowth by Nogo-66, compared with the Nogo-66 alone group (P

The effects of pH values, nutrients, and preserving temperature on akinete formation in Anabaena flos-aquae were examined at 30 ± 2°C exposed to fluorescent light at an intensity of 3000 lux. A marked effect of pH 6 and pH 7 on akinete formation was observed. At the third day, A. flos-aquae subjected to pH 6 and pH 7 developed the maximum frequency of akinetes. Saccharose and glucose are also important factors inducing akinete formation, with the frequency of 15% and 11%, respectively. Besides, lower temperature (4°C) makes a great contribution for the strain to possess the potential to form akinetes.

The effects of monosulfuron on growth and photosynthetic pigments of the nitrogen-fixing cyanobacterium Anabaena flos-aquae grown mixotrophically exposed to 0.05, 0.20, and 0.80 mg/L N-content were studied. Exposed to three N content, the five concentrations of monosulfuron tested can significantly inhibit algal growth in a dose-dependent manner. The growth rate were decreased with the increase in monosulfuron concentration, and A. flos-aquae had different degrees of sensitivity to monosulfuron with the most sensitive N-content being 0.80 mg/L followed by 0.20 and 0.05 mg/L. The herbicide monosulfuron appeared to have markedly inhibition effects on the synthesis of photosynthetic pigments. The chlorophyll a, caroteniod, and biliprotein appeared to tackle monosulfuron concentration and N-content.

Nogo-A is a myelin-associated neuronal growth inhibitory molecule in central nervous system after trauma. However, the physiological functions of Nogo-A in neural development and in healthy oligodendrocytes are largely unknown. In this study, we investigated the expression of Nogo-66 receptor (NgR) protein in 60 cases of human astrocytoma by Western blot RT-PCR and immunohistochemistry. The correlation between the expression of NgR and pathologic grades of astrocyoma was further analyzed. The results showed that the expression of NgR protein and NgR mRNA immunoreactivity score, were decreased markedly with the increasing pathological grades. Double immunostaining results showed that Nogo-A and NgR were colocalized at the interface of astrocytoma cells and extracellular matrix. Our results indicated that NgR may have inhibitory effects on tumor activity and Nogo-A may restrict migration of tumor cells via NgR.

Data from laboratory studies are presented to show that organic solvent acetone exhibited a stimulatory effect on the growth of both ubiquitous nitrogen-fixing cyanobacteria, Anabaena azotica and Anabaena flos-aquae at lower concentrations (0.001~1%) and inhibitory effect at a higher concentration (10%). The similar effect was also observed for acetone on photosynthetic functions which may be suggested that use of the growth and photosynthetic pigments of the both cyanobacteria as sensitive indicator index of toxicity to microorganism in environment.

Exposed to three light intensities, the six concentrations of monosulfuron tested can significantly inhibit the growth of Auabaena flos-aquae in a dose-dependent manner. The herbicide monosulfuron appeared to have different effects on the synthesis of photosynthetic pigments. A. flos-aquae had different degrees of sensitivity to monosulfuron with the most sensitive light intensity being 4000-lux followed by 3000-lux and 2000-lux.

Application of monosulfuron, a new sulfonylurea herbicide, produced a simulative effect on heterocyst formation and nitrogenase activity but an inhibitory effect on photosynthesis, i.e., a lower net photosynthetic rate, fewer photosynthetic pigments, and a smaller Fv/Fm ratio at increasingly higher monosulfuron concentrations (0.001-10 mg/l) for three nonspecific filamentous nitrogen-fixing cyanobacteria: Anabaena azollae, A. flos-aquae, and A. azotica. The decrease in biliprotein of algal cells was less than that of carotenoid and chlorophyll-a. Monosulfuron was more readily degraded and less accumulated in A. azotica compared with A. azollae and A. flos-aquae. The three algae exhibited varying degrees of sensitivity to monosulfuron: Calculated 50% inhibition concentrations (IC(50)s) of algal growth and no observed-effect concentration (NOEC) values after 4 days of treatment were 0.014 and 0.005, 0.029 and 0.019, and 0.22 and 0.075 mg/l for A. flos-aquae, A. azollae, and A. azotica, respectively. Normal agricultural use of monosulfuron at postemergence rates of 0.3-0.8 mg/l in rice fields will likely be toxic to these three ubiquitous nitrogen-fixing cyanobacteria. Low-dose monosulfuron application (

Widespread herbicide applications during the last 20 years have altered prevailing weed species in rice fields; many nitrogen-fixing cyanobacteria were not found even though they were not targets of the herbicides. Four types of herbicides exhibited different toxicity to algae, and the algae displayed different degree of sensitivity to herbicides. A novel theory of the order parameter of rice-weed ecological system may be utilized to establish a better herbicide application strategy based on control parameters lambda of the rice-weed system.

The ubiquitin-proteasome system (UPS) plays an indispensable role in inducing MHC class I-restricted CD8(+) T cells. In this study, we exploited UPS to induce CD8(+) T cells specific for mycobacterial HSP65 (mHSP65), one of the leading vaccine candidates against infection with Mycobacterium tuberculosis. A chimeric DNA termed pU-HSP65 encoding a fusion protein between murine ubiquitin and mHSP65 was constructed, and C57BL/6 (B6) mice were immunized with the DNA using gene gun bombardment. Mice immunized with the chimeric DNA acquired potent resistance against challenge with the syngeneic B16F1 melanoma cells transfected with the mHSP65 gene (HSP65/B16F1), compared with those immunized with DNA encoding only mHSP65. Splenocytes from the former group of mice showed a higher grade of cytotoxic activity against HSP65/B16F1 cells and contained a larger number of granzyme B- or IFN-gamma-producing CD8(+) T cells compared with those from the latter group of mice.

Huntingtin-associated protein 1 (HAP1) was originally found to be localized in neurons and is thought to play an important role in neuronal vesicular trafficking and/or organelle transport. Based on functional similarity between neuron and endocrine cell in vesicular trafficking, we examined the expression and localization of HAP1 in the rat endocrine system using immunohistochemistry. HAP1-immunoreactive cells are widely distributed in the anterior lobe of the pituitary, scattered in the wall of the thyroid follicles, or clustered in the interfollicular space of the thyroid gland, exclusively but diffusely distributed in the medullae of adrenal glands, and selectively located in the pancreas islets. HAP1-containing cells were also found in the mucosa of stomach and small intestine with a distributive pattern similar to that of gastrointestinal endocrine cells. However, no HAP1-immunoreactive cell was found in the cortex of the adrenal gland, the testis, and the ovary. In the posterior lobe of the pituitary, HAP1-immunoreactive products were not detected in the cell bodies but in many stigmoid bodies, one kind of non-membrane-bound cytoplasmic organelle with a central or eccentric electron-lucent core. HAP1-immunoreactive stigmoid bodies were also found in the cytoplasm of endocrine cells in the thyroid gland, the medullae of adrenal gland, the pancreas islets, the stomach, and small intestine. The present study demonstrates that HAP1 is selectively expressed in part of the small peptide-, protein-, and amino-acid analog and derivative-secreting endocrine cells but not in steroid hormone-secreting cells, suggesting that HAP1 is also involved in intracellular trafficking in certain types of endocrine cells.

The T4 bacteriophage gene e.1 was cloned into an expression vector and expressed in Escherichia coli, and the purified protein was identified as a Nudix hydrolase active on FAD, adenosine 5'-triphospho-5'-adenosine (Ap(3)A), and ADP-ribose. Typical of members of the Nudix hydrolases, the enzyme has an alkaline pH optimum (pH 8) and requires a divalent cation for activity that can be satisfied by Mg(2+) or Mn(2+). For all substrates, AMP is one of the products, and unlike most of the other enzymes active on Ap(3)A, the T4 enzyme hydrolyzes higher homologues including Ap(4-6)A. This is the first member of the Nudix hydrolase gene superfamily identified in bacterial viruses and the only one present in T4. Although the protein was predicted to be orthologous to E. coli MutT on the basis of a sequence homology search, the properties of the gene and of the purified protein do not support this notion because of the following. (a) The purified enzyme hydrolyzes substrates not acted upon by MutT, and it does not hydrolyze canonical MutT substrates. (b) The e.1 gene does not complement mutT1 in vivo. (c) The deletion of e.1 does not increase the spontaneous mutation frequency of T4 phage. The properties of the enzyme most closely resemble those of Orf186 of E. coli, the product of the nudE gene, and we therefore propose the mnemonic nudE.1 for the T4 phage orthologue.

Background: Neurotrophins (NTs) and their receptors play crucial roles in the development, functions and maintenance of nervous systems. It is widely believed that NT-induced dimerization of the receptors initiates the transmembrane signaling. However, it is still controversial whether the receptor molecule has a monomeric or dimeric structure on the cell surface before its ligand binding. Findings: Using chemical cross-linking, bimolecular fluorescence complementation (BiFC) and luciferase fragment complementation (LFC) assays, in this study, we show the brain-derived neurotrophic factor (BDNF) receptor TrkB exists as a homodimer before ligand binding. We have also found by using BiFC and LFC that the dimer forms in the endoplasmic reticulum (ER), and that the receptor lacking its intracellular domain cannot form the dimeric structure. Conclusions: Most, if not all, of the TrkB receptor has a preformed, yet inactive, homodimeric structure before BDNF binding. The intracellular domain of TrkB plays a crucial role in the spontaneous dimerization of the newly synthesized receptors, which occurs in ER. These findings provide new insight into an understanding of a molecular mechanism underlying transmembrane signaling mediated by NT receptors.

ygdP, a gene associated with the invasion of brain microvascular endothelial cells by Escherichia coli K1 (Badger, J. L., Wass, C. A., and Kim, K. S. (2000) Mol. Microbiol. 36, 174-182), the primary Gram-negative bacterium causing meningitis in newborns, has been cloned and expressed in E. coli. The protein, YgdP, was purified to near homogeneity and identified as a member of the Nudix hydrolase subfamily of dinucleoside oligophosphate pyrophosphatases. It catalyzes the hydrolysis of diadenosine tetra-, penta-, and hexa-phosphates with a preference for diadenosine penta-phosphate, from which it forms ATP and ADP. The enzyme has a requirement for a divalent metal cation that can be met with Mg2+, Zn2+, or Mn2+ and, like most of the Nudix hydrolases, has an alkaline pH optimum between 8.5 and 9. This is the second identification of a gene associated with the invasiveness of a human pathogen as a member of the Nudix hydrolase subfamily of dinucleoside oligophosphate pyrophosphatases, and an examination of homologous proteins in other invasive bacteria suggests that this may be a common feature of cellular invasion.

Malaria is still a life-threatening infectious disease that continues to produce 2 million deaths annually. Malaria parasites have acquired immune escape mechanisms and prevent the development of sterile immunity. Regulatory T cells (Tregs) have been reported to contribute to immune evasion during malaria in mice and humans, suggesting that activating Tregs is one of the mechanisms by which malaria parasites subvert host immune systems. However, little is known about how these parasites activate Tregs. We herein show that TLR9 signaling to dendritic cells (DCs) is crucial for activation of Tregs. Infection of mice with the rodent malaria parasite Plasmodium yoelii activates Tregs, leading to enhancement of their suppressive function. In vitro activation of Tregs requires the interaction of DCs with parasites in a TLR9-dependent manner. Furthermore, TLR9−/− mice are partially resistant to lethal infection, and this is associated with impaired activation of Tregs and subsequent development of effector T cells. Thus, malaria parasites require TLR9 to activate Tregs for immune escape.