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Our previous research revealed that androgen receptor (AR) activation reduces endothelial cell proliferation via non-genomic pathways. We hypothesized that AR activation might also affect endothelial cell migration, a critical step in angiogenesis. Our data demonstrates that treatment of human umbilical vein endothelial cells (HUVECs) with AR agonists, metribolone (R1881) or dihydrotestosterone (DHT), results in a dose-dependent reduction in migration, which can be reversed by AR antagonists or AR knockdown. Mechanistically, R1881 inhibits HUVEC migration by suppressing RhoA activity through the cSrc/FAK/paxillin pathway and promoting RhoA degradation via RhoA-p27 complex formation, ultimately resulting in RhoA ubiquitination. Transfection with constitutively active RhoA-V14 rescues the inhibitory effect of R1881 on HUVEC migration. Furthermore, R1881 elevates intracellular vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF) levels but reduces VEGF secretion from HUVECs. This reduction is attributed to the formation of VEGF-CTGF complexes in the cytosol induced by R1881. Transfection with RhoA-V14 reduces CTGF levels and VEGF-CTGF complex formation, leading to enhanced VEGF secretion. Pre-treatment with WP631, a CTGF inhibitor, mitigates the R1881-induced reduction in VEGF secretion and HUVECs migration. In vivo assessments using zebrafish angiogenesis and mouse matrigel plug assays validate the anti-angiogenic effects of R1881. These findings provide insight into the molecular mechanisms through which AR activation modulates endothelial cell migration and angiogenesis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Lipopolysaccharide (LPS) triggers an inflammatory response, causing impairment of cardiomyocyte Ca 2+ and Na  +  regulation. This study aimed to determine whether piscidin-1 (PCD-1), an antimicrobial peptide, improves intracellular Ca 2+ and Na  +  regulation in LPS-challenged atrial cardiomyocytes. Rabbit atrial cardiomyocytes were enzymatically isolated from the left atria. Patch-clamp ionic current recording, intracellular Ca 2+ monitoring using Fluo-3, and detection of cytosolic reactive oxygen species production were conducted in control, LPS-challenged, and LPS + PCD-1-treated atrial cardiomyocytes. LPS-challenged cardiomyocytes showed shortened durations of action potential at their 50% and 90% repolarizations, which was reversed by PCD-1 treatment. LPS-challenged cardiomyocytes showed decreased L-type Ca 2+ channel currents and larger Na + /Ca 2+ exchange currents compared to controls. While LPS did not affect the sodium current, an enhanced late sodium current with increased cytosolic Na + levels was observed in LPS-challenged cardiomyocytes. These LPS-induced alterations in the ionic current were ameliorated by PCD-1 treatment. LPS-challenged cardiomyocytes displayed lowered Ca 2+ transient amplitudes and decreased Ca 2+ stores and greater Ca 2+ leakage in the sarcoplasmic reticulum compared to the control. Exposure to PCD-1 attenuated LPS-induced alterations in Ca 2+ regulation. The elevated reactive oxygen species levels observed in LPS-challenged myocytes were suppressed after PCD-1 treatment. The protein levels of NF-κB and IL-6 increased following LPS treatment. Decreased sarcoplasmic/endoplasmic reticulum Ca 2+ ATPase 2a protein levels were observed in LPS-challenged cardiomyocytes. PCD-1 modulates LPS-induced alterations in inflammatory and Ca 2+ regulatory protein levels. Our results suggest that PCD-1 modulates LPS-induced alterations in intracellular Ca 2+ and Na  +  homeostasis, reactive oxygen species production, and the NF-κB inflammatory pathway in atrial cardiomyocytes., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Chih-Yuan Lin reports article publishing charges, equipment, drugs, or supplies, statistical analysis, and writing assistance were provided by Ministry of Science and Technology of Taiwan. Hsiang-Yu Yang reports article publishing charges, equipment, drugs, or supplies, statistical analysis, and writing assistance were provided by Ministry of National Defense Medical Affairs Bureau. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Second-generation antipsychotics increase the risk of atrial fibrillation. This study explores whether the atypical antipsychotic ziprasidone triggers inflammasome signaling, leading to atrial arrhythmia., Methods: Electromechanical and pharmacological assessments were conducted on the rabbit left atria (LA). The patch-clamp technique was used to measure ionic channel currents in single cardiomyocytes. Detection of cytosolic reactive oxygen species production was performed in atrial cardiomyocytes., Results: The duration of action potentials at 50 % and 90 % repolarization was dose-dependently shortened in ziprasidone-treated LA. Diastolic tension in LA increased after ziprasidone treatment. Ziprasidone-treated LA showed rapid atrial pacing (RAP) triggered activity. PI3K inhibitor, Akt inhibitor and mTOR inhibitor abolished the triggered activity elicited by ziprasidone in LA. The NLRP3 inhibitor MCC950 suppressed the ziprasidone-induced post-RAP-triggered activity. MCC950 treatment reduced the reverse-mode Na + /Ca 2+ exchanger current in ziprasidone-treated myocytes. Cytosolic reactive oxygen species production decreased in ziprasidone-treated atrial myocytes after MCC950 treatment. Protein levels of inflammasomes and proinflammatory cytokines, including NLRP3, caspase-1, IL-1β, IL-18, and IL-6 were observed to be upregulated in myocytes treated with ziprasidone., Conclusions: Our findings suggest ziprasidone induces atrial arrhythmia, potentially through upregulation of the NLRP3 inflammasome and enhancement of reactive oxygen species production via the PI3K/Akt/mTOR pathway., Competing Interests: Declaration of Competing Interest All authors declare that they have no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Targeting key enzymes that generate oxalate precursors or substrates is an alternative strategy to eliminate primary hyperoxaluria type I (PH1), the most common and life-threatening type of primary hyperoxaluria. The compact Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) from the Prevotella and Francisella 1 (Cpf1) protein simplifies multiplex gene editing and allows for all-in-one adeno-associated virus (AAV) delivery. We hypothesized that the multiplex capabilities of the Cpf1 system could help minimize oxalate formation in PH1 by simultaneously targeting the hepatic hydroxyacid oxidase 1 ( Hao1 ) and lactate dehydrogenase A ( Ldha ) genes. Study cohorts included treated PH1 rats ( Agxt Q84X rats injected with AAV-AsCpf1 at 7 days of age), phosphate-buffered saline (PBS)-injected PH1 rats, untreated PH1 rats, and age-matched wild-type (WT) rats. The most efficient and specific CRISPR RNA (crRNA) pairs targeting the rat Hao1 and Ldha genes were initially screened ex vivo . In vivo experiments demonstrated efficient genome editing of the Hao1 and Ldha genes, primarily resulting in small deletions. This resulted in decreased transcription and translational expression of Hao1 and Ldha . Treatment significantly reduced urine oxalate levels, reduced kidney damage, and alleviated nephrocalcinosis in rats with PH1. No liver toxicity, ex-liver genome editing, or obvious off-target effects were detected. We demonstrated the AAV-AsCpf1 system can target multiple genes and rescue the pathogenic phenotype in PH1, serving as a proof-of-concept for the development of multiplex genome editing-based gene therapy.

Aim: To report a novel splicing mutation in the RPGR gene (encoding retinitis pigmentosa GTPase regulator) in a three-generation Chinese family with X-linked retinitis pigmentosa (XLRP)., Methods: Comprehensive ophthalmic examinations including best corrected visual acuity, fundus photography, vision field, and pattern-visual evoked potential were performed to identify the disease phenotype of a six-year-old boy from the family (proband). Genomic DNA was extracted from peripheral blood of five available members of the pedigree. Whole-exome sequencing (WES), Sanger sequencing, and pSPL3-based exon trapping were used to investigate the aberrant splicing of RPGR . Human Splice Finder v3.1 and NNSPLICE v0.9 were used for in silico prediction of splice site variants., Results: The proband was diagnosed as having retinitis pigmentosa (RP). He had severe symptoms with early onset. A novel splicing mutation, c.619+1G>C in RPGR was identified in the proband by WES and in four family members by Sanger sequencing. Minigene splicing assays verified that c.619+1G>C in RPGR would result in the formation of a damaging alternative transcript in which the last 91 bp of exon 6 were skipped, leading to the subsequent deletion of 623 correct amino acids (c.529_619del p.Val177Glnfs*16)., Conclusion: We identify a novel splice donor site mutation causing aberrant splicing of RPGR . Our findings add to the catalog of pathological mutations of RPGR and further emphasize the functional importance of RPGR in RP pathogenesis and its complex clinical phenotypes., (International Journal of Ophthalmology Press.)

Purpose: Fruit intake is beneficial to several chronic diseases, but controversial in diabetes. We aimed to investigate prospectively the associations of whole fresh fruit intake with risk of incident type 2 diabetes (T2D) in subjects with different glucose regulation capacities., Methods: The present study included 79,922 non-diabetic participants aged ≥ 40 years from an ongoing nationwide prospective cohort in China. Baseline fruit intake information was collected by a validated food frequency questionnaire. Plasma HbA1c, fasting and 2 h post-loading glucose levels were measured at both baseline and follow-up examinations. Cox proportional hazards models were used to calculate hazard ratio (HR) and 95% confidence intervals (CI) for incident diabetes among participants with normal glucose tolerance (NGT) and prediabetes, after adjusted for multiple confounders. Restricted cubic spline analysis was applied for dose-response relation., Results: During a median 3.8-year follow-up, 5886 (7.36%) participants developed diabetes. Overall, we identified a linear and dose-dependent inverse association between dietary whole fresh fruit intake and risk of incident T2D. Each 100 g/d higher fruit intake was associated with 2.8% lower risk of diabetes (HR 0.972, 95%CI [0.949-0.996], P = 0.0217), majorly benefiting NGT subjects with 15.2% lower risk (HR 0.848, 95%CI [0.766-0.940], P = 0.0017), while not significant in prediabetes (HR 0.981, 95%CI 0.957-4.005, P = 0.1268). Similarly, the inverse association was present in normoglycemia individuals with a 48.6% lower risk of diabetes when consuming fruits > 7 times/week comparing to those < 1 time/week (HR 0.514, 95% CI [0.368-0.948]), but not in prediabetes (HR 0.883, 95% CI [0.762-1.023])., Conclusion: These findings suggest that higher frequency and amount of fresh fruit intake may protect against incident T2D, especially in NGT, but not in prediabetes, highlighting the dietary recommendation of higher fresh fruit consumption to prevent T2D in normoglycemia population., (© 2022. The Author(s).)

We previously demonstrated that activation of progesterone receptor (PR) is essential for folic acid (FA)-inhibited proliferation in colorectal cancer cell lines. In the present study, we further investigated whether the requirement of PR activation for the FA-regulated cell proliferation and migration is a general phenomenon for all cancer cell lines or specific for colorectal cancer cell lines only. Initially, we examined the expression of PR in various cancer cell lines using Western blot analyses and RT-PCR technique, and then investigated the effects of FA on these cancer cell lines. Our data showed that the effects of FA on proliferation and migration only occurred in the PR positive (+) cancer cell lines, but not the PR negative (-) cancer cell lines, and these effects were abolished by pre-treatment with the PR specific inhibitor, Org 31710. On the other hand, FA significantly reduced the proliferation and migration in the PR (-) cancer cell lines transfected with PR pcDNA. However, FA did not significantly affect the proliferation and migration in the PR-transefected Hep-3B cell line, which does not express endogenous PR and FA receptor (FR). Since we previously showed that FA-regulated proliferation in colorectal and breast cancer cell lines through the cSrc-mediated pathway, we conducted immunoprecipitation assay to demonstrate that PR formed a complex with FR and cSrc, but FR did not directly associate with cSrc. Taken together, these findings suggest that the requirement of PR activation for the FA-regulated cell proliferation and migration is a general phenomenon for all cancer cell lines., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The current study evaluated the efficacy and safety of a denosumab biosimilar, QL1206 (60 mg), compared to placebo in postmenopausal Chinese women with osteoporosis and high fracture risk. At 31 study centers in China, a total of 455 postmenopausal women with osteoporosis and high fracture risk were randomly assigned to receive QL1206 (60 mg subcutaneously every 6 months) or placebo. From baseline to the 12-month follow-up, the participants who received QL1206 showed significantly increased bone mineral density (BMD) values (mean difference and 95% CI) in the lumbar spine: 4.780% (3.880%, 5.681%), total hip :3.930% (3.136%, 4.725%), femoral neck 2.733% (1.877%, 3.589%) and trochanter: 4.058% (2.791%, 5.325%) compared with the participants who received the placebo. In addition, QL1206 injection significantly decreased the serum levels of C-terminal crosslinked telopeptides of type 1 collagen (CTX): -77.352% (-87.080%, -66.844%), and N-terminal procollagen of type l collagen (P1NP): -50.867% (-57.184%, -45.217%) compared with the placebo over the period from baseline to 12 months. No new or unexpected adverse events were observed. We concluded that compared with placebo, QL1206 effectively increased the BMD of the lumbar spine, total hip, femoral neck and trochanter in postmenopausal Chinese women with osteoporosis and rapidly decreased bone turnover markers. This study demonstrated that QL1206 has beneficial effects on postmenopausal Chinese women with osteoporosis and high fracture risk., (© 2022. The Author(s), under exclusive licence to Shanghai Institute of Materia Medica, Chinese Academy of Sciences and Chinese Pharmacological Society.)

Sertoli cells (SCs) provide structural and nutritional support for developing germ cells. Normal glucose metabolism of SCs is necessary for spermatogenesis. Melatonin could alleviate the effects of heat stress on spermatogenesis. However, the influences of heat stress on glucose metabolism in SCs remain unclear, and the potential protective mechanisms of melatonin on SCs need more exploration. In this study, boar SCs were treated at 43°C for 30 min, and different concentrations of melatonin were added to protect SCs from heat stress-induced impairment. These results showed that heat stress-induced oxidative stress caused cell apoptosis, inhibited the pentose phosphate pathway, and decreased the ATP content. Furthermore, heat stress increased the expressions of glucose intake- and glycolytic-related enzymes, which enhanced the glycolysis activity to compensate for the energy deficit. Melatonin relieved heat stress-induced oxidative stress and apoptosis by activating the Kelch-like ECH-associated protein 1 (KEAP1)/NF-E2-related factor 2 signaling pathway to increase the capacity of antioxidants. In addition, melatonin enhanced heat-shock protein 90 (HSP90) expression through melatonin receptor 1B (MTNR1B), thereby stabilizing hypoxia-inducible factor-1α (HIF-1α). Activation of the HIF-1α signaling pathway enhanced glycolysis, promoted the pentose phosphate pathway, and increased cell viability. Our results suggest that melatonin reprograms glucose metabolism in SCs through the MTNR1B-HSP90-HIF-1α axis and provides a theoretical basis for preventing heat stress injury., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background and Aims: Inflammatory bowel disease (IBD) places a heavy medical burden on countries and families due to repeated and prolonged attacks, and the incidence and prevalence of IBD are increasing worldwide. Therefore, finding an effective treatment is a matter of great urgency. Glycerol monolaurate (GML), which has a twelve-carbon chain, is a compound naturally found in human breast milk. Some studies have shown that GML has antibacterial and anti-inflammatory effects. However, the specific mechanism of action remains unclear., Methods: Acute colitis was established in mice using 3% DSS, and glycerol monolaurate (500 mg·kg- 1 ) was administered for two weeks. QPCR and western blotting were performed to examine the inflammatory status. Mice described were subjected to flow cytometry analysis for immune cell activation., Results: GML treated alleviated macroscopic symptoms such as shortened colons, increased spleen weight, and caused weight loss in mice with DSS-induced colitis. In addition, GML decreased the expression of pro-inflammatory factors (NF-α, IL-1β and IL-1α) and increased the expression of anti-inflammatory factors (IL-10 and TGF-β). GML inhibited the activation of the MAPK and NF-κB signalling pathways, improved tissue damage, and increased the expression of intestinal tight junction proteins. In addition, LPMCs extracted from intestinal tissue via flow cytometry showed that GML treatment led to a decrease of Th17 cells, Neutrophils and Macrophages. 16S rDNA sequencing showed that GML increased the abundance of commensal bacterium such as Akkermansia and Lactobacillus murinus., Conclusions: We showed that oral administration of GML ameliorated DSS-induced colitis by inhibiting infiltration of Th17 cells, Neutrophils, and Macrophages, protecting the intestinal mucosal barrier and altered the abundance of commensal bacterium. This study provides new insights into the biological function and therapeutic potential of GML in the treatment of IBD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 He, Dong, Ouyang, Huo, Cheng, Lin, Li, Gong, Liu, Ren and Guleng.)

Background: This study aims to identify the underlying genetic cause of a Chinese patient with Leber congenital amaurosis (LCA)., Methods: Detailed clinical data and family history were collected. A medical diagnostic panel sequencing covering 4450 genes was conducted. Two candidate disease-causing mutations detected in CEP290 were then validated with Sanger sequencing and bioinformatic analysis. Reverse transcription polymerase chain reaction (RT-PCR) and cDNA sequencing were performed to understand the effect of the novel CEP290 mutation on CEP290 mRNA splicing., Results: A five-month-old LCA patient with both parents was enrolled. Medical diagnostic panel sequencing revealed that the patient is a compound heterozygote for two potentially pathogenic CEP290 mutations. Among them, c.1666dupA (p.I556NfsX20) was previously reported and has a significant association with LCA phenotype. A novel CEP290 mutation (c.3310-1&#95;3313delGCTTA) was confirmed in both the patient and her father. RT-PCR and cDNA sequencing confirmed that the novel mutation affects the CEP290 mRNA splicing and results in a complete skipping of exon 29. The frameshift resulted in an early stop codon and truncation of the mutant protein by 1371 amino acid residues (p.L1104fsX6)., Conclusions: Our report provided a new mutation to the spectrum of CEP290 -related diseases. The data suggested that the c.3310-1&#95;3313delGCTTA mutation affects the CEP290 mRNA splicing and the CEP290 protein function. This valuable information is important for future studies on the mRNA splicing of CEP290 and the pathogenesis of CEP290 -related diseases.

Background: Lipotoxicity causes endoplasmic reticulum (ER) stress, leading to cell apoptosis. Sirtuin 1 (Sirt1) regulates gene transcription and cellular metabolism. In this study, we investigated the role of Sirt1 in palmitate-induced ER stress., Methods: Both H9c2 myoblasts and heart-specific Sirt1 knockout mice fed a palmitate-enriched high-fat diet were used., Results: The high-fat diet induced C/EBP homologous protein (CHOP) and activating transcription factor 4 (ATF4) expression in both Sirt1 knockout mice and controls. The Sirt1 knockout mice showed higher CHOP and ATF4 expression compared to those in the control. Palmitic acid (PA) induced ATF4 and CHOP expression in H9c2 cells. PA-treated H9c2 cells showed decreased cytosolic NAD + /NADH alongside reduced Sirt1's activity. The H9c2 cells showed increased ATF4 and CHOP expression when transfected with plasmid encoding dominant negative mutant Sirt1. Sirt1 activator SRT1720 did not affect CHOP and ATF4 expression. Although SRT1720 enhanced the nuclear translocation of ATF4, the extent of the binding of ATF4 to the CHOP promoter did not increase in PA treated-H9c2 cells., Conclusion: PA-induced ER stress is mediated through the upregulation of ATF4 and CHOP. Cytosolic NAD + concentration is diminished by PA-induced ER stress, leading to decreased Sirt1 activity. The Sirt1 activator SRT1720 promotes the nuclear translocation of ATF4 in PA-treated H9c2 cells.

Zoledronic acid (ZOL) is a therapy inhibiting bone resorption. In this study, generic ZOL (Yigu®) showed its clinical efficacy consistency with original ZOL (Aclasta®) in Chinese postmenopausal women with osteoporosis. This study provides a practical basis for the application of Yigu® in Chinese population., Introduction: Yigu® has been approved its bioequivalence to Aclasta®. However, the clinical efficacy and safety of Yigu® have not been evaluated yet. Here, we compared the effectiveness and safety between Yigu® and Aclasta® in Chinese postmenopausal women with osteoporosis and assessed the efficacy of intravenous infusion of ZOL., Methods: This was a randomized open-label, active-controlled study in postmenopausal women with osteoporosis of 14 clinical centers in China. Postmenopausal women with osteoporosis were recruited and randomized to receive a single infusion of 5 mg Yigu® or Aclasta®. The primary endpoint was the percentage change in bone mineral density (BMD) at lumbar spine after 12 months of treatment and was assessed for equivalence. The secondary endpoint was the percentage change in BMD at proximal femur after 12 months. Additional secondary endpoints were percentage changes in BMD at the above sites after 6 months of treatment and changes in bone turnover biomarkers during ZOL treatment. Safety was also evaluated and compared between two groups., Results: A total of 458 postmenopausal women with osteoporosis were enrolled (n = 227, Yigu®; n = 231, Aclasta®). The mean percentage change in the BMD had no statistical difference at the lumbar spine (5.32% vs 5.18%), total hip (2.72% vs 2.83%), and femoral neck (2.37% vs 2.81%) between Yigu® and Aclasta® groups after 12 months of treatment. The mean difference of BMD change at the lumbar spine after 12 months between two groups was 0.15% (95% CI: - 0.71 to 1.00, equivalence margin: - 1.5%, 1.5%), demonstrating the treatments were equivalent. Meanwhile, the decreases in the P1NP and β-CTX showed no difference between two groups after 14 days and 6 and 12 months of treatment. As regards the whole sample, BMD significantly increased after 12 months of treatment. Also, serum C-terminal telopeptide of type 1 collagen (β-CTX) and procollagen 1 N-terminal peptide (P1NP) significantly decreased at each visit period. The overall adverse events were comparable and quite well between two groups., Conclusion: Intravenous infusion of zoledronic acid achieved the potent anti-resorptive effects which led to significant increase in BMD of Chinese postmenopausal women with osteoporosis. Yigu® was equivalent to Aclasta® with respect to efficacy and safety., (© 2021. The Author(s).)

Objective: The relationship between serum uric acid (SUA) levels and glycemic indices, including plasma glucose (FPG), 2-hour postload glucose (2h-PG), and glycated hemoglobin (HbA1c), remains inconclusive. We aimed to explore the associations between glycemic indices and SUA levels in the general Chinese population., Methods: The current study was a cross-sectional analysis using the first follow-up survey data from The China Cardiometabolic Disease and Cancer Cohort Study. A total of 105,922 community-dwelling adults aged ≥ 40 years underwent the oral glucose tolerance test and uric acid assessment. The nonlinear relationships between glycemic indices and SUA levels were explored using generalized additive models., Results: A total of 30,941 men and 62,361 women were eligible for the current analysis. Generalized additive models verified the inverted U-shaped association between glycemic indices and SUA levels, but with different inflection points in men and women. The thresholds for FPG, 2h-PG, and HbA1c for men and women were 6.5/8.0 mmol/L, 11.0/14.0 mmol/L, and 6.1/6.5, respectively (SUA levels increased with increasing glycemic indices before the inflection points and then eventually decreased with further increases in the glycemic indices)., Conclusion: An inverted U-shaped association was observed between major glycemic indices and uric acid levels in both sexes, while the inflection points were reached earlier in men than in women., (Copyright © 2020 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.)

Purpose: We previously demonstrated that progesterone (P4) interacted with folic acid (FA) and abolished the FA-reduced endothelial cell proliferation and migration. These findings led us to investigate whether FA can interfere with the P4-promoted breast cancer cell proliferation and migration., Methods: We conducted MTT and wound healing assay to evaluate cell proliferation and migration, respectively. Western blot analysis and immunoprecipitation were performed to examine the protein expression and protein-protein interaction, respectively., Results: We demonstrated that P4 promoted proliferation and migration of breast cancer cell lines (T47D, MCF-7, BT474, and BT483). However, co-treatment with P4 and FA together abolished these promotion effects. Treatment with P4 alone increased the formation of PR-cSrc complex and the phosphorylation of cSrc at tyrosine 416 (Tyr416). However, co-treatment with P4 and FA together increased the formations of cSrc-p140Cap, cSrc-Csk, and cSrc-p-Csk complex, and the phosphorylation of cSrc at tyrosine 527 (Tyr527). Co-treatment with P4 and FA together also abolished the activation of cSrc-mediated signaling pathways involved in the P4-promoted breast cancer cell proliferation and migration., Conclusions: Co-treatment with FA and P4 together abolished the P4-promoted breast cancer cell proliferation and migration through decreasing the formation of PR-cSrc complex and increasing the formations of cSrc-p140Cap and cSrc-Csk complex, subsequently activating Csk, which in turn suppressed the phosphorylation of cSrc at Tyr416 and increased the phosphorylation of cSrc at Tyr527, hence inactivating the cSrc-mediated signaling pathways. The findings from this study might provide a new strategy for preventing the P4-promoted breast cancer progress.

Mindin is important in broad spectrum of immune responses. On the other hand, we previously reported that mindin attenuated human colon cancer development by blocking angiogenesis through Egr-1-mediated regulation. However, the mice original mindin directly suppressed the syngenic colorectal cancer (CRC) growth in our recent study and we aimed to further define the role of mindin during CRC development in mice. We established the mouse syngeneic CRC CMT93 and CT26 WT cell lines with stable mindin knock-down or overexpression. These cells were also subcutaneously injected into C57BL/6 and BALB/c mice as well as established a colitis-associated colorectal cancer (CAC) mouse model treated with lentiviral-based overexpression and knocked-down of mindin. Furthermore, we generated mindin knockout mice using a CRISPR-Cas9 system with CAC model. Our data showed that overexpression of mindin suppressed cell proliferation in both of CMT93 and CT26 WT colon cancer cell lines, while the silencing of mindin promoted in vitro cell proliferation via the ERK and c-Fos pathways and cell cycle control. Moreover, the overexpression of mindin significantly suppressed in vivo tumour growth in both the subcutaneous transplantation and the AOM/DSS-induced CAC models. Consistently, the silencing of mindin reversed these in vivo observations. Expectedly, the tumour growth was promoted in the CAC model on mindin-deficient mice. Thus, mindin plays a direct tumour suppressive function during colon cancer progression and suggesting that mindin might be exploited as a therapeutic target for CRC., (© 2020 Zhongshan Hospital affiliated to Xiamen University, China. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Objective: MiRNA has been found to have therapeutic effect on corneal damage. This paper aimed to study the effect of miR-205-3p on corneal damage induced by ultraviolet (UV) radiation., Materials and Methods: HCE cells were exposed to UV light and transfected. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot were used to determine miRNA/mRNA and protein expression. CCK-8 assay, Edu incorporation experiment, and flow cytometry were used to separately measure cell activity, proliferation and apoptosis. LC3 puncta were researched by immunofluorescence experiment. TNF-α, IL-6 and IL-1β levels in cells were detected by enzyme-linked immunosorbent assay (ELISA) kit. MDA, SOD, and GSH-PX levels were measured using detection kits. Reactive oxygen species (ROS) level was reflected by detecting DCFH-DA density. Luciferase activity assay was performed to verify the regulating relationship between miR-205-3p and TLR4., Results: UV radiation decreased HCE cell viability, proliferation, and increased HCE cell apoptosis and autophagy (all p < 0.01). When exposed UV radiation, the overexpression of miR-205-3p group elevated HCE cells viability, proliferation and weakened HCE cells apoptosis and autophagy (all p < 0.01). MiR-205-3p inhibited inflammation and oxidative stress in HCE cells induced by UV radiation (p < 0.01). MiR-205-3p directly inhibited TLR4 expression. The upregulation of TLR4 significantly reversed the effects of miR-205-3p on HCE cells phenotypes induced by UV radiation (p < 0.01)., Conclusions: MiR-205-3p protected HCE cells from UV damage by inhibiting autophagy via targeting TLR4.

The effect of androgen on angiogenesis has been documented. However, its underlying molecular mechanisms have not been well illustrated. Here, we show that treatment with an androgen receptor (AR) agonist, metribolone (R1881; 0.05-5 nM), or dihydrotestosterone (DHT; 0.5-2 nM), concentration- and time-dependently inhibited proliferation in human umbilical venous endothelial cells (HUVEC). This inhibitory effect was confirmed in human microvascular endothelial cells (HMEC-1). Flow cytometric analysis demonstrated that R1881 induced G0/G1 phase cell cycle arrest in HUVEC. Blockade of the AR activity by pre-treatment with an AR antagonist, hydroxyflutamide (HF), or knockdown of AR expression using the shRNA technique abolished the R1881-induced HUVEC proliferation inhibition, suggesting that AR activation can inhibit endothelial cell proliferation. We further investigated the signaling pathway contributing to the proliferation inhibition induced by AR activation. Our data suggest that R1881 reduced the proliferation rate of HUVEC through activating the AR/cSrc/AKT/p38/ERK/NFκB pathway, subsequently up-regulating p53 expression, which in turn increased the levels of p21 and p27 protein, hence decreasing the activities of cyclin-dependent kinase 2 (CDK2) and CDK4, and finally reduced the cell proliferation rate. An extra-nuclear pathway involved in the proliferation inhibition induced by AR activation in vascular endothelial cells was confirmed by showing that membrane-impermeable testosterone-bovine serum albumin (BSA) treatment significantly increased the levels of p53, p27 and p21 protein and reduced cell proliferation. These data highlight the underlying molecular mechanisms by which AR activation induced proliferation inhibition in vascular endothelial cells., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

As an important gram-negative bacterial outer membrane component, lipopolysaccharide (LPS) plays an important role in bacterial-induced endometritis in sows. However, how LPS induces endometritis is unclear. We stimulated sow endometrial epithelial cells (EECs) with LPS and detected cell viability and tumour necrosis factor-α (TNF-α) and interleukin-1 (IL-1) secretion. LPS affected EEC viability and TNF-α and IL-1 secretion in a dose-dependent manner. LPS induced differential expression in 10 of 393 miRNAs in the EECs (downregulated, nine; upregulated, one). MicroRNA (miRNA) high-throughput sequencing of the LPS-induced EECs plus bioinformatics analysis and the dual-luciferase reporter system revealed a novel miRNA target gene: mitogen-activated protein kinase kinase kinase 14 (MAP3K14). Ssc-novel-miR-106-5p mimic, inhibitor and the nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) phosphorylation inhibitor Bay11-7085 were used to detect EEC nuclear factor-κB phosphorylation levels (p-NF-κB) and TNF-α and IL-1 secretion. MiR-106-5p mimic downregulated MAP3K14 mRNA and protein expression levels, inhibited p-NF-κB levels and decreased IL-1 and TNF-α secretion, whereas miR-106-5p inhibitor had the opposite effect. Bay11-7085 inhibited p-NF-κB expression and TNF-α and IL-1 secretion. These results suggest that LPS downregulates ssc-novel-miR-106-5p expression in sow EECs to increase MAP3K14 expression, which increases p-NF-κB to promote IL-1 and TNF-α secretion.

Mindin has a broad spectrum of roles in the innate immune system, including in macrophage migration, antigen phagocytosis and cytokine production. Mindin functions as a pattern-recognition molecule for microbial pathogens. However, the underlying mechanisms of mindin-mediated phagocytosis and its exact membrane receptors are not well established. Herein, we generated mindin-deficient mice using the CRISPR-Cas9 system and show that peritoneal macrophages from mindin-deficient mice were severely defective in their ability to phagocytize E  coli. Phagocytosis was enhanced when E  coli or fluorescent particles were pre-incubated with mindin, indicating that mindin binds directly to bacteria or non-pathogen particles and promotes phagocytosis. We defined that 131 I-labelled mindin binds with integrin Mac-1 (CD11b/CD18), the F-spondin (FS)-fragment of mindin binds with the α M -I domain of Mac-1 and that mindin serves as a novel ligand of Mac-1. Blockade of the α M -I domain of Mac-1 using either a neutralizing antibody or si-Mac-1 efficiently blocked mindin-induced phagocytosis. Furthermore, mindin activated the Syk and MAPK signalling pathways and promoted NF-κB entry into the nucleus. Our data indicate that mindin binds with the integrin Mac-1 to promote macrophage phagocytosis through Syk activation and NF-κB p65 translocation, suggesting that the mindin/Mac-1 axis plays a critical role during innate immune responses., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

We recently showed that folic acid (FA) could decrease the proliferation rate of colorectal cancer cells in vitro and reduce the volume of COLO-205 tumor in vivo. Since cancer cell proliferation and migration are two major events during cancer development, we further examined whether FA could also affect the migration of colorectal cancer cells. Transwell invasion assays demonstrated that FA reduced the invasion ability of colorectal cancer cell lines, COLO-205, LoVo and HT-29. Using COLO-205 as a cell model, we further delineated the molecular mechanism underlying FA-inhibited colorectal cancer cell invasion. Western blot analyses showed that FA (10 μM) activated cSrc, ERK1/2, NFκB, and p27 at serine 10 (Ser10), and up-regulated p53, p27, and KIS protein. Subcellular fractionation illustrated that FA treatment increased cytosolic translocation of p27, formation of the p27-RhoA complex, and RhoA degradation. The FA-induced migration inhibition in COLO-205 was abolished by blockade of the cSrc or ERK1/2 activity, knockdown of p27 or KIS using the siRNA technique, or over-expression of a constitutive active RhoA cDNA. Our results suggest that FA up-regulated p27 through increasing the cSrc/ERK1/2/NFκB/p53-mediated pathway. In the nucleus, FA up-regulated KIS, which in turn increased p27 phosphorylation at serine 10 (Ser10), subsequently resulting in cytosolic translocation of p27 and forming the p27-RhoA complex, thereby causing RhoA degradation, and eventually inhibited COLO-205 cell migration. Together with our previous findings suggest that FA reduced colorectal cancer development through inhibiting colorectal cancer cell proliferation and migration., (Copyright © 2018 Elsevier Inc. All rights reserved.)

We previously showed that overexpression of Thy-1 inhibited and knock-down of Thy-1 enhanced endothelial cell migration. Here, we used phorbol-12-myristate-13-acetate (PMA) as an inducer for Thy-1 expression to investigate molecular mechanisms underlying Thy-1 up-regulation. Our data showed that increased levels of Thy-1 mRNA and protein in endothelial cells were observed at 14-18 hours and 20-28 hours after PMA treatment, respectively. Treatment with PMA for 32 hours induced Thy-1 up-regulation and inhibited capillary-like tube formation and endothelial cell migration. These effects were abolished by Röttlerin (a PKC-δ inhibitor), but not Gö6976 (a PKC-α/β inhibitor). Moreover, pre-treatment with Bay 61-3606 (a Syk inhibitor) or Bay 11-7082 (a NF-κB inhibitor) abolished the PMA-induced Thy-1 up-regulation and migration inhibition in endothelial cells. Using the zebrafish model, we showed that PMA up-regulated Thy-1 and inhibited angiogenesis through the PKC-δ-mediated pathway. Surprisingly, we found that short-term (8-10 hours) PMA treatment enhanced endothelial cell migration. However, this effect was not observed in PMA-treated Thy-1-overexpressed endothelial cells. Taken together, our results suggest that PMA initially enhanced endothelial cell migration, subsequently activating the PKC-δ/Syk/NF-κB-mediated pathway to up-regulate Thy-1, which in turn inhibited endothelial cell migration. Our results also suggest that Thy-1 might play a role in termination of angiogenesis.

Cornea is at the outermost surface of eye globe, and it easily receives damage from ultraviolet light exposure, physiology wounding, and infections. It is essential to understand the mechanisms controlling human corneal epithelial (HCE) cell proliferation and wound healing. Epidermal growth factor (EGF) could stimulate cell proliferation and migration in various cell types. Therefore, we investigated the roles and mechanisms of EGF on HCE cell proliferation and migration. CCK-8 kit and wound healing experiment were used to investigate HCE cell proliferation and cell migration, respectively. ROS activity was quantified by DCFDA and flow cytometry. Western blot and Q-PCR were performed to examine protein and RNA levels. EGF could promote HCE cell proliferation and migration in both physiology status and UV irradiation conditions, which is used to mimic the disease condition in human corneal epithelial cells. Interestingly, the promotion effect of EGF on HCE cell proliferation is mainly mediated by activated ROS signaling under disease condition. However, the EGF function is mediated by ROS and MAPK/ERK pathway in EGF-treated corneal epithelial cells in physiology status, in which ROS and MAPK/ERK pathway have no mutual influence on the other signaling pathway in EGF-stimulated corneal epithelial cells. We also revealed that MAPK/ERK pathway instead of ROS mediates EGF-stimulated HCE cell migration. Interestingly, we found that PKC proteins were downregulated by EGF in HCE cells that is partially mediated by ROS signaling, while PKC pathway was not involved in EGF-stimulated corneal cell proliferation and migration. EGF promotes human corneal cell proliferation and migration both in physiology and disease conditions, and ROS, MAPK/ERK and PKC pathways play different roles in these processes.

We previously showed that progesterone (P4) could inhibit the proliferation of human umbilical venous endothelial cells (HUVECs) through the p53-dependent pathway. In the present study, we further demonstrated that P4 at physiologic levels (5-500 nM) concentration-dependently inhibited migration of HUVECs. This effect was blocked by pre-treatment with the P4 receptor (PR) agonist-antagonist, RU486, suggesting that the P4-induced migration inhibition in HUVECs was through the PR-mediated signaling pathway. Western blot analyses demonstrated that the levels of RhoA and Rac-1 protein were reduced in the P4-treated HUVECs. P4 also inhibited the membrane translocation of RhoA and Rac-1 protein. Moreover, the P4-induced migration inhibition in HUVECs was prevented by over-expression of the constitutively active RhoA construct (RhoA V14). However, pre-treatment with the ROCK (a kinase associated with RhoA for transducing RhoA signaling) inhibitor, Y27632, abolished the over-expression of RhoA-induced prevention effect on the P4-induced migration inhibition in HUVECs. These data suggest that the inhibition of Rho GTPases might account for the P4-induced migration inhibition of HUVECs. Pre-treatment with the cSrc inhibitor, PP2, prevented the P4-induced migration inhibition in HUVEC. The levels of phosphorylated focal adhesion kinase (FAK) and paxillin protein were also decreased by P4 treatment. Taken together, these results suggest that suppression of the Rho-mediated pathway might be involved in the signal transduction leading to the inhibition of cell migration caused by P4 in HUVECs., (© 2015 Wiley Periodicals, Inc.)

Aim: To examine the association between depression and impaired glucose regulation, newly diagnosed diabetes and previously diagnosed diabetes in middle-aged and elderly Chinese people, and whether depression was associated with different treatment regimens or durations of diabetes., Methods: A cross-sectional study was performed among 229,047 adults living in the community aged ≥ 40 years from 25 centres in China. The self-reported depression rating scale Patient Health Questionnaire 9 (PHQ-9) was used to diagnose probable and sub-threshold depression. Glucose metabolism status was determined according to World Health Organization 1999 diagnostic criteria., Results: The numbers of participants with normal glucose regulation, impaired glucose regulation, newly diagnosed diabetes and previously diagnosed diabetes were 120,458, 59,512, 24,826 and 24,251, respectively. The prevalence of sub-threshold depression in the total sample of participants was 4.8% (4.8%, 4.8%, 4.4% and 5.6% from normal glucose regulation to previously diagnosed diabetes, respectively), and the prevalence of probable depression was 1.1% (1.1%, 1.0%, 0.9% and 1.8% from normal glucose regulation to previously diagnosed diabetes, respectively). Compared with participants with normal glucose regulation, those with previously diagnosed diabetes had increased odds of probable depression [odds ratio (OR) = 1.61, 95% confidence interval (CI) 1.39-1.87] and sub-threshold depression (OR = 1.14, 95% CI 1.06-1.24), after adjustment for multiple confounding factors. Newly diagnosed diabetes or impaired glucose regulation was not associated with depression. Among those with previously diagnosed diabetes, insulin treatment was associated with greater odds of depression compared with no treatment or oral anti-diabetic medicine., Conclusion: Previously diagnosed diabetes, but not newly diagnosed diabetes or impaired glucose regulation, was associated with a higher prevalence of depression. Patients receiving insulin were more likely to have depression than those not receiving treatment or being treated with oral anti-diabetic medicine., (© 2014 The Authors. Diabetic Medicine © 2014 Diabetes UK.)

We previously demonstrated that progesterone (P4) up-regulates p53 expression in human umbilical venous endothelial cells (HUVECs) through P4 receptor (PR) activation of extranuclear signaling pathways. However, the involvement of nuclear PR in P4-increased p53 expression is still unclear. Here, the molecular mechanism underlying PR-regulated p53 expression in HUVECs was investigated. Treatment with P4 increased nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor, α phosphorylation (IκBα and nuclear factor-κB (NFκB) nuclear translocation. Interestingly, P4 also increased PR-A, but not PR-B, nuclear translocation in HUVECs. Immunoprecipitation assay illustrated that P4 increased the formation of PR-A-NFκB complex in both the cytosol and the nucleus of HUVEC. Chromatin immunoprecipitation assay showed an interaction between PR and the NFκB binding motif on the p53 promoter. Ablation of the NFκB binding motif in the p53 promoter completely abolished P4-increased p53 promoter activity. In the absence of P4, overexpression of NFκB did not increase NFκB nuclear translocation. In contrast, treatment of NFκB-overexpressing HUVECs with P4 for only 4 hours, which is much shorter than the time (21.5 h) required for P4-induced IκBα phosphorylation, increased NFκB nuclear translocation. Blockade of PR activity abolished this effect. Taken together, these results uncover a novel role of PR for P4-induced NFκB nuclear translocation and suggest that PR-A-NFκB complex formation is required for NFκB nuclear translocation and binding onto the p53 promoter in HUVECs. Our data indicate that both nuclear and extranuclear signaling pathways of PR are involved in P4-regulated p53 expression in HUVECs.

Our previous study indicated that Thy-1, which is expressed on blood vessel endothelium in settings of pathological and a specific of physiological, but not during embryonic, angiogenesis, may be used as a marker for angiogenesis. However, the function of Thy-1 during angiogenesis is still not clear. Here, we demonstrate that knock-down of the endogenous Thy-1 expression by Thy-1 siRNA transfection promoted the migration of human umbilical vein endothelial cells (HUVEC). In contrast, treatment with interleukin-1β (IL-1β) or phorbol-12-myristate-13-acetate (PMA) increased the level of Thy-1 protein and reduced the migration of HUVEC. These effects were abolished by pre-transfection of HUVEC with Thy-1 siRNA to knock-down the expression of Thy-1. Moreover, over-expression of Thy-1 by transfection of HUVEC with Thy-1 pcDNA3.1 decreased the activity of RhoA and Rac-1 and inhibited the adhesion, migration and capillary-like tube formation of these cells. These effects were prevented by co-transfection of the cell with constitutively active RhoA construct (RhoA V14). On the other hand, pre-treatment with a ROCK (a kinase associated with RhoA for transducing RhoA signaling) inhibitor, Y27632, abolished the RhoA V14-induced prevention effect on the Thy-1-induced inhibition of endothelial cell migration and tube formation. Taken together, these results indicate that suppression of the RhoA-mediated pathway might participate in the Thy-1-induced migration inhibition in HUVEC. In the present study, we uncover a completely novel role of Thy-1 in endothelial cell behaviors.

Objective: To investigate gene mutations associated with three different types of corneal dystrophies (CDs), and to establish a phenotype-genotype correlation., Methods: Two patients with Avellino corneal dystrophy (ACD), four patients with lattice corneal dystrophy type I (LCD I) from one family, and three patients with macular corneal dystrophy type I (MCD I) were subjected to both clinical and genetic examinations. Slit lamp examination was performed for all the subjects to assess their corneal phenotypes. Genomic DNA was extracted from peripheral blood leukocytes. The coding regions of the human transforming growth factor β-induced (TGFBI) gene and carbohydrate sulfotransferase 6 (CHST6) gene were amplified by polymerase chain reaction (PCR) and subjected to direct sequencing. DNA samples from 50 healthy volunteers were used as controls., Results: Clinical examination showed three different phenotypes of CDs. Genetic examination identified that two ACD subjects were associated with homozygous R124H mutation of TGFBI, and four LCD I subjects were all associated with R124C heterozygous mutation. One MCD I subject was associated with a novel S51X homozygous mutation in CHST6, while the other two MCD I subjects harbored a previously reported W232X homozygous mutation., Conclusions: Our study highlights the prevalence of codon 124 mutations in the TGFBI gene among the Chinese ACD and LCD I patients. Moreover, we found a novel mutation among MCD I patients.

Purpose: Intracellular reactive oxygen species have been reported to associate with growth factor and integrin signalings in promoting cell adhesion in many cell types. This study is to explore if exogenous H(2)O(2) at low levels can be beneficial to cell adhesion, migration, and wound healing., Methods: Primary rabbit corneal epithelial cells treated with 0-70 μM H(2)O(2) were tested for viability by MTT assay, adhesion by centrifugation assay, focal contacts of vinculin and F-actin by immunofluorescence, activated Src(pY416), EGF receptor (pY845), vinculin(pY1065), FAK(pY397), and FAK(pY576) by immunoblotting. Cell migration was examined with 0-50 μM H(2)O(2) using the scratch wound technique. Corneal wound healing of ex vivo pig model and in vivo mouse model was examined using H(2)O(2) with and without antioxidant N-acetylcysteine (NAC)., Results: Compared with the untreated control, H(2)O(2) at 10-50 μM stimulated cell viability and facilitated adhesion and migration with clear induction of vinculin-rich focal adhesions and F-actin-containing stress fibers by increasing activated Src, FAK(pY576), and vinculin(pY1065). H(2)O(2) also increased phosphorylation of EGFR(Y845) parallel to that of activated Src, but both were eliminated by NAC and PP1 (Src inhibitor). Finally, H(2)O(2) induced faster wound healing in cornea both in vitro and in vivo, but the healing was diminished by NAC., Conclusions: These findings suggest that H(2)O(2) at low levels promotes cell adhesion, migration, and wound healing in cornea cells or tissue, and the interaction of H(2)O(2) with Src plays a major role.

Gelatinous drop-like corneal dystrophy (GDLD) is an autosomal recessive hereditary disease, which may result in bilateral loss of vision. The gene responsible for GDLD, M1S1 is mapped on the short arm of chromosome 1 (1p), but the possible etiology of this disease remains unclear. Corneal transplantation is the only treatment for visual rehabilitation. The detection of the mutations of the M1S1 gene and the possible etiological involvement of the amyloid deposits are discussed. The current literatures are extensively reviewed in this article.

Objectives: To explore intervention with electromagnetic noise for co-suppression effect on gap-junctional intercellular communication (GJIC) induced or strengthened by low intensity magnetic field with carcinogen 12-O-tetradecanoylphorbol-13-acetate (TPA)., Methods: Fibroblast cells from NIH 3T3 mice were exposed to extremely low intensity magnetic field (MF) 0.2 mT, 0.2 mT + TPA or/and electromagnetic noise with the same intensity of MF for 24 h, and GJIC was determined using fluorescence recovery analysis after photobleaching (FRAP) with a laser-scanning confocal microscope (Leica, Germany)., Results: GJIC function could be co-suppressed by MF of 0.2 mT with TPA, with fluorescence recovery of (23 +/- 11)%, lower than that in the control group [(46 +/- 19)%] and in the group with TPA only [(34 +/- 17) %] (P < 0.01), indicating 0.2 mT MF plus TPA could co-inhibit GJIC (P < 0.01). Superposition of 0.2 mT noise MF could get a fluorescence recovery of (35 +/- 19)% and significantly antagonize its co-suppression by TPA., Conclusion: Electromagnetic noise of 0.2 mT could block the intensifying effect of power frequency magnetic field on TPA-induced GJIC inhibition.

OBJECTIVE: To evaluate the relationship between katG gene mutation and isoniazid (INH) resistance and to develop a rapid screening method of point mutation in the katG gene associated with MTB resistance. METHODS: Twenty-four clinical isolates of MTB with 8 INH resigtance isolates and 16 INH-sensitive isolates were analyzed by PCR-RFLP, with the H(37)Rv reference strain as the control. RESULTS: G-->C point mutations were detected in 7 of 8 isoniazid-resistant strains and no gene mutation was shown in 16 isoniazid-sensitive isolates. The sensitivity and specificity were 87.5 % and 100 % respectively. No katG gene sequence deletion was observed in any specimen. CONCLUSION: Our results suggest katG gene mutation is one of the most important mechanisms of INH-resistant TB. PCR-RFLP may be useful in detection of katG gene mutation.

Zinc ions at micromolar levels exhibited a significant inhibitory activity toward platelet activating factor (AGEPC)- and thrombin-induced serotonin release from washed rabbit platelets. In the ranges from 25 to 30 microM and 10 to 50 microM, respectively, zinc essentially prevented any serotonin release from 1.25 X 10(8) cells/microliter by 1 X 10(-10) M AGEPC and by 0.2 unit thrombin/ml. This inhibition by zinc ions, in micromolar range, occurred in the presence of 1.0 mM Ca2+. The amount of zinc needed for inhibition was inversely proportional to the amount of AGEPC present and further zinc must be added prior to or at the same time as the AGEPC to be effective. Introduction of zinc ions after the AGEPC essentially abolished the inhibitory properties of this divalent cation. Other cations such as Cu2+, La3+, Cd2+, and Mg2+ were ineffective as inhibitors at concentrations where zinc showed its maximal effects. Under conditions similar to those noted above, aggregation induced by AGEPC was blocked only to the extent of 25% of a control. No inhibitory action by zinc on thrombin-induced aggregation was noted. It is apparent that zinc ions influence a site(s) on the rabbit platelet of considerable importance to the activation (or signaling) process by AGEPC and thrombin in these cells, as expressed by serotonin release. Zinc should provide a suitable probe to explore the mechanism of action of these agonists in their interaction with sensitive cells and to define in more specific biochemical terms the putative receptor for these molecules.