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The herbal formula Sinisan (SNS) is a commonly used treatment for depression; however, its mechanism of action remains unclear. This article uses a combination of the GEO database, network pharmacology and molecular docking technologies to investigate the mechanism of action of SNS. The aim is to provide new insights and methods for future depression treatments. The study aims to extract effective compounds and targets for the treatment of depression from the T CMSP database. Relevant targets were searched using the GEO, Disgenet, Drugbank, PharmGKB and T T D databases, followed by screening of core targets. In addition, GO and KEGG pathway enrichment analyses were performed to explore potential pathways for the treatment of depression. Molecular docking was used to evaluate the potential targets and compounds and to identify the optimal core protein-compound complex. Molecular dynamics was used to further investigate the dynamic variability and stability of the complex. The study identified 118 active SNS components and 208 corresponding targets. Topological analysis of P P I networks identified 11 core targets. GO and KEGG pathway enrichment analyses revealed that the mechanism of action for depression involves genes associated with inflammation, apoptosis, oxidative stress, and the MAP K3 and P I3K-Akt signalling pathways. Molecular docking and dynamics simulations showed a strong binding affinity between these compounds and the screened targets, indicating promising biological activity. The present study investigated the active components, targets and pathways of SNS in the treatment of depression. Through a preliminary investigation, key signalling pathways and compounds were identified. These findings provide new directions and ideas for future research on the therapeutic mechanism of SNS and its clinical application in the treatment of depression.Communicated by Ramaswamy H. Sarma.

It has been recognized that depth of invasion (DOI) is closely associated with patient survival for most types of cancer. The purpose of this study was to determine the DOI optimal cutoff value and its prognostic value in laryngeal squamous carcinoma (LSCC). Most importantly, we evaluated the prognostic performance of five candidate modified T-classification models in patients with LSCC. LSCC patients from Harbin Medical University Cancer Hospital and Chinese Academy of Medical Sciences Cancer Hospital were divided into training group (n = 412) and validation group (n = 147). The primary outcomes were overall survival (OS) and relapse-free survival (RFS), and the effect of DOI on prognosis was analyzed using a multivariable regression model. We identified the optimal model based on its simplicity, goodness of fit and Harrell's consistency index. Further independent testing was performed on the external validation queue. The nomograms was constructed to predict an individual's OS rate at one, three, and five years. In multivariate analysis, we found significant associations between DOI and OS (Depth of Medium-risk invasion HR, 2.631; P < .001. Depth of high-risk invasion: HR, 5.287; P < .001) and RFS (Depth of high-risk invasion: HR, 1.937; P = .016). Model 4 outperformed the American Joint Committee on Cancer (AJCC) staging system based on a low Akaike information criterion score, improvement in the concordance index, and Kaplan-Meier curves. Inclusion of DOI in the current AJCC staging system can improve the differentiation of T classification in LSCC patients.

Diabetic encephalopathy is a chronic complication of diabetes that lacks an optimized treatment strategy. The present study sought to elucidate the potential molecular mechanism of Qi Fu Yin in improving diabetic encephalopathy through network pharmacology. The active components and target information of Qi Fu Yin were obtained from the TCMSP and Swiss target databases, while the target information of diabetic encephalopathy was sourced from Gene cards, OMIM, and Pharm Gkb databases. Enrichment analyses of KEGG and GO were conducted utilizing drug-disease common targets, while protein-protein interactions were predicted through the utilization of the STRING database platform. Subsequently, molecular docking was executed via Auto Dock Vina to authenticate the interaction between core components and core targets. The findings revealed that Qi Fu Yin exhibited 178 common targets with diabetic encephalopathy, and the enrichment analyses demonstrated that these targets were associated with lipid and atherosclerosis, AGE-RAGE signaling pathways, and other related pathways. The findings of the molecular docking indicated a favorable binding affinity between the active components of drug and the core targets, with EGF and quercetin exhibiting the most notable docking score. Additionally, the molecular dynamics simulation corroborated this high affinity. These results suggested that the active ingredients of Qi Fu Yin, including quercetin and kaempferol, may modulated the expression of genes such as IL10, TNF, EGF, and MMP2, thereby activating the AGE-RAGE signaling pathways and potentially serving as a therapeutic intervention for diabetic encephalopathy.Communicated by Ramaswamy H. Sarma.

In this study, we recombinantly expressed the V protein of the peste des petits ruminants virus (PPRV) and evaluated its diagnostic value for PPRV infection using an indirect ELISA (i-ELISA). The optimal concentration of the coated antigen of V protein was 15 ng/well at a serum dilution of 1:400, and the optimal positive threshold value was 0.233. A cross-reactivity assay showed that the V protein-based i-ELISA was specific to PPRV with consistent reproducibility and showed a specificity of 82.6% and a sensitivity of 100% with a virus neutralization test. Using the recombinant V protein as an antigen in ELISA is useful for seroepidemiological studies of PPRV infections.

The colchicine binding site of tubulin is a promising target for discovering novel antitumour agents. Previously, we identified 2-aryl-4-amide-quinoline derivatives displayed moderate tubulin polymerisation inhibitory activity and broad-spectrum in vitro antitumour activity. In this study, structure based rational design and systematic structural optimisation were performed to obtain analogues C1∼J2 bearing diverse substituents and scaffolds. Among them, analogue G13 bearing a hydroxymethyl group displayed good tubulin polymerisation inhibitory activity (IC 50  =  13.5 μM) and potent antiproliferative activity (IC 50 values: 0.65 μM∼0.90 μM). G13 potently inhibited the migration and invasion of MDA-MB-231 cells, and displayed potent antiangiogenic activity. It efficiently increased intracellular ROS level and decreased MMP in cancer cells, and obviously induced the fragmentation and disassembly of the microtubules network. More importantly, G13 exhibited good in vivo antitumour efficacy in MDA-MB-231 xenograft model (TGI  =  38.2%; i.p., 30 mg/kg).

Context: Alzheimer's disease (AD) is a neurodegenerative disorder that affects millions of people worldwide. Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) are promising therapeutic targets for AD., Objective: To evaluate the inhibitory effects of aaptamine on two cholinesterases and investigate the in vivo therapeutic effect on AD in a zebrafish model., Materials and Methods: Aaptamine was isolated from the sponge Aaptos suberitoides Brøndsted (Suberitidae). Enzyme inhibition, kinetic analysis, surface plasmon resonance (SPR) and molecular docking assays were used to determine its inhibitory effect on AChE and BuChE in vitro . Zebrafish were divided into six groups: control, model, 8 μM donepezil, 5 , 10  and 20 μM aaptamine. After three days of drug treatment, the behaviour assay was performed., Results: The IC 50 values of aaptamine towards AChE and BuChE were 16.0 and 4.6 μM. And aaptamine directly inhibited the two cholinesterases in the mixed inhibition type, with K i values of 6.96 ± 0.04 and 6.35 ± 0.02 μM, with K d values of 87.6 and 10.7 μM. Besides, aaptamine interacts with the crucial anionic sites of AChE and BuChE. In vivo studies indicated that the dyskinesia recovery rates of 5 , 10  and 20 μM aaptamine group were 34.8, 58.8 and 60.0%, respectively, and that of donepezil was 63.7%., Discussion and Conclusions: Aaptamine showed great potential to exert its anti-AD effects by directly inhibiting the activities of AChE and BuChE. Therefore, this study identified a novel medicinal application of aaptamine and provided a new structural scaffold for the development of anti-AD drugs.

This study was designed to study functions of Circ&#95;0000020 during osteogenic differentiation. First, we used RT-qPCR to detect the expression of Circ&#95;0000020, miR-142-5p and osteogenesis-related genes, whereas western blot analysis detected the expression of osteogenesis markers after the osteogenic differentiation of primary BMSCs isolated from rats. Alkaline phosphatase (ALP) activity and alizarin red Sstaining validated osteoblast phenotypes. Flow cytometry was used to detect cell apoptosis. Sh-Circ&#95;0000020 was used to study the function of Circ&#95;0000020 in osteogenic differentiation of BMSCs. Luciferase assay and RNA immunoprecipitation were used to validate the interaction between Circ&#95;0000020 and miR-142-5p, and BMP2 and miR-142-5p. Co-transfection of miR-142-5p and sh-Circ&#95;0000020 was used to verify the downstream signaling pathway. Circ&#95;0000020 expression was up-regulated during osteogenic differentiation, whereas miR-142-5p expression was significantly decreased. Silencing Circ&#95;0000020 inhibited osteogenic differentiation and promoted apoptosis, and inhibited ALP activity and mineralization ability. Moreover, Circ&#95;0000020 interacts directly with miR-142-5p which binds to the BMP2 3'UTR and inhibits its expression. Additionally, co-transfection of miR-142-5p inhibitors and sh-Circ&#95;0000020 rescued down-regulated BMP2, increased the expression osteogenesis-related gene expressions, and thereby rescued the inhibition of osteogenic differentiation induced by Circ&#95;0000020 silencing. Furthermore, co-transfection of miR-142-5p inhibitors and sh-Circ&#95;0000020 reversed Circ&#95;0000020 silencing-induced downregulation of p-Smad1/5/8, Runx2, and Osterix protein levels. Circ&#95;0000020 regulates BMP2 expression through sponging miR-142-5p as ceRNA, thereby positively regulating BMSCs osteogenic differentiation through Circ&#95;0000020/miR-142-5p/BMP2/SMAD-dependent signaling pathway.

Overexpression of DTYMK is related with tumorigenesis and progression in several human tumors. However, the role of upregulated DTYMK in hepatocellular carcinoma (HCC) patients still remains unclear. In this study, the DTYMK expression in HCC tumors was evaluated in three GEO series (GSE14520, GSE54236, GSE63898), TCGA-LIHC, and ICGC-IRLR-JP cohorts. Survival analysis of DTYMK based on TCGA-LIHC and ICGC-LIRI-JP cohorts was conducted. We found that DTYMK was dramatically upregulated in tumor tissue compared with that in adjacent liver tissue. Kaplan-Meier analysis revealed that high expression of DTYMK in HCC patients' tumor tissue was significantly corresponded to worse overall survival (OS) (P < 0.05). Further analysis showed that overexpressing DTYMK led to poor relapse free survival (RFS) and disease-specific survival (DSS) (all P < 0.05). In conclusion, DTYMK is upregulated in tumors and correlated with poor prognosis in HCC patients. In our report, DTYMK is higher expression in HCC cancer tissue and cell line than tumor adjacent tissue and normal liver cell line. Knocking down DTYMK can inhabit tumor cell proliferation by interfering cell cycle, whereas overexpression of DTYMK can promote tumor cell proliferation. These findings indicate that upregulation of DTYMK enhances tumor growth and proliferation by promoting cell cycle.

Pentasachme caudatum Wallich ex Wight is considered as one of the Asian enigmatic genera classified in the Asclepiadoideae (Apocynaceae). To determine its evolutionary relationship in the family, we sequenced and characterized the complete chloroplast genome of P. caudatum . The plastid genome of P. caudatum is 158,487 bp in length, containing a large single-copy (90,380 bp), a small single-copy (18,585 bp), and a pair of inverted repeats (24,761 bp). It has 127 annotated genes, consisting of 83 protein-coding, eight rRNA and 36 tRNA genes. Phylogenetic analysis using 76 protein-coding regions of the plastid genomes of related taxa showed that P. caudatum was resolved in a fully supported clade with Orthanthera albida . The newly sequenced P. caudatum provides essential genetic information that is useful for future phylogenetic studies in the family Apocynaceae., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

Gynochthodes cochinchinensis previously known as Morinda cochinchinensis is considered as potential medicinal plant in family Rubiaceae. In this paper, the complete chloroplast genome of G. cochinchinensis was sequenced and characterized for the first time. The cp genome of G. cochinchinensis was 153,022 bp in length containing a large single copy region (83,799 bp), a small single copy region (17,591 bp), and a pair of inverted repeat regions (25,816 bp). It has a total of 131 genes, comprising of 86 protein-coding genes, eight rRNA genes, and 37 tRNA genes. Phylogenetic analysis revealed that Gynochthodes cochinchinensis together with Gynochthodes officinalis were closely related to genus Morinda ., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

Context: Aaptamine is a potent ocean-derived non-traditional drug candidate against human cancers. However, the underlying molecular mechanisms governing aaptamine-mediated repression of lung cancer cells remain largely undefined., Objective: To examine the inhibitory effect of aaptamine on proliferation and progression of non-small cell lung carcinoma (NSCLC) and dissect the potential mechanisms involved in its anticancer functions., Materials and Methods: In vitro assays of cell proliferation, cell cycle analysis, clonal formation, apoptosis and migration were performed to examine the inhibitory effects of aaptamine (8, 16 and 32 μg/mL) on NSCLC cells. The expression levels of proteins were analysed using western blotting analysis when cells were treated with a single drug or a combination treatment for 48 h., Results: Aaptamine significantly inhibited A549 and H1299 cells proliferation with IC 50 values of 13.91 and 10.47 μg/mL. At the concentrations of 16 and 32 μg/mL, aaptamine significantly reduced capacities in clonogenicity, enhanced cellular apoptosis and decreased the motile and invasive cellular phenotype. In addition, aaptamine arrested cell cycle at G1 phase via selectively abating cell cycle regulation drivers (CDK2/4 and Cyclin D1/E). Western blotting results showed that aaptamine attenuated the protein expression of MMP-7, MMP-9 and upregulated the expression of cleaved-PARP and cleaved-caspase 3. Moreover, aaptamine inhibited PI3K/AKT/GSK3β signalling cascades through specifically degrading the phosphorylated AKT and GSK3β., Discussion and Conclusions: Aaptamine retarded the proliferation and invasion of NSCLC cells by selectively targeting the pathway PI3K/AKT/GSK3β suggesting it as a potential chemotherapeutic agent for repressing tumorigenesis and progression of NSCLC in humans.

Apocynum venetum L. (Apocynaceae) or Luobuma is a widely known traditional medicine use to treat hypertension, relieve anxiety, soothe the nerves and promote diuresis. In this study, the complete chloroplast genome of this medicinal plant was determined through Illumina sequencing method. The A. venetum cp genome is 150,897 bp in length, containing a small single copy region (17,256 bp), a large single copy region (81,957 bp), and a pair of IR regions (25,842 bp). It encodes for a total of 131 genes, including 86 protein-coding genes, 8 rRNA genes, and 37 tRNA genes. Phylogenetic analysis also reveals that A. venetum is relatively close to Aganosma cymosa., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

Although the process by which the cortical tissues of the brain fold has been the subject of considerable study and debate over the past few decades, a single mechanistic description of the phenomenon has yet to be fully accepted. Rather, two competing explanations of cortical folding have arisen in recent years; known as the axonal tension and the differential tangential expansion models. In the present review, these two models are introduced by analyzing the computational, theoretical, materials-based, and cell studies which have yielded them. Then Four-dimensional bioprinting is presented as a powerful technology which can not only be used to test both models of cortical folding de novo , but can also be used to explore the reciprocal effects that folding associated mechanical stresses may have on neural development. Therein, the fabrication of "smart" tissue models which can accurately simulate the in vivo folding process and recapitulate physiologically relevant stresses are introduced. We also provide a general description of both cortical neurobiology as well as the cellular basis of cortical folding. Our discussion also entails an overview of both 3D and 4D bioprinting technologies, as well as a brief commentary on recent advancements in printed central nervous system tissue engineering., Competing Interests: Disclosure Statement The authors declare no conflicts of interest.

Chemotherapy resistance represents a major issue associated with gastric cancer (GC) treatment, and arises through multiple mechanisms, including modulation of the cell-cycle check point. Several ubiquitin kinases, including RING finger protein 138 (RNF138), have been reported to mediate the G2/M phase arrest. In this study, we investigated the role of RNF138 in the development of cisplatin resistance of two GC cell lines. We show that RNF138 levels are higher in cisplatin-resistant cell lines, compared with cisplatin-sensitive cells, and RNF138 expression was elevated during drug withdrawal following the cisplatin treatment. Using gene overexpression and silencing, we analyzed the impact of altering RNF138 level on GC cell viability, apoptosis, and cell cycle phenotypes in two isogenic cisplatin-sensitive and resistant cell lines. We show that RNF138 overexpression increased GC cell viability, decreased apoptosis and delayed cell cycle progression in the cisplatin-sensitive GC cells. Conversely, RNF138 silencing produced opposite phenotypes in the cisplatin-resistant cells. Moreover, RNF138-dependent phosphorylation of Chk1 was seen in GC cells, indicating a novel connection between cisplatin-induced DNA damage and apoptosis. Collectively, these data suggest that RNF138 modulates the cisplatin resistance in the GC cells, thus serving as a potential drug target to challenge chemotherapy failure. In addition, RNF138 can also be used as a marker to monitor the development of cisplatin resistance in GC treatment.

Previous studies have established that hedgehog (Hh) signaling mediates tumor-stroma interaction and promotes hepatocellular carcinoma progression. Here, we demonstrated that activation of Hh signaling in hepatic stellate cell (HSC) line LX-2 by Huh-7-derived sonic Hh led to increased secretion of angiogenic factors and promoted angiogenesis in vitro. The activated LX-2 also enhanced vascular mimicry of hepatoma cells. Furthermore, co-injection of Huh-7 and LX-2 significantly accelerated tumor growth with enhanced angiogenesis compared with Huh-7 alone, which could be partly abrogated by Hh signaling inhibitor. Collectively, our data showed that paracrine Hh signaling mediated pro-angiogenic function of HSC and enhanced hepatoma growth.

Many studies have suggested that polymorphisms of three key genes (ACE, AGT and CYP11B2) in the renin-angiotensin-aldosterone system (RAAS) play important roles in the development of blood pressure (BP) salt sensitivity, but they have revealed inconsistent results. Thus, we performed a meta-analysis to clarify the association. PubMed and Embase databases were searched for eligible published articles. Fixed- or random-effect models were used to pool odds ratios and 95% confidence intervals based on whether there was significant heterogeneity between studies. In total, seven studies [237 salt-sensitive (SS) cases and 251 salt-resistant (SR) controls] for ACE gene I/D polymorphism, three studies (130 SS cases and 221 SR controls) for AGT gene M235T polymorphism and three studies (113 SS cases and 218 SR controls) for CYP11B2 gene C344T polymorphism were included in this meta-analysis. The results showed that there was no significant association between polymorphisms of these three polymorphisms in the RAAS and BP salt sensitivity under three genetic models (all p > 0.05). The meta-analysis suggested that three polymorphisms (ACE gene I/D, AGT gene M235T, CYP11B2 gene C344T) in the RAAS have no significant effect on BP salt sensitivity.

Objectives: Lipoxin A4 (LXA4) is a potent anti-inflammatory mediator that exerts a neuroprotective effect following cerebral ischaemia/reperfusion (I/R) injury. However, little is known about the underlying mechanisms. Upregulation of nuclear factor erythroid 2-related factor 2 (Nrf2) is generally considered to reduce cerebral I/R injury. Nuclear factor erythroid 2-related factor 2 can induce haeme oxygenase-1 (HO-1) and glutathione (GSH) expression to combat increased oxidative stress. The present study aimed to investigate the effects of Nrf2 signalling on LXA4-mediated neuroprotection., Methods: Adult male Sprague Dawley rats were subjected to 2-hour middle cerebral artery occlusion followed by 24-hour reperfusion. Rats were randomly divided into four groups: Sham, I/R, LXA4, and LXA4+butoxycarbonyl-Phe-Leu-Phe-Leu-Phe (Boc2) (all n = 24). Brain infarction was detected by 2,3,5-triphenyltetrazolium chloride staining. After 24 hours of reperfusion, Nrf2, HO-1, and p62 expression levels were determined by western blot, and GSH synthesis was assessed., Results: Lipoxin A4 effectively reduced infarct volumes and improved neurological scores. These effects were partially blocked by Boc2, a specific antagonist of the LXA4 receptor (ALXR). Lipoxin A4 induced Nrf2 expression and its nuclear translocation, as well as HO-1 expression and GSH synthesis; Boc2 did not block these effects. The excess p62 accumulation induced by LXA4 might be closely related to Nrf2 activation., Discussion: Overall, our data suggest that Nrf2 upregulation is involved in the neuroprotective effects of LXA4 and may be ALXR independent.

Bisphenol A (BPA) is an industrial chemical with estrogenic activity. In our study, 40 rats were given BPA at 4, 40, and 400 mg/kg per day. Controls were treated with corn oil of same volume until the offspring were 30 d olds. At the end of the experiment, the expression of estrogen receptor (ER)-alpha and the mRNA levels of inerleukin (IL)-2, IL-12, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha in spleens were investigated by Western blotting and real-time reverse-transcription polymerase chain reaction (RT-PCR). The histopathological change of organs was observed. The results showed that bisphenol A reduced the expression of ER-alpha in males of the high-dose group in the F(0) generation and in middle- and high-dose groups of the F(1) generation (p < .05), but increased it in females in the high-dose group of the F(0) generation and in middle- and high-dose groups of the F(1) generation (p < .05). The levels of IL-2, IL-12, IFN-gamma, and TNF-alpha in spleens were downregulated in all groups in contrast to controls (p < .05). We also found some histological changes in spleens, livers, and kidneys. These findings demonstrated that bisphenol A has estrogen-like activity and might affect some immune organs and parameters.

The YWK-II component of human sperm membrane is related to the betaA4-amyloid precursor protein (APP) of Alzheimer's disease. A yeast 2-hybrid system was used to screen a mouse testis cDNA expression library for potential ligands capable of interacting with the extracellular domain of the YWK-II component. One of the bound proteins was identified as hDIA1, which has 96% identity with p140mDia. These proteins are members of the formin homology family and participate in cytokinesis and organization of the actin cytoskeleton. By interacting with these diaphanous proteins, the YWK-II component may be involved in germ cell differentiation and in the structural formation of the acrosome.

The HSD-I gene codes a human sperm membrane protein (hSMP-1) and has been assigned the accession number U12978. The gene is located on human chromosome 9, region p12-p13. When the 1.7-kb cDNA of HSD-I was digested sequentially with EcoRI, BamHI, and HindIII, a 550-bp cDNA fragment was formed, which codes for the extracellular domain. This fragment was cloned into the asd+ vector pYA3149 to construct pYA3149R. The recombinant plasmid was used to transform an avirulent deltacva, deltacrp, deltaasd vaccine strain of Salmonella typhimurium chi4550. The hSMP-1 component was localized on the surface of the head of mature rat spermatozoa by an immunofluorescence technique using polyclonal anti-hSMP-1 antibodies. Since rat sperm contain hSMP-1, this rodent can be used to assay the immunogenicity of pYA3149R. Female Wistar rats were immunized by oral administration of the recombinant Salmonella. Anti-hSMP-1 antibodies in blood and vaginal washes of immunized animals were determined. Both body fluids contained significant amounts of the antibodies, showing that the recombinant Salmonella is an effective oral immunogen in rats.

Serum was obtained from an infertile woman having antibodies with sperm agglutinating activity. The antibodies interacted with a human sperm membrane protein (hSMP-1) with an estimated Mr of 55 kD. The gene (HSD-1) coding hSMP-1 was isolated from a human testis cDNA expression library and assigned the accession number U12978. The cDNA was conjugated to a prokaryotic expression vector to construct the recombinant vector, pRSET-HSD-I, which was expressed in Escherichia coli. The recombinant hSMP-1 was isolated and used to immunize rabbits to raise polyclonal antibodies. Usingan immunocytochemical technique, hSMP-1 protein was immunolocalized in germ cells of human testis at all stages of spermatogenesis. mRNAs were prepared from 16 different human tissues and analyzed by Northern blot using HSD-1 as probe. A positive reaction was elicited only with testis mRNA. The present findings suggest that the expression of hSMP-1 gene is testis-specific and occurs during the early stages of germ cell differentiation. In a comparative study, the location of the hSMP-I protein in sperm and in germ cells of the seminiferous tubules of rats was determined. The target antigen was immunolocated on the head and tail of rat sperm and in late spermatids and spermatozoa of rat testis. These results suggest that, in the rat, the HSD-1 gene is expressed during spermiogenesis.

The cDNA encoding a human sperm membrane designated as HSD-1 was isolated from a human testis lambda gt11 cDNA expression library and assigned the accession number U12978 by GenBank. HSD-1 was conjugated to an eukaryotic expression plasmid (pRSV) to construct the recombinant plasmid pRSV-HSD-1. Female mice were inoculated intramuscularly with the plasmid DNA and the expression of HSD-1 was determined. HSD-1 mRNAs were detected in myocytes and endomysial connective tissue cells of the quadriceps muscle by in situ hybridization. Spleen of inoculated animals contained an increased number of cytotoxic T lymphocytes, phagocytes, and plasma cells. Fertility of the treated animals was not affected. Thus, intramuscular inoculation of female mice with the plasmid DNA (pRSV-HSD-1) results in the expression of HSD-1 and may elicit a tissue-mediated immune response.

A 20-kDa sperm membrane protein cDNA, designated as RSD-1, was isolated by epitope selection from a rat testis lambda gtll expression library. RSD-1 was used as a probe to screen a human testis lambda ZAPII cDNA expression library. A cDNA designated as BS-63 was isolated and found to consist of 1933 bp with an open reading frame of 1824 bp and assigned the accession number U64675 by GenBank. The deduced polypeptide consisted of 608 amino acid residues containing XFXFG or FG motifs that are characteristic of nuclear pore complex (NPC) proteins and act as potential binding sites for Ran. The N-terminal region has high homology with RanBP2/Nup358, a nucleoporin component, showing that BS-63 is a member of the NPC family. Northern blot analysis of mRNAs prepared from various human tissues shows that BS-63 is transcribed in two forms: 6.0 and 8.5 kb. The 8.5-kb transcript was present in low amounts in several somatic tissues, whereas the 6.0-kb transcript is expressed only in testis. In situ hybridization analysis of human testis sections showed that BS-63 mRNA is expressed only in germ cells at all stages of spermatogenesis. Sertoli cells did not transcribe the gene.

A truncated cDNA coding a rabbit epididymal protein (BE-20) was identified in a previous study. In the present study the full-length cDNA was isolated by the method of rapid amplification of cDNA ends. The BE-20 cDNA consisted of 585 bp with a poly(A) tail of 26 residues and an open reading frame composed of 369 bp encoding a deduced polypeptide containing 123 amino acid residues with a calculated molecular mass of 13 kDa. The N-terminus-contained a leucine-rich segment. BE-20 cDNA has about 76.8% homology with the HE4 gene of human epididymis. Northern blot analysis of mRNAs prepared from 17 different human tissues was performed using as probe a 0.5-kb DNA fragment corresponding to a segment of BE-20 cDNA. Positive reaction was elicited only with epididymal mRNA. A DNA fragment corresponding to a section of the open reading frame of BE-20 cDNA was cloned in Escherichia coli under the control of the T7 promoter. The cellular content of the expressed recombinant protein comprised about 55% of the total protein. The chromatographically purified bacterial product migrated as a single band with an estimated M(r) of 15 kDa on analysis by SDS-PAGE. In conclusion, BE-20 cDNA is expressed only in the epididymis. It is structurally related to the four-disulfide core family of extracellular proteinase inhibitors and may be involved in sperm maturation.

The cDNA (HED-2) encoding a 20 kDa protein found in mammalian epididymal fluid was isolated from a human testis expression library. It is composed of 1908 bp, containing a reading frame of 1479 bp, coding a polypeptide consisting of 493 amino acids, and assigned the accession number: U15158 by GenBank (Biochem. Mol. Biol. Int. 34, 1131-1136, 1994). HED-2 has 99% identity with the zyxin gene in amino acid sequence, a component of cell junction matrix and a member of the LIM domain protein family. Northern blot analysis of RNAs prepared from various human tissues showed that the HED-2 gene was expressed in all tissues analyzed. Sertoli cells of human testis expressed the gene as determined by an in situ hybridization method. The present study shows that the HED-2 gene is a member of the LIM domain protein family.

A protein designated as BE-20 was purified from cauda epididymal fluid of male rabbits and the amino acid sequence of the N-terminus was determined. A 23-mer oligonucleotide coding the N-terminal eight amino acids of the BE-20 protein was synthesized. The oligonucleotide was used as sense primer with rabbit epididymal mRNA as template in the RT-PCR system. The BE-20 cDNA consisted of 499 bp with an open reading frame of 285 bp encoding a deduced polypeptide composed of 95 amino acids. Digoxigenin-labeled BE-20 cDNA was prepared and used as a hybridization probe to detect the specific mRNA. The probe interacted with a 1.2-kb mRNA prepared from rabbit epididymis; mRNAs prepared from rabbit testis gave negative reaction. Using tissue sections, the BE-20 mRNA was located in the epithelial cells of the cauda epididymis and proximal segment of the ductus deferens by in situ hybridization method. Sections of the corpus and caput epididymis, testis, and liver gave negative reaction. Polyclonal anti-BE-20 antibodies were raised and found to inhibit in vitro the capacity of human sperm to penetrate zona-free hamster ova. The results suggest that BE-20 protein may influence maturation of spermatozoa during its movement through the epididymis and/or the capacity of sperm to fertilize ova.

A protein designated as BE-20 was purified from cauda epididymal fluid of the rabbit by preparative polyacrylamide gel electrophoresis and HPLC on a mono Q HR5/5 anion exchange column. The purified protein migrated with an estimated Mt of 20,000 when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The amino acid sequence of the N-terminus of the BE-20 protein was determined. The initial eight amino acid residues were His-Gly-Ala-Asp-Lys-Pro-Gly-Val. The corresponding 23 mer oligonucleotide (5'-CATGGCGCTGACAAGCCTGGGGT-3') was synthesized and used as sense primer with rabbit epididymal mRNA as template in the RT-PCR system. The purified BE-20 cDNA consisted of 499 bp with an open reading frame of 285 bp encoding a deduced polypeptide composed of 95 amino acids. The BE-20 cDNA had 78.5% identity in 479 bp overlap with human epididymis-specific HE4 cDNA. The amino acid sequences of the initial 30 amino acid residues of the N-terminus of the purified protein and the deduced polypeptides were as follows: N-His-Gly-Ala-Asp-Lys-Pro-Gly-Val-Cys-Pro-Gln-Leu-Ser-Ala-Asp-Leu-Asn-Cy s- Thr-Gln-Asp-Cys-Arg-Ala-Asp-Gln-Asp-Cys-Ala-Glu. The deduced polypeptide contained 16 cysteine residues and had partial sequence homology with proteins belonging to the four-disulfide core family of extracellular proteinase inhibitors. The BE-20 protein may play a role in sperm maturation and/or capacitation.

Serum obtained from an infertile woman contained antibodies that agglutinate human sperm. The antibodies interacted with a sperm protein with an estimated M(r) of 17.5 kD. The cDNA coding the 17.5-kD protein was isolated from a human testis lambda gt11 expression library and identified as a segment of the calpastatin gene. Single-stranded 35S-labeled RNA probes were prepared from the calpastatin cDNA segment. Using the techniques of in situ hybridization, the calpastatin mRNA was located in spermatids of human testis. The results support a previous observation that the calpastatin segment is produced during spermiogenesis and suggest that transcription of the calpastatin gene occurred during the postmeiotic haploid stage of spermatogenesis.

Polyclonal antibodies raised against a 20 kD epididymal protein (EP20) were used to isolate the cDNA from a human testis lambda gt11 expression library. The nucleotide sequence of the cDNA consisted of 1908 base pairs (bp) containing an open reading frame composed of 1479 bp encoding a polypeptide of 493 amino acid residues. The nucleotide sequence of EP-20 cDNA had 97% identities (282/288) with ESTO 0991 Homo Sapiens cDNA clone HHC M14 in the reverse orientation. The HHC M14 sequence corresponded to a segment in the non-translatable 3'end of EP-20 cDNA. The amino acid sequence of the deduced polypeptide showed no homology with reported polypeptides. The epididymal protein may be involved in sperm maturation and/or capacitation.

Antibodies present in a serum obtained from an infertile woman interacted with a 17.5 kD glycoprotein (BS-17 component) extracted from human sperm by Western blot. Polyclonal antibodies were raised against the BS-17 component and used to identify positive staining clones from a human testis lambda gt11 expression library. The cDNA encoding the BS-17 component was isolated and its nucleotide sequence determined. The BS-17 cDNA contained 758 nucleotides with an open reading frame of 558 nucleotides encoding a polypeptide consisting of 186 amino acids. The BS-17 cDNA showed 99.7% homology in 758 nucleotides overlap with the 3' terminus of the gene coding calpastatin and 99.5% identity in 186 amino acid overlap with the carboxyl terminus of calpastatin. The BS-17 component of human sperm corresponds to the carboxyl terminus of calpastatin. This conclusion is supported by the finding that the polyclonal antibodies also interacted with a 84 kD protein corresponding to the M(r) of calpastatin.

Serum obtained from an infertile subject possessed antibodies that interacted with a human sperm glycoprotein with an estimated M(r) of 17,550 and pI of 5.65 containing 17.7% neutral hexoses and designated as the BS-17 component. Polyclonal antibodies raised against the BS-17 antigen blocked the capacity of human sperm to fertilize zona-free hamster ova in vitro; however, the antibodies did not influence the binding of human sperm to zone-free ova or alter the motility of human sperm. The antibodies inhibited the capacity of mouse sperm to fertilize ova upon in vivo insemination. The BS-17 antigen was detected in human, rat, mouse, rabbit, and hamster sperm by an immunocytochemical method, using polyclonal anti-BS-17 antibodies. Intense staining occurred over the surface of the acrosomal region of all mammalian sperm. The results suggest that the production of anti-BS-17 antibodies contribute to infertility by preventing the capacitation of sperm and/or by blocking the ability of capacitated sperm to fertilize the egg.

The cDNA (RSD-2) encoding a human sperm protein (YWK-II) was isolated from a rat testis lambda gt11 expression library. Its nucleotide sequence was determined. The deduced polypeptide showed high identity with the transmembrane-cytoplasmic domains of A4 amyloid precursor protein of Alzheimer's disease. The RSD-2 was inserted into the EcoRI site of the pSV2-EP vector to construct the pSVRS-2 vector. Chinese hamster ovarian (CHO-dhfr) cells were cotransformed with pSV2-neo and pSVRS-2. mRNA and chromosomal DNA prepared from the transformed cells interacted with [32P]RSD-2 as probe by dot hybridization. The production of the YWK-II protein was determined by staining with the YWK-II mAb by an indirect immunofluorescence technique. There was marked staining of the cytoplasm. The RSD-2 cDNA encoding the YWK-II sperm was expressed in the transformed CHO cells. The pSV2-EP vector and the CHO cell expression system can be utilized to produce sperm proteins for antifertility studies.

Monoclonal antibodies were raised against a specific human sperm protein and designated as the YWK-II mAb. The partial cDNA encoding the protein was isolated from a rat testis lambda gt11 expression library and the amino acid sequence of the protein was deduced. The cytoplasmic-transmembrane domains of the deduced protein had high homology with the A4 amyloid precursor protein of Alzheimer's disease. To evaluate the stage of spermatogenesis when the gene was expressed, single-stranded 35S-labeled RNA probes were prepared from the cDNA. By an in situ hybridization technique the mRNA for the antigen was detected in germ cells at all stages of spermatogenesis. The finding that the gene is expressed in spermatogonia suggests possible involvement in the initiation of germ cell differentiation or in the detachment of spermatogonia from the basement membrane.

Rabbit sperm tails were obtained by a nitrogen cavitation procedure and separated by discontinuous sucrose gradient centrifugation. Proteins were extracted from sperm tails with 3[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS) and separative disk electrophoresis and DEAE-trisacryl column chromatography. A protein with an estimated mol wt of 20.1 +/- 1.1 kD was isolated and found to be homogeneous by SDS-PAGE and designated as rSMP-B. The isoelectric point of rSMP-B was in the range of pH 4.4-4.7. The amino acid composition was determined, and glycine was identified as the N-terminal residue. Antisera were raised against purified rSMP-B. Using a peroxidase-antiperoxidase method, the rSMP-B antigen was located on the surface of the midpiece and tail of the sperm. Testis sections showed intense staining of late spermatids located within the seminiferous tubules. Adult male rabbits were inoculated with rSMP-B protein and Freund's adjuvant. The testis and epididymis of the immunized animals showed absence of sperm. The immunolocalization findings and the immunization data suggest that rSMP-B is formed in late spermatid and that it is an essential structural component of sperm.