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Later onset of apoptosis in the bulbourethral glands after castration compared to that in the seminal vesicles.

Androgens affect many different target organs within the male reproductive tract to stimulate their development and secretory cytodifferentiation, and to maintain structure and function in adulthood. Castration causes regression of these organs via apoptosis. However, not all organs of the reproductive tract are equally sensitive to androgen withdrawal. The effects of castration on the mouse seminal vesicles (SVs) and bulbourethral glands (BUGs) were compared in terms of protein and DNA contents, epithelial apoptosis, and proliferative response of epithelial cells to androgen. Castration induced similar, marked decreases in protein contents in the SV and BUG by 2 days after castration which reached a minimum at 16 days post castration. Both organs underwent a decrease in DNA content, but the kinetics of this decline differed. In the SV, DNA content was significantly decreased by 4 days whereas in the BUG this did not occur until 16 days post castration. By day 16 both organs had regressed to roughly the same degree. The apoptotic index in the epithelium reflected this difference in timing as well. Apoptotic index of the SV epithelium was highest on day 3 after castration and declined thereafter. On the other hand, the apoptotic index in the BUG didn't begin to increase until 7 days after castration and became maximal on day 12. Daily injections of testosterone propionate (TP) from day 8, 16, or 30 after castration all increased epithelial labelling index in the SVs to a similar degree. However, the TP-induced increase in the epithelial labelling index in the BUG beginning on day 8 after castration was considerably less than that in BUGs receiving TP treatment from day 16 or 30 after castration. Thus, the proliferative response of the epithelium depended upon prior apoptosis in the gland, with the timing being delayed in the BUG as compared with the SV. The present results indicate that castration induces epithelial apoptosis and reduction in glandular DNA content considerably later in the BUG than in the SV though reduction in protein content in the BUG fell simultaneously with that in the SV.

Animals↗

Analysis and optimization of structure-based virtual screening protocols (1): exploration of ligand conformational sampling techniques.

Ligand conformational flexibility has long been recognized as an important issue in virtual screening (VS). To this end, a number of different methodologies have been adapted to tackle the problem. Many of said techniques were originally designed for ligand derived pharmacophore screens, but have subsequently been fashioned for application within structure-based virtual screening (SVS). A popular adaptation is the pre-calculation of diverse ligand conformations for subsequent docking in target active sites. In this paper, we study a number of the software programs currently being used in conformer generation, analyzing their ability to regenerate known ligand binding conformations. The implications of these studies are discussed, from the perspective of VS in general and SVS in particular.

Computational Biology↗

Structural basis of Smad1 activation by receptor kinase phosphorylation.

Phosphorylation of Smad1 at the conserved carboxyl terminal SVS sequence activates BMP signaling. Here we report the crystal structure of the Smad1 MH2 domain in a conformation that reveals the structural effects of phosphorylation and a molecular mechanism for activation. Within a trimeric subunit assembly, the SVS sequence docks near two putative phosphoserine binding pockets of the neighboring molecule, in a position ready to interact upon phosphorylation. The MH2 domain undergoes concerted conformational changes upon activation, which signal Smad1 dissociation from the receptor kinase for subsequent heteromeric assembly with Smad4. Biochemical and modeling studies reveal unique favorable interactions within the Smad1/Smad4 heteromeric interface, providing a structural basis for their association in signaling.

Allosteric Regulation↗

Urothelial transformation into functional glandular tissue in situ by instructive mesenchymal induction.

It is generally believed that adult tissue is terminally differentiated. The ureter is derived from the metanephric diverticulum which, along with the derivatives of the metanephric mesoderm, forms the kidney. In our experiments, the left ureters of adult male athymic mouse hosts were severed below the kidney, and mesenchyme from neonatal rat seminal vesicles (SVM) was grafted to the cut end of the ureter, thus bringing adult mouse ureter epithelium (URE) in contact with neonatal rat SVM. After four to eight weeks, the in situ tissue recombinants were harvested, and the epithelial secretory proteins recovered. In 5 of 11 cases, an induction occurred, resulting in an in situ transformation of the non-glandular transitional epithelium of the adult mouse ureter into the simple columnar epithelium of the seminal vesicle (SV). Functional cytodifferentiation was examined in these neonatal rat SVM + adult mouse URE tissue recombinants using antibodies against SV-specific secretory (SVS) proteins of the mouse and rat. From the cut end of the ureter, the adult URE was induced to undergo SV morphogenesis, to express SV cytodifferentiation, and to produce the complete spectrum of major SVS proteins characteristic of the mouse. The induced seminal vesicle epithelium (SVE) also expressed androgen receptors (AR) which are not seen in urothelial tissue. Staining with Hoechst dye 33258, which can distinguish cells of mouse and rat origin, further demonstrated that the induced SVE was indeed of mouse origin and not a contaminant of the inducing rat SVM. in addition, neonatal mouse vaginal mesenchyme was grafted in situ beneath the bladder mucosa of adult male mice, and the host animals were killed after three months. The vaginal mesenchyme implanted into the bladders induced prostate-like acini, indicating that the above reprogramming of adult organs in situ is not an isolate occurrence. These results set a precedent for the "recreation" of new vital organs, such as the kidney, in situ by demonstrating that adult epithelial cells retain a developmental plasticity equivalent to their undifferentiated fetal counterparts and are capable of being reprogrammed in situ to express a completely new morphological, biochemical, and functional phenotype.

Animals↗

Optimizing GRIDSS for clinical use: A targeted NGS filtering strategy for germline structural variant detection.

Detecting intermediate-sized structural variants (SVs) remains challenging in diagnostics, as tools for single-nucleotide and copy-number variants, particularly read-depth-based methods, are often insufficient. GRIDSS addresses this gap by integrating paired-end mapping, split-read analysis, and assembly-based approaches. However, its use in targeted sequencing and diagnostic workflows remains complex. NGS panel data from 9726 patients with suspected hereditary cancer were analyzed using GRIDSS. A filtering strategy was developed to prioritize clinically relevant germline SVs. Multiple parameter settings were tested to optimize performance. The initial dataset of 1,307,592 variants was reduced to 89 candidates after applying the selected filtering strategy. Of these, 24 had been previously detected by routine callers and were not further analyzed. Among the remaining 65, 13 were considered likely true positives after visual inspection using IGV. Experimental validation was performed by Sanger/Nanopore long-read sequencing for these variants, all of which were confirmed. Eight were classified as (likely) pathogenic, including two frameshift duplications in MSH6, one splicing variant in BARD1, and five mobile element insertions in APC, BRCA2, and PALB2. Altogether, GRIDSS implementation increased diagnostic yield while maintaining feasibility for diagnostic workflows. Comprehensive workflow scheme for germline structural variant detection and results in our diagnostic setting.

Humans↗

Genomic and genetic dissection underlying seedling drought resilience in oats.

Drought threatens global crop yields, and common oat, a vital nutritional source for food and feed, is particularly constrained in the semi‑arid regions where it is widely cultivated. Here, we report two high-quality genome assemblies for drought-resilient (Borris37) and drought-sensitive (XymC06) oat accessions with distinct seedling survival rates and genome sizes of 10.92 Gb and 10.96 Gb, and construct comprehensive landscapes of insertion‑deletions (InDels) and structural variants (SVs). Integrating population-level genomic, transcriptomic and phenotypic (seedling survival rate), we demonstrate that InDels and SVs underpin divergent drought resilience and identify 52 candidate genes associated with drought resistance whose expression is significantly modulated by these variants. Borris37 accumulates 36 favorable alleles of these genes. An InDel in the AsNF-YB3 promoter enhances binding to AsARF1, upregulating AsNF‑YB3 under drought, and overexpression of AsNF‑YB3 reduces ROS accumulation. Our findings provide resources and targets for drought‑resistance breeding in oat, thereby supporting global food security.

Drought Resistance↗

Clozapine but not haloperidol Re-establishes normal task-activated rCBF patterns in schizophrenia within the anterior cingulate cortex.

Our previous work has identified that unmedicated volunteers with schizophrenia have regional cerebral blood flow (rCBF) activation patterns inappropriately related to the cognitive demand of a task in anterior cingulate cortex (ACC). Using positron emission tomography (PET) with (15)O water, we compared task-induced rCBF patterns induced by haloperidol or clozapine in individuals with schizophrenia. We hypothesized that clozapine, given its superior clinical action, would tend to normalize the abnormal task-activated response in ACC more than haloperidol. Schizophrenia volunteers (SVs) (n=6) and normal volunteers (NVs) (n=12) were trained to perform a tone discrimination task with 70-80% accuracy. They were then scanned during three task conditions: (1). Rest, (2). sensory motor control (SMC) task, and (3). decision task (DEC). SVs were initially scanned after withdrawal of all psychotropic medication and again after treatment with therapeutic doses of haloperidol (n=5) and/or clozapine (n=5). rCBF values, sampled in the grown maxima of the task-activated ACC cluster, were analyzed between groups and task conditions. Task performance was similar across the unmedicated, haloperidol- and clozapine-medicated SV groups. There was a reduction in accuracy in the haloperidol SV group compared to the NVs. Group and task conditions affected rCBF in the ACC. Clozapine, but not haloperidol, reversed the abnormal ACC rCBF pattern in unmedicated SV to normal. The clozapine-treated SV group showed a rCBF pattern similar to the NV group in that ACC activation was not observed during the control task but occurred during the decision condition. The pattern seen in the haloperidol-treated SV group was similar to the unmedicated SV group in that ACC activation was seen during the control task and no further activation was seen during the DEC. We report that clozapine, but not haloperidol, normalizes anterior cingulate rCBF patterns in schizophrenia during a cognitive task. Based on these preliminary data, we propose that this pattern may account for the superior therapeutic effect of clozapine and represents a surrogate marker of this action.

Acoustic Stimulation↗

Parkin is associated with cellular vesicles.

We recently identified a novel gene, parkin, as a pathogenic gene for autosomal recessive juvenile parkinsonism. Parkin encodes a 52-kDa protein with a ubiquitin-like domain and two RING-finger motifs. To provide a insight into the function of parkin, we have examined its intracellular distribution in cultured cells. We found that parkin was localized in the trans-Golgi network and the secretory vesicles in U-373MG or SH-SY5Y cells by immunocytochemical analyses. In the subsequent subcellular fractionation studies of rat brain, we showed that parkin was copurified with the synaptic vesicles (SVs) when we used low ionic conditions throughout the procedure. An immunoelectromicroscopic analysis indicated that parkin was present on the SV membrane. Parkin was readily released from SVs into the soluble phase by increasing ionic strength at neutral pH, but not by a non-ionic detergent. To elucidate its responsible region for membrane association, we transfected with green fluorescent protein-tagged deletion mutants of parkin into COS-1 cells followed by subcellular fractionation. We demonstrated the ability of parkin to bind to the membranes through a broad region except for the ubiquitin-like domain. The significance of SV localization of parkin is discussed.

Animals↗

The utilisation of superficial venous surgery for chronic venous insufficiency by the U.K. Asian population.

BACKGROUND: The epidemiology of superficial venous disease is relatively well defined in the U.K. Caucasian population. By contrast, there are currently no data available for Asians, who comprise 3.6% of the U.K., and 14.1% of this institution's catchment population. The aim of this study was to compare surgery for superficial venous disease in Caucasians and Asians in this institution, in the context of our local population. METHODS: A prospectively gathered database of all 2011 superficial venous operations performed between January 1997 and April 2002 was retrospectively analysed with regard to ethnicity. The ethnic, gender and age composition of our catchment area was determined from U.K. census data. The full institutional records of 100 Asian and 100 randomly selected age and sex-matched Caucasian patients were compared in a case control study. RESULTS: After adjusting for age and gender according to census data, Asians were 40% less likely to undergo superficial venous surgery (SVS). Considering the 2011 operated patients as a whole, Asians were significantly younger and more likely to be male. In the case control study, Asians were significantly less likely to be operated for recurrent disease and significantly more likely to be operated for advanced disease. CONCLUSIONS: Although Asians are significantly less likely to undergo SVS; those that do are more likely to be young, male and operated for skin changes and ulcers. This strongly suggests that the under-representation of Asians is due to cultural, genetic or environmental factors and unmet health care need, rather than a lower prevalence of clinically significant venous disease in the Asian population.

Adult↗

Isolation and sequencing of cDNAs for splice variants of growth hormone-releasing hormone receptors from human cancers.

The proliferation of various tumors is inhibited by the antagonists of growth hormone-releasing hormone (GHRH) in vitro and in vivo, but the receptors mediating the effects of GHRH antagonists have not been identified so far. Using an approach based on PCR, we detected two major splice variants (SVs) of mRNA for human GHRH receptor (GHRH-R) in human cancer cell lines, including LNCaP prostatic, MiaPaCa-2 pancreatic, MDA-MB-468 breast, OV-1063 ovarian, and H-69 small-cell lung carcinomas. In addition, high-affinity, low-capacity binding sites for GHRH antagonists were found on the membranes of cancer cell lines such as MiaPaCa-2 that are negative for the vasoactive intestinal peptide/pituitary adenylate cyclase-activating polypeptide receptor (VPAC-R) or lines such as LNCaP that are positive for VPAC-R. Sequence analysis of cDNAs revealed that the first three exons in SV(1) and SV(2) are replaced by a fragment of retained intron 3 having a new putative in-frame start codon. The rest of the coding region of SV(1) is identical to that of human pituitary GHRH-R, whereas in SV(2) exon 7 is spliced out, resulting in a 1-nt upstream frameshift, which leads to a premature stop codon in exon 8. The intronic sequence may encode a distinct 25-aa fragment of the N-terminal extracellular domain, which could serve as a proposed signal peptide. The continuation of the deduced protein sequence coded by exons 4-13 in SV(1) is identical to that of pituitary GHRH-R. SV(2) may encode a GHRH-R isoform truncated after the second transmembrane domain. Thus SVs of GHRH-Rs have now been identified in human extrapituitary cells. The findings support the view that distinct receptors are expressed on human cancer cells, which may mediate the antiproliferative effect of GHRH antagonists.

Amino Acid Sequence↗

Cholera toxin and cell growth: role of membrane gangliosides.

The binding of cholera toxin to three transformed mouse cell lines derived from the same parent strain, and the effects of the toxin on DNA synthesis and adenylate cyclase activity, vary in parallel with the ganglioside composition of the cells. TAL/N cells of early passage, which contain large quantities of gangliosides G(M3), G(M2), G(M1), and G(Dla), as well as the glycosyltransferases necessary for the synthesis of these gangliosides, bind the most cholera toxin and are the most sensitive to its action. TAL/N cells of later passage, which lack chemically detectable G(M1) and G(Dla) and which have no UDP-Gal:G(M2) galactosyltransferase activity, are intermediate in binding and response to the toxin. SVS AL/N cells, which lack G(M2) in addition to G(M1) and G(Dla) and which have little detectable UDP-GalNAc:G(M3)N-acetylgalactosaminyltransferase activity, bind the least amount of toxin. The SVS AL/N cells are the least responsive to inhibition of DNA synthesis and stimulation of adenylate cyclase activity by cholera toxin. Gangliosides (especially G(M1)), which appear to be the natural membrane receptors for cholera toxin, may normally have important roles in the regulation of cell growth and cAMP-mediated responses.

Adenylyl Cyclases↗

Keratinocyte growth factor functions in epithelial induction during seminal vesicle development.

Development of the seminal vesicle (SV) is elicited by androgens and is dependent on epithelial-mesenchymal interactions. Androgenic signal transmission from the androgen-receptor-positive mesenchyme to the epithelium has been postulated to involve paracrine factors. Keratinocyte growth factor (KGF), a member of the fibroblast growth factor family, is produced by stromal/mesenchymal cells and acts specifically on epithelial cells. The KGF transcript was detected by reverse transcription-polymerase chain reaction in newborn mouse SVs and by Northern blot analysis of RNA from cultured neonatal SV mesenchymal cells. Newborn SVs placed in organ culture undergo androgen-dependent growth and differentiation. Addition of a KGF-neutralizing monoclonal antibody to this system caused striking inhibition of both SV growth and branching morphogenesis. This inhibition was due to a decline in epithelial proliferation and differentiation, as the mesenchymal layer was not affected by anti-KGF treatment. When KGF (100 ng/ml) was substituted for testosterone in the culture medium, SV growth was approximately 50% that observed with an optimal dose of testosterone (10(-7) M). All of these findings suggest that KGF is present during a time of active SV morphogenesis and functions as an important mediator of androgen-dependent development.

Animals↗

Endothelin-converting enzyme-1, abundance of isoforms a-d and identification of a novel alternatively spliced variant lacking a transmembrane domain.

Endothelin-converting enzyme-1 (ECE-1) cleaves big endothelins, as well as bradykinin and beta-amyloid peptide. Several isoforms of ECE-1 (a-d) have been identified to date; they differ only in their NH(2) terminus but share the catalytic domain located in the COOH-terminal end. Using quantitative PCR, we found ECE-1d to be the most abundant type in several endothelial cells (EC) types. In addition to full-length ECE-1 forms we have identified novel, alternatively spliced mRNAs of ECE-1 b-d. These splice variants (SVs) lack exon 3', which codes for the transmembrane region and is present in full-length forms. SVs mRNA were highly expressed in EC derived from macro and microvascular beds but much less so in other, non-endothelial cells expressing ECE-1, which suggests that the splicing mechanism is cell-specific. Analyses of ECE-1d and its SV form in stably transfected HEK-293 cells revealed that both proteins were recognized by anti COOH-terminal ECE-1 antibodies, but anti NH(2)-terminal antibodies only bound ECE-1d. The novel protein, designated ECE-1 sv, has an apparent molecular mass of 75 kDa; by using site-directed mutagenesis its start site was identified in a region common to all ECE-1 forms suggesting that ECE-1 b-d SV mRNAs are translated into the same protein. In agreement with the findings demonstrating common COOH terminus for ECE-1sv and ECE-1d, both exhibited a similar catalytic activity. However, immunofluorescence staining and differential centrifugation revealed a distinct intracellular localization for these two proteins. The presence of ECE-1sv in different cellular compartments than full-length forms of the enzyme may suggest a distinct physiological role for these proteins.

Alternative Splicing↗

Induction of apoptosis by castration in epithelium of the mouse seminal vesicles.

Castration on days 0, 5, 10, 20, 40, and 60 caused increases in an apoptotic index (% of apoptotic cells) in seminal vesicle (SV) epithelium, peaking 1-3 days after castration. The peak apoptotic indices after castration on days 0, 5, 10, and 20 were significantly lower than peak apoptotic indices observed after castration on days 40 and 60. DNA extracted from mouse SVs 2 days after castration on days 0, 5, 10, and 60 showed a ladder pattern on agarose gel electrophoresis. The secretion of androgen by testes was confirmed by the growth retardation of the SVs after castration on days 0, 5, 10, and 20. It would appear that a proportion of SV epithelial cells dependent on testicular androgens for survival is smaller before day 20 than after day 20.

Animals↗

Design and in vitro evaluation of polymeric formulae of simvastatin for local bone induction.

Simvastatin (SVS), a cholesterol-lowering drug, has been shown to stimulate bone formation. This study deals with the design and in vitro evaluation of local delivery systems for simvastatin. They are intended to treat bony defects resulting from periodontitis or to induce osteogenesis around the titanium implants. Granules and gels were formulated using bioerodible/biocompatible polymers, namely hydroxypropylmethyl cellulose (H), sodium carboxymethyl cellulose (C), and chitosan (Ch). The in vitro release profiles and kinetics were evaluated and the swelling and/or erosion was monitored. Differential scanning calorimetry (DSC) and infrared (IR) were used to detect any SVS/polymer interactions that may affect drug release. The results revealed variable extents of controlled drug release from the designed formulae depending on the polymer nature. About 50% cumulative SVS was released from both H granules and gel formulae within 24 h and approximately 66% and approximately 88% from C granules and gel, respectively. Ch formulae exhibited approximately 50% release from granules and approximately 30% from gel.

Biocompatible Materials↗

Colocalization of synaptophysin with transferrin receptors: implications for synaptic vesicle biogenesis.

We have reported previously that the synaptic vesicle (SV) protein synaptophysin, when expressed in fibroblastic CHO cells, accumulates in a population of recycling microvesicles. Based on preliminary immunofluorescence observations, we had suggested that synaptophysin is targeted to the preexisting population of microvesicles that recycle transferrin (Johnston, P. A., P. L. Cameron, H. Stukenbrok, R. Jahn, P. De Camilli, and T. C. Südhof. 1989. EMBO (Eur. Mol. Biol. Organ.) J. 8:2863-2872). In contrast to our results, another group reported that expression of synaptophysin in cells which normally do not express SV proteins results in the generation of a novel population of microvesicles (Leube, R. E., B. Wiedenmann, and W. W. Franke. 1989. Cell. 59:433-446). We report here a series of morphological and biochemical studies conclusively demonstrating that synaptophysin and transferrin receptors are indeed colocalized on the same vesicles in transfected CHO cells. These observations prompted us to investigate whether an overlap between the distribution of the two proteins also occurs in endocrine cell lines that endogenously express synaptophysin and other SV proteins. We have found that endocrine cell lines contain two pools of membranes positive for synaptophysin and other SV proteins. One of the two pools also contains transferrin receptors and migrates faster during velocity centrifugation. The other pool is devoid of transferrin receptors and corresponds to vesicles with the same sedimentation characteristics as SVs. These findings suggest that in transfected CHO cells and in endocrine cell lines, synaptophysin follows the same endocytic pathway as transferrin receptors but that in endocrine cells, at some point along this pathway, synaptophysin is sorted away from the recycling receptors into a specialized vesicle population. Finally, using immunofluorescent analyses, we found an overlap between the distribution of synaptophysin and transferrin receptors in the dendrites of hippocampal neurons in primary cultures before synapse formation. Axons were enriched in synaptophysin immunoreactivity but did not contain detectable levels of transferrin receptor immunoreactivity. These results suggest that SVs may have evolved from, as well as coexist with, a constitutively recycling vesicular organelle found in all cells.

Animals↗

Synaptic vesicle proteins and early endosomes in cultured hippocampal neurons: differential effects of Brefeldin A in axon and dendrites.

The pathways of synaptic vesicle (SV) biogenesis and recycling are still poorly understood. We have studied the effects of Brefeldin A (BFA) on the distribution of several SV membrane proteins (synaptophysin, synaptotagmin, synaptobrevin, p29, SV2 and rab3A) and on endosomal markers to investigate the relationship between SVs and the membranes with which they interact in cultured hippocampal neurons developing in isolation. In these neurons, SV proteins are detected as punctate immunoreactivity that is concentrated in axons but is also present in perikarya and dendrites. In the same neurons, the transferrin receptor, a well established marker of early endosomes, is selectively concentrated in perikarya and dendrites. In the perikaryal-dendritic region, BFA induced a dramatic tubulation of transferrin receptors as well as a cotubulation of the bulk of synaptophysin. Synaptotagmin, synaptobrevin, p29 and SV2 immunoreactivities retained a primarily punctate distribution. No tubulation of rab3A was observed. In axons, BFA did not produce any obvious alteration of the distribution of SV proteins, nor of peroxidase- or Lucifer yellow-labeled early endosomes. The selective effect of BFA on dendritic membranes suggests the existence of functional differences between the endocytic systems in dendrites and axons. Cotubulation of transferrin receptors and synaptophysin in the perikaryal-dendritic region is consistent with a functional interconnection between the traffic of SV proteins and early endosomes. The heterogeneous effects of BFA on SV proteins in this cell region indicates that SV proteins are differentially sorted upon exit from the TGN and are coassembled into SVs at the cell periphery.

Amino Acid Sequence↗

Direct spectroscopic evidence for ionized polycyclic aromatic hydrocarbons in the interstellar medium.

Long-slit 8-13 micrometers spectroscopy of the nebula around NGC 1333 SVS 3 reveals spatial variations in the strength and shape of emission features that are probably produced by polycyclic aromatic hydrocarbons (PAHs). Close to SVS 3, the 11.2 micrometers feature develops an excess at approximately 10.8-11.0 micrometers and a feature appears at approximately 10 micrometers. These features disappear with increasing distance from the central source, and they show striking similarities to recent laboratory data of PAH cations, providing the first identification of emission features arising specifically from ionized PAHs in the interstellar medium.

Astronomical Phenomena↗