Silencing of dedicator of cytokinesis (DOCK180) obliterates pregnancy by interfering with decidualization due to blockage of nuclear entry of autoimmune regulator (AIRE).

PROBLEM: Dedicator of cytokinesis (DOCK 180) involved in cytoskeletal reorganization is primarily a cytosolic molecule. It is recently shown to be nuclear in HeLa cells but its nuclear function is not known. METHOD OF STUDY: The spatiotemporal distribution of DOCK180 in uterus was studied in uterine cytoplasmic and nuclear compartments during the "window of implantation." The functional significance of nuclear DOCK180 was explored by homology modeling, co-immunoprecipitation assays, and mass spectrometric analysis. Dock180's role in early pregnancy was ascertained by Dock 180 silencing and subsequent quantitative real-time PCR and Western blotting analysis. RESULTS: Our study shows a nuclear DOCK180 in the uterus during "window of implantation." Estrogen and progesterone mediate expression and nuclear translocation of DOCK180. The nuclear function of DOCK180 is attributed to its ability to import autoimmune regulator (AIRE) into the nucleus. Silencing of Dock180 inhibited AIRE nuclear shuttling which influenced its downstream targets, thereby affecting decidualization with AIRE and HOXA-10 as the major players as well as lack of implantation site formation due to impact on angiogenesis-associated genes. CONCLUSION: DOCK180 has an indispensable role in pregnancy establishment as knocking down Dock180 abrogates pregnancy by a consolidated impact on decidualization and angiogenesis by regulating AIRE nuclear entry.

Expression of Cnnm1 and Its Association with Stemness, Cell Cycle, and Differentiation in Spermatogenic Cells in Mouse Testis.

Cyclin M1 (CNNM1) functions as a copper storage protein in neuronal cells. We report that Cnnm1 is expressed in mouse testis and brain and has a coding sequence of 1761 bp that encodes a 586 amino acid protein with a molecular weight of 66 kDa. Cnnm1 is expressed in the testes of mice from neonatal to adult stages with relatively higher levels in neonates. CNNM1 expression appeared to be restricted to c-KIT- and OCT3/4-positive cells in the testis, indicating that they are early spermatogonial cells. Spermatogonial stem cells in primary culture expressed Cnnm1, and their differentiation into embryoid body-like clusters in vitro resulted in the loss of Cnnm1 expression. Silencing of Cnnm1 in GC1-spg cells resulted in a significant reduction in the number of cells in G1 phase with concomitant increase in the numbers of cells in both S and G2/M phases. Further, retinoic acid downregulated the expression of Cnnm1 in GC1-spg cells. We conclude that CNNM1 is associated with stemness and self-renewal, and its downregulation triggers differentiation in spermatogonial cells in mouse.

Aberrant Expression of Dynein light chain 1 (DYNLT1) is Associated with Human Male Factor Infertility.

DYNLT1 is a member of a gene family identified within the t-complex of the mouse, which has been linked with male germ cell development and function in the mouse and the fly. Though defects in the expression of this gene are associated with male sterility in both these models, there has been no study examining its association with spermatogenic defects in human males. In this study, we evaluated the levels of DYNLT1 and its expression product in the germ cells of fertile human males and males suffering from spermatogenic defects. We screened fertile (n = 14), asthenozoospermic (n = 15), oligozoospermic (n = 20) and teratozoospermic (n = 23) males using PCR and Western blot analysis. Semiquantitative PCR indicated either undetectable or significantly lower levels of expression of DYNLT1 in the germ cells from several patients from across the three infertility syndrome groups, when compared with that of fertile controls. DYNLT1 was localized on head, mid-piece, and tail segments of spermatozoa from fertile males. Spermatozoa from infertile males presented either a total absence of DYNLT1 or its absence in the tail region. Majority of the infertile individuals showed negligible levels of localization of DYNLT1 on the spermatozoa. Overexpression of DYNLT1 in GC1-spg cell line resulted in the up-regulation of several cytoskeletal proteins and molecular chaperones involved in cell cycle regulation. Defective expression of DYNLT1 was associated with male factor infertility syndromes in our study population. Proteome level changes in GC1-spg cells overexpressing DYNLT1 were suggestive of its possible function in germ cell development. We have discussed the implications of these observations in the light of the known functions of DYNLT1, which included protein trafficking, membrane vesiculation, cell cycle regulation, and stem cell differentiation.

Wounds due to a modified shot gun (home-made): a case report.

In a case of firearm fatality, the autopsy surgeon is required to opine as to the range of fire in addition to the cause of death which will help in reconstruction of the events. Problems may arise in estimating the range of fire based on wound ballistics when there is an alteration or modification in the internal ballistics. We encountered such a case in the department of Forensic Medicine, Kasturba Medical College, Manipal, which is discussed.

Thalidomide attenuates nitric oxide-driven angiogenesis by interacting with soluble guanylyl cyclase.

BACKGROUND AND PURPOSE: Nitric oxide (NO) promotes angiogenesis by activating endothelial cells. Thalidomide arrests angiogenesis by interacting with the NO pathway, but its putative targets are not known. Here, we have attempted to identify these targets. EXPERIMENTAL APPROACH: Cell-based angiogenesis assays (wound healing of monolayers and tube formation in ECV304, EAhy926 and bovine arterial endothelial cells), along with ex vivo and in vivo angiogenesis assays, were used to explore interactions between thalidomide and NO. We also carried out in silico homology modelling and docking studies to elucidate possible molecular interactions of thalidomide and soluble guanylyl cyclase (sGC). KEY RESULTS: Thalidomide inhibited pro-angiogenic functions in endothelial cell cultures, whereas 8-bromo-cGMP, sildenafil (a phosphodiesterase inhibitor) or a NO donor [sodium nitroprusside (SNP)] increased these functions. The inhibitory effects of thalidomide were reversed by adding 8-bromo-cGMP or sildenafil, but not by SNP. Immunoassays showed a concentration-dependent decrease of cGMP in endothelial cells with thalidomide, without affecting the expression level of sGC protein. These results suggested that thalidomide inhibited the activity of sGC. Molecular modelling and docking experiments revealed that thalidomide could interact with the catalytic domain of sGC, which would explain the inhibitory effects of thalidomide on NO-dependent angiogenesis. CONCLUSION AND IMPLICATIONS: Our results showed that thalidomide interacted with sGC, suppressing cGMP levels in endothelial cells, thus exerting its anti-angiogenic effects. These results could lead to the formulation of thalidomide-based drugs to curb angiogenesis by targeting sGC.

Fluorescence resonance energy transfer between polyphenolic compounds and riboflavin indicates a possible accessory photoreceptor function for some polyphenolic compounds.

The photoreceptive extreme tip of the wheat coleoptile exhibits intense green-yellow fluorescence under UV light, suggesting the presence of UV-absorbing materials. Fluorescence spectra of the intact coleoptile tip and tip homogenate showed the presence of the known photoreceptor pigments flavin and carotene, and a preponderance of phenolic compounds. Absorption spectra and fluorescence spectra of various phenolic compounds showed close overlap with the absorption and fluorescence spectra of the wheat coleoptile tip homogenate. Fluorescence spectra of several phenolic compounds showed close overlap with the absorption bands of flavin, carotene and pterine, suggesting possible energy transduction from phenols to these photoreceptors. Excitation of gentisic acid and ferulic acid with 340 nm light in the presence of flavin showed enhancement of flavin fluorescence in a concentration- and viscosity-dependent fashion, indicating fluorescence resonance energy transfer between them and riboflavin. Furthermore, several phenolic compounds tested generated superoxide anion on excitation at 340 nm, suggesting that superoxide-dependent signal cascades could operate in a polyphenol-mediated pathway. Phenolic compounds thus may act as accessory photoreceptors bringing about excitation energy transfer to the reactive photoreceptor molecules, or they may take over the function of the normal photoreceptor in genetic mutations lacking the system, or both processes may occur. The responses of plants to UV-B and UV-A light in mutants may be explained in terms of various phenolics acting as energy transducers in photoreceptor functioning.

Mifepristone (Ru486) antagonizes monocyte chemotactic protein-3 down-regulation at early mouse pregnancy revealing immunomodulatory events in Ru486 induced abortion.

PROBLEM: The survival of an embryo bearing the paternal antigens within the immunocompetent environment of the maternal uterus renders 'pregnancy' to be a state of immunological paradox. The ratio of Th1/Th2 responses is crucial for pregnancy maintenance. Monocyte Chemotactic Protein-3 (MCP3) is a pro-inflammatory, CC chemokine and a Th1 effector which is capable of eliciting significant anti-tumoral immune responses. METHOD OF STUDY: MCP3 expression was investigated in the murine uterine tissue at different days of initial pregnancy and the effect of RU 486 in immature and delayed implantation model studied using Western blotting and Immunocytochemical techniques. RESULTS AND CONCLUSION: Our results show very high uterine MCP3 expression during pre-implantation followed by a significant MCP3 down-regulation at peri-implantation and low levels of MCP3 during post-implantation period. At the peri-implantation stage, embryos exhibited lowered MCP3 expression when compared with the pre-implantation stage. Ru486, a progesterone antagonist when given in a competitive mode with progesterone resulted in a massive surge in MCP3 expression in both immature mice and delayed implantation models. We hypothesize that it is imperative for MCP3 expression to be down-regulated for the success of pregnancy. The cross-talk between Ru486 and amplified MCP3 expression may be one of the mechanisms by way of which RU486 performs its abortificient and anti tumor role.

17Beta-estradiol induces nuclear translocation of CrkL at the window of embryo implantation.

Crk family adaptors are widely expressed and mediate the timely formation of signal transduction protein complexes upon a variety of extracellular stimuli, including various growth and differentiation factors. The window of implantation is the favorable time period when the uterus develops a receptive approach to the invading embryo. Various signaling cascades are likely to become active at the window of implantation both in the uterus and the embryo. This helps create maternal embryo dialogue leading to successful embryo implantation. In this study we report for the first time the presence and nuclear translocation of the adaptor molecule CrkL both in the uterine and embryonic partners at the window of implantation. We also report that estrogen, which initiates and guides crucial changes in the uterus and the embryo at the window of receptivity, causes a massive surge in the expression and subsequent nuclear translocation of CrkL. We have also identified the existence of one LXXLL motif in the CrkL amino acid sequence and a single LXD is sufficient for activation by the estrogen receptor. This is suggestive that CrkL can bind to estrogen receptors and act as a coactivator.

Formation and dynamic alterations of horizontal microdomains in sperm membranes during progesterone-induced acrosome reaction.

Capacitated mammalian spermatozoa undergo a fusion response of their head plasma membrane and the outer acrosomal membrane leading to vesiculation classically known as acrosome reaction. Acrosome reaction occurs in response to various acrosome reaction inducers including zona pellucida proteins, calcium ionophore, dibutyryl cAMP, progesterone, etc. All the acrosome reaction inducers cause a transient of calcium influx into the sperm through voltage-dependent cation channels. Efflux of chloride, stimulation of activity of phospholipases, and phosphorylation of proteins are other known changes introduced by acrosome reaction inducers. Macromolecular organization and dynamics of sperm membranes during the progression of this vesiculation are largely unexplored. In this study, we report that progesterone induced the formation of horizontal microdomains within the exofacial surfaces of sperm membranes, which showed progressive and independent alterations in molecular dynamics. In the light of this observation, we propose that sperm membrane rafts may contain both horizontal and vertical microdomains.