Handedness of duplex DNA: role of the glycosydic torsion

Model building studies on helical structures of polynucleotides indicate the glycosydic torsion, χ, and the sugar pucker to be the most important conformational parameters in determining the handedness of the models. Sugar puckers in two typical regions of the pseudorotational space namely, C2′ endo and C3′ endo and the glycosydic torsion in four ranges, namely, anti, high anti, syn and low anti lead to eight conformational combinations for the model bulding of base-paired double helical structures of polynucleotides. Interestingly, not all combinations of sugar pucker and glycosidic torsion lead to structures with Watson-Crick base-pairing. The stereochemical details of the double stranded Watson-Crick base-paired models and their consistency with X-ray diffraction data are discussed.     

Coexistence of γ-Aminobutyric Acid Type A and Type B Receptors in Testicular Interstitial Cells

The existence of specific gamma-aminobutyric acid (GABA)ergic receptors in testicular interstitial cells was investigated in the present study. Specific binding of [3H]GABA to interstitial cell membranes was found to be time- and temperature-dependent and varied according to Ca2+ concentration present in the incubation medium. We analyzed the ability of different GABAergic agonists and antagonists to displace the bound radioactivity. In the absence of Ca2+ (1 mM EDTA), GABA and the GABAergic agonist isoguvacine displaced the bound radioactivity. When the radioligand assay was performed in the presence of 2.5 mM CaCl2, the [3H]GABA specifically bound increased twofold. Under such conditions, the specific GABAergic agonist baclofen, as well as GABA and isoguvacine, displaced the [3H]GABA bound. Saturation analysis revealed the presence of a population of GABAA binding sites with a KD value of 45.2 nM and a maximal number of binding sites of 57.4 fmol/mg of protein. The maximal binding increased on addition of 2.5 mM CaCl2 to 102 fmol/mg of protein, indicating the existence of a second population of GABAergic receptors, i.e., type B, with essentially the same affinity. In addition, the incubation of testicular interstitial cells with GABA and baclofen resulted in an increase in androgen production. These results support a functional role of GABA in the neuroendocrine control of the male gonad.     

A comparison of the annual changes in testicular activity and serum androgen levels between the early and delayed maturing groups of male Cottus hangiongensis

Synopsis Annual changes in testicular activity and concentration of two serum androgens were monitored in two groups of the river-sculpin Cottus hangiongensis collected from the upper and lower reaches of a river at southern Hokkaido, Japan. One of them (early maturing group) underwent testicular maturation with aberrant spermatids and spermatid masses produced during the reproductive cycle. Moreover, regular seasonal changes in serum testosterone and 11-ketotestosterone concentrations were observed. On the other hand, in the other group (delayed maturing group), although body size of the fish was large enough to undergo reproduction, annual changes in gonadosomatic index and testicular activity did not vary much. During the months of active testicular development in the early maturing group, spermatogenesis was observed to begin in some regions of the testes of delayed maturing fish, but always resulted in the formation of aberrant spermatids and spermatid masses. Moreover, concentration of serum androgens did not significantly vary throughout the year. Results suggest that low androgen production is a proximal factor for delayed sexual maturity in the delayed maturing group, and that the occurrence of aberrant spermatids and spermatid masses during spermatogenesis is not linked to the delayed maturity.     

Ultrastructure of the pseudohermaphrodite rat testis

Summary The interstitial cells of the pseudohermaphrodite rat testis are both hypertrophic and hyperplastic. The cytoplasm is characterized by smooth endoplasmic reticulum which is abundant and variable in form. Mitochondria are numerous and large with tubular cristae and occasional inclusions. Structural features of the Leydig cells indicate potential for increased steroid synthesis. The presence of large numbers of mast cells in the intertubular area is confirmed.Small seminiferous tubules lack advanced germinal elements. Additional connective tissue and myoepithelial layers produce a thickening of the limiting membrane. Some myoepithelial cells are atypical with an electron translucent cytoplasm and nuclei with dense peripheral chromatin. No spermatogenic cells beyond the cap phase of the spermatid are observed. The cytoplasm of Sertoli cells contains large lipid droplets and degenerating germ cells.The authors are greatly indebted to Drs. A. J. Stanley, J. E. Allison, and L. G. Gumbreck for kindly providing the animals for this study.     

Nuclei in testicular feminization (Tfm) are not activated by intact testosterone receptor complexes: A morphometric study in striated urethral muscle of mosaic mice

Summary Sex reversed mice heterozygous for the X-linked Tfm mutation are mosaics with respect to the Tfm locus. In the androgen-dependent striated urethral muscle, nuclei coding for the intact testosterone receptor protein and nuclei coding for the defective Tfm receptor protein are incorporated in the same multinucleate muscle fibres. The intact testosterone receptor complex can thus be expected to enter the Tfm nuclei. Our measurements show that the fibre diameters of the mosaic muscle form a homogeneous population, intermediate in size between induced male and non-inducible Tfm phenotypes. By contrast, the nuclear size shows a bimodal distribution, the subpopulations corresponding to Tfm and wild type nuclei. The results indicate that the Tfm nuclei are not activated by the intact testosterone receptor complex.     

The impact of protein flexibility on protein-protein docking

Accounting for protein flexibility in protein-protein docking algorithms is challenging, and most algorithms therefore treat proteins as rigid bodies or permit side-chain motion only. While the consequences are obvious when there are large conformational changes upon binding, the situation is less clear for the modest conformational changes that occur upon formation of most protein-protein complexes. We have therefore studied the impact of local protein flexibility on protein-protein association by means of rigid body and torsion angle dynamics simulation. The binding of barnase and barstar was chosen as a model system for this study, because the complexation of these 2 proteins is well-characterized experimentally, and the conformational changes accompanying binding are modest. On the side-chain level, we show that the orientation of particular residues at the interface (so-called hotspot residues) have a crucial influence on the way contacts are established during docking from short protein separations of approximately 5 A. However, side-chain torsion angle dynamics simulations did not result in satisfactory docking of the proteins when using the unbound protein structures. This can be explained by our observations that, on the backbone level, even small (2 A) local loop deformations affect the dynamics of contact formation upon docking. Complementary shape-based docking calculations confirm this result, which indicates that both side-chain and backbone levels of flexibility influence short-range protein-protein association and should be treated simultaneously for atomic-detail computational docking of proteins.     

Revised structure of the AbrB N-terminal domain unifies a diverse superfamily of putative DNA-binding proteins

New relationships found in the process of updating the structural classification of proteins (SCOP) database resulted in the revision of the structure of the N-terminal, DNA-binding domain of the transition state regulator AbrB. The dimeric AbrB domain shares a common fold with the addiction antidote MazE and the subunit of uncharacterized protein MraZ implicated in cell division and cell envelope formation. It has a detectable sequence similarity to both MazE and MraZ thus providing an evolutionary link between the two proteins. The putative DNA-binding site of AbrB is found on the same face as the DNA-binding site of MazE and appears similar, both in structure and sequence, to the exposed conserved region of MraZ. This strongly suggests that MraZ also binds DNA and allows for a consensus model of DNA recognition by the members of this novel protein superfamily.     

Structural determinants of unexpected agonist activity in a retro-peptide analogue of the SDF-1alpha N-terminus

We have synthesised two retro-peptide analogues of the stromal cell derived growth factor 1 (SDF-1alpha) segment known to be critical for CXCR4 receptor binding, corresponding to the sequences HSEFFRCPCRFFESH and HSEFFRGGGRFFESH. We have assayed the ability of these peptides to activate extracellular signal-regulated kinase 1/2 phosphorylation in cells over expressing the SDF-1alpha receptor, finding that the first variant was able to serve as an agonist of CXCR4, whereas the second one was inactive. Finally, by comparing representative solution structures of the two peptides, we have found that the biological response of HSEFFRCPCRFFESH may be ascribed to a beta-beta-type turn motif centred on Phe(4)-Phe(5).     

Fundamental cryobiology of reproductive cells and tissues

During the last half of the 20th century there have been considerable advancements in mammalian reproductive technologies, including in vitro production of pre-implantation embryos and embryo sexing, and even cloning in some species. However, in most cases, management of non-cryopreserved reproductive cells (i.e., spermatozoa or oocytes) and tissues (i.e., testicular tissue or ovarian tissue) is problematic due to difficulties in donor-recipient synchronization and the potential for transmission of infectious pathogens, which cumulatively limits widespread application of these techniques. Therefore, there is an urgent need for the development of optimum cryopreservation methods for reproductive cells and tissues from many species. Today frozen-thawed spermatozoa and embryos have become an integral component of animal agriculture, laboratory animal genome banking, and human sperm banking and infertility programs. However, although widely implemented, the protocols currently used to cryopreserve bull sperm, for example, are still suboptimal, and cannot readily be extrapolated to other species' sperm. Similarly, embryo-freezing protocols successfully used for mouse and cattle have yielded little success when applied to some other species' embryos, or to a related cell type, oocytes. To date, with the exception of mouse oocytes, almost all mammalian species' oocytes studied have proven very difficult to successfully cryopreserve. Currently, there is a growing interest to understand the underlying cryobiological fundamentals responsible for these low survival rates in an effort to develop better cryopreservation methods for oocytes. Additionally, there is growing interest in developing technologies for the optimal isolation and cryopreservation of the earliest stage of male (spermatogonia, spermatids) and female (primordial follicle) germ cells, with subsequent maturation to the desired stage in vitro. Female gamete maturation, fertilization, and embryo development entirely under in vitro conditions from primordial follicles has been achieved in mice, however techniques for this and other species are still very early in their development. Furthermore, with the recent advances made in intracytoplasmic sperm injection (ICSI), and gamete isolation and maturation, close attention has been given to cryopreservation of gametes in the form of gonadal tissue (i.e., testicular tissue and ovarian tissue) containing various developmental stages of male (spermatogonia, spermatids, and spermatozoa) and female (primordial, secondary) germ lines.     

Regulation of the initial segment of the murine epididymis by dihydrotestosterone and testicular exocrine secretions studied by expression of specific proteins and gene expression

The murine caput epididymidis responded to deprivation of luminal fluid from the testis by regression of the initial segment but maintenance of the adjacent proximal and distal caput regions, as judged by immuno-histochemical staining of the glutamate transporter EAAC1 and the lipocalin MEP17 and enzymatic activity of -galactosidase (-Gal). Additional removal of circulating androgens by bilateral castration similarly led to loss of the initial segment and of the proximal caput but the distal caput was transformed into an epithelium containing more apical than principal cells staining for EAAC1; this epithelium resembled the precursor epithelium usually only seen in prepubertal juveniles. Administration of dihydrotestosterone (DHT) to the castrates maintained the proximal and distal caput epithelia and induced a proximal epithelium, which resembled the initial segment in its prominent staining for Golgi, EAAC1 and -Gal activity, although it was short and exhibited no MEP17 expression. DHT was present in the c-ros knockout caput epididymidis lacking the initial segment and in the heterozygous organ but the DHT concentration was lower in the knockout corpus. The maintenance of the full complement of epithelia in the murine caput epididymidis in the adult thus requires a combination of luminal fluid from the testis, tissue DHT and the presence of the c-ros oncogene.