Masking of sperm maturation antigen by sialic acid in the epididymis of the mouse

Summary Sperm antigen expression during epididymal transit was examined in 4- to 16-week-old intact and castrated ICR mice, using the avidin-biotin complex (ABC) immunohistochemical method with monoclonal antibody T21 against a flagellar surface antigen. On untreated sections, the antigen was first expressed weakly on sperm in the proximal part of the corpus epididymis, and intraluminal components were stained in 4-week-old mice. Epididymal epithelial cells and their stereocilia, and cells in other reproductive organs were not stained. In contrast, on sections treated with neuramainidase, (1) the initial site of antigen appearance is a more proximal position in treated than in untreated sections, (2) stereocilia stained strongly, (3) the staining intensity of sperm and intraluminal components increased, and (4) some clear cells in the epithelium from the distal position of the caput to the corpus epididymis were stained. These results indicate that the antigen is produced by clear cells of the epididymal epithelium, that the antigenic determinant is masked initially by sialic acid residues, and that expression of the antigenic determinant on the sperm surface during epididymal maturation apparently involves desialylation.     

Presence of atrial natriuretic polypeptide in the pulmonary vein and vena cava

Striated muscle cells and storage granules observed in the atria were found in main branches of the pulmonary veins and superior and inferior venae cavae of the rat, pig, and ox. The presence of atrial natriuretic polypeptide (ANP) in these veins was examined by reverse-phase high-performance liquid chromatography coupled with a radioimmunoassay for ANP. The veins contained 0.6 to 8.0 ng ANP/mg wet tissue with the major molecular form being gamma-ANP. ANP was detected in the peripheral lung tissue in a small quantity, but was not detected in the pulmonary artery. The identification of gamma-ANP and storage granules stained with an anti-ANP antiserum in the pulmonary vein and vena cava suggest that the veins may participate in regulating volume status, blood pressure, and cardiovascular homeostasis through the release of ANP.     

Secretion of Bacillus subtilis alpha-amylase in the periplasmic space of Escherichia coli

The Bacillus subtilis alpha-amylase structural gene (amyE) lacking its own signal peptide coding sequence was joined to the end of the Escherichia coli alkaline phosphatase (phoA) signal peptide coding sequence by using the technique of oligonucleotide-directed site-specific deletion. On induction of the phoA promoter, the B. subtilis alpha-amylase was expressed and almost all the activity was found in the periplasmic space of E. coli. The sequence of the five amino-terminal amino acids of the secreted polypeptide was Glu-Thr-Ala-Asn-Lys-, and thus the fused protein was correctly processed by the E. coli signal peptidase at the end of the phoA signal peptide.     

Identification of a protein in the fibrous sheath of the sperm flagellum

The fibrous sheath is a unique cytoskeletal component in the principal-piece segment of the mammalian sperm flagellum. Monoclonal antibody ATC was shown by indirect immunofluorescence (IIF) to bind to the principal piece of the flagellum of permeabilized mouse, rat, and hamster sperm, but not to that region of guinea pig, rabbit, or human sperm. IIF on isolated fibrous sheaths confirmed that the antigen was present in the fibrous sheath of mouse, rat, and hamster sperm. On Western blots of mouse spermatozoa, ATC identified a relatively insoluble major antigen with an apparent molecular weight of 67,000 (Mr 67,000). Hamster sperm fibrous sheaths contain an antigen of Mr 66,000, while rat sperm fibrous sheaths contain an antigen of Mr 65,500. The antigen was first detected in late spermatids, as determined by immunohistochemical procedures on sections of mouse, rat, and hamster testis. The antigen was not detected on Western blots of mouse brain, kidney, liver, or thymus. These results indicate that ATC recognizes a protein integral to the fibrous sheath of the principal piece of sperm detected by immunohistochemistry late in spermiogenesis that is probably restricted to the male germ cell line.     

Survivability and reactivity of glycine and alanine in early oceans: effects of meteorite impacts

Prebiotic oceans might have contained abundant amino acids, and were subjected to meteorite impacts, especially during the late heavy bombardment. It is so far unknown how meteorite impacts affected amino acids in the early oceans. Impact experiments were performed under the conditions where glycine was synthesized from carbon, ammonia, and water, using aqueous solutions containing ¹³C-labeled glycine and alanine. Selected amino acids and amines in samples were analyzed with liquid chromatography-mass spectrometry (LC/MS). In particular, the ¹³C-labeled reaction products were analyzed to distinguish between run products and contaminants. The results revealed that both amino acids survived partially in the early ocean through meteorite impacts, that part of glycine changed into alanine, and that large amounts of methylamine and ethylamine were formed. Fast decarboxylation was confirmed to occur during such impact processes. Furthermore, the formation of n-butylamine, detected only in the samples recovered from the solutions with additional nitrogen and carbon sources of ammonia and benzene, suggests that chemical reactions to form new biomolecules can proceed through marine impacts. Methylamine and ethylamine from glycine and alanine increased considerably in the presence of hematite rather than olivine under similar impact conditions. These results also suggest that amino acids present in early oceans can contribute further to impact-induced reactions, implying that impact energy plays a potential role in the prebiotic formation of various biomolecules, although the reactions are complicated and depend upon the chemical environments as well.     

Interaction mode between catalytic and regulatory subunits in glucosidase II involved in ER glycoprotein quality control

The glycoside hydrolase family 31 (GH31) α‐glucosidases play vital roles in catabolic and regulated degradation, including the α‐subunit of glucosidase II (GIIα), which catalyzes trimming of the terminal glucose residues of N‐glycan in glycoprotein processing coupled with quality control in the endoplasmic reticulum (ER). Among the known GH31 enzymes, only GIIα functions with its binding partner, regulatory β‐subunit (GIIβ), which harbors a lectin domain for substrate recognition. Although the structural data have been reported for GIIα and the GIIβ lectin domain, the interaction mode between GIIα and GIIβ remains unknown. Here, we determined the structure of a complex formed between GIIα and the GIIα‐binding domain of GIIβ, thereby providing a structural basis underlying the functional extension of this unique GH31 enzyme.     

Gel filtration studies of methylene blue–heparin interactions in aqueous solution

The gel filtration method was applied to the study of the interaction of the metachromatic cationic dye methylene blue (MB) with heparin in aqueous solution. This method requires the determination of the total MB concentration in the effluent. It was found that (i) MB in aqueous solution can be extracted quantitatively with benzyl alcohol (BzlOH), (ii) the metachromatic effect of MB is not observed in BzlOH, and (iii) the presence of heparin in aqueous solution does not interfere in the BzlOH extraction of MB. Accurate determination of MB concentration in the effluent can be made by extraction of MB with BzlOH, followed by the usual absorption spectroscopy. In addition, the use of the spectrophotometric method with this procedure entailed no serious problem relating to adsorption of MB on the glass walls. Gel filtration binding experiments were successfully carried out on a Sephadex LH-20 column using 0.005M acetate buffer as effluent. The binding data obtained were analyzed in terms of the cooperativity parameter according to the method of Schwarz. The thermodynamic parameters for the binding process of MB were also evaluated. It is shown that the binding of MB to heparin is highly cooperative, exothermic, and stabilized by entropic factors.     

Racemization of Valine by Impact-Induced Heating

Homochirality plays an important role in all living organisms but its origin remains unclear. It also remains unclear whether such chiral molecules survived terrestrial heavy impact events. Impacts of extraterrestrial objects on early oceans were frequent and could have affected the chirality of oceanic amino acids when such amino acids accumulated during impacts. This study investigated the effects of shock-induced heating on enantiomeric change of valine with minerals such as olivine ([Mg_0.9, Fe_0.1]₂SiO₄), hematite (Fe₂O₃), and calcite (CaCO₃). With a shock wave generated by an impact at ~0.8 km/s, both d- and l-enriched valine were significantly decomposed and partially racemized under all experimental conditions. Different minerals had different shock impedances; therefore, they provided different P-T conditions for identical impacts. Furthermore, the high pH of calcite promoted the racemization of valine. The results indicate that in natural hypervelocity impacts, amino acids in shocked oceanic water would have decomposed completely, since impact velocity and the duration of shock compression and heating are typically greater in hypervelocity impact events than those in experiments. Even with the shock wave by the impact of small and decelerated projectiles in which amino acids survive, the shock heating may generate sufficient heat for significant racemization in shocked oceanic water. However, the duration of shock induced heating by small projectiles is limited and the population of such decelerated projectiles would be limited. Therefore, even though impacts of asteroids and meteorites were frequent on the prebiotic Earth, impact events would not have significantly changed the ee of proteinogenic amino acids accumulated in the entire ocean.     

Effects of prostaglandin E2 on the micronucleus formation in mouse bone marrow cells by various mutagens

The effects of prostaglandin E₂ (PGE)₂, as trigger of erythroid progenitor cells into the cell cycle, were studied on the induction of micronucleu by various mutagens; with mitomicin C (MMC) the optimal protocol was established. dose-response relationship between PGE₂ doses and micronucleus frequency were observed 30 h after injection of MMC to mice administered PGE₂ 24 h previously. Sensitazion by PGE₂ pretreatment was also found for other mutagens, such as vincristine, 5-fluorouracil, benzo[a]pyrene, 1,1-dimethylhydrazine and 2-naphthylamine. These results support the hypothesis that accelerating the erythropoiesis increases the frequency of micronucleic induced by mutagens.     

Integration of the mouse sperm fertilization-related protein equatorin into the acrosome during spermatogenesis as revealed by super-resolution and immunoelectron microscopy

Spermatids must precisely integrate specific molecules into structurally supported domains that develop during spermatogenesis. Once established, the architecture of the acrosome contributes to the acrosome reaction, which occurs prior to gamete interaction in mammals. The present study aims to clarify the morphology associated with the integration of the mouse fertilization-related acrosomal protein equatorin (mEQT) into the developing acrosome. EQT mRNA was first detected by in situ hybridization in round spermatids but disappeared in early elongating spermatids. The molecular size of mEQT was approximately 65 kDa in the testis. Developmentally, EQT protein was first detected on the nascent acrosomal membrane in round spermatids at approximately step 3, was actively integrated into the acrosomal membranes of round spermatids in the following step and then participated in acrosome remodeling in elongating spermatids. This process was clearly visualized by high-resolution fluorescence microscopy and super-resolution stimulated emission depletion nanoscopy by using newly generated C-terminally green-fluorescent-protein-tagged mEQT transgenic mice. Immunogold electron microscopy revealed that mEQT was anchored to the acrosomal membrane, with the epitope region observed as lying 5–70 nm away from the membrane and was associated with the electron-dense acrosomal matrix. This new information about the process of mEQT integration into the acrosome during spermatogenesis should provide a better understanding of the mechanisms underlying not only acrosome biogenesis but also fertilization and male infertility.