On the selective elimination of Y-bearing sperm

The potential use of antibodies that selectively recognize either X-bearing or Y-bearing sperm is self-evident. Thus our attention was directed to the fact that under optimal conditions, H-Y antibody lyses 50% of mouse spermatozoa. Accordingly, we asked whether expression of H-Y antigen is haploid in spermatozoa from XY male mice heterozygous for the autosomal dominantSxr gene, for if H-Y expression were haploid, H-Y antibody would be expected to kill 75% of spermatozoa derived from these XY,Sxr/- males. However, maximal lysis remained at the 50% level, which indicates that haploid expression of H-Y antigen and the potential immunoselection of Y-(or X-) bearing spermatozoa are unlikely.     

Effect of the removal of the COOH-terminal region of bacteriorhodopsin on its light-induced H+ changes.

Removal of the COOH-terminal region of bacteriorhodopsin by digestion with trypsin or papain reduces the yield of light-induced H+ release by 50-70%. The rate of H+ release is not affected significantly, but the half time of H+ uptake increases almost twofold. However, there is no effect on the photocycle of bacteriorhodopsin as judged by the yield and decay kinetics of the M412 photointermediate. The H+:M ratio in enzyme-digested membranes is approximately 0.4-0.8, whereas untreated membranes have a H+:M ratio of approximately 2. Purple membrane sheets stored in distilled water at 4 degrees C for prolonged periods also have a low H+:M ratio, probably due to protease activity associated with bacterial contamination. Electrophoresis on sodium dodecylsulfate-polyacrylamide gels showed that both the enzyme-treated and the stored purple membrane samples have a higher electrophoretic mobility compared to the fresh preparation. The reduction in molecular weight can be accounted for by the loss of several residues from the COOH-terminal portion of the bacteriorhodopsin. We propose that the COOH-terminal region is partially responsible for the high yield of H+ release by the purple membrane.     

Interaction with phospholipid bilayers, ion channel formation, and antimicrobial activity of basic amphipathic alpha-helical model peptides of various chain lengths.

Basic amphipathic alpha-helical peptides Ac-(Leu-Ala-Arg-Leu)3 or 4-NHCH3 (4(3) or 4(4)) and H-(Leu-Ala-Arg-Leu)3-(Leu-Arg-Ala-Leu)2 or 3-OH (4(5) or 4(6)) were synthesized and studied in terms of their interactions with phospholipid membranes, biological activity, and ion channel-forming ability. CD study of the peptides showed that they form alpha-helical structures in the presence of phospholipid liposomes and thus they have amphipathic distribution of the side chains along the axis of the helix. A leakage study of carboxyfluorescein encapsulated in phospholipid vesicles indicated that the peptides possess a highly potent ability to perturb the membrane structure. Membrane current measurements using the planar lipid bilayer technique revealed that the peptide 4(6), which was long enough to span the lipid bilayer in the alpha-helical structure, formed cation-selective ion channels at a concentration of 0.5 microM in a planar diphytanoylphosphatidylcholine bilayer. In contrast, other shorter peptides failed to form discrete and stable channels though they occasionally induced an increase in the membrane current with erratic conductance levels. The probability of detecting a conductance increase was in the order of 4(6) greater than 4(5) greater than 4(4) greater than 4(3), which corresponds to the order of the peptide chain lengths. Furthermore, 4(6) but not 4(5) showed an antimicrobial activity against both Gram-positive and -negative bacteria. The structure of ion channels formed by 4(6) and the relationship between the peptide chain length and biological activity of the synthetic peptides are discussed.     

CD2 can mediate TCR/CD3-independent T cell activation.

T lymphocytes can be activated clonotypically through TCR/CD3 complex or polyclonally via the CD2 molecule. Whether CD2-mediated activation is dependent on TCR/CD3 expression or signaling is controversial. We have re-explored this issue by using a series of CD2-transfected, TCR/CD3 surface membrane-negative human and mouse T cells. Our results clearly show that such T cells can be triggered for IL-2 secretion and increases in intracellular Ca2+ through the CD2 molecule in the absence of surface expression of TCR/CD3 complexes. These responses are only observed when cells express high levels of CD2 and there is a critical threshold of CD2 expression necessary for such activation in the absence of CD3. Concomitant expression of TCR/CD3 complex markedly lowers the level of CD2 required for activation via the latter pathway. These results provide a clear resolution of the controversy concerning the requirement for surface CD3 expression in T cell activation through CD2 and further suggest a possible role for CD2 in activation of TCR/CD3-negative cells.     

Redescription of Caprogammarus gurjanovae Kudrjaschov & Vassilenko, 1966 (Crustacea: Amphipoda) from Hokkaido,Japan, with notes on the taxonomic status of Caprogammarus

Caprogammarus gurjanovae Kudrjaschov & Vassilenko, 1966 (Amphipoda: Caprellidea: Caprogammaridae) was redescribed based on the materials newly collected off Kushiro and Akkeshi, Hokkaido, Japan, which represents the southernmost record of this genus. Caprogammarus and Caprella share an identical feature, that of having the head and perionite I partially fused. Thus, Caprogammarus is considered to be a member of the suborder Caprellidea.     

Postnatal changes in fatty acids composition of brown adipose tissue

It has been demonstrated that thermogenic activity of brown adipose tissue (BAT) is higher during the early postnatal period, decreasing towards a low adult level. The present study examined postnatal changes in the lipid composition of BAT. BAT from pre-weaning rats at 4 and 14 days old showed the following differences in lipid composition compared to that from adults of 12 weeks old. (i) Relative weight of interscapular BAT to body weight was markedly greater. (ii) BAT-triglyceride (TG) level was lower, while BAT-phospholipid (PL)level was higher. (iii) In TG fatty acids (FA) polyunsaturated fatty acids (PU; mol %), arachidonate index (AI), unsaturation index (UI) and PU/saturated FA (SA) were higher; rare FA such as eicosadienoate, bishomo--linolenic acid and lignoceric acid in mol % were also higher. (iv) In PL-FA monounsaturated FA (MU) in mol % was lower; PU mol %, AI and UI were higher. These features in BAT of pre-weaning rats resembled those in the cold-acclimated adults, suggesting a close relationship of the PL-FA profile to high activity of BAT.     

Structure and regulated expression of Kunitz chymotrypsin inhibitor genes in winged bean [Psophocarpus tetragonolobus (L.) DC.]

We analyzed the structure and the expression of Kunitz chymotrypsin inhibitor (WCI) genes in winged bean. WCI was encoded by a multigene family which comprised at least seven members. From their primary structures, four genes (WCI-2, WCI-3a, WCI-3b, and WCI-x) were expected to be functional ones and the other three (WCI-P1, WCI-P2, and WCI-P3) to be pseudogenes. The nucleotide sequences of the WCI-3a and WCI-3b genes were nearly identical, and they encoded the WCI-3 protein, the major chymotrypsin inhibitor in seeds. The WCI-2 gene also encoded the chymotrypsin inhibitor found in seeds and the WCI-x gene was expected to encode an unidentified chymotrypsin inhibitor. WCI messenger RNA and protein accumulated mainly in developing seeds and tuberous roots, small amounts of WCI mRNA being present in stems and pericarps. In seeds, transient accumulation of WCI mRNA was observed during the seed maturation period. These results suggest that the expression of WCI genes is regulated organ-specifically and developmentally in winged bean.     

Localization of laminin in nephritic glomeruli as revealed by a quick-freezing and deep-etching method with immunohistochemistry

Summary The three-dimensional localization of laminin in rat glomeruli at the chronic phase of Masugi nephritis was investigated by a quick-freezing and deep-etching method combined with immunohistochemistry. Light-microscopically, laminin was localized in increased mesangial matrix and thickened glomerular basement membrane. The quick-freezing and deep-etching method revealed that the increased mesangial matrix, which was newly formed in axial portions and areas of mesangial interposition, was composed of fine fibrillar networks. They were revealed with the 3,3-diaminobenzidine tetrahydrochloride (DAB) reaction products of peroxidase-labelled secondary antibody following anti-laminin antibody. However, these reaction products were not uniformly distributed in the newly formed matrix. Although the fibrils organizing lamina densa were also immunostained with anti-laminin antibody, the fibrils connected to mesangial cells, podocytes and endothelial cells had smaller amounts of DAB reaction products for laminin. These results indicate that one of the components of fibrils in the mesangial matrix and lamina densa is laminin, which is heterogeneously distributed in the newly formed matrix.     

Estrogen modulates Ca(2+)-independent lipid-stimulated kinase in the rabbit corpus luteum of pseudopregnancy. Identification of luteal estrogen-modulated lipid-stimulated kinase as protein kinase C delta.

Rabbit corpora lutea were tested for the presence of phosphorylative responses sensitive to estrogen. Luteal Ca(2+)-independent lipid-stimulated kinase activity was detected by phosphorylation of the endogenous substrate, p76. Estrogen treatment, by way of estradiol-17 beta implant, increased levels of the lipid-stimulated phosphoprotein 2-3-fold throughout pseudopregnancy. Midpseudopregnant rabbit luteal extracts were further evaluated to determine the identity of the lipid-stimulated kinase. Results of low pH-activated phosphorylation were consistent with the identification of p76 as an autophosphorylated member of the protein kinase C (PKC) family. Partial purification of the luteal lipid-stimulated kinase was performed using sequential DEAE-cellulose/hydroxylapatite chromatographies and using gel filtration. Western immunoblot with type-specific anti-PKC delta antiserum showed coelution of kinase p76 activity with immunoreactive PKC delta. Immunoblot analysis confirmed that luteal levels of PKC delta were increased by estrogen treatment.