Protein carboxyl methyltransferase activity specific for age-modified aspartyl residues in mouse testes and ovaries: Evidence for translation during spermiogenesis

An antiserum prepared against the purified protein carboxyl methltransferase (PCMT) from bovine brain has been used to compare testicular and ovarian levels of the enzyme and to study the regulation of PCMT concentrations during spermatogenesis. The PCMT, which specifically modifies age-damaged aspartyl residues, is present at a significantly higher concentration in mature mouse testis than in ovary. However, the PCMT is present at nearly equal concentrations in extracts of germ cell-deficient ovaries and testes obtained from mutant atrichosislatrichosis mice. In normal testis, the concentration of the PCMT increases severalfold during the first 4–5 weeks after birth, paralleling the appearance and maturation of testicular germ cells. Both immunochemical and enzymatic measurements of PCMT specific activities in purified spermatogenic cell preparations indicate that PCMT levels are twofold and 3.5-fold higher in round spermatids and residual bodies, respectively, than in pachytene spermatocytes. The results are consistent with the enhanced synthesis and/or stability of the PCMT in spermatogenic cells and with the continued translation of the PCMT during the haploid portion of spermatogenesis. The relatively high levels of PCMT in spermatogenic cells may be important for the extensive metabolism of proteins accompanying spermatid condensation or for the repair of damaged proteins in translationally inactive spermatozoa.     

Inhibition of in Vitro Pollen Tube Growth by Isolated S-Glycoproteins of Nicotiana alata

Pollen from three S-genotypes of Nicotiana alata was grown in vitro in the presence of S-glycoproteins isolated from styles of the same three genotypes. Pollen germination was not affected by the presence of the S-glycoproteins, but pollen tube growth of all genotypes was inhibited. S2 pollen was preferentially inhibited by the S2-glycoprotein and S3 pollen by the S3-glycoprotein. The S6-glycoprotein preferentially inhibited growth of both S2 and S6 pollen over S3 pollen. Heat treatment dramatically increased the inhibitory activity of the S-glycoproteins as inhibitors both of pollen germination and tube growth; after heat treatment, S-allele specificity of pollen tube inhibition was not detected.     

A common theme in the amino acid sequences of actin and many actin-binding proteins?

The amino acid sequences of several actin regulatory proteins have recently been determined. Do these proteins function by mimicking actin-actin interaction sites?     

Role of carbohydrate moiety of IL-5. Effect of tunicamycin on the glycosylation of IL-5 and the biologic activity of deglycosylated IL-5

IL-5 is a T cell-derived lymphokine that induces B cell growth and differentiation in murine systems. In this study, we examined the role of carbohydrate moiety of IL-5 in the expression of biological function. IL-5 polypeptides translated in Xenopus oocytes were heterogeneous in terms of isoelectric point (pI 4.7 to 8.0) and m.w. (45,000 to 60,000 under nonreducing conditions) and yielded m.w. of 25,000 to 30,000 under reducing conditions. Treatment of rIL-5 with N-glycanase under reducing conditions yielded an IL-5 monomer of m.w. 12,000 to 14,000. Furthermore, deglycosylated rIL-5 that had been translated in the presence of tunicamycin showed very limited heterogeneity by two-dimensional gel electrophoresis (first dimension, nonequilibrium pH gradient electrophoresis; second dimension, SDS-PAGE). The m.w. was 27,000 to 28,000 under non-reducing conditions and migrated to m.w. 13,000 to 14,000 under reducing conditions. These results indicate that IL-5 is a glycoprotein carrying the N-glycosidically-linked carbohydrates. Treatment of IL-5 with sialidase caused the decrease in the heterogeneity in isoelectric point of IL-5. Deglycosylated rIL-5 that had been obtained from tunicamycin-treated oocytes could bind to IL-5-responding cells (T88-M), which express both high- and low-affinity IL-5 receptors, as efficient as intact rIL-5 under high-affinity conditions. Scatchard plot analysis of equilibrium binding of 35S-labeled rIL-5 to T88-M cells revealed that the dissociation constants (Kd) of glycosylated rIL-5 and deglycosylated rIL-5 were 127 pM and 110 pM, respectively. IL-5 activities determined by both B cell growth and differentiation assays were not affected by deglycosylation. These results indicate that N-linked glycoside moiety of IL-5 molecules may not play an essential role in the expression of its activity.     

Purification of the microsomal Ca2+-ATPase from rat liver

Summary The Ca²⁺-ATPase from rat liver microsomes has been solubilized in Triton X-100 and purified to homogeneity by ficollsucrose treatment, column chromatography with agarose-hexane adenosine 5-triphosphate Type 2, and high pressure liquid chromatography (HPLC). The purified enzyme obtained by this sequential procedure exhibited a 183-fold increase in specific activity. After ficoll-sucrose treatment, the activity of the Ca²⁺-ATPase was stable for at least two weeks when stored at –70°C. In SDS-polyacrylamide gels, several fractions from HPLC chromatography showed a single band at a position corresponding to a molecular weight of about 107 kDa. This value is consistent with the molecular weight of the phosphoenzyme intermediate of endoplasmic reticulum (ER) Ca²⁺-ATPase. Further characterization of the ER Ca²⁺-ATPase was performed by western immunoblots. Antiserum raised against the 100-kDa sarcoplasmic reticulum (SR) Ca²⁺-ATPase cross-reacted with the purified Ca²⁺-ATPase from rat liver ER membranes.     

Localization of a 24-kilodalton glycoprotein in adult Schistosoma mansoni using immunofluorescence and immunoelectron microscopy

Studies on schistosome protective immune responses have focused mainly on antigens of the parasite's syncytial surface. One of the characterized schistosome antigens, a 24-kDa glycoprotein, has been considered important in mechanisms of immune evasion by the parasites. In the present study, using affinity-purified antibodies to the 24-kDa protein for immunofluorescence and immunoelectron microscopy, we demonstrated an association of the 24-kDa antigen with the discoid bodies (the major syncytial inclusion bodies; DBs) and the surface membrane complex (most likely the apical plasma membrane) of adult Schistosoma mansoni. This is consistent with previous observations that the 24-kDa antigen appeared to be localized to the syncytial membrane and DB fractions. The present results also support the suggestion that the DBs are the precursor organelles of the apical plasma membrane.     

Platelet-activating factor and serum components from oestrous mice co-operate to mimic the activity of 'early pregnancy factor' in the rosette inhibition assay

When male mouse spleen cells were incubated with a combination of platelet activating factor (PAF, 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) and sera from female mice in oestrus, the cells displayed a markedly increased rosette inhibition titre (RIT) when subsequently tested in the rosette inhibition assay. Neither PAF nor oestrous mouse sera alone could induce this effect, the combined action was required. Lyso-PAF could not substitute for the PAF, nor could male mouse sera nor the sera from females in dioestrus or metoestrus substitute for the oestrous mouse serum requirement. Pro-oestrous mouse sera could replace oestrous mouse sera but were less effective in their dose-responses. Studies on the mechanism of action of the PAF and oestrous mouse serum components suggested that the PAF stimulated the production and release of soluble factors (termed S2 factors) which by themselves could induce increased RIT values when applied to fresh spleen cells. The PAF-stimulated cell populations were rendered refractory to the action of these S2 factors and did not display increased RIT values, unless oestrous mouse serum was added. This serum acted to reverse the refractory state, allowing the S2 factors to exert their effect, and so cells treated with PAF and oestrous mouse serum displayed increased RIT values.     

Avoidance of used endometrial sites by blastocysts of the bank vole, Clethrionomys glareolus

In bank voles blastocysts arising from mating immediately after parturition do not implant in used zones of the preceding pregnancy but in the regions between them. However, if mating is postponed until 12 or more days after parturition blastocysts do implant in used zones of the previous pregnancy. Implantations also occur at zones used two pregnancies previously. It seems likely that the absence of implantations at recently used zones of the uterus is determined by the uterus rather than blastocysts.