Characterization of three mouse monoclonal antibodies that react with high-molecular-mass glycopeptides isolated from F9 mouse teratocarcinoma cells

Three IgM mouse monoclonal antibodies, NL-9, Thy-22, and HL-5, which were produced primarily against human hematopoietic cells, were tested for their reactivity with various mouse cell lines and were found to react predominantly with mouse embryonal carcinoma cells. Thy-22 reacted with 2-cell-stage mouse embryos, whereas the other two antibodies were not reactive at this stage. All three antibodies, however, reacted with 8-cell-stage embryos. At the blastocyst stage, Thy-22 reacted with the entire surface of the trophectoderm cells, whereas the reactivity of NL-9 and HL-5 was weaker and was polarized on the mural trophectoderm. Immunohistological examination of 6th-day mouse embryos using anti-complement immunofluorescence demonstrated that the embryonic ectoderm was positive for all three antibodies: the reaction of NL-9 and Thy-22 was uniformly distributed over these cells, whereas HL-5 predominantly stained the luminal aspects of the cells lining the proamniotic cavity. Visceral-endoderm cells and trophoblastic cells were positive with all three monoclonal antibodies, whereas the parietal endoderm, extraembryonic ectoderm, and ectoplacental cone were negative. In 19th-day fetuses and adult tissues, certain epithelial cells were stained by these three antibodies. The biochemical nature of the antigens detected was also investigated. Farr's assay showed that both NL-9 and Thy-22 precipitated approximately 10% of the high-molecular-mass glycopeptides isolated from F9 cells, while HL-5 reacted with about 5% of these glycopeptides. The reactivity of the three antibodies against the glycopeptides was completely inhibited by the presence of X-hapten-conjugated silica.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alteration of sperm thiol-disulfide status and capacitation in the guinea pig

Epididymal spermatozoa of the guinea pig were incubated under conditions known to promote a rapid synchronous capacitation in a large proportion of the spermatozoa (Ca²⁺-free medium with lysophosphatidylcholine, LC) or in Ca ²⁺-free medium without LC. To study the effects of altered thiol-disulfide status and content, incubations were conducted with reagents that maintain and increase thiol groups (DTT, GSH), maintain and increase disulfide groups (diamide, GSSG), or which irreversibly block thiol groups by alkylation (NEM). The permeable DTT inhibited LC-induced capacitation and at high concentrations diminished the percentage of acrosome reactions in capacitated spermatozoa. The permeable diamide exhibited a stimulatory effect upon capacitation. The largely impermeable GSH and GSSG exhibited effects similar to their respective permeable counterparts but their effects were moderate and required extremely high concentrations. The DTT inhibition of LC-induced capacitation was reversible by washing and a further 1 hr incubation. In this final incubation after removal of DTT by washing, LC was absent too so its stimulatory effect must have been accomplished prior to washing and in the presence of DTT. NEM-alkylation of the existing thiol population did not affect LC-induced capacitation but alkylation of the increased thiol population after prior DTT treatment was inhibitory of capacitation. These results suggest that the maintenance and/or formation of disulfide groups on enzymes or structural proteins may be a component of the capacitation process. In contrast, the formation and maintenance by alkylation of increased thiol groups but not the maintenance of existing thiol groups, is inhibitory of capacitation. The relevance of these findings to a role for a thiol-sensitive proteinase in capacitation is discussed.     

The structure of the phytoplankton community in the subsurface chlorophyll maxima in the western North Pacific Ocean

The phytoplankton taxonomic composition of the subsurface chlorophyllmaximum layer (SCM) was compared with that of the surface layerin July, September and November 1979 in the western North PacificOcean. Besides the use of chemical fixatives for preservablephytoplankton, serial dilution culture method was employed fornon-preservable flagellates and monads, i.e., fragile forms.The SCM was characterized by a high species diversity of preservablephytoplankton and by the numerical dominance of fragile forms.Among the fragile forms, Micromonas and Ochromonas were dominant.This dominance is consistent with their viability at low lightintensity. Cluster analysis revealed that, for blue-green algae,coccolithophorids, silicoflagellates and diatoms, a marked differenceexisted in species assemblages between the SCM and the surfacebut there was no distinct difference in dinoflagellate assemblages.The difference between both layers is discussed, as relatedto the mechanisms of formation of the SCM. ¹Present address: Nodai Research Institute, Tokyo Universityof Agriculture, 1-1-1, Sakuragaoka, Setagaya-ku, Tokyo 156,Japan.     

Import of a putative precursor of rat liver mitochondrial glutamic oxaloacetic transaminase into mitochondria

A putative precursor of rat liver mitochondrial glutamic oxaloacetic transaminase which was about 2,000 daltons larger than the subunits of the mature enzyme synthesized in vitro was sensitive to proteases (trypsin and chymotrypsin). When this precursor was incubated with isolated mitochondria in the absence of protein synthesis, it was processed to the mature form; the mature form co-sedimented with mitochondria and was resistant to externally added proteases. Mature enzyme did not compete with this transport.     

Quick-freeze,deep-etch visualization of the ‘cytoskeletal spring’ of cochlear outer hair cells

Summary The lateral membrane system of the cochlear outer hair cell, consisting of the lateral plasma membrane, pillars, filamentous lattice and subsurface cisternae, is considered to be involved in the contractile movement of the isolated cochlear outer hair cell. The filamentous lattice, called the cytoskeletal spring, has been identified in the demembranated cochlear outer hair cell treated with the detergent Triton X-100. In this study, the quick-freeze, deep-etch method was applied to demonstrate the three-dimensional organization of both the filamentous and membranous structures of the lateral membrane system of cochlear outer hair cells. Treatment with saponin revealed that the inner leaflet of the lateral plasma membrane of the cochlear outer hair cell possesses more membrane particles than the outer leaflets, and that the pillars are closely associated with membrane particles in the inner leaflet of the lateral membrane. The presence of filamentous bridges between the filamentous lattice and the subsurface cisternae was also detected. We propose that the lateral membrane system in the cochlear outer hair cell may play an important role in the tuning mechanisms within the cochlea in normal hearing.     

Superovulation in immature and mature Chinese hamsters

Mature female Chinese hamsters ovulate an average of 8.8 ± 1.0 (mean ± SD) eggs per female in each estrous cycle. Superovulation can be induced in both immature and mature females by subcutaneous or intraperitoneal injections of pregnant mare serum gonadotropin (PMSG) and either human chorionic gonadotropin (hCG) or pituitary luteinizing hormone (PLH). The best superovulation in immature females was induced by the administration of 15 IU of PMSG followed 72 hr later by injection of 15 IU of hCG (about 25 eggs per female) or 0.2 mg (200 IU) PLH (about 46 eggs per female). Ovulation started about 13–15 hr after administration of hCG (or PLH) and was completed during the next 5–6 hr. Superovulation in mature females could be induced by injecting PMSG any day of the estrous cycle, but the best superovulation (about 39 eggs per female) was induced by injecting 15 IU of PMSG on day 1 (day of ovulation) followed by the injection of 0.4 mg of PLH 72 hr later. When immature females treated with the best superovulatory protocol were mated on the evening of PLH injection, only 5% of the eggs were found fertilized 50 hr after PLH administration. On the other hand, about 60% of the eggs were found fertilized in mature females mated following treatment with the best superovulatory protocol. The majority (83–85%) of superovulated eggs obtained from both immature and mature females were normally fertilized in vitro.     

Interaction of Yeast Phosphoglyceromutase with Nucleotides

Moderate cell growth occurred after a long lag phase of about 100 hr when oxygen-sensitive hydrogen bacterium N34 was cultivated chemoautotrophically under 40% O₂. A decrease in cell growth or viable count was not observed during the lag phase. These cells grown under 40 % O₂ were oxygen-resistant because when used as inocula for fresh 40 % O₂-culture, the growth lag period was less than 10 hr. Nine oxygen-sensitive colonies developed from a single oxygen-sensitive cell respectively. When these colonies were inoculated into 40% O₂-culture, they showed an almost equal lag period and growth rate. These results suggest that cell growth in 40% O₂-culture inoculated with oxygen-sensitive strain N34 occurred not by selection of oxygen-resistant variants which might preexist but by adaptation of very oxygen-sensitive cells to high oxygen tension. Oxygen-resistance thus developed was maintained after successive subcultures under 10% O₂ for more than one year.     

Identification of GABAA Receptor Subunits in Rat Retina: Cloning of the Rat GABAA Receptor ρ2-Subunit cDNA

Abstract: We identified GABA_A receptor subunits in rat retina using PCR. The high degree of conservation among previously described members of ligand-gated anion channels in transmembrane domains was used to design degenerate sense and antisense oligonucleotides. These oligonucleotides were used as primers for PCR, which was applied to the rat retina cDNA. Analysis of clones derived from the PCR amplification identified the GABA_Aα₁, β₁, β₃, and γ₂ subunits and the glycine α₁ subunit. In addition, two clones closely related to the human GABA_Aρ-subunit class were obtained. Molecular cloning revealed one of them as the rat counterpart of the human ρ₂ subunit. Northern blot analysis demonstrated the expression of mRNAs for ρ subunits in retina. These results further support the hypothesis that bicuculline-insensitive GABA channels in rat retina are comprised of ρ subunits.     

Acarbose, an alpha-glucosidase inhibitor, improves endothelial dysfunction in Goto-Kakizaki rats exhibiting repetitive blood glucose fluctuation

Several epidemiological studies have revealed that subjects with postprandial hyperglycemia are at increased risk of cardiovascular disease. However, the impact of postprandial hyperglycemia and its treatment on endothelial function has not been clarified yet. In this study, Goto-Kakizaki (GK) rats, a non-obese type 2 diabetes model, fed twice daily were used as a model of repetitive postprandial glucose spikes. We investigated the endothelial function in these rats treated or untreated with acarbose, an alpha-glucosidase inhibitor. Administration of acarbose for 12 weeks markedly improved postprandial hyperglycemia, postprandial insulin level, total cholesterol, triglyceride, and free fatty acid level in GK rats. Furthermore, acarbose efficiently reduced the number of monocytes adherent to aortic endothelial layer, improved acetylcholine-dependent vasodilatation, and reduced intimal thickening of the aorta. While it is generally regarded that repetitive postprandial hyperglycemia is associated with the onset of cardiovascular diseases, our data demonstrated that acarbose treatment efficiently ameliorated endothelial dysfunction and reduced intimal thickening, thus adding support to the protective effect of acarbose against the onset of cardiovascular disease.     

Specific characteristics of family 45 endoglucanases from Mucorales in the use of textiles and laundry

We examined the characteristics of family 45 endoglucanases (glycoside hydrolases family 45; GH45) from Mucorales belonging to Zygomycota in the use of textiles and laundry. The defibrillation activities on lyocell fabric of family 45 endoglucanases from Mucorales, such as RCE1 and RCE2 from Rhizopus oryzae, MCE1 and MCE2 from Mucor circinelloides, and PCE1 from Phycomyces nitens, were much higher than those of the other family 45 endoglucanases. By contrast, family 45 endoglucanases from Mucorales were less resistant to anionic surfactant and oxidizing agent, main components in detergents, than the other family 45 endoglucanases. RCE1 consists of two distinct modules, a catalytic module and a carbohydrate-binding module family 1 (CBM1), and these common specific characteristics were considered to due to the catalytic module, but not to the CBM1.