Effect of temperature and cell density on cellular protein content in temperature-sensitive mutants of rat 3Y1 diploid fibroblasts

Summary We examined cellular protein content in four temperature-sensitive (ts) mutants of rat 3Y1 fibroblasts (3Y1tsD123, 3Y1tsF121, 3Y1tsG125, and 3Y1tsH203) under various conditions of culture that affect cell proliferation. When proliferation of the ts mutants was inhibited at a nonpermissive temperature (39.8°C) in the G₁ phase, prominent accumulation of cellular protein occurred in three mutants (3Y1tsF121, 3Y1tsG125, and 3Y1tsH203) but not in 3Y1tsD123. The over-accumulation of protein at 39.8°C in the former three mutants was inhibited at high cell densities. At low cell densities there was an upper limit in the protein accumulation at 39.8°C. When the three mutants, proliferation-arrested at high cell densities at 33.8°C, were replated sparsely in fresh medium and shifted to 39.8°C, proliferation was completely inhibited whereas over-accumulation of protein occurred. These results indicating dissociation of protein accumulation and cell proliferation suggest that the two events are regulated by different mechanisms. This work was supported in part by a Grant-in-Aid for Encouragement of Young Scientists (1984) to K. Y. from the Ministry of Education, Science, and Culture, Japan.     

Differential Degradation of Different Benzodiazepine Binding Proteins by Incubation of Membranes from Cerebellum or Hippocampus with Trypsin

When rat brain membranes were incubated with [3H]flunitrazepam in the presence of UV light, predominantly one protein (P51) was irreversibly labeled in cerebellum and at least two proteins (P51 and P55) were labeled in hippocampus. On digestion of membranes with increasing concentrations of trypsin up to 40% of radioactivity irreversibly bound to proteins was removed from the membranes. In addition, P51 was nearly completely degraded to a peptide with apparent molecular weight 39,000 and this peptide was further degraded to a peptide with apparent molecular weight 25,000. In contrast, protein P55 was only partially degraded by trypsin and yielded two proteolytic peptides with apparent molecular weights 42,000 and 45,000 which seemed to be rather stable against further attack by trypsin. Membranes treated with trypsin still had the capacity to bind [3H]-flunitrazepam reversibly with an affinity similar to that of membranes not previously treated with trypsin. When these membranes were irradiated with UV light, the same proteolytic peptides were detected as in membranes first photolabeled and then digested with trypsin. These results suggest a close association between reversible and irreversible benzodiazepine binding sites and indicate that membrane-associated proteins P51 and P55 are differentially protected against degradation by trypsin.     

Effect of 6-hydroxydopamine on neuropeptides in the rat female genitourinary tract

The occurrence and distribution of neuropeptide Y has been determined in the rat female genitourinary tract by radioimmunoassay and chromatographic analysis. Within the bladder, higher concentrations of neuropeptide Y were found in the trigone (48.8±5.2 pmol/g) than in the dome (36.0±2.1 pmol/g). In the genital tract, highest concentrations were identified in the vagina (41.4±2.1 pmol/g). Treatment of rats with 6-hydroxydopamine resulted in significant depletion of neuropeptide Y concentrations in both parts of the bladder, together with vagina, uterine horn and fallopian tube. No change was observed in the cervix, uterine body and ovary. Concentrations of vasoactive intestinal polypeptide were unaffected by treatment with 6-hydroxydopamine except in the area of the cervix where concentrations rose from 64.1±5.7 pmol/g to 133.6±15.1 pmol/g (p<0.05). There was a generalised, but statistically insignificant rise in substance P concentrations.     

球针壳属一新种和一新组合

本文报道了球针壳属(Phyllactinia)的一个新种和一个新组合,即寄生于杜鹃花科(Ericaceae)吊钟花属(ENkianthus)灯笼花(E.chinensis Franch)上的灯笼花球针壳(Phyllactiniaenkianthi z.y. zhao sp. nov.)和寄生于茶藨子科(Grossulariaceae)茶藨子(Ribes sp.)上的茶藨子球针壳(Phyllactinia ribesii(Jacz.)z. y. zhao comb. nov.)。上述新分类单位均有汉文和拉丁文描述,新分类单位与近似种有汉文讨论。     

Isolation of tetraploid clones with high efficiency from diploid 3Y1 rat fibroblasts treated with sodium butyrate

Summary Sodium butyrate causes proliferation arrest with a G2 (4C) DNA content and induces formation of tetraploid cells upon removal of the inhibitor, in rat 3Y1 diploid fibroblasts. We isolated tetraploid clones from the butyrate-treated 3Y1 cells with high efficiency; among 21 clones randomly isolated, 5 were pure diploid, 7 were mainly tetraploid with a small contaminating diploid population, and 7 were pure tetraploid. Among the pure tetraploid clones, two showed doubled chromosome numbers with slightly broader distributions than that seen in parental 3Y1 cells. Butyrate further induced polyploid formation in the tetraploid cells thus produced, but octaploid cells that resulted could not be maintained for prolongeed, cultivation. We found no difference between the tetraploid and the (parental and parallel isolated) diploid clones in terms of colony-forming ability, proliferation rate, and sensitivity to density-dependent inhibition of proliferation. These results suggest that doubling of chromosome number by itself does not cause a change in proliferation property. The tetraploid clones had lower average saturation densities possibly due to enlargement of cell size represented by higher cellular protein content.     

Production of auto-anti-idiotypic antibody during the normal immune response: IX. Characteristics of the auto-anti-idiotype antibody and its production

Using hapten-reversible inhibition of plaque formation as an assay for auto-anti-idiotype antibody (anti-Id) and as a means for following idiotype (Id) expression, we have obtained evidence that following immunization with trinitrophenyl (TNP) conjugates (a) there are differences in Id expression in the anti-TNP antibody response to different TNP conjugates although there is some overlap; (b) different strains, although showing some differences in Id expression, tend to produce cross-reactive Ids, thus no obvious allotype linked inheritance of Id expression is observed in this heterogeneous immune response; (c) the auto-anti-Id produced following immunization with TNP-Brucella abortus or TNP-Ficoll tends to be of the IgG_2a and IgG_2b isotypes.     

Neuropeptide Y,enkephalin and noradrenaline coexist in sympathetic neurons innervating the bovine spleen

Summary The subcellular distribution of noradrenaline (NA), neuropeptide Y (NPY), Met and Leu-enkephalin (ENK), substance P (SP), somatostatin (SOM), and vasoactive intestinal polypeptide (VIP) was investigated in homogenates of bovine splenic nerve. The distribution of noradrenergic peptide-containing nerves in the bovine celiac ganglion, splenic nerve and terminal areas in spleen was studied by indirect immunofluorescence histochemistry using antisera to tyrosine hydroxylase (TH), dopamine--hydroxylase (DBH), NPY, enkephalin peptides, SP, SOM, VIP and peptide HI (PHI).After density gradient centrifugation, high levels of NPY and ENK-like immunoreactivity (LI) were found in high-density gradient fractions, coinciding with the main NA peak. SP, SOM and VIP were found in fractions with a lower density, VIP being also enriched in a heavy fraction; the latter three peptides were present in low concentrations.Immunohistochemistry revealed that staining for NPYLI and ENK-LI partly overlapped that for TH and DBH in celiac ganglia, splenic nerve axons and terminal areas of spleen. Almost all principal ganglion cells were TH- and DBH-immunoreactive. Many were also NPY-immunoreactive, whereas a smaller number were ENK-positive. In the celiac ganglion patches of dense SP-positive networks and some VIP/PHI- and ENK-immunoreactive fibers were seen around cell bodies.The results indicate that NPY and ENK are stored with NA in large dense-cored vesicles in unmyelinated axons of bovine splenic nerve. SP, SOM and VIP appear in different organelles in axon populations separate from sympathetic noradrenergic nerves.     

Factors affecting the oligomeric structure of yeast external invertase

It has been assumed that yeast external invertase is a dimer, with each subunit composed of a 60-kDa polypeptide chain. We now present evidence that at its optimal pH of 5.0, the predominant form of external invertase is an octamer with an average size of 8 X 10(5) Da. During ultracentrifugation the octamer dissociated to lower molecular weight forms, including a hexamer, tetramer, and dimer. All forms of the enzyme were shown to possess identical specific activities and to contain a similar carbohydrate to protein ratio. Although the monomer subunits (1 X 10(5) Da) were heterogenous in carbohydrate content, each subunit possessed nine oligosaccharide chains. When stained for protein and enzyme activity following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, only the oligomeric form of the enzyme appeared to be active. Thus, on partially inactivating invertase with 4 M guanidine hydrochloride both octamer and monomer were evident on the gels but only the former was active. Similarly, incubating at pH 2.5 in the presence of sodium dodecyl sulfate yielded only inactive monomer. The monomer, unlike the active oligomeric aggregate, was unable to hydrolyze sucrose after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Consistent with the in vitro studies, freshly prepared yeast lysate was shown to contain the octameric species of external invertase as the major active form of this enzyme. From these studies and others which employed deglycosylated invertase, it is concluded that the carbohydrate component of external invertase contributes not only to stabilizing enzyme activity, but also to maintaining its oligomeric structure.     

Induction of 7-ethoxycoumarin o-deethylase activity in cultured human epithelial cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD): evidence for TCDD receptor

The responsiveness of 5 human squamous cell carcinoma (SCC) lines derived from tumors of the epidermis and tongue to 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) was assessed by measuring the induction of the cytochrome P1-450-mediated monooxygenase activity, 7-ethoxycoumarin O-deethylase (ECOD). In 4 of the SCC lines the EC50 for this response was approximately 10(-9)M, whereas in one line the EC50 was 10(-10)M. In each of the less sensitive lines a concentration of 10(-10)M TCDD elicited less than 5% of the maximal enzyme activity. Specific binding of radiolabeled TCDD was detected in the cytosol fraction from all the SCC lines. The relative amount of receptor measured in each line correlated with maximally-induced ECOD activity. The data indicate that human cell lines derived from a target tissue for TCDD toxicity contain the TCDD receptor and show differential sensitivity to TCDD analogous to the murine strain differences in sensitivity regulated by the Ah locus.