Changes in protein phosphorylation during the cell cycle of Chinese hamster ovary cells

The phosphorylation patterns of proteins were examined during the cell cycle of Chinese hamster ovary cells. This was accomplished by labeling synchronized cells at various times with [32P]orthophosphate and separating the proteins by both isoelectric focusing and nonequilibrium pH gradient two-dimensional gel electrophoresis. The most dramatic changes occurred during late G2/M when approximately eight proteins (including vimentin, lamin B, and histones 1 and 3) showed increased phosphorylation. Ten other proteins appeared to be uniquely phosphorylated during late G2/M. Of these 10 proteins, seven were no longer phosphorylated shortly after mitosis. There is also at least one protein which showed a relative decrease in phosphorylation during late G2/M.     

N-terminal sequence analysis of human placental protein 14, purified in high yield from decidual cytosol

Human placental protein 14 (PP14) has been purified in high yield from first trimester decidual cytosol. High-performance liquid chromatography on anion exchange, gel filtration and reverse-phase chromatography were used. The protein obtained is approximately 97% pure with an overall recovery of about 50% from the original tissue extract. The first 24 amino acids of the N-terminal were found to be Met-Asp-Ile-Pro-Gln-Thr-Lys-Gln-Asp-Leu-Glu-Leu-Pro-Lys-Leu-Ala-Gly-Thr-Glu-His - Glu-Met-Ala-Met. PP14 has been characterized in this study to be a dimeric glycoprotein of Mr 60,000, with homologous subunits having an Mr of 28,000.     

Evolutionary conservation of the chromosomal configuration and regulation of amylase genes among eight species of the Drosophila melanogaster species subgroup

Nuclear DNA was extracted from each of the eight species comprising the Drosophila melanogaster species subgroup. Southern hybridization of this DNA by using a molecular probe specific for the alpha-amylase coding region showed that the duplicated structure of the amylase locus, first found in D. melanogaster, is conserved among all species of the melanogaster subgroup. Evidence is also presented for the concerted evolution of the duplicated genes within each species. In addition, it is shown that the glucose repression of amylase gene expression, which has been extensively studied in D. melanogaster, is not confined to this species but occurs in all eight members of the species subgroup. Thus, both the duplicated gene structure and the glucose repression of Drosophila amylase gene activity are stable over extended periods of evolutionary time.      

The use of deoxyfluoro-d-galactopyranoses in a study of yeast galactokinase specificity

1. 2-Deoxy-2-fluoro-d-galactose, 3-deoxy-3-fluoro-d-galactose, 4-deoxy-4-fluoro-d-galactose, 6-deoxy-6-fluoro-d-galactose and 2-deoxy-d-lyxo-hexose are substrates for yeast galactokinase. 2. The variation in K(m) values for the d-hexose derivatives was not associated with a variation in the value of K(m) for MgATP(2-) indicating that the binding of MgATP(2-) is not modified by the binding of the sugar substrate. 3. Donated H bonds from OH-3, OH-4 and OH-6 and an accepted H bond to OH-2 of the d-hexose are important for the binding of the sugar substrate to galactokinase. 4. Yeast galactokinase exhibits similar kinetics to the galactokinase from Escherichia coli and operates by a similar random sequential mechanism. 5. 4-Deoxy-4-fluoro-d-glucose was neither a substrate for nor an inhibitor of yeast galactokinase.     

PHOTORESPONSES OF THE WOODPIGEON COLUMBA PALUMBUS IN RELATION TO THE BREEDING SEASON

• 1 The gametogenetic response following photo-stimulation was tested in Woodpigeons under controlled laboratory and aviary conditions. Subjects were wild-caught adult or first-winter birds or were hand-reared from wild-taken nestlings. The natural seasonal gonad cycle is described and experiments were related to this cycle.
• 2 Gonad recrudescence could be initiated in adults with regressed organs by exposure to summer photoperiods, while the normal vernal recrudescence could be prevented if birds were artificially kept on short (winter) daylengths. Thus Woodpigeons could be stimulated towards reproductive condition at seasons when the gonads of wild-living birds remained inactive, viz. late September to mid-February.
• 3 Juvenile males were unresponsive to extra photo-stimulation until they were about six months old. This meant that some spermatogonial division could be procured in a few individuals in late February, but juveniles were not very noticeably affected by an extended photoperiod until mid-late March. This is the time when juveniles first begin gametogenetic development in the wild, though spermatogenesis normally accelerates in April after which time the rate of gonad development is constant. First-winter females were judged to resemble males in their photoperiodic responses but the sample examined was small.
• 4 The results confirm that the natural gonad cycle in the Woodpigeon is controlled (via gonadotrophic hormones) by seasonal changes in daylength. More important is the finding that the species appears to possess no post-nuptial refractory period. Thus, at the end of the breeding season in September birds kept on summer daylengths were maintained in full reproductive condition until December. The testes of controls placed on winter daylengths regressed within one month, but they were immediately responsive to a summer daylength which stimulated spermatogenesis within a month.
• 5 The discussion argues that the avian refractory period is not a necessary period for gonad rehabihtation and reorganization and that it has not evolved as a fixed period serving to time the breeding season; views which until now have been current. Instead, it functions only as a safety mechanism, preventing unseasonal reproduction in those species for which natural selection favours seasonal breeding. In such species the evolution of sensitivity to a particular daylength could result in breeding taking place at the wrong time, for example spring responding species might also respond in the autumn. Because natural selection permits Woodpigeons to remain in breeding condition from March until September, without disadvantage, a direct response to natural daylength can regulate the cycle, and there is no need for a period of pituitary refractoriness. It is not known if some pigeons have lost the refractory period or whether they never possessed one.

     

Effects of heat and other inducers of the stress response on protein degradation in Chinese hamster and Drosophila cells

Many recent studies have suggested that heat and other inducers of the heat shock (stress) response in eukaryotic cells might result in the generation of abnormal proteins which would result in the overloading of protein degradation systems and the stabilization of proteins involved in positively regulating heat shock (hs) gene expression. In this study we have examined the effects different heat treatments and other hs inducers have on protein degradation in Chinese hamster ovary (CHO) and Drosophila Kc and Schneider cells. We have found that intermediate temperatures which induced the hs response (42 degrees C in CHO and 34 degrees C in Kc cells) did increase protein degradation rates whereas, higher temperatures which also induced the hs response (45 degrees C in CHO and 37 degrees C in Kc cells) initially increased but then decreased protein degradation rates. While these results are consistent with a model in which the protein degradation system is being overloaded and/or components of it are being depleted, we have found several conditions which induce hs proteins which rule out this mechanism. Exposure of either cell type to amino acid analogs (5 mM canavanine or 5 mM S-aminoethyl cysteine) resulted in the rapid degradation of those proteins which had incorporated the analogs in both CHO and Drosophila cells. However, the addition of analogs had little or no effect on the degradation of preexisting proteins, indicating that the introduction of abnormal proteins probably didn't overload the protein degradation system(s). The addition of 100 microM cadmium sulfate or 100 microM sodium arsenite had little or no effect on protein degradation rates in CHO cells even though both were good inducers of the hs proteins. Thus, exposure to inducers of the hs response does not universally increase protein degradation rates nor does it stabilize preexisting proteins. Therefore, the degradation of abnormal proteins is probably not involved in inducing the hs genes.     

Growth of Phytomonas serpens in a Defined Medium; Nutritional Requirements

ABSTRACT. Three strains of Phytomonas serpens two from tomatoes, Lycopersicon esculentum one from the insect Phtia picta (Hemiptera, Coreidae), were cultivated in a chemically defined medium developed from a defined medium for cultivating insect flagellates. Besides organic growth factors required by other insect trypanosomatids this flagellate requires, serine and inositol. Glutamine stimulates growth, and, surprisingly, does not require heme.     

Membrane Skeletal Proteins and Their Integral Membrane Protein Anchors are Targets for Tyrosine and Threonine Kinases in Euglena

ABSTRACT. Proteins of the membrane skeleton of Euglena gracilis were extensively phosphorylated in vivo and in vitro after incubation with [³²P]-orthophosphate or γ-[³²P] ATP. Endogenous protein threonine/serine activity phosphorylated the major membrane skeletal proteins (articulins) and the putative integral membrane protein (IP39) anchor for articulins. The latter was also the major target for endogenous protein tyrosine kinase activity. A cytoplasmic domain of IP39 was specifically phosphorylated, and removal of this domain with papain eliminated the radiolabeled phosphoamino acids and eliminated or radically shifted the PI of the multiple isoforms of IP39. In gel kinase assays IP39 autophosphorylated and a 25 kDa protein which does not autophosphorylate was identified as a threonine/serine (casein) kinase. Plasma membranes from the membrane skeletal protein complex contained threonine/serine (casein) kinase activity, and cross-linking experiments suggested that IP39 was the likely source for this membrane activity. pH optima, cation requirements and heparin sensitivity of the detergent solubilized membrane activity were determined. Together these results suggest that protein kinases may be important modulators of protein assembly and function of the membrane skeleton of these protistan cells.     

Sequence variation in the outer-surface-protein genes of Borrelia burgdorferi

Borrelia burgdorferi is a spirochete pathogen transmitted among warm- blooded hosts by ixodid ticks. Frequency-dependent selection for variant outer-surface proteins might be expected to arise in this species, since rare variants are more likely to avoid immune surveillance in previously infected hosts. We sequenced the OspA and OspB genes of nine North American strains and compared them with nine strains previously described. For each gene, the mean number of synonymous substitutions per synonymous site and the mean number of nonsynonymous substitutions per nonsynonymous site show only a twofold excess of silent mutations. Synonymous rates vary widely along the OspB protein. Some regions show a significant excess of silent substitutions, while divergence in other regions is constrained by biased base composition or selection. The presence, in antigenically important regions of the protein, of significant variation among strains, as well as evidence for recombination among strains, should be considered in attempts to develop vaccines against this disease.