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.     

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.     

Phosphorylation in vivo of the P light chain of myosin in rabbit fast and slow skeletal muscles.

The P light chain of myosin is partially phosphorylated in resting slow and fast twitch skeletal muscles of the rabbit in vivo. The extent of P light-chain phosphorylation increases in both muscles on stimulation. Rabbit slow-twitch muscles contain two forms of the P light chain that migrate with the same electrophoretic mobilities as the two forms of P light chain in rabbit ventricular muscle. The rate of phosphorylation of the P light chain in slow-twitch muscle is slower than its rate of phosphorylation in fast-twitch muscles during tetanus. The rate of P light-chain dephosphorylation is slow after tetanic contraction of fast-twitch muscles in vivo. The time course of dephosphorylation does not correlate with the decline of post-tetanic potentiation of peak twitch tension in rabbit fast-twitch muscles. The frequency of stimulation is an important factor in determining the extent of P light-chain phosphorylation in fast- and slow-twitch muscles.     

High frequency of the Gaucher disease mutation at nucleotide 1226 among Ashkenazi Jews.

Reliable estimates of the frequency of Gaucher disease-producing mutations are not available. The high frequency of Gaucher disease in the Ashkenazi Jewish population is due to the occurrence of a mutation at nucleotide (nt) 1226. We have screened 593 DNA samples from normal Ashkenazi Jews, as well as 62 DNA samples from all our Ashkenazi Jewish patients with Gaucher disease, for the presence of the 1226 mutation. In the 593 presumed normal Ashkenazi Jewish individuals the 1226 mutation was identified in the heterozygous state in 37 and in the homozygous state in two, giving a gene frequency of .035 for the mutation. This 1226 mutation represented 73% of the 124 Gaucher disease alleles in Jewish Gaucher disease patients. Accordingly we estimate that the gene frequency for Gaucher disease among the Ashkenazi Jewish population is .047, which is equivalent to a carrier frequency of 8.9% and a birth incidence of 1:450.     

Erythropoietic progenitor cells from premature neonates studied using a validated serum-deprived medium

We have examined a serum-deprived culture system in order to verify that it is suitable for the study of burst forming unit erythroid (BFU-E) progenitor cells from premature neonates. Optimum growth of BFU-E from premature neonates was observed with each media constituent using the same concentration as that previously described for adult subjects. Growth of immature BFU-E from premature neonates were highly dependant upon a source of Burst Promoting Activity and mature BFU-E derived colonies emerged at day 12 compared to day 14 in adults. Our preliminary results with the validated medium suggest that premature infants have increased peripheral blood concentrations of BFU-E compared to healthy adult controls.Abbreviations Ad Adherent cells - BPA Burst promoting activity - BFU-E Burst forming unit erythroid - Epo Erythropoietin - IL3 Interleukin-3 - LDC Low density (<1.077 g ml¹) peripheral blood mononuclear cells     

Viral Pollution of Surface Waters Due to Chlorinated Primary Effluents

The role of chlorinated primary effluents in viral pollution of the Ottawa River (Ontario) was assessed by examining 282 field samples of wastewaters from two different sewage treatment plants over a 2-year period. The talc-Celite technique was used for sample concentration, and BS-C-1 cells were employed for virus detection. Viruses were detected in 80% (75/94) of raw sewage, 72% (68/94) of primary effluent, and 56% (53/94) of chlorinated effluent samples. Both raw sewage and primary effluent samples contained about 100 viral infective units (VIU) per 100 ml. Chlorination produced a 10- to 50-fold reduction in VIU and gave nearly 2.7 VIU/100 ml of chlorinated primary effluent. With a combined daily chlorinated primary effluent output of approximately 3.7 × 10⁸ liters, these two plants were discharging 1.0 × 10¹⁰ VIU per day. Because the river has a mean annual flow of 8.0 × 10¹⁰ liters per day, these two sources alone produced a virus loading of 1.0 VIU/8 liters of the river water. This river also receives at least 9.0 × 10⁷ liters of raw sewage per day and undetermined but substantial amounts of storm waters and agricultural wastes. It is used for recreation and acts as a source of potable water for some 6.0 × 10⁵ people. In view of the potential of water for disease transmission, discharge of such wastes into the water environment needs to be minimized.