Factors reducing and promoting the effectiveness of proline as an osmoprotectant in Escherichia coli K12

Proline accumulation in Escherichia coli is mediated by three proline porters. Proline catabolism is effected by proline porter I (PPI) and proline/delta 1-pyrroline carboxylate dehydrogenase. Proline did not accumulate cytoplasmically when E. coli was subjected to osmotic stress in minimal salts medium. Although PPI is induced when proline is provided as carbon or nitrogen source, its activity decreased following growth of the bacteria in minimal salts medium of high osmotic strength. Proline dehydrogenase was induced by proline in low or high osmotic strength media. Proline porter II (PPII) was both activated and induced in osmotically stressed bacteria, though the dependencies of the two responses on medium osmolarity differed. Osmotic downshift during the transport measurement decreased the uptake of proline, serine and glutamine by bacteria cultured in media of high osmotic strength. Thus, while osmotic upshift caused specific activation of PPII, osmotic downshift caused a non-specific reduction in amino acid uptake. Glycine betaine inhibited the uptake of [14C]proline via PPII and PPIII but not via PPI. The dependence of that inhibition on glycine betaine concentration was similar when PPII was uninduced, induced or activated by osmotic stress, or induced by amino acid limited growth. Thus PPII and PPIII, not PPI, contribute to the mechanism of osmoprotection by proline and glycine betaine. The tendency for exogenous proline to accumulate in the cytoplasm of bacteria exposed to osmotic stress would, however, be countered by increased proline catabolism.     

Glucose uptake in Saccharomyces cerevisiae grown under anaerobic conditions: effect of null mutations in the hexokinase and glucokinase structural genes.

Glucose uptake was investigated in a set of isogenic strains carrying a single glucose kinase structural gene, the other two kinase genes having been rendered nonfunctional through the construction of null mutations. Any one of the three kinases was sufficient for growth and glucose utilization aerobically or anaerobically. Under anaerobic conditions, substrate inhibition and regulation of carrier activity varied and depended upon the particular kinase present in the cell.     

Proline transport and osmotic stress response in Escherichia coli K-12.

Proline is accumulated in Escherichia coli via two active transport systems, proline porter I (PPI) and PPII. In our experiments, PPI was insensitive to catabolite repression and was reduced in activity twofold when bacteria were subjected to amino acid-limited growth. PPII, which has a lower affinity for proline than PPI, was induced by tryptophan-limited growth. PPII activity was elevated in bacteria that were subjected to osmotic stress during growth or the transport measurement. Neither PPI nor uptake of serine or glutamine was affected by osmotic stress. Mutation proU205, which was similar in genetic map location and phenotype to other proU mutations isolated in E. coli and Salmonella typhimurium, influenced the sensitivity of the bacteria to the toxic proline analogs azetidine-2-carboxylate and 3,4-dehydroproline, the proline requirements of auxotrophs, and the osmoprotective effect of proline. This mutation did not influence proline uptake via PPI or PPII. A very low uptake activity (6% of the PPII activity) observed in osmotically stressed bacteria lacking PPI and PPII was not observed when the proU205 lesion was introduced.     

Regulation of cardiac contractile proteins by phosphorylation

Several of the contractile proteins of the heart can be phosphorylated, but in studies with isolated proteins only phosphorylation of the inhibitory subunit of troponin (TnI) produces a major change in the properties of the contractile system. As TnI is phosphorylated, the concentration of calcium required for activation of contraction is increased. Phosphorylation of the tropomyosin-binding subunit of troponin (TnT) or of the light chain of myosin fails to change ATPase activity of the isolated protein system. Phosphorylation of TnI is stimulated by the beta-adrenergic system and inhibited by the cholinergic system. Maximum calcium-activated force produced by the contractile system can be increased in hyperpermeable cardiac cells by cyclic AmP (cAMP) or agents that stimulate cAMP synthesis. This change in the contractile system, which appears to be part of the physiological response to beta-adrenergic stimulation, is mediated by phosphorylation of an intermediate that then modifies the contractile system. Phosphorylation of the contractile proteins is not involved.     

The induction of liver tumors by 239Pu citrate or 239PuO2 particles in the Chinese hamster

The influence of radiation dose distribution on the frequency of 239Pu-induced liver tumors was evaluated in the Chinese hamster. Different concentrations of 239Pu citrate 239PuO2 particles of known sizes were injected intravenously via the jugular vein. About 60% of the injected 239Pu citrate was deposited in the liver and 40% in the bone. The 239Pu citrate was rather uniformly distributed throughout the liver parenchyma. Injected plutonium oxide particles were taken up by the reticuloendothelial system with 90% of the body burden deposited in the liver. The 239PuO2 particles were localized in the Kupffer cells and produced nonuniform dose distributions that were dependent on particle size. There was an activity- and dose-dependent increase in the incidence of total liver parenchymal cell tumors following injection with either plutonium particles or citrate. For animals that received 14.0-, 2.7-, 0.3-, and 0.04-Gy dose to liver from 239Pu citrate the cumulative tumor incidence was 39, 32, 5, and 0%, respectively. Animals that were injected with the 0.24 micron 239PuO2 particles had doses of 42.0, 7.2, and 0.8 Gy to the liver and tumor incidences of 34, 26, and 5%, respectively. Plutonium citrate also produced hemangiosarcomas of the liver and tumors in bone and bone marrow. The latent period for liver tumor appearance in animals exposed to 239Pu citrate or 239PuO2 particles increased as the injected activity decreased. For animals injected with a similar total activity (7.4 Bq/g), the lifetime cumulative liver tumor incidence was similar for animals exposed to either 239Pu citrate (32%) or 239PuO2 (26%). There was little effect of particle size on liver tumor incidence. These data indicate that, in Chinese hamster liver, local radiation dose distribution is less important in altering tumor incidence than injected activity or average dose. However, the more uniform irradiation from 239Pu citrate administration was more effective in cancer production than the nonuniform irradiation from 239PuO2 particles.     

Isolation and characterization of a large,neurite-associated glycoconjugate from neuroblastoma cells

A high molecular weight glycoconjugate has been isolated from neurite-producing neuronal tumor cells in culture and has been designated as I(0) based on its elution characteristics in gel filtration chromatography. This molecule cannot be found in a variety of nonneuronal cells. I(0) is found in the substratum-attached material or cell fraction of neurite-producing neuroblastoma cells, depending upon culture conditions. It is found in the substratum-bound fraction of B104 rat neuroblastoma cells during serum starvation and in the EGTA-detached cell fraction of B104 cells grown in chemically defined N2 medium. It occurs only in the cell fraction of the human neuroblastoma line Platt. Examination of behavioral variants of the B104 rat line further strengthens the association of I(0) with neurite production; the constitutive neurite-producing E(R)B9 variant contains I(0) while the non-neurite-producing E(R)A11 variant does not. I(0) is large, eluting in the void volume of sepharose-CL2B columns. Radioiodination of intact cells with lactoperoxidase shows I(0) to be a cell surface component. Metabolic radiolabeling studies show that it contains a high proportion of polysaccharide to protein, does not contain mannose, and is unsulfated. Alkaline borohydride reduction release two size classes of large polysaccharide chain. The alkaline reduction results, along with the mannose incorporation studies, show the presence of O-glycosidic linkages and few, if any, N-linkages. Resistance to nitrous acid deamination, insensitivity to glycosaminoglycan lyases, and the absence of sulfation, indicate that I(0) does not contain the glycosaminoglycans hyaluronic acid, chondroitin-, dermatan-, or heparin- sulfates. Affinity column chromatography reveals high binding affinity of I(0) to polyornithine and no binding to gelatin (collagen) or the glycosaminoglycans hyaluronate and heparin. These studies describe a unique high molecular weight glycoconjugate on the surface of neurite-producing neuroblastoma cell lines from two species.     

Relative apparent synapomorphy analysis (RASA). I: The statistical measurement of phylogenetic signal

We have developed a new approach to the measurement of phylogenetic signal in character state matrices called relative apparent synapomorphy analysis (RASA). RASA provides a deterministic, statistical measure of natural cladistic hierarchy (phylogenetic signal) in character state matrices. The method works by determining whether a measure of the rate of increase of cladistic similarity among pairs of taxa as a function of phenetic similarity is greater than a null equiprobable rate of increase. Our investigation of the utility and limitations of RASA using simulated and bacteriophage T7 data sets indicates that the method has numerous advantages over existing measures of signal. A first advantage is computational efficiency. A second advantage is that RASA employs known methods of statistical inference, providing measurable sensitivity and power. The performance of RASA is examined under various conditions of branching evolution as the number of characters, character states per character, and mutations per branch length are varied. RASA appears to provide an unbiased and reliable measure of phylogenetic signal, and the general approach promises to be useful in the development of new techniques that should increase the rigor and reliability of phylogenetic estimates.      

Molecular evolution of voltage-sensitive ion channel genes: on the origins of electrical excitability

We have analyzed nucleic acid and amino acid sequence alignments of a variety of voltage-sensitive ion channels, using several methods for phylogenetic tree reconstruction. Ancient duplications within this family gave rise to three distantly related groups, one consisting of the Na+ and Ca++ channels, another the K+ channels, and a third including the cyclic nucleotide-binding channels. A series of gene duplications produced at least seven mammalian homologues of the Drosophila Shaker K+ channel; clones of only three of these genes are available from all three mammalian species examined (mouse, rat, and human), pointing to specific genes that have yet to be recovered in one or another of these species. The Shaw-related K+ channels and the Na+ channel family have also undergone considerable expansion in mammals, relative to flies. These expansions presumably reflect the needs of the high degree of physiological and neuronal complexity of mammals. Analysis of the separate domains of the four-domain channels (Ca++ and Na+) supports their having evolved by two sequential gene duplications and implies the historical existence of a functional two-domain channel.      

mtDNA diversity in rhesus monkeys reveals overestimates of divergence time and paraphyly with neighboring species

Reconstructions of the human-African great ape phylogeny by using mitochondrial DNA (mtDNA) have been subject to considerable debate. One confounding factor may be the lack of data on intraspecific variation. To test this hypothesis, we examined the effect of intraspecific mtDNA diversity on the phylogenetic reconstruction of another Plio- Pleistocene radiation of higher primates, the fascicularis group of macaque (Macaca) monkey species. Fifteen endonucleases were used to identify 10 haplotypes of 40-47 restriction sites in M. mulatta, which were compared with similar data for the other members of this species group. Interpopulational, intraspecific mtDNA diversity was large (0.5%- 4.5%), and estimates of divergence time and branching order incorporating this variation were substantially different from those based on single representatives of each species. We conclude that intraspecific mtDNA diversity is substantial in at least some primate species. Consequently, without prior information on the extent of genetic diversity within a particular species, intraspecific variation must be assessed and accounted for when reconstructing primate phylogenies. Further, we question the reliability of hominoid mtDNA phylogenies, based as they are on one or a few representatives of each species, in an already depauperate superfamily of primates.