Evidence of histidine phosphorylation in isocitrate lyase from Escherichia coli

Escherichia coli isocitrate lyase (EC 4.1.3.1.) can be phosphorylated in vitro by an ATP-dependent reaction. The enzyme becomes phosphorylated by an endogenous kinase when partially purified sonic extracts are incubated with [gamma-32P]ATP. Treatment of isocitrate lyase with diethyl pyrocarbonate, a histidine-modifying reagent, blocked incorporation of [32P]phosphate from [gamma-32P]ATP. The isoelectric point of the enzyme was altered by treatment with phosphoramidate, a histidine phosphorylating agent, which suggests that isocitrate lyase can be phosphorylated at a histidine residue(s). Immunoprecipitated 32P-labeled isocitrate lyase was subjected to alkaline hydrolysis, mixed with chemically synthesized phosphohistidine standards, and analyzed by anion exchange chromatography. Characterization of the phosphoamino acid was based on the demonstration that the 32P-labeled product from alkali-hydrolyzed isocitrate lyase comigrated with synthetic 1-phosphohistidine. In addition, loss of catalytic activity after treatment with potato acid phosphatase indicates that catalytically active isocitrate lyase is the phosphorylated form of the enzyme.     

Estimated feeding rates and energy requirements of gentoo penguins,Pygoscelis papua,at Macquarie Island

Summary Water and sodium turnovers of 6–7 week old gentoo penguin chicks and breeding adults were measured using isotopically labelled water and sodium. Influx rates for chicks averaged 188 ml·kg^-1·day^-1 and 13.9 mmol·kg^-1·day^-1 for water and sodium, respectively. Chicks consumed an estimated 228 g·kg^-1·day^-1 fresh food or 886 kJ kg^-1 day. These values correspond to 761 g·day^-1 or 2945 kJ·day^-1 for a gentoo chick mid-way through the growth period. Flux rates for adults attending chicks ranged from 199 to 428 ml·kg^-1·day^-1 for water and from 15 to 36 mmol·kg^-1·ay^-1 for sodium.     

Genotyping and sequence analysis of apolipoprotein E isoforms

Apolipoprotein E (apoE), a polymorphic plasma protein, is essential for catabolism of lipoproteins by receptor-mediated endocytosis. One of the apoE isoforms (E2) differs in its binding affinity to specific receptors and contributes to variations in lipoprotein metabolism. Diagnosis of apoE isoforms is done by isoelectric focusing, but it is hindered by various degrees of post-translational sialylation of the apoE protein. Electrophoretically silent structural variations may also escape detection by this technique. We describe a method for genotyping apoE based on hybridization of allele-specific oligonucleotides with enzymatically amplified genomic DNA, which permits unambiguous diagnosis of six common apoE phenotypes within 24 h. Among 100 E2 alleles present in 81 unrelated individuals genotyped by this technique, we found two rare structural mutants of apoE in addition to the common E2 form, E2(158Arg----Cys). Automated sequencing of amplified DNA identified the rare mutants as E2(136Arg----Ser) and E2(145Arg----Cys). The genotypic method may complement or even replace isoelectric focusing for routine determination of apoE phenotypes and for identification of rare structural variants.     

Mice homozygous for the ablm1 mutation show poor viability and depletion of selected B and T cell populations

The c-abl gene, originally identified as the cellular homolog of the transforming gene of the Abelson murine leukemia virus, encodes a protein-tyrosine kinase of unknown function that is expressed in all mammalian tissues. We have previously described the introduction of a mutation in the c-abl gene into the mouse germline via targeted gene disruption of embryonic stem cells. We now show that mice homozygous for this mutation are severely affected, displaying increased perinatal mortality, runtedness, and abnormal spleen, head, and eye development. We have examined components of the immune system and have found major reductions in B cell progenitors in the adult bone marrow, with less dramatic reductions in developing T cell compartments.     

The use of natural bacterial populations for the treatment of sulphur-containing wastewater

Pollution by inorganic and organic sulphur compounds is increasing and, because of the many environmental hazards associated with these compounds (e.g. toxicity, acidification of rain and freshwater, increase of COD, the greenhouse effect), must be taken seriously. There is a wide variety of sulphur oxidizing bacteria available in nature, and these can be used for the effective control of such pollution. The best way to break the sulphur cycle is to stop it at sulphur which, being insoluble, can be easily recovered (e.g.SO₄ ^2- S^2- S⁰). (Eco)physiological knowledge about the sulphur oxidizing bacteria has proved very useful in the prediction of the performance of sulphur oxidizing communities in actual wastewater treatment systems. Appropriate reactor design, based on this type of study, is essential if such bacterial communities are to function efficiently, especially when toxic sulphides must be treated. This paper reviews the natural and anthropogenic sources of sulphur pollution, its consequences and possible solutions.     

Segmental release of amino acid neurotransmitters from transcranial stimulation

The present study used microdialysis techniques in an intact rabbit model to measure the release of amino acids within the lumbar spinal cord in response to transcranial electrical stimulation. Dialysis samples from the extracellular space were obtained over a stimulation period of 90 minutes and were examined using high pressure liquid chromatography. Neuronal excitation was verified by recerding corticomotor evoked potentials (CMEPs) from the spinal cord. A significant increase in the release of glycine and taurine compared to sham animals was measured after 90 minutes of transcranial stimulation. Glutamate and aspartate release was not significantly elevated. GABA concentrations were consistently low. CMEP components repeatedly showed adequate activation of descending fiber pathways and segmental interneuron pools during dialysis sampling. Since glycine, and to a lesser extent taurine, have been shown to inhibit motor neuron activity and are closely associated with segmental interneuron pools, suprasegmental modulation of motor activity may be, in part, through these inhibitory amino acid neurotransmitters in the rabbit lumbar spinal cord.     

Molecular cloning and expression of the Bacillus anthracis edema factor toxin gene: a calmodulin-dependent adenylate cyclase.

The Bacillus anthracis exotoxin is composed of a lethal factor, a protective antigen, and an edema factor (EF). EF is a calmodulin-dependent adenylate cyclase which elevates cyclic AMP levels within cells. The entire EF gene (cya) has been cloned in Escherichia coli, but EF gene expression by its own B. anthracis promoter could not be detected in E. coli. However, when the EF gene was placed downstream from the lac or the T7 promoter, enzymatically active EF was produced. The EF gene, like the protective antigen (pag) and lethal factor (lef) genes, was present on the large B. anthracis toxin plasmid pXO1.     

Product inhibition of immobilized Escherichia coli arising from mass transfer limitation.

Mass transfer-limited removal of metabolic products led to product-inhibited growth of Escherichia coli that was immobilized in a model system. Comparison of the growth kinetics of immobilized and free-living cells revealed no further physiological differences between cells in these two modes of existence beyond those manifested in the local concentrations of substrate and product. Bacteria were retained on a microporous membrane in a dense, planar aggregate and were grown anaerobically on a glucose-based minimal medium. Radioisotope labeling of the immobilized cell mass with 35S was used to determine growth kinetic parameters. Growth rates in the immobilized cell layer were measured by an autoradiographic technique which allowed comparison of the size of the growing region with the rate of cell convection caused by growth. Immobilized cell growth rates and growth yields ranged from near maximal (0.56 h-1 and 39 g of dry cell weight/mol of glucose, respectively) to substantially reduced (0.15 h-1 and 15 g/mol). The depression of these kinetic parameters was attributed to product inhibition arising from mass transfer-limited removal of acidic waste products from the cell mass. A simple one-dimensional reaction-diffusion model, which incorporated data on the product-inhibited growth kinetics of free-living cells collected in a product-limited chemostat, satisfactorily predicted product inhibition of immobilized cell growth.     

Structure of the hepatitis A virion: peptide mapping of the capsid region.

Milligram amounts of highly purified hepatitis A virus (HAV) were obtained from persistently infected cell cultures. The HAV polypeptides were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose for detection by an enzyme-linked immunotransfer blot procedure. The HAV nucleotide-derived amino acid sequence was subjected to computer analysis to identify potential immunogenic regions within the HAV capsid polypeptides. Synthetic peptides corresponding to selected regions of each of the larger putative capsid polypeptides were coupled to keyhole limpet hemocyanin and used to immunize rabbits. Four of six anti-HAV peptide sera were strongly reactive. Antipeptide serum generated against amino acids (a.a.) 75 through 82 reacted with the 27,000-molecular-weight (MW) polypeptide; serum against a.a. 279 through 285 reacted with the 29,000-MW HAV polypeptide; and sera against a.a. 591 through 602 and 606 through 618 reacted with the 33,000-MW HAV polypeptide. These reactions enabled the identification of the gene order of the larger HAV P1 region gene products. Our data indicate the following molecular weights: HAV VP2 or 1B, 27,000; HAV VP3 or 1C, 29,000; and HAV VP1 or 1D, 33,000.