Methionine transport in Salmonella typhimurium: evidence for at least one low-affinity transport system.

The systems which transport methionine in Salmonella typhimurium LT2 have been studied. Fourteen mutants, isolated by three different selection procedures, had similar growth characteristics and defects in the specific transport process showing a Km of 0.3 microM for L-methionine, and therefore lack the high-affinity, metP transport system. The sites of mutation in four of the mutants were shown by P1-mediated transduction to be linked (0.3 to 1.1%) with a proline marker located at unit 7 on the S. typhimurium chromosome. The high-affinity system was subject to both repression and transinhibition by methionine, and it may also be regulated by the metJ and metK genes. There appeared to be at least two additional transport systems with relatively low affinities for methionine in the metP763 mutant strain, with apparent Km values for methionine of 24 microM and approximately 1.8 mM. The latter system, with a very low affinity for methionine, was inhibited by leucine. In addition, methionine inhibited leucine transport, suggesting that one of the low-affinity methionine transport systems may actually be a leucine transport system.     

A specific kinetic assay for phenylalanine hydroxylase

An assay procedure is given which is speedy, accurate, and specific, permitting direct recording of velocities, and obviating the use of reagents other than those necessary for the enzymatic reaction itself. The method is suitable for the study of enzyme mechanism and inhibition and also offers distinct advantages when used for other purposes, e.g., assay during purification of enzymes or for measurement of phenylalanine hydroxylase activity in the liver of hyperphenylalaninemics.The method is based on the phenylalanine-dependent change in absorbance of the tetrahydropteridine cofactor as it is oxidized to the dihydro form. The reaction rate measured by this procedure is linear over a wide range of enzyme concentration. The K_m and V for both tetrahydropteridine and for phenylalanine were the same as the values determined by the old procedure. Measurement of the stoichiometry of the reaction showed that one dihydropteridine is formed per tyrosine formed, or per DPNH consumed. The rate of reaction was identical to that measured by a coupled assay using DPNH and purified dihydropteridine reductase.     

Habitat partitioning and vulnerability of sharks in the Great Barrier Reef Marine Park

Sharks present a critical conservation challenge, but little is known about their spatial distribution and vulnerability, particularly in complex seascapes such as Australia’s Great Barrier Reef Marine Park (GBRMP). We review (1) the distribution of shark species among the primary habitats of the GBRMP (coral reefs, inshore/shelf, pelagic and deep-water habitats) (2) the relative exploitation of each species by fisheries, and (3) how current catch rates interact with their vulnerability and trophic index. Excluding rays and chimaeras, we identify a total of 82 shark species in the GBRMP. We find that shark research in the GBRMP has yielded little quantitative information on most species. Reef sharks are largely site-fidelic, but can move large distances and some regularly use non-reef habitats. Inshore and shelf sharks use coastal habitats either exclusively or during specific times in their life cycle (e.g. as nurseries). Virtually nothing is known about the distribution and habitat use of the GBRMP’s pelagic and deep-water sharks. At least 46 species (53.5 %) are caught in one or more fisheries, but stock assessments are lacking for most. At least 17 of the sharks caught are considered highly vulnerable to exploitation. We argue that users of shark resources should be responsible for demonstrating that a fishery is sustainable before exploitation is allowed to commence or continue. This fundamental change in management principle will safeguard against stock collapses that have characterised many shark fisheries.     

Harmonised Australian Reference Intervals for Serum PINP and CTX in Adults

Bone turnover markers (BTMs) are classified as either formation or resorption markers. Their concentrations in blood or urine of adults are considered to reflect the rate of bone remodelling and may be of use in the management of patients with bone disease. Major inter-method differences exist for BTMs, and harmonisation of methods is currently being pursued at an international level. Based on published data, this article describes age- and sex-specific Australian consensus reference intervals for adults for serum procollagen type I amino-terminal propeptide (s-PINP) and serum β-isomerised carboxy-terminal cross-linking telopeptide of type I collagen (s-CTX).     

Palmitoylation targets AKAP79 protein to lipid rafts and promotes its regulation of calcium-sensitive adenylyl cyclase type 8

PKA anchoring proteins (AKAPs) optimize the efficiency of cAMP signaling by clustering interacting partners. Recently, AKAP79 has been reported to directly bind to adenylyl cyclase type 8 (AC8) and to regulate its responsiveness to store-operated Ca(2+) entry (SOCE). Although AKAP79 is well targeted to the plasma membrane via phospholipid associations with three N-terminal polybasic regions, recent studies suggest that AKAP79 also has the potential to be palmitoylated, which may specifically allow it to target the lipid rafts where AC8 resides and is regulated by SOCE. In this study, we have addressed the role of palmitoylation of AKAP79 using a combination of pharmacological, mutagenesis, and cell biological approaches. We reveal that AKAP79 is palmitoylated via two cysteines in its N-terminal region. This palmitoylation plays a key role in targeting the AKAP to lipid rafts in HEK-293 cells. Mutation of the two critical cysteines results in exclusion of AKAP79 from lipid rafts and alterations in its membrane diffusion behavior. This is accompanied by a loss of the ability of AKAP79 to regulate SOCE-dependent AC8 activity in intact cells and decreased PKA-dependent phosphorylation of raft proteins, including AC8. We conclude that palmitoylation plays a key role in the targeting and action of AKAP79. This novel property of AKAP79 adds an unexpected regulatory and targeting option for AKAPs, which may be exploited in the cellular context.     

Antibody repertoires of four- and five-feature translocus mice carrying human immunoglobulin heavy chain and kappa and lambda light chain yeast artificial chromosomes

We have produced mice that carry the human Ig heavy (IgH) and both kappa and lambda light chain transloci in a background in which the endogenous IgH and kappa loci have been inactivated. The B lymphocyte population in these translocus mice is restored to about one-third of normal levels, with preferential (3:1) expression of human lambda over human kappa. Human IgM is found in the serum at levels between 50 and 400 microg/ml and is elevated following immunization. This primary human Ab repertoire is sufficient to yield diverse Ag-specific responses as judged by analysis of mAbs. The use of DH and J segments is similar to that seen in human B cells, with an analogous pattern of N nucleotide insertion. Maturation of the response is accompanied by somatic hypermutation, which is particularly effective in the light chain transloci. These mice therefore allow the production of Ag-specific repertoires of both IgM,kappa and IgM,lambda Abs and should prove useful for the production of human mAbs for clinical use.     

Role of C6 chirality of tetrahydropterin cofactor in catalysis and regulation of tyrosine and phenylalanine hydroxylases.

The chiral specificities of bovine striatal tyrosine hydroxylase (TH) (unphosphorylated and phosphorylated by cAMP-dependent protein kinase) and rat liver phenylalanine hydroxylase (PH) were examined at physiological pH using the pure C6 stereoisomers of 6-methyl- and 6-propyl-5,6,7,8-tetrahydropterin (6-methyl-PH4 and 6-propyl-PH4) and (6R)- and (6S)-tetrahydrobiopterin (BH4). Both PH and phosphorylated TH have substantially higher Vmax values with the unnatural (6R)-propyl-PH4 than the natural (6S)-propyl-PH4 (approximately 6- and 11-fold, respectively). However, the Km's are also higher such that Vmax/Km is almost unaffected by C6 chirality. Unphosphorylated TH has equal Km values for both isomers of 6-propyl-PH4, but has about a 6 times greater Vmax with the unnatural isomer, making it the fastest cofactor yet for this form of the enzyme. With the shorter 6-methyl group, chiral differences are still recognized by phosphorylated TH but hardly at all by PH. Inhibition of both PH and TH by amino acid substrate which occurs with (6R)-BH4 as cofactor is also observed with (6S)-propyl-PH4 but not with (6S)-BH4, (6R)-propyl-PH4, or (6R)- or (6R,S)-methyl-PH4. The Km for (6S)-BH4 with phosphorylated TH is nearly 3 times higher than with (6R)-BH4, but Vmax is unchanged. With unphosphorylated TH, (6S)-BH4 produces very low decelerating rates, which was shown not to be due to irreversible inactivation of the enzyme. The Km for (6R)-BH4 with either hydroxylase is 10 times higher than for the equivalently configured (6S)-propyl-PH4. Comparison of these two cofactors reveals that the 1' and 2' side-chain hydroxyl groups of the natural cofactor promote different regulatory functions in PH than in TH.     

Nucleotide polymorphism at the alcohol dehydrogenase locus of pocket gophers, genus Geomys

Using the strictly neutral model as a null hypothesis, we tested for deviations from expected levels of nucleotide polymorphism at the alcohol dehydrogenase locus (Adh-1) within and among four species of pocket gophers (Geomys bursarius major, G. knoxjonesi, G. texensis llanensis, and G. attwateri). The complete protein-encoding region was examined, and 10 unique alleles, representing both electromorphic and cryptic alleles, were used to test hypotheses (e.g., the neutral model) concerning the maintenance of genetic variation. Nineteen variable sites were identified among the 10 alleles examined, including 9 segregating sites occurring in synonymous positions and 10 that were nonsynonymous. Several statistical methods, including those that test for within-species variation as well as those that examine variation within and among species, failed to reject the null hypothesis that variation (both within and between species of Geomys) at the Adh locus is consistent with the neutral theory. However, there was significant heterogeneity in the ratio of polymorphism to divergence across the gene, with polymorphisms clustered in the first half of the coding region and fixed differences clustered in the second half of the gene. Two alternative hypotheses are discussed as possible explanations for this heterogeneity: an old balanced polymorphism in the first half of the gene or a recent selective sweep in the second half of the gene.      

Recommendations for Reporting and Flagging of Reference Limits on Pathology Reports

Surveys by the RCPA PITUS Project have shown significant variations in report rendering between Australasian Pathology Providers. The same project collected anecdotal evidence that this variation has led to the misunderstanding and misreading of results - a clinical safety issue. Recommendations are given for the rendering of reference limits on pathology reports, determination and rendering of result flags, and the documentation of sub-population partitions for reference intervals. These recommendations apply equally for paper or electronic reporting, but should not limit the use of novel techniques within electronic reports to convey additional meaning. PITUS Working Group 4 will publish draft recommendations for peer review and comment in relation to the above in the second half of 2014.