Rapid determination of [guanidino-15N]arginine in plasma with gas chromatography--mass spectrometry: application to human metabolic studies

A rapid gas chromatographic-mass spectrometric method for the determination of 15N in the guanidino nitrogens of arginine is described. The method is based on formation of the N-tetratrifluoroacetyl-arginine derivative. Approximately 0.15 mol% excess 15N can be detected in as little as 50 microliters of plasma with an average coefficient of variation of 8.8%. The possible fragmentation pattern of the N-tetra-trifluoroacetyl-arginine derivative is described. The method was applied to determine the appearance of 15N enrichment in plasma arginine in a healthy adult volunteer during a constant infusion of 15NH4Cl. A plateau level of 0.7 atom% excess was observed 2 h after the 15NH4Cl infusion was started.     

Two-trait-locus linkage analysis: a powerful strategy for mapping complex genetic traits.

Recent advances in molecular biology have provided geneticists with ever-increasing numbers of highly polymorphic genetic markers that have made possible linkage mapping of loci responsible for many human diseases. However, nearly all diseases mapped to date follow clear Mendelian, single-locus segregation patterns. In contrast, many common familial diseases such as diabetes, psoriasis, several forms of cancer, and schizophrenia are familial and appear to have a genetic component but do not exhibit simple Mendelian transmission. More complex models are required to explain the genetics of these important diseases. In this paper, we explore two-trait-locus, two-marker-locus linkage analysis in which two trait loci are mapped simultaneously to separate genetic markers. We compare the utility of this approach to standard one-trait-locus, one-marker-locus linkage analysis with and without allowance for heterogeneity. We also compare the utility of the two-trait-locus, two-marker-locus analysis to two-trait-locus, one-marker-locus linkage analysis. For common diseases, pedigrees are often bilineal, with disease genes entering via two or more unrelated pedigree members. Since such pedigrees often are avoided in linkage studies, we also investigate the relative information content of unilineal and bilineal pedigrees. For the dominant-or-recessive and threshold models that we consider, we find that two-trait-locus, two-marker-locus linkage analysis can provide substantially more linkage information, as measured by expected maximum lod score, than standard one-trait-locus, one-marker-locus methods, even allowing for heterogeneity, while, for a dominant-or-dominant generating model, one-locus models that allow for heterogeneity extract essentially as much information as the two-trait-locus methods. For these three models, we also find that bilineal pedigrees provide sufficient linkage information to warrant their inclusion in such studies. We also discuss strategies for assessing the significance of the two linkages assumed in two-trait-locus, two-marker-locus models.     

Oxygen affinities of maternal and fetal hemoglobins of the viviparous seaperch,Embiotoca lateralis

Summary The striped seaperch,Embiotoca lateralis, is a viviparous teleost. The hemoglobins of adult and fetal seaperch are both tetrameric proteins which in their native state appear to be indistinguishable from one another by electrophoresis. However, differences in the subunit structure of maternal versus fetal seaperch hemoglobins can be detected by electrophoresis in urea with a reducing agent, amino acid analyses and peptide maps of the respective proteins. Furthermore, stripped adult and fetal hemoglobins have different oxygen binding affinities at all pH's tested between pH 6.8 and 8.0. Mid-gestation fetal hemoglobin has a higher oxygen affinity than late-gestation fetal hemoglobin which in turn has a higher affinity than that of the adult hemoglobin. All three stripped hemoglobins show a similar Bohr effect (=–0.9). These data suggest that a difference in oxygen affinities exists in vivo between the adult and fetal blood of the seaperchEmbiotoca lateralis and that it can be explained in part by the presence of a structurally unique fetal hemoglobin. This report is the first to provide evidence for a mechanism of maternal-fetal oxygen transfer in a teleost fish.Abbreviations A adult - LF late-gestation fetal - MF mid-gestation fetal (hemoglobins)     

Coordinate Regulation of the Cyclic AMP System with Firing Rate and Expression of Tyrosine Hydroxylase in the Rat Locus Coeruleus: Effects of Chronic Stress and Drug Treatments

Recent studies have demonstrated that chronic stress increases the firing rate and expression of tyrosine hydroxylase (TH) in neurons of the locus coeruleus (LC), the major noradrenergic nucleus in brain. The present study was undertaken to examine the influence of chronic stress and other treatments known to influence the activity of LC neurons on the cyclic AMP (cAMP) second messenger system in these neurons. Chronic (5 days) cold exposure significantly increased levels of TH immunoreactivity in the LC, as previously reported, but not in substantia nigra (SN) or ventral tegmentum (VT), two dopaminergic nuclei studied for comparison. Chronic cold exposure increased levels of cAMP-dependent protein kinase activity in soluble, but not particulate, fractions of the LC, and increased basal and GTP- and forskolin-stimulated adenylate cyclase activity in this brain region. In contrast, levels of the protein kinase and adenylate cyclase in VT, SN, and frontal cortex were not significantly influenced by cold exposure. To study further the relationship between regulation of LC firing rate, TH expression, and the cAMP system in the LC, other treatments known to influence TH were examined. Reserpine treatment, shown previously to increase levels of TH, was found to increase both LC firing rate and levels of soluble cAMP-dependent protein kinase activity in the LC. 6-Hydroxydopamine, shown previously to increase levels of TH and firing rate of LC neurons, also increased soluble levels of protein kinase activity. Other treatments known to either increase (adrenalectomy) or decrease (chronic imipramine) levels of TH in the LC were also found to increase or decrease, respectively, levels of cAMP-dependent protein kinase activity in this brain region.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heme catabolism in the causative agent of anthrax

A challenge common to all bacterial pathogens is to acquire nutrients from hostile host environments. Iron is an important cofactor required for essential cellular processes such as DNA repair, energy production and redox balance. Within a mammalian host, most iron is sequestered within heme, which in turn is predominantly bound by hemoglobin. While little is understood about the mechanisms by which bacterial hemophores attain heme from host‐hemoglobin, even less is known about intracellular heme processing. Bacillus anthracis, the causative agent of anthrax, displays a remarkable ability to grow in mammalian hosts. Hypothesizing this pathogen harbors robust ways to catabolize heme, we characterize two new intracellular heme‐binding proteins that are distinct from the previously described IsdG heme monooxygenase. The first of these, HmoA, binds and degrades heme, is necessary for heme detoxification and facilitates growth on heme iron sources. The second protein, HmoB, binds and degrades heme too, but is not necessary for heme utilization or virulence. The loss of both HmoA and IsdG renders B. anthracis incapable of causing anthrax disease. The additional loss of HmoB in this background increases clearance of bacilli in lungs, which is consistent with this protein being important for survival in alveolar macrophages.     

Simple and highly efficient site-specific mutagenesis, by ligation of an oligodeoxyribonucleotide into gapped heteroduplex DNA in which the template strand contains deoxyuridine

A simple and highly efficient procedure for oligodeoxynucleotide (oligo)-directed mutagenesis has been developed. In this procedure, a gapped heteroduplex DNA is first constructed and purified. The gapped heteroduplex consists of a circular 'template' strand of DNA, which contains some misincorporated deoxyuridine nucleotides, and a complementary strand which does not contain deoxyuridine, and which lacks a defined segment. Making a specific change in the sequence of the DNA within the gapped region then only requires ligation and transformation. An oligo, exactly the same length as the gap, and with the desired sequence, is synthesized, purified, and ligated directly into the gap in the heteroduplex. When this DNA is used to transform wt (ung+) Escherichia coli, about 80% of the resulting plasmids contain the sequence determined by the synthetic oligo. One gapped heteroduplex preparation can be used for many mutagenesis experiments, so that this procedure is well-suited for producing a series of defined mutations within a defined target region flanked by sites for restriction enzyme cleavage. As the method does not require a polymerase, the effects of primer displacement and polymerase infidelity are avoided.     

Rapid protein-folding assay using green fluorescent protein.

Formation of the chromophore of green fluorescent protein (GFP) depends on the correct folding of the protein. We constructed a "folding reporter" vector, in which a test protein is expressed as an N-terminal fusion with GFP. Using a test panel of 20 proteins, we demonstrated that the fluorescence of Escherichia coli cells expressing such GFP fusions is related to the productive folding of the upstream protein domains expressed alone. We used this fluorescent indicator of protein folding to evolve proteins that are normally prone to aggregation during expression in E. coli into closely related proteins that fold robustly and are fully soluble and functional. This approach to improving protein folding does not require functional assays for the protein of interest and provides a simple route to improving protein folding and expression by directed evolution.     

Genomewide scan for familial combined hyperlipidemia genes in finnish families, suggesting multiple susceptibility loci influencing triglyceride, cholesterol, and apolipoprotein B levels

Familial combined hyperlipidemia (FCHL) is a common dyslipidemia predisposing to premature coronary heart disease (CHD). The disease is characterized by increased levels of serum total cholesterol (TC), triglycerides (TGs), or both. We recently localized the first locus for FCHL, on chromosome 1q21-q23. In the present study, a genomewide screen for additional FCHL loci was performed. In stage 1, we genotyped 368 polymorphic markers in 35 carefully characterized Finnish FCHL families. We identified six chromosomal regions with markers showing LOD score (Z) values >1.0, by using a dominant mode of inheritance for the FCHL trait. In addition, two more regions emerged showing Z>2.0 with a TG trait. In stage 2, we genotyped 26 more markers and seven additional FCHL families for these interesting regions. Two chromosomal regions revealed Z>2.0 in the linkage analysis: 10p11.2, Z=3.20 (theta=.00), with the TG trait; and 21q21, Z=2.24 (theta=.10), with the apoB trait. Furthermore, two more chromosomal regions produced Z>2.0 in the affected-sib-pair analysis: 10q11.2-10qter produced Z=2.59 with the TC trait and Z=2.29 with FCHL, and 2q31 produced Z=2.25 with the TG trait. Our results suggest additional putative loci influencing FCHL in Finnish families, some potentially affecting TG levels and some potentially affecting TC or apoB levels.