apbA, a new genetic locus involved in thiamine biosynthesis in Salmonella typhimurium.

In Salmonella typhimurium, the synthesis of the pyrimidine moiety of thiamine can occur by utilization of the first five steps in de novo purine biosynthesis or independently of the pur genes through the alternative pyrimidine biosynthetic, or APB, pathway (D. M. Downs, J. Bacteriol. 174:1515-1521, 1992). We have isolated the first mutations defective in the APB pathway. These mutations define the apbA locus and map at 10.5 min on the S. typhimurium chromosome. We have cloned and sequenced the apbA gene and found it to encode a 32-kDa polypeptide whose sequence predicts an NAD/flavin adenine dinucleotide-binding pocket in the protein. The phenotypes of apbA mutants suggest that, under some conditions, the APB pathway is the sole source of the pyrimidine moiety of thiamine in wild-type S. typhimurium, and furthermore, the pur genetic background of the strain influences whether this pathway can function under aerobic and/or anaerobic growth conditions.     

The VietNam war as an exemplar of American culture

Glenn Petersen is a Professor in the Department of Sociology and Anthropology at Baruch College and its Graduate Center, City University of New York, New York City.     

Dynamics of a pasture soil microbial community after deposition of cattle urine amended with [13C]urea

Within grazed pastures, urine patches are hot spots of nitrogen turnover, since dietary N surpluses are excreted mainly as urea in the urine. This short-term experiment investigated 13C uptake in microbial lipids after simulated deposition of cattle urine at 10.0 and 17.1 g of urea C m(-2). Confined field plots without or with cattle urine amendment were sampled after 4 and 14 days, and soil from 0- to 5-cm and 10- to 20-cm depths was analyzed for content and composition of phospholipid fatty acids (PLFAs) and for the distribution of urea-derived 13C among individual PLFAs. Carbon dioxide emissions were quantified, and the contributions derived from urea were assessed. Initial changes in PLFA composition were greater at the lower level of urea, as revealed by a principal-component analysis. At the higher urea level, osmotic stress was indicated by the dynamics of cyclopropane fatty acids and branched-chain fatty acids. Incorporation of 13C from [13C]urea was low but significant, and the largest amounts of urea-derived C were found in common fatty acids (i.e., 16:0, 16:1omega7c, and 18:1omega7) that would be consistent with growth of typical NH4(+)-oxidizing (Nitrosomonas) and NO2(-)-oxidizing (Nitrobacter) bacteria. Surprisingly, a 20 per thousand depletion of 13C in the cyclopropane fatty acid cy17:0 was observed after 4 days, which was replaced by a 10 to 20 per thousand depletion of that in cy19:0 after 14 days. Possible reasons for this pattern are discussed. Autotrophic nitrifiers could not be implicated in urea hydrolysis to any large extent, but PLFA dynamics and the incorporation of urea-derived 13C in PLFAs indicated a response of nitrifiers which differed between the two urea concentrations.     

Missense mutations in the N-terminal domain of human phenylalanine hydroxylase interfere with binding of regulatory phenylalanine

Hyperphenylalaninemia due to a deficiency of phenylalanine hydroxylase (PAH) is an autosomal recessive disorder caused by >400 mutations in the PAH gene. Recent work has suggested that the majority of PAH missense mutations impair enzyme activity by causing increased protein instability and aggregation. In this study, we describe an alternative mechanism by which some PAH mutations may render PAH defective. Database searches were used to identify regions in the N-terminal domain of PAH with homology to the regulatory domain of prephenate dehydratase (PDH), the rate-limiting enzyme in the bacterial phenylalanine biosynthesis pathway. Naturally occurring N-terminal PAH mutations are distributed in a nonrandom pattern and cluster within residues 46-48 (GAL) and 65-69 (IESRP), two motifs highly conserved in PDH. To examine whether N-terminal PAH mutations affect the ability of PAH to bind phenylalanine at the regulatory domain, wild-type and five mutant (G46S, A47V, T63P/H64N, I65T, and R68S) forms of the N-terminal domain (residues 2-120) of human PAH were expressed as fusion proteins in Escherichia coli. Binding studies showed that the wild-type form of this domain specifically binds phenylalanine, whereas all mutations abolished or significantly reduced this phenylalanine-binding capacity. Our data suggest that impairment of phenylalanine-mediated activation of PAH may be an important disease-causing mechanism of some N-terminal PAH mutations, which may explain some well-documented genotype-phenotype discrepancies in PAH deficiency.     

Metabolism of 4-hydroxynonenal by rat Kupffer cells

Kupffer cells are known to participate in the early events of liver injury involving lipid peroxidation. 4-Hydroxy-2,3-(E)-nonenal (4-HNE), a major aldehydic product of lipid peroxidation, has been shown to modulate numerous cellular systems and is implicated in the pathogenesis of chemically induced liver damage. The purpose of this study was to characterize the metabolic ability of Kupffer cells to detoxify 4-HNE through oxidative (aldehyde dehydrogenase; ALDH), reductive (alcohol dehydrogenase; ADH), and conjugative (glutathione S-transferase; GST) pathways. Aldehyde dehydrogenase and GST activity was observed, while ADH activity was not detectable in isolated Kupffer cells. Additionally, immunoblots demonstrated that Kupffer cells contain ALDH 1 and ALDH 2 isoforms as well as GST A4-4, P1-1, Ya, and Yb. The cytotoxicity of 4-HNE on Kupffer cells was assessed and the TD50 value of 32.5+/-2.2 microM for 4-HNE was determined. HPLC measurement of 4-HNE metabolism using suspensions of Kupffer cells incubated with 25 microLM 4-HNE indicated a loss of 4-HNE over the 30-min time period. Subsequent production of 4-hydroxy-2-nonenoic acid (HNA) suggested the involvement of the ALDH enzyme system and formation of the 4-HNE-glutathione conjugate implicated GST-mediated catalysis. The basal level of glutathione in Kupffer cells (1.33+/-0.3 nmol of glutathione per 10(6) cells) decreased significantly during incubation with 4-HNE concurrent with formation of the 4-HNE-glutathione conjugate. These data demonstrate that oxidative and conjugative pathways are primarily responsible for the metabolism of 4-HNE in Kupffer cells. However, this cell type is characterized by a relatively low capacity to metabolize 4-HNE in comparison to other liver cell types. Collectively, these data suggest that Kupffer cells are potentially vulnerable to the increased concentrations of 4-HNE occurring during oxidative stress.     

The DNA-binding protein of Pf1 filamentous bacteriophage: amino-acid sequence and structure of the gene

The amino-acid sequence of the single-stranded DNA-binding protein of bacteriophage Pf1 and the nucleotide sequence of the corresponding gene have been determined. The protein has 144 amino acids and a molecular weight of 15 400; the gene consists of 435 nucleotides. The amino-acid sequence was determined by Edman degradation, carboxypeptidase A, B, and P digestion of intact protein and of peptides derived by chymotrypsin, Staphylococcus aureus V8 protease, and trypsin digestion. The nucleotide sequence was determined by the dideoxy method after random cloning of fragments of Pf1 DNA into M13. No sequence homology could be established between the amino-acid sequence of the DNA-binding protein of Pseudomonas aeruginosa-specific bacteriophage Pf1 and bacteriophage fd of Escherichia coli.     

Cod (Gadus morhua) cardiorespiratory physiology and hypoxia tolerance following acclimation to low-oxygen conditions

Previous research has shown that hypoxia-acclimated Atlantic cod (Gadus morhua) have significantly reduced cardiac function but can consume more oxygen for a given cardiac output (Q). However, it is not known (1) which physiological changes permit a greater "oxygen pulse" (oxygen consumed per mL of blood pumped) in hypoxia-acclimated individuals or (2) whether chronic exposure to low-oxygen conditions improves the hypoxia tolerance of cod. Thus, we exposed normoxia- and hypoxia-acclimated (> 6 wk at a water oxygen partial pressure [P(w)O(2)] ~8-9 kPa) cod to a graded normoxia challenge until loss of equilibrium occurred while recording the following cardiorespiratory variables: oxygen consumption (MO(2)), ventilatory rate, cardiac function (Q, heart rate f(H), and stroke volume S(V)), ventral aortic blood pressure (P(VA)), venous oxygen partial pressure (P(v)O(2)) and oxygen content (C(v)O(2)), plasma catecholamines, and blood hemoglobin ([Hb]) and hematocrit (Hct). In addition, we performed in vitro hemoglobin oxygen binding curves to examine whether hypoxia acclimation influences hemoglobin functional properties. Numerous physiological adjustments occurred in vivo during the > 6 wk of hypoxia acclimation: that is, increased f(H), decreased S(V) and Q, elevated [Hb], enhanced tissue oxygen extraction (by 10% at a P(w)O(2) of 20 kPa), and a more robust stress response as evidenced by circulating catecholamine levels that were two to eight times higher when fish were acutely exposed to severe hypoxia. In contrast, chronic hypoxia had no significant effect on the affinity of hemoglobin for oxygen, on in vitro hemoglobin oxygen carrying capacity, or on the cod's hypoxia tolerance (H(crit); the P(w)O(2) at which the fish lost equilibrium, which was 4.3 ± 0.2 and 4.8 ± 0.3 kPa in normoxia- and hypoxia-acclimated fish, respectively). These data suggest that while chronic hypoxia results in numerous physiological adjustments, these changes do not improve the cod's capacity to tolerate low-oxygen conditions.     

Activation of the extracellular signal-regulated kinase pathway contributes to the behavioral deficit of fragile x-syndrome

Fragile X syndrome (FXS) is a developmental disorder caused by the loss of Fragile X Mental Retardation 1 (FMR1) gene function because of a CGG repeat expansion (> 200 repeats) in the gene. The molecular mechanism(s) linking loss of FMR1 function to the molecular pathology and cognitive/behavioral disability remain unclear. Given the critical role of extracellular signal-regulated kinase (ERK) in synaptic plasticity and neurodevelopment, a number of recent studies have investigated ERK phosphorylation under basal conditions or upon mGluR-induction using neuronal and peripheral tissues from Fmr1 knockout mice and peripheral tissues from FXS patients. However, these reports have presented conflicting results. The current study is the first to focus on the levels of ERK phosphorylation in brain tissue from human FXS patients. In both human brain tissue and brain tissue from Fmr1 knockout mice there was significantly increased phosphorylation of MEK1/2 and ERK. Indeed, treating Fmr1 knockout mice with the MEK1/2 inhibitor SL327 abrogated audiogenic seizure activity, a feature of the Fmr1 knockout mice that replicates the symptom in patients with FXS. These findings suggest that activation of the ERK pathway results in some cardinal cognitive and clinical features in FXS patients and likely have profound translational implications.     

Limited genetic differentiation among breeding,molting, and wintering groups of the threatened Steller’s eider: the role of historic and contemporary factors

Due to declines in the Alaska breeding population, the Steller’s eider (Polysticta stelleri) was listed as threatened in North America in 1997. Periodic non-breeding in Russia and Alaska has hampered field-based assessments of behavioral patterns critical to recovery plans, such as levels of breeding site fidelity and movements among three regional populations: Atlantic-Russia, Pacific-Russia and Alaska. Therefore, we analyzed samples from across the species range with seven nuclear microsatellite DNA loci and cytochrome b mitochondrial (mt)DNA sequence data to infer levels of interchange among sampling areas and patterns of site fidelity. Results demonstrated low levels of population differentiation within Atlantic and Pacific nesting areas, with higher levels observed between these regions, but only for mtDNA. Bayesian analysis of microsatellite data from wintering and molting birds showed no signs of sub-population structure, even though band-recovery data suggests multiple breeding areas are present. We observed higher estimates of F-statistics for female mtDNA data versus male data, suggesting female-biased natal site fidelity. Summary statistics for mtDNA were consistent with models of historic population expansion. Lack of spatial structure in Steller’s eiders may result largely from insufficient time since historic population expansions for behaviors, such as natal site fidelity, to isolate breeding areas genetically. However, other behaviors such as the periodic non-breeding observed in Steller’s eiders may also play a more contemporary role in genetic homogeneity, especially for microsatellite loci.