Normal N-acetylglutamate concentration measured in liver from a new patient with N-acetylglutamate synthetase deficiency: physiologic and biochemical implications.

N-Acetyl-L-glutamate synthetase (NAG synthetase) is a mitochondrial matrix enzyme which catalyzes the synthesis of N-acetyl-Lglutamate (NAG), a physiologic activator of the urea cycle enzyme carbamylphosphate synthetase I. Deficiency of NAG synthetase in humans has been reported only three times previously. Two cases presented with uncontrolable neonatal hyperammonemia leading to death, while a third child presented with hyperammonemia and a neurodegenerative picture at 15 months of age after previously being healthy. We report here a new case of NAG synthetase deficiency who presented at 4 years, 10 months of age with an episode of hyperammonemia. Diagnosis was made at age 5 years, 6 months when a liver biopsy showed 9.7% of normal activity. Urine orotic acid was low, and total NAG content in liver was normal. Liver pathology revealed micro- and macrovesicular fat and mitochondria of irregular size and shape with intracristae crystallizations. NAG content in liver in patients with NAG synthetase deficiency has not previously been reported. Its normal value in the face of NAG synthetase deficiency suggests an abnormal localization of NAG to the cytoplasm and the likelihood of aberrant cytoplasmic synthesis of this compound. Additional physiologic implications of this speculative abnormal compartmentalization are discussed.     

Effects of increased salinity on the diatom assemblage in Fonda Lake,Michigan

A salt storage facility has been located adjacent to Fonda Lake since 1953. In February 1981 a core was taken from the profundal sediments of the lake and analyzed to determine the effects of salt perturbation on the diatom community over a 32-year period. Diatom assemblages from different levels were compared using multivariate techniques including cluster analysis and principal component analysis. Shifts in diatom composition related to salinification were revealed most clearly by subdominant taxa. Five distinct groups of diatom taxa were found to correspond with 5 depth intervals. The diatom component of the lake up to 1960 included two groups of taxa which were alkaliphilous and chloride indifferent. A reduction in species diversity beginning in 1960 may indicate a salt effect. By 1968, when diversity reached a minimum, a variety of halophilic taxa (including Diatoma tenue, Navicula gregaria and Synedra fasciculata) attained their highest relative abundances. At the top of the core, diversity increased slightly and some halophilic taxa decreased in relative abundance, which suggests a possible decrease in salt loading to the lake.     

Ontogeny of ovine fetal liver and kidney plasma membrane insulin receptors and fetal growth

This investigation was performed to define certain characteristics of insulin-receptor interaction during the last 2 months of gestation in fetal sheep liver and kidney. Twenty-one sheep carrying a total of 46 fetuses were sacrificed at various gestational ages from 94 days to term; fetal and maternal livers and kidneys were analyzed by a radioreceptor assay for insulin binding characteristics. Specific binding of insulin to partially purified ovine fetal liver and kidney plasma membranes increased as gestation approached term, at which time specific binding was two- to fourfold greater to fetal than to maternal tissues. Associated with increased specific binding were late gestational increases in affinity of insulin for receptors in both fetal liver and kidney and an earlier increase in insulin receptor concentration in fetal kidney. These observations in fetal sheep liver and kidney are similar to reported observations in other species. However, the increase in specific binding of insulin to male fetal liver membranes was exponential; in contrast, there was no apparent increase in specific binding to female fetal liver membranes during the gestational interval surveyed. Both the weights and the vertebral column lengths of these fetuses were shown by multivariate analysis to be significantly affected by the interaction between specific binding of insulin and fetal sex. However, in 30 additional sheep fetuses we observed no difference between male and female fetuses in the increase with time in liver glycogen content. The lack of sex difference in this postreceptor event is consonant with the demonstrated dissociation between liver insulin receptors and glycogen synthesis in the late fetal rat. Our observations suggest that late gestational differences between male and female sheep fetuses in insulin specific binding to liver and, possibly, to other tissues such as cartilage, muscle, and/or fat, that are coupled to postreceptor events may account for differences in fetal growth between the sexes.     

Flow rate and vertical position influence ingestion rates of colonial zebra mussels (Dreissena polymorpha)

1. Zebra mussels aggregate to form dense colonies where, depending on the flow rate, individuals in different vertical locations within the colony may experience restricted food availability. 2. Using ³²P‐labelled Chlamydomonas angulosa, we found ingestion rates of individual mussels located at the surface to exceed those in the bottom of a 6 cm thick colony by up to 75%. 3. Higher velocities (10 and 20 cm s^?1) increased algal delivery to the colony's middle layer (2–4 cm depth), subsequently increasing ingestion rates to equal those in the surface layer, while increasing ingestion only for the smallest mussels in the bottom (4–6 cm). 4. At all vertical locations within the colonies, smaller mussels showed higher ingestion rates per unit mass than larger mussels, particularly at higher flow rates.     

X-chromosome inactivation in the liver of female heterozygous OTC-deficient sparse-furash mice

X-chromosome inactivation patterns were investigated in livers of nine spfash female heterozygous ornithine transcarbamylase (OTC)-deficient mice. Quantitative morphometric analysis of cellular mosaicism was performed on sections of frozen liver reacted with purified anti-OTC antibody and prepared for immunofluorescent microscopy. Analysis of enzymatic OTC activity was performed on sections of these livers using a radiochromatographic technique. Several areas of cellular mosaicism were seen in each of the histological sections that were studied. The distribution of the volume fraction of the liver tissue cells having cells with normal OTC content among the nine mice ranged from 20 to 70% and it correlated (r = 0.8, P = 0.005) with the enzymatic activities of the respective livers. The extreme variegation of mosaic patches in the liver suggests the high probability that a single needle biopsy will be diagnostic in females heterozygous for an OTC mutation. This study also suggests that at the time of X inactivation, the number of primordial liver embryonic cells is small and the observed variegation of liver mosaicism probably results from complex migration patterns of liver cells during fetal development. This study shows that the spfash mouse is a suitable animal model for quantitative studies of X-chromosome inactivation in liver using immunohistochemical staining of OTC protein.     

Intercellular communication in the rat anterior pituitary gland: an in vivo and in vitro study

The concept of "stimulus-secretion coupling" suggested by Douglas and co-workers to explain the events related to monamine discharge by the adrenal medulla (5, 7) may be applied to other endocrine tissues, such as adrenal cortex (36), pancreatic islets (4), and magnocellular hypothalamic neurons (6), which exhibit a similar ion-dependent process of hormone elaboration. In addition, they share another feature, that of joining neighbor cells via membrane junctions (12, 26, and Fletcher, unpublished observation). Given this, and the reports that hormone secretion by the pars distalis also involves a secretagogue-induced decrease in membrane bioelectric potential accompanied by a rise in cellular [Ca++] (27, 34, 41), it was appropriate to test the possibility that cells of the anterior pituitary gland are united by junctions.     

Crystal Structure of the N-Acetyltransferase Domain of Human N-Acetyl-L-Glutamate Synthase in Complex with N-Acetyl-L-Glutamate Provides Insights into Its Catalytic and Regulatory Mechanisms

N-acetylglutamate synthase (NAGS) catalyzes the conversion of AcCoA and L-glutamate to CoA and N-acetyl-L-glutamate (NAG), an obligate cofactor for carbamyl phosphate synthetase I (CPSI) in the urea cycle. NAGS deficiency results in elevated levels of plasma ammonia which is neurotoxic. We report herein the first crystal structure of human NAGS, that of the catalytic N-acetyltransferase (hNAT) domain with N-acetyl-L-glutamate bound at 2.1 Å resolution. Functional studies indicate that the hNAT domain retains catalytic activity in the absence of the amino acid kinase (AAK) domain. Instead, the major functions of the AAK domain appear to be providing a binding site for the allosteric activator, L-arginine, and an N-terminal proline-rich motif that is likely to function in signal transduction to CPS1. Crystalline hNAT forms a dimer similar to the NAT-NAT dimers that form in crystals of bifunctional N-acetylglutamate synthase/kinase (NAGS/K) from Maricaulis maris and also exists as a dimer in solution. The structure of the NAG binding site, in combination with mutagenesis studies, provide insights into the catalytic mechanism. We also show that native NAGS from human and mouse exists in tetrameric form, similar to those of bifunctional NAGS/K.     

Use of Inducible Feedback-Resistant N-Acetylglutamate Synthetase (argA) Genes for Enhanced Arginine Biosynthesis by Genetically Engineered Escherichia coli K-12 Strains

The goal of this work was to construct Escherichia coli strains capable of enhanced arginine production. The arginine biosynthetic capacity of previously engineered E. coli strains with a derepressed arginine regulon was limited by the availability of endogenous ornithine (M. Tuchman, B. S. Rajagopal, M. T. McCann, and M. H. Malamy, Appl. Environ. Microbiol. 63:33–38, 1997). Ornithine biosynthesis is limited due to feedback inhibition by arginine of N-acetylglutamate synthetase (NAGS), the product of the argA gene and the first enzyme in the pathway of arginine biosynthesis in E. coli. To circumvent this inhibition, the argA genes from E. coli mutants with feedback-resistant (fbr) NAGS were cloned into plasmids that contain “arg boxes,” which titrate the ArgR repressor protein, with or without the E. coli carAB genes encoding carbamyl phosphate synthetase and the argI gene for ornithine transcarbamylase. The free arginine production rates of “arg-derepressed” E. coli cells overexpressing plasmid-encoded carAB, argI, and fbr argA genes were 3- to 15-fold higher than that of an equivalent system overexpressing feedback-sensitive wild-type (wt) argA. The expression system with fbr argA produced 7- to 35-fold more arginine than a system overexpressing carAB and argI genes on a plasmid in a strain with a wt argA gene on the chromosome. The arginine biosynthetic capacity of arg-derepressed DH5α strains with plasmids containing only the fbr argA gene was similar to that of cells with plasmids also containing the carAB and argI genes. Plasmids containing wt or fbr argA were stably maintained under normal growth conditions for at least 18 generations. DNA sequencing identified different point mutations in each of the fbr argA mutants, specifically H15Y, Y19C, S54N, R58H, G287S, and Q432R.     

Biomass harvest of invasive Typha promotes plant diversity in a Great Lakes coastal wetland

Ecological and financial constraints limit restoration efforts, preventing the achievement of desired ecological outcomes. Harvesting invasive plant biomass for bioenergy has the potential to reduce feedback mechanisms that sustain invasion, while alleviating financial limitations. Typha × glauca is a highly productive invasive wetland plant that reduces plant diversity, alters ecological functioning, its impacts increase with time, and is a suitable feedstock for bioenergy. We sought to determine ecological effects of Typha utilization for bioenergy in a Great Lakes coastal wetland by testing plant community responses to harvest‐restoration treatments in stands of 2 age classes and assessing community resilience through a seed bank study. Belowground harvesting increased light penetration, diversity, and richness and decreased Typha dominance and biomass in both years post‐treatment. Aboveground harvesting increased light and reduced Typha biomass in post‐year 1 and in post‐year 2, increased diversity and richness and decreased Typha dominance. Seed bank analysis revealed that young stands (<20 years) had greater diversity, richness, seedling density, and floristic quality than old stands (>30 years). In the field, stand‐age did not affect diversity or Typha dominance, but old stands had greater Typha biomass and slightly higher richness following harvest. Harvesting Typha achieved at least 2 desirable ecological outcomes: reducing Typha dominance and increasing native plant diversity. Younger stands had greater potential for native recovery, indicated by more diverse seed banks. In similar degraded wetlands, a single harvest of Typha biomass would likely result in significant biodiversity and habitat improvements, with the potential to double plant species richness.     

Natural products as starting points for future anti-malarial therapies: going back to our roots?

Background

Population movements along the Thailand-Cambodia border, particularly among highly mobile and hard-to-access migrant groups from Cambodia and Myanmar, are assumed to play a key role in the spread of artemisinin resistance. Data on treatment-seeking behaviours, knowledge and perceptions about malaria, and use of preventive measures is lacking as characteristics of this population prevent them from being represented in routine surveillance and the lack of a sampling frame makes reliable surveys challenging.

Methods

A survey of migrant populations from Cambodia and Myanmar was implemented in five selected rural locations in Thailand along the Thai-Cambodian border using respondent driven sampling (RDS) to determine demographic characteristics of the population, migratory patterns, knowledge about malaria, and health-care -seeking behaviours.

Results

The majority of migrants from Myanmar are long-term residents (98%) with no plans to move back to Myanmar, understand spoken Thai (77%) and can therefore benefit from health messages in Thai, have Thai health insurance (99%) and accessed public health services in Thailand (63%) for their last illness. In comparison, the majority of Cambodian migrants are short-term (72%). Of the short-term Cambodian migrants, 92% work in agriculture, 18% speak Thai, 3.4% have Thai health insurance, and the majority returned to Cambodia for treatment (45%), self-treated (11%), or did not seek treatment for their last illness (27%).

Conclusion

Most highly mobile migrants along the Thai-Cambodia border are not accessing health messages or health treatment in Thailand, increasing their risk of malaria and facilitating the spread of potentially resistant Plasmodium falciparum as they return to Cambodia to seek treatment. Reaching out to highly mobile migrants with health messaging they can understand and malaria diagnosis and treatment services they can access is imperative in the effort to contain the spread of artemisinin-resistant P. falciparum.