Neurotransmitter alterations in embryonic succinate semialdehyde dehydrogenase (SSADH) deficiency suggest a heightened excitatory state during development

Background  

SSADH (aldehyde dehydrogenase 5a1 (Aldh5a1); γ-hydroxybutyric (GHB) aciduria) deficiency is a defect of GABA degradation in which the neuromodulators GABA and GHB accumulate. The human phenotype is that of nonprogressive encephalopathy with prominent bilateral discoloration of the globi pallidi and variable seizures, the latter displayed prominently in Aldh5a1^-/- mice with lethal convulsions. Metabolic studies in murine neural tissue have revealed elevated GABA [and its derivatives succinate semialdehyde (SSA), homocarnosine (HC), 4,5-dihydroxyhexanoic acid (DHHA) and guanidinobutyrate (GB)] and GHB [and its analogue D-2-hydroxyglutarate (D-2-HG)] at birth. Because of early onset seizures and the neurostructural anomalies observed in patients, we examined metabolite features during Aldh5a1^-/- embryo development.     

High-pressure studies of aggregation of recombinant human interleukin-1 receptor antagonist: thermodynamics, kinetics, and application to accelerated formulation studies

Recombinant human interleukin-1 receptor antagonist (IL-1ra) in aqueous solutions unfolds and aggregates when subjected to hydrostatic pressures greater than about 180 MPa. This study examined the mechanism and thermodynamics of pressure-induced unfolding and aggregation of IL-1ra. The activation free energy for growth of aggregates (DeltaG-/+(aggregation)) was found to be 37 +/- 3 kJ/mol, whereas the activation volume (DeltaV-/+(aggregation)) was -120 +/- 20 mL/mol. These values compare closely with equilibrium values for denaturation: The free energy for denaturation, DeltaG(denaturation), was 20 +/- 5 kJ/mol, whereas the partial specific volume change for denaturation, DeltaV(denaturation), was -110 +/- 30 mL/mol. When IL-1ra begins to denature at pressures near 140 MPa, cysteines that are normally buried in the native state become exposed. Under oxidizing conditions, this results in the formation of covalently cross-linked aggregates containing nonnative, intermolecular disulfide bonds. The apparent activation free energy for nucleation of aggregates, DeltaG-/+(nuc), was 42 +/- 4 kJ/mol, and the activation volume for nucleation, DeltaV-/+(nuc),was -175 +/- 37 mL/mol, suggesting that a highly solvent-exposed conformation is needed for nucleation. We hypothesize that the large specific volume of IL-1ra, 0.752 +/- 0.004 mL/g, coupled with its relatively low conformational stability, leads to its susceptibility to denaturation at relatively low pressures. The positive partial specific adiabatic compressibility of IL-1ra, 4.5 +/- 0.7 +/- 10(-12) cm2/dyn, suggests that a significant component of the DeltaV(denaturation) is attributable to the elimination of solvent-free cavities. Lastly, we propose that hydrostatic pressure is a useful variable to conduct accelerated formulation studies of therapeutic proteins.     

The oxidation of chlorophyll a in alcohols

Oxidation potentials for the chlorophyll (Chl/Chl⁺) couple, relative to that of the Fe²⁺/Fe³⁺ couple, were determined in methanol, ethanol, and isopropanol from the initial extent of reaction of chlorophyll with FeCl₃. The presence of 4,7-dimethylphenanthroline was necessary to get sufficient oxidation of chlorophyll in isopropanol. The values were +0.75 V, +0.865 V, and approx. +1.0 V, respectively, based on an assumed value of +0.77 V for the Fe²⁺/Fe³⁺ potential. It is suggested that alcohols, especially methanol, may stabilize Chl⁺ by adding to the carbonyl group or the conjugated double bond system.     

Transport of Fluorescence through Highly Scattering Media: Corrections to the Determination of Quantum Yields

The equations for transport of fluorescence, generated within a highly scattering medium, are solved within the boundary conditions of the Kubelka and Munk treatment. Expressions are derived in closed form for fluorescence fluxes emanating from the front and back surfaces of a highly scattering infinite slab, whether or not fluorescence is absorbed within the sample. An “apparent” quantum yield, calculated from observed intensities of fluorescence and of back-scattered light from the front surface of the slab, can be corrected by these expressions to return the true quantum yield of fluorescence. Allowance for re-emission of reabsorbed fluorescence can be made, but may not be applicable in some experimental arrangements. Calculations performed on the fluorescence of rhodamine 101 suggest that in typical practical situations the correction factor may not be far from unity.     

Pharmacokinetics, immunogenicity, and efficacy of dimeric TNFR binding proteins in healthy and bacteremic baboon

Immunogenicity, pharmacokinetics, and therapeutic efficacy ofthree novel dimeric soluble tumor necrosis factor (TNF)-receptor Iconstructs [TNF-binding protein (bp)] were evaluated in 28 baboons, 12 of which were healthy and 16 were challenged with a lethal Escherichia coli bacteremia. The threeconstructs differed only in the number of extracellular domains of theTNF receptor I and were dimerized with polyethylene glycol. Althoughall three constructs had generally similar pharmacokinetics whenadministered to a naive animal, they differed quantitatively in theirimmunogenicity. Antibodies were detected more frequently, and titerswere significantly higher (P < 0.05)in both healthy and septic baboons that received the 4.0-domain TNF-bpconstruct, compared with animals receiving the 2.6-domain construct.When the TNF-bp constructs were administered a second time (21 dayslater), the half-lives of the three constructs were significantlyshorter in animals that had an antibody response after the firstinjection. In contrast, all three TNF-bp constructs were equallyeffective at improving outcome, blocking a systemic TNF- response,and attenuating the cytokine responses when administered at a dose of1.0 mg/kg body wt 1 h before a lethal E. coli infusion. The findings suggest that immunogenicityof TNF-bp constructs can be altered by changing the number offunctional domains, without affecting their capacity to neutralizeTNF- and to abrogate TNF-mediated pathology.

     

Distribution of cytochrome-like respiration in streptococci.

The electron transport systems of 134 strains of streptococci were studied after aerobic growth on glucose in the presence of haematin, by examining the inhibition of electron transport as well as the cellular site of NADH oxidation. Each strain was placed into one of three possible groups: cytochrome-like NADH oxidase; flavin-like NADH oxidase; or no NADH oxidase. Most (88%) of the strains of Streptococcus faecalis and its variants liquefaciens and zymogenes and a few strains of S. lactis and its variant diacetylactis contained cytochrome-like respiratory systems. Other streptococci including S. faecium fell into one of the other groups but did not contain cytochrome-like NADH oxidases.     

Tyrosine kinase-defective insulin receptors undergo insulin-induced microaggregation but do not concentrate in coated pits

Biologically active colloid-gold complexes were used to compare ligand-induced microaggregation, redistribution, and internalization of insulin receptors on Rat 1 fibroblasts expressing wild type (HIRc) or tyrosine kinase-defective (HIR A/K1018) human insulin receptors. Insulin-like growth factor I (IGF I) and alpha 2-macroglobulin receptors also were compared. On both cell types, all four unoccupied receptor types occurred predominantly as single receptors. Ligand binding caused receptor microaggregation. Microaggregation of wild type or kinase-defective insulin receptors or IGF I receptors was not different. alpha 2-Macroglobulin receptors formed larger microaggregates. Compared to wild type insulin or IGF I receptors, accumulation of kinase-defective insulin receptor microaggregates in endocytic structures was decreased, and the size of microaggregates in coated pits was significantly smaller. As a result, receptor-mediated internalization of gold-insulin by HIR A/K1018 cells was less than 6% of the cell-associated particles compared to approximately 60% of the particles in HIRc cells. On HIR A/K1018 cells, alpha 2-macroglobulin and IGF I were internalized via coated pits demonstrating that those structures were functional. These results suggest that: 1) ATP binding, receptor autophosphorylation, and activation of receptor kinase activity are not required for receptor microaggregation; 2) receptor microaggregation per se is not sufficient to cause ligand-induced receptor-mediated internalization or the biological effects of insulin; and 3) autophosphorylation of the beta-subunit or activation of the receptor kinase activity is required for the insulin-induced concentration of occupied receptors in coated pits.     

Localized chromosomal mosaicism as a cause of dysmorphic development

Regional chromosomal mosaicism has been found in tissue from the clitoral mass of an infant presenting with ambiguous genitalia. Chromosome analysis of skin from the clitoral mass was interpreted as 46,XX/52,XX, +2, +7, +8, +12, +13, +20, whereas study of ovarian tissue and peripheral lymphocytes found each to have normal 46,XX karyotypes with no indication of mosaicism. We believe that somatic cell mutation led to a hyperdiploid clonal cell line resulting in maldevelopment of this patient's external genitalia. This observation, which to our knowledge has not been previously reported, indicates that localized chromosomal abnormalities in some cases may be etiologic of isolated congenital malformations.     

Competition and habitat selection in Namib desert tenebrionid beetles

Summary We tested whether intra- and interspecific competition could affect habitat selection in the two most abundant tenebrionid beetles,Physadesmia globosa andOnymacris rugatipennis, in a dry riverbed in the Namib desert. The spatial distributions of these beetles at the microhabitat scale were negatively correlated. We performed a removal experiment, progressively removing first 25% and then a further 25% of the population of the most abundant species,P. globosa, under the trees where most of the preferred food of both species is concentrated. There was no response ofO. rugatipennis to this removal in the tree habitat. In the open, barely-vegetated habitat where mostO. rugatipennis are found, the number of this species caught in pitfall traps increased following both removals and decreased followingP. globosa replacement under the trees. It appears that intraspecific competition forces someP. globosa to occupy the open habitat. Interspecific competition betweenP. globosa andO. rugatipennis in the open habitat reduces the number ofO. rugatipennis that can co-exist withP. globosa there. Removal ofP. globosa under the trees allows conspecifics in the open habitat to move under the trees, releasingO. rugatipennis in the open habitat from competition. This then results in an increase in the numbers ofO. rugatipennis in the open habitat as a result of immigration from neighbouring areas. We found that differences in foraging efficiency, measured as giving-up times in artificial food patches, create a likely mechanism of co-existence that explains the distinct preferences of these two species for tree and open habitats.     

Cyanobacteria drive community composition and functionality in rock–soil interface communities

Most ecological research on hypoliths, significant primary producers in hyperarid deserts, has focused on the diversity of individual groups of microbes (i.e. bacteria). However, microbial communities are inherently complex, and the interactions between cyanobacteria, heterotrophic bacteria, protista and metazoa are likely to be very important for ecosystem functioning. Cyanobacterial and heterotrophic bacterial communities were analysed by pyrosequencing, while metazoan and protistan communities were assessed by T‐RFLP analysis. Microbial functionality was estimated using carbon substrate utilization. Cyanobacterial community composition was significant in shaping community structure and function in hypoliths. Ecological network analysis showed that most significant co‐occurrences were positive, representing potential synergistic interactions. There were several highly interconnected associations (modules), and specific cyanobacteria were important in driving the modular structure of hypolithic networks. Together, our results suggest that hypolithic cyanobacteria have strong effects on higher trophic levels and ecosystem functioning.