Cyanobacterial Diversity and Halotolerance in a Variable Hypersaline Environment

The Great Salt Plains (GSP) in north-central Oklahoma, USA is an expansive salt flat (∼65 km²) that is part of the federally protected Salt Plains National Wildlife Refuge. The GSP serves as an ideal environment to study the microbial diversity of a terrestrial, hypersaline system that experiences wide fluctuations in freshwater influx and diel temperature. Our study assessed cyanobacterial diversity at the GSP by focusing on the taxonomic and physiological diversity of GSP isolates, and the 16S rRNA phylogenetic diversity of isolates and environmental clones from three sites (north, central, and south). Taxonomic diversity of isolates was limited to a few genera (mostly Phormidium and Geitlerinema), but physiological diversity based on halotolerance ranges was strikingly more diverse, even between strains of the same phylotype. The phylogenetic tree revealed diversity that spanned a number of cyanobacterial lineages, although diversity at each site was dominated by only a few phylotypes. Unlike other hypersaline systems, a number of environmental clones from the GSP were members of the heterocystous lineage. Although a number of cyanobacterial isolates were close matches with prevalent environmental clones, it is not certain if these clones reflect the same halotolerance ranges of their matching isolates. This caveat is based on the notable disparities we found between strains of the same phylotype and their inherent halotolerance. Our findings support the hypothesis that variable or poikilotrophic environments promote diversification, and in particular, select for variation in ecotype more than phylotype.     

GISP binding to TSG101 increases GABA receptor stability by down-regulating ESCRT-mediated lysosomal degradation

The neuron-specific G protein-coupled receptor interacting scaffold protein (GISP) is a multidomain, brain-specific protein derived from the A-kinase anchoring protein-9 gene. We originally isolated GISP as an interacting partner for the GABA(B) receptor subunit GABA(B1). Here, we show that the protein tumour susceptibility gene 101 (TSG101), an integral component of the endosomal sorting machinery that targets membrane proteins for lysosomal degradation, also interacts with GISP. TSG101 co-immunoprecipitates with GISP from adult rat brain, and using GST pull-downs, we identified that the eighth coiled-coiled region of GISP is critical for TSG101 association. Intriguingly, although there is no direct interaction between GISP and the GABA(B2) subunit, their co-expression in HEK293 cells increases levels of GABA(B2). GISP also inhibits TSG101-dependent GABA(B2) down-regulation in human embryonic kidney 293 cells whereas over-expression of a mutant GISP lacking the TSG101 binding domain has no effect on GABA(B2) degradation. These data suggest that GISP can function as a negative regulator of TSG101-dependent lysosomal degradation of transmembrane proteins in neurons to promote receptor stability.     

The effect of ampicillin plus streptomycin on growth and photosynthesis of two halotolerant chlorophyte algae

Streptomycin and ampicillin are antibiotics commonly used to eliminate prokaryotes from the cultures of eukaryotic algae. We studied the effects of 25 mg l⁻¹ streptomycin plus 50 mg l⁻¹ ampicillin on the growth and photosynthesis of two broadly halotolerant algae, Picochlorum oklahomensis and Dunaliella sp. (Chlorophyceae). We measured growth rate, oxygen evolution, chlorophyll fluorescence kinetics, and pigment content in low (150 μmol photons m⁻² s⁻¹) and high (600 μmol photons m⁻² s⁻¹) light grown batch cultures. Our results show only a minor effect of the antibiotics on P. oklahomensis, and none on Dunaliella sp., so this combination of antibiotics is suitable for maintenance of stock cultures for physiological experiments. We also show that these antibiotics can be used in turbidostat cultures of P. oklahomensis, which otherwise tend to succumb to bacteria.     

Hippocampal LTD expression involves a pool of AMPARs regulated by the NSF-GluR2 interaction

We investigated whether the interaction between the N-ethyl-maleimide-sensitive fusion protein (NSF) and the AMPA receptor (AMPAR) subunit GluR2 is involved in synaptic plasticity in the CA1 region of the hippocampus. Blockade of the NSF-GluR2 interaction by a specific peptide (pep2m) introduced into neurons prevented homosynaptic, de novo long-term depression (LTD). Moreover, saturation of LTD prevented the pep2m-induced reduction in AMPAR-mediated excitatory postsynaptic currents (EPSCs). Minimal stimulation experiments indicated that both pep2m action and LTD were due to changes in quantal size and quantal content but were not associated with changes in AMPAR single-channel conductance or EPSC kinetics. These results suggest that there is a pool of AMPARs dependent on the NSF-GluR2 interaction and that LTD expression involves the removal of these receptors from synapses.     

S-Adenosylmethionine Decarboxylase Activity Is Decreased in the Rat Cortex After Traumatic Brain Injury

Abstract: S -Adenosyl- l -methionine decarboxylase (SAMdc) and l -ornithine decarboxylase (ODC) are major enzymes regulating polyamine synthesis. Following ischemia, putrescine content increases as a result of post-traumatic activation of ODC and inhibition of SAMdc. These alterations are thought to mediate edema and cell death. The purpose of this study was to quantify SAMdc activity and edema in the brain following controlled cortical impact injury. Anesthetized adult male rats underwent a right parietal craniectomy and were subjected to cortical impact injury. Tissues were obtained from three bilateral regions: parietal cortex, motor area (CPm); parietal cortex, somatosensory area (CPs); and the pyriform cortex (CPF). SAMdc activity was determined in the postmitochondrial fraction from homogenates of fresh, unfrozen tissues by measuring the decarboxylation of S -adenosyl- l -[ carboxyl -¹⁴C]methionine. Basal SAMdc activity was determined in unoperated rats, and regional differences were noted: Activity was lower in the CPF than in the CPm and CPs. SAMdc activity decreased to the greatest extent in the ipsilateral CPm (impact site) from 1 to 72 h following traumatic brain injury. Significant edema was found in the ipsilateral CPm 1, 8, 16, 24, and 48 h after injury. Decreased SAMdc activity impairs the conversion of putrescine to polyamines and may contribute to delayed pathological changes in the brain after traumatic injury.     

Effect of chronic treatment with deoxycorticosterone acetate on content of a natriuretic substance in atria of rats

Chronic (72 days) administration of deoxycorticosterone acetate (DOCA), with or without saline as the sole drinking fluid, depleted atria of rats of their diuretic and natriuretic activities. Chronic ingestion of saline as the sole drinking fluid did not affect the diuretic, natriuretic, and kaliuretic activities of atria compared with those of rats receiving water to drink. Since systolic blood pressure of the DOCA-treated group did not differ significantly from that of the untreated control group, the decrease in potency of atrial extract from DOCA-treated rats most likely occurred in response to increases in extracellular and vascular volumes. The ability of DOCA to decrease diuretic and natriuretic activities of atria was dose dependent. The decreased activities of the atria of DOCA-treated rats could reflect an increased production and turnover of atrial natriuretic factor. Additional studies revealed an increased diuretic and natriuretic responsiveness of DOCA-treated recipients to atrial extract from untreated rats. Thus, the results of these studies suggest that chronic treatment with DOCA reduced the natriuretic and diuretic potencies of atrial extract and increased renal responsiveness to it.     

Influence of modifying agents on enzyme inactivation studies. An analysis using a series mechanism and a form of the Hill-type equations

A series deactivation model is utilized to theoretically examine the influence of different modifying agents on enzyme deactivation kinetics. A form of the Hill-type equation is used to describe the effect of the modifying agents on the model parameters. Modification-induced inactivation equations are presented for the acetylation and succinylation of E. Coli asparaginase, for the site-specific reagent and substrate modification of flavocytochrome b(2) from Baker's yeast, and for the guanidinium chloride inactivation of cathepsin D. The analysis of more data for these and other enzymes would help further substantiate the technique presented and enhance the applicability of the model.     

Chronic dietary administration of tryptophan prevents the development of deoxycorticosterone acetate salt induced hypertension in rats

Hypertension developed within 3 to 5 weeks in uninephrectomized rats administered deoxycorticosterone acetate (DOCA) at a dose of 850 micrograms X kg-1 X day-1 via Silastic tubes and given isotonic saline to drink. Chronic dietary administration of tryptophan (25 and 50 g/kg of food) to DOCA-treated rats reduced their exaggerated intake of NaCl solution and attenuated the elevation of blood pressure induced by treatment with DOCA alone. Treatment with tryptophan also protected against the reduction in urinary concentrating ability during a 24-h dehydration that is characteristic of DOCA-treated rats. Other tests assessed the responsiveness to the beta-adrenergic agonist, isoproterenol. These included measurement of drinking and heart rate following acute administration of isoproterenol. The characteristically depressed drinking and chronotropic responses of DOCA-treated rats to acute administration of isoproterenol were unaffected by tryptophan. Responsiveness to angiotensin II (AII) was also tested by assessment of dipsogenic and metabolic responses to acute administration of AII. The increased drinking and tail skin temperature responses to administration of AII, characteristic of DOCA-treated rats, were reduced in a graded fashion by treatment with graded doses of tryptophan. The specific binding of AII to its receptors in membranes form the diencephalon of the brain was increased by treatment with DOCA but was returned to control level by concomitant treatment with tryptophan. The content of serotonin in the mesencephalon of the brain was not changed significantly by treatment with tryptophan, but the content of 5-hydroxyindole acetic acid in the same region increased significantly, suggesting that turnover of serotonin was increased by chronic treatment with tryptophan. The cardiac hypertrophy characteristic of treatment with DOCA was attenuated significantly by chronic treatment with tryptophan, while the low, resting plasma renin activity of the DOCA-treated group was unchanged. These results suggest that tryptophan provides significant protection against the development of DOCA-induced hypertension, polydipsia, polyuria, and cardiac hypertrophy in rats. It also reduces the hyperresponsiveness to treatment with AII, possibly by decreasing the specific binding of AII to its receptors. It also appears to increase the turnover of serotonin in the brain. Whether either one or all of these is responsible for the antihypertensive effect of tryptophan remains for further study.     

Solubilization and characterization of kainate receptors from goldfish brain

The binding of [3H]kainate to goldfish brain membrane fragments was investigated. Scatchard analysis revealed a single class of binding sites in Tris-HCl buffer with a Kd of 352 nM and a Bmax of 3.1 pmol/mg wet weight. In Ringer's saline, [3H]kainate bound with a Bmax of 1.8 pmol/mg wet weight and a Kd of 214 nM. Binding in Ringer's saline, but not Tris-HCl buffer, displayed positive cooperativity with a Hill coefficient of 1.15. The [3H]kainate binding sites were solubilized in Ringer's saline using the nonionic detergent n-octyl-beta-D-glucopyranoside. Approximately 30-50% of the total number of membrane-bound binding sites were recovered on solubilization. The Kd of [3H]kainate for solubilized binding sites was approximately 200 nM. The rank order of potency for glutamatergic ligands at inhibiting [3H]kainate binding was identical and the competitive ligands had similar Ki values in both membranes and solubilized extracts. In membrane preparations, [3H]kainate displayed a two component off-rate with koff values of 0.97 min-1 and 0.07 min-1; in solubilized extracts, however, only a single off-rate (koff = 0.52 min-1) was observed. The hydrodynamic properties of n-octyl-beta-D-glucopyranoside solubilized [3H]kainate binding sites was investigated by sucrose density centrifugation. A single well defined peak was detected which yielded a sedimentation coefficient of 8.3 S. The results presented in this report suggest that goldfish brain may provide an ideal system in which to study kainate receptor biochemistry.