The rate of action of tetrodotoxin on sodium conductance in the squid giant axon.

When tetrodotoxin is applied to or washed away from the squid giant axon, the rates at which the sodium conductatnce is blocked and unblocked are an order of magnitude smaller than those reported for the isolated node of Ranvier. This slowing is to be expected if in squid the tetrodotoxin binding sites act as a saturable sink in series with the barrier to free diffusion imposed by the presence of the Schwann cell. A comparison has been made between the rates observed experimentally and those calculated for a computer model of the system, in order to estimate the apparent density in the membrane of both specific and non-specific tetrodotoxin binding sites. The figure thus obtained for the number of sodium channels in the squid giant axon, several hundred per square micrometre, agrees well with those derived from other lines of argument.     

Small molecule glutaminase inhibitors block glutamate release from stimulated microglia

Glutaminase plays a critical role in the generation of glutamate, a key excitatory neurotransmitter in the CNS. Excess glutamate release from activated macrophages and microglia correlates with upregulated glutaminase suggesting a pathogenic role for glutaminase. Both glutaminase siRNA and small molecule inhibitors have been shown to decrease excess glutamate and provide neuroprotection in multiple models of disease, including HIV-associated dementia (HAD), multiple sclerosis and ischemia. Consequently, inhibition of glutaminase could be of interest for treatment of these diseases. Bis-2-(5-phenylacetimido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) and 6-diazo-5-oxo-l-norleucine (DON), two most commonly used glutaminase inhibitors, are either poorly soluble or non-specific. Recently, several new BPTES analogs with improved physicochemical properties were reported. To evaluate these new inhibitors, we established a cell-based microglial activation assay measuring glutamate release. Microglia-mediated glutamate levels were significantly augmented by tumor necrosis factor (TNF)-α, phorbol 12-myristate 13-acetate (PMA) and Toll-like receptor (TLR) ligands coincident with increased glutaminase activity. While several potent glutaminase inhibitors abrogated the increase in glutamate, a structurally related analog devoid of glutaminase activity was unable to block the increase. In the absence of glutamine, glutamate levels were significantly attenuated. These data suggest that the in vitro microglia assay may be a useful tool in developing glutaminase inhibitors of therapeutic interest.     

Cryopreserved Morulae can be used to Efficiently Generate Germline-transmitting Chimeras by Blastocyst Injection

The production of chimeric mice is a complex process, requiring the careful coordination of tissue culture cell growth, production of a large number (30–75) of competent blastocysts and the availability of appropriately timed pseudo pregnant female mice. Failure at any of these steps can impinge upon the rapid production of chimeras. One potential improvement for the efficient generation of chimeric mice would be the utilization of cryopreserved embryos suitable for injection. C57Bl/6 morulae were frozen using a standard 2-step protocol with ethylene glycol as the cryopreservation agent. We determined that cryopreserved morulae could thaw, culture to blastocyst stage in KSOM media and survive injection at rates equivalent to control embryos. Cryopreserved morulae were also equivalent to controls at all later stages in the process of production of chimeric mice, including birth rate, percentage chimerism of resulting animals and ability to produce germline progeny. Hence, cryopreservation of morulae for blastocyst injection is a suitable option to enhance the efficiency of chimeric mouse generation.     

Stabilizing proteins from sequence statistics: the interplay of conservation and correlation in triosephosphate isomerase stability

Understanding the determinants of protein stability remains one of protein science's greatest challenges. There are still no computational solutions that calculate the stability effects of even point mutations with sufficient reliability for practical use. Amino acid substitutions rarely increase the stability of native proteins; hence, large libraries and high-throughput screens or selections are needed to stabilize proteins using directed evolution. Consensus mutations have proven effective for increasing stability, but these mutations are successful only about half the time. We set out to understand why some consensus mutations fail to stabilize, and what criteria might be useful to predict stabilization more accurately. Overall, consensus mutations at more conserved positions were more likely to be stabilizing in our model, triosephosphate isomerase (TIM) from Saccharomyces cerevisiae. However, positions coupled to other sites were more likely not to stabilize upon mutation. Destabilizing mutations could be removed both by removing sites with high statistical correlations to other positions and by removing nearly invariant positions at which "hidden correlations" can occur. Application of these rules resulted in identification of stabilizing mutations in 9 out of 10 positions, and amalgamation of all predicted stabilizing positions resulted in the most stable yeast TIM variant we produced (+8 °C). In contrast, a multimutant with 14 mutations each found to stabilize TIM independently was destabilized by 2 °C. Our results are a practical extension to the consensus concept of protein stabilization, and they further suggest the importance of positional independence in the mechanism of consensus stabilization.     

High prevalence of low HDL cholesterol concentrations and mixed hyperlipidemia in a Mexican nationwide survey

The prevalence of lipid abnormalities revealed in a survey done in 417 Mexican cities is described. Information was obtained on 15,607 subjects, aged 20 to 69 years. In this report, only samples obtained after a 9- to 12-h fast were included (2,256 cases: 953 men and 1,303 women). The population is representative of Mexican urban adults. Mean lipid concentrations were: cholesterol, 4.80 mmol/l; triglycerides, 2.39 mmol/l; HDL cholesterol, 1.00 mmol/l; and LDL cholesterol, 3.06 mmol/l. The most prevalent abnormality was HDL cholesterol below 0.9 mmol/l (46.2% for men and 28.7% for women). Hypertriglyceridemia (>2.26 mmol/l) was the second most prevalent abnormality (24.3%). Severe hypertriglyceridemia (>11.2 mmol/l) was observed in 0.42% of the population. Increased LDL cholesterol (> or =4.21 mmol/l) was observed in 11.2% of the sample. Half of the hypertriglyceridemic subjects had a mixed dyslipidemia or low HDL cholesterol. More than 50% of the low HDL cholesterol cases were not related to hypertriglyceridemia. Insulin resistance was found in 59% of them. In conclusion, the prevalence of hypoalphalipoproteinemia and other forms of dyslipidemia in Mexican adults is very high and it is among the highest previously reported worldwide.     

Sulphatide content in a membrane fraction isolated from rabbit gastric mucosal: its possible role in the enzyme involved in H+ pumping

The sulphatide content of vesicular membrane fraction from rabbit mucosal gastric microsomes was analyzed. This vesicular membrane fraction, in addition to a high sulphatide content, was enriched in an ouabain-insensitive (H+ + K+)-ATPase, a (Mg+2 + K+)-activated phosphatase, and a H+ pumping activity. The enzyme system involved in the process of acid secretion and the translocation of K+ was studied in these membrane preparations treated with arylsulphatase A, an enzyme that specifically hydrolyzes sulphatide. The results indicate that the breakdown of sulphatides of the vesicular membrane fraction inactivated both the (H+ + K+)-ATPase activity and the H+ pumping. Both activities were partially restored by the sole addition of sulphatide. The K+-stimulated ouabain-insensitive phosphatase activity, suggested as a partial reaction of the (H+ + K+)-ATPase sequence, was unaffected by arylsulphatase. These results suggest that sulphatides may play a function in the high activity binding site for K+ of the enzyme involved in H+ pumping.     

Substrate specificity, inhibition and enzymological analysis of recombinant human glutamate carboxypeptidase II

Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a membrane peptidase expressed in a number of tissues such as kidney, prostate and brain. The brain form of GCPII (also known as NAALADase) cleaves N-acetyl-aspartyl glutamate to yield free glutamate. Animal model experiments show that inhibition of GCPII prevents neuronal cell death during experimental ischaemia. GCPII thus represents an important target for the treatment of neuronal damage caused by excess glutamate. In this paper we report expression of an extracellular portion of human glutamate carboxypeptidase II (amino acids 44-750) in Drosophila Schneider's cells and its purification to homogeneity. A novel assay for hydrolytic activity of recombinant human GCPII (rhGCPII), based on fluorimetric detection of released alpha-amino groups was established, and used for its enzymological characterization. rhGCPII does not show dipeptidylpeptidase IV-like activity assigned to the native form of the enzyme previously. Using a complete set of protected dipeptides, substrate specificity of rhGCPII was elucidated. In addition to the previously described substrates, four novel compounds, Ac-Glu-Met, Ac-Asp-Met and, surprisingly, Ac-Ala-Glu and Ac-Ala-Met were identified as substrates for GCPII, and their respective kinetic constants determined. The glycosylation of rhGCPII was found indispensable for the enzymatic activity.     

New Ecuadorian records of the eyeless banjo catfish Micromyzon akamai (Siluriformes: Aspredinidae) expand the species range and reveal intraspecific morphological variation

Two specimens of Micromyzon akamai, an eyeless and miniaturized species previously known only from the deep channels of the eastern Amazon basin in Brazil, are reported from the Curaray River, a tributary of the Napo River in Ecuador. The new specimens are the first records of Micromyzon in the headwaters of the Amazon River and the first records of M. akamai outside Brazil. External morphological characters and a phylogenetic analysis of cytochrome c oxidase I (coI) gene support the identification of the new specimens as M. akamai. Nevertheless, the new specimens also indicate that some features previously hypothesized to be apomorphic for M. akamai are intraspecifically variable.