Molecular epidemiology of Vibrio cholerae in the U.S. Gulf Coast.

Enterotoxigenic strains of Vibrio cholerae O-1, biotype El Tor, isolated from a case of cholera in Texas in 1973, an outbreak of cholera in Louisiana in 1978, and Louisiana sewage samples in 1980 and 1981 were analyzed for their genetic similarities. Chromosomal DNA was isolated from each strain, digested with restriction endonuclease, and analyzed by the Southern blot technique. A radioactive probe consisting of Escherichia coli heat-labile enterotoxin DNA detected cholera toxin gene sequences in these strains and demonstrated that the toxin gene sequence, if not the entire chromosomal DNA, is identical in these strains and distinctly different from other strains of V. cholerae isolated throughout the world. In addition, two strains of enterotoxigenic V. cholerae non-O-1 isolated from clinical cases, were analyzed and found to possess cholera toxin genes which differed in the DNA sequence from the V. cholerae O-1 strains. We concluded that a single strain of enterotoxigenic V. cholerae O-1 is resident in the U.S. Gulf Coast and that a second reservoir of cholera toxin genes exists in V. cholerae non-O-1 strains in Louisiana.     

Changes in transmitter release patterns in vitro induced by tremorgenic mycotoxins

The neurochemical effects of the tremorgenic mycotoxins Verruculogen and Penitrem A, which produce a neurotoxic syndrome characterized by sustained tremors, were studied using sheep and rat synaptosomes. The toxins were administered in vivo, either by chronic feeding (sheep) or ip injection 45 min prior to sacrifice (rat). Synaptosomes were subsequently prepared from cerebrocortical and spinal cord/medullary regions of rat, and corpus striatum of sheep. Penitrem A (400 mg mycelium/kg) increased the spontaneous release of endogenous glutamate, GABA, and aspartate by 213%, 455%, and 227%, respectively, from cerebrocortical synaptosomes. Verruculogen (400 mg mycelium/kg) increased the spontaneous release of glutamate and aspartate by 1,300% and 1,200% respectively, but not that of GABA, from cerebrocortical synaptosomes. The spontaneous release of the transmitter amino acids or other amino acids was not increased by the tremorgens in spinal cord/medullary synaptosomes. Penitrem A pretreatment reduced the Veratrine (75 microM) stimulated release of glutamate, aspartate and GABA from cerebrocortical synaptosomes by 33%, 46%, and 11% respectively, and the stimulated release of glycine and GABA from spinal cord/medulla synaptosomes by 67% and 32%, respectively. Verruculogen pretreatment did not alter the Veratrine-induced release of transmitter amino acids from cerebrocortex and spinal cord/medulla synaptosomes. Penitrem A pretreatment increased the spontaneous release of aspartate, glutamate and GABA by 68%, 62%, and 100%, respectively, from sheep corpus striatum synaptosomes but did not alter the synthesis and release of dopamine in this tissue. Verruculogen was shown to cause a substantial increase (300-400%) in the miniature-end-plate potential frequency at the locust neuromuscular junction. The response was detectable within 1 min, rose to a maximum within 5-7 min, and declined to the control rate over a similar period. No change in the amplitude of the m.e.p.p.s was observed. These effects of the tremorgens on transmitter release are interpreted in terms of their mode of action.     

The orchestration of the sensory-motor systems: Clues from Neuropsychology

Research over the last several decades has led to clear and empirically tractable proposals about the representation of conceptual knowledge in the brain. Here we argue that there are already sufficient data from neuropsychology to strongly constrain extant hypotheses about the representation of conceptual knowledge. One constraint imposed by these neuropsychological data is that recognition of actions and understanding of objects do not necessarily depend on the ability to produce object-associated actions. This conclusion compels a reconsideration of the role played by motor planning and/or execution processes in action and object recognition and understanding.     

Molecular diagnosis of Beckwith-Wiedemann syndrome using quantitative methylation-sensitive polymerase chain reaction.

PURPOSE: Beckwith-Wiedemann Syndrome is caused by defects in imprinted gene expression at 11p15. Currently, quantitative Southern analysis using DNA methylation-sensitive restriction enzymes is used in molecular diagnosis of this syndrome. METHODS: We describe a rapid and highly quantitative test for assessing DNA methylation at 11p15 using sodium bisulfite treatment of genomic DNA coupled with quantitative TaqMan methylation-sensitive polymerase chain reaction. RESULTS: TaqMan MSP can assess DNA methylation at both differentially methylated region (DMR)1 and DMR2 at 11p15. In addition, by using TaqMan MSP we were able to determine the parent of origin of a duplication of 11p15 by quantification of both DMR1 and DMR2 DNA methylation. CONCLUSION: TaqMan MSP method is a robust and rapid method for detecting changes in DNA methylation that compares favorably to the current standard of Southern blot for DNA methylation analysis. Assessment of DMR1 and DMR2 provides the most comprehensive assay for methylation defects in Beckwith Wiedemann Syndrome, accounting for more than 70% of the cases. The advantages of TaqMan MSP are that it requires less DNA and that it is rapid, less labor-intensive, and amenable to high-throughput analysis. Moreover, this approach can be modified to assess DNA methylation changes anywhere in the genome.     

A new radioimmunoassay for human mast cell tryptase using monoclonal antibodies

A solid phase immunoradiometric assay was developed for the quantitation of tryptase released from activated human mast cells. Tryptase exhibits a linear dose-response curve over the standard range of 2-50 micrograms/l in buffer, serum, and plasma. The dose-response curve approached a plateau at a tryptase concentration of 100 micrograms/l and exhibited partial inhibition at concentrations above 10,000 micrograms/l. The sensitivity of the assay was 0.2-0.4 micrograms/l, and the intra-assay and interassay coefficients of variation were below 4% at 2 micrograms/l or higher tryptase concentrations. The recovery of known amounts of purified tryptase added to serum ranged from 91 to 115%. Detection of tryptase was evaluated with several body fluids and was accurate in sera, plasma, bronchoalveolar lavage fluid, nasal lavage fluid, and saliva. The concentration of tryptase was examined in serum samples from 100 healthy controls; in each case the level was less than 2 micrograms/l. The immunoassay also was utilized to examine serum levels of tryptase after the onset of a hypotensive reaction in one patient receiving general anesthesia. A maximally elevated level of tryptase (25 micrograms/l) was detected at the first time point, 0.5 h, and elevated levels persisted to 6 h before a return to normal levels was documented at 24 h. Thus, the involvement of mast cell activation in hypotensive subjects can be ascertained by this new tryptase radioimmunoassay.     

Lumbar spine osteolysis: an entity caused by spinal instability

Presented is a new entity in the spectrum of complications following spinal decompression. It occurs in patients with spinal instability after decompression. Thereafter, instability accelerates, leading to a rapid clinical and radiologic deterioration. The presenting symptoms are back pain and lower extremity weakness, resembling spinal stenosis. Erythrocyte sedimentation rate and temperature remain normal. Radiologically, a distinct form of progressing osteolysis is present. This entity may present a difficult diagnostic problem and is usually diagnosed erroneously as a destructive osteomyelitis. Craig needle biopsy results are normal. Treatment consists of stabilization rather than antibiotic therapy and debridement. It is apparent that patients with spinal instability are to be considered "at risk" as further decompression may accelerate the instability, resulting in progressive osteolysis.     

The Effect of Basic Fibroblast Growth Factor on the Reversion of Omental Adipocytes from Lean and Obese Patients

Background: this study was designed to characterize some of the biochemical and molecular genetic changes during reversion of human fat cells. Methods: mature adipocytes were isolated from greater omental fat tissue of eight lean and 14 massively obese persons by established methodology. Results: at day 7 of adherence to Leighton tubes, there was appreciable depletion of triacylglycerol, as well as assumption of an elongated contour. Relatedly, there was an increase in the expression of β-actin mRNA and a significant decrease in the specific activity of cytosolic glycerophosphate dehydrogenase. The decrement in the specific activity of glycerophosphate dehydrogenase, after 7 days in culture, was significant at p < 0.001. Basic fibroblast growth factor at 10 ngml^-1 accelerated significantly (p < 0.03) the decrease in the specific activity of glycerophosphate dehydrogenase in adipose cells from lean subjects. In contrast, basic fibroblast growth factor had no significant influence on cells from massively obese persons. Conclusion: such resistance may contribute to the intractability of massive obesity.