Rapid chemical kinetic techniques for investigations of neurotransmitter receptors expressed in Xenopus oocytes

Xenopus laevis oocytes have been used extensively during the past decade to express and study neurotransmitter receptors of various origins and subunit composition and also to express and study receptors altered by site-specific mutations. Interpretations of the effects of structural differences on receptor mechanisms were, however, hampered by a lack of rapid chemical reaction techniques suitable for use with oocytes. Here we describe flow and photolysis techniques, with 2-ms and 100-μs time resolution, respectively, for studying neurotransmitter receptors in giant (≈20-μm diameter) patches of oocyte membranes, using muscle and neuronal acetylcholine receptors as examples. With these techniques, we find that the muscle receptor in BC₃H1 cells and the same receptor expressed in oocytes have comparable kinetic properties. This finding is in contrast to previous studies and raises questions regarding the interpretations of the many studies of receptors expressed in oocytes in which an insufficient time resolution was available. The results obtained indicate that the rapid reaction techniques described here, in conjunction with the oocyte expression system, will be useful in answering many outstanding questions regarding the structure and function of diverse neurotransmitter receptors.     

More expanded access for DMP 266

An expanded access program for DMP 266 (Sustiva/efavirenz) is now available to people with less than 400 T-cells and on failing therapy. Studies show DMP 266 is effective with Combivir (AZT/3TC), a combination that does not include a protease inhibitor. DuPont Merck warns that it may not be effective in people who have taken other non-nucleoside analogues that have similar resistance patterns.     

Thalidomide's comeback

Thalidomide shows a significant ability to eliminate painful mouth ulcers in HIV patients and is also effective in reversing wasting and clearing up severe diarrhea caused by microsporidiosis. However, the drug comes with several side effects. Side effects include peripheral neuropathy, neutropenia, sedation, rash, and a small but statistically significant rise in HIV viral loads. Many physicians are not using Thalidomide due to the publicity that was generated from the birth defects that it caused. Thalidomide is available free through a compassionate use program and through an Investigational New Drug (IND) protocol. Contact information is included for both of these programs.     

Martin-Gruber anastomosis revisited

Based on a study of 70 human cadavers (31 male, 39 female) and on cases described previously, we propose a new classification of the Martin-Gruber anastomosis, a neural connection between the median and ulnar nerves in the forearm. The anastomosis was found in 16 (22.9%) cadavers, being bilateral in three (18.7%) and unilateral in 13 (81.3%), five right and eight left. It occurred in eight (25.8%) of the 31 male cadavers and in eight (20.5%) of the 39 females. Therefore, the anastomosis was found in 19 (13.6%) of the 140 forearms. In Pattern I (89.5%) the anastomosis was made by only one branch, whereas in Pattern II (10.5%) it was made by two. The individual branches were classified as Types a, b, and c based on the nature of their origin from the median nerve. Type a (47.3%) arose from the branch to the superficial forearm flexor muscles, Type b (10.6%) from the common trunk, and Type c (31.6%) from the anterior interosseous nerve. Pattern II was a duplication of Type c (10.5%). The anastomotic branch took an oblique or arched course before joining the ulnar nerve, undivided in 15 cases, but divided into two branches in four cases. The anastomosis passed in front of the ulnar artery in four cases, behind it in six, and in nine cases it was related to the anterior ulnar recurrent artery.     

Currant allergy and the Rosaceae-grass pollen allergy syndrome: a case report.

BACKGROUND: Despite the increasing use of currants in culinary recipes, currant allergy has rarely been reported. OBJECTIVES: To study a case of currant allergy and to explore cross-reactivity between grass pollen and Rosaceae family fruit allergens. METHODS: Skin prick tests to pollen and skin prick-to-prick tests with currants and peach were performed. Specific IgE levels were determined using the CAP method. We prepared a protein extract of 0.1 mg/mL in phosphate-buffered saline using red currant in the presence of protease inhibitors. Immunoblot inhibition studies were performed to explore cross-reactivity between grass pollen and currant allergens. RESULTS: Skin prick test results were positive to Dactylis, arizonic, and olive pollens. Results of skin prick-to-prick tests with fresh red and black currants were negative and positive, respectively, to peach. The specific IgE level was 5.7 KU/L to red currant and 2.92 KU/L to peach (CAP). Western blot analysis with red currant extract revealed specific IgE protein bands of 37 and 26 kDa. Preincubation of sera with extracts from red currant and peach inhibited both IgE bands, and preincubation with Dactylis pollen inhibited the 37-kDa band only. CONCLUSIONS: We report a case of allergy to grass pollen with an oral allergy syndrome involving several fruits from 2 different families of the Rosidae subclass confirmed by in vitro tests. Inhibition studies demonstrated cross-reactivity between different fruits (currant and raspberry) from the Rosidae subclass and were incomplete with grass pollen allergens.     

Microplate technique to determine hemolytic activity for routine typing of Listeria strains.

Because the hemolysis produced by Listeria monocytogenes and Listeria seeligeri on blood agar is frequently difficult to interpret, we developed a microplate technique for the routine determination of hemolytic activity with erythrocyte suspensions. This microtechnique is a simple and reliable test for distinguishing clearly between hemolytic and nonhemolytic strains and could be used instead of the CAMP (Christie-Atkins-Munch-Petersen) test with Staphylococcus aureus in the routine typing of Listeria strains. Furthermore, our results suggest that the quantitation of the hemolytic activity of the Listeria strains, along with the D-xylose, L-rhamnose, and alpha-methyl-D-mannoside acidification tests, allows the differentiation of L. monocytogenes, L. seeligeri, and Listeria ivanovii. We also observed that the treatment of erythrocytes with crude exosubstances of rhodococcus equi, Pseudomonas fluorescens, Acinetobacter calcoaceticus, and S. aureus enhanced the hemolytic activity of all Listeria strains with this characteristic.     

Three highly polymorphic microsatellites at the human myelin oligodendrocyte glycoprotein locus, 100 kb telomeric to HLA-F : Characterization and relation to HLA haplotypes

The MOG locus, located on chromosomal bands 6p21.3-p22 and mapped about 100 kb telomeric to HLA-F, was isolated from cosmid ICRFcl09A2434 and shown to contain three microsatellites. These CA-repeat polymorphic markers were characterized in a sample of 173 healthy unrelated individuals and 84 DNAs from the HLA Workshop reference panel, by a method combining fluorescence labeling of PCR products and use of an automated DNA sequencer. For the three markers, frequencies of heterozygotes are well predicted from allele frequencies by the Hardy—Weinberg rule, which suggests that problems of allele nonamplification are unlikely. Typing of cell lines homozygous in the HLA region allowed unambiguous definition of 81 HLA-MOG haplotypes and showed that several HLA ancestral haplotypes extended to the MOG region. The high degree of polymorphism (59%, 51%, and 81% at the three loci, respectively, and 87% at the haplotype level) makes these new markers informative for association or linkage studies with diseases such as hemochromatosis or multiple sclerosis, and for studies aimed at precisely delineating the site of crossover in chromosomes in which recombination occurred in the distal part of the HLA class I region.     

Saquinavir plus ritonavir reduce viral load by 99.9 percent

A combination of two protease inhibitors, saquinavir (Invirase) and ritonavir (Norvir), may produce a median drop in viral load of 99.9 percent. Several dosage variations were tested. The most common side effects of the combination regimen included tingling around the mouth, diarrhea, fatigue, and nausea. A low level of toxicity was found. This combination may be used for people who have failed other protease inhibitor therapy or have developed a resistance to nucleoside analogues.