Gastrointestinal tolerance of erythritol-containing beverage in young children: a double-blind, randomised controlled trial.

BACKGROUND/OBJECTIVE: To determine gastrointestinal (GI) responses and maximum tolerated dose of erythritol in young children given as a single oral dose in a 250-ml non-carbonated fruit-flavoured beverage in between meals. This is a multicentre double-blind study with sequential design for multiple dose groups and randomised crossover for comparators of placebo vs dose. SUBJECTS/METHODS: A total of 185 healthy young children aged 4-6 years were recruited at three clinical investigation centres after informed consent of both parents; 184 children completed the study. Children were included in one of the four dose groups (5, 15, 20 or 25 g erythritol) and exposed randomly to only one single dose vs an isosweet sucrose placebo. After consumption in the clinic and an observation period, GI symptoms and stooling patterns were recorded during the next 48 h. RESULTS: Statistically significantly more episodes of diarrhoea and/or severe GI symptoms were observed in the 20 and 25 g groups compared with placebo, but not in the 5 and 15 g groups. Stool consistency, as measured by Bristol stool scale, was lower in the 15-, 20- and 25 g groups for the first 24 -h period, but not at later time points. Incidences of nausea, vomiting, borborygmi, excess flatus and abdominal pain were not significantly different from the placebo controls at all doses of erythritol. CONCLUSIONS: Rapid ingestion of up to and including 15 g (6% w/v) of erythritol in a beverage in between meals by young children aged 4-6 years was well tolerated. The no observed effect level for diarrhoea and/or severe GI symptoms was 15 g (0.73 g/kg body weight (bw)). Children appeared not to be more sensitive to the GI effects of erythritol than published for adults on a g/kg bw basis.

[Maternal diabetic ketosic decompensation which occurred during corticotherapy for fetal lung maturation. A report of three cases]. / Décompensation cétosique d'un diabète maternel au décours d'une corticothérapie de maturation pulmonaire fœtale. À propos de trois cas.

Three cases of ketosis decompensation occurring immediately in type I diabetic after corticotherapy for lung foetal maturation (LFM) are reported. Few of observations have been published. Increasing doses of insulin is mandatory under close monitoring of blood glucose levels, in particular according to the protocol proposed by Kaushal et al.: infusion of insulin adapted to the results of glucose levels, as a supplementation to the usual doses in each patient. Diabetes does not lead to hesitate prescribing a corticotherapy for LFM, but requires a strict control of needs in insulin to avoid a ketosis decompensation.

Chronic consumption of rebaudioside A, a steviol glycoside, in men and women with type 2 diabetes mellitus.

This trial evaluated the effects of 16 weeks of consumption of 1000mg rebaudioside A (n=60) a steviol glycoside with potential use as a sweetener, compared to placebo (n=62) in men and women (33-75 years of age) with type 2 diabetes mellitus. Mean+/-standard error changes in glycosylated hemoglobin levels did not differ significantly between the rebaudioside A (0.11+/-0.06%) and placebo (0.09+/-0.05%; p=0.355) groups. Changes from baseline for rebaudioside A and placebo, respectively, in fasting glucose (7.5+/-3.7mg/dL and 11.2+/-4.5mg/dL), insulin (1.0+/-0.64microU/mL and 3.3+/-1.5microU/mL), and C-peptide (0.13+/-0.09ng/mL and 0.42+/-0.14ng/mL) did not differ significantly (p>0.05 for all). Assessments of changes in blood pressure, body weight, and fasting lipids indicated no differences by treatment. Rebaudioside A was well-tolerated, and records of hypoglycemic episodes showed no excess vs. placebo. These results suggest that chronic use of 1000mg rebaudioside A does not alter glucose homeostasis or blood pressure in individuals with type 2 diabetes mellitus.

Overview: the history, technical function and safety of rebaudioside A, a naturally occurring steviol glycoside, for use in food and beverages.

Rebaudioside A is a sweet tasting steviol glycoside extracted and purified from Stevia rebaudiana (Bertoni). Steviol glycosides can currently be used as a food ingredient in only a handful of countries. Questions on specifications, safety and special population effects have prevented steviol glycosides from obtaining a legal status permitting their use as a sweetener in most countries. A set of papers reporting results of research studies and reviews has been compiled in this Supplement to definitively answer unresolved questions. Specifically, recently completed studies on the general and reproductive toxicity of rebaudioside A corroborate studies carried out with purified steviol glycosides demonstrating safety at high dietary intake levels. Comparative metabolism studies provide further affirmation of the common metabolic pathway for all steviol glycosides and the common metabolism between rats and humans. Finally, clinical studies provide further evidence that purified rebaudioside A has no effect on either blood pressure or glucose homeostasis. This paper summarizes the information used to conclude that high purity rebaudioside A (rebiana) produced to food-grade specifications and according to Good Manufacturing Practices is safe for human consumption under its intended conditions of use as a general purpose sweetener.

The hemodynamic effects of rebaudioside A in healthy adults with normal and low-normal blood pressure.

Rebaudioside A and stevioside are steviol glycosides extracted from the plant Stevia rebaudiana Bertoni and are used in several countries as food and beverage sweeteners. This randomized, double-blind trial evaluated the hemodynamic effects of 4weeks consumption of 1000mg/day rebaudioside A vs. placebo in 100 individuals with normal and low-normal systolic blood pressure (SBP) and diastolic blood pressure (DBP). Subjects were predominantly female (76%, rebaudioside A and 82%, placebo) with a mean age of approximately 41 (range 18-73) years. At baseline, mean resting, seated SBP/DBP was 110.0/70.3mmHg and 110.7/71.2mmHg for the rebaudioside A and placebo groups, respectively. Compared with placebo, rebaudioside A did not significantly alter resting, seated SBP, DBP, mean arterial pressure (MAP), heart rate (HR) or 24-h ambulatory blood pressures responses. These results indicate that consumption of as much as 1000mg/day of rebaudioside A produced no clinically important changes in blood pressure in healthy adults with normal and low-normal blood pressure.

Pharmacokinetics of rebaudioside A and stevioside after single oral doses in healthy men.

This randomized, double-blind, cross-over study assessed the comparative pharmacokinetics of steviol and steviol glucuronide following single oral doses of rebaudioside A and stevioside in healthy adult male subjects. Steviol glucuronide appeared in the plasma of all subjects after administration of rebaudioside A or stevioside, with median tmax values of 12.0 and 8.00h post-dose, respectively. Steviol glucuronide was eliminated from the plasma, with similar t1/2 values of approximately 14h for both compounds. Administration of rebaudioside A resulted in a significantly (approximately 22%) lower steviol glucuronide geometric mean Cmax value (1472ng/mL) than administration of stevioside (1886ng/mL). The geometric mean AUC0-t value for steviol glucuronide after administration of rebaudioside A (30,788ngh/mL) was approximately 10% lower than after administration of stevioside (34,090ngh/mL). Steviol glucuronide was excreted primarily in the urine of the subjects during the 72h collection period, accounting for 59% and 62% of the rebaudioside A and stevioside doses, respectively. No steviol glucuronide was detected in feces. Pharmacokinetic analysis indicated that rebaudioside A and stevioside underwent similar metabolic and elimination pathways in humans with steviol glucuronide excreted primarily in the urine and steviol in the feces. No safety concerns were noted as determined by reporting of adverse events, laboratory assessments of safety or vital signs.

Effects of gamma2S subunit incorporation on GABAA receptor macroscopic kinetics.

GABA(A) receptors, the major inhibitory neurotransmitter receptors in the mammalian central nervous system, are heteropentameric proteins. We are interested in understanding the contribution of the gamma subunit to the kinetic properties of GABA(A) receptors. Studies in Xenopus oocytes have suggested that co-expression of alpha1, beta2, and gamma 2S subunits results in the formation of both alpha beta and alpha betagamma receptors (Boileau et al. 2002a; Boileau et al., 1998). Here, we have used an excess of the gamma 2S subunit in transfections of HEK293 cells to bias expression toward alpha beta gamma-containing receptors. Using rapid application and whole cell patch clamp techniques, we found that incorporation of the gamma subunit eliminated the rapid phases of desensitization and accelerated deactivation, consistent with a proposed role of desensitization in slowing deactivation. In addition, alpha betagamma receptors had an increased GABA EC(50), reduced sensitivity to block by Zn(2+), and did not display outward rectification as compared to alpha beta receptors.

The relative amount of cRNA coding for gamma2 subunits affects stimulation by benzodiazepines in GABA(A) receptors expressed in Xenopus oocytes.

Benzodiazepine (BZD) potentiation of GABA-activated Cl(-)-current (I(GABA)) in recombinant GABA(A) receptors requires the presence of the gamma subunit. When alpha1, beta2 and gamma2S cRNA are expressed in a 1:1:1 ratio in Xenopus oocytes, BZD potentiation of I(GABA) is submaximal, variable and diminishes over time. Potentiation by BZDs is increased, more reproducible and is stabilized over time by increasing the relative amount of cRNA coding for the gamma2S subunit. In addition, GABA EC(50) values for alpha1beta2gamma2 (1:1:1) receptors are intermediate to values measured for alpha1beta2 (1:1) and alpha1beta2gamma2 (1:1:10) receptors. We conclude that co-expression of equal ratios of alpha1, beta2 and gamma2 subunits in Xenopus oocytes produces a mixed population of alpha1beta2 and alpha1beta2gamma2 receptors. Therefore, for accurate measurements of BZD potentiation it is necessary to inject a higher ratio of gamma2 subunit cRNA relative to alpha1 and beta2 cRNA. This results in a purer population of alpha1beta2gamma2 receptors.

Compensatory phospholipid digestion is required for cholesterol absorption in pancreatic phospholipase A(2)-deficient mice.

BACKGROUND AND AIMS: Numerous studies have suggested phospholipid inhibition of dietary cholesterol absorption through the gastrointestinal tract. This study addressed the importance of luminal phospholipid hydrolysis in this process. METHODS: The effect of phospholipase inhibition on cholesterol transport from intestinal lumen to the lymphatics was evaluated in lymph fistula rats. Cholesterol and phospholipid absorption efficiency in intact animals was evaluated in control and phospholipase A(2) (PLA2) gene-targeted mice. RESULTS: The PLA2 inhibitor FPL 67047XX retarded cholesterol absorption in a lymph fistula rat model. Under basal chow-fed dietary conditions, cholesterol absorption efficiency from a single bolus meal, and plasma lipid levels, were similar among PLA2+/+, PLA2+/-, and PLA2-/- mice. Interestingly, the nonhydrolyzable phospholipid dioleoyl ether phosphatidylcholine suppressed cholesterol absorption by 10% to 18% in mice without regard to their PLA2 genotype. When 1-palmitoyl-2-[(14)C]oleoyl-phosphatidylcholine was used as the substrate, the radiolabeled phospholipid was found to be hydrolyzed and absorbed with equal efficiency in PLA2+/+, PLA2+/-, and PLA2-/- mice. CONCLUSIONS: These results suggested that although phospholipid digestion in the intestinal lumen is a prerequisite for efficient cholesterol absorption, additional enzyme(s) can compensate for pancreatic PLA2 in catalyzing phospholipid digestion and facilitating cholesterol absorption in PLA2 knockout mice.

Vitamin A deficiency reduces uptake of beta-carotene by brush border membrane vesicles but does not alter intestinal retinyl ester hydrolase activity in the rat.

Vitamin A deficiency has been reported to result in mild structural and functional changes within the small intestine. The objective of this study was to measure the impact of vitamin A deficiency in the rat on several functional aspects of beta-carotene uptake and intestinal retinyl ester hydrolysis. These included uptake of (14)C-beta-carotene by brush border membrane vesicles (BBMV) and in vitro activity of intrinsic retinyl ester hydrolase (REH). Rats (n = 33) were randomly assigned to receive one of three dietary treatments: vitamin A deficient (-VA), vitamin A sufficient pair-fed (PF), or vitamin A sufficient free access-fed (FA). Liver, serum retinol, and growth data were used to verify clinical vitamin A deficiency. Rats in the -VA group were clinically vitamin A deficient by Day 56 on a vitamin A-free diet and, at that point, all rats were randomly assigned to one of two experimental treatments: BBMV studies or REH activity assays. Uptake of (14)C-beta-carotene by BBMV was significantly suppressed (P < 0.05) in -VA rats when compared to both PF and FA control rats during early passive uptake equilibration (10-20 sec). Uptake was also significantly decreased by BBMV isolated from -VA rats compared to PF controls, but not FA controls, after a 10-min incubation (P < 0.05). In vitro activity of REH was not impacted by vitamin A deficiency in rats, although a trend for greater activity from -VA rats was noted. These data suggest that vitamin A deficiency impairs enterocyte membrane uptake of beta-carotene without altering the enzymatic activity of intrinsic REH.

Amount of dietary fat and type of soluble fiber independently modulate postabsorptive conversion of beta-carotene to vitamin A in mongolian gerbils.

Current dietary guidelines recommend a decrease in fat intake and an increase in fiber consumption. Decreased bioavailability (BV) of carotenoids is thought to be associated with both of these recommendations. A 2 x 4 factorial design was used to test the effects of dietary fat level at 10 or 30% of total energy and fiber type using no fiber, silica, citrus pectin or oat gum (7 g/100 g) on beta-carotene (betaC) BV in 4- to 5-wk-old Mongolian gerbils. We assessed BV as both accumulation of betaC and bioconversion of betaC to vitamin A (VA) in tissues. A VA- and betaC-deficient diet was fed for 1 wk followed by one of eight isocaloric, semipurified diets supplemented with carrot powder [ approximately 1 microgram betaC, 0.5 microgram alpha-carotene (alphaC)/kJ diet] for 2 wk (n = 12/group). Increasing dietary fat resulted in higher VA (P: = 0.074) and lower betaC (P: = 0.0001) stores in the liver, suggesting that consumption of high fat diets enhances conversion of betaC to VA. The effect of soluble fiber on hepatic VA storage was dependent on fiber type. Consumption of citrus pectin resulted in lower hepatic VA stores and higher hepatic betaC stores compared with all other groups, suggesting less conversion of betaC to VA. In contrast, consumption of oat gum resulted in hepatic VA and betaC stores that were higher (P = 0.012) and lower (P = 0.022), respectively, than those of citrus pectin-fed gerbils. The level of dietary fat consumed with soluble fiber had no interactive effects on hepatic VA, betaC or alphaC stores. Results demonstrate that betaC BV is independently affected by dietary fat level and type of soluble fiber, and suggest that these dietary components modulate postabsorptive conversion of betaC to VA. This study confirms the negative effects of citrus pectin on betaC BV, and suggests that oat gum does not adversely affect betaC BV.

Identification of benzodiazepine binding site residues in the gamma2 subunit of the gamma-aminobutyric acid(A) receptor.

gamma-Aminobutyric acid(A) receptor gamma-subunits are important for benzodiazepine (BZD) binding and modulation of the gamma-aminobutyric acid-mediated Cl(-) current. Previously, by using gamma2/alpha1 chimeric subunits, we identified two domains of the gamma2-subunit, Lys-41-Trp-82 and Arg-114-Asp-161, that are, in conjunction, necessary and sufficient for high-affinity BZD binding. In this study, we generated additional gamma2/alpha1 chimeric subunits and gamma2 point mutants to identify specific residues within the gamma2 Lys-41-Trp-82 region that contribute to BZD binding. Mutant gamma2 and gamma2/alpha1 chimeric subunits were expressed with wild-type alpha1 and beta2 subunits in HEK 293 cells, and the binding of several BZDs was measured. We present evidence that the gamma2 region Met-57-Ile-62 is important for flunitrazepam binding and that, in particular, gamma2 Met-57 and gamma2 Tyr-58 are essential determinants for conferring high-affinity binding. Furthermore, we identify an additional residue, gamma2 Ala-79, that not only is important for high-affinity binding by flunitrazepam (a strong positive modulator) but also plays a crucial role in the binding of the imidazobenzodiazepines Ro15-1788 (a zero modulator) and Ro15-4513 (a weak negative modulator) in the BZD binding pocket. Results from site-directed mutagenesis of gamma2 Ala-79 suggest that this residue may be part of a microdomain within the BZD binding site that is important for binding imidazobenzodiazepines. This separation of drug-specific microdomains for competitive BZD ligands lends insight into the structural determinants governing the divergent effects of these compounds.

Review of animal models in carotenoid research.

Foods containing provitamin A carotenoids are the primary source of vitamin A in many countries, despite the poor bioavailability of carotenoids. In addition, epidemiologic studies suggest that dietary intake of carotenoids influences the risk for certain types of cancer, cardiovascular disease and other chronic diseases. Although it would be ideal to use humans directly to answer critical questions regarding carotenoid absorption, metabolism and effects on disease progression, appropriate animal models offer many advantages. This paper will review recent progress in the development of animal models with which to study this class of nutrients. Each potential model has strengths and weaknesses. Like humans, gerbils, ferrets and preruminant calves all absorb beta-carotene (betaC) intact, but only gerbils and calves convert betaC to vitamin A with efficiency similar to that of humans. Mice and rats efficiently convert betaC to vitamin A but absorb carotenoids intact only when they are provided in the diet at supraphysiologic levels. Mice, rats and ferrets can be used to study cancer, whereas primates and gerbils are probably more appropriate for studies on biomarkers of heart disease. No one animal model completely mimics human absorption and metabolism of carotenoids; thus the best model must be chosen with consideration of the specific application being studied, characteristics of the model, and the available funding and facilities.

Identification of transduction elements for benzodiazepine modulation of the GABA(A) receptor: three residues are required for allosteric coupling.

Modulation of GABA(A) receptors by benzodiazepines (BZDs) is believed to involve two distinct steps: a recognition step in which BZDs bind and a conformational transition step in which the affinity of the receptor for GABA changes. Previously, using gamma(2)/alpha(1) chimeric subunits (chi), we demonstrated that although the N-terminal 167 gamma(2) amino acid residues confer high-affinity BZD binding, other gamma(2) domains couple BZD binding to potentiation of the GABA-mediated Cl(-) current (I(GABA)). To determine which gamma(2) regions couple binding to potentiation, we generated chis with longer N-terminal gamma(2) segments for voltage-clamp experiments in Xenopus oocytes. Chimeras containing greater than the N-terminal 167 gamma(2) residues showed incremental gains in maximal potentiation for diazepam enhancement of I(GABA). Residues in gamma(2)199-236, gamma(2)224-236 (pre-M1), and particularly gamma(2)257-297 (M2 and surrounding loops) are important for BZD potentiation. For several positive BZD modulators tested, the same regions restored potentiation of I(GABA). In contrast, beta-carboline inverse-agonism was unaltered in chimeric receptors, suggesting that structural determinants for positive and negative BZD allosteric modulation are different. Dissection of the gamma(2)257-297 domain revealed that three residues in concert, gamma(2)T281, gamma(2)I282 (M2 channel vestibule), and gamma(2)S291 (M2-M3 loop) are necessary to impart full BZD potentiation to chimeric receptors. Thus, these residues participate in coupling distant BZD-binding events to conformational changes in the GABA(A) receptor. The location of these novel residues provides insight into the mechanisms underlying allosteric coupling for other members of the ligand-gated ion channel superfamily.

Cis-lycopene is more bioavailable than trans-lycopene in vitro and in vivo in lymph-cannulated ferrets.

Lycopene is the predominant carotenoid in tomatoes and tomato-based foods and is also a predominant carotenoid in human serum and tissues. Intake of lycopene-rich foods was recently associated with decreased risk for several chronic diseases. The observation that serum and tissue lycopene is more than 50% cis-lycopene, whereas tomatoes and tomato-based foods contain mainly all-trans-lycopene, has led to the hypothesis that cis-isomers of lycopene are more bioavailable. We tested this hypothesis both in vitro (study 1) and in vivo (study 2). In study 1, bile acid micelles containing crystalline lycopene were prepared. The crystalline lycopene used for these analyses was 54.4% cis-lycopene. The optically clear micelle preparation contained 75.9% cis-lycopene in repeated analyses. In study 2, mesenteric lymph duct cannulated ferrets were used to study the in vivo absorption of lycopene from LycoredTM (an ethyl acetate extract of tomatoes containing 5% lycopene by weight; of which 91% was all-trans lycopene). Before being anesthetized, male ferrets (n = 7) were dosed orally with 40 mg lycopene per kg body weight in soybean oil. Lymph secretions were collected, on ice, for 2 h. The residual stomach and small intestinal contents, mucosa lining, lymph secretion and serum were analyzed by HPLC. Whereas the dose, stomach and intestinal contents contained 6.2-17.5% cis-lycopene, the mesenteric lymph secretions contained significantly more, 77.4%, cis-lycopene (P < 0.01). These studies demonstrate that in ferrets, cis-isomers of lycopene are more bioavailable than trans-lycopene probably because cis-isomers are more soluble in bile acid micelles and may be preferentially incorporated into chylomicrons.

Mapping the agonist binding site of the GABAA receptor: evidence for a beta-strand.

GABAA receptors, along with the receptors for acetylcholine, glycine, and serotonin, are members of a ligand-gated ion channel superfamily (Ortells and Lunt, 1995). Because of the paucity of crystallographic information for these ligand-gated channels, little is known about the structure of their binding sites or how agonist binding is transduced into channel gating. We used the substituted cysteine accessibility method to obtain secondary structural information about the GABA binding site and to systematically identify residues that line its surface. Each residue from alpha1 Y59 to K70 was mutated to cysteine and expressed with wild-type beta2 subunits in Xenopus oocytes or HEK 293 cells. The sulfhydryl-specific reagent N-biotinylaminoethyl methanethiosulfonate (MTSEA-Biotin) was used to covalently modify the cysteine-substituted residues. Receptors with cysteines substituted at positions alpha1 T60, D62, F64, R66, and S68 reacted with MTSEA-Biotin, and alpha1 F64C, R66C, and S68C were protected from reaction by agonist. We conclude that alpha1 F64, R66, and S68 line part of the GABA binding site. The alternating pattern of accessibility of consecutive engineered cysteines to reaction with MTSEA-Biotin indicates that the region from alpha1 Y59 to S68 is a beta-strand.

Transformation of Paramecium tetraurelia by electroporation or particle bombardment.

Methods for mass transformation of Paramecium tetraurelia were established using plasmids bearing neomycin-resistance or calmodulin gene fragments. Phenotypic and molecular analyses showed that, although variable, up to 5% transformation can be achieved by electroporation. Concentrations of divalent cations Ca2+ and Mg2+ in the electroporation medium were crucial for efficient transformation. Strong neomycin-resistance transformation using bioballistic particle bombardment with gold particles was observed. For both methods, hybridization to transformant DNA revealed plasmid signals consistent with macronuclear transformation and correlated with transformed phenotypes. Complementation of a known calmodulin gene mutation was also achieved by mass transformation. Possible sources of variation and the general utility of these methods are discussed.

A GABAA receptor alpha1 subunit tagged with green fluorescent protein requires a beta subunit for functional surface expression.

gamma-Aminobutyric acid, type A (GABAA) receptors, the major inhibitory neurotransmitter receptors in the central nervous system, are heteropentameric proteins assembled from distinct subunit classes with multiple subtypes, alpha(1-6), beta(1-4), gamma(1-3), delta(1), and epsilon(1). To examine the process of receptor assembly and targeting, we tagged the carboxyl terminus of the GABAA receptor alpha1 subunit with red-shifted enhanced green fluorescent protein (EGFP). Xenopus oocytes were injected with cRNA of this fusion protein, alpha1-EGFP, alone or in combination with cRNA of GABAA receptor beta2, gamma2, or beta2+gamma2 subunits. Within 72 h after injection, EGFP fluorescence was visible in all fusion protein-injected cells. The fluorescence was associated with the plasmalemma only when the beta2 subunit was co-injected with alpha1-EGFP. Texas Red-conjugated immunolabeling of EGFP on nonpermeabilized cells demonstrated that EGFP was localized extracellularly. Hence, the COOH terminus of the alpha1 subunit is extracellular. Two-electrode voltage clamp of alpha1-EGFPbeta2- and alpha1-EGFPbeta2 gamma2-injected oocytes demonstrates that these cells express functional receptors, with EC50 values for GABA and diazepam similar to wild-type receptors. Thus, a COOH-terminal tag of the alpha1 subunit appears to be functionally silent, providing a useful marker for studies of GABAA receptor expression, assembly, transport, targeting, and clustering. Moreover, the beta2 subunit is required for receptor assembly and surface expression.

Molecular dissection of benzodiazepine binding and allosteric coupling using chimeric gamma-aminobutyric acidA receptor subunits.

Although gamma-aminobutyric acid (GABA)A receptor alpha subunits are important for benzodiazepine (BZD) binding and GABA-current potentiation by BZDs, the presence of a gamma subunit is required for high affinity BZD effects. To determine which regions unique to the gamma2S subunit confer BZD binding and potentiation, we generated chimeric protein combinations of rat gamma2S and alpha1 subunits using a modified protocol to target crossover events to the amino-terminal extracellular region of the subunits. Several chimeras with full open reading frames were constructed and placed into vectors for either voltage-clamp experiments in Xenopus laevis oocytes or radioligand binding experiments in human embryonic kidney 293 cells. Chimeras (chi) containing at least the amino-terminal 161 amino acids of gamma2S bound BZDs with wild-type affinity when coexpressed with alpha1 and beta2 subunits. Further analysis of the gamma2S binding site region uncovered two areas, gamma2S K41-W82 and gamma2S R114-D161, that together are necessary and sufficient for high affinity BZD binding. Surprisingly, although the 161-amino acid residue amino terminus of the gamma2S subunit is sufficient for high affinity BZD binding, it is not sufficient for efficient allosteric coupling of the GABA and BZD binding sites, as demonstrated by reduced diazepam potentiation of the GABA-gated current and GABA potentiation of [3H]flunitrazepam binding. Thus, by using gamma/alpha chimeras, we identified two gamma2 subunit regions required for BZD binding that are distinct from domain or domains responsible for allosteric coupling of the BZD and GABA binding sites.

Osmotaxis in Escherichia coli.

The escape of motile organisms from high concentrations of chemicals was studied in Escherichia coli. We have found all chemicals tested to be osmorepellents. It was shown in both a spatial assay and a temporal assay that the known sensory receptors for chemotaxis are not used for osmotaxis, so a different sensory mechanism appears to be employed. According to the temporal assay, the mechanism between sensory receptors and flagella is also not used for tumbling response (at least in solutions above 0.4 osmolar).