The beta subunit of tryptophan synthase. Clarification of the roles of histidine 86, lysine 87, arginine 148, cysteine 170, and cysteine 230

Our studies, which are aimed at understanding the catalytic mechanism of the beta subunit of tryptophan synthase from Salmonella typhimurium, use site-directed mutagenesis to clarify the functional roles of several putative active site residues. Although previous chemical modification studies have suggested that histidine 86, arginine 148, and cysteine 230 are essential residues in the beta subunit, our present findings that beta subunits with single amino acid replacements at these positions have partial activity show that these 3 residues are not essential for catalysis or substrate binding. These conclusions are consistent with the recently determined three-dimensional structure of the tryptophan synthase alpha 2 beta 2 complex. Amino acid substitution of lysine 87, which forms a Schiff base with pyridoxal phosphate in the wild type beta subunit, yields an inactive form of the beta subunit which binds alpha subunit, pyridoxal phosphate, and L-serine. We also report a rapid and efficient method for purifying wild type and mutant forms of the alpha 2 beta 2 complex from S. typhimurium from an improved enzyme source. The enzyme, which is produced by a multicopy plasmid encoding the trpA and trpB genes of S. typhimurium expressed in Escherichia coli, is crystallized from crude extracts by the addition of 6% poly(ethylene glycol) 8000 and 5 mM spermine. This new method is also used in the accompanying paper to purify nine alpha 2 beta 2 complexes containing mutant forms of the alpha subunit.     

Wild carnivore acceptance of baits for delivery of liquid rabies vaccine

A series of experiments are described on the acceptance, by red foxes (Vulpes vulpes) and other species, of two types of vaccine-baits intended to deliver liquid rabies vaccine. The baits consisted of a cube of sponge coated in a mixture of tallow and wax, or a plastic blister-pack embedded in tallow. All baits contained tetracycline as a biological marking agent: examination of thin sections of carnivore canines under an ultraviolet microscope revealed a fluorescent line of tetracycline if an individual had eaten baits. Baits were dropped from fixed-wing aircraft flying about 100 m above ground at approximately 130 km/h. Flight lines followed the edges of woodlots midway between parallel roads. Baits were dropped at one/sec, resulting in one bait/36 m on the ground, or 17 to 25 baits per km2. Crows (Corvus brachyrhynchos) removed many baits, but did not appear to lower the percent of the fox population which took bait. Dropping baits only into corn and woodland to conceal baits, to reduce depredation by crows, reduced acceptance by foxes. Acceptance by foxes ranged between 37 and 68%. Meat added as an attractant did not raise acceptance. Presence, absence, color and perforations of plastic bags did not alter bait acceptance. Dispersal by juvenile foxes probably lowered the estimates of bait acceptance. It took 7 to 17 days for 80% (n = 330) of foxes to eat their first bait. The rapidity with which foxes picked up their first bait appeared more affected by unknown characteristics of years or study areas than by experimental variables. Skunks (Mephitis mephitis) and raccoons (Procyon lotor) also ate these baits, but acceptance was lower. Small mammals contacted baits, but rarely contacted the vaccine, which had the potential for vaccine-induced rabies in some species. Aerial distribution of baits was more cost-effective than ground distribution as practiced in Europe. This system has potential for field control of rabies, although higher acceptance will be desirable.     

Ethanol-responsive gene expression in neural cell cultures.

We have studied the molecular mechanisms underlying neuronal adaptation to chronic ethanol exposure. NG108-15 neuroblastoma cells were used to perform a detailed analysis of ethanol-induced changes in neuronal gene expression. High resolution, quantitative two-dimensional (2-D) gel electrophoresis of in vitro translation products showed both dose-dependent increases and decreases in specific mRNA abundance following treatment with ethanol at concentrations seen in actively drinking alcoholics (50-200 mM). Dose response curves for representative members of the increasing or decreasing response groups had very similar profiles, suggesting that similar mechanisms may regulate members of a response group. Some mRNAs that increased with ethanol treatment appeared identical to species induced by heat shock while other mRNAs were only induced by ethanol. We conclude that chronic ethanol exposure can produce specific coordinate changes in expression of neuronal mRNAs, including some members of the stress protein response. However, the overall pattern of ethanol-responsive gene expression is distinct from the classical heat shock subgroup of stress proteins response. Changes in gene expression and specifically, mechanisms regulating a subset of stress protein expression, could be an important aspect of neuronal adaptation to chronic ethanol seen in alcoholics.     

Fatigue of single motor units in human masseter

The spike-triggered averaging technique was used to determine the time course and extent of fatigue of single motor unit twitches in the human masseter. This is the first report of a fatigue test having been applied to masseter motor units in either animals or humans. The human masseter was found to be comprised predominantly of fast-twitch motor units with a broad spectrum of fatigability. Very few physiological type S units were found, despite histochemical evidence for a substantial population of type I fibers in the masseter. In addition, there was no significant correlation between fatigability and either twitch amplitude or contractile speed in the motor units studied. The latter observations are consistent with the unusual histological features of the masseter. Comparison with other human fatigue data suggests that the extent of fatigue in the present population of masseter motor units after approximately 3,000 activations is similar to that reported for populations of units in first dorsal interosseous and medial gastrocnemius.     

Projection pattern of sensory neurons in the central nervous system of a homeotic mutation of the moth Manduca sexta

Octopod (Octo) is a mutation of the moth Manduca sexta, which transforms the first abdominal segment (A1) in the anterior direction. Mutant animals are characterized by the appearance of homeotic thoracic-like legs on A1. We exploited this mutation to determine what rules might be used in specifying the fates of sensory neurons located on the body surface of larval Manduca. Mechanical stimulation of homeotic leg sensilla did not cause reflexive movements of the homeotic legs, but elicited responses similar to those observed following stimulation of ventral A1 body wall hairs. Intracellular recordings demonstrated that several of the motoneurons in the A1 ganglion received inputs from the homeotic sensory hairs. The responses of these motoneurons to stimulation of homeotic sensilla resembled their responses to stimulation of ventral body wall sensilla. Cobalt fills revealed that the mutation transformed the segmental projection pattern of only the sensory neurons located on the ventral surface of A1, resulting in a greater number with intersegmental projection patterns typical of sensory neurons found on the thoracic body wall. Many of the sensory neurons on the homeotic legs had intersegmental projection patterns typical of abdominal sensory neurons: an anteriorly directed projection terminating in the third thoracic ganglion (T3). Once this projection reached T3, however, it mimicked the projections of the thoracic leg sensory neurons. These results demonstrate that the same rules are not used in the establishment of the intersegmental and leg-specific projection patterns. Segmental identity influences the intersegmental projection pattern of the sensory neurons of Manduca, whereas the leg-specific projections are consistent with a role for positional information in determining their pattern. © 1995 John Wiley & Sons, Inc.     

Thermal denaturation and loss of viability in Escherichia coli and Bacillus stearothermophilus

When Escherichia coli was heated at 10°C/min in a differential scanning calorimeter, the onset of irreversible thermal denaturation occurred at 51°C, about 5°C above the maximum growth temperature. The temperature at which death rate was maximal (63°C) coincided with the thermogram peak caused by denaturation of the 30S ribosomal subunit. The maximum death rate in vegetative cells of Bacillus stearothermophilus occurred at the higher temperature of 71°C which also coincided with the leading edge of the main thermogram peak.     

The lepidopteran mitochondrial control region: structure and evolution

For several species of lepidoptera, most of the approximately 350-bp mitochondrial control-region sequences were determined. Six of these species are in one genus, Jalmenus; are closely related; and are believed to have undergone recent rapid speciation. Recent speciation was supported by the observation of low interspecific sequence divergence. Thus, no useful phylogeny could be constructed for the genus. Despite a surprising conservation of control-region length, there was little conservation of primary sequences either among the three lepidopteran genera or between lepidoptera and Drosophila. Analysis of secondary structure indicated only one possible feature in common--inferred stem loops with higher-than-random folding energies-- although the positions of the structures in different species were unrelated to regions of primary sequence similarity. We suggest that the conserved, short length of control regions is related to the observed lack of heteroplasmy in lepidopteran mitochondrial genomes. In addition, determination of flanking sequences for one Jalmenus species indicated (i) only weak support for the available model of insect 12S rRNA structure and (ii) that tRNA translocation is a frequent event in the evolution of insect mitochondrial genomes.      

Solid-phase synthesis of triple-helical collagen-model peptides

Summary The triple-helical conformation of collagen has been proposed to be important for mediation of cellular activities, such as adhesion and activation, extracellular matrix assembly, and enzyme function. We have developed synthetic protocols that allow for the study of biological activities of specific collagen sequences in triple-helical conformation. These methods primarily involve solid-phase assembly and covalent linkage of three peptide chains. The resultant triple-helical peptides have sufficient thermal stabilities to permit structural and biological characterization under physiological conditions. The present article critically reviews the various approaches for constructing synthetic triple-helices.This paper is based on a presentation given at the Symposium on Peptide Structure and Design as part of the 31st Annual ACS Western Regional Meeting held in San Diego, CA, USA, October 18–21, 1995.     

Comparison of denaturation of tryptophan synthase alpha-subunits from Escherichia coli, Salmonella typhimurium, and an interspecies hybrid

Guanidine hydrochloride-induced denaturation and thermal denaturation of three kinds of tryptophan synthase alpha subunit have been compared by circular dichroism measurements. The three alpha subunits are from Escherichia coli, Salmonella typhimurium, and an interspecies hybrid in which the C-terminal domain comes from E. coli (alpha-2 domain) and the N-terminal domain comes from S. typhimurium (alpha-1 domain). Analysis of denaturation by guanidine hydrochloride at 25 degrees C showed that the alpha-2 domain of S. typhimurium was more stable than the alpha-2 domain of E. coli, but the alpha-1 domain of S. typhimurium was less stable than the alpha-1 domain of the E. coli protein; overall, the hybrid protein was slightly less stable than the two original proteins. It is concluded that the stability to guanidine hydrochloride denaturation of each of the domains of the interspecies hybrid is similar to the stability of the domain of the species from which it originated. The E. coli protein was more stable to thermal denaturation than the other proteins near the denaturation temperature, but the order of their thermal stability was reversed at 25 degrees C and coincided with that obtained from guanidine hydrochloride-induced denaturation.     

Determination of ketone body turnover in vivo with stable isotopes, utilizing gas chromatography/mass spectrometry

JH imple and reliable method for the determination of ketone body turnover in vivo using a primed, continuous infusion of [3,4-13C2]acetoacetate is described. Mole percent enrichment of beta-[13C2]hydroxybutyrate and [13C2]acetoacetate is determined by gas chromatography/mass spectrometry using electron-impact ionization and selected ion monitoring. Ketone body flux data are provided from preliminary dog experiments. The method is readily applicable to the study of ketone body metabolism in both laboratory animals and humans.