Transfer of the first arabinofuranose residue to galactan is essential for Mycobacterium smegmatis viability

The mycobacterial arabinan is an elaborate component of the cell wall with multiple glycosyl linkages and no repeating units. In Mycobacterium spp., the Emb proteins (EmbA, EmbB, and EmbC) have been identified as putative mycobacterial arabinosyltransferases implicated in the biogenesis of the cell wall arabinan. Furthermore, it is now evident that the EmbA and EmbB proteins are involved in the assembly of the nonreducing terminal motif of arabinogalactan and EmbC is involved in transferring arabinose, perhaps in the early stage of arabinan synthesis in lipoarabinomannan. It has also been shown that the Emb proteins are a target of the antimycobacterial drug ethambutol (EMB). In the search for additional mycobacterial arabinosyltransferases in addition to the Emb proteins, we disrupted MSMEG_6386 (an orthologue of Rv3792 and a gene upstream of embC) in Mycobacterium smegmatis. Allelic exchange at the chromosomal MSMEG_6386 locus of M. smegmatis could only be achieved in the presence of a rescue plasmid carrying a functional copy of MSMEG_6386 or Rv3792, strongly suggesting that MSMEG_6386 is essential. An in vitro arabinosyltransferase assay using a membrane preparation from M. smegmatis expressing Rv3792 and synthetic beta-d-Galf-(1-->5)-beta-D-Galf-(1-->6)-beta-D-Galf-octyl and beta-D-Galf-(1-->6)-beta-D-Galf-(1-->5)-beta-D-Galf-octyl showed that Rv3792 gene product can transfer an arabinose residue to the C-5 position of the internal 6-linked galactose. The reactions were insensitive to EMB, and when alpha-d-Manp-(1-->6)-alpha-D-Manp-(1-->6)-alpha-D-Manp-octylthiomethyl was used as an acceptor, no product was formed. These observations indicate that transfer of the first arabinofuranose residue to galactan is essential for M. smegmatis viability.     

Suppression of Proline Requirement of proA and proAB Deletion Mutants in Salmonella typhimurium by Mutation to Arginine Requirement

Eleven variants able to grow without proline (provided arginine was absent) were obtained by spontaneous mutation from Salmonella typhimurium LT7 proA and proAB deletion mutants. Suppression resulted from mutation at argG, which specifies N(alpha)-acetylornithine delta-transaminase. In the absence of exogenous arginine, deficiency of this enzyme would cause derepression of the arginine pathway and accumulation of N-acetylglutamic gamma-semialdehyde. N-acetylglutamic gamma-semialdehyde, if deacetylated, would produce glutamic gamma-semialdehyde, the proline precursor whose synthesis from glutamate is blocked in proA and proAB mutants. All of the mutants grew only slowly (some very slowly) if not supplied with arginine. Sonic-treated preparations of eight mutants had no measurable acetylornithine delta-transaminase activity, but those of the three mutants least dependent on arginine had 0.11, 0.28, and 1.48 of wild-type activity; presumably, their enzymes have low specific activity, at least in vivo. Phage P22 cotransduced argG and strA. Genetic analysis showed that the minor degree of arginine dependence of the mutant with greater than wild-type in vitro enzyme activity was a characteristic of its argG allele, not the result of modification of the argG phenotype by mutation elsewhere.     

Possible mechanisms responsible for the increased ascorbic acid content of Plasmodium vinckei-infected mouse erythrocytes

The possible mechanisms underlying the acquisition of an increased ascorbic acid content by mouse erythrocytes containing the malarial parasite Plasmodium vinckei were investigated. Ascorbic acid was taken up readily by parasitized red blood cells but not by controls, whilst its partly oxidized form, dehydroascorbic acid, entered both. The uptake of both ascorbic acid and dehydroascorbic acid into erythrocytes was increased as a result of malarial infection. Lysates prepared from parasitized red blood cells reduced exogenous dehydroascorbic acid to ascorbic acid at a higher rate than control red blood cell lysates; this difference was abolished following dialysis of the lysates, a process which removes endogenous reduced glutathione (GSH). The rates of chemical and enzymatic reduction of dehydroascorbic acid to ascorbic acid by GSH were of similar magnitude, thus calling into question the existence of a specific dehydroascorbate reductase in erythrocytes and parasites. These observations suggest that the increased uptake of dehydroascorbic acid into parasitized red blood cells may be a result of enhanced dehydroascorbate-reducing capacity, whilst the presence of the parasite induces a selective increase in the permeability of the erythrocyte plasma membrane to ascorbic acid. The endogenous ascorbic acid content of livers obtained from infected mice was 55% below the normal concentration and its relative rate of destruction during incubation in vitro was enhanced in comparison with that of control livers. Furthermore, the capacity of liver homogenates to synthesize ascorbic acid from glucuronic acid was greatly reduced in infected mice. Therefore it is unlikely that the increase in ascorbic acid content of parasitized red blood cells is a consequence of increased biosynthesis and release of ascorbic acid by the host liver. We have not been able to exclude the possibility that the malarial parasite itself may be capable of de novo synthesis of ascorbic acid.     

Developmental studies in Drosophila

A study of the puffing patterns of the salivary gland chromosomes of D. pseudoobscura was carried out through several larval, prepupal, and pupal stages of development. A total of 176 puffs were found, 111 of which changed during the stages studied. As described in previous investigations with other Drosophila species there are two major peaks of puffing activity. These two peaks occur during puparium formation and pupation. Additionally, a minor activity-peak occurs during mid-prepupal life. Attempts have been made to establish correlations between the puffing data and those obtained from electrophoretic and ultrastructural studies.Supported in part by grants GM-16736-03 and FR-05426-09 from the U.S. Department of Health, Education, and Welfare. Ann Jacob Stocker was a holder of a University of Texas predoctoral fellowship.     

Biochemical characterization and electron-transfer reactions of sym1, a Rhodobacter capsulatus reaction center symmetry mutant which affects the initial electron donor.

A 51 bp section of the Rhodobacter capsulatus photosynthetic reaction center M subunit gene (nucleotides M562-M612 of the pufM structural sequence) encoding amino acids M187-M203 was replaced by the homologous region of the L subunit gene. This resulted in the symmetrization of much of the amino acid environment of the reaction center initial electron donor, P. This is the first in a series of large-scale symmetry mutations and is referred to as sym1. The sym1 mutant was able to grow photosynthetically, indicating that reaction center function was largely intact. Isolated reaction centers showed an approximately 10-nm blue shift in the QY band of P. The standard free energy change between P* and P+BphA- determined from analysis of the long-lived fluorescence from quinone-reduced reaction centers decreased from about -120 meV in the wild-type to about -75 meV in the sym1 mutant. A 65-70% quantum yield of electron transfer from P* to P+QA- was observed, most of the yield loss occurring between P* and P+BphA-. The decay of the stimulated emission from P* was about 3-fold slower in this mutant than in the wild-type. Time-resolved spectral analysis of the charge-separated intermediates formed in sym1 reaction centers indicated that the major product was P+BphA-. A model-dependent analysis of the observed rates and electron-transfer yields gave the following microscopic rate constants for sym1 reaction centers (wild-type values under the same conditions are given in parentheses): [formula: see text] Analysis of the sym1 mutant, mutants near P made by other groups, and interspecies variation of amino acids in the vicinity of P suggests that the protein asymmetry in the environment of the initial electron donor is important for optimizing the rate and yield of electron transfer, but is not strictly required for overall reaction center function.     

An amino acid outside the pore region influences apamin sensitivity in small conductance Ca2+-activated K+ channels

Small conductance calcium-activated potassium channels (SK, K(Ca)) are a family of voltage-independent K+ channels with a distinct physiology and pharmacology. The bee venom toxin apamin inhibits exclusively the three cloned SK channel subtypes (SK1, SK2, and SK3) with different affinity, highest for SK2, lowest for SK1, and intermediate for SK3 channels. The high selectivity of apamin made it a valuable tool to study the molecular makeup and function of native SK channels. Three amino acids located in the outer vestibule of the pore are of particular importance for the different apamin sensitivities of SK channels. Chimeric SK1 channels, enabling the homomeric expression of the rat SK1 (rSK1) subunit and containing the core domain (S1-S6) of rSK1, are apamin-insensitive. By contrast, channels formed by the human orthologue human SK1 (hSK1) are sensitive to apamin. This finding hinted at the involvement of regions beyond the pore as determinants of apamin sensitivity, because hSK1 and rSK1 have an identical amino acid sequence in the pore region. Here we investigated which parts of the channels outside the pore region are important for apamin sensitivity by constructing chimeras between apamin-insensitive and -sensitive SK channel subunits and by introducing point mutations. We demonstrate that a single amino acid situated in the extracellular loop between the transmembrane segments S3 and S4 has a major impact on apamin sensitivity. Our findings enabled us to convert the hSK1 channel into a channel that was as sensitive for apamin as SK2, the SK channel with the highest sensitivity.     

Functional Analysis of Missense Mutations in Kv8.2 Causing Cone Dystrophy with Supernormal Rod Electroretinogram

Mutations in KCNV2 have been proposed as the molecular basis for cone dystrophy with supernormal rod electroretinogram. KCNV2 codes for the modulatory voltage-gated potassium channel α-subunit, Kv8.2, which is incapable of forming functional channels on its own. Functional heteromeric channels are however formed with Kv2.1 in heterologous expression systems, with both α-subunit genes expressed in rod and cone photoreceptors. Of the 30 mutations identified in the KCNV2 gene, we have selected three missense mutations localized in the potassium channel pore and two missense mutations localized in the tetramerization domain for analysis. We characterized the differences between homomeric Kv2.1 and heteromeric Kv2.1/Kv8.2 channels and investigated the influence of the selected mutations on the function of heteromeric channels. We found that two pore mutations (W467G and G478R) led to the formation of nonconducting heteromeric Kv2.1/Kv8.2 channels, whereas the mutations localized in the tetramerization domain prevented heteromer generation and resulted in the formation of homomeric Kv2.1 channels only. Consequently, our study suggests the existence of two distinct molecular mechanisms involved in the disease pathology.     

Cardiovascular responses to light isometric and aerobic exercise in 21- and 59-year-old males

The effects of aging on the cardiovascular response to continuous light isometric and aerobic exercise remains to be determined. Thus, the purpose of this study was to compare the cardiovascular response of young and older males during light handgrip and cycle ergometry exercise. Blood pressure, heart rate, rate pressure product, as well as pre-ejection period (derived from impedance cardiography) were obtained for 15 young [mean (SE) age: 21 (0.7) years] and 15 older males [59 (0.8) years] during and after light handgrip exercise and cycle ergometry. The parasympathetic influence on the heart was also assessed through a time-series analysis of heart period variability (HPVts). Both during and when recovering from the handgrip exercise and cycle ergometry, the older subjects exhibited a significantly higher absolute systolic and diastolic blood pressure, and rate pressure product, and a lower HRVts than the young subjects. Relative to baseline, the change in pre-ejection period was lower for the young subjects during the handgrip and cycle ergometry, tasks. These results indicate that although the sympathetic influence on both the myocardium and the vasculature was less pronounced in the older males, the aging cardiovasculature was under greater hemodynamic stress both during rest and during exposure to light isometric and aerobic challenge.     

Drosophila P[Gal4] lines reveal that motor neurons involved in feeding persist through metamorphosis

Two P[Gal4] insertion lines in Drosophila melanogaster, MT11 and MT26, express GAL4 specifically in two to three pairs of pharyngeal motor neurons (PMN) in the suboesophageal ganglion. By using various secondary reporters, the architecture of the PMN, including their efferent axons in the pharyngeal nerve, was visualized. This allowed us to identify a pharyngeal dilator muscle as their target. To study the function of these neurons, we crossed line MT11 with a UAS-tetanus toxin gene construct (TNT-C) that inhibits all synaptic transmission. The offspring shows a reduction in food ingestion of 75% compared to the MT11 and TNT-C controls, demonstrating that PMN control food uptake. More important, lines MT11 and MT26 enabled us to follow PMN and their processes through metamorphosis, since labeling appears in the late third larval instar and persists up to adulthood. The motor axons innervate a pharyngeal muscle in the larva as well and extend through the maxillary nerve, proving that this nerve is homologous to the adult pharyngeal nerve. Efferent arborizations persist throughout metamorphosis, even though the larval muscle histolyzes by 20% of pupal life. Yet, some dedifferentiated structures remain, which may serve as a template for the formation of the adult muscle. Labeling of line MT26 with bromodeoxyuridine at embryonic or larval stages suggests that these neurons undergo their terminal mitosis in the mid to late embryo. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 237–250, 1998