Membrane topology and mutational analysis of the TolQ protein of Escherichia coli required for the uptake of macromolecules and cell envelope integrity.

TolQ is a 230-amino-acid protein required to maintain the integrity of the bacterial envelope and to facilitate the import of both filamentous bacteriophage and group A colicins. Cellular fractionation experiments showed TolQ to be localized to the cytoplasmic membrane. Bacteria expressing a series of TolQ-beta-galactosidase and TolQ-alkaline phosphatase fusion proteins were analyzed for the appropriate enzyme activity, membrane location, and sensitivity to exogenously added protease. The results are consistent with TolQ being an integral cytoplasmic membrane protein with three membrane-spanning regions. The amino-terminal 19 residues as well as a small loop in the 155 to 170 residue region appear exposed in the periplasm, while the carboxy terminus and a large loop after the first transmembrane region are cytoplasmic. Amino-terminal sequence analysis of TolQ purified from the membrane revealed the presence of the initiating formyl methionine group, suggesting a rapid translocation of the amino-terminal region across the cytoplasmic membrane. Analysis of various tolQ mutant strains suggests that the third transmembrane region as well as parts of the large cytoplasmic loop are necessary for activity.     

Crop/Weed gene flow:Cucurbita argyrosperma Huber andC. fraterna L. H. Bailey (Cucurbitaceae)

A mixed population of Cucurbita at Vado El Mow in northern Tamaulipas, Mexico showed an anomalous pattern of fruit bitterness. Some domesticated plants (C. argyrosperma andC. moschata) expressed cucurbitacin bitterness whereas some sympatric free-living plants produced non-bitter fruits. This reversal of typical cucurbitacin expression suggested gene flow between crop and weed at the site. Isozyme analysis provided little insight as to taxa involved in gene exchange, although progeny from a single free-living plant carried IDH allozymes that are associated with Mexican landraces ofC. pepo. Synthetic hybridization revealed that fertile F, hybrids are produced from crosses involvingC. fraterna as the pistillate parent andC. argyrosperma as the staminate parent. Interspecific crop/weed hybrids can produce viable progeny upon self-pollination or backcrossingto either parent, andF₂ families display normal allozyme segregation. Hybrid fertility, as indicated by pollen stainability, increases in progeny produced by backcrossingfrom theC. argyrosperma parent. Interspecific hybridfertility represents a potential for crop/weed gene flow that would be realized under natural conditions if pollen flow occurs betweenC. fraterna andC. argyrosperma in the fields of Tamaulipas. Oligolectic “squash bees” (Teponapis), efficientCucurbita pollen vectors, are present at the site. Thus, it is likely that natural interspecific crop/weed hybridization has occurred at Vado El Moro and this might at least partially explain the anomalous distribution of fruit bitterness among extant populations at the site.     

Mutations in somePolycomb group genes ofDrosophila interfere with regulation of segmentation genes

Mutations in severalPolycomb (Pc) group genes cause maternal-effect or zygotic segmentation defects, suggesting thatPc group genes may regulate the segmentation genes ofDrosophila. We show that individuals doubly heterozygous for mutations inpolyhomeotic and six otherPc group genes show gap, pair rule, and segment polarity segmentation defects. We examined double heterozygous combinations ofPc group and segmentation mutations for enhancement of adult and embryonic segmentation defects.Posterior sex combs andpolyhomeotic interact withKrüppel ² and enhance embryonic phenotypes ofhunchback andknirps, andpolyhomeotic enhanceseven-skipped. Surprisingly, flies carrying duplications ofextra sex combs (esc), that were heterozygous for mutations ofeven-skipped (eve), were extremely subvital. Embryos and surviving adults of this genotype showed strong segmentation defects in even-numbered segments. Antibody studies confirm that expression ofeve is suppressed by duplications ofesc. However,esc duplications have no effect on other gap or pair rule genes tested. To our knowledge, this is only the second triplo-abnormal phenotype associated withPc group genes. Duplications of nine otherPc group genes have no detectable effect oneve. Expression ofengrailed (en) was abnormal in the central nervous systems of mostPc group mutants. These results support a role forPc genes in regulation of some segmentation genes, and suggest thatesc may act differently from otherPc group genes.     

Alzheimer's disease-like phosphorylation of the microtubule-associated protein tau by glycogen synthase kinase-3 in transfected mammalian cells

Background: Paired helical filaments (PHFs) are a characteristic pathological feature of Alzheimer's disease; their principal component is the microtubule-associated protein tau. The tau in PHFs (PHF-tau) is hyperphosphorylated, but the cellular mechanisms responsible for this hyperphosphorylation have yet to be elucidated. A number of kinases, including mitogen-activated protein (MAP) kinase, glycogen synthase kinase (GSK)-3α, GSK-3β and cyclin-dependent kinase-5, phosphorylate recombinant tau in vitro so that it resembles PHF-tau as judged by its reactivity with a panel of antibodies capable of discriminating between normal tau and PHF-tau, and by a reduced electrophoretic mobility that is characteristic of PHF-tau. To determine whether MAP kinase, GSK-3α and GSK-3β can also induce Alzheimer's disease-like phosphorylation of tau in mammalian cells, we studied the phosphorylation status of tau in primary neuronal cultures and transfected COS cells following changes in the activities of MAP kinase and GSK-3.Results Activating MAP kinase in cultures of primary neurons or transfected COS cells expressing tau isoforms did not increase the level of phosphorylation for any PHF-tau epitope investigated. But elevating GSK-3 activity in the COS cells by co-transfection with GSK-3α or GSK-3β decreased the electrophoretic mobility of tau so that it resembled that of PHF-tau, and induced reactivity with eight PHF-tau-selective monoclonal antibodies.Conclusion Our data indicate that GSK-3α and/or GSK-3β, but not MAP kinase, are good candidates for generating PHF-type phosphorylation of tau in Alzheimer's disease. The involvement of other kinases in the generation of PHFs cannot, however, be eliminated. Our results suggest that aberrant regulation of GSK-3 may be a pathogenic mechanism in Alzheimer's disease.     

Chiral pharmacokinetics of rac-flurbiprofen and pharmacodynamics of anabolic bone response in the normal rat

The route of administration of the NSAID, flurbiprofen (sq vs. po) resulted in positive and negative results respectively with regard to enhanced cancellous and cortical bone accumulation in the immature rat. This pharmacokinetic study was an effort to understand the pharmacodynamic difference between the two routes of administration observed when the same dose range of drug, given as single daily doses, had been employed in both studies. Conventional chiral pharmacokinetics were evaluated in young rats. A significant difference was observed in the T_max of the active (S)-enantiomer by both administration routes (sq 4 h and po 1 h). The bioavailability, as evaluated by AUCs favored the sq route as expected. The plasma concentrations over 18 h, at steady state, for one po dose group (0.5 mg/kg/day) fell well within the therapeutic window described by the 0.1 and 0.5 mg/kg sq doses which had demonstrated anabolic bone activity. Oral dosing had exhibited no significant bone activity. We conclude that the pharmacodynamic difference between routes of administration cannot be simply explained on a pahrmacokinetic basis. Consequently, experiments detailing the pharmacodynamics and pharmacokinetics of single and multiple dose administration of aryl-propionic acids in normal and osteopenic states need further pharmacologic study. © 1994 Wiley-Liss, Inc.     

Orientation by helical motion—II. Changing the direction of the axis of motion

We analyse the helical motion of organisms, concentrating on the means by which organisms change the direction in space of the axis of the helical trajectory, which is the net direction of motion. We demonstrate that the direction of the axis is determined largely by the direction of the organism's rotational velocity. Changes in direction of the rotational velocity, with respect to the organism's body, change the direction in space of the axis of the helical trajectory. Conversely, changes in direction of the translational velocity, with respect to the body of the organism, have little effect on the direction in space of the axis of the trajectory. Because the axis of helical motion is the net direction of motion, it is likely that organisms that move in helices change direction by pointing their rotational velocity, not their translational velocity, in a new direction.     

A comparative study of glucose conversion to ethanol by Zymomonas mobilis and a recombinant Escherichia coli

Summary Zymomonas mobilis and recombinant Escherichia coli B (pLOI297) were compared in side-by-side batch fermentations using a synthetic cellulose hydrolysate (glucose/salts) medium with pH control at 6.0 and an inoculation cell density of 35–50 mg dry wt. cells/L. At a nominal glucose concentration of 6%, both cultures achieved near maximal theoretical ethanol yields; however, the Z. mobilis fermentation was complete at 13h compared to 33h for the E.coli fermentation. With approx.12% glucose, the Z. mobilis fermentation was complete in 20h with a process yield of 0.49 g ethanol/g added glucose compared to the E. coli fermentation which remained 20% incomplete after 6 days resulting in a process yield of only 0.32 g/g. Nutrient supplementation (10g tryptone/L) resulted in complete fermentation of 12% glucose (pH 6.3) by the recombinant E. coli in 4 days, with a yield of 0.48 g/g.     

Aluminum alters calcium transport in plasma membrane and endoplasmic reticulum from rat brain

Calcium is actively transported into intracellular organelles and out of the cytoplasm by Ca²⁺/Mg²⁺-ATPases located in the endoplasmic reticulum and plasma membranes. We studied the effects of aluminum on calcium transport in the adult rat brain. We examined ⁴⁵Ca-uptake in microsomes and Ca²⁺-ATPase activity in microsomes and synaptosomes isolated from the frontal cortex and cerebellum of adult male Long-Evans rats. ATP-dependent⁴⁵Ca-uptake was similar in microsomes from both brain regions. The addition of 50-800 μM AICI₃ resulted in a concentration-dependent inhibition of ⁴⁵Ca-uptake. Mg²⁺-dependent Ca²⁺-ATPase activity was significantly lower in synaptosomes compared to microsomes in both frontal cortex and cerebellum. In contrast to the uptake studies, AICI³ stimulated Mg²⁺-dependent Ca²⁺-ATPase activity in both microsomes and synaptosomes from both brain regions. To determine the relationship between aluminum and Mg²⁺, we measured ATPase activity in the presence of increasing concentrations of Mg²⁺ or AICI³. Maximal ATPase activity was obtained between 3 and 6 mM Mg²⁺. When we substituted AICI³ for Mg²⁺, ATPase activity was also stimulated in a concentration-dependent manner, but to a greater extent than with Mg²⁺. One interpretation of these data is that aluminum acts at multiple sites to displace both Mg²⁺ and Ca²⁺, increasing the activity of the Ca²⁺-ATPase, but disrupting transport of calcium.