Regulation of fatty acid degradation in Escherichia coli: dominance studies with strains merodiploid in gene fadR.

Strains stably merodiploid in the 25-min region of the chromosome of Escherichia coli were constructed and used in dominance tests between various wild-type and mutant alleles of the fadR gene. Whereas the monoploid fadR+ and fadR strains were inducible and constitutive, respectively, for the enzymes involved in fatty acid degradation (fad), merodiploids with at least one fadR+ allele were inducible. This observation was true whether the fadR+ allele resided on the main chromosome or on the episome. These results show that fadR+ is trans dominant to fadR, and they are consistent with the proposal that the fadR gene product is a repressor protein. Complementation tests were also performed by constructing 24 merodiploids harboring fadR alleles on both the main chromosome and the episome. All of these fadR/fadR diploids were able to utilize the noninducing substrate decanoate as sole carbon source, suggesting that only one polypeptide is encoded by the fadR gene.     

Transfection of protoplasts from Streptomyces lividans 66 with actinophage SH10 DNA

Summary The slightly modified procedure for the transformation of protoplasts of S. coelicolor A3 (2) with SCP2 plasmid DNA and polyethylenglycol (PEG) (Bibb et al., 1978) was extented to infection of protoplasts of S. lividans 66 with actinophage SH10 DNA (Klaus et al., 1979).Maximal yield of transfected protoplasts was obtained at 20% PEG, 3 mM sodium-citrate and 150 mM NaCl final concentrations. The efficiency of transfection was determined to be about 2×10^-8 to 2×10^-7. The average value of competent protoplasts was about 1–2×10^-4 of regenerating protoplasts.In comparison with outgrowing spores infected with phage particles the average burst size of transfected protoplasts was reduced from 100 to 10 pfu/infected cell, the latent period prolonged from 45 min to 120 min and the rise period was not affected.     

Sex differences in tooth wear ofMacaca fuscata,the Arashiyama-A troop in Texas

The incidence of tooth wear was studied in a wild troop ofM. fuscata, that had previously been transplanted from Arashiyama, Japan, to Texas. This study was undertaken to determine differences of attrition between males and females, and between maxillary and mandibular dentitions. Contrary to other findings, the rate of wear was not found to be an expression of sex difference, but seemed rather related to function. The following observations may suffice as examples: The mandibular third premolars function as a honing surface for the maxillary canines, and experience greater wear over time in males due to their proximity to smaller canines which leave their neighbors more vulnerable to wear. The degree of attrition intensity is neither the same for males and females, nor the maxillary and mandibular dentitions. Certain maxillary and mandibular teeth “pair up”; although all “pairs” are identical in males and females, they rank differently in the degree of wear experienced. Overall, females express greater attrition in the maxillary, and males in the mandibular dentitions.     

Über das Aktionsspektrum der phototaktischen Reaktionen von Euglena

Zusammenfassung Bei Euglena gracilis zeigt das Aktionsspektrum der positiv-phototaktischen Reaktion, gemessen an Reizstärken, die eben über der Schwelle liegen, ein Hauptmaximum bei etwa 490–500 m, ein Nebenmaximum wurde bei etwa 415–430 m gefunden.Das Aktionsspektrum für die negativ-phototaktische Reaktion, gemessen an den Reizstärken, die eben über der Schwelle für diesen Reaktionstyp liegen, hat sein Hauptmaximum bei etwa 415 m; es stimmt ungefähr mit dem eben erwähnten Nebenmaximum der positivphototaktischen Reaktion überein.Die Empfindlichkeitsgrenze zum Langwelligen liegt für beide Reaktionstypen bei etwa 550 m.Die Befunde legen folgende Deutung nahe: Die negativ-phototaktische Reaktion erfolgt ohne Mitwirkung des Stigmas rein phobisch. Die positiv-phototaktische Reaktion ist an die gleiche strahlungsabsorbierende Substanz gebunden wie die negative, für sie ist dabei aber eine periodische Verdunkelung dieser Photoreceptorsubstanz durch die photochemisch nicht wirksamen Carotinoide des Stigmas erforderlich. Wo diese Verdunkelung wegen geringer Absorption der Stigma-Carotinoide (etwa bei 400 m) nicht möglich ist, kann nur die negative (phobotaktische) Reaktion entstehen. Wo umgekehrt zwar das Stigma absorbiert (etwa zwischen 550–600 m), nicht aber die lichtempfindliche Substanz des Photoreceptors, sind beide Reaktionen ausgeschlossen.     

Cholesterol Is Required for Surface Transport of Influenza Virus Hemagglutinin

Transport from the TGN to the basolateral surface involves a rab/N-ethylmaleimide–sensitive fusion protein (NSF)/soluble NSF attachment protein (SNAP)/SNAP receptor (SNARE) mechanism. Apical transport instead is thought to be mediated by detergent-insoluble sphingolipid–cholesterol rafts. By reducing the cholesterol level of living cells by 60–70% with lovastatin and methyl-β-cyclodextrin, we show that the TGN-to-surface transport of the apical marker protein influenza virus hemagglutinin was slowed down, whereas the transport of the basolateral marker vesicular stomatitis virus glycoprotein as well as the ER-to-Golgi transport of both membrane proteins was not affected. Reduction of transport of hemagglutinin was accompanied by increased solubility in the detergent Triton X-100 and by significant missorting of hemagglutinin to the basolateral membrane. In addition, depletion of cellular cholesterol by lovastatin and methyl-β-cyclodextrin led to missorting of the apical secretory glycoprotein gp-80, suggesting that gp-80 uses a raft-dependent mechanism for apical sorting. Our data provide for the first time direct evidence for the functional significance of cholesterol in the sorting of apical membrane proteins as well as of apically secreted glycoproteins.     

The small GTPase rab5 functions as a regulatory factor in the early endocytic pathway.

We have investigated the in vivo functional role of rab5, a small GTPase associated with the plasma membrane and early endosomes. Wild-type rab5 or rab5-ile133, a mutant protein defective in GTP binding, was overexpressed in baby hamster kidney cells. In cells expressing the rab5ile 133 protein, the rate of endocytosis was decreased by 50% compared with normal, while the rate of recycling was not significantly affected. The morphology of early endosomes was also drastically changed by the mutant protein, which induced accumulation of small tubules and vesicles at the periphery of the cell. Surprisingly, overexpression of wild-type rab5 accelerated the uptake of endocytic markers and led to the appearance of atypically large early endosomes. We conclude that rab5 is a rate-limiting component of the machinery regulating the kinetics of membrane traffic in the early endocytic pathway.     

Role of cysteines 640, 656, and 661 in steroid binding to rat glucocorticoid receptors.

The involvement of a vicinally spaced dithiol group in steroid binding to the glucocorticoid receptor has been deduced from experiments with the thiol-specific reagent methyl methanethiolsulfonate and the vicinal dithiol-specific reagent sodium arsenite. The vicinally spaced dithiol appears to reside in the 16-kDa trypsin fragment of the receptor, which is thought to contain 3 cysteines (Cys-640, -656, and -661 of the rat receptor) and binds hormone with an approximately 23-fold lower affinity than does the intact 98-kDa receptor. We now report that the steroid binding specificity of preparations of this 16-kDa fragment and the intact receptor are virtually identical. This finding supports our designation of the 16-kDa fragment as a steroid-binding core domain and validates our continued use of this tryptic fragment in studies of steroid binding. To identify the cysteines which comprise the vicinally spaced dithiol group, and to examine further the role of cysteines in steroid binding, a total of five point mutant receptors were prepared: cysteine-to-serine for each suspected cysteine, cysteine-to-glycine for Cys-656, and the C656,661S double mutant. Unexpectedly, each receptor with a single point mutation still bound steroid. Even the double mutant (C656,661S) bound steroid with wild type affinity. These results suggest that none of these cysteines are directly required either for steroid binding to the glucocorticoid receptor or for heat shock protein 90 association with the receptor. However, the presence of Cys-656 was obligatory for covalent labeling of the receptor by [3H]dexamethasone 21-mesylate. Studies with preparations of the 98 and 16 kDa forms of these mutant receptors revealed both that Cys-656 and -661 comprise the vicinally spaced dithiols reacting with arsenite and that any two of the three thiols could form an intramolecular disulfide after treatment with low concentrations of methyl methanethiolsulfonate. These data, in conjunction with those from experiments on the effects of steric bulk on various receptor functions, support a model for the ligand binding cavity of the receptor that involves all three thiols in a flexible cleft but where thiol-steroid interactions are not essential for binding.     

Differential roles of Fc gamma RII and Fc gamma RIII in immune complex stimulation of human neutrophils.

Insoluble immune complexes (IIC) stimulate human neutrophils through Fc gamma receptors. Freshly isolated human neutrophils express two FcR subclasses, FcRII and FcRIII. We explored the role of FcRII and FcRIII in this activation process by selectively binding each FcR subclass with the Fab fragments of the respective anti-FcR monoclonal antibodies (MFab) before exposure to IIC. Correlation among liganded FcR subclass, IIC binding, and ensuant IIC stimulation was achieved with multiparameter flow cytometry. We utilized rhodamine-labeled anti-FcRIII and fluorescein-labeled IIC to study binding and observed the change in [Ca2+]i in the same cell with a Ca2+ indicator, Indo-1. Treatment with either anti-FcRII (IV.3) or anti-FcRIII (3G8) MFab decreased both the fraction of cells exhibiting a Ca2+ transient and the magnitude of that transient, although only anti-FcRIII but not anti-FcRII significantly inhibited the subsequent IIC binding. In addition, cells treated with anti-FcRII and then stimulated with IIC exhibited a decrease in both the intracellular Ca2+ transient and the later Ca2+ influx, whereas anti-FcRIII totally abolished the mobilization of intracellular Ca2+ without affecting the Ca2+ influx. Treatment with either anti-FcR MFab decreased the IIC-stimulated transmembrane potential change, oxidative burst, and elastase release. These studies indicate that freshly isolated neutrophils' Fc receptor subclasses have unique roles in the IIC-initiated stimulation and that full activation can only be achieved when both FcR subclasses are available.     

Nucleotide sequence and expression in Escherichia coli of the Lactococcus lactis citrate permease gene.

The plasmid-encoded citrate determinant of the Lactococcus lactis subsp. lactis var. diacetylactis NCDO176 was cloned and functionally expressed in a Cit- Escherichia coli K-12 strain. From deletion derivative analysis, a 3.4-kilobase region was identified which encodes the ability to transport citrate. Analysis of proteins encoded by the cloned fragment in a T7 expression system revealed a 32,000-dalton protein band, which correlated with the ability of cells to transport citrate. Energy-dependent [1,5-14C]citrate transport was found with membrane vesicles prepared from E. coli cells harboring the citrate permease-expressing plasmid. The gene encoding citrate transport activity, citP, was located on the cloned fragment by introducing a site-specific mutation that abolished citrate transport and resulted in a truncated form of the 32,000-dalton expression product. The nucleotide sequence for a 2.2-kilobase fragment that includes the citP gene contained an open reading frame of 1,325 base pairs coding for a very hydrophobic protein of 442 amino acids, which shows no sequence homology with known citrate carriers.     

Arsenite and cadmium(II) as probes of glucocorticoid receptor structure and function

Low concentrations of arsenite, but not arsenate, and Cd2+ blocked steroid binding to the glucocorticoid receptors of HTC cells. Inhibition by arsenite was faster and occurred at lower concentrations than for Cd2+. Half-maximal inhibition of [3H]dexamethasone binding was seen after a 30-min preincubation with approximately 7 microM arsenite. The effect of arsenite and of Cd2+ appears to be mediated by a reaction with vicinal dithiols of the receptor as shown by (a) the reversal of arsenite inhibition by much lower concentrations of dithiothreitol (approximately 0.1 mM) than of beta-mercaptoethanol (approximately 10 mM); (b) the ability of both arsenite and Cd2+ to block [3H]dexamethasone 21-mesylate labeling of receptors but not of other thiol-containing proteins; and (c) the known selectivity of arsenite and of Cd2+ for reactions with vicinal dithiols. Arsenite forms a tight complex with these vicinal dithiols since the removal of loosely associated arsenite by gel exclusion chromatography did not reverse the inhibition of steroid binding. The effect of other ions on steroid binding was also examined. Half-maximal inhibition of binding occurred with approximately 5 microM selenite, whereas up to 300 microM Zn2+ was without effect. Much higher concentrations of arsenite were required for effects on unactivated and activated complexes. Arsenite slowly induced a loss of unactivated complexes but rapidly inhibited a portion of the DNA binding of activated complexes. Any effect on activation occurred at arsenite concentrations equal to or higher than those that inhibited DNA binding. In contrast, Cd2+ concentrations similar to those that block steroid binding caused a biphasic loss of unactivated complexes and a marginal loss of activated complexes. This is the first report of effects of arsenite on glucocorticoid receptors. These results confirm directly our earlier hypothesis that steroid binding to rat glucocorticoid receptors involves a vicinal dithiol (Miller, N. R., and Simons, S. S., Jr. (1988) J. Biol. Chem. 263, 15217-15225) and show that arsenite is a potent new reagent for probing receptor structure and function.