Mutational analysis of the connector segment in the HAMP domain of Tsr, the Escherichia coli serine chemoreceptor

HAMP domains are approximately 50-residue motifs, found in many bacterial signaling proteins, that consist of two amphiphilic helices joined by a nonhelical connector segment. The HAMP domain of Tsr, the serine chemoreceptor of Escherichia coli, receives transmembrane input signals from the periplasmic serine binding domain and in turn modulates output signals from the Tsr kinase control domain to elicit chemotactic responses. We created random amino acid replacements at each of the 14 connector residues of Tsr-HAMP to identify those that are critical for Tsr function. In all, we surveyed 179 connector missense mutants and identified three critical residues (G235, L237, and I241) at which most replacements destroyed Tsr function and another important residue (G245) at which most replacements impaired Tsr function. The region surrounding G245 tolerated 1-residue deletions and insertions of up to 10 glycines, suggesting a role as a relatively nonspecific, flexible linker. The critical connector residues are consistent with a structural model of the Tsr-HAMP domain based on the solution structure of an isolated thermophile HAMP domain (M. Hulko, F. Berndt, M. Gruber, J. U. Linder, V. Truffault, A. Schultz, J. Martin, J. E. Schultz, A. N. Lupas, and M. Coles, Cell 126:929-940, 2006) in which G235 defines a critical turn at the C terminus of the first helix and L237 and I241 pack against the helices, perhaps to stabilize alternative HAMP signaling conformations. Most I241 lesions locked Tsr signal output in the kinase-on mode, implying that this residue is responsible mainly for stabilizing the kinase-off signaling state. In contrast, lesions at L237 resulted in a variety of aberrant output patterns, suggesting a role in toggling output between signaling states.     

Identification of a Functional Connectome for Long-Term Fear Memory in Mice

Long-term memories are thought to depend upon the coordinated activation of a broad network of cortical and subcortical brain regions. However, the distributed nature of this representation has made it challenging to define the neural elements of the memory trace, and lesion and electrophysiological approaches provide only a narrow window into what is appreciated a much more global network. Here we used a global mapping approach to identify networks of brain regions activated following recall of long-term fear memories in mice. Analysis of Fos expression across 84 brain regions allowed us to identify regions that were co-active following memory recall. These analyses revealed that the functional organization of long-term fear memories depends on memory age and is altered in mutant mice that exhibit premature forgetting. Most importantly, these analyses indicate that long-term memory recall engages a network that has a distinct thalamic-hippocampal-cortical signature. This network is concurrently integrated and segregated and therefore has small-world properties, and contains hub-like regions in the prefrontal cortex and thalamus that may play privileged roles in memory expression.     

Syntaxin 7 is localized to late endosome compartments, associates with Vamp 8, and Is required for late endosome-lysosome fusion

Protein traffic from the cell surface or the trans-Golgi network reaches the lysosome via a series of endosomal compartments. One of the last steps in the endocytic pathway is the fusion of late endosomes with lysosomes. This process has been reconstituted in vitro and has been shown to require NSF, alpha and gamma SNAP, and a Rab GTPase based on inhibition by Rab GDI. In Saccharomyces cerevisiae, fusion events to the lysosome-like vacuole are mediated by the syntaxin protein Vam3p, which is localized to the vacuolar membrane. In an effort to identify the molecular machinery that controls fusion events to the lysosome, we searched for mammalian homologues of Vam3p. One such candidate is syntaxin 7. Here we show that syntaxin 7 is concentrated in late endosomes and lysosomes. Coimmunoprecipitation experiments show that syntaxin 7 is associated with the endosomal v-SNARE Vamp 8, which partially colocalizes with syntaxin 7. Importantly, we show that syntaxin 7 is specifically required for the fusion of late endosomes with lysosomes in vitro, resulting in a hybrid organelle. Together, these data identify a SNARE complex that functions in the late endocytic system of animal cells.     

Signalling‐dependent interactions between the kinase‐coupling protein CheW and chemoreceptors in living cells

Chemical signals sensed on the periplasmic side of bacterial cells by transmembrane chemoreceptors are transmitted to the flagellar motors via the histidine kinase CheA, which controls the phosphorylation level of the effector protein CheY. Chemoreceptor arrays comprise remarkably stable supramolecular structures in which thousands of chemoreceptors are networked through interactions between their cytoplasmic tips, CheA, and the small coupling protein CheW. To explore the conformational changes that occur within this protein assembly during signalling, we used in vivo cross‐linking methods to detect close interactions between the coupling protein CheW and the serine receptor Tsr in intact Escherichia coli cells. We identified two signal‐sensitive contacts between CheW and the cytoplasmic tip of Tsr. Our results suggest that ligand binding triggers changes in the receptor that alter its signalling contacts with CheW (and/or CheA).     

System Development for Linked-Fermentation Production of Solvents from Algal Biomass

Five species of the genus Dunaliella (D. tertiolecta, D. primolecta, D. parva, D. bardawil, and D. salina) were examined for glycerol accumulation, growth rate, cell density, and protein and chlorophyll content. The suitability of each algal species for use as a fermentation substrate was judged according to glycerol accumulation and quantities of neutral solvents produced after sequential bacterial fermentations. When grown in 2 M NaCl, with 24 mM NaHCO₃ or 3% CO₂ at 28°C and with 10,000 to 15,000 lx of incident light on two sides of a glass aquarium, four of the five species tested produced ca. 10 to 20 mg of glycerol per liter of culture. Clostridium pasteurianum was found to convert an algal biomass mixture supplemented with 4% glycerol to ca. 16 g of mixed solvents (n-butanol, 1,3-propanediol, and ethanol) per liter. Acetone was not detected. Additionally, it has been demonstrated that Dunaliella concentrates of up to 300-fold can be directly fermented to an identical pattern of mixed solvents. Overall solvent yields were reduced by >50% when fermentations were performed in the presence of 2% NaCl. These results are discussed in terms of practical application in tropical coastal zones.     

Acid denaturation of recombinant porcine growth hormone: formation and self-association of folding intermediates

We have investigated the conformational changes incurred during the acid-induced unfolding and self-association of recombinant porcine growth hormone (pGH). Acidification (pH 8 to pH 2) of pGH resulted in intrinsic fluorescence, UV absorbance, and near-UV CD transitions centered at pH 4.10. At pH 2.0, a red shift in the fluorescence emission maximum of approximately 3 nm and a 15% loss of the far-UV CD signal at 222 nm imply that the protein did not become extensively unfolded. Acidification in the presence of 4 M urea resulted in similar pH-dependent transitions. However, these occurred at a higher pH (approximately 5.2). At pH 2.0 + 4 M urea, an 8 nm red shift in the fluorescence emission maximum suggests that unfolding was greater than in the absence of urea. The presence of a prominent peak centered at 298 nm in the near-UV CD spectrum, which is absent without urea, signifies further differences in the intermediates generated at pH 2. Sedimentation equilibrium experiments in the analytical ultracentrifuge showed that native pGH and the partially unfolded intermediates reversibly self-associate. Self-association was strongly promoted at pH 2 while urea reduced self-association at both pH 8 and pH 2. These results demonstrate that acidification of pGH in the absence or presence of 4 M urea induced the formation of molten globule-like states with measurable differences in conformation. Similarities and differences in these structural conformations with respect to other growth hormones are discussed.     

Functional bitter taste receptors are expressed in brain cells

Hepatic stellate cells (HSC) store retinoids and upon activation differentiate into myofibroblast-like cells, a process whereby they lose their retinoid-containing lipid droplets. We reported earlier, activation of tissue factor (TF) in our MCT/LPS hepatotoxicity model. We now report the involvement of TF in the release of retinoid receptors RAR-α and RXR-α as accumulated lipid droplet during monocrotaline/lipopolysaccharide (MCT/LPS)-liver injury. Constitutive expression of RAR-α was observed in HSCs and endothelial cells of bile duct and portal vein, while expression of RXR-α was observed in certain pericentral hepatocytes and HSCs. Administration of sub-toxic doses of MCT or LPS strongly increased TF and RXR-α but not RAR-α expressions in HSCs and hepatocytes. However MCT/LPS co-treatment showed insoluble droplets containing RAR-α and RXR-α in the vicinity of the necrotic areas. Blocking TF with TF antisense oligonucleotides (TF-AS ODN) led to normal hepatocyte expression of RXR-α and upregulated the expression of RAR-α in HSCs. This study shows clear evidence of in vivo release of RAR-α and RXR-α as insoluble lipid droplets in liver injury. It is possible that these insoluble droplets of RAR-α and RXR-α could be used as markers for liver injury in general and activation of HSCs in particular. RXR-α appears to be a more sensitive than RAR-α as it was affected by even the subtoxic doses of MCT or LPS. The fact that TF-AS treatment not only down-regulated TF but also obliterated the release of RAR-α and RXR-α as insoluble lipid droplets in hepatocytes points towards TF being an important regulatory molecule for RAR-α and RXR-α.     

Quadruplex DNA crystal structures and drug design

Crystallographic studies of G-quadruplex nucleic acids have resulted in a small group of structures to date. Their morphological and detailed conformational features are described here, emphasizing the stability of the G-tetrad core and the flexibility of loops, especially upon ligand binding. Implications for drug design are discussed, in the context of the druggability of both telomeric and non-telomeric quadruplex DNAs.     

Polychlorinated biphenyls as inducers of hepatic microsomal enzymes: Structure-activity rules

A number of highly purified polychlorinated biphenyl (PCB) isomers and congeners were synthesized and administered to male Wistar rats at dosage levels of 30 and 150 μmol · kg⁻¹. The effects of this in vivo treatment on the drug-metabolizing enzymes were determined by measuring the microsomal benzo[a]pyrene (B[a]P) hydroxylase, dimethylaminoantipyrine (DMAP) N-demethylase and NADPH-cytochrome c reductase enzyme activities, the cytochrome b₅ content and the relative peak intensities and spectral shifts of the reduced microsomal cytochrome P-450: CO and ethylisocyanide (EIC) binding difference spectra. The results were compared to the effects of administering phenobarbitone (PB), 3-methylcholanthrene (MC) and PB plus MC (coadministered) to the test animals. The synthetic PCB congeners used in this study included 3,4,4′,5-tetrachlorobiphenyl (TCBP-1), 2,3′,4,4′-tetrachlorobiphenyl (TCBP-2), 2,3′,4,4′,5′-pentachlorobiphenyl (PCBP-1), 2,3,4,4′,5-pentachlorobiphenyl (PCBP-2), 2,3,3′,4,4′,5-hexachlorobiphenyl (HCBP-1), 2,3,3′,4′,5,6-hexachlorobiphenyl (HCBP-2), 2,3,3′,5,5′,6-hexachlorobiphenyl (HCBP-3), 2,2′,3,5,5′,6-hexachlorobiphenyl (HCBP-4) and 2,3,3′,4,5,5′-hexachlorobiphenyl (HCBP-5) and were used to reappraise the structure-activity rules for PCBs as hepatic microsomal enzyme inducers. The results suggested that (a) PCBs which induce MC or mixed-type activity must be substituted at both para positions, at least two meta positions but not necessarily on the same phenyl ring and can also contain one ortho chloro substituent; (b) due to the considerable structural diversity of the PB-type inducers the rules for induction of this activity by PCB congeners are not readily defined.     

Species differences and interindividual variation in liver microsomal cytochrome P450 2A enzymes: effects on coumarin, dicumarol, and testosterone oxidation.

Antibody against purified CYP2A1 recognizes two rat liver microsomal P450 enzymes, CYP2A1 and CYP2A2, that catalyze the 7 alpha- and 15 alpha-hydroxylation of testosterone, respectively. In human liver microsomes, this antibody recognizes a single protein, namely CYP2A6, which catalyzes the 7-hydroxylation of coumarin. To examine species differences in CYP2A function, liver microsomes from nine mammalian species (rat, mouse, hamster, rabbit, guinea pig, cat, dog, cynomolgus monkey, and human) were tested for their ability to catalyze the 7 alpha- and 15 alpha-hydroxylation of testosterone and the 7-hydroxylation of coumarin. Antibody against rat CYP2A1 recognized one or more proteins in liver microsomes from all mammalian species examined. However, liver microsomes from cat, dog, cynomolgus monkey, and human catalyzed negligible rates of testosterone 7 alpha- and/or 15 alpha-hydroxylation, whereas rat and cat liver microsomes catalyzed negligible rates of coumarin 7-hydroxylation. Formation of 7-hydroxycoumarin accounted for a different proportion of the coumarin metabolites formed by liver microsomes from each of the various species examined. 7-Hydroxycoumarin was the major metabolite (greater than 70%) in human and monkey, but only a minor metabolite (less than 1%) in rat. The 7-hydroxylation of coumarin by human liver microsomes was catalyzed by a single, high-affinity enzyme (Km 0.2-0.6 microM), which was markedly inhibited (greater than 95%) by antibody against rat CYP2A1. The rate of coumarin 7-hydroxylation varied approximately 17-fold among liver microsomes from 22 human subjects. This variation was highly correlated (r2 = 0.956) with interindividual differences in the levels of CYP2A6, as determined by immunoblotting. These results indicate that CYP2A6 is largely or entirely responsible for catalyzing the 7-hydroxylation of coumarin in human liver microsomes. Treatment of monkeys with phenobarbital or dexamethasone increased coumarin 7-hydroxylase activity, whereas treatment with beta-naphthoflavone caused a slight decrease. These results suggest that environmental factors can increase or decrease CYP2A expression in cynomolgus monkeys, which implies that environmental factors may be responsible for the large variation in CYP2A6 levels in humans, although genetic factors may also be important. In contrast to rats and mice, the expression of CYP2A enzymes in cynomolgus monkeys and humans was not sexually differentiated. Despite their structural similarity to coumarin, the anticoagulants dicumarol and warfarin do not appear to be substrates for CYP2A6. The overall rate of dicumarol metabolism varied approximately 5-fold among the human liver microsomal samples, but this variation correlated poorly (r2 = 0.126) with the variation observed in CYP2A6 levels and coumarin 7-hydroxylase activity.(ABSTRACT TRUNCATED AT 400 WORDS)