The coiled-coil domain of Escherichia coli FtsLB is a structurally detuned element critical for modulating its activation in bacterial cell division

The FtsLB complex is a key regulator of bacterial cell division, existing in either an off state or an on state, which supports the activation of septal peptidoglycan synthesis. In Escherichia coli, residues known to be critical for this activation are located in a region near the C-terminal end of the periplasmic coiled-coil domain of FtsLB, raising questions about the precise role of this conserved domain in the activation mechanism. Here, we investigate an unusual cluster of polar amino acids found within the core of the FtsLB coiled coil. We hypothesized that these amino acids likely reduce the structural stability of the domain and thus may be important for governing conformational changes. We found that mutating these positions to hydrophobic residues increased the thermal stability of FtsLB but caused cell division defects, suggesting that the coiled-coil domain is a “detuned” structural element. In addition, we identified suppressor mutations within the polar cluster, indicating that the precise identity of the polar amino acids is important for fine-tuning the structural balance between the off and on states. We propose a revised structural model of the tetrameric FtsLB (named the “Y-model”) in which the periplasmic domain splits into a pair of coiled-coil branches. In this configuration, the hydrophilic terminal moieties of the polar amino acids remain more favorably exposed to water than in the original four-helix bundle model (“I-model”). We propose that a shift in this architecture, dependent on its marginal stability, is involved in activating the FtsLB complex and triggering septal cell wall reconstruction.     

Centrin in Giardia lamblia - ultrastructural localization

Giardia lamblia is a multiflagellar parasite and one of the earliest diverging eukaryotic cells. It possesses a complex cytoskeleton based on different groups of microtubular structures - a ventral adhesive disc, four pairs of flagella, a median body and funis. Centrin is an important member of the EF-hand family of calcium-binding proteins, and it is known to show calcium-sensitive contractile behaviour. In the present study, we performed an ultrastructural localization of centrin in G. lamblia using several monoclonal antibodies to centrin. Microtubular structures such as the basal bodies, all the flagella axonemes, the adhesive disc, funis, and the median bodies presented positive labelling to centrin. In addition, the dense rods also demonstrated positive labelling. These results show that centrin is located in key positions related to microtubules. The role of centrin in these dynamic regions is discussed.     

Ecological role of energy taxis in microorganisms

Motile microorganisms rapidly respond to changes in various physico-chemical gradients by directing their motility to more favorable surroundings. Energy generation is one of the most important parameters for the survival of microorganisms in their environment. Therefore it is not surprising that microorganisms are able to monitor changes in the cellular energy generating processes. The signal for this behavioral response, which is called energy taxis, originates within the electron transport system. By coupling energy metabolism and behavior, energy taxis is fine-tuned to the environment a cell finds itself in and allows efficient adaptation to changing conditions that affect cellular energy levels. Thus, energy taxis provides cells with a versatile sensory system that enables them to navigate to niches where energy generation is optimized. This behavior is likely to govern vertical species stratification and the active migration of motile cells in response to shifting gradients of electron donors and/or acceptors which are observed within microbial mats, sediments and soil pores. Energy taxis has been characterized in several species and might be widespread in the microbial world. Genome sequencing revealed that many microorganisms from aquatic and soil environments possess large numbers of chemoreceptors and are likely to be capable of energy taxis. In contrast, species that have a fewer number of chemoreceptors are often found in specific, confined environments, where relatively constant environmental conditions are expected. Future studies focusing on characterizing behavioral responses in species that are adapted to diverse environmental conditions should unravel the molecular mechanisms underlying sensory behavior in general and energy taxis in particular. Such knowledge is critical to a better understanding of the ecological role of energy taxis.     

Stanniocalcin 1 is an autocrine modulator of endothelial angiogenic responses to hepatocyte growth factor

Stanniocalcin 1 (STC1) is a secreted glycoprotein originally described as a hormone involved in calcium and phosphate homeostasis in bony fishes. We recently identified the mammalian homolog of this molecule to be highly up-regulated in an in vitro model of angiogenesis, as well as focally and intensely expressed at sites of pathological angiogenesis (e.g. tumor vasculature). In the present study, we report that STC1 is a selective modulator of hepatocyte growth factor (HGF)-induced endothelial migration and morphogenesis, but not proliferation. STC1 did not inhibit proliferative or migratory responses to vascular endothelial growth factor or basic fibroblast growth factor. The mechanism of STC1 inhibitory effects on HGF-induced endothelial migration seem to occur secondary to receptor activation because STC1 did not inhibit HGF-induced c-met receptor phosphorylation, but did block HGF-induced focal adhesion kinase activation. In the mouse femoral artery ligation model of angiogenesis, STC1 expression closely paralleled that of the endothelial marker CD31, and the peak level of STC1 expression occurred after an increase in HGF expression. We propose that STC1 may play a selective modulatory role in angiogenesis, possibly serving as a "stop signal" or stabilizing factor contributing to the maturation of newly formed blood vessels. HGF is a mesenchyme-derived pleiotropic factor with mitogenic, motogenic, and morphogenic activities on a number of different cell types. HGF effects are mediated through a specific tyrosine kinase, c-met, and aberrant HGF and c-met expression are frequently observed in a variety of tumors. Recent studies have shown HGF to be a potent growth factor implicated in wound healing, tissue regeneration, and angiogenesis.     

Structural features of an arabinogalactan gum exudates from Spondias dulsis (Anacardiaceae)

The tree Spondias dulcis, located in Venezuela, exudes a light-brown gum. The polysaccharide, isolated from the original gum, contains galactose, arabinose, mannose, rhamnose, glucuronic acid, and its 4-O-methyl derivative. Application of chemical methods, in combination with 1D and 2D NMR spectroscopy afforded interesting structural features of the gum polysaccharide. The unequivocal presence of rhamnose in the polymer structure was confirmed by chemical and spectral data [1H (1.03 ppm); 13C (16.92 ppm)]. Also confirmed was the existence of 3-O- and 6-O-substitutes galactose residues by the spectral data correlations observed in Heteronuclear Multiple Quantum Coherence (HMQC) and Heteronuclear Multiple Bond Correlation (HMBC). Also observed were unequivocal resonances for beta-D-glucuronic acid and its 4-O-methyl derivative, and the presence of 3-O-alpha-L-arabinofuranose and 3-O-beta-L-arabinopyranose residues.     

Different evolutionary constraints on chemotaxis proteins CheW and CheY revealed by heterologous expression studies and protein sequence analysis

CheW and CheY are single-domain proteins from a signal transduction pathway that transmits information from transmembrane receptors to flagellar motors in bacterial chemotaxis. In various bacterial and archaeal species, the cheW and cheY genes are usually encoded within homologous chemotaxis operons. We examined evolutionary changes in these two proteins from distantly related proteobacterial species, Escherichia coli and Azospirillum brasilense. We analyzed the functions of divergent CheW and CheY proteins from A. brasilense by heterologous expression in E. coli wild-type and mutant strains. Both proteins were able to specifically inhibit chemotaxis of a wild-type E. coli strain; however, only CheW from A. brasilense was able to restore signal transduction in a corresponding mutant of E. coli. Detailed protein sequence analysis of CheW and CheY homologs from the two species revealed substantial differences in the types of amino acid substitutions in the two proteins. Multiple, but conservative, substitutions were found in CheW homologs. No severe mismatches were found between the CheW homologs in positions that are known to be structurally or functionally important. Substitutions in CheY homologs were found to be less conservative and occurred in positions that are critical for interactions with other components of the signal transduction pathway. Our findings suggest that proteins from the same cellular pathway encoded by genes from the same operon have different evolutionary constraints on their structures that reflect differences in their functions.     

A major chemotaxis gene cluster in Azospirillum brasilense and relationships between chemotaxis operons in alpha-proteobacteria

Azospirillum brasilense shows chemotaxis to a variety of nutrients and oxygen. Genes encoding the central signal transduction pathway in chemotaxis were identified by phenotypic complementation of generally non-chemotactic mutants. Sequencing of a DNA fragment, which complemented two different mutants, revealed a region of five open reading frames translated in one direction and encoding homologs of known genes comprising excitation and adaptation pathways for chemotaxis in other bacterial species. The major chemotaxis gene cluster appears to be essential for all known behavioral responses that direct swimming motility in A. brasilense. Phylogenetic and genomic analysis revealed three groups of chemotaxis operons in alpha-proteobacterial species and assigned the A. brasilense operon to one of them. Interestingly, operons that are shown to be major regulators of behavior in several alpha-proteobacterial species are not orthologous.     

Maize HSP101 plays important roles in both induced and basal thermotolerance and primary root growth

HSP101 belongs to the ClpB protein subfamily whose members promote the renaturation of protein aggregates and are essential for the induction of thermotolerance. We found that maize HSP101 accumulated in mature kernels in the absence of heat stress. At optimal temperatures, HSP101 disappeared within the first 3 days after imbibition, although its levels increased in response to heat shock. In embryonic cells, HSP101 concentrated in the nucleus and in some nucleoli. Hsp101 maps near the umc132 and npi280 markers on chromosome 6. Five maize hsp101-m-::Mu1 alleles were isolated. Mutants were null for HSP101 and defective in both induced and basal thermotolerance. Moreover, during the first 3 days after imbibition, primary roots grew faster in the mutants at optimal temperature. Thus, HSP101 is a nucleus-localized protein that, in addition to its role in thermotolerance, negatively influences the growth rate of the primary root. HSP101 is dispensable for proper embryo and whole plant development in the absence of heat stress.     

Regulation of hepatic connexins in cholestasis: possible involvement of Kupffer cells and inflammatory mediators

Hepatocyte gap junction proteins, connexins (Cxs) 26 and 32, are downregulated during obstructive cholestasis (OC) and lipopolysaccharide hepatocellular cholestasis (LPS-HC). We investigated rat hepatic Cxs during ethynylestradiol hepatocellular cholestasis (EE-HC) and choledochocaval fistula (CCF) and compared them with OC and LPS-HC. Levels (immunoblotting) and cellular distribution (immunofluorescence) of Cx26, -32, and -43, as well as macrophage infiltration, were studied in livers of rats under each condition. Cx26 and -32 were reduced in LPS-HC, OC, and CCF. However, in EE-HC, Cx26 did not change and Cx32 was increased. Prominent inflammation occurred in LPS-HC, OC, and CCF, which was associated with increased levels of Cx43 in LPS-HC and OC but not CCF. No inflammation nor changes in Cx43 levels occurred during EE-HC. In cultured hepatocytes, dye coupling was reduced by tumor necrosis factor-alpha and interleukins-1beta and -6, whereas reduction induced by LPS required coculture with Kupffer cells. Thus hepatocyte gap junctions are downregulated in forms of cholestasis associated with inflammation, and reduced intercellular communication might be induced in part by proinflammatory mediators.     

Impaired neutrophil function in patients with pulmonary tuberculosis and its normalization in those undergoing specific treatment,except the HIV-coinfected cases

Our study investigated whether the respiratory burst (RB) of polymorphonuclear neutrophils from tuberculosis (TB) patients was related with the disease severity or treatment, as well as the circulating levels of TNF-alpha. The sample comprised 57 patients with moderate (n=21) or advanced disease (n=36, 13 of them with HIV coinfection, TB-HIV) and 12 controls. Patients were newly diagnosed (n=27) or under treatment (moderate=14, advanced=10, TB-HIV=6). Cytometric analysis showed that untreated patients had a depressed RB in response to Candida albicans, being more pronounced in the advanced group and nearly absent in TB-HIV cases. A recovered RB was observed in treated patients, except for the TB-HIV cases that continued to show a poor response. TNF-alpha serum levels were increased in untreated patients, mostly in the advanced and TB-HIV groups, and showed an inverse and significant correlation with the RB. Disease severity and anti-TB therapy exerted negative and positive influences on the reactive oxygen intermediates production, respectively.