Contractions associated with the salivary glands of the blood-feeding bug, Rhodnius prolixus: evidence for both a neural and neurohormonal coordination

The salivary glands of the blood-feeding bug, Rhodnius prolixus, are composed of a single epithelial layer of binucleate cells and a double layer of visceral muscle cells surrounding a large secretory cavity. The saliva contains substances which counteract the hemostasis of the host, and injection of saliva into the host is an essential component of successful and efficient gorging.The muscles surrounding the salivary glands of Rhodnius are under polyneuronal control from the salivary nerve projecting out of the hypocerebral ganglion. The amplitude of contractions induced by neural stimulation is dependent upon both intensity and frequency of nerve stimulation.Serotonin and FMRFamide-related peptides (FaRPs) are delivered in the nerve supply to the salivary glands, and both classes of neuroactive chemicals increase frequency and amplitude of phasic contractions in a dose-dependent manner. A member of the FaRP myosuppressin subfamily, however, inhibits contractions. CRF-related and Leucokinin-like peptides are not delivered in the nerve supply but may be present in the hemolymph during feeding. Leucokinin 1 and Zoone DH (a CRF-related peptide) both induce a dose-dependent increase in basal tonus, with phasic contractions superimposed. Zoone DH is more active than Leucokinin 1. Factors are present in the CNS of Rhodnius which mimic the effects of serotonin and the stimulatory peptides.     

Effects of MCH/NEI in the striatum and interactions between them and with alpha-MSH on IP3 levels

In this study, we investigated: (a) the effect of melanocyte concentrating hormone (MCH) and neuropeptide glutamine (E)-isoleusine-(I) (NEI) on IP(3) production on an "in vitro" model using slices containing caudate putamen (CP) and accumbens nuclei (ACC); and (b) the interaction between these peptides and with alpha-melanocyte-stimulating-hormone (alpha-MSH) on the production of this second messenger. Only MCH at the highest dose studied (3.6 microM) increased the production of IP(3), whereas at the low concentration (0.6 microM) it did not affect IP(3) levels. NEI and alpha-MSH at both concentrations tested (0.6 and 3.6 microM), did not affect IP(3) production either. However, when NEI or alpha-MSH (at 3.6 microM) were added together with 3.6 microM MCH, the increase in the IP(3) content induced by this last peptide was blocked.     

Cooperativity and flexibility of cystic fibrosis transmembrane conductance regulator transmembrane segments participate in membrane localization of a charged residue

Polytopic protein topology is established in the endoplasmic reticulum (ER) by sequence determinants encoded throughout the nascent polypeptide. Here we characterize 12 topogenic determinants in the cystic fibrosis transmembrane conductance regulator, and identify a novel mechanism by which a charged residue is positioned within the plane of the lipid bilayer. During cystic fibrosis transmembrane conductance regulator biogenesis, topology of the C-terminal transmembrane domain (TMs 7-12) is directed by alternating signal (TMs 7, 9, and 11) and stop transfer (TMs 8, 10, and 12) sequences. Unlike conventional stop transfer sequences, however, TM8 is unable to independently terminate translocation due to the presence of a single charged residue, Asp(924), within the TM segment. Instead, TM8 stop transfer activity is specifically dependent on TM7, which functions both to initiate translocation and to compensate for the charged residue within TM8. Moreover, even in the presence of TM7, the N terminus of TM8 extends significantly into the ER lumen, suggesting a high degree of flexibility in establishing TM8 transmembrane boundaries. These studies demonstrate that signal sequences can markedly influence stop transfer behavior and indicate that ER translocation machinery simultaneously integrates information from multiple topogenic determinants as they are presented in rapid succession during polytopic protein biogenesis.     

The cell biology of Listeria monocytogenes infection: the intersection of bacterial pathogenesis and cell-mediated immunity

Listeria monocytogenes has emerged as a remarkably tractable pathogen to dissect basic aspects of cell biology, intracellular pathogenesis, and innate and acquired immunity. In order to maintain its intracellular lifestyle, L. monocytogenes has evolved a number of mechanisms to exploit host processes to grow and spread cell to cell without damaging the host cell. The pore-forming protein listeriolysin O mediates escape from host vacuoles and utilizes multiple fail-safe mechanisms to avoid causing toxicity to infected cells. Once in the cytosol, the L. monocytogenes ActA protein recruits host cell Arp2/3 complexes and enabled/vasodilator-stimulated phosphoprotein family members to mediate efficient actin-based motility, thereby propelling the bacteria into neighboring cells. Alteration in any of these processes dramatically reduces the ability of the bacteria to establish a productive infection in vivo.     

Identification and characterization of the<Emphasis Type="Italic"> carAB</Emphasis> genes responsible for encoding carbamoylphosphate synthetase in<Emphasis Type="Italic"> Halomonas eurihalina</Emphasis>

Halomonas eurihalina is a moderately halophilic bacterium which produces exopolysaccharides potentially of great use in many fields of industry and ecology. Strain F2-7 of H. eurihalina synthesizes an anionic exopolysaccharide known as polymer V2-7, which not only has emulsifying activity but also becomes viscous under acidic conditions, and therefore we consider it worthwhile making a detailed study of the genetics of this strain. By insertional mutagenesis using the mini-Tn 5 Km2 transposon we isolated and characterized a mutant strain, S36 K, which requires both arginine and uracil for growth and does not excrete EPS. S36 K carries a mutation within the carB gene that encodes the synthesis of the large subunit of the carbamoylphosphate synthetase enzyme, which in turn catalyzes the synthesis of carbamoylphosphate, an important precursor of arginine and pyrimidines. We describe here the cloning and characterization of the carAB genes, which encode carbamoylphosphate synthetase in Halomonas eurihalina, and discuss this enzyme's possible role in the pathways for the synthesis of exopolysaccharides in strain F2-7.     

Dynamics of bacterial growth and distribution within the liver during Salmonella infection

Salmonella enterica causes severe systemic diseases in humans and animals and grows intracellularly within discrete tissue foci that become pathological lesions. Because of its lifestyle Salmonella is a superb model for studying the in vivo dynamics of bacterial distribution. Using multicolour fluorescence microscopy in the mouse typhoid model we have studied the interaction between different bacterial populations in the same host as well as the dynamic evolution of foci of infection in relation to bacterial growth and localization. We showed that the growth of Salmonella in the liver results in the spread of the microorganisms to new foci of infection rather than simply in the expansion of the initial ones. These foci were associated with independently segregating bacterial populations and with low numbers of bacteria in each infected phagocyte. Using fast-growing and slow-growing bacteria we also showed that the increase in the number of infected phagocytes parallels the net rate of bacterial growth of the microorganisms in the tissues. These findings suggest a novel mechanism underlying growth of salmonellae in vivo with important consequences for understanding mechanisms of resistance and immunity.     

Role of the promyelocytic leukemia body in the dynamic interaction between the androgen receptor and steroid receptor coactivator-1 in living cells

The dynamic interaction between the androgen receptor (AR) and steroid receptor coactivator-1 (SRC-1) was explored in living cells expressing chimeric forms of the receptor and the coactivator containing two spectral variants of jellyfish fluorescent protein. Laser scanning confocal imaging of transfected cells expressing fluorescently labeled SRC-1 revealed that in an unsynchronized cell population, the coactivator is distributed in approximately 40% cells as nuclear bodies of 0.2-1.0 microm in diameter. Immunostaining of cyan fluorescent protein-labeled SRC-1 (CFP-SRC1)-expressing cells with antibody to promyelocytic leukemia (PML) protein showed significant overlap of the CFP fluorescence with the antibody stain. Cotransfection of cells with a plasmid expressing the CFP conjugate of Sp100 (another marker protein for the PML nuclear body) also showed colocalization of the yellow fluorescent protein (YFP)-SRC1 containing nuclear foci with the PML bodies in living cells. Analysis of the three-dimensional structure revealed that the PML bodies are round to elliptical in shape with multiple satellite bodies on their surface. Some of these satellite bodies contain the SRC-1. Activation and nuclear import of CFP-AR by the agonistic ligand 5alpha-dihydrotestosterone, but not by the antagonist casodex, transferred YFP-SRC1 from the PML bodies to an interlacing filamentous structure. In a single living cell, agonist-activated AR caused a time-dependent movement of YFP-SRC1 from the PML bodies to this filamentous structure. Additionally, coexpression of a constitutively active mutant of AR (AR-deltaligand binding domain) also displaced YFP-SRC1 from the PML bodies to this intranuclear filamentous structure. The fluorescence recovery after photobleaching approach was used to examine changes in the kinetics of movement of YFP-SRC1 during its mobilization from the PML bodies to the intranuclear filamentous structure by the agonist-activated AR. Results of the relative half-times (t(1/2)) of replacement of YFP-SRC1 within the photobleached region of a single PML body from its surrounding nuclear space supported the conclusion that SRC-1 is actively transported from the PML bodies to the intranuclear filamentous structure by the ligand-activated AR. This observation also suggests an interaction between AR and SRC-1 before its binding to the target gene. The PML bodies have been implicated as a cross-road for multiple regulatory pathways that control cell proliferation, cellular senescence, and apoptosis. Our present results along with other recent reports expand the role of this subnuclear structure to include the regulation of steroid hormone action.     

Spatial and temporal effects of free-air CO2 enrichment (POPFACE) on leaf growth,cell expansion,and cell production in a closed canopy of poplar

Leaf expansion in the fast-growing tree, Populus x euramericana was stimulated by elevated [CO(2)] in a closed-canopy forest plantation, exposed using a free air CO(2) enrichment technique enabling long-term experimentation in field conditions. The effects of elevated [CO(2)] over time were characterized and related to the leaf plastochron index (LPI), and showed that leaf expansion was stimulated at very early (LPI, 0-3) and late (LPI, 6-8) stages in development. Early and late effects of elevated [CO(2)] were largely the result of increased cell expansion and increased cell production, respectively. Spatial effects of elevated [CO(2)] were also marked and increased final leaf size resulted from an effect on leaf area, but not leaf length, demonstrating changed leaf shape in response to [CO(2)]. Leaves exhibited a basipetal gradient of leaf development, investigated by defining seven interveinal areas, with growth ceasing first at the leaf tip. Interestingly, and in contrast to other reports, no spatial differences in epidermal cell size were apparent across the lamina, whereas a clear basipetal gradient in cell production rate was found. These data suggest that the rate and timing of cell production was more important in determining leaf shape, given the constant cell size across the leaf lamina. The effect of elevated [CO(2)] imposed on this developmental gradient suggested that leaf cell production continued longer in elevated [CO(2)] and that basal increases in cell production rate were also more important than altered cell expansion for increased final leaf size and altered leaf shape in elevated [CO(2)].     

Connexin-30 deletion analysis in connexin-26 heterozygotes

Mutations in the Connexin-26 gene (Cx 26, GJB2) are the most common cause of hereditary nonsyndromic sensorineural hearing loss (SNHL). DNA analysis of the Cx 26 gene in deaf or hard-of-hearing individuals frequently demonstrates heterozygosity despite the fact that most mutations are known to be recessive. A 342-kb deletion in a gene adjacent to Cx 26, the Connexin-30 gene (Cx 30, GJB6), has been reported to cause deafness in the homozygous state or in combination with heterozygous mutations in Cx 26 (digenic inheritance). We have analyzed deaf or hard-of-hearing Cx 26 heterozygotes and individuals with no mutations in Cx 26 for this Cx 30 deletion. We found that 4/20 (20%) of the Cx 26 heterozygotes are heterozygous for this deletion and that no individuals were homozygous for the Cx 30 deletion. Cx 30 deletion analysis is recommended for all individuals with nonsyndromic SNHL following Cx 26 sequencing that does not demonstrate two recessive mutations.