Genetic analysis of radiation sensitive and chemical-mutagen sensitive mutants of Pseudomonas aeruginosa.

Thirty-five mutants of Pseudomonas aeruginosa sensitive to methyl methanesulfonate (MMS) have been genetically characterized. They constitute ten separable groups as defined by transduction and conjugation. Three of the groups have been shown to be cotransducible with auxotrophic markers.     

Nicotiana chloroplast genome

Summary RuBPCase, the enzyme responsible for carboxylation and oxidation of RuBP in a wide variety of photosynthetic organisms, is the major protein found in the chloroplast. Here we present the first evidence for direct expression in E. coli and B. subtilis of tobacco and Chlamydomonas ct-DNA sequences coding for the LS of RuBPCase as demonstrated by a simple in situ immunoassay.     

Ecological dynamics of emerging bat virus spillover

Viruses that originate in bats may be the most notorious emerging zoonoses that spill over from wildlife into domestic animals and humans. Understanding how these infections filter through ecological systems to cause disease in humans is of profound importance to public health. Transmission of viruses from bats to humans requires a hierarchy of enabling conditions that connect the distribution of reservoir hosts, viral infection within these hosts, and exposure and susceptibility of recipient hosts. For many emerging bat viruses, spillover also requires viral shedding from bats, and survival of the virus in the environment. Focusing on Hendra virus, but also addressing Nipah virus, Ebola virus, Marburg virus and coronaviruses, we delineate this cross-species spillover dynamic from the within-host processes that drive virus excretion to land-use changes that increase interaction among species. We describe how land-use changes may affect co-occurrence and contact between bats and recipient hosts. Two hypotheses may explain temporal and spatial pulses of virus shedding in bat populations: episodic shedding from persistently infected bats or transient epidemics that occur as virus is transmitted among bat populations. Management of livestock also may affect the probability of exposure and disease. Interventions to decrease the probability of virus spillover can be implemented at multiple levels from targeting the reservoir host to managing recipient host exposure and susceptibility.     

LPS receptor subunits have antagonistic roles in epithelial apoptosis and colonic carcinogenesis

Colorectal carcinoma (CRC) is characterized by unlimited proliferation and suppression of apoptosis, selective advantages for tumor survival, and chemoresistance. Lipopolysaccharide (LPS) signaling is involved in both epithelial homeostasis and tumorigenesis, but the relative roles had by LPS receptor subunits CD14 and Toll-like receptor 4 (TLR4) are poorly understood. Our study showed that normal human colonocytes were CD14⁺TLR4⁻, whereas cancerous tissues were CD14⁺TLR4⁺, by immunofluorescent staining. Using a chemical-induced CRC model, increased epithelial apoptosis and decreased tumor multiplicity and sizes were observed in TLR4-mutant mice compared with wild-type (WT) mice with CD14⁺TLR4⁺ colonocytes. WT mice intracolonically administered a TLR4 antagonist displayed tumor reduction associated with enhanced apoptosis in cancerous tissues. Mucosa-associated LPS content was elevated in response to CRC induction. Epithelial apoptosis induced by LPS hypersensitivity in TLR4-mutant mice was prevented by intracolonic administration of neutralizing anti-CD14. Moreover, LPS-induced apoptosis was observed in primary colonic organoid cultures derived from TLR4 mutant but not WT murine crypts. Gene silencing of TLR4 increased cell apoptosis in WT organoids, whereas knockdown of CD14 ablated cell death in TLR4-mutant organoids. In vitro studies showed that LPS challenge caused apoptosis in Caco-2 cells (CD14⁺TLR4⁻) in a CD14-, phosphatidylcholine-specific phospholipase C-, sphingomyelinase-, and protein kinase C-ζ-dependent manner. Conversely, expression of functional but not mutant TLR4 (Asp299Gly, Thr399Ile, and Pro714His) rescued cells from LPS/CD14-induced apoptosis. In summary, CD14-mediated lipid signaling induced epithelial apoptosis, whereas TLR4 antagonistically promoted cell survival and cancer development. Our findings indicate that dysfunction in the CD14/TLR4 antagonism may contribute to normal epithelial transition to carcinogenesis, and provide novel strategies for intervention against colorectal cancer.Colorectal tumorigenesis proceeds via the accumulation of genetic and epigenetic alterations that promote unlimited cell proliferation, self-sufficient growth signaling, neovascularization, tissue invasion, and resistance to cell death.¹ The transformation of normal epithelium into colorectal carcinomas (CRC) is associated with the progressive inhibition of apoptosis; this confers a selective advantage for tumor cell survival and chemoresistance.^{2, 3} It is generally believed that sufficient epithelial apoptosis may hamper colon cancer formation in terms of incidence and growth rate.^{4, 5, 6} Direct evidence for this was recently reported in mice deficient in pro-apoptotic molecules.^{7, 8} To date, the regulatory mechanisms of physiological apoptosis to eliminate premalignant cells in the gut remain incompletely understood.Intestinal homeostasis is maintained by the dynamic, yet strictly regulated, turnover of epithelial cells. An imbalance in epithelial death versus survival/proliferative responses may lead to barrier dysfunction, chronic inflammation, and tumorigenesis.^{9, 10} Accumulating evidence indicates that gut microbiota and bacterial lipopolysaccharide (LPS) have critical roles in epithelial cell renewal under baseline conditions and on injury,^{11, 12} and are involved in the pathogenesis of colitis-associated CRC as well.^{13, 14, 15} Given the juxtaposition of commensal bacteria and the gut mucosa, it has been assumed that normal epithelial cells are not equipped with LPS receptor complexes (CD14/TLR4/MD2) or express altered forms of receptors and signaling molecules to achieve immunotolerance.¹⁵ Constitutive expression of CD14 was reported in the presence of negligible-to-low levels of Toll-like receptor 4 (TLR4) in normal human colonocytes,^{16, 17, 18} whereas strong TLR4 immunoreactivity was detected in CRC.^{18, 19} Nevertheless, divergent cellular responses to LPS (death versus survival) have been reported among human CRC cell lines. Several laboratories, using Caco-2 cells, have described increases in apoptotic cell death following apical LPS challenge,^{20, 21} whereas others have documented enhanced survival and proliferative responses of HT29 and SW480 cells to LPS.^{22, 23} Here we hypothesize that differing expression patterns of LPS receptor subunits on epithelial surfaces may have a determining role in cell death versus survival.CD14, as the membrane-bound subunit of LPS receptor complex and lacking a cytoplasmic tail, has traditionally been regarded as merely a binding component for transferring LPS to TLR4. TLR4 subsequently activates downstream adaptors and signaling pathways, such as myeloid differentiation factor (MyD88), mitogen-activated protein kinases (MAPKs), inhibitor of κB (IκB)/nuclear factor-κB (NFκB), and interferon regulatory factor 3 (IRF3).^{24, 25} Recent findings in monocytes have indicated that LPS/CD14 binding triggers a cascade of lipid messenger signals before TLR4 trafficking to lipid rafts for complex formation. CD14-dependent lipid signaling includes the conversion of membranous phosphatidylcholine (PC) to diacylglcerol by PC-specific phospholipase C (PC-PLC) and the activation of sphingomyelinase (SMase) for sphingolipid metabolism and ceramide production. This process leads to the phosphorylation of protein kinase C (PKC) ζ, which recruits TLR4 to interact with CD14 (Cuschieri et al.²⁶ and Triantafilou et al.²⁷). Lipid messengers, such as sphingolipids and ceramides, and their downstream PKCζ signals have been implicated in pro-apoptotic pathways and are considered tumor suppressors.^{28, 29, 30} Decreased SMase activity and PKCζ levels have been observed in human colorectal tumors, correlated with poor prognosis.^{31, 32} In contrast, the TLR4/MyD88 and IκB/NFκB pathways are associated with anti-apoptotic and hyperproliferative responses.^{5, 33, 34, 35} Reduced colorectal tumor formation has been documented in TLR4(−/−), MyD88(−/−), and epithelial-specific IκB kinase β-deficient mice as compared with wild-type (WT) mice.^{5, 19, 36} These findings led us to speculate that the expression of CD14 and TLR4 on epithelial cell surfaces may provide antagonistic signals to counteract apoptotic responses to LPS and to influence tumor progression.The aims of this study were to (1) investigate the expression patterns of LPS receptor subunits in normal and cancerous colonic epithelia in human and murine tissues; (2) examine the individual roles of CD14 and TLR4 in epithelial apoptosis and tumor formation using a mouse model of colitis-associated CRC; (3) assess the involvement of CD14-mediated lipid messengers and/or TLR4-dependent signaling in the mechanism of LPS-induced apoptosis using human carcinoma cell lines; and (4) evaluate whether TLR4 has an opposing role against CD14-mediated apoptosis to promote tumor cell survival.     

Identification of a novel multiple environmental factor‐responsive 1‐aminocyclopropane‐1‐carboxylate synthase gene,NT‐ACS2, from tobacco

Many abiotic environmental factors elicit the production of stress‐ethylene in higher plants. To elucidate the molecular mechanisms underlying the regulation of stress‐ethylene production and the physiological roles played by stress‐ethylene in stress responses of plants, we studied the gene expression of ACC synthase in tobacco plants that had been subjected to environmental stresses. Four new tobacco ACC synthase cDNA fragments, NT‐ACS2, NT‐ACS3, NT‐ACS4 and NT‐ACS5, were identified and sequenced. It was found that NT‐ACS2 could be induced by wounding, cold temperature and, especially, sunlight. NT‐ACS4 was induced at a faster kinetics by wounding. The multiple environmental stress‐responsive (MESR) NT‐ACS2 gene was found to contain three introns and four exons and encode a polypeptide of 484 amino acids, 54·6 kDa and pI 6·87. Computer analysis of the 3·4 kb 5 ′ flanking region upstream of the ACS coding region revealed the existence of a group of putative cis‐acting regulatory elements potentially conferring wounding, chilling, and UV light inducibility. Phylogenetic analysis of ACC synthase genes of different plant origins indicated that the chill‐inducible NT‐ACS2 gene is closely related to a chilling‐inducible citrus ACS gene.     

Transformation of chicken embryo fibroblasts by direct DNA transfection of single oncogenes: comparative analyses of src, erbB, myc, and ras.

Chicken embryo fibroblasts (CEF) have been used extensively to study the transformation parameters of a number of avian sarcoma-leukemia viruses. Previously, oncogene transformation of CEF has been conducted almost exclusively with replicating viruses, because of perceived difficulties with direct DNA transfection. Here, we show that CEF can be efficiently and stably transfected by selection for the neomycin resistance gene (neo). Cotransfection of neo with various oncogenes resulted in CEF transformation in vitro and, in several instances, sarcoma formation in vivo. Transfection of src, myc, erbB, and ras, either singly or in combination, resulted in soft-agar colonies with unique morphologies. Transfection of a family of v-src, c-src, and v/c-src chimeric constructs demonstrated the ability of the assay to discriminate between transforming and nontransforming genes. Transfection of a number of erbB variants showed that internal mutations, primarily in the kinase domain, contribute significantly to the ability to transform fibroblasts. The tumorigenic potential detected by transfection of oncogenes faithfully reproduced those previously reported by using viral infections. Our studies establish the utility of CEF transformation by direct DNA transfection. This method should prove useful in analyzing oncogenes, (e.g., myc) that do not readily transform rodent cell lines and in studying host-range mutants of oncogenes, such as those recently identified for src and erbB.     

Chemically charging the pore constriction opens the mechanosensitive channel MscL

MscL is a bacterial mechanosensitive channel that protects the cell from osmotic downshock. We have previously shown that substitution of a residue that resides within the channel pore constriction, MscL's Gly-22, with all other 19 amino acids affects channel gating according to the hydrophobicity of the substitution (). Here, we first make a mild substitution, G22C, and then attach methanethiosulfonate (MTS) reagents to the cysteine under patch clamp. Binding MTS reagents that are positively charged ([2-(trimethylammonium)ethyl] methanethiosulfonate and 2-aminoethyl methanethiosulfonate) or negatively charged (sodium (2-sulfonatoethyl)methanethiosulfonate) causes MscL to gate spontaneously, even when no tension is applied. In contrast, the polar 2-hydroxyethyl methanethiosulfonate halves the threshold, and the hydrophobic methyl methanethiolsulfonate increases the threshold. These observations indicate that residue 22 is in a hydrophobic environment before gating and in a hydrophilic environment during opening to a substate, a finding consistent with our previous study. In addition, we have found that cysteine 22 is accessible to reagents from the cytoplasmic side only when the channel is opened whereas it is accessible from the periplasmic side even in the closed state. These results support the view that exposure of hydrophobic surfaces to a hydrophilic environment during channel opening serves as the barrier to gating.     

Identification of the allosteric regulatory site in bacterial phosphoribulokinase

Bacterial phosphoribulokinases (PRKs) are octameric members of the adenylate kinase family of enzymes. The enzyme is allosterically activated by NADH and allosterically inhibited by AMP. We have determined the crystal structure of PRK from Rhodobacter sphaeroides bound to the ATP analogue AMP-PCP to a resolution of 2.6 A. The structure reveals that the ATP analogue does not bind to the canonical ATP site found in adenylate kinase family members. Rather, the AMP-PCP binds in two different orientations at the interface of three of the monomers in the octamer. This interface was previously characterized as having an unusually large number of arginine residues. Of the five arginine residues that are near the bound nucleotide, one (Arg 221) is highly conserved in both prokaryotic and eukaryotic (nonallosterically regulated) PRKs, two (Arg 234 and Arg 257) are on a second subunit and conserved in only prokaryotic PRKs, and two (Arg 30 and Arg 31) are on a third subunit with only one of them (Arg 31) conserved in prokaryotic PRKs. Each of these arginine residues was converted by site-directed mutagenesis to alanine. Fluorescence binding data suggest that none of these arginines are involved in active site ATP binding and that Arg 234 and Arg 257 on the second subunit are directly involved in NADH binding, while the other arginines have a minimal effect on NADH binding. While the wild-type enzyme exhibits low maximal activity and hyperbolic kinetics with respect to ATP in the absence of NADH and high maximal activity and sigmoidal kinetics in the presence of NADH, the R31A mutant exhibits identical hyperbolic kinetics with respect to ATP in the presence or absence of NADH. Thus, the transmission of allosteric information from one subunit to another is conducted through a single path that includes NADH and Arg 31.     

The severe acute respiratory syndrome (SARS) coronavirus NTPase/helicase belongs to a distinct class of 5' to 3' viral helicases

The putative NTPase/helicase protein from severe acute respiratory syndrome coronavirus (SARS-CoV) is postulated to play a number of crucial roles in the viral life cycle, making it an attractive target for anti-SARS therapy. We have cloned, expressed, and purified this protein as an N-terminal hexahistidine fusion in Escherichia coli and have characterized its helicase and NTPase activities. The enzyme unwinds double-stranded DNA, dependent on the presence of a 5' single-stranded overhang, indicating a 5'o 3' polarity of activity, a distinct characteristic of coronaviridae helicases. We provide the first quantitative analysis of the polynucleic acid binding and NTPase activities of a Nidovirus helicase, using a high throughput phosphate release assay that will be readily adaptable to the future testing of helicase inhibitors. All eight common NTPs and dNTPs were hydrolyzed by the SARS helicase in a magnesium-dependent reaction, stimulated by the presence of either single-stranded DNA or RNA. The enzyme exhibited a preference for ATP, dATP, and dCTP over the other NTP/dNTP substrates. Homopolynucleotides significantly stimulated the ATPase activity (15-25-fold) with the notable exception of poly(G) and poly(dG), which were non-stimulatory. We found a large variation in the apparent strength of binding of different homopolynucleotides, with dT24 binding over 10 times more strongly than dA24 as observed by the apparent Km.