Rapid detection of Shigella species in environmental sewage by an immunocapture PCR with universal primers

In the present study, we developed a quick, highly specific method for detection of Shigella species by combining immunocapturing of the bacteria and a universal primer PCR. The method drastically enhances test sensitivity, and it can be used not only for identification of Shigella species in the environment but also for rapid detection of other pathogens.     

A neural network approach to approximating MAP in belief networks

Bayesian belief networks (BBN) are a widely studied graphical model for representing uncertainty and probabilistic interdependence among variables. One of the factors that restricts the model's wide acceptance in practical applications is that the general inference with BBN is NP-hard. This is also true for the maximum a posteriori probability (MAP) problem, which is to find the most probable joint value assignment to all uninstantiated variables, given instantiation of some variables in a BBN. To circumvent the difficulty caused by MAP's computational complexity, we suggest in this paper a neural network approximation approach. With this approach, a BBN is treated as a neural network without any change or transformation of the network structure, and the node activation functions are derived based on an energy function defined over a given BBN. Three methods are developed. They are the hill-climbing style discrete method, the simulated annealing method, and the continuous method based on the mean field theory. All three methods are for BBN of general structures, with the restriction that nodes of BBN are binary variables. In addition, rules for applying these methods to noisy-or networks are also developed, which may lead to more efficient computation in some cases. These methods' convergence is analyzed, and their validity tested through a series of computer experiments with two BBN of moderate size and complexity. Although additional theoretical and empirical work is needed, the analysis and experiments suggest that this approach may lead to effective and accurate approximation for MAP problems.     

Bollworm responses to release of genetically modified Helicoverpa armigera nucleopolyhedroviruses in cotton

Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HaSNPV) has been developed as a commercial biopesticide to control the cotton bollworm, H. armigera, in China. The major limitation to a broader application of this virus has been the relative long time to incapacitate the target insect. Two HaSNPV recombinants with improved insecticidal properties were released in bollworm-infested cotton. One recombinant (HaCXW1) lacked the ecdysteroid UDP-glucosyltransferase (egt) gene and in another recombinant (HaCXW2), an insect-selective scorpion toxin (AaIT) gene replaced the egt gene. In a cotton field situation H. armigera larvae treated with either HaCXW1 or HaCXW2 were killed faster than larvae in HaSNPV-wt treated plots. Second instar H. armigera larvae, which were collected from HaCXW1 and HaCXW2 treated plots and further reared on artificial diet, showed reduced ST(50) values of 15.3 and 26.3%, respectively, as compared to larvae collected from HaSNPV-wt treated plots. The reduction in consumed leaf area of field collected larvae infected with HaCXW1 and HaCXW2 was approximated 50 and 63%, respectively, as compared to HaSNPV-wt infected larvae at 108 h after treatment. These results suggest that in a cotton field situation the recombinants will be more effective control agents of the cotton bollworm than wild-type HaSNPV.     

Integrative nuclear FGFR1 signaling (INFS) pathway mediates activation of the tyrosine hydroxylase gene by angiotensin II,depolarization and protein kinase C

The integrative nuclear FGFR1 signaling (INFS) pathway functions in association with cellular growth, differentiation, and regulation of gene expression, and is activated by diverse extracellular signals. Here we show that stimulation of angiotensin II (AII) receptors, depolarization, or activation protein kinase C (PKC) or adenylate cyclase all lead to nuclear accumulation of fibroblast growth factor 2 (FGF-2) and FGFR1, association of FGFR1 with splicing factor-rich domains, and activation of the tyrosine hydroxylase (TH) gene promoter in bovine adrenal medullary cells (BAMC). The up-regulation of endogenous TH protein or a transfected TH promoter-luciferase construct by AII, veratridine, or PMA (but not by forskolin) is abolished by transfection with a dominant negative FGFR1TK-mutant which localizes to the nucleus and plasma membrane, but not by extracellularly acting FGFR1 antagonists suramin and inositolhexakisphosphate (IP6). Mechanism of TH gene activation by FGF-2 and FGFR1 was further investigated in BAMC and human TE671 cultures. TH promoter was activated by co-transfected HMW FGF-2 (which is exclusively nuclear) but not by cytoplasmic FGF-1 or extracellular FGFs. Promoter transactivation by HMWFGF-2 was accompanied by an up-regulation of FGFR1 specifically in the cell nucleus and was prevented FGFR1(TK-) but not by IP6 or suramin. The TH promoter was also transactivated by co-transfected wild-type FGFR1, which localizes to both to the nucleus and the plasma membrane, and by an exclusively nuclear, soluble FGFR1(SP-/NLS) mutant with an inserted nuclear localization signal. Activation of the TH promoter by nuclear FGFR1 and FGF-2 was mediated through the cAMP-responsive element (CRE) and was associated with induction of CREB- and CBP/P-300-containing CRE complexes. We propose a new model for gene regulation in which nuclear FGFR1 acts as a mediator of CRE transactivation by AII, cell depolarization, and PKC.     

Structural requirements within the lipoyl domain for the Ca2+-dependent binding and activation of pyruvate dehydrogenase phosphatase isoform 1 or its catalytic subunit

The inner lipoyl domain (L2) of the dihydrolipoyl acetyltransferase (E2) 60-mer forms a Ca(2+)-dependent complex with the pyruvate dehydrogenase phosphatase 1 (PDP1) or its catalytic subunit, PDP1c, in facilitating large enhancements of the activities of PDP1 (10-fold) or PDP1c (6-fold). L2 binding to PDP1 or PDP1c requires the lipoyl-lysine prosthetic group and specificity residues that distinguish L2 from the other lipoyl domains (L1 in E2 and L3 in the E3-binding component). The L2-surface structure contributing to binding was mapped by comparing the capacities of well folded mutant or lipoyl analog-substituted L2 domains to interfere with E2 activation by competitively binding to PDP1 or PDP1c. Our results reveal the critical importance of a regional set of residues near the lipoyl group and of the octanoyl but not the dithiolane ring structure of the lipoyl group. At the other end of the lipoyl domain, substitution of Glu(182) by alanine or glutamine removed L2 binding to PDP1 or PDP1c, and these substitutions for the neighboring Glu(179) also greatly hindered complex formation (E179A > E179Q). Among 11 substitutions in L2 at sites of major surface residue differences between the L1 and L2 domains, only the conversion of Val-Gln(181) located between the critical Glu(179) and Glu(182) to the aligned Ser-Leu sequence of the L1 domain greatly reduced L2 binding. Certain modified L2 altered E2 activation of PDP1 differently than PDP1c, supporting significant impact of the regulatory PDP1r subunit on PDP1 binding to L2. Our results indicate hydrophobic binding via the extended aliphatic structure of the lipoyl group and required adjacent L2 structure anchor PDP1 by acting in concert with an acidic cluster at the other end of the domain.     

SS Bond-activation of diorganyl disulfide by anionic [Mn(CO)5]: crystal structures of [Mn(SC5H4NO)3] and [(CO)3Mn(μ-SR)3Co(μ-SR)3Mn(CO)3] (R=C6H4NHCOPh)

The SS bond-activation of diorganyl disulfide by the anionic metal carbonyl fragment [Mn(CO)₅]⁻ gives rise to an extensive chemistry. Oxidative decarbonylation addition of 2,2′-dithiobis(pyridine-N-oxide) to [Mn(CO)₅]⁻, followed by chelation and metal-center oxidation, led to the formation of [Mn^II(SC₅H₄NO)₃]⁻ (1). The effective magnetic moment in solid state by SQUID magnetometer was 5.88 μ_B for complex 1, which is consistent with the Mn^II having a high-spin d⁵ electronic configuration in an octahedral ligand field. The average Mn(II)S, SC and NO bond lengths of 2.581(1), 1.692(4) and 1.326(4) Å, respectively, indicate that the negative charge of the bidentate 1-oxo-2-thiopyridinato [SC₅H₄NO]⁻ ligand in complex 1 is mainly localized on the oxygen atom. The results are consistent with thiolate-donor [SC₅H₄NO]⁻ stabilization of the lower oxidation state of manganese (Mn(I)), while the O,S-chelating [SC₅H₄NO]⁻ ligand enhances the stability of manganese in the higher oxidation state (Mn(II)). Activation of SS bond as well as OH bond of 2,2′-dithiosalicylic acid by [Mn(CO)₅]⁻ yielded [(CO)₃Mn(μ-SC₆H₄C(O)O)₂Mn(CO)₃]²⁻ (4). Oxidative addition of bis(o-benzamidophenyl) disulfide to [Mn(CO)₅]⁻ resulted in the formation of cis-[Mn(CO)₄(SR)₂]⁻ (R=C₆H₄NHCOPh) which was employed as a chelating metallo ligand to synthesize heterotrinuclear [(CO)₃Mn(μ-SR)₃Co(μ-SR)₃Mn(CO)₃]⁻ (8) possessing a homoleptic hexathiolatocobalt(III) core.     

Tumor-induced L-selectinhigh suppressor T cells mediate potent effector T cell blockade and cause failure of otherwise curative adoptive immunotherapy

Tumor-specific effector T cells (T(E)) are naturally sensitized within the L-selectin(low) (CD62L(low)) fraction of tumor-draining lymph nodes (TDLN). Whether isolated from day 9 (D9) or day 12 (D12) TDLN, 5 million L-selectin(low) T(E) could be culture activated and adoptively transferred to achieve complete rejection of established intradermal, pulmonary, and brain tumors. Surprisingly, although 25 million unfractionated T cells from D9 TDLN were equally effective, even 100 million unfractionated T cells from D12 TDLN seldom prevented lethal intradermal tumor progression, despite a pronounced therapeutic excess of T(E). This highly reproducible treatment failure was due to cotransfer of tumor-induced, L-selectin(high) suppressor T cells (T(S)) which were also present in D12 TDLN. In contrast, D9 TDLN and normal spleens lacked L-selectin(high) T(S). Only those L-selectin(high) D12 TDLN T cells that down-regulated L-selectin during culture activation were suppressive in vivo and in vitro, and, like L-selectin(low) T(E), trafficked promptly into tumors following i.v. administration. This is the first demonstration that adoptive immunotherapy can fail as a direct result of passenger T(S) that share certain phenotypic and trafficking features of T(E), even when otherwise curative doses of T(E) have been administered. Furthermore, in contrast to recently described CD4(+)CD25(+) T(S) and plasmacytoid dendritic cell-activated T(S), tumor-induced L-selectin(high) T(S) prevent tumor rejection via blockade of sensitized, activated T(E) rather than via afferent blockade.     

A mitogen-activated protein kinase gene (MGV1) in Fusarium graminearum is required for female fertility,heterokaryon formation,and plant infection

Fusarium graminearum is an important pathogen of small grains and maize in many areas of the world. Infected grains are often contaminated with mycotoxins harmful to humans and animals. During the past decade, F. graminearum has caused several severe epidemics of head scab in wheat and barley. In order to understand molecular mechanisms regulating fungal development and pathogenicity in this pathogen, we isolated and characterized a MAP kinase gene, MGV1, which is highly homologous to the MPS1 gene in Magnaporthe grisea. The MGV1 gene was dispensable for conidiation in F. graminearum but essential for female fertility during sexual reproduction. Vegetative growth of mgv1 deletion mutants was normal in liquid media but reduced on solid media. Mycelia of the mgv1 mutants had weak cell walls and were hypersensitive to cell wall degrading enzymes. Interestingly, the mgv1 mutants were self-incompatible when tested for heterokaryon formation, and their virulence was substantially reduced. The ability of the mutants to accumulate trichothecene mycotoxins on inoculated wheat was also greatly reduced. Our data suggest that MGV1 in F. graminearum is involved in multiple developmental processes related to sexual reproduction, plant infection, and cell wall integrity.     

Electrochemical and peroxidase oxidation study of N'-hydroxyguanidine derivatives as NO donors

The electrochemical properties of a series of N-substituted-N'-hydroxyguanidines were studied. Two oxidation potentials of each compound were obtained by cyclic voltammetry. The E(ox1) values were from 0.51 to 0.62V, while the E(ox2) values were from 1.14 to 1.81V in acetonitrile solution. Next, their enzymatic controlled NO release abilities were evaluated. All N'-hydroxyguanidines exhibited efficient NO release abilities under the oxidation by horseradish peroxidase in the presence of H(2)O(2).