The PecM protein of the phytopathogenic bacterium Erwinia chrysanthemi, membrane topology and possible involvement in the efflux of the blue pigment indigoidine

The pecS regulatory locus negatively modulates the expression of many virulence genes in Erwinia chrysanthemi. This locus consists of two genes, pecS and pecM, divergently transcribed. Previous studies have shown that PecS down-regulates the expression of both pecSand pecMgenes and that PecM is required for full PecS activity. Computer-aided hydropathy analysis of PecM predicted the presence of between 8 to 10 potential transmembrane segments. We analyzed the membrane topology of PecM using the beta-lactamase gene fusion system and obtained the following unique characteristics. PecM contains 10 membrane spanning segments, with both the amino and carboxyl termini located in the cytoplasmic side of the inner membrane. The fourth periplasmic loop, which has a relatively long hydrophilic domain containing 17 amino acid residues, may play an important role in PecM function. The topological model obtained for PecM can be applied to PecM homologues in other bacteria. Measurement of the extrusion of the blue pigment indigoidine by the E. chrysanthemi derivative isogenic mutants pecS, pecM and pecS-pecM revealed that PecM is required for complete efflux of the pigment. Its relation to other efflux systems and its potential physiological role are discussed.     

Relationship between long-term resistance to Trypanosoma cruzi and latent infection,examined by antibody production and polymerase chain reaction in mice

Trypanosoma cruzi infections persist for the lifetime of humans and laboratory animals as either latent or pathogenic parasitism. Mice inoculated with a nonpathogenic, attenuated strain (TCC) display resistance against virulent challenge, with a strong control of parasitemia and protection against tissue lesions for more than 12 mo. Three main approaches were used to test whether protection by TCC inocula is based on a latent infection or on a "sterile" immunological memory: curative Benznidazole (Bzl) treatment, serological reactions, and detection of infection by polymerase chain reaction (PCR). If resistance is maintained in the absence of infection, it should not be reduced by Bzl treatment and TCC-inoculated animals should not maintain long-term serological or PCR reactivity. The Bzl treatment after TCC inoculations did not reduce, after periods of up to 420 days, TCC-induced resistance to challenge. But TCC inocula given during Bzl treatment conferred short-term, but not long-term. protection. Maintenance of high antibody levels and protection were better in the virulent Tulahuen (TUL) strain than in the attenuated TCC strain infections, and trypomastigote inocula of either strain were better inducers of antibodies and resistance than epimastigotes. PCR detection of T. cruzi DNA was positive in almost all TUL strain-inoculated animals and negative in immunocompetent animals inoculated with TCC epimastigotes, although high numbers of TCC trypomastigotes produced persistent PCR signals of infection in newborn BALB mice. Thus, 2 polar models were developed, where latent infection by TCC was either demonstrated or excluded. In both, resistance to virulent challenge was maintained during long periods. But late declination of antibody titers (>200 days) and resistance to challenge (>350 days) was observed in animals displaying clearance of all signals of infection.     

Theoretical studies of biliverdin: energetics of the reduction pathways to bilirubin

Geometries and energies of formation of bilirubin formed by reduction of biliverdin via three meso carbon sites, the , and positions, have been calculated using semiempirical methods. It has been shown that -bilirubin with a ridge-tile conformation forms six intramolecular hydrogen bonds and is the most stable of the three above mentioned positions by at least 22 kcal mol^–1. Reduction pathways for -, - and -bilirubin formations from biliverdin are studied in detail. The roles of loss of conjugation and hydrogen bond formations in stability of different conformers have been discussed. -Bilirubin was fully optimized by using ab initio methods. Fine refinements of calculated results show excellent agreement with experimental results. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00894-002-0078-9.Electronic Supplementary Material available.     

Mechanomyogram and electromyogram responses of upper limb during sustained isometric fatigue with varying shoulder and elbow postures

To investigate the behavior of mechanomyogram (MMG) and electromyogram (EMG) signals in the time and frequency domains during sustained isometric contraction, MMG and surface EMG were obtained simultaneously from four muscles: upper trapezius (TP), anterior deltoid (DL), biceps brachii (BB), and brachioradialis (BR) of 10 healthy male subjects. Experimental conditions consisted of 27 combinations of 9 postures [3 shoulder angles (SA): 0 degree, 30 degrees, 60 degrees and 3 elbow angles (EA): 120 degrees, 90 degrees, 60 degrees] and 3 contraction levels: 20%, 40%, and 60% of maximum voluntary contraction (MVC). Subjective evaluations of fatigue were also assessed using the Borg scale at intervals of 60, 30, and 10 sec at 20%, 40%, and 60% MVC tests, respectively. The mean power frequency (MPF) and root mean square (RMS) of both signals were calculated. The current study found clear and significant relationships among physiological and psychological parameters on the one hand and SA and EA on the other. EA's effect on MVC was found to be significant. SA had a highly significant effect on both endurance time and Borg scale. In all experimental conditions, significant correlations were found between the changes in MPF and RMS of EMG in BB with SA and EA (or muscle length). In all four muscles, MMG frequency content was two or three times lower than EMG frequency content. During sustained isometric contraction, the EMG signal showed the well-known shift to lower frequencies (a continuous decrease from onset to completion of the contraction). In contrast, the MMG spectra did not show any shift, although its form changed (generally remaining about constant). Throughout the contraction, increased RMS of EMG was found for all tests, whereas in the MMG signal, a significant progressive increase in RMS was observed only at 20% MVC in all four muscles. This supports the hypothesis that the RMS amplitude of the MMG signal produced during contraction is highly correlated with force production. Possible explanations for this behavioral difference between the MMG and EMG signals are discussed.     

Muscular performance modeling of the upper limb in static postures

The purpose of the present paper is to describe and evaluate the polynomial models for predicting the muscular work capacity of the upper limb during sustained holding tasks. This research was concerned with the relationship between indicators of performance, i.e., specific posture or specific level of maximum voluntary contraction (MVC), and then modeling the functional data based on experimental results to estimate factors that may have an effect on task performance. To this end, we designed an experiment using 10 subjects in which each subject performed sustained isometric shoulder and elbow flexion endurance exercise under 27 conditions [3 shoulder angles (SA)x3 elbow angles (EA)x3 levels of %MVC]. Throughout all experiments, subjective perception of effort was assessed using the Borg scale, every 60, 30, and 10 s during the 20%, 40%, and 60% MVC tests, respectively. Proposal models were represented by three approaches: model A: estimation of endurance time (ET), with input variables such as SA, EA, and %MVC; model B: estimation of recommendation time (RT, the time during which the operator was able to maintain a position under the desired condition), with input variables such as SA, EA, %MVC, and required rate on the Borg scale; and model C: estimation of limit strength or %MVC, with input variables such as SA, EA, request limit time for work (LT), and required rate on the Borg scale. Statistical analysis indicated that the three proposal estimation models based on polynomial regression functions showed high significance (p<0.0001). The proposal models suggested and recommended the possibility of finding the best positions entailing the reduction and minimization of total muscular strain from manual material handling tasks in different work situations, with the consequent increase in work efficiency.     

Tetrahydroisoquinoline alkaloids and 2-deoxyribonolactones from Aristolochia arcuata

2-Deoxyribonolactones and four tetrahydroisoquinoline alkaloids were isolated from the acetone extract of the leaves of Aristolochia arcuata Mast., together with pinitol, sequoyitol, glycerol, fructose, sucrose, eupomatenoid-7, salsolinol, and 6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydroisoquinoline. Their structures were determined on the basis of spectroscopic methods, mainly using 1H, 13C, 15N, and 31P NMR.     

A maize defense-inducible gene is a major facilitator superfamily member related to bacterial multidrug resistance efflux antiporters

A defense-inducible maize gene was discovered through global mRNA profiling analysis. Its mRNA expression is induced by pathogens and defense-related conditions in various tissues involving both resistant and susceptible interactions. These include Cochliobolus heterostrophus and Cochliobolus carbonum infection, ultraviolet light treatment, the Les9 disease lesion mimic background, and plant tissues engineered to express flavonoids or the avirulence gene avrRxv. The gene was named Zm-mfs1 after it was found to encode a protein related to the major facilitator superfamily (MFS) of intregral membrane permeases. It is most closely related to the bacterial multidrug efflux protein family, typified by the Escherichia coli TetA, which are proton motive force antiporters that export antimicrobial drugs and other compounds, but which can be also involved in potassium export/proton import or potassium re-uptake. Other related plant gene sequences in maize, rice, and Arabidopsis were identified, three of which are introduced here. Among this new plant MFS subfamily, the characteristic MFS motif in cytoplasmic TM2-TM3 loop, and the antiporter family motif in transmembrane domain TM5 are both conserved, however the TM7 and the cytoplasmic TM8-TM9 loop are divergent from those of the bacterial multidrug transporters. We hypothesize that Zm-Mfs1 is a prototype of a new class of plant defense-related proteins that could be involved in either of three nonexclusive roles: (1) export of antimicrobial compounds produced by plant pathogens; (2) export of plant-generated antimicrobial compounds; and (3) potassium export and/or re-uptake, as can occur in plant defense reactions.     

Synthesis of some N-galactosides of 3-aryl-5-benzyl (or substituted benzyl)-1,2,4-triazin-6(1H)-/ones or thiones of expected biological activity

The 1-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-3-aryl-5-benzyl (or substituted benzyl)-1,2,4-triazin-6(1H)-/ones or thiones were prepared via galactosidation of 3-aryl-5-benzyl (or substituted benzyl)-1,2,4-triazin-6(1H)-/ones or thiones with 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromide. The structure of the new galactosyl derivatives was based on both spectroscopic and chemical evidences.     

Synthesis of some new 2-alpha-L-arabinopyranosyl-1,2,4-triazines as potential antitumor chemotherapeutics

Synthetic routes towards different 2-alpha-L-arabinopyranosyl-3-thioxo-2,3-dihydro-1,2,4-triazin-5(4H)-/ones or thiones were investigated. Primary human anticancer screening of two selected compounds resulted in an active compound against SF-268 (CNS) cell line.     

Vasoactive Intestinal Polypeptide Promotes Intestinal Barrier Homeostasis and Protection Against Colitis in Mice

Inflammatory bowel disease is a chronic gastrointestinal inflammatory disorder associated with changes in neuropeptide expression and function, including vasoactive intestinal peptide (VIP). VIP regulates intestinal vasomotor and secretomotor function and motility; however, VIP’s role in development and maintenance of colonic epithelial barrier homeostasis is unclear. Using VIP deficient (VIPKO) mice, we investigated VIP’s role in epithelial barrier homeostasis, and susceptibility to colitis. Colonic crypt morphology and epithelial barrier homeostasis were assessed in wildtype (WT) and VIPKO mice, at baseline. Colitic responses were evaluated following dinitrobenzene sulfonic acid (DNBS) or dextran-sodium sulfate (DSS) exposure. Mice were also treated with exogenous VIP. At baseline, VIPKO mice exhibited distorted colonic crypts, defects in epithelial cell proliferation and migration, increased apoptosis, and altered permeability. VIPKO mice also displayed reduced goblet cell numbers, and reduced expression of secreted goblet cell factors mucin 2 and trefoil factor 3. These changes were associated with reduced expression of caudal type homeobox 2 (Cdx2), a master regulator of intestinal function and homeostasis. DNBS and DSS-induced colitis were more severe in VIPKO than WT mice. VIP treatment rescued the phenotype, protecting VIPKO mice against DSS colitis, with results comparable to WT mice. In conclusion, VIP plays a crucial role in the development and maintenance of colonic epithelial barrier integrity under physiological conditions and promotes epithelial repair and homeostasis during colitis.