Structural and immunochemical relationship between the O-antigenic polysaccharides from the enteroaggregative Escherichia coli strain 396/C-1 and Escherichia coli O126

The structure of the O-antigen polysaccharide (PS) from the enteroaggregative Escherichia coli strain 396/C-1 has been determined. Sugar and methylation analyses together with 1H and 13C NMR spectroscopy were the main methods used. Inter-residue correlations were determined by 1H,1H-NOESY, 1H,13C-heteronuclear multiple-bond correlation and dipole-dipole cross-correlated relaxation experiments. The PS is composed of pentasaccharide repeating units with the following structure: [structure: see text]. Analysis of NMR data reveals that on average the PS consists of approximately 13 repeating units and indicates that the biological repeating unit contains an N-acetylglucosamine residue at its reducing end. This structure is different to that reported for the O-antigen polysaccharide from E. coli O126. Monospecific anti-E. coli O126 rabbit serum from The International Escherichia and Klebsiella Centre did not distinguish between the E. coli strain 396/C-1 and the E. coli O126 reference strain, neither in slide agglutination nor in an indirect enzyme immunoassay. Subsequent successful serotyping of the E. coli strain 396/C-1 showed it to be E. coli O126:K+:H27.     

Amlodipine decreases fibrosis and cardiac hypertrophy in spontaneously hypertensive rats: persistent effects after withdrawal

Our objective was to examine the effect of chronic treatment with amlodipine on blood pressure, left ventricular hypertrophy, and fibrosis in spontaneously hypertensive rats and the persistence of such an effect after drug withdrawal. We investigated the effects of treatment with 2, 8 and 20 mg/kg/day of amlodipine given orally for six months and at three months after drug withdrawal. Systolic blood pressure was measured using the tail-cuff method. At the end of the study period, the heart was excised, the left ventricle was isolated, and the left ventricle weight/body weight ratio was calculated as a left ventricular hypertrophy index. Fibrosis, expressed as collagen volume fraction, was evaluated using an automated image-analysis system on sections stained with Sirius red. Age-matched untreated Wistar-Kyoto and SHR were used as normotensive and hypertensive controls, respectively. Systolic blood pressure was reduced in the treated SHR in a dose-dependent way and after amlodipine withdrawal it increased progressively, without reaching the values of the hypertensive controls. Cardiac hypertrophy was reduced by 8 and 20 mg/kg/day amlodipine, but when treatment was withdrawn only the group treated with 8 mg/kg/day maintained significant differences versus the hypertensive controls. All three doses of amlodipine reduced cardiac fibrosis and this regression persisted with the two highest doses after three months without treatment. We concluded that antihypertensive treatment with amlodipine is accompanied by a reduction in left ventricular hypertrophy and regression in collagen deposition. Treatment was more effective in preventing fibrosis than in preventing ventricular hypertrophy after drug withdrawal.     

Size and age structure of the striped marlin Tetrapturus audax (Pisces: Xiphidae), at Cabo San Lucas, Baja California sur, México

The striped marlin (Tetrapturus audax) is found in temperate and tropical waters of the Pacific and Indian Ocean. It is particularly abundant in the southern tip of the Baja California peninsula. Specimens brought to Cabo San Lucas port (22 degrees 53' N y 109 degrees 54' W) by the sport fishery fleet that operates withing a radius of 54 km from 1988 to 1993 were sexes, measured and weighed. A total 1030 individuals were sampled. Dorsal fin spines (389) were collected for age estimation. The spines were sectioned and hyaline-opaque bands counted. The relation of length to spine radius was similar for males and females. The fourth dorsal spine reflected the growth of the fish. The rhythm of growth mark formation is yearly and related to the sea surface temperature. Ten age groups were found and the seventh group was the most abundant, followed by groups "6" and "8". The age structure of the striped marlin was stable in the study period. The observed sizes were 160-280 cm jaw length with 90% of the organisms measuring between 190 and 225 cm. The small individuals recruit to the fishery from June to December.     

Counterselection and Co-Delivery of Transposon and Transposase Functions for <Emphasis Type="Italic">Sleeping Beauty</Emphasis>-Mediated Transposition in Cultured Mammalian Cells

Sleeping Beauty (SB) is a gene-insertion system reconstructed from transposon sequences found in teleost fish and is capable of mediating the transposition of DNA sequences from transfected plasmids into the chromosomes of vertebrate cell populations. The SB system consists of a transposon, made up of a gene of interest flanked by transposon inverted repeats, and a source of transposase. Here we carried out a series of studies to further characterize SB-mediated transposition as a tool for gene transfer to chromosomes and ultimately for human gene therapy. Transfection of mouse 3T3 cells, HeLa cells, and human A549 lung carcinoma cells with a transposon containing the neomycin phosphotransferase (NEO) gene resulted in a several-fold increase in drug-resistant colony formation when co-transfected with a plasmid expressing the SB transposase. A transposon containing a methotrexate-resistant dihydrofolate reductase gene was also found to confer an increased frequency of methotrexate-resistant colony formation when co-transfected with SB transposase-encoding plasmid. A plasmid containing a herpes simplex virus thymidine kinase gene as well as a transposon containing a NEO gene was used for counterselection against random recombinants (NEO+TK+) in medium containing G418 plus ganciclovir. Effective counterselection required a recovery period of 5 days after transfection before shifting into medium containing ganciclovir to allow time for transiently expressed thymidine kinase activity to subside in cells not stably transfected. Southern analysis of clonal isolates indicated a shift from random recombination events toward transposition events when clones were isolated in medium containing ganciclovir as well as G418. We found that including both transposon and transposase functions on the same plasmid substantially increased the stable gene transfer frequency in Huh7 human hepatoma cells. The results from these experiments contribute technical and conceptual insight into the process of transposition in mammalian cells, and into the optimal provision of transposon and transposase functions that may be applicable to gene therapy studies.     

Molecular evolution and geographic origins of type 1 human lymphotrophic virus in Colombia detected by RFLP polymorphism

The human T-lymphotropic virus type I (HTLV-I) infection is a public health problem in many endemic areas of Colombia. The subtyping of HTLV-I was based on the analysis of restriction fragment length polymorphisms (RFLP) in 3'LTR proviral DNA. From 31 HTLV-I isolates collected throughout Colombia, a RFLP analysis in a 737 bp 3'LTR fragment was performed. Fifty-eight percent (18/31) were identified as the Cosmopolitan subtype a, 19.4% (6/31) in the West African subtype b, 12.9% (4/31) in the Cosmopolitan subtype b and 9.6% (3/31) in the West African subtype c. The phylogenetic analysis of 3'LTR nucleotide sequences indicated that all the isolates in the current study were in the subgroup B or Japanese, in contrast with the highly divergent isolates from native Amerindians grouped in subgroup a or Transcontinental. The supported hypothesis was that of a post-Columbus introduction of virus represented in the African-American communities of the Colombian South Pacific. Some viral isolates from Colombian native Amerindians exhibited a nucleotide variation compatible with a Paleolithic introduction of the virus. The genetic diversity of HTLV-I in Colombia is complex and probably represents several independent introductions of lymphotropic virus.     

Effect of acute global ischemia on the upper limit of vulnerability: a simulation study

The goal of this modeling research is to provide mechanistic insight into the effect of altered membrane kinetics associated with 5-12 min of acute global ischemia on the upper limit of cardiac vulnerability (ULV) to electric shocks. We simulate electrical activity in a finite-element bidomain model of a 4-mm-thick slice through the canine ventricles that incorporates realistic geometry and fiber architecture. Global acute ischemia is represented by changes in membrane dynamics due to hyperkalemia, acidosis, and hypoxia. Two stages of acute ischemia are simulated corresponding to 5-7 min (stage 1) and 10-12 min (stage 2) after the onset of ischemia. Monophasic shocks are delivered in normoxia and ischemia over a range of coupling intervals, and their outcomes are examined to determine the highest shock strength that resulted in induction of reentrant arrhythmia. Our results demonstrate that acute ischemia stage 1 results in ULV reduction to 0.8A from its normoxic value of 1.4A. In contrast, no arrhythmia is induced regardless of shock strength in acute ischemia stage 2. An investigation of mechanisms underlying this behavior revealed that decreased postshock refractoriness resulting mainly from 1) ischemic electrophysiological substrate and 2) decrease in the extent of areas positively-polarized by the shock is responsible for the change in ULV during stage 1. In contrast, conduction failure is the main cause for the lack of vulnerability in acute ischemia stage 2. The insight provided by this study furthers our understanding of mechanisms by which acute ischemia-induced changes at the ionic level modulate cardiac vulnerability to electric shocks.     

Hydroxyl radical activation of a Ca(2+)-sensitive nonselective cation channel involved in epithelial cell necrosis

In a previous work the involvement of a fenamate-sensitive Ca²⁺-activated nonselective cation channel (NSCC) in free radical-induced rat liver cell necrosis was demonstrated (5). Therefore, we studied the effect of radical oxygen species and oxidizing agents on the gating behavior of a NSCC in a liver-derived epithelial cell line (HTC). Single-channel currents were recorded in HTC cells by the excised inside-out configuration of the patch-clamp technique. In this cell line, we characterize a 19-pS Ca²⁺-activated, ATP- and fenamate-sensitive NSCC nearly equally permeable to monovalent cations. In the presence of Fe²⁺, exposure of the intracellular side of NSCC to H₂O₂ increased their open probability (P_o) by 40% without affecting the unitary conductance. Desferrioxamine as well as the hydroxyl radical (·OH) scavenger MCI-186 inhibited the effect of H₂O₂, indicating that the increase in P_o was mediated by ·OH. Exposure of the patch membrane to the oxidizing agent 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB) had a similar effect to ·OH. The increase in P_o induced by ·OH or DTNB was not reverted by preventing formation or by DTNB washout, respectively. However, the reducing agent dithiothreitol completely reversed the effects on P_o of both ·OH and DTNB. A similar increase in P_o was observed by applying the physiological oxidizing molecule GSSG. Moreover, GSSG-oxidized channels showed enhanced sensitivity to Ca²⁺. The effect of GSSG was fully reversed by GSH. These results suggest an intracellular site(s) of action of oxidizing agents on cysteine targets on the fenamate-sensitive NSCC protein implicated in epithelial cell necrosis. Ca²⁺-activated channels; radical oxygen species; oxidative stress     

Retroviral restriction factor TRIM5alpha is a trimer

The retrovirus restriction factor TRIM5alpha targets the viral capsid soon after entry. Here we show that the TRIM5alpha protein oligomerizes into trimers. The TRIM5alpha coiled-coil and B30.2(SPRY) domains make important contributions to the formation and/or stability of the trimers. A functionally defective TRIM5alpha mutant with the RING and B-box 2 domains deleted can form heterotrimers with wild-type TRIM5alpha, accounting for the observed dominant-negative activity of the mutant protein. Trimerization potentially allows TRIM5alpha to interact with threefold pseudosymmetrical structures on retroviral capsids.     

Tumor cells deactivate human monocytes by up-regulating IL-1 receptor associated kinase-M expression via CD44 and TLR4

Although blood monocytes possess significant cytotoxic activity against tumor cells, tumor-infiltrating monocytes are commonly deactivated in cancer patients. Monocytes pre-exposed to tumor cells show significantly decreased expression levels of TNF-alpha, IL-12p40, and IL-1R-associated kinase (IRAK)-1. Activation of the Ser/Thr kinase IRAK-1 is an important event in several inflammatory processes. By contrast, another IRAK family member, IRAK-M, negatively regulates this pathway, and is up-regulated in cultures of endotoxin-tolerant monocytes and in monocytes from septic patients within the timeframe of tolerance. In this study, we show that IRAK-M expression is enhanced at the mRNA and protein level in human monocytes cultured in the presence of tumor cells. IRAK-M was induced in monocytes upon coculturing with different tumor cells, as well as by fixed tumor cells and medium supplemented with the supernatant from tumor cell cultures. Moreover, blood monocytes from patients with chronic myeloid leukemia and patients with metastasis also overexpressed IRAK-M. Low concentrations of hyaluronan, a cell surface glycosaminoglycan released by tumor cells, also up-regulated IRAK-M. The induction of IRAK-M by hyaluronan and tumor cells was abolished by incubation with anti-CD44 or anti-TLR4 blocking Abs. Furthermore, down-regulation of IRAK-M expression by small interfering RNAs specific for IRAK-M reinstates both TNF-alpha mRNA expression and protein production in human monocytes re-exposed to a tumor cell line. Altogether, our findings indicate that deactivation of human monocytes in the presence of tumor cells involves IRAK-M up-regulation, and this effect appears to be mediated by hyaluronan through the engagement of CD44 and TLR4.