IN VIVO EFFECTS OF CARDIOTROPHIN-1

Cardiotrophin-1 (CT-1) is a recently discovered cytokine that was isolated based on its ability to induce cardiac myocyte hypertrophy in vitro. In this study, the effects of chronic administration of CT-1 to mice (0.5 or 2 μg by intraperitoneal injection, twice a day for 14 days) were determined. A dose-dependent increase in both the heart weight and ventricular weight to body ratios was observed in the treated groups. The body weights of the animals were unaffected. These results indicate that CT-1 can induce cardiac hypertrophy in vivo. CT-1 was not specific for the heart, however. It stimulated the growth of the liver, kidney, and spleen, and caused atrophy of the thymus. CT-1 administration also increased the platelet counts by 70%, with no change in mean platelet volume. Red blood cell counts were increased in the treated animals, and there was a concomitant increase in haemoglobin concentration. Thus, CT-1 has a broad spectrum of biological activities in vivo. This observation is consistent with previous in-vitro findings showing that the mRNA for CT-1 is expressed in several tissues, and that CT-1 can function through binding to the leukaemia inhibitory factor (LIF) receptor and signalling through the gp130 pathway.     

Treatment of Helicobacter pylori Infection: A Review of the World Literature

Background. None of the currently used anti- Helicobacter pylori drug regimens cures the infection 100%, and cure results still vary considerably. The present article reviews the effectiveness of currently used antimicrobial regimens, aimed to cure H. pylori infection.
Methods. Data collection started from the beginning of the anti- H. pylori -therapy era until May 1995. No attempt at formal metanalysis has been made, because many studies have been published only in abstract form. Attempts were made to exclude duplicates of studies by comparison to previously reported ones; the authors of suspected duplicates were contacted. After amalgamation of the number of included patients and the number of successfully treated patients, the mean values of eradication rates and the 95% confidence intervals were calculated.
Results. A total of 237 treatment arms were analyzed. Bismuth triple therapy continues to reach high eradication rates worldwide (78–89%). Side effects leading to diminished patient compliance and the marked decline of eradication efficacy in cases of metronidazole resistance are considered to be the major drawbacks of this therapy. Proton pump inhibitor (PPI) dual therapy is better tolerated with fewer side effects than is bismuth triple therapy. The mean eradication rates vary from 55 to 75%, and the extremes lie between 24 and 93%. PPI triple therapies have been shown to be very effective against H. pylori (eradication rates, 80–89%). Quadruple therapy leads to a mean eradication rate of 96%.
Conclusion. Based on efficacy, PPI triple or bismuth triple therapy are recommended as first-line treatment for H. pylori infection. Quadruple therapy could serve as second-line treatment for eradication of initial failures and in case of metronidazole resistance.     

Identification of molecular markers linked to the Agropyron elongatum-derived leaf rust resistance gene Lr24 in wheat

The objective of this study was to identify molecular markers linked to the wheat leaf rust resistance gene Lr24 derived from Agropyron elongatum (3DL/3Ag translocation). Two near isogenic lines (NILs), ‘Arina’ and Lr24/7 ^* “Arina”, were screened for polymorphism at the DNA level with 115 RFLP probes. Twenty-one of these probes map to the homoeologous group 3. In addition, 360 RAPD primers were tested on the NILs. Six RFLP probes showed polymorphism between the NILs, and 11 RAPD primers detected one additional band in the resistant NIL. The genetic linkage of the polymorphic markers with Lr24 was tested on a segregating F₂ population (150 plants) derived from a cross between the leaf rust resistant Lr24/7 ^* “Arina” and the susceptible spelt (Triticum spelta) variety ‘Oberkulmer’. All 6 RFLP markers were completely linked to Lr24: one was inherited as a codominant marker (PSR1205), one was in coupling phase (PSR1203) and 4 were in repulsion phase (PSR388, PSR904, PSR931, PSR1067) with Lr24. The localization of these probes on chromosome 3D was confirmed by nulli-tetrasomic analysis. Distorted genotypic segregation was found for the Codominant RFLP marker PSR1205. This distortion can be explained by the occurrence of hemizygous plants. One of the 11 RAPD markers (OPJ-09) also showed complete linkage to theLr24 resistance gene. The polymorphic RAPD fragment was cloned and sequenced. Specific primers were synthesized, and they produced an amplification product only in the resistant plants. This specific marker allows a reliable and rapid screening of a large number of genotypes in practical breeding. Analysis of 6 additional lines containing Lr24 revealed that 3 lines have a smaller chromosomal segment of A. elongatum than lines derived from ‘Agent’, a commonly used gene donor for the Lr24 resistance gene.     

Increased chilling tolerance and altered carbon metabolism in tomato leaves following application of mechanical stress

We investigated the effects of brushing on the chilling tolerance and metabolism of nonstructural carbohydrates (soluble sugars and starch) in tomato leaves before, during and after a chilling stress. Tomato plants ( Lycopersicon esculentum Mill. cv. Caruso) were cultivated either without mechanical stress application (control plants) or with daily brushing treatments for 15 days (brushed plants), prior to a 7-day chilling treatment (8/5°C day/night). Brushing resulted in shorter plants with a 34% reduction in leaf dry weight per area and a 59% reduction of soluble sugars and starch, on a dry weight basis. The sugar to starch ratio was not affected by brushing. A greater chilling tolerance in the brushed plants was demonstrated by the maintenance of a significantly higher PSII efficiency in brushed plants (42%) compared to that of the control plants (30%) after 7 days of chilling treatment, less visible damage to the leaf tissue, and a more rapid resumption of growth during 3 days of recovery as compared to control plants. During the chilling treatment levels of soluble sugars per leaf dry weight increased 15-fold in the brushed plants and 5-fold in control plants. In the present study we have demonstrated that brushing can increase chilling tolerance in tomato plants. The observed differences in chilling tolerance and concentration of soluble sugars in the leaves may indicate an involvement of soluble sugar levels in acclimation to chilling.     

Molecular cloning of a new receptor-like kinase gene encoded at the Lr10 disease resistance locus of wheat

More than 100 resistance genes against wheat rust pathogens have been described in wheat and its relatives. Although many of them have been extensively used in wheat resistance breeding, none of these resistance loci has yet been analyzed at the molecular level. By screening a set of near-isogenic lines carrying different leaf rust resistance genes with a wheat probe encoding a serine/threonine protein kinase, we detected a polymorphic DNA fragment in the line with the Lr10 resistance gene. This fragment mapped to the Lr10 disease resistance locus and encodes a receptor-like protein kinase which we called LRK10. LRK10 contains a new type of extracellular domain not found in known plant or animal receptor kinases. Several conserved amino acids in S-domain glycoproteins and receptor-like kinases were also found in LRK10, suggesting that LRK10 and S-domain proteins belong to the same superfamily of specific recognition molecules in plants. Lrk10 was expressed at low levels in young seedlings and belongs to a gene family. Analysis of wheat lines with and without the Lr10 gene demonstrated that Lrk10 and Lr10 belong to the same genetic locus. We conclude that gene isolation based on protein kinase homology can identify new receptor domains and provide candidates for disease resistance genes in the complex wheat genome.     

Hierarchical Analysis of Genetic Structure in Native Fire Ant Populations: Results from Three Classes of Molecular Markers

We describe genetic structure at various scales in native populations of the fire ant Solenopsis invicta using two classes of nuclear markers, allozymes and microsatellites, and markers of the mitochondrial genome. Strong structure was found at the nest level in both the monogyne (single queen) and polygyne (multiple queen) social forms using allozymes. Weak but significant microgeographic structure was detected above the nest level in polygyne populations but not in monogyne populations using both classes of nuclear markers. Pronounced mitochondrial DNA (mtDNA) differentiation was evident also at this level in the polygyne form only. These microgeographic patterns are expected because polygyny in ants is associated with restricted local gene flow due mainly to limited vagility of queens. Weak but significant nuclear differentiation was detected between sympatric social forms, and strong mtDNA differentiation also was found at this level. Thus, queens of each form seem unable to establish themselves in nests of the alternate type, and some degree of assortative mating by form may exist as well. Strong differentiation was found between the two study regions using all three sets of markers. Phylogeographic analyses of the mtDNA suggest that recent limitations on gene flow rather than longstanding barriers to dispersal are responsible for this large-scale structure.     

The Lipid Peroxidation Product, 4-Hydroxy-2-trans-Nonenal, Alters the Conformation of Cortical Synaptosomal Membrane Proteins

Abstract: Alzheimer's disease (AD) is widely held to be a disorder associated with oxidative stress due, in part, to the membrane action of amyloid β-peptide (Aβ). Aβ-associated free radicals cause lipid peroxidation, a major product of which is 4-hydroxy-2- trans -nonenal (HNE). We determined whether HNE would alter the conformation of synaptosomal membrane proteins, which might be related to the known neurotoxicity of Aβ and HNE. Electron paramagnetic resonance spectroscopy, using a protein-specific spin label, MAL-6(2,2,6,6-tetramethyl-4-maleimidopiperidin-1-oxyl), was used to probe conformational changes in gerbil cortical synaptosomal membrane proteins, and a lipid-specific stearic acid label, 5-nitroxide stearate, was used to probe for HNE-induced alterations in the fluidity of the bilayer domain of these membranes. Synaptosomal membranes, incubated with low concentrations of HNE, exhibited changes in protein conformation and bilayer order and motion (fluidity). The changes in protein conformation were found to be concentration- and time-dependent. Significant protein conformational changes were observed at physiologically relevant concentrations of 1–10 µ M HNE, reminiscent of similar changes in synaptosomal membrane proteins from senile plaque- and Aβ-rich AD hippocampal and inferior parietal brain regions. HNE-induced modifications in the physical state of gerbil synaptosomal membrane proteins were prevented completely by using excess glutathione ethyl ester, known to protect neurons from HNE-caused neurotoxicity. Membrane fluidity was found to increase at higher concentrations of HNE (50 µ M ). The results obtained are discussed with relevance to the hypothesis of Aβ-induced free radical-mediated lipid peroxidation, leading to subsequent HNE-induced alterations in the structure and function of key membrane proteins with consequent neurotoxicity in AD brain.     

Cloning of cDNAs encoding the 160 kDa subunit of the bovine cleavage and polyadenylation specificity factor.

3'-processing of mRNA precursors depends on several protein factors. One of them, cleavage and polyadenylation specificity factor (CPSF) is required for the cleavage of the mRNA precursor or as well as for the tail elongation reaction. We have obtained complementary DNA encoding the 160 kDa subunit, which had previously been shown to interact with the AAUAAA polyadenylation signal. The cDNAs code for an open reading frame of 1444 amino acids. The translated protein has a calculated molecular weight of 161 kDa and a predicted pl of 6.2. Polyclonal antibodies raised against a bacterially expressed fragment of the cDNA recognise 160 kDa subunit of purified calf thymus CPSF. The sequence contains a possible nuclear localisation signal but none of the known RNA binding motifs. It does, however, show sequence similarities to a UV-damaged DNA binding protein (UVdDb).     

Open-loop simulations of the primate saccadic system using burst cell discharge from the superior colliculus

 Saccade-related burst neurons (SRBNs) in the monkey superior colliculus (SC) have been hypothesized to provide the brainstem saccadic burst generator with the dynamic error signal and the movement initiating trigger signal. To test this claim, we performed two sets of open-loop simulations on a burst generator model with the local feedback disconnected using experimentally obtained SRBN activity as both the driving and trigger signal inputs to the model. First, using neural data obtained from cells located near the middle of the rostral to caudal extent of the SC, the internal parameters of the model were optimized by means of a stochastic hill-climbing algorithm to produce an intermediate-sized saccade. The parameter values obtained from the optimization were then fixed and additional simulations were done using the experimental data from rostral collicular neurons (small saccades) and from more caudal neurons (large saccades); the model generated realistic saccades, matching both position and velocity profiles of real saccades to the centers of the movement fields of all these cells. Second, the model was driven by SRBN activity affiliated with interrupted saccades, the resumed eye movements observed following electrical stimulation of the omnipause region. Once again, the model produced eye movements that closely resembled the interrupted saccades produced by such simulations, but minor readjustment of parameters reflecting the weight of the projection of the trigger signal was required. Our study demonstrates that a model of the burst generator produces reasonably realistic saccades when driven with actual samples of SRBN discharges. Received: 25 October 1994/Accepted in revised form: 20 June 1995     

A TnphoA insertion within the Bradyrhizobium japonicum sipS gene, homologous to prokaryotic signal peptidases, results in extensive changes in the expression of PBM-specific nodulins of infected soybean (Glycine max) cells

Bradyrhizobium japonicum mutant 132 was obtained by random TnphoA mutagenesis of strain 110spc4. A 6.5 kb BamHI kanamycin-resistance-coding DNA fragment of mutant 132 was used as a hybridization probe to clone the corresponding wild-type fragment. DNA sequence analysis of a 3213 bp BamHI—ClaI fragment revealed that three open reading frames (ORFs) were encoded in the same orientation. Based on sequence similarities to other proteins in the database, the second ORF was called sipS (signal peptidase). The TnphoA insertion in mutant 132 was found to be in frame near the 3′ end of sipS. The resulting SipS—PhoA hybrid polypeptide was shown to be expressed in free-living B. japonicum and in Escherichia coli cultures. An immunoblot analysis with a polyclonal antibody directed against the alkaline phosphatase revealed the appearance of a weak signal of a 70 kDa polypeptide both in B. Japonicum and in E. coli, in agreement with the calculated size of the hybrid polypeptide. A much stronger 52 kDa band was also detected. Mutant 132 was specifically disturbed in the interaction with soybean (Glycine max) when the bacteria were released from the infection threads. The bacteroids were not stably maintained within the symbiosome. Numerous vesicles were found in the plant cytosol, which finally resulted in the formation of large vacuoles within the infected nodule cells. Immunohistochemical analyses with antibodies directed against nodulins of the peribacteroid membrane indicated a lower expression of these proteins. The mutant phenotype was genetically complemented by 4.4 kb BamHI fragment including sipS. Subfragments thereof also complemented a temperature-sensitive E. coli lepB mutant, demonstrating that the B. japonicum fragment was functionally replacing Lep^ts in E. coli. Genetic studies suggested that the three genes are organized in one common operon which is expressed from a promoter upstream of the sequenced region. Inactivation of the gene downstream of sipS did not result in a detectable phenotype.