Semen characteristics of genetically identical quadruplet bulls

Modern cloning methods have become an important technology in artificial insemination which is used to create and maintain pools of genetically superior bull semen. In this study, semen from four identical quadruplet bulls (Q(1), Q(2), Q(3), and Q(4)) produced by blastomere separation was analyzed to evaluate the differences in reproductive potential, if any, that existed between the identical quadruplet siblings. Analysis of fresh semen collected from 1994 to 1996, showed lower progressive motility and lower sperm concentration for one bull (Q(3)) compared to his identical brothers (P<0.05). Semen characteristics following freezing-thawing procedures have also been tested for these quadruplet bulls. The percentage of motility, progressive motility, and mean path velocity were lower in Q(4) compared with Q(1). Moreover, intracellular calcium level and P25b level (P25b is a sperm surface protein proposed to be a potential bull fertility marker) were lower in Q(4) compared with his siblings (P<0.05). Cryodamage to Q(4)'s frozen-thawed spermatozoa were confirmed by a lower percentage of embryo development after in vitro fertilization. Thus, the higher instability of cryopreserved spermatozoa from Q(4) and the lower semen production of Q(3), compared to their siblings, indicate that differences in semen characteristics can indeed exist among genetically identical animals produced by blastomere separation.     

Mercury methylation independent of the acetyl-coenzyme A pathway in sulfate-reducing bacteria

Sulfate-reducing bacteria (SRB) in anoxic waters and sediments are the major producers of methylmercury in aquatic systems. Although a considerable amount of work has addressed the environmental factors that control methylmercury formation and the conditions that control bioavailability of inorganic mercury to SRB, little work has been undertaken analyzing the biochemical mechanism of methylmercury production. The acetyl-coenzyme A (CoA) pathway has been implicated as being key to mercury methylation in one SRB strain, Desulfovibrio desulfuricans LS, but this result has not been extended to other SRB species. To probe whether the acetyl-CoA pathway is the controlling biochemical process for methylmercury production in SRB, five incomplete-oxidizing SRB strains and two Desulfobacter strains that do not use the acetyl-CoA pathway for major carbon metabolism were assayed for methylmercury formation and acetyl-CoA pathway enzyme activities. Three of the SRB strains were also incubated with chloroform to inhibit the acetyl-CoA pathway. So far, all species that have been found to have acetyl-CoA activity are complete oxidizers that require the acetyl-CoA pathway for basic metabolism, as well as methylate mercury. Chloroform inhibits Hg methylation in these species either by blocking the methylating enzyme or by indirect effects on metabolism and growth. However, we have identified four incomplete-oxidizing strains that clearly do not utilize the acetyl-CoA pathway either for metabolism or mercury methylation (as confirmed by the absence of chloroform inhibition). Hg methylation is thus independent of the acetyl-CoA pathway and may not require vitamin B(12) in some and perhaps many incomplete-oxidizing SRB strains.     

Fate of 4-hydroxynonenal in vivo: disposition and metabolic pathways

Due to the cytotoxicity of 4-hydroxynonenal (HNE), and to the fact that this major product of lipid peroxidation is a rather long-living compound compared with reactive oxygen species, the capability of organisms to inactivate and eliminate HNE has received increasing attention during the last decade. Several recent in vivo studies have addressed the issue of the diffusion, kinetics, biotransformation and excretion of HNE. Part of these studies are primarily concerned with the toxicological significance of HNE biotransformation and more precisely with the metabolic pathways by which HNE is inactivated and eliminated. The other aim of in vivo metabolic study is the characterisation of end-metabolites, especially in urine, in order to develop specific and non-invasive biomarkers of lipid peroxidation. When HNE is administered intravenously or intraperitoneally, it is mainly excreted into urine and bile as conjugated metabolites, in a proportion that is dependent on the administration route. However, biliary metabolites undergo an enterohepatic cycle that limits the final excretion of faecal metabolites. Only a very low amount of metabolites is found to be bound to macromolecules. The main urinary metabolites are represented by two groups of compounds. One comes from the mercapturic acid formation from (i) 1,4 dihydroxynonene-glutathione (DHN-GSH) which originates from the conjugation of HNE with GSH by glutathione-S-transferases and the subsequent reduction of the aldehyde by a member of aldo-keto reductase superfamily; (ii) the lactone of 4-hydroxynonanoic-GSH (HNA-lactone-GSH) which originates from the conjugation of HNE followed by the oxidation of the aldehyde by aldehyde dehydrogenase; (iii) HNA-GSH which originates from the hydrolysis of the corresponding lactone. The other one is a group of metabolites issuing from the omega-hydroxylation of HNA or HNA-lactone by cytochromes P450 4A, followed eventually, in the case of omega-oxidized-HNA-lactone, by conjugation with GSH and subsequent mercapturic acid formation. Biliary metabolites are GSH or mercapturic acid conjugates of DHN, HNE and HNA. Stereochemical aspects of HNE metabolism are also discussed.     

Characterization of the RpoS status of clinical isolates of Salmonella enterica

The stationary-phase-inducible sigma factor, sigma(S) (RpoS), is the master regulator of the general stress response in Salmonella and is required for virulence in mice. rpoS mutants can frequently be isolated from highly passaged laboratory strains of Salmonella: We examined the rpoS status of 116 human clinical isolates of Salmonella, including 41 Salmonella enterica serotype Typhi strains isolated from blood, 38 S. enterica serotype Typhimurium strains isolated from blood, and 37 Salmonella serotype Typhimurium strains isolated from feces. We examined the abilities of these strains to produce the sigma(S) protein, to express RpoS-dependent catalase activity, and to resist to oxidative stress in the stationary phase of growth. We also carried out complementation experiments with a cloned wild-type rpoS gene. Our results showed that 15 of the 41 Salmonella serotype Typhi isolates were defective in RpoS. We sequenced the rpoS allele of 12 strains. This led to identification of small insertions, deletions, and point mutations resulting in premature stop codons or affecting regions 1 and 2 of sigma(S), showing that the rpoS mutations are not clonal. Thus, mutant rpoS alleles can be found in freshly isolated clinical strains of Salmonella serotype Typhi, and they may affect virulence properties. Interestingly however, no rpoS mutants were found among the 75 Salmonella serotype Typhimurium isolates. Strains that differed in catalase activity and resistance to hydrogen peroxide were found, but the differences were not linked to the rpoS status. This suggests that Salmonella serotype Typhimurium rpoS mutants are counterselected because rpoS plays a role in the pathogenesis of Salmonella serotype Typhimurium in humans or in the transmission cycle of the disease.     

Membrane fluidity adjustments in ethanol-stressed Oenococcus oeni cells

The effect of ethanol on the cytoplasmic membrane of Oenococcus oeni cells and the role of membrane changes in the acquired tolerance to ethanol were investigated. Membrane tolerance to ethanol was defined as the resistance to ethanol-induced leakage of preloaded carboxyfluorescein (cF) from cells. To probe the fluidity of the cytoplasmic membrane, intact cells were labeled with doxyl-stearic acids and analyzed by electron spin resonance spectroscopy. Although the effect of ethanol was noticeable across the width of the membrane, we focused on fluidity changes at the lipid-water interface. Fluidity increased with increasing concentrations of ethanol. Cells responded to growth in the presence of 8% (vol/vol) ethanol by decreasing fluidity. Upon exposure to a range of ethanol concentrations, these adapted cells had reduced fluidity and cF leakage compared with cells grown in the absence of ethanol. Analysis of the membrane composition revealed an increase in the degree of fatty acid unsaturation and a decrease in the total amount of lipids in the cells grown in the presence of 8% (vol/vol) ethanol. Preexposure for 2 h to 12% (vol/vol) ethanol also reduced membrane fluidity and cF leakage. This short-term adaptation was not prevented in the presence of chloramphenicol, suggesting that de novo protein synthesis was not involved. We found a strong correlation between fluidity and cF leakage for all treatments and alcohol concentrations tested. We propose that the protective effect of growth in the presence of ethanol is, to a large extent, based on modification of the physicochemical state of the membrane, i.e., cells adjust their membrane permeability by decreasing fluidity at the lipid-water interface.     

Insertional mutagenesis of a fungal biocontrol agent led to discovery of a rare cellobiose lipid with antifungal activity

Insertional mutagenesis was applied for the first time to a fungal biocontrol agent, Pseudozyma flocculosa, in an attempt to obtain mutants with altered antagonistic properties. Transformants were obtained via DNA-mediated transformation. Molecular analyses of the transformants revealed that multiple copies of the plasmid were integrated in tandem at one to many chromosomal loci. The transformants were screened for their biocontrol properties using standard bioassays, and the 160 tested transformants were classified into four groups: group I mutants (22 transformants) showed a stronger antagonistic effect than the wild type (WT) while those of group II (107 transformants) had a comparable antagonistic effect; group III mutants (17 transformants) had a decreased antagonistic effect relative to WT and group IV mutants (14 transformants) had lost their biocontrol properties. Culture extracts of the mutants (group IV) and WT were analyzed and compared for the presence of active metabolites which were then separated by solid-phase extraction and purified using conventional methods. Nuclear magnetic resonance experiments and analytical studies on a metabolite specifically produced by the WT revealed the presence of 2-(2',4'-diacetoxy-5'-carboxy-pentanoyl) octadecyl cellobioside (flocculosin), a novel glycolipid with strong antifungal properties; the production of this compound would account for the biocontrol activity of P. flocculosa.     

Influence of putrescine on anthocyanin production in callus cultures of <Emphasis Type="Italic">Daucus carota</Emphasis> mediated through calcium ATPase

The influence of Putrescine (Put) on the growth and elicitation of anthocyanin in callus cultures of Daucus carota var. Nantes scarlet was investigated through the use of α-DL-difluoromethylarginine (DFMA), the polyamine (PA) biosynthetic inhibitor. It was observed that the addition of Put (0.05 mM) resulted in enhancement of growth and anthocyanin content. The anthocyanin content was found to be enhanced by 1.68 fold on the 21^st day as compared to the untreated controls. The PA inhibitor was found to result in lowering of the growth and the anthocyanin accumulation, which could be partially restored by the addition of Put in combination with this inhibitor. The levels of Ca²⁺ ATPase were also found to be elevated in treatment with Put suggesting the involvement of calcium in the elicitation of anthocyanin. The endogenous titres of PAs and the ethylene production under these treatments were also studied. The treatment with DFMA resulted in lower levels of endogenous PAs and higher levels of ethylene. Lowering of ethylene by putrescine treatment shows that PA treatment also inhibited ethylene formation, which would also imply that endogenous ethylene does not influence anthocyanin production in carrot callus cultures.     

Differential antioxidant enzyme and thiol responses of tolerant and non-tolerant clones of <Emphasis Type="Italic">Chloris barbata</Emphasis> to cadmium-stress

Tolerant and non-tolerant clones of Chloris barbata Sw. obtained, respectively, from an erstwhile mercury contaminated solid waste dump site near a chloralkali plant and a non-contaminated (control) site were subjected to cadmium-stress by growing the rooted cuttings in water containing CdSO₄, 13 and 130 μM. Differences between the two clones in their response to cadmium-stress were noted in root growth, and also with respect to certain biochemical parameters. Whereas catalase activity decreased and non protein-thiol levels increased in the non-tolerant clone, the level of protein-thiol alone increased significantly in the tolerant clone in response to cadmium-stress. No remarkable differences between the clones, however, were noted with respect to total soluble protein, peroxidase activity and lipid peroxidation. Remarkably the two clones responded differently to buthionine sulfoximine, an inhibitor of glutathione and/or phytochelatin synthesis, which inhibited root growth significantly in non-tolerant clone but not in the tolerant clone. Buthionine sulfoximine, nonetheless, could potentate cadmium toxicity in either of the clones, but more effectively in the tolerant clone. The high sensitivity of tolerant-clone to the combined treatment of BSO and Cd in the present study could, therefore, be attributed to the cumulative oxidative stress generated synergistically by BSO and Cd.     

Effects of inflammation and antiinflammatory treatment on serum trace elements concentrations

We investigated the serum concentrations of zinc and copper during the inflammatory process together with the effect of treatment with a nonsteroid anti-inflammatory agent on these trace elements concentrations. In the present study, we used 92 guinea pigs, 12 of which constituted the control group; the remaining 80 were the experimental group. To start with, proquazone (as anti-inflammatory agent) was administered orally to 40 guinea pigs of the experimental group at 20-mg/kg doses 2 h before the surgery. Throughout the experimental period, the above dose was administered to the animals twice a day. We produced inflammation in all animals of the experimental group by using carrageenan (inflammatory agent) dropped into mandibular surgical defects. Serum concentrations of zinc and copper were determined by atomic absorption spectrophotometry in both groups at the 6th, 48th, 120th, 168th, and 240th h. The serum zinc concentrations of the carrageenan-administered group decreased significantly (p<0.01). When comparing the serum zinc concentrations of the carrageenan plus proquazone-administered group with those of control group, the decrease (p<0.05) at the 6th, 48th, and 120th h were statistically significant. When the copper serum concentrations of the carrageenan-administered group were compared with those of the control group, at the 48th, 120th, and 168th h, a statistically significant increase (p<0.01) was observed. However, there was no significant change in the carrageenan plus proquazone-administered group at the 168th and 240th h. As a result during the acute phase of inflammation, serum zinc concentrations decreased, whereas serum copper concentrations increased. The alterations in zinc concentrations were more rapid than those in copper concentrations, but the administration of proquazone slowed the rate of decrease in serum zinc concentrations. This work was presented at the Fourth International Congress of Pathophysiology, June 29–July 5, 2002, Budapest, Hungary.     

Real-time PCR for chloroquine sensitivity assay and for pfmdr1-pfcrt single nucleotide polymorphisms in Plasmodium falciparum

Plasmodium falciparum drug resistance is a major problem in malaria endemic areas. Molecular markers and in vitro tests have been developed to study and monitor drug resistance. However, none, used alone, can provide sufficient data concerning the level of drug resistance and to issue precise guidelines for drug use policies in endemic areas. We propose real-time PCR for the simultaneous detection of pfcrt and pfmdr1 genes mutations and to determine the half-maximal inhibitory response (IC(50)) of antimalarial drug. Using hybridization probes and SybrGreen technology on LightCycler instrument, point mutations of pfcrt and pfmdr1 genes have been successfully detected in 161 human blood samples and determination of IC values was applied to chloroquine-sensitive and chloroquine-resistant strains. Moreover, mixed infections caused by P. falciparum clones with wild-type or mutant alleles could be efficiency separated. The aim of this study was not to provide definitive data concerning the rate of mutations in an endemic area, but to describe a powerful method allowing the quantification of DNA for IC(50) determination and the detection of major pfmdr1 and pfcrt mutations.