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PubMed2002

Demecolcine-induced oocyte enucleation for somatic cell cloning: coordination between cell-cycle egress, kinetics of cortical cytoskeletal interactions, and second polar body extrusion.

Studies were designed to further explore the use of pharmacological agents to produce developmentally competent enucleated mouse oocytes for animal cloning by somatic cell nuclear transfer. Metaphase II oocytes from CF-1 and B6D2F1 strains were activated with ethanol and subsequently exposed to demecolcine at various times postactivation. Chromosome segregation, spindle dynamics, and polar body (PB) extrusion were monitored by fluorescence microscopy using DNA-, microtubule-, and microfilament-selective probes. Exposure to demecolcine did not affect rates of oocyte activation induced by ethanol but did disrupt the coordination of cytokinesis and karyokinesis, suppressing the extent and completion of spindle rotation and second PB extrusion in a strain-dependent manner. Moreover, strain- and treatment-specific variations in the rate of oocyte enucleation were also detected. In particular, CF1 oocytes were more efficiently enucleated relative to B6D2F1 oocytes, and demecolcine treatments initiated early after activation resulted in higher enucleation rates than when treatment was delayed. The observed strain differences are possibly caused by a combination of factors, such as the time course of meiotic cell-cycle progression after ethanol activation, the degree of spindle rotation, and the extent of second PB extrusion. These results suggest that developmentally competent cytoplasts can be produced by timely exposure of activated oocytes to agents that disrupt spindle microtubules. However, the utility of the demecolcine-induced enucleation protocol will require further investigation into factors linking karyokinesis to cytokinesis at the levels of cell-cycle control and oocyte cytoskeletal remodeling following artificial or natural means of egg activation.

Animals↗

PubMed2006

An operon in Streptococcus pneumoniae containing a putative alkylhydroperoxidase D homologue contributes to virulence and the response to oxidative stress.

Analysis of the pneumococcal genome sequences from strains R6 and TIGR4 identified a putative alkylhydroperoxidase homologue RT-PCR showed this gene to be expressed in an operon with the downstream open reading frame. No probable function for this second gene is suggested although it appears to be an integral membrane protein. An allelic replacement mutant lacking this two-gene operon in strain D39 was attenuated in competitive infections with the wild type parent. This operon is, therefore, a novel pneumococcal virulence determinant. In line with a role in the response to oxidative stress, this mutant showed enhanced resistance to killing by hydrogen peroxide, a phenotype shared by alkylhydroperoxidase mutants in other bacterial species. The analysis of non-polar single mutants shows that both genes contribute to these phenotypes. Finally, an important role in pneumococcal biology is suggested by the presence of this operon in all 20 clinical isolates examined and the highly conserved sequence of the two genes.

Amino Acid Sequence↗

PubMed2003

Upstream short sequence repeats regulate expression of the alpha C protein of group B Streptococcus.

Group B streptococci (GBS) express a family of repeat-containing surface proteins, the prototype of which is the alpha C protein expressed in type Ia/C strain A909. We have isolated a series of mutant GBS strains by mouse-passage of A909 that do not produce normal levels of the alpha C protein. Polymerase chain reaction amplification and sequencing of the gene encoding the alpha C protein, bca, from four mutant strains revealed the presence of a full-length gene in each strain. However, Northern and RT-PCR analysis revealed greatly reduced levels of RNA encoding the alpha C protein. Sequence analysis of the mutant genes found the coding region unchanged from the wild-type gene in each case, but variation was observed in a specific locus located 110 bp upstream of the start codon. The presence of a 5-nucleotide repeat, AGATT, and a string of adenine residues mark this locus. Both deletion and expansion of the AGATT motif were associated with the complete null phenotype. Deletions in the string of adenine residues were associated with both a decreased-production phenotype and a complete null phenotype. Cloning of this upstream region into a green-fluorescent protein (GFP) reporter system in GBS demonstrated promoter activity that was completely abolished by changes in the pentanucleotide repeat or adenine string. Primer extension studies of the wild-type strain revealed one dominant and two minor transcription start sites. Primer extension studies of the null and low-expression mutant strains revealed that the dominant transcript is completely absent in each mutant. The short sequence repeat locus is located at position - 55 to - 78 relative to the start site of the dominant transcript. We have demonstrated in vitro phase variation in expression of the alpha C protein associated with variation at the pentanucleotide repeat locus. We conclude that this short sequence repeat motif is located upstream of the dominant promoter for the alpha C protein and represents a regulatory site for alpha C protein expression. This is the first evidence of transcriptional regulation by short-sequence repeats in a Gram-positive organism.

Animals↗

PubMed2003

Genetic component of sensorimotor capacity in rats.

Creation of generalized genetic models for low and high sensorimotor capacity would be important tools for resolution of this complex trait. As proof-of-principle we estimated phenotypic variation and narrow-sense heritability (h(2)) of sensorimotor capacity in 19 families of genetically heterogeneous N:NIH rats and in 11 strains of inbred rats. Sensorimotor capacity was defined as the time a rat remained on an accelerating rotorod. N:NIH rats recorded variation in rotorod scores that ranged from 3- to 7-fold. The value of h(2), estimated from offspring-parent regression across one generation, was 0.68 for females and 0.74 for males in N:NIH rats. In inbred rats, h(2) was estimated by partitioning phenotypic variation into additive genetic and environmental components and averaged 0.39 in females and 0.48 in males. These results demonstrate a heritable component to sensorimotor capacity sufficient for success in developing contrasting genetic models by divergent artificial selection in rats.

Animals↗

PubMed2003

Oxygen regulates invasiveness and virulence of group B streptococcus.

The facultative anaerobe group B Streptococcus (GBS) is an opportunistic pathogen of pregnant women, newborns, and the elderly. Although several virulence factors have been identified, environmental factors that regulate the pathogenicity of GBS have not been well characterized. Using the dynamic in vitro attachment and invasion system (DIVAS), we examined the effect of oxygen on the ability of GBS to invade immortalized human epithelial cells. GBS type III strain M781 invaded human epithelial cells of primitive neurons, the cervix, the vagina, and the endometrium in 5- to 400-fold higher numbers when cultured at a cell mass doubling time (t(d)) of 1.8 h than at a slower t(d) of 11 h. Invasion was optimal when GBS was cultured at a t(d) of 1.8 h in the presence of >or=5% oxygen and was significantly reduced without oxygen. Moreover, GBS grown in a chemostat under highly invasive conditions (t(d) of 1.8 h, with oxygen) was more virulent in neonatal mice than was GBS grown under suboptimal invasion conditions (t(d) of 1.8 h, without oxygen), suggesting a positive association between in vitro invasiveness with DIVAS and virulence.

Animals↗

PubMed2004

GNA33 of Neisseria meningitidis is a lipoprotein required for cell separation, membrane architecture, and virulence.

GNA33 is a membrane-bound lipoprotein with murein hydrolase activity that is present in all Neisseria species and well conserved in different meningococcal isolates. The protein shows 33% identity to a lytic transglycolase (MltA) from Escherichia coli and has been shown to be involved in the degradation of both insoluble murein sacculi and unsubstituted glycan strands. To study the function of the gene and its role in pathogenesis and virulence, a knockout mutant of a Neisseria meningitidis serogroup B strain was generated. The mutant exhibited retarded growth in vitro. Transmission electron microscopy revealed that the mutant grows in clusters which are connected by a continuous outer membrane, suggesting a failure in the separation of daughter cells. Moreover, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of culture supernatant revealed that the mutant releases several proteins in the medium. The five most abundant proteins, identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis, belong to the outer membrane protein family. Finally, the mutant showed an attenuated phenotype, since it was not able to cause bacteremia in the infant rat model. We conclude that GNA33 is a highly conserved lipoprotein which plays an important role in peptidoglycan metabolism, cell separation, membrane architecture, and virulence.

Animals↗

PubMed2000

Fragilysin, the enterotoxin from Bacteroides fragilis, enhances the serum antibody response to antigen co-administered by the intranasal route.

Fragilysin, an extracellular zinc metalloprotease produced by enterotoxigenic strains of the anaerobic bacterium Bacteroides fragilis, disrupts the paracellular barrier by cleavage of the intercellular proteins between epithelial cells resulting in fluid secretion. Intranasal immunization of mice with fragilysin and co-administered ovalbumin (Ova) resulted in an Ova-specific serum IgG response that was over 18000-fold higher than Ova alone, as well as detectable levels of serum IgA. Serum IgG titers were comparable with those seen when whole cholera toxin was used as the adjuvant, although the responses obtained with fragilysin showed more variability between mice. Metalloproteases to which fragilysin is structurally related were ineffective as mucosal adjuvants. Our results and similar studies with enterotoxins that affect the paracellular barrier suggest that alteration of mucosal permeability may play an important role in the mechanisms of adjuvanticity.

Adjuvants, Immunologic↗

PubMed2004

The pmrF polymyxin-resistance operon of Yersinia pseudotuberculosis is upregulated by the PhoP-PhoQ two-component system but not by PmrA-PmrB, and is not required for virulence.

The Yersinia pseudotuberculosis chromosome contains a seven-gene polycistronic unit (the pmrF operon) whose products share extensive homologies with their pmrF counterparts in Salmonella enterica serovar Typhimurium (S. typhimurium), another Gram-negative bacterial enteropathogen. This gene cluster is essential for addition of 4-aminoarabinose to the lipid moiety of LPS, as demonstrated by MALDI-TOF mass spectrometry of lipid A from both wild-type and pmrF-mutated strains. As in S. typhimurium, 4-aminoarabinose substitution of lipid A contributes to in vitro resistance of Y. pseudotuberculosis to the antimicrobial peptide polymyxin B. Whereas pmrF expression in S. typhimurium is mediated by both the PhoP-PhoQ and PmrA-PmrB two-component regulatory systems, it appears to be PmrA-PmrB-independent in Y. pseudotuberculosis, with the response regulator PhoP interacting directly with the pmrF operon promoter region. This result reveals that the ubiquitous PmrA-PmrB regulatory system controls different regulons in distinct bacterial species. In addition, pmrF inactivation in Y. pseudotuberculosis has no effect on bacterial virulence in the mouse, again in contrast to the situation in S. typhimurium. The marked differences in pmrF operon regulation in these two phylogenetically close bacterial species may be related to their dissimilar lifestyles.

Animals↗

PubMed2006

Immunogenicity of West Nile virus infectious DNA and its noninfectious derivatives.

The exceptionally high virulence of the West Nile NY99 strain makes its suitability in the development of a live WN vaccine uncertain. The aim of this study is to investigate the immunogenicity of noninfectious virus derivatives carrying pseudolethal mutations, which preclude virion formation without affecting preceding steps of the viral infectious cycle. When administered using DNA immunization, such constructs initiate an infectious cycle but cannot lead to a viremia. While the magnitude of the immune response to a noninfectious replication-competent construct was lower than that of virus or infectious DNA, its overall quality and the protective effect were similar. In contrast, a nonreplicating construct of similar length induced only a marginally detectable immune response in the dose range used. Thus, replication-competent noninfectious constructs derived from infectious DNA may offer an advantageous combination of the safety of noninfectious formulations with the quality of the immune response characteristic of infectious vaccines.

Amino Acid Sequence↗

PubMed2003

Development of an improved selective agar medium for isolation of Yersinia pestis.

Existing media designed for selective isolation of clinically important members of the genus Yersinia were found to be unsatisfactory for the growth and isolation of Yersinia pestis. We report the development of a new selective agar medium (termed BIN) that supports the growth of Y. pestis. The development of the formulation of this medium was based on a fluorescence screening system designed for monitoring bacterial growth on semisolid media, using a green fluorescent protein-expressing strain. High-throughput combinatorial experiments can be conducted for the quantitative evaluation of the effect of different medium components on growth. Generation of fluorescence plots in this system, using microplates, allowed the quantitative evaluation of the growth rate of Y. pestis EV76 cultures in different agar compositions. The final BIN formulation is based on brain heart infusion agar, to which the selective agents irgasan, cholate salts, crystal violet, and nystatin were introduced. It was found that BIN agar is more efficient in supporting colony formation and recovery of Y. pestis than are the conventional semisolid media MacConkey agar and Yersinia-selective agar (cefsulodin-irgasan-novobiocin agar). The advantage of BIN over other media has been also demonstrated in recovering virulent Y. pestis from the mixed bacterial populations found in decaying carcasses of infected mice. The BIN medium is suggested as a selective medium for isolation and recovery of Y. pestis from various backgrounds.

Agar↗

PubMed2002

Global transcriptional analysis of clpP mutations of type 2 Streptococcus pneumoniae and their effects on physiology and virulence.

Streptococcus pneumoniae is an important human pathogen that contains single copies of genes encoding the ClpP and FtsH ATP-dependent proteases but lacks the Lon and HslV proteases. We constructed and characterized the phenotypes of clpP, clpC, and clpX deletion replacement mutants, which lack the ClpP protease subunit or the putative ClpC or ClpX ATPase specificity factor. A DeltaclpP mutant, but not a DeltaclpC or DeltaclpX mutant, of the virulent D39 type 2 strain of S. pneumoniae grew poorly at 30 degrees C and failed to grow at 40 degrees C. Despite this temperature sensitivity, transcription of the heat shock regulon determined by microarray analysis was induced in a DeltaclpP mutant, which was also more sensitive to oxidative stress by H2O2 and to puromycin than its clpP+ parent strain. A DeltaclpP mutant, but not a DeltaclpC mutant, was strongly attenuated for virulence in the murine lung and sepsis infection models. All of these phenotypes were complemented in a DeltaclpP/clpP+ merodiploid strain. Consistent with these complementation patterns, clpP was found to be in a monocistronic operon, whose transcription was induced about fivefold by heat shock in S. pneumoniae as determined by Northern and real-time reverse transcription-PCR analyses. Besides clpP, transcription of clpC, clpE, and clpL, but not clpX or ftsH, was induced by heat shock or entry into late exponential growth phase. Microarray analysis of DeltaclpP mutants showed a limited change in transcription pattern (approximately 80 genes) consistent with these phenotypes, including repression of genes involved in oxidative stress, metal ion transport, and virulence. In addition, transcription of the early and late competence regulon was induced in the DeltaclpP mutant, and competence gene expression and DNA uptake seemed to be constitutively induced throughout growth. Together, these results indicate that ClpP-mediated proteolysis plays a complex and central role in numerous pneumococcal stress responses, development of competence, and virulence.

ATPases Associated with Diverse Cellular Activitie↗

PubMed2005

Specificity of serum neutralizing antibodies induced by transient immune suppression of inapparent carrier ponies infected with a neutralization-resistant equine infectious anemia virus envelope strain.

It has been previously reported that transient corticosteroid immune suppression of ponies experimentally infected with a highly neutralization resistant envelope variant of equine infectious anemia virus (EIAV), designated EIAV(DeltaPND), resulted in the appearance of type-specific serum antibodies to the infecting EIAV(DeltaPND) virus. The current study was designed to determine if this induction of serum neutralizing antibodies was associated with changes in the specificity of envelope determinants targeted by serum antibodies or caused by changes in the nature of the antibodies targeted to previously defined surface envelope gp90 V3 and V4 neutralization determinants. To address this question, the envelope determinants of neutralization by post-immune suppression serum were mapped. The results demonstrated that the neutralization sensitivity to post-immune suppression serum antibodies mapped specifically to the surface envelope gp90 V3 and V4 domains, individually or in combination. Thus, these data indicate that the development of serum neutralizing antibodies to the resistant EIAV(DeltaPND) was due to an enhancement of host antibody responses caused by transient immune suppression and the associated increase in virus replication.

Amino Acid Sequence↗

PubMed2001

Chronic toxicity of thiabendazole (TBZ) in CD-1 mice.

Male and female CD-1 mice (50 mice per group) were administered thiabendazole (TBZ) in diet at levels of 0 (control), 0.031, 0.125 and 0.5% for 78 weeks. A life time study was terminated after 78 weeks due to enhanced strain specific mortality. There were no significant differences in mortality between the control and treated groups. Mean body weights of high-dose groups showed significant decreases compared with the controls. The bladder weights of male and female mice of the 0.5% group were significantly higher than those of the control mice. Gross findings in treated mice included the renal atrophy, hydronephrosis, calculi in renal pelvis and/or bladder and ovarian atrophy. Microscopic findings in the kidneys of treated mice included the nephrosis, hydronephrosis or hyperplasia of transitional epithelium of renal pelvis or papilla. In the bladder of treated mice, hyperplasia or squamous metaplasia of transitional epithelium and one transitional cell papilloma were observed. Dose-dependent decreases in the incidence of spontaneous lesion in the male or female reproductive system were recognized. It is concluded that TBZ is not carcinogenic to CD-1 mice of both sexes. However, caution should be exercised in the long-term application of high TBZ doses.

Administration, Oral↗

PubMed2005

Reactive nitrogen intermediate production and tolerance variability in different mouse strains after in vivo treatment with lipopolysaccharide from Salmonella abortus equi.

Lipopolysaccharide (LPS) stimulation in animal models generates a large number of immune factors including cytokines and mediators. It also acts as a potent inducer of macrophage reactive oxygen intermediates and reactive nitrogen intermediates (RNI). RNI as stable metabolites of nitric oxide (NO) are produced by cells stimulated with LPS and cytokines. In this study, LPS from Salmonella abortus equi was investigated as an inducer of RNI in untreated controls and test groups of white Naval Medical Research Institute (NMRI) mice. Animals were humanely killed at 30, 60, 120 and 180 min after LPS injection, and plasma RNI was measured by Griess microassay. In a further experiment, host tolerance against bacterial LPS was evaluated by sequential intravenous injection of LPS concentrations of 4, 1 and 0.5 mg/kg at 24 h intervals in NMRI and with the same schedule but via subcutaneous injection in Balb/c mice. Statistical analysis of RNI values using analysis of variance test indicated that in vivo LPS stimulation induced high levels of NO in murine hosts (p<0.001). Comparison of RNI levels at different times after administration revealed the largest amount of RNI at 180 min after inoculation. Analysis of the time course until maximum RNI induction indicated that NMRI mice had the longest delay, suggesting a difference in tolerance of NMRI and Balb/c mice to LPS stimulation dependent on LPS concentration, dose, and route of inoculation.

Animals↗

PubMed2000

Group B Streptococcus induces apoptosis in macrophages.

Group B Streptococcus (GBS) is a pathogen that has developed some strategies to resist host immune defenses. Because phagocytic killing is an important pathogenetic mechanism for bacteria, we investigated whether GBS induces apoptosis in murine macrophages. GBS type III strain COH31 r/s (GBS-III) first causes a defect in cell membrane permeability, then at 24 h, apoptosis. Apoptosis was confirmed by several techniques based on morphological changes and DNA fragmentation. Cytochalasin D does not affect apoptosis, suggesting that GBS-III needs not be within the macrophage cytoplasm to promote apoptosis. Inhibition of host protein synthesis prevents apoptosis, whereas inhibition of caspase-1 or -3, does not. Therefore, GBS can trigger an apoptotic pathway independent of caspase-1 and -3, but dependent on protein synthesis. Inhibition of apoptosis by EGTA and PMA, and enhancement of apoptosis by calphostin C and GF109203X suggests that an increase in the cytosolic calcium level and protein kinase C activity status are important in GBS-induced apoptosis. Neither alteration of plasma membrane permeability nor apoptosis were induced by GBS grown in conditions impeding hemolysin expression or when we used dipalmitoylphosphatidylcholine, which inhibited GBS beta-hemolytic activity, suggesting that GBS beta-hemolysin could be involved in apoptosis. beta-Hemolysin, by causing membrane permeability defects, could allow calcium influx, which initiates macrophage apoptosis. GBS also induces apoptosis in human monocytes but not in tumor lines demonstrating the specificity of its activity. This study suggests that induction of macrophage apoptosis by GBS is a novel strategy to overcome host immune defenses.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2002

The beneficial effect of interleukin-12 on arthritis induced by group B streptococci is mediated by interferon-gamma and interleukin-10 production.

OBJECTIVE: To assess the effect of interleukin-12 (IL-12) administration on the evolution of systemic infection and septic arthritis induced by group B streptococci (GBS) in mice. METHODS: CD1 mice were inoculated intravenously with arthritogenic strain 1/82 of type IV GBS. Exogenous murine IL-12 was administered intraperitoneally 18 hours or 5 days after infection with 1 x 10(7) GBS, at doses ranging from 0.5 to 2.5 microg per mouse. Mice were monitored daily for survival and for signs of arthritis. In a subsequent set of experiments, mice were killed at selected times for examination of bacterial clearance, histopathologic changes in the joints, and cytokine production. RESULTS: IL-12 administration before the onset of clinical signs had a beneficial effect on GBS-induced arthritis and was clearly dose-dependent. The 2.5-microg dose per mouse totally prevented death from GBS-induced arthritis. The decrease in pathology was associated with a reduction of the bacterial burden and a change in the cytokine profile. In particular, systemic and joint levels of interferon-gamma (IFN gamma) and IL-10 significantly increased in mice treated with IL-12, whereas a decrease in IL-6 and IL-1 beta production was observed. The beneficial effects of IL-12, in terms of the incidence and severity of articular lesions, were reversed by coadministration of anti-IFN gamma or anti-IL-10-neutralizing antibodies. CONCLUSION: The findings of this study demonstrate that IL-12 is important in controlling the cytokine production that leads to the evolution of GBS-induced experimental arthritis. The amelioration of articular lesions is mostly attributable to IL-12-induced IFN gamma, but with a relevant participation of IL-12-induced IL-10.

Adjuvants, Immunologic↗

PubMed2002

Sexual dimorphism in digit-length ratios of laboratory mice.

The lengths of the index finger (2D) and ring finger (4D) are sexually dimorphic in humans, and men have a smaller 2D:4D ratio compared to women. Prenatal androgens appear to be important in the development of the 2D:4D sex difference, since it has been reported in children as young as 2 years old, and since humans exposed to supernormal prenatal androgen levels display a smaller 2D:4D ratio. We tested whether another mammalian species in which the process of peripheral sexual differentiation is androgen-dependent might also show a sex difference in digit ratios. The 2D:4D ratio of adult outbred mice was calculated for both the left and right rear paws. A sex difference was observed in the right rear paw: female mice had a larger 2D:4D ratio than did males. We also found this difference in prepubescent weanling mice. This sex difference is in the same direction as that observed in humans, and suggests that sexual dimorphism in digit length ratios is a feature common to many, if not all, mammals. The mouse may therefore be a useful animal model for studying the factors that influence finger length patterns, which have recently been correlated with several specific behaviors and disease predispositions in humans.

Age Factors↗

PubMed2006

Developmental toxicity evaluation of berberine in rats and mice.

BACKGROUND: Berberine, a plant alkaloid, is found in some herbal teas and health-related products. It is a component of goldenseal, an herbal supplement. Berberine chloride dihydrate (BCD) was evaluated for developmental toxicity in rats and mice. METHODS: Berberine chloride dihydrate was administered in the feed to timed-mated Sprague-Dawley (CD) rats (0, 3,625, 7,250, or 14,500 ppm; on gestational days [GD] 6-20), and Swiss Albino (CD-1) mice (0, 3,500, 5,250, or 7,000 ppm; on GD 6-17). Ingested doses were 0, 282, 531, and 1,313 mg/kg/day (rats) and 0, 569, 841, and 1,155 mg/kg/day (mice). RESULTS: There were no maternal deaths. The rat maternal lowest observed adverse effect level (LOAEL), based on reduced maternal weight gain, was 7,250 ppm. The rat developmental toxicity LOAEL, based on reduced fetal body weight per litter, was 14,500 ppm. In the mouse study, equivocal maternal and developmental toxicity LOAELs were 5,250 ppm. Due to scattering of feed in the high dose groups, a gavage study at 1,000 mg/kg/day was conducted in both species. CONCLUSIONS: In rats, maternal, but not fetal adverse effects were noted. The maternal toxicity LOAEL remained at 7,250 ppm (531 mg/kg/day) based on the feed study and the developmental toxicity NOAEL was raised to 1,000 mg/kg/day BCD based on the gavage study. In the mouse, 33% of the treated females died. Surviving animals had increased relative water intake, and average fetal body weight per litter decreased 5-6% with no change in live litter size. The maternal toxicity LOAEL remained at 5,250 ppm (841 mg/kg/day) BCD, based on increased water consumption. The developmental toxicity LOAEL was raised to 1,000 mg/kg/day BCD based on decreased fetal body weight.

Animals↗