Preparation of PCR-quality mouse genomic DNA with hot sodium hydroxide and tris (HotSHOT).
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Aging↗
Biomedical subjects
J A Walker
Publications and source records attributed to J A Walker.
Comparison of the phenotypes of Streptococcus zooepidemicus isolated from tonsils of healthy horses and specimens obtained from foals and donkeys with pneumonia.
OBJECTIVE: To determine whether streptococcal pneumonia is caused by strains of Streptococcus zooepidemicus similar to those obtained from the tonsils of healthy horses. SAMPLE POPULATION: 5 tonsils from healthy horses, 8 tracheal washes and 6 lung specimens from foals with pneumonia, and 5 nasopharyngeal swab specimens from donkeys with acute bronchopneumonia. PROCEDURE: Variable M-like protectively immunogenic SzP proteins of 5 isolates of S. zooepidemicus from each tonsil and clinical specimen were compared, using immunoblots. The SzP gene of 13 isolates representative of various SzP immunoblot phenotypes from 1 healthy horse and 9 horses and donkeys with pneumonia were sequenced and compared. Cell-associated hyaluronic acid concentration and resistance to phagocytosis of some isolates were measured. RESULTS: Tonsils of each healthy horse were colonized by several SzP phenotypes similar to those of foals or donkeys with pneumonia. In contrast, multiple isolates from animals with pneumonia had the same SzP phenotype, indicating infection by a single strain or clone. Analysis of the SzP sequence confirmed that differences in immunoblot phenotype were associated with sequence differences and that several SzP genotypes were in healthy horses and animals with pneumonia. Isolates with high concentrations of cell-associated hyaluronic acid were more resistant to phagocytosis. CONCLUSIONS AND CLINICAL RELEVANCE: An SzP-specific immunoblot is a useful, sensitive measure of diversity among strains of S. zooepidemicus. Single strains with SzP phenotypes similar to those found in tonsils of healthy horses cause pneumonia. Because of the diversity of SzP phenotype and genotype among isolates from animals with pneumonia, SzP phenotype is not an important determinant of invasiveness or epizootic capabilities.
Animals↗
Eradication of infection with Helicobacter spp. by use of neonatal transfer.
BACKGROUND AND PURPOSE: Efficient methods for detection and elimination of Helicobacter spp. infections are needed to facilitate the development of Helicobacter-free mouse colonies. We developed an inexpensive, high-throughput method for preparation of fecal DNA for Helicobacter polymerase chain reaction (PCR) assays. METHODS: Fecal DNA was prepared by heating fecal pellets to 95 degrees C for 10 minutes in an alkaline solution, then adjusting the pH by addition of Tris buffer. This solution is used for PCR assays without purification of DNA. We then tested fostering as a method of generating Helicobacter-free mice. Litters born to Helicobacter-positive dams were transferred to Helicobacter-negative foster dams on the first day of life. RESULTS: Fostered pups tested Helicobacter negative up to 89 days of age, whereas pups raised by Helicobacter-positive dams were all test positive by 19 days of age. CONCLUSION: These simple methods provide an efficient system for the development of Helicobacter-free mouse colonies.
Animals↗
Smaug, a novel RNA-binding protein that operates a translational switch in Drosophila.
During Drosophila embryogenesis, a gradient of Nanos protein emanating from the posterior pole organizes abdominal segmentation. This gradient arises from translational regulation of nanos mRNA, which is activated in the specialized cytoplasm at the posterior pole of the embryo and repressed elsewhere. Previously, we have defined cis-acting elements in the mRNA that mediate this translational switch. In this report, we identify a factor named Smaug that binds to these elements and represses translation in the bulk cytoplasm. Smaug interacts gentically and biochemically with Oskar, a key component of the pole plasm for activation of nanos mRNA and specification of the germline precursors. These observations suggest that Smaug operates a translational switch that governs the distribution of Nanos protein.
Abdomen↗
Hormonal changes throughout the menstrual cycle and increased anterior cruciate ligament laxity in females.
OBJECTIVE: To determine whether women experience significantly greater anterior cruciate ligament (ACL) laxity in conjunction with estrogen and progesterone surges during a normal 28- to 30-day menstrual cycle. DESIGN AND SETTING: Serial estrogen and progesterone levels were measured via radioimmunoassay procedures to identify the follicular and luteal phases of a subject's menstrual cycle and to determine periods of peak hormonal surges. Concomitant ACL laxity measures were taken using a knee arthrometer. Hormone levels and ACL laxity were assessed on days 1, 10, 11, 12, 13, 20, 21, 22, and 23 of the menstrual cycle. Day 1 corresponds to the menstrual phase, when estrogen and progesterone levels are at their lowest. Days 10 through 13 correspond to peak estrogen surge (follicular phase), and days 20 through 23 correspond to peak progesterone surge (luteal phase). SUBJECTS: Seven active females between the ages of 21 and 32 years with at least one apparently healthy knee (no known knee anomalies) volunteered for participation in this study. Each subject stated that she experienced a normal (28- to 30-day) menstrual cycle and was not currently taking any type of hormone therapy (eg, birth control medication). MEASUREMENTS: Blood was drawn on days 1, 10, 11, 12, 13, 20, 21, 22, and 23 of each subject's menstrual cycle, and ACL laxity measurements were assessed immediately after the blood draws. Estrogen and progesterone levels were determined via radioimmunoassay procedures, and ACL laxity was determined using a knee arthrometer. RESULTS: A within-subjects, repeated-measures analysis of variance was applied to determine the presence or absence of significant differences in ACL laxity values over the course of a subject's menstrual cycle. We found a significant difference in ACL laxity when comparing baseline levels of estrogen with peak levels of estrogen. A significant increase in ACL laxity was also noted when comparing baseline levels of progesterone with peak levels of progesterone. CONCLUSIONS: ACL laxity increased significantly throughout the menstrual cycle when comparing baseline with peak levels of estrogen and progesterone.
Journal Article↗
Synthesis and antiviral evaluation of certain novel pyrazinoic acid C-nucleosides.
Pyrazine (1,4-diazine) C-nucleosides constitute a rare class of nucleic acid analogues that has only recently been reported in the literature. As part of our ongoing investigation into the synthesis and reactivity of these compounds, we have developed an electrophilic esterification of a lithiated pyrazine C-nucleoside (1) to give, following deprotection, the versatile intermediate ethyl 3,5-dichloro-6-(beta-d-ribofuranosyl)pyrazine-2-carboxylate (4). This intermediate was subjected to a variety of reaction conditions to generate a series of pyrazinoic acid C-nucleosides. These compounds, along with 3, 5-dichloro-2-(beta-d-ribofuranosyl)pyrazine (2) and 4, were evaluated for antiviral activity and cytotoxicity. No significant activity was observed for compounds 2 and 5-9, but 4 was active against two herpes viruses and cytotoxic in the micromolar range.
Antiviral Agents↗
Design, synthesis, and antiviral activity of alpha-nucleosides: D- and L-isomers of lyxofuranosyl- and (5-deoxylyxofuranosyl)benzimidazoles.
Several 2-substituted alpha-D- and alpha-L-lyxofuranosyl and 5-deoxylyxofuranosyl derivatives of 5,6-dicholro-2-(isopropylamino)-1-(beta-L-ribofuranosyl) benzimidazole (1263W94) and 2,5,6-trichloro-1(beta-D-ribofuranosyl)benzimidazole (TCRB) were synthesized and evaluated for activity against two herpesviruses (HSV-1 and HCMV) and for their cytotoxicity against HFF and KB cells. Condensation of 1,2,3,5-tetra-O-acetyl-L-lyxofuranose (2a) with 2,5,6-trichlorobenzimidazole (1) yielded the alpha-nucleoside 3a. The 2-bromo derivative and 2-methylamino derivative were prepared by treatment of 3a with HBr followed by deprotection or from methylamine, respectively. Compound 3a was deprotected and the resultant nucleoside used to prepare the 2-cyclopropylamino and 2-isopropylamino derivatives. The 2-alkylthio nucleosides were prepared by condensing 2a with 5,6-dichlorobenzimidazole-2-thione followed by deprotection. Alkylation of this adduct gave the 2-methylthio and 2-benzylthio derivatives. Condensation of 5-deoxy-1,2,3-tri-O-acetyl-L-lyxofuranosyl, prepared from L-lyxose, with 1 or 2-bromo-5,6-dichlorobenzimidazole (15), followed by deprotection, gave the 2-chloro or 2-bromo-5'-deoxylyxo-furanosyl derivative, respectively. The cyclopropylamino derivative was prepared from the 2-chloro derivative. All D-isomers were prepared in an analogous fashion from D-lyxose. Either compounds were inactive against HSV-1 or weak activity was poorly separated from cytotoxicity. In contrast, the 2-halogen derivatives in both the alpha-lyxose and 5-deoxy-alpha-lyxose series were active against the Towne strain of HCMV. The 5-deoxy alpha-L analogues were the most active, IC50's = 0.2-0.4 microM, plaque assay; IC90's = 0.2-2 microM, yield reduction assay. All of the 2-isopropylamino or 2-cyclopropylamino derivatives were less active (IC50's = 60-100 microM, plaque assay; IC90's = 17-100 microM, yield reduction assay) and were not cytotoxic. The methylamino, thio, and methylthio derivatives were neither active nor cytotoxic. The benzylthio derivatives were weakly active, but this activity was poorly separated from cytotoxicity. The alpha-lyxose L-isomers were more active in a plaque assay against the AD169 strain of HCMV compared to the Towne strain, thereby providing additional evidence of antiviral specificity.
Antiviral Agents↗
ELISA detection of restriction site polymorphisms in the pig ryanodine receptor locus.
We compare two strategies for ELISA detection of restriction site polymorphisms (EDRSP) that are suitable for high-throughput genotyping of the pig ryanodine receptor point mutation (RYR1(hal)). In both procedures, target DNA is amplified by PCR with one primer that is 5' biotinylated and a second primer that is 5' fluoresceinylated. PCR products are captured in duplicate wells on a streptavidin-coated, 96-well plate. The duplicates may be treated in two ways. In a single restriction enzyme assay, one duplicate is exposed to a restriction enzyme that cuts one allele specifically, and the second duplicate is exposed to no restriction enzyme. In a dual restriction enzyme assay, the second replicate is exposed to a second restriction enzyme that cuts the alternate allele specifically. Thereafter, the two procedures are similar; anti-fluorescein antibodies conjugated to peroxidase are allowed to bind to the fluoresceinylated ends, the plate is washed, and a substrate is converted to a colored end product. The ratio of the absorbances in the two wells is used to classify subjects by genotype. When the dual restriction enzyme assay is run, three genotype groups are easily distinguishable. When the single restriction enzyme assay is run, heterozygotes generate values that may overlap with those of the homozygotes that are not cut by the restriction enzyme. Dual restriction enzyme assays are more accurate than single restriction enzyme assays; however, single restriction enzyme assays are sufficient for identifying pigs that carry RYR1(hal).
Animals↗
Misty (m) affects growth traits.
The misty (m) coat color mutation is commonly maintained in linkage disequilibrium with the obesity mutation diabetes (Leprdb) to serve as a marker for Leprdb genotype. Comparisons among Leprdb genotypes are made under the untested assumption that m has no effects on traits under investigation. We tested this assumption in a population segregating m in the absence of db. Analysis of growth curves revealed that m/m mice are smaller than M/M mice by the 2nd wk of life and remain smaller through the 5th wk of life. Analysis of variance of three traits measured at 35 days of age revealed that m/m mice are 8% shorter than M/M mice, weigh 15% less, and have 21% less inguinal adipose mass. These results indicate that m affects growth traits. Therefore, when m and Leprdb segregate in the same cross, interpretation of their effects is confounded by linkage. More accurate estimates of Leprdb genotype effects can be made by removing m from populations segregating Leprdb and using a direct assay to measure Leprdb genotype.
Aging↗
Relation of lipoprotein subclasses as measured by proton nuclear magnetic resonance spectroscopy to coronary artery disease.
Although each of the major lipoprotein fractions is composed of various subclasses that may differ in atherogenicity, the importance of this heterogeneity has been difficult to ascertain owing to the labor-intensive nature of subclass measurement methods. We have recently developed a procedure, using proton nuclear magnetic resonance (NMR) spectroscopy, to simultaneously quantify levels of subclasses of very low density (VLDL), low density (LDL), and high density (HDL) lipoproteins; subclass distributions determined with this method agree well with those derived by gradient gel electrophoresis. The objective of the current study of 158 men was to examine whether NMR-derived lipoprotein subclass levels improve the prediction of arteriographically documented coronary artery disease (CAD) when levels of lipids and lipoproteins are known. We found that a global measure of CAD severity was positively associated with levels of large VLDL and small HDL particles and inversely associated with intermediate size HDL particles; these associations were independent of age and standard lipid measurements. At comparable lipid and lipoprotein levels, for example, men with relatively high (higher than the median) levels of either small HDL or large VLDL particles were three to four times more likely to have extensive CAD than were the other men; the 27 men with high levels of both large VLDL and small HDL were 15 times more likely to have extensive CAD than were men with low levels. In contrast, adjustment for levels of triglycerides or HDL cholesterol greatly reduced the relation of small LDL particles to CAD. These findings suggest that large VLDL and small HDL particles may play important roles in the development of occlusive disease and that their measurement, which is not possible with routine lipid testing, may lead to more accurate risk assessment.
Adult↗
Normal postprandial lipemia and chylomicron clearance in offspring of parents with early coronary artery disease.
To assess the importance of postprandial lipemia and delayed chylomicron clearance as early atherogenic risk factors, 60 male offspring of parents with early coronary artery disease (CAD) and 41 controls were administered a fat-rich meal containing vitamin A. There were no significant differences between CAD-positive (CAD+) offspring and CAD-negative controls for areas under the postprandial curves for triglyceride and plasma, chylomicron, and chylomicron remnant retinyl palmitate. Older CAD+ offspring, aged 31-45 yr, had significantly increased very low density lipoprotein (VLDL) cholesterol, VLDL triglyceride, VLDL apoprotein B, and areas under postprandial curves for triglyceride and plasma, chylomicron, and chylomicron remnant retinyl palmitate than younger CAD+ offspring, aged 15-30 yr. Correcting for waist/hip ratio eliminated significant differences between the two groups for VLDL and areas under the triglyceride and chylomicron remnant curves, but this was not the case for the insulin sensitivity index. We conclude that neither increased postprandial lipemia nor abnormalities of chylomicron clearance are important early atherogenic risk factors in this population. An increase in age is associated with increased VLDL and postprandial lipemia and decreased chylomicron remnant clearance. This is due mainly to an increase in the waist/hip ratio and not to a change in insulin sensitivity.
Adolescent↗
Pharmacokinetics of vancomycin when administered during high flux hemodialysis.
This study was undertaken to evaluate the pharmacokinetics of relatively high-dose vancomycin when administered during high-flux hemodialysis using a polysulfone membrane (F-80, Fresenius). Five noninfected, anuric patients received a single dose of 25 mg/kg of vancomycin infused during hemodialysis at a rate of one gram per hour and timed such that the end of the infusion coincided with the end of dialysis. Blood samples were drawn during the infusion, up to six hours after the end of dialysis and then prior to the next three dialysis treatments. Spent dialysate was collected during the infusion. Samples were analyzed using the EMIT assay. The percent of vancomycin lost during the first dialysis session ranged from 39.1 to 55.1% (mean, 45.7+/-6.4). The concentration of vancomycin at 6 hours after hemodialysis ranged from 18.2 to 45.1 mg/L (mean, 29.6+/-10.0 mg/l). Dialysis clearance ranged from 96.1 to 158.1 ml/min (mean, 130.7 +/-30.0 ml/min). One week after dosing, serum concentrations ranged from 8.14 mg/l to 10.1 mg/l (mean, 9.0+/-1.0 mg/l). This study suggests than an initial dose of 25 mg/kg of vancomycin, given during high-flux dialysis, may provide adequate serum concentrations in anuric hemodialysis patients for up to seven days. This dosing scheme reduces inconvenience to the patient and staff, and potentially can reduce nursing costs associated with post-dialysis administration; its cost is minimal. At this point, subsequent dosing is best determined by therapeutic drug monitoring.
Adult↗
Molecular basis of variation in protective SzP proteins of Streptococcus zooepidemicus.
OBJECTIVES: To characterize, on a molecular basis, variable regions of the SzP proteins of the Moore and Bryans serovars of Streptococcus zooepidemicus and specificity of opsonic responses. SAMPLE POPULATION: 14 Moore and Bryans serovars of S zooepidemicus. PROCEDURE: Using polymerase chain reaction analysis and primers from the 5' and 3' sequences of the prototype gene SzPW60, the SzP genes of each Moore and Bryans serovar were sequenced and translated, then the amino acid sequences were compared. RESULTS: Comparison of the amino acid sequences revealed 2 variations at the N terminus; a hypervariable (HV) region from residue 106 to 166, approximately; and proline-glutamic acid-proline-lysine repeats in the carboxy terminus that ranged in number from 7 to 12. Five distinct motifs, HV 1 to 5, which varied independently of the N termini were found in the internal HV region. All serovars were opsonized by antiserum to the prototype SzPW60 protein, indicating that opsonogenic epitopes are on the conserved regions of the protein. CONCLUSION AND CLINICAL RELEVANCE: Variant motifs may be valuable in epizootiologic and pathogenesis studies of S zooepidemicus infections of the respiratory tract of young horses and in determining whether there are populations of S zooepidemicus unique to specific animal hosts. It is also clear from the opsonic responses to SzP that at least a portion of the protective responses are probably not serovar specific.
Amino Acid Sequence↗
An assay for the Cpefat obesity mutation created with a modified primer.
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Animals↗
Low-density lipoprotein particle size is not a discriminating marker for atherogenic risk in male offspring of parents with early coronary artery disease.
The aim of this study was to assess the importance of low-density lipoprotein (LDL) particle size as a marker of atherogenic risk in male offspring of a parent with early coronary artery disease (CAD) before the age of 60 years. CAD-positive (CAD+) offspring were recruited into two groups based on age, 15 to 30 years (n = 20) and 31 to 45 years (n = 41), and matched to CAD-negative (CAD-) offspring by age and body mass index (BMI) (n = 20 and 21 per group). LDL peak particle diameter was assessed by polyacrylamide gradient gel electrophoresis. There was no significant difference in LDL peak particle diameter between CAD+ and CAD- offspring (26.2 +/- 0.1 v 26.2 +/- 0.1 nm, mean +/- SE). There was also no difference between CAD+ offspring and CAD- offspring when comparisons were made within their own age group (26.5 +/- 0.1 nm in younger CAD+ offspring v 26.2 +/- 0.1 nm in younger CAD- offspring, and 26.0 +/- 0.1 nm in older CAD+ offspring v 26.1 +/- 0.2 nm in older CAD- offspring). Peak particle diameter was significantly greater in younger CAD+ offspring than in older CAD+ offspring (26.5 +/- 0.1 v 26.0 +/- 0.1 nm, P < .05). We conclude that small LDL particle size is not a discriminating marker for early atherogenic risk, and that measurement of LDL particle size has limited value in the assessment of coronary risk, at least in the age ranges we studied.
Adolescent↗
Decreased glucose tolerance, not decreased insulin sensitivity, is a maturational abnormality in the male offspring of a parent with early coronary artery disease.
We investigated whether the male offspring of a parent with early coronary artery disease (before the age of 60; n = 61) exhibit decreased insulin sensitivity compared with controls matched for age and body mass index (BMI) (n = 39). The insulin sensitivity index (S[I]) was determined by the minimal modeling method of Bergman from a frequently sampled intravenous glucose tolerance test with intravenous tolbutamide. Offspring and controls had a similar S[I], insulin-independent glucose utilization (S[G]), first-phase insulin response (AIR[G]), and area under the glucose curve. When subjects were separated into two age groups, younger subjects aged 15 to 30 years and older subjects aged 31 to 45 years, important differences were seen. S[G] was significantly increased in younger offspring compared with controls (22.8 +/- 2.3 v 16.8 +/- 2.3 x 10(-3) x min(-1), P < .05). Older offspring had a significantly increased area under the glucose curve compared with controls (18,250 +/- 322 v 17,225 +/- 347 mg/dL x min(-1), P < .05). Older offspring also had decreased S[I] compared with younger offspring (5.0 +/- 0.4 v 6.6 +/- 0.9 x 10(-4) x min(-1) x micro U/mL, P < .05), but this difference was eliminated after adjusting for BMI and waist to hip ratio (5.5 +/- 0.4 v5.8 +/- 0.9 x 10(-3) x min(-1), nonsignificant). This study does not support the concept that insulin resistance is an early atherogenic risk factor in offspring at risk for coronary disease because of their family history. However, it does point to the importance of maturational changes in glucose homeostasis in these offspring.
Adolescent↗
Insulin resistance is not a major determinant of low-density lipoprotein particle size.
The relationship between low-density lipoprotein (LDL) peak particle diameter and insulin sensitivity, very-low-density lipoprotein (VLDL) + intermediate-density lipoprotein (LDL) triglyceride, cholesterol, and apoprotein B, postprandial lipemia, and LDL + high-density lipoprotein (HDL) triglyceride was assessed. The subjects were 101 healthy males aged 15 to 45 years. Sixty-one subjects (60.4%) were offspring of a parent with coronary artery disease before age 60, and 40 subjects (39.6%) had no parental history of coronary artery disease. LDL peak particle diameter was measured following polyacrylamide gradient gel electrophoresis. An insulin sensitivity index (Si) was determined from a frequently sampled intravenous glucose tolerance test using a minimal modeling method. A fat tolerance test was performed with a test meal containing 70 g/m2 fat, with triglyceride concentrations measured hourly for 12 hours. LDL peak particle diameter was significantly correlated with body mass index (BMI) (r = -.282, P < .01), waist to hip ratio (r = -.291, P < .01), fasting triglyceride (logarithmically [log] transformed) (r = -.566, P < .001), area under the postprandial triglyceride curve (log transformed) (r = -.562, P < .001), VLDL + IDL triglyceride (log transformed) (r = -.462, P < .001), VLDL + IDL cholesterol (log transformed) (r = -.477, P < .001), VLDL + IDL apoprotein B (log transformed) (r = -.321, P < .001), LDL + HDL triglyceride (log transformed) (r = .583, P < .001), and HDL cholesterol (r = .347, P < .001), but there was no significant correlation with Si. Using stepwise regression analysis, LDL + HDL triglyceride showed the strongest relationship to LDL peak particle diameter, accounting for 34% of the variation in size. Si was not an independent predictor of LDL particle size. In conclusion, insulin sensitivity appears to have little influence on LDL particle size. The importance of LDL + HDL triglyceride should be considered a preliminary finding warranting verification in this and other populations.
Adolescent↗
Late pancreas allograft rejection. Preliminary experience with factors predisposing to rejection.
A case series of 31 cadaveric pancreas transplant recipients who were insulin-independent at least for one year was analyzed for the factors predisposing to late acute rejection (> 12 months posttransplant). Sixty-two pancreas transplants were performed in 61 patients, of whom 53 had functioning allografts 3 months posttransplant; 31 of these had a follow-up > 12 months. Twenty had no evidence of late rejection, whereas 11 had evidence of acute rejection after 12 months. All patients received quadruple induction immunosuppression. No demographic or clinical factors-including donor age, organ cold time, HLA mismatch, age, sex, or race-could distinguish the late acute rejection group. The presence of acute rejection in the first year posttransplant was similar in the late rejectors (21 episodes in 9 of 11 patients) compared with patients without late rejection (31 episodes in 16 of 20 patients). Antilymphocyte induction therapy type had no influence, but the amount of immunosuppression with prednisone and cyclosporine (CsA) at 3 months posttransplant was significantly lower in those patients who experienced late rejection. After the first year posttransplant, CsA 12-hr trough levels were significantly lower in late rejection months (121 +/- 7 ng/ml) compared with each patient's own stable months (183 +/- 5 ng/ml, P < 0.0001). Neither prednisone nor azathioprine dosages differed between teh two groups after the first year posttransplant. Our preliminary results suggest that early under immunosuppression with prednisone and CsA in the first year and 12-hr trough CsA levels less than approximately 180 ng/ml after the first year posttransplant predispose to late pancreatic rejection.
Adult↗