Application of a label-free, gel-free quantitative proteomics method for ecotoxicological studies of small fish species.

Although two-dimensional electrophoresis (2D-GE) remains the basis for many ecotoxicoproteomic analyses, newer non-gel-based methods are beginning to be applied to overcome throughput and coverage limitations of 2D-GE. The overall objective of our research was to apply a comprehensive, liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic approach to identify and quantify differentially expressed hepatic proteins from female fathead minnows exposed to fadrozole, a potent inhibitor of estrogen synthesis. Female fathead minnows were exposed to 0 (control), 0.04, and 1.0 µg of fadrozole/L of water for 4 days, and proteomic analysis was performed. Proteins were extracted and digested, and proteolytic peptides were separated via high-resolution one- or two-dimensional (1-D or 2-D) ultrapressure liquid chromatography (UPLC) and analyzed by tandem mass spectrometry. Mass spectra were searched against the National Center for Biotechnology Information (NCBI) ray-finned fish ( Actinopterygii ) database, resulting in identification of 782 unique proteins by single-dimension UPLC. When multidimensional LC analysis (2-D) was performed, an average increase of 1.9× in the number of identified proteins was observed. Differentially expressed proteins in fadrozole exposures were consistent with changes in liver function, including a decline in concentrations of vitellogenin as well as other proteins associated with endocrine function and cholesterol synthesis. Overall, these results demonstrate that a gel-free, label-free proteomic analysis method can successfully be utilized to determine differentially expressed proteins in small fish species after toxicant exposure.

Sequence-based typing of HLA class I alleles in Alaskan Yupik Eskimo.

In comparison to South America, native North Americans tend to be less diverse in their repertoire of HLA class I alleles. Based upon this observation, we hypothesized that the Yupik Eskimo would exhibit a limited number of previously identified class I HLA alleles. To test this hypothesis, sequence-based typing was performed at the HLA-A, -B and -C loci for 99 Central Yupik individuals from southwestern Alaska. Two new class I alleles, A*2423 and Cw*0806, were identified. While A*2423 was observed in only one sample, Cw*0806 was present in 26 of the 99 individuals and all of the Cw*0806 samples contained B*4801. Allele Cw*0806 differs from Cw*0803 by a single nucleotide substitution such that Cw*0803 may be the progenitor of Cw*0806. Allele Cw*0803 was originally characterized as unique to South America, but detection of Cw*0803 in the Yupik indicates that Cw*0803 was a founding allele of the Americas. The presence of new alleles and previously unrecognized founding alleles in the Yupik population show that natives of North America are more diverse than previously envisioned.

Non-conservative substitutions distinguish previously uncharacterized HLA-A molecules.

The extent of class I HLA polymorphism is not yet realized, and to provide a glimpse of the HLA-A polymorphism which remains undetected, we have analyzed approximately 3,700 National Marrow Donor Program (NMDP) Donor/Recipient Pair Retrospective Study Samples with HLA-A DNA sequence-based typing (SBT). Seventeen new HLA-A alleles were detected, with a total of 19 nucleotide substitutions distinguishing these new alleles from their closest HLA-A relatives. Nearly all of the new alleles differ by single nucleotide substitutions; a majority of these substitutions can be explained by gene conversion events but 6 alleles likely originated by point mutation. Fifteen of the 19 nucleotide substitutions translate into amino acid differences in the molecule. Structurally, the inferred amino acid alterations were non-conservative in terms of chemical property, and most substitutions were positioned in 1 or more of the specificity pockets which determine peptide binding. Although these new alleles were identified in a primarily Caucasian sample population, 9 of the 17 new HLA-A alleles were found in samples of non-Caucasoid origin. A new allele detection rate of 1 in approximately 200 individuals in our data set would, therefore, be higher in a non-Caucasoid sample population. In summary, the single nucleotide substitutions that distinguish undetected HLA-A alleles translate into functionally distinct HLA-A molecules. Further studies of the role of HLA-A in transplantation, in disease association, and in evolution must therefore accommodate the discovery of new alleles differing by single nucleotides.

Correlates of total plasma homocysteine: folic acid, copper, and cervical dysplasia.

We examined correlates of total plasma homocysteine (tHcy) in 294 subjects with cervical intraepithelial neoplasia and 170 control subjects. Associations of tHcy with risk factors for cervical intraepithelial neoplasia and 24-h intakes and biochemical indices of nutrients were examined. Plasma and red blood cell folate and plasma B(12) were strong inverse correlates of tHcy (r = -0.35, -0. 31, and -0.27, respectively). Plasma copper and severity of dysplasia were positively correlated with tHcy (r = 0.14 and 0.21, respectively). A stepwise regression model that included red blood cell folate, plasma copper, grade of dysplasia, ethnicity, intake of polyunsaturated fatty acids, plasma vitamin B(12), intake of fat, and oral contraceptive use explained 29% of the variation in tHcy. Two hundred thirty-five subjects with cervical intraepithelial neoplasia were randomized to receive folic acid (10 mg/d) or placebo for 6 mo. After 2, 4, and 6 mo, mean tHcy in the folate-supplemented group (7.2 +/- 1.8, 7.0 +/- 1.9, and 7.0 +/- 2.3 micromol/L, respectively) was significantly lower than baseline and the placebo group at 2, 4, and 6 mo (8.9 +/- 3.1, 8.4 +/- 3.0, and 8.9 +/- 3.1 micromol/L, respectively). Supplementation lowered tHcy even in subjects in the highest quintile of baseline folate. Folate, vitamin B(12), copper, and severity of dysplasia are associated with tHcy. Folate supplementation significantly lowers tHcy even in folate-replete subjects.

Effect of total plasma homocysteine on cervical dysplasia risk.

We investigated whether total plasma homocysteine (tHcy) is associated with risk for cervical intraepithelial neoplasia (CIN). tHcy was evaluated, along with numerous risk factors for CIN and biochemical indexes of nutrients, in a previously reported study population of 294 subjects with CIN and 170 female controls without CIN. tHcy was significantly higher in cases than in controls (9.1 vs. 8.3 mumol/l, p = 0.002). Human papillomavirus type 16 infection [odds ratio (OR) = 6.7], oral contraceptive use (OR = 6.0), parity (OR = 2.2), and cigarette smoking (OR = 1.9) were significantly associated with CIN after adjustment for each other and for age, number of sexual partners, and plasma tHcy, folate, iron, and zinc. Human papillomavirus type 16 positivity increased risk for CIN more when tHcy was > 9.12 mumol/l (OR = 4.7) than when it was < or = 9.12 mumol/l (OR = 3.0). Cigarette use increased risk for CIN when tHcy was > 9.12 mumol/l (OR = 3.9), but not when tHcy was < or = 9.12 mumol/l (OR = 1.5). Parity increased risk for CIN more when tHcy was > 9.12 mumol/l (OR = 4.0) than when tHcy was < or = 9.12 mumol/l (OR = 2.0). These results suggest that elevated plasma tHcy is a risk factor for cervical dysplasia and that it enhances the effects of other risk factors. It is unknown whether tHcy is serving as a marker of folate deficiency or is acting through other mechanisms.

Female Wistar-Kyoto and SHR/y rats have the same genotype but different patterns of expression of renin and angiotensinogen genes.

OBJECTIVES: To evaluate whether renin and angiotensinogen gene expression in females from two strains of rats that share the same autosomes and X chromosomes differs. Female SHR/y rats have the parental Wistar-Kyoto rat autosomes and X chromosomes and have no chromosomes of spontaneously hypertensive rat origin; thus they are genetically equivalent to female Wistar-Kyoto rats. DESIGN AND METHODS: Because these genes are regulated by steroid hormones, we investigated the effects of removal of estrogen (ovariectomy) and addition of androgen (testosterone implants) on three groups of female SHR/y rats and the parental rat strain Wistar-Kyoto rat with groups of intact (control) rats, rats subjected to ovariectomy at age 3 weeks, and rats subjected to ovariectomy with a testosterone implant at age 3 weeks. RESULTS: The combination of removing estrogen early in development and supplementing the ovariectomized females with testosterone revealed strain differences in response of blood pressure. Renin and angiotensinogen messenger RNA levels appear to be regulated coordinately within each strain, although actual levels of messenger RNA differ between the strains. CONCLUSIONS: Similar patterns of responses of renin and angiotensinogen genes to ovariectomy and ovariectomy plus testosterone suggest that regulation of the genes is likely to be similar or coordinate. Differences in regulation of renin-angiotensin system genes between strains may result from epigenetic mechanisms such as genome imprinting of these genes or of another gene that functions as a common regulator of renin and angiotensinogen.

Influence of study goals on study design and execution.

From the viewpoint of a clinician who makes recommendations to patients about choosing from the multiple possible management schemes, quantitative information derived from statistical analyses of observational studies is useful. Although random assignment of therapy is optimal, appropriately performed studies in which therapy has been nonrandomly "assigned" are considered acceptable, albeit occasionally with limitations in inferences. The analyses are considered most useful when they generate multivariable equations suitable for predicting time-related outcomes in individual patients. Graphic presentations improve communication with patients and facilitate truly informed consent.

A typical hemolytic uremic syndrome in human immunodeficiency virus-1-infected children.

We describe the clinical and pathological findings of the hemolytic uremic syndrome (HUS) in two children with human immunodeficiency virus (HIV) infection. Both patients presented with microangiopathic hemolytic anemia, thrombocytopenia, and subsequently developed renal failure. The diagnosis of HUS was confirmed by renal histopathology in both patients. None of these children presented with bloody diarrhea, evidence of circulating antibody response to Escherichia coli O157 lipopolysaccharide, or other known risk factors for HUS, except for the presence of HIV infection. Each patient was treated with intravenous plasma infusion and renal replacement therapy. Their clinical course was characterized by non-oliguria and lack of significant hypertension throughout the acute phase of the disease. Despite these favorable clinical parameters, both patients developed end-stage renal failure. The etiology of this atypical HUS characterized by poor renal survival remains unknown and the role of HIV infection in its pathogenesis, although possible, is unclear.

Renal autotransplantation for renovascular hypertension caused by midaortic syndrome.

Midaortic syndrome (MAS) is a well-recognized but rare cause of renovascular hypertension (RVH). Several techniques have been described to treat RVH caused by MAS. The authors recently treated two children with MAS and RVH. In both patients the right kidney had two renal arteries. A 13-year-old boy presented with severe headaches, pain in his lower extremities with exertion, and marked hypertension (blood pressure, 170/110). An aortogram demonstrated 70% narrowing of his abdominal aorta from the suprarenal region to 5 cm above the iliac bifurcation. There was significant stenosis of the celiac axis, superior mesenteric artery, and left renal artery. The right kidney had two renal arteries, and the upper pole artery was stenotic at its origin. A 10-year-old girl, known to have hypertension for several years had an aortogram that demonstrated 70% narrowing of the abdominal aorta from the suprarenal region to 3 cm above the iliac bifurcation. There was involvement of the left renal artery at its orifice. She also had two renal arteries to the right kidney with the right upper pole artery being stenotic at its origin and in the mid-portion of the vessel. Aortic reconstruction was accomplished with a polytetrafluoroehtylene (PTFE) bypass graft in each case. The first case also involved patch angioplasty of the celiac axis. In both cases, the right kidney was autotransplanted. It was removed intraoperatively, cold perfused, and the two renal arteries reconstructed followed by transplantation to the right iliac vessels. In both cases the left renal artery was reimplanted into the PTFE graft. Both patients had uncomplicated postoperative courses. The 13-year-old boy had evidence of renal ischemia in a portion of the lower pole of the autotransplanted kidney by DTPA scan. He has mild hypertension controlled with antihypertensive medication. The 10-year-old girl has a normal DTPA scan and is normotensive. MAS is a rare and challenging congenital vascular anomaly that causes RVH. In the presence of double renal arteries the technique of autotransplantation with cold perfusion and "bench" vascular reconstruction reduces the warm ischemia time and should produce satisfactory results.

Diabetes as a complication of tacrolimus (FK506) in pediatric renal transplant patients.

Three cases of insulin-requiring diabetes mellitus associated with tacrolimus (FK506) therapy in pediatric renal transplant patients are presented. New-onset diabetes mellitus has been reported with tacrolimus therapy post liver and kidney transplants in up to 12% of adult patients, but is thought to be rare in pediatrics. Although insulin requirement with tacrolimus therapy has been occasionally reported in adolescent patients post liver transplant, only a single case in a pediatric kidney transplant recipient has been previously documented. These cases illustrate the significant diabetogenic effect of tacrolimus in pediatric renal transplant patients. As the use of tacrolimus becomes more prevalent in pediatric kidney transplantation, pediatric nephrologists should be aware of this potential complication.

Steroid sulfatase and the Y chromosome hypertensive locus of the spontaneously hypertensive rat.

The spontaneously hypertensive rat (SHR) has a Y chromosome locus that increases blood pressure. This locus requires an androgen receptor and testosterone for maximum expression. Steroid sulfatase (STS) catalyzes the conversion of steroid sulfates to their active nonconjugated form. In some mammals the steroid sulfatase locus (Sts) is on the Y chromosome, although the rat Sts is on the X chromosome. We measured STS activity levels in SHR and normotensive Wistar Kyoto (WKY) males. SHR had significantly higher STS activity in testes, adrenal gland, liver, and hypothalamus. The Km values for STS in the two strains were not significantly different; thus, activity differences were likely due to differences in enzyme amounts. STS activity was measured in the backcross strains SHR/y and SHR/a to test and/or confirm a Y chromosome influence on STS. STS activity levels in these strains were intermediate between those of SHR and WKY. Because the blood pressures of SHR/y and SHR/a were also intermediate between SHR and WKY, the STS activity could be a secondary response to the hypertension. An alternative hypotheses is that a regulatory locus in addition to the structural locus is responsible for STS activity levels, and this regulatory locus is on the rat Y chromosome. Further study is needed to discriminate between these possibilities, and until the second hypothesis can be eliminated, the Sts locus or its modifier loci remain a potential component of the Y chromosome hypertensive locus.

The implementation of a continuous quality improvement program in a pediatric peritoneal dialysis unit.

Continuous quality improvement (CQI) principles and practices were utilized to evaluate the outpatient peritoneal dialysis follow-up process and the routine monthly laboratory testing of chronic dialysis patients. CQI enabled us to reduce total clinic visit time from a mean of 161 min to 90.8 min. Waiting was decreased from a mean of 51.6 min to 15.8 min. The number of routine monthly laboratory tests performed on patients undergoing chronic dialysis was also reduced, resulting in decreased charges of at least $429.50 per patient per month.

The value of urinalysis in differentiating acute pyelonephritis from lower urinary tract infection in febrile infants.

There is an ongoing debate on the best way to screen febrile infants for urinary tract infection. We examined the urinanalysis (UA) findings on admission among infants less than 16 weeks old, with and without acute pyelonephritis (APN), as defined by the dimercaptosuccinic acid (DMSA) renal scan findings, performed during a 57-month period. Forty-nine cases with a positive DMSA scan were compared with 79 negative study cases. A negative UA for leukocytes (< 5 white blood cells/high power field) was found in 4 of 49 (8.1%) cases with APN by DMSA, and in 34 of 79 (43%) cases with a negative DMSA (odds ratio 10.88 (95% confidence interval, 2.31 to 70.3; P < 0.001)). Three of the 4 infants not suspected to have APN by their UA findings would have been admitted for a full sepsis workup based on their clinical presentation and/or their laboratory findings on admission. We conclude that a fresh UA may be a sufficient screening method for the exclusion of APN in infants assessed for fever of no obvious origin.

Efficacy of once- versus thrice-weekly subcutaneous recombinant human erythropoietin in children receiving continuous cycling peritoneal dialysis.

The subcutaneous administration of recombinant human erythropoietin is effective in the treatment of anemia of end-stage renal disease. Single-dose pharmacokinetic studies suggest the possibility of less frequent dosing via the subcutaneous route. In this study, dosing once-weekly was as effective as thrice-weekly subcutaneous dosing in maintaining the corrected hematocrit in a group of children receiving continuous cycling peritoneal dialysis. This regimen was preferred by and beneficial to both patients and their parents.

Recombinant erythropoietin (Epogen) improves cardiac exercise performance in children with end-stage renal disease.

To determine the effects of anemia in children with end-stage renal disease, we studied cardiac performance before and 1 and 6 months after recombinant erythropoietin (Epogen). Children with end-stage renal disease were included if they had significant anemia [hematocrit (Hct) < 30%]. Epogen 50 U/kg was given subcutaneously or intravenously three times per week until the Hct was > or = 33%. Echocardiography, cardiac output (acetylene rebreathing), and treadmill (modified Bruce) tests were performed. Boys (9) and girls (9), 11.9 +/- 5.6 years, were given Epogen and the Hct increased (from 21.7 +/- 2.7% to 33.4 +/- 2.1%, P = 0.001). Heart rate decreased (P = 0.04) and stroke volume did not change. Blood pressure did not change. Cardiac thickness, chamber dimensions, left ventricular wall stress, velocity of circumferential fiber shortening, and indices of diastolic function were normal and did not change after Epogen. Exercise time increased (from 10.3 +/- 1.9 to 11.2 +/- 1.9 min, P = 0.01) after 1 month of Epogen. Resting oxygen consumption (VO2) decreased (from 7.8 +/- 1.8 to 6.9 +/- 1.4 ml/min per kg, P = 0.01) 1 month after Epogen and peak exercise VO2 did not change after Epogen. There were no differences in exercise tests between the 1 and 6 month measurements. Exercise tolerance improves after the short-term correction of anemia and there is no further improvement after long-term correction.

Inconsistent divergence of mitochondrial DNA in the spontaneously hypertensive rat.

We have recently shown that the spontaneously hypertensive rat (SHR) and the Wistar-Kyoto (WKY) rat differ at a frequency of 1 per 62 bases in their nuclear DNA (Hypertension 1992; 19:425-427). Given the origin of these strains this level of divergence was unexpected. To investigate the origin of this nuclear divergence we have examined mitochondrial DNA. Mitochondrial DNA was isolated from SHR and WKY rats, digested with several restriction enzymes, electrophoresed in 1.0% agarose gels, and the fragments visualized with ethidium bromide staining. This approach allowed us to analyze 220 base pairs of mitochondrial DNA. No differences were detected between SHR and WKY rats. Comparison with the King-Holtzman rat strain produced differences at an average of 1 per 52 base pairs. We also examined several SHR and WKY rats from within our colonies and found no differences suggesting intrastrain homogeneity for mitochondrial DNA phenotypes. These data indicate that the SHR and WKY rat share a recent, common maternal ancestor. This result is consistent with the published origins of the SHR and WKY rat strains. Together with the nuclear divergence results, the data suggest that the original Wistar colony from which SHR and WKY rats were derived was probably highly polymorphic for nuclear genes.

The hypertensive Y chromosome elevates blood pressure in F11 normotensive rats.

Our laboratory has shown that the Y chromosome has a significant effect on blood pressure in the spontaneously hypertensive rat (SHR) model of hypertension and that the testes and androgen receptor contribute to the blood pressure rise. As an extension of our research, we have developed two new rat strains, SHR/a and SHR/y (F11) to study the Y chromosome. The objectives of the following research were 1) to study the blood pressure of rats with an SHR Y chromosome in a normotensive genetic background (SHR/y) or a normotensive Y chromosome in an SHR genetic background (SHR/a), 2) to determine the effect of male sex phenotype on the blood pressure of these rats, 3) to determine if testosterone replacement in castrated rats would restore blood pressure, and 4) to determine whether the Y chromosome from the SHR/y strain when crossed with a normotensive female can induce hypertension in androgen receptor-deficient male offspring. Blood pressure of male SHR/y rats was significantly higher than that of normotensive Wistar-Kyoto males (p < 0.01), and SHR/a males had significantly lower blood pressure compared with that of the parent SHR strain (p = 0.05). Testosterone replacement in castrated rats of both strains (SHR/a and SHR/y) restored blood pressure to control levels. Normotensive female King-Holtzman rats heterozygous for the testicular feminization gene were crossed with F11 SHR/a and SHR/y males.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic divergence between the Wistar-Kyoto rat and the spontaneously hypertensive rat.

A method of restriction fragment length polymorphism (RFLP) analysis was used to estimate the amount of genetic divergence between the spontaneously hypertensive rat (SHR) strain and the Wistar-Kyoto (WKY) strain. DNA from each strain was digested with eight restriction endonucleases and hybridized with six single copy gene sequences. The number of hybridization bands in each digestion was used to estimate the total number of bases analyzed and RFLPs were scored as single mutations. Divergence was then estimated by dividing the number of mutations by the number of bases analyzed. In a total of 808 bases analyzed in WKY rats, a minimum of 13 mutations were scored in SHR, which yields a nucleotide divergence of 1 change per 62 bp. This is an extremely high amount of divergence given the known origin of these two strains and is comparable to the maximum divergence possible between unrelated humans.

Purification of neuropathy target esterase from avian brain after prelabelling with [3H]diisopropyl phosphorofluoridate.

Neuropathy target esterase from hen brains was radiolabelled at the active site with [3H]diisopropyl phosphorofluoridate. The labelled protein was purified by differential centrifugation and Nonidet P40 solubilization, detergent phase partitioning, anion exchange, and preparative sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The volatilizable counts assay and analytical SDS-PAGE were used to monitor the protein. The 150-kDa subunit polypeptide appears as a single band on analytical SDS-PAGE.