Assessment of the clinical significance of anti-Dob

BACKGROUND: Anti-Dob is an uncommon antibody, and there are few data regarding its clinical importance. In the present case, the patient's transfusion management was based on both in vivo and in vitro assay results. CASE REPORT: A delayed hemolytic transfusion reaction was suspected in a 64-year-old white woman awaiting cardiac surgery when the transfusion of 1 unit of red cells failed to raise her hematocrit. Although direct antiglobulin tests were negative, antibody screening tests on samples drawn 9 days after transfusion were positive, and anti-Dob was identified, reacting to a titer of 4. 51Cr in vivo survival studies with incompatible Do(b+) red cells showed poor survival: 83.2 percent at 1 hour, 43 percent at 24 hours, and 29.6 percent at 48 hours and t1/2 = 19 hours (normal t1/2 = 25-35 days). A monocyte monolayer assay performed with the same incompatible Do(b+) donor red cells also indicated poor survival: 22 percent and 30 percent reactive monocytes, respectively, with and without the addition of complement (normal, 0-3%). The patient was given 4 Do(b-) red cell units without clinical signs or symptoms of a reaction. CONCLUSIONS: This example of anti-Dob was implicated in a delayed hemolytic transfusion reaction. The 51Cr survival studies and monocyte monolayer assay results indicated that the anti-Dob was clinically significant, requiring the use of Do(b-) red cells for transfusion.     

Inhibitors of glycogen phosphorylase b: synthesis, biochemical screening, and molecular modeling studies of novel analogues of hydantocidin

The synthesis and biochemical screening of four novel spironucleosides 1-4 against rabbit liver glycogen phosphorylase b (Gpb), along with molecular modeling studies on compound 2 and its 4-hydroxy analogue VII, have been presented. Gpb is a key enzyme of glycogen metabolism, and is known to be involved in the control of diabetes mellitus. The general strategy for synthesis involved base-catalyzed condensation of diethyl 2,4-dioxoimidazolidine-5-phosphonate (5) with either 2-deoxy-D-ribose or D-ribose, followed by sequential reactions involving ring-closure with phenylselenenyl chloride and reduction with tri-n-butyltin hydride catalyzed by azobisisobutyronitrile. Compounds 2 and 4 were found to be weak competitive inhibitors of Gpb, whereas 1 and 3 were inactive.     

Diet, plasma lipoproteins and experimental atherosclerosis in baboons (Papio sp.)

Diet-induced hyperlipidaemia in baboons is similar to that in humans. As in humans, the ratio between low density lipoprotein (LDL) and high density lipoprotein (HDL) cholesterol is a major determinant of atherosclerosis. Baboons, like humans and other non-human primates, vary in their lipaemic responses to dietary lipids. By selective breeding based on variability in plasma and lipoprotein cholesterol response to diet, lines of baboons with high and low responses of various lipoproteins have been developed. Genetic analyses suggest that lipoprotein patterns in response to dietary cholesterol and fat are heritable. Metabolic and molecular studies of high and low LDL and HDL cholesterol responses to dietary lipids have suggested that different mechanisms regulate plasma LDL cholesterol on the chow and on the high cholesterol-high fat (HCHF) diet. On the chow diet, plasma LDL cholesterol levels are positively associated with cholesterol absorption and negatively associated with hepatic LDL receptor levels and, thus, cholesterol absorption and LDL receptors seem to regulate plasma LDL cholesterol levels. However, when the animals consume a human-like fat- and cholesterol-enriched diet, plasma LDL cholesterol levels are not associated with either cholesterol absorption or hepatic LDL receptor mRNA levels, but are negatively associated with plasma 27-hydroxycholesterol concentrations, hepatic sterol 27-hydroxylase activity, and mRNA levels. Hepatic sterol 27-hydroxylase activity and mRNA levels are induced by dietary cholesterol and fat in low responding baboons more than in high responding baboons. Thus, the ability to induce sterol 27-hydroxylase determines the LDL cholesterol response in baboons. High HDL response baboons often have high levels of HDL1 in their plasma. Our studies suggest that the N-terminal fragment of apo C-I with 38 amino acids and a molecular weight of approximately 4 kDa acts as a cholesteryl ester transfer inhibitor peptide in high HDL1 baboons. The inhibitor peptide associates with apo A-1 in HDL to produce a modified apo A-1 protein with a molecular weight of approximately 31 kDa. The inhibitor peptide is a gene product and the presence of this peptide produces an antiatherogenic high HDL1 phenotype.     

Improved hemodynamics shown by continuous monitoring of electrical impedance during external counterpulsation with the end-diastolic pneumatic boot and improved ambulatory EKG monitoring after 3 weeks of therapy

Some investigators have reported previously that phorbol esters inhibit in vitro erythropoietin production stimulated by hypoxia; whereas others have reported that phorbol esters enhanced Epo production during exposure to hypoxia. We have demonstrated in the present experiments that hypoxia significantly increased diacylglycerol levels in cultured human hepatocellular carcinoma (Hep3B) cells. 1-oleoyl-2-acetyl-ras-glycerol (OAG) and N-(6-phenylhexyl)-5-chloro-1-naphthalenesulfonamide (SC-9), two well-known protein kinase C activators, significantly increased medium levels of erythropoietin as well as erythropoietin messenger RNA levels in normoxic Hep3B cells. A potent protein kinase C inhibitor, chelerythrine chloride, significantly decreased hypoxia-induced increases in medium levels of erythropoietin as well as erythropoietin messenger RNA levels in Hep3B cells. A cis-unsaturated free fatty acid, oleic acid, significantly enhanced OAG-induced medium levels of erythropoietin in normoxic Hep3B cells, whereas a phospholipase A2 inhibitor, mepacrine, significantly decreased hypoxia-induced erythropoietin production in Hep3B cells. These results provide strong support for a positive role for protein kinase C in the hypoxic regulation of erythropoietin production.     

Revision rates after knee replacement in the United States

We present a force plate system which measures low-magnitude vertical reaction forces generated by small laboratory animals. The force plate mechanical design minimizes radiated transverse waves, acoustic reverberation, and standing waves caused by impacts on the force plate surface. A secondary force plate and PC-based software algorithm minimize floor vibrational artifact. The force plate was used to measure function of rats during two tests: forelimb/hindlimb hopping reaction and surface righting reaction. In control rats, forelimb hopping rate exceeded hindlimb hopping rate during 16 weeks of repeated testing. Subchronic intraperitoneal (i.p.) dosing of 10 mg/kg/day acrylamide produced a selective impairment of hindlimb hopping. In contrast, single doses of haloperidol (1-5 mg/kg, i.p.) slowed the righting reaction and produced a relatively selective impairment of forelimb hopping. The force plate system presents new opportunities for performing quantitative neurological assessments of small laboratory animals when previously such tests had been performed subjectively and qualitatively.     

Protein kinase C--catalyzed calponin phosphorylation in swine carotid arterial homogenate

Calponin, a thin filament-associated protein, inhibits actin-activated myosin ATPase activity, and this inhibition is reversed by phosphorylation. Calponin phosphorylation by protein kinase C and Ca2+/calmodulin-dependent protein kinase II has been shown in purified protein systems but has been difficult to demonstrate in more physiological preparations. We have previously shown that calponin is phosphorylated in a cell-free homogenate of swine carotid artery. The goal of this study was to determine whether protein kinase C and/or Ca2+/calmodulin-dependent protein kinase II catalyzes calponin phosphorylation. Ca2+-dependent calponin phosphorylation was not inhibited by calmodulin antagonists. In contrast, both Ca2+- and phorbol dibutyrate/1-oleoyl-2-acetyl-sn-glycerol dependent calponin phosphorylation were inhibited by the pseudosubstrate inhibitor of protein kinase C and staurosporine. Our results also demonstrate that stimulation with either Ca2+, phorbol dibutyrate, or 1-oleoyl-2-acetyl-sn-glycerol activates endogenous protein kinase C. We interpret our results as clearly demonstrating that the physiological kinase for calponin phosphorylation is protein kinase C and not Ca2+/calmodulin-dependent protein kinase II. We also present data showing that the direct measurement of 32P incorporation into calponin and the indirect measurement of calponin phosphorylation using nonequilibrium pH gradient gel electrophoresis provide similar quantitative values of calponin phosphorylation.     

Sequence and evolution of the Drosophila pseudoobscura glycerol-3-phosphate dehydrogenase locus

The Gpdh genomic region has been cloned and sequenced in Drosophila pseudoobscura. A total of 6.8 kb of sequence was obtained, encompassing all eight exons of the gene. The exons have been aligned with the sequence from D. melanogaster, and the rates of synonymous and nonsynonymous substitution have been compared to those of other genes sequenced in these two species. Gpdh has the lowest rate of nonsynonymous substitution yet seen in genes sequenced in both D. pseudoobscura and D. melanogaster. No insertion/deletion events were observed, and the overall architecture of the gene (i.e., intron sites, etc.) is conserved. An interesting amino acid reversal was noted between the D. melanogaster Fast allele and the D. pseudoobscura gene.     

Clinical denouement and mutation analysis of patients with cystic fibrosis undergoing liver transplantation for biliary cirrhosis

OBJECTIVE: To describe the clinical characteristics of patients with cystic fibrosis considered for liver transplantation and the clinical outcome after transplantation. METHODS: Patient charts were reviewed. Mutation analysis was performed on blood or liver tissue samples with a panel of 17 mutations. RESULTS: Eight patients (five girls) with cystic fibrosis have undergone orthotopic liver transplantation for biliary cirrhosis. Mean age at transplantation was 12.0 years +/- 7.7 years (range, 9 months to 23 years). Preoperatively, seven patients had mild to moderate pulmonary dysfunction and one moderate to severe pulmonary dysfunction. All patients required pancreatic enzyme replacement, and four patients required insulin for diabetes mellitus. The 1-year survival rate was 75%, with no deaths related to septic events. Mean time of follow-up the six operative survivors was 4.1 years +/- 1.9 years. Pulmonary function testing, in those serially tested, showed that forced expiratory volume in 1 second was maintained or improved and that forced vital capacity improved after transplantation. Mutation analysis showed the following genotypes: four patients, delta F508/delta F508; one patient, delta F508/N1303K; and three patients, delta F508/unknown. CONCLUSIONS: Despite the high risk of transplantation, these encouraging results indicate that liver transplantation should be considered for patients with cystic fibrosis and complications of end-stage liver disease. We could not demonstrate an unusual pattern of CF gene mutations in these patients with severe liver disease. It appeared that immunosuppressive agents did not have a deleterious effect on pulmonary function.