One-third-the-sites transition-state inhibitors for purine nucleoside phosphorylase

Genetic defects in human purine nucleoside phosphorylase cause T-cell deficiency as the major phenotype. It has been proposed that efficient inhibitors of the enzyme might intervene in disorders of T-cell function. Compounds with features of the transition-state structure of purine nucleoside phosphorylase were synthesized and tested as inhibitors. The transition-state structure for purine nucleoside phosphorylase is characterized by (1) an elevated pKa at N7 of the purine ring for protonation or favorable H-bond interaction with the enzyme and (2) oxocarbenium ion formation in the ribosyl ring (Kline, P. C., and Schramm, V. L. (1995) Biochemistry 34, 1153-1162). Both features have been incorporated into the stable transition-state analogues, (1S)-1-(9-deazahypoxanthin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol (immucillin-H) and (1S)-1-(9-deazaguanin-9-yl)-1,4-dideoxy-1, 4-imino-D-ribitol (immucillin-G). Both inhibitors exhibit slow-onset tight-binding inhibition of calf spleen and human erythrocyte purine nucleoside phosphorylase. The inhibitors exhibit equilibrium dissociation constants (Ki) from 23 to 72 pM and are the most powerful inhibitors reported for the enzyme. Complete inhibition of the homotrimeric enzyme occurs at one mole of inhibitor per mole of enzymic trimer. Binding of the transition-state inhibitor at one site per trimer prevents inhibitor binding at the remaining two sites of the homotrimer. A mechanism of sequential catalysis at each subunit, similar to that of F1 ATPase, is supported by these results. Slow inhibitor dissociation (e.g., t1/2 of 4.8 h) suggests that these compounds will have favorable pharmacologic properties. Interaction of transition-state inhibitors with purine nucleoside phosphorylase is different from reactant-state (substrate and product analogue) inhibitors of the enzyme which bind equally to all subunits of the homotrimer.     

Common office problems in pediatric urology and gynecology

The number of genital problems that pediatricians encounter is substantial. The most common ones have been reviewed in this article. Perhaps the most important point to reinforce is the appropriateness of nonintervention in uncircumcised boys whose foreskins have not become retractile during early school years. Without infections or pathologic phimosis, these boys do well, and most foreskins become retractile as they approach puberty. Abnormalities beyond those discussed or those not fitting the anticipated pattern probably warrant specialty referral.     

Solution structure of the C-terminal SH2 domain of the p85 alpha regulatory subunit of phosphoinositide 3-kinase

Heterodimeric class IA phosphoinositide 3-kinase (PI 3-kinase) plays a crucial role in a variety of cellular signalling events downstream of a number of cell-surface receptor tyrosine kinases. Activation of the enzyme is effected in part by the binding of two Src homology-2 domains (SH2) of the 85 kDa regulatory subunit to specific phosphotyrosine-containing peptide motifs within activated cytoplasmic receptor domains. The solution structure of the uncomplexed C-terminal SH2 (C-SH2) domain of the p85 alpha subunit of PI 3-kinase has been determined by means of multinuclear, double and triple-resonance NMR experiments and restrained molecular-dynamics simulated-annealing calculations. The solution structure clearly indicates that the uncomplexed C-SH2 domain conforms to the consensus polypeptide fold exhibited by other SH2 domains, with an additional short helical element at the N terminus. In particular, the C-SH2 structure is very similar to both the p85 alpha N-terminal SH2 domain (N-SH2) and the Src SH2 domain with a root mean square difference (rmsd) for 44 C alpha atoms of 1.09 and 0.89 A, respectively. The canonical BC, EF and BG loops are less well-defined by the experimental restraints and show greater variability in the ensemble of C-SH2 conformers. The lower level of definition in these regions may reflect the presence of conformational disorder, an interpretation supported by the absence or broadening of backbone and side-chain NMR resonances for some of these residues. NMR experiments were performed, where C-SH2 was titrated with phosphotyrosine-containing peptides corresponding to p85 alpha recognition sites in the cytoplasmic domain of the platelet-derived growth-factor receptor. The ligand-induced chemical-shift perturbations indicate the amino-acid residues in C-SH2 involved in peptide recognition follow the pattern predicted from homologous complexes. A series of C-SH2 mutants was generated and tested for phosphotyrosine peptide binding by surface plasmon resonance. Mutation of the invariant Arg36 (beta B5) to Met completely abolishes phosphopeptide binding. Mutation of each of Ser38, Ser39 or Lys40 in the BC loop to Ala reduces the affinity of C-SH2 for a cognate phosphopeptide, as does mutation of His93 (BG5) to Asn. These effects are consistent with the involvement of the BC loop and BG loops regions in ligation of phosphopeptide ligands. Mutation of Cys57 (beta D5) in C-SH2 to Ile, the corresponding residue type in the p85 alpha N-SH2 domain, results in a change in peptide binding selectivity of C-SH2 towards that demonstrated by p85 alpha N-SH2. This pattern of p85 alpha phosphopeptide binding specificity is interpreted in terms of a model of the p85 alpha/PDGF-receptor interaction.     

Efficient gene transfer to human endothelial cells using DNA complexed to adenovirus particles

Human endothelial cells have been found to be relatively refractory to various methods of DNA transfection currently in common use. By using a transfection method involving DNA complexed with replication-deficient adenovirus particles, we have shown that 20% of a population of cultured endothelial cells can be transfected and high levels of transient expression achieved. Both early-passage human umbilical vein endothelial cells and the continuous differentiated line of human endothelium-derived EA.hy926 cells are responsive to this method of transfection. Efficient DNA transfection of endothelial cells is important for studies of endothelium-specific promoters and is a potentially useful route for transgenic therapy.     

Low levels of eicosapentaenoic and docosahexaenoic acids mimic the effects of fish oil upon rat lymphocytes

Fish oil is rich in the long chain n-3 polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA); typically these fatty acids constitute 20 to 25 g/100 g total fatty acids in fish oil. Feeding rodents diets rich in fish oil has been shown to decrease lymphocyte proliferation and natural killer cell activity. It is not known what level of EPA + DHA is required in the diet to exert these effects. This question was addressed in the current study. Weanling rats were fed on high fat (178 g/kg) diets which contained 4.4 g alpha-linolenic acid (control) or 4.4 g EPA + DHA (4.4 EPA + DHA) or 6.6 g EPA + DHA (6.6 EPA + DHA)/100 g total fatty acids. The n-6 to n-3 polyunsaturated fatty acid ratio was maintained at approximately 7. The fatty acid compositions of the serum and of spleen leukocytes were markedly influenced by that of the diet. Spleen lymphocyte proliferation in response to concanavalin A, spleen natural killer cell activity and PGE2 production by spleen leukocytes were reduced by feeding the EPA + DHA diets compared with feeding the control diet; the 4.4 and 6.6 EPA + DHA diets caused very similar reductions. The 4.4 EPA + DHA diet reduced popliteal lymph node weight following a localised graft versus host response; this response was not investigated in rats fed the 6.6 EPA + DHA diet. The reductions in lymphocyte functions and in the in vivo graft versus host response caused by the EPA + DHA diets were similar to those previously reported following the feeding of diets rich in fish oil. Thus, this study shows that diets containing relatively low levels of EPA + DHA (20 to 25% of the level found in fish oil) exert immunomodulatory effects. Furthermore, this study suggests that the maximal effect of EPA + DHA is exerted when these fatty acids constitute a level of less than or equal to 4.4 g/100 g total dietary fatty acids.     

The economic cost of musculoskeletal disorders in Canada

OBJECTIVE: This study estimated the total cost of musculoskeletal disorders for Canadians in 1994 and assessed the sensitivity of these cost estimates to variations in the definition of musculoskeletal disorders. METHODS: Disease-related costs, from a societal perspective, were measured using a prevalence-based analysis. First, direct treatment costs, including expenditures on hospitals and other institutions, physicians and other health professionals, drugs, research, and other items were assessed. Second, indirect costs associated with lost (or foregone) productivity due to disability and premature mortality were evaluated using the human capital approach. RESULTS: The total cost of musculoskeletal disorders in Canada was $25.6 billion (in 1994 Canadian dollars, $1.00 CDN approximately $0.75 US) or 3.4% of the gross domestic product. Direct and indirect costs were estimated at $7.5 billion and $18.1 billion, respectively. Lower and upper bound estimates of the total cost of musculoskeletal disorders, derived from the sensitivity analysis, were $19.9 billion and $30.8 billion, respectively. Wide variations were reported in the total cost of various musculoskeletal disorder subcategories, with the highest costs reported for injuries ($10.7 billion), back and spine disorders ($8.1 billion), and arthritis and rheumatism ($5.9 billion). CONCLUSIONS: The economic cost of musculoskeletal disorders was substantial and was sensitive to the definition of musculoskeletal disorders and other underlying assumptions. The hallmark of this study was the variation between subcategories in their cost, pattern of health resource use, and sequelae. The cost estimates may provide guidance in setting priorities for research and prevention activities.     

Outcomes research: a review

PURPOSE: The purpose of this article is to review the history of the medical outcomes movement as well as the methodologies used in outcomes research. CONCEPT: Outcomes research refers to a genre of clinical investigation that emphasizes the measurement of patient health outcomes, including the patient's symptoms, functional status, quality of life, satisfaction with treatment, and health care costs. RATIONALE: Outcomes research evolved from studies that demonstrated the presence of wide geographic variations in the practice of medicine and surgery. Such differences in utilization were unaccompanied by any discernible difference in patient outcomes. With escalating health care costs, there has been a growing interest in measuring the outcomes of medical intervention to determine the quality and appropriateness of medical care. DISCUSSION: Outcomes may be measured both directly and indirectly, over differing periods of time, and with varying degrees of objectivity, reliability, and validity. Current research has focused on quality of life issues, which include the extent to which a patient's usual or expected physical, emotional, and social well-being have been affected by a medical condition or treatment. The true value of health care can be determined only by a systematic examination of patient outcomes. To accomplish this goal, methods are required that are relatively unfamiliar to many clinical researchers. Future clinical research should include patient-oriented outcome measures that would otherwise focus solely on physiological or anatomic outcomes. Such information will be essential in determining which medical and surgical treatment strategies should be abandoned and which will gain acceptance in the future.     

Effects of intraduodenal administration of tarazepide on pancreatic secretion and duodenal EMG in neonatal calves

The influence of CCK-A receptor antagonism on pancreatic exocrine secretion and duodenal EMG, and the mechanism(s) involved in CCK-induced pancreatic secretion were studied in conscious calves. Seven 1-week-old calves were fitted with a pancreatic duct catheter, duodenal cannula and duodenal electrodes. Pancreatic exocrine secretion and duodenal EMG were studied following intraduodenal CCK-A receptor antagonist (Tarazepide), intravenous atropine, and intravenous or intraduodenal CCK-8 administrations. Tarazepide decreased duodenal electric activity, reduced interdigestive pancreatic secretion, especially protein; reduced cephalic and early postprandial (milk) induced secretion of bicarbonate and protein. Pancreatic protein secretion to intravenous CCK-8 was little affected by atropine, but was significantly reduced by Tarazepide+/-atropine; in contrast, protein secretion to intraduodenal CCK-8 was abolished by Tarazepide or atropine. We conclude that pre- and especially early postprandial pancreatic secretion are partly controlled via CCK-A (mainly mucosal) mediated mechanisms.