A one-bead, one-stock solution approach to chemical genetics: part 2.

BACKGROUND: Chemical genetics provides a systematic means to study biology using small molecules to effect spatial and temporal control over protein function. As complementary approaches, phenotypic and proteomic screens of structurally diverse and complex small molecules may yield not only interesting individual probes of biological function, but also global information about small molecule collections and the interactions of their members with biological systems. RESULTS: We report a general high-throughput method for converting high-capacity beads into arrayed stock solutions amenable to both phenotypic and proteomic assays. Polystyrene beads from diversity-oriented syntheses were arrayed individually into wells. Bound compounds were cleaved, eluted, and resuspended to generate 'mother plates' of stock solutions. The second phase of development of our technology platform includes optimized cleavage and elution conditions, a novel bead arraying method, and robotic distribution of stock solutions of small molecules into 'daughter plates' for direct use in chemical genetic assays. This library formatting strategy enables what we refer to as annotation screening, in which every member of a library is annotated with biological assay data. This phase was validated by arraying and screening 708 members of an encoded 4320-member library of structurally diverse and complex dihydropyrancarboxamides. CONCLUSIONS: Our 'one-bead, multiple-stock solution' library formatting strategy is a central element of a technology platform aimed at advancing chemical genetics. Annotation screening provides a means for biology to inform chemistry, complementary to the way that chemistry can inform biology in conventional ('investigator-initiated') small molecule screens.

The ketogenic diet in refractory epilepsy: the experience of Children's Hospital of Pittsburgh.

The ketogenic diet appears to be effective in reducing seizure frequency in patients with epilepsy refractory to antiepileptic drug therapy. Reported seizure frequencies before and after the diet was initiated were obtained for 48 patients started on the ketogenic diet between December 1994 and January 1998 at Children's Hospital of Pittsburgh. The majority of patients (71%) were able to achieve > or = 50% reduction in seizure activity. Of these, more than half (53%) had > 90% reduction in seizures after 45 days of diet therapy. Complications included gastrointestinal complaints and infrequent lipid abnormalities. The ketogenic diet appears to be an effective method of treatment for children with epilepsy refractory to drug therapy.

Coordinated interdisciplinary management of pediatric intestinal failure: a 2-year review.

BACKGROUND/PURPOSE: Intestinal failure is a complex metabolic process that results from malabsorption and malnutrition and provides challenges for a variety of pediatric subspecialists. The purpose of this study was to evaluate the effect of coordinated interdisciplinary team management of children with intestinal failure on nutritional outcome measures. METHODS: The Intestinal Care Center (ICC) is staffed with an interdisciplinary team of pediatric specialists including a gastroenterologist, pediatric surgeon, transplant surgeon, clinical dietitians, and a nutrition support nurse. Using an established registry, the authors conducted a comprehensive evaluation of patient data including anthropometric measures, organ system function, and mode of nutrition support. Disease-associated complications including micronutrient deficiencies, growth delay, and death also were monitored. Nutritional outcome was assessed by transition from enteral to oral feeding, cessation of total parenteral nutrition (TPN), and maintenance of linear growth. RESULTS: Since the inception of the ICC in 1996, 103 patients (69 boys, 34 girls) with intestinal failure have been evaluated with a median age of 2.6 years (range, 0.2 to 21.3 years). Mode of nutritional therapy on initial consultation included TPN (n = 76, 74%), enteral feedings (n = 6, 6%) and oral intake (n = 21, 20%). After intensive management of the 76 patients who were TPN dependent, 22 (29%) subsequently have been weaned from TPN (duration, 0.2 to 17.5 years) to oral (n = 14), oral-enteral (n = 4) or enteral feedings (n = 4). Of the 6 patients who were receiving enteral feedings, 4 (67%) were transitioned to oral feedings. Sixty-eight patients (66%) had evidence of hepatic disease. Of these, 10 underwent transplant, and 23 died (2 posttransplant). Linear growth velocity of neither pre- nor postpubescent patients significantly improved during the 2-year study period. CONCLUSION: Data registry establishment and concurrent interdisciplinary team management of children with intestinal failure provides for innovative treatment approaches and a foundation for retrospective or prospective assessment of children with disease.

The role of lysine 55 in determining the specificity of the purine repressor for its operators through minor groove interactions.

The interaction of the dimeric Escherichia coli purine repressor (PurR) with its cognate sequences leads to a 45 degrees to 50 degrees kink at a central CpG base step towards the major groove, as dyad-related leucine side-chains interdigitate between these bases from the minor groove. The resulting broadening of the minor groove increases the accessibility of the six central base-pairs towards minor groove interactions with residues from PurR. It has been shown that lysine 55 of PurR makes a direct contact with the adenine base (Ade8) directly 5' to the central CpG base-pair step in the high-affinity purF operator sequence. We have investigated the importance of this interaction in the specificity and affinity of wild-type PurR (WT) for its operators and we have studied a mutant of PurR in which Lys55 is replaced with alanine (K55A). Complexes of WT and K55A with duplex DNA containing pur operator sequences varied at position 8 were investigated crystallographically, and binding studies were performed using fluorescence anisotropy. The structures of the protein-DNA complexes reveal a relatively unperturbed global conformation regardless of the identity of the base-pair at position 8 or residue 55. In all structures the combination of higher resolution and a palindromic purF operator site allowed several new PurR.DNA interactions to be observed, including contacts by Thr15, Thr16 and His20. The side-chain of Lys55 makes productive, though varying, interactions with the adenine, thymine or cytosine base at position 8 that result in equilibrium dissociation constants of 2.6 nM, 10 nM and 35 nM, respectively. However, the bulk of the lysine side-chain apparently blocks high-affinity binding of operators with guanine at position 8 (Kd620 nM). Also, the high-affinity binding conformation appears blocked, as crystals of WT bound to DNA with guanine at position 8 could not be grown. In complexes containing K55A, the alanine side-chain is too far removed to engage in van der Waals interactions with the operator, and, with the loss of the general electrostatic interaction between the phosphate backbone and the ammonium group of lysine, K55A binds each operator weakly. However, the mutation leads to a swap of specificity of PurR for the base at position 8, with K55A exhibiting a twofold preference for guanine over adenine. In addition to defining the role of Lys55 in PurR minor groove binding, these studies provide structural insight into the minor groove binding specificities of other LacI/GalR family members that have either alanine (e.g. LacI, GalR, CcpA) or a basic residue (e.g. RafR, ScrR, RbtR) at the comparable position.

Insulinlike growth factor 1 and insulinlike growth factor 3: indices of intestinal failure in children.

BACKGROUND/PURPOSE: A number of pediatric patients with short bowel syndrome (SBS) manifest growth failure despite aggressive nutritional support. Exogenous growth hormone (GH) therapy in children with SBS has proved disappointing. The purpose of this study was to determine if there were characteristic patterns of GH, IGF-1, or IGFBP-3 levels in pediatric SBS patients with profound growth failure in an effort to elucidate an early strategic approach to management of SBS in the subpopulation. METHODS: Forty patients (29 boys, 11 girls; mean age, 5.3 years; range, 0.5 to 18.6 years) with SBS (<30% total bowel length) who received intensive nutrition support and follow-up underwent serological tests for GH, IGF-1, IGFBP-3, and thyroid function. Height (HT), weight (WT), and bone age were assessed relative to age-appropriate percentiles. Growth failure was defined as a HT and WT at less than the fifth percentile and bone age > or = 2 standard deviations below actual age. Residual small bowel length was determined by review of pathological and operative reports. Comparisons between the growth factors, bowel length, and anthropometric data were analyzed by chi2. RESULTS: Two distinct subgroups of patients emerged from our study. Thirty-eight percent of patients (n = 11) had growth failure by anthropometry that was associated significantly with low IGF-1 independently and with both IGF-1 and IGFBP-3 levels (P< 0.05). There were no significant associations with GH level, thyroid function, small bowel length, or the amount of parenteral versus enteral intake in either subgroup of these patients. Low IGF-1 and IGFBP-3 but not GH levels may be indices of intestinal failure in pediatric SBS. Growth in this subpopulation is refractory to aggressive standard approaches to nutritional support and may require early interventions. CONCLUSION: Exogenous IGF-1 and IGFBP-3, not GH, may be beneficial to treat this subpopulation.

Association of retinol binding protein in multiple-case families with insulin-dependent diabetes.

We performed a family study to investigate the heritability of reduced serum retinol levels observed in type 1 diabetes cases. Diet and serum factors, including retinol, total carotene, malondialdehyde, and retinol binding protein levels, were measured in 11 multiple-case families. The mean serum retinol level of the diabetics (46 ug/dl) was significantly less than the mean serum retinol level of the nondiabetics (60.9 ug/dl). The level of retinol binding protein was also significantly lower in diabetics (6.2 mg/dl) than in nondiabetics (7.6 mg/dl). The serum values of retinol binding protein were closely related within families, including both diabetic and nondiabetic family members. A characteristic shared between diabetics and one-third of their family members was a low ratio of serum retinol to total carotene, suggesting a low conversion of dietary carotene into retinol. Analysis of food frequency reports showed no difference between dietary retinol or total carotene level in diabetics or their relatives. This study offers evidence that heritability and the reduced conversion of carotene may play a role in the level of serum retinol in type 1 diabetes cases.

Molecular epidemiology of autoimmune thyroid disease.

This paper presents preliminary data regarding the prevalence and risk factors for autoimmune thyroid disease in IDDM probands ascertained from the Children's Hospital of Pittsburgh IDDM Registry for 1950-1965 (n = 669). Living IDDM probands who participated in the 1990 follow-up survey (n = 380) were recruited for the Familial Autoimmune and Diabetes Study. Siblings and parents were also invited to participate. To date, 255 IDDM probands and 597 parents and siblings have been evaluated. The diagnosis of autoimmune thyroid disease was based on a clinical evaluation, medical history, and laboratory determinations. Graves disease was rare in this cohort (n = 5). However, Hashimoto's thyroiditis was common among women. Prevalence rates ranged from 54% for IDDM women age < 40 years to 75% for those > 50 years. Corresponding age-specific estimates for female relatives were 22% and 44%, respectively. Approximately one-half of the Hashimoto's individuals were euthyroid; they were more likely to have other autoantibodies and a positive family history than those who were hypothyroid or had no thyroid disease. Genetic analyses revealed a 2-fold increase in DQA1*0501-DQB1*0201 among the Hashimoto's compared to the non-Hashimoto's haplotypes. These findings suggested that Hashimoto's thyroiditis was common in IDDM families, which may be due, in part, to common disease susceptibility genes.

Nutrition in the pediatric double lung transplant patient with cystic fibrosis.

Cystic fibrosis (CF) is the most common lethal genetic disease in the white population. The pulmonary infections and pancreatic insufficiency make CF a medically challenging disease. Although the importance of nutrition in the CF patient is known, approximately 50% of CF patients are in less than the 10th percentile for weight and height as reported by the 1991 CF Foundation Registry of 114 CF Centers in the United States. This paper addresses the nutritional status of 10 pediatric CF patients who underwent double lung transplant at Children's Hospital of Pittsburgh between August 1991 and May 1993. Patients who survived beyond 1 year gained a significant amount of weight sooner after transplant than those who survived less than 1 year. Gastrostomy tube feedings were more effective than oral intake for weight gain after transplant. CF patients with pancreatic insufficiency have more difficulty with adjustment of doses of immunosuppressive agents for reasons that are not clearly understood.