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Biomedical subjects

W N Kelley

Publications and source records attributed to W N Kelley.

At least 19 recordsLinked to original sources
PubMed1992

Faculty tracks and academic success.

In 1977, the Department of Internal Medicine at the University of Michigan implemented two specific faculty career tracks, the physician-scientist and clinician-scholar, to define more clearly the goals and expectations to which individuals should strive to achieve academic success. In response to the changing environment, a leadership track and a full-time clinical track were added. Although concerns about comparability, transfer between tracks, and research productivity were raised initially, they were alleviated as it became apparent that the ability to achieve tenure was similar in the physician-scientist and clinician-scholar tracks. The development of well-defined faculty tracks has facilitated the alignment of talents, training, and effort with career goals. It has also enabled us to protect the time of young investigators to pursue their research activities and to define the expectations for promotion for clinicians with a major commitment to patient care.

Administrative Personnel↗
PubMed1991

Determination of the mutations responsible for the Lesch-Nyhan syndrome in 17 subjects.

Hypoxanthine--guanine phosphoribosyltransferase (HPRT) is a purine salvage enzyme that catalyzes the conversion of hypoxanthine to inosine monophosphate and guanine to guanosine monophosphate. Previous studies of mutant HPRT proteins analyzed at the molecular level have shown a significant heterogeneity. This investigation further verifies this heterogeneity and identifies insertions, deletions, and point mutations. The direct sequencing of the polymerase chain reaction-amplified product of reverse-transcribed HPRT mRNA enabled the rapid identification of the mutations found in 17 previously uncharacterized cell lines derived from patients with the Lesch-Nyhan syndrome.

Cell Line↗
PubMed1991

A mutant allele common to the type I adenine phosphoribosyltransferase deficiency in Japanese subjects.

Adenine phosphoribosyltransferase (APRT) deficiency is a genetic disorder which causes 2,8-dihydroxy-adenine urolithiasis. The estimated incidence of heterozygosity in Caucasian and Japanese populations is 1%. Mutant alleles responsible for the disease have been classified as APRT*Q0 (type I) and APRT* (type II). In our previous study, we demonstrated in APRT*J a single common base change which accounts for 70% of the Japanese mutants. The present report describes the analysis of an APRT*Q0 mutation in Japanese subjects. Two nucleotide substitutions common to all seven affected alleles from four unrelated subjects (three homozygotes and a heterozygote) were identified: G----A at nucleotide position 1453 and C----T at 1456. The G----A altered the amino acid Trp98 to a stop codon. The C----T did not alter Ala99. These point mutations were demonstrated by sequence analysis of polymerase chain reaction (PCR)-amplified genomic DNA and cDNA. The G----A change at 1453 results in the elimination of a PflMI site in the APRT gene. PflMI digests, which were used to confirm the G----A transition, can be useful in screening for this specific mutation.

Adenine Phosphoribosyltransferase↗
PubMed1991

Identification of 17 independent mutations responsible for human hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency.

Complete hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency causes the Lesch-Nyhan syndrome, an X-linked, purine metabolism disorder manifested by hyperuricemia, hyperuricaciduria, and neurologic dysfunction. Partial HPRT deficiency causes hyperuricemia and gout. One requirement for understanding the molecular basis of HPRT deficiency is the determination of which amino acids in this salvage enzyme are necessary for structural or catalytic competence. In this study we have used the PCR coupled with direct sequencing to determine the nucleotide and subsequent amino acid changes in 22 subjects representing 17 unrelated kindreds from the United Kingdom. These mutations were confirmed by using either RNase mapping or Southern analyses. In addition, experiments were done to determine enzyme activity and electrophoretic mobility, and predictive paradigms were used to study the impact of these amino acid substitutions on secondary structure.

Chromosome Deletion↗
PubMed1989

The integrated inpatient management model. A new approach to clinical practice.

The delivery and financing of health care have undergone a metamorphosis over the past 10 years. These changes have been particularly dramatic for hospital care. The new health care environment, with more prospective-payment and managed-care systems and less fee-for-service payment for both physicians and hospitals, has made physicians and hospitals mutually dependent. A hospital's long-term financial viability is now dependent largely on the practice style of its physicians. The Department of Internal Medicine at The University of Michigan has developed a new clinical management system called the Integrated Inpatient Management Model (IIMM). This new system includes a major revision of the hospital organization structure, new administrative information systems, and new clinical information systems. Physicians in the Department of Internal Medicine have assumed for the first time formal organizational responsibility for many aspects of the operations of the inpatient medical service. The IIMM represents a prototype of a system that we believe offers considerable promise for involving physicians to a much greater extent in the management of the nation's hospitals. We hope that describing the system in detail will facilitate the development of other systems for the management of the inpatient practice of internal medicine.

Diagnosis-Related Groups↗
PubMed1989

Expression of human HPRT mRNA in brains of mice infected with a recombinant herpes simplex virus-1 vector.

Complete deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) results in a devastating neurological disease, the Lesch-Nyhan syndrome. This disorder has been identified as a candidate for initial attempts at somatic cell gene therapy. We have previously reported the construction of a recombinant herpes simplex virus type 1 (HSV-1) vector containing human hprt cDNA sequences under the regulatory control of the viral thymidine kinase gene (tk) [Palella et al., Mol. Cell. Biol. 8 (1988) 457-460]. Infection of HPRT- cultured rat neuronal cells with these vectors resulted in transient expression of human hprt. In this paper, we report the expression of human hprt mRNA transcripts in the brains of mice infected in vivo with this vector by direct intracranial inoculation. Human hprt transcripts were distinguished from endogenous mouse transcripts by RNase A mapping using riboprobes transcribed from human hprt cDNA. These initial studies demonstrate the transfer and transcription of a human gene in brain cells by direct in vivo infection with recombinant HSV-1 vectors.

Animals↗
PubMed1989

Human hypoxanthine-guanine phosphoribosyltransferase deficiency. The molecular defect in a patient with gout (HPRTAshville).

The genetic basis of hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency has been identified by nucleotide sequence analysis of HPRT cDNAs cloned from a patient with gout. A single nucleotide change was identified in two independent clones: an A to G transition at nucleotide 602. Confirmation of a mutation at this site was provided by RNase mapping analysis. The predicted consequence of this transition is an aspartic acid to glycine substitution at amino acid 201. We have designated this variant HPRTAshville. Prior to this report, enzyme activity in HPRTAshville had not been detected by routine assay. Using more sensitive techniques, including an in situ gel assay for HPRT activity, we were able to demonstrate electrophoretic, kinetic, and structural differences between HPRTAshville and normal HPRT. Electrophoretic migration of HPRTAshville has elevated Michaelis constants for 5-phosphoribosyl-1-pyrophosphate and hypoxanthine. Predicted secondary structural alterations may result from the aspartic acid to glycine substitution.

Amino Acid Sequence↗
PubMed1989

Lesch-Nyhan syndrome due to a single nucleotide change in the hypoxanthine-guanine phosphoribosyltransferase gene (HPRTYale).

We have cloned and sequenced a full length cDNA for HPRT cDNA for HPRTYale isolated from Lesch-Nyhan subject and identified a single nucleotide substitution which results in amino acid substitution of glycine to arginine. Since most HPRT mutants have normal levels of specific HPRT messenger RNA, mutant cDNA analysis is the method of choice to define the mutation in HPRT deficient subjects.

Amino Acid Sequence↗
PubMed1989

Identification of a single nucleotide change in the hypoxanthine-guanine phosphoribosyltransferase gene (HPRTYale) responsible for Lesch-Nyhan syndrome.

Complete deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT) causes the Lesch-Nyhan syndrome. Previous characterization of a mutant form of HPRT, HPRTYale, from a subject with the Lesch-Nyhan syndrome revealed normal mRNA and protein concentrations, no residual catalytic activity, and cathodal migration upon PAGE. We have cloned and sequenced HPRTYale cDNA. The nucleotide sequence of full-length HPRTYale cDNA revealed a single nucleotide substitution compared with normal HPRT cDNA: G----C at nucleotide position 211. This transversion predicts substitution of arginine for glycine at amino acid position 71, explaining the cathodal migration of HPRTYale. Chou-Fasman secondary structure analysis predicts a change in the probability of beta-turn formation in the region containing the mutation. Inclusion of the bulky arginine side chain in place of glycine probably disrupts protein folding as well. Cloning mutant forms of cDNA allows identification of specific mutations, provides insight into mutational mechanisms, and facilitates structure-function analysis of mutant proteins.

DNA↗
PubMed1989

Molecular basis of hypoxanthine-guanine phosphoribosyltransferase deficiency in ten subjects determined by direct sequencing of amplified transcripts.

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency is an inborn error of purine metabolism. Mutant HPRT gene sequences from patients deficient in enzyme activity have previously been characterized by cDNA cloning or amino acid sequencing techniques. The presence of HPRT-specific mRNA in nearly all deficient subjects, as well as the small size of the HPRT mRNA (1,400 bp), make the polymerase chain reaction (PCR) an alternative for the identification of mutations at this locus. In this report we use the PCR to identify previously undetermined mutations in HPRT mRNA from B lymphoblasts derived from 10 deficient individuals. Six of these variants contain single point mutations, three contain deletions, and one contains a single nucleotide insertion. Several of these mutations map near previously identified HPRT variants, and are located in evolutionarily conserved regions of the molecule.

Amino Acid Sequence↗
PubMed1988

Human hypoxanthine-guanine phosphoribosyltransferase: a single nucleotide substitution in cDNA clones isolated from a patient with Lesch-Nyhan syndrome (HPRTMidland).

We have determined the molecular basis for hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency in a patient, J.H., with Lesch-Nyhan syndrome. Radioimmunoassay of lysates of erythrocytes or cultured B-lymphoblasts showed that this patient had no detectable HPRT enzyme activity or HPRT protein. HPRT-specific mRNA levels were normal by Northern analysis. We created a cDNA library from mRNA isolated from cultured lymphoblasts derived from this patient. Nucleotide sequencing of full-length HPRT cDNA clones revealed a single nucleotide (nt) substitution: a T-to-A transversion at nt 389. We have designated this variant HPRTMidland. The predicted amino acid (aa) substitution in HPRTMidland is a valine to aspartic acid at aa 130. This substitution is within 2 aa of the amino acid substitution in a previously defined HPRT variant, HPRTAnn Arbor. Both mutations are within a highly conserved sequence in the putative 5-phosphoribosyl-1-pyrophosphate-binding domain. The amino acid substitution in HPRTMidland causes a significant perturbation in the predicted secondary structure of this region. The HPRTMidland mutation affects a different domain of HPRT than the HPRTFlint mutation located at 167 nt away.

Adenine↗
PubMed1988

Genetic basis of hypoxanthine guanine phosphoribosyltransferase deficiency in a patient with the Lesch-Nyhan syndrome (HPRTFlint).

The molecular basis for complete hypoxanthine guanine phosphoribosyltransferase (HPRT) deficiency has been determined in a patient with Lesch-Nyhan syndrome. A B-lymphoblastoid cell line derived from this patient expresses normal amounts of HPRT mRNA yet no detectable immunoreactive protein as determined by radioimmunoassay. These findings suggest either a decreased rate of translation or accelerated degradation due to enhanced proteolytic susceptibility. cDNAs synthesized from this patient's RNA have a single nucleotide (nt) substitution, a C----A transversion at nt 222. RNase A cleavage analysis confirms the presence of a mutation at this position within mRNA isolated from lymphoblasts from patient A.C. This transversion predicts a phenylalanine to leucine replacement at amino acid position 73 in the translated protein. We have designated this mutant HPRTFlint. The mutation in HPRTFlint disrupts a strongly conserved region among PRTases from Escherichia coli, rodents and man, suggesting an important role for this region for the normal function of HPRT. Since it is unlikely that this amino acid substitution alters the translational rate, we hypothesize that disruption of the secondary structure within this region renders HPRTFlint more susceptible to proteolysis.

Amino Acid Sequence↗
PubMed1988

Identification of a single nucleotide change in a mutant gene for hypoxanthine-guanine phosphoribosyltransferase (HPRT Ann Arbor).

HPRT Ann Arbor is a variant of hypoxanthine (guanine) phosphoribosyl-transferase (HPRT: EC 2.4.2.8), which was identified in wo brothers with hyperuricemia and nephrolithiasis. In previous studies, this mutant enzyme was characterized by an increased Km for both substrates, a normal Vmax, a decreased intracellular concentration of enzyme protein, a normal subunit molecular weight and an acidic isoelectric point under native isoelectric focusing conditions. We have cloned a full-length cDNA for HPRT Ann Arbor and determined its complete nucleotide sequence. A single nucleotide change (T----G) at nucleotide position 396 has been identified. This transversion predicts an amino acid substitution from isoleucine (ATT) to methionine (ATG) in codon 132, which is located within the putative 5'-phosphoribosyl-1-pyrophosphate (PRPP)-binding site of HPRT.

Base Sequence↗