5'-UTR structural organization, transcript expression, and mutational analysis of the human Rab geranylgeranyl transferase alpha-subunit (RABGGTA) gene.

Hermansky-Pudlak syndrome (HPS) is a recessively inherited disease with dysfunction of several related subcellular organelles including platelet-dense granules, melanosomes, and lysosomes. Our recent identification of the mutation in murine Rab geranylgeranyl transferase alpha-subunit gene (Rabggta) in one mouse model of HPS, the gunmetal mouse, suggested that human patients with similar phenotypes might have mutations in the human orthologous RABGGTA gene. This prompted reanalysis of the 5'-untranslated structure of the human RABGGTA gene in normal individuals and in patients with deficiencies of platelet-dense granules (alphadelta-SPD), alpha granules (alpha-SPD or gray platelet syndrome, GPS) or alpha plus dense granules (alphadelta-SPD).

Rab geranylgeranyl transferase alpha mutation in the gunmetal mouse reduces Rab prenylation and platelet synthesis.

Few molecular events important to platelet biogenesis have been identified. Mice homozygous for the spontaneous, recessive mutation gunmetal (gm) have prolonged bleeding, thrombocytopenia, and reduced platelet alpha- and delta-granule contents. Here we show by positional cloning that gm results from a G-->A substitution mutation in a splice acceptor site within the alpha-subunit of Rab geranylgeranyl transferase (Rabggta), an enzyme that attaches geranylgeranyl groups to Rab proteins.

Automated identification of amino acid sequence variations in proteins by HPLC/microspray tandem mass spectrometry.

Amino acid sequence variations resulting from single-nucleotide polymorphisms (SNPs) were identified using a novel mass spectrometric method. This method obtains 99+% protein sequence coverage for human hemoglobin in a single LC-microspray tandem mass spectrometry (microLC-MS/MS) experiment. Tandem mass spectrometry data was analyzed using a modified version of the computer program SEQUEST to identify the sequence variations.

Chromosomal localization and complete genomic sequence of the murine autoimmune regulator gene (Aire).

We have recently cloned the murine autoimmune regulator (Aire) gene, the homologue of human AIRE responsible for the autoimmune polyglandular syndrome type 1 (APS1) or autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED). Here, we report the genomic sequence (18,413 bp) for the entire Aire gene and its 5' flanking region, which contains putative regulatory sequences. Comparison of the genomic and cDNA sequences indicates that the Aire gene is composed of 14 exons and the coding sequence shares high similarities between mouse and human.

Identification of novel simple sequence length polymorphisms (SSLPs) in mouse by interspersed repetitive element (IRE)-PCR.

Interspersed repetitive element (IRE)-PCR is a useful method for identification of novel human or mouse sequence tagged sites (STSs) from contigs of genomic clones. We describe the use of IRE-PCR with mouse B1 repetitive element primers to generate novel, PCR amplifiable, simple sequence length polymorphisms (SSLPs) from yeast artificial chromosome (YAC) clones containing regions of mouse chromosomes 13 and 14. Forty-two IRE-PCR products were cloned and sequenced from eight YACs.

Delayed bottle weaning and iron deficiency in southeast Asian toddlers.

We undertook this study to determine if culturally influenced feeding practices are associated with iron deficiency in infants and toddlers from low-income families. We obtained a dietary survey, illness history, hematocrit, and zinc protoporphyrin-to-heme ratio (ZPP/H) from 35 Southeast Asian children and 73 children of other ethnicities between ages 5 and 30 months. We confirmed iron deficiency by serum ferritin measurement in children with ZPP/H > 80 mmol/mol or evaluated them after a 3-month iron treatment.

Genetic mapping of 20 novel expressed sequence tags from midgestation mouse embryos suggests chromosomal clustering.

Current knowledge of genes that regulate pattern formation and differentiation processes during mammalian embryonic development is limited. In an effort to isolate developmentally relevant genes, 20 novel, end-sequenced cDNAs selected from a Day 10.5 postcoitum mouse embryo library were genetically mapped in intersubspecific backcross mice.

Positional cloning of the Chediak-Higashi syndrome gene: genetic mapping of the beige locus on mouse chromosome 13.

Background: Chediak-Higashi syndrome (CHS) is a systemic disorder of human and mouse (beige, bg) that is characterized by aberrant intracellular protein kinesis and lysosomal trafficking. Affected individuals exhibit a severe primary immune deficiency that principally affects the function of granulocytes and cytolytic lymphocytes and partial oculocutaneous albinism, platelet dysfunction, and neurodegeneration. Chediak-Higashi syndrome is inherited as an autosomal recessive Mendelian trait in human and mouse and maps on proximal mouse Chromosome 13.

Identification of the homologous beige and Chediak-Higashi syndrome genes.

Vesicular transport to and from the lysosome and late endosome is defective in patients with Chediak-Higashi syndrome (CHS) and in mutant beige (bg) mice. CHS and bg cells have giant, perinuclear vesicles with characteristics of late endosomes and lysosomes that arise from dysregulated homotypic fusion. CHS and bg lysosomes also exhibit compartmental missorting of proteins, such as elastase, glucuronidase and cathepsin G.

Elevated zinc protoporphyrin associated with thalassemia trait and hemoglobin E.

Objective: Increased zinc protoporphyrin/heme (ZPP/H) ratio has been used in pediatrics to screen for iron deficiency and lead poisoning. This study was conducted to determine whether common hereditary hemoglobin disorders (alpha- and beta-thalassemia traits, hemoglobin E) found in U.S.

A novel deletion of the entire alpha globin locus causing alpha-thalassemia-1 in a northern European family.

alpha-Thalassemia, though one of the most common genetic abnormalities in humans, is uncommon outside of tropical or subtropical regions of the world. The authors describe a family of northern European ancestry with a clinical presentation consistent with alpha-thalassemia-1, or deletion of both alpha globin genes on chromosome 16 in cis. Genomic mapping studies show that the deletion in this family spans 36.

Screening for alpha-thalassemia. Correlation of hemoglobin H inclusion bodies with DNA-determined genotype.

We evaluated potential screening protocols for alpha-thalassemia in a group of 80 patients whose genotypes were determined by Southern blot analysis with alpha- and zeta-globin DNA probes. Erythrocyte inclusion bodies were measured by a modified brilliant cresyl blue test. Erythrocyte indices and iron status were also measured.

Obtaining reliable plasma sodium and glucose determinations from pulmonary artery catheters.

In critically ill patients, blood specimens are frequently withdrawn from intravascular catheters without consideration of possible infusate contamination, of test accuracy, or of undue blood loss. The reliability of sodium (Na+) and glucose determinations withdrawn from the venous infusion port of a pulmonary artery catheter was studied in 30 patients after cardiac surgery. Catheter patency was maintained by a saline and heparin infusate.

Accurate coagulation studies from heparinized radial artery catheters.

We studied the feasibility of obtaining accurate coagulation studies from indwelling, heparinized radial artery catheters in 28 patients after cardiac surgery. PTT assay was chosen because of its frequent clinical use. Thrombin time assay was chosen because it is a component of the coagulation screening panel and because it is useful in assessing heparin contamination of specimens.

Hereditary sideroblastic anaemia and ataxia: an X linked recessive disorder.

We report two families in which a non-progressive spinocerebellar syndrome and a sideroblastic anaemia are segregating together in an X linked recessive fashion. Four males in two generations of one family and a fifth male from an unrelated family had both conditions. Both the sideroblastic anaemia and the spinocerebellar syndrome differ from those which have previously been reported to be inherited in an X linked recessive manner.

A screening protocol for a prenatal population at risk for inherited hemoglobin disorders: results of its application to a group of Southeast Asians and blacks.

Several inherited hemoglobin disorders are present among certain racial subgroups of the United States population, particularly among blacks and Southeast Asians. Many of these disorders are unfamiliar to the obstetrician-gynecologist but may have important implications for care in pregnancy, including genetic counseling and prenatal diagnosis. A simple, effective screening tool was devised for detection of thalassemias and hemoglobinopathies in a prenatal clinic population.

Abnormal plasma amino acid profiles in patients undergoing bone marrow transplant.

Amino acid concentrations in plasma have been measured during total parenteral nutrition in patients undergoing bone marrow transplantation. Profound hyperaminoacidemia was noted in the immediate para-transplant period. The increase was due mainly to high levels of phenylalanine and methionine.

Hemoglobin G trait and S trait in the same patient.

We describe a subject who had a positive sickle solubility test and a routine alkaline electrophoretic pattern showing three major hemoglobulin bands migrating to the A, S, and A2 positions. In addition, the non-heme protein carbonic anhydrase appeared to be increased, suggesting the presence of a split A2. Two major hemoglobin bands separated on agar gel electrophoresis using a citrate buffer, pH 6.

Successful long-term use of a plasmapheresis reactor system in dogs with lymphoma.

The successful long-term use of asparaginase-glutaminase reactor in dogs with lymphoma is described. The limitations of the system consisting of an activation of enzymes leading to rapid neosynthesis of asparagine and glutamine are outlined. The possible beneficial effect of future combination therapy with amino acid analogues is discussed.

Successful in vivo experiments in sheep using a new nonhydrolyzable enzyme reactor resistant against tryptic digestion.

Blockage of E-amino groups of asp-ase bound to cellulose by succinylation results in a greatly enhanced resistance against tryptic digestion by "plasma enzymes." Plasmapheresis in sheep by plasma filters connected to asp-ase Cellulose cartridges led to decrease of asp-NH2 and glut-NH2 in plasma over 21 hrs with undetectable enzyme leakage from the solid phase.

The reliability of laboratory data from blood samples collected through pulmonary artery catheters.

Blood samples obtained through quadruple-lumen Swan-Ganz catheters in ten critically ill patients provided reliable results after withdrawal of a total of 2.2 mL (including 1.2 mL of dead space) for hematocrit tests and 3.

An improved method for detection of red cell hemoglobin H inclusions.

Supravital staining of red blood cells with brilliant cresyl blue (BCB) is used to detect hemoglobin H (Hb H). This simple test is useful in the diagnosis of alpha thalassemia trait; however, the search for Hb H containing cells in often very tedious. A technique is described that enriches BCB preparations for Hb H containing cells about ten times over conventional methods.

Physiologic studies in normal and uremic sheep. II. Changes in erythropoiesis and oxygen transport.

Red cell production and survival, cardiac output (CO), renal blood flow (RBF), serum erythropoietin (ESF), oxygen (O2) consumption, 2,3-diphosphoglycerate (2,3-DPG), P50, and circulating hemoglobin (HbC) were measured in normal sheep and in animals made chronically uremic and anemic by subtotal nephrectomy. The erythropoiesis characterizing uremic anemia was hypoproliferative as quantitated by ferrokinetics, but was still subject to normal feedback control in that erythropoiesis in normal and uremic animals increased with phlebotomy and was suppressed with transfusion. ESF levels were only measurable when the hematocrit fell below 25 in normal animals and were undetected in most stable uremics (hematocrit, 18 to 10%).