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

Summary HPRT_{Ann Arbor} is a variant of hypoxanthine (guanine) phosphoribosyl-transferase (HPRT: EC 2.4.2.8), which was identified in two brothers with hyperuricemia and nephrolithiasis. In previous studies, this mutant enzyme was characterized by an increased K_m for both substrates, a normal V_max, 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 (TG) 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.     

G6PD Huntsville: a new glucose-6-phosphate dehydrogenase associated with chronic hemolytic anemia

Summary We describe a previously unreported glucose-6-phosphate dehydrogenase (G6PD) variant. G6PD Huntsville was found in a Caucasian male, resident of Huntsville, Alabama who was investigated for otherwise unexplained chronic hemolytic anemia. An unusual feature of this unique, apparently hemolytic, G6PD mutant is that its red cell enzymatic activity has not been decreased. The mutant enzyme is unstable. Additionally, the enzyme variant is characterized by normal electrophoretic mobility, biphasic and slightly alkaline pH optimum, and abnormal kinetics for the natural substrates G6PD and NADP as well as the artificial substrates deamino NADP. Its activity for another artificial substrate 2-deoxy G6PD is normal. The inhibition constant for NADPH is normal. The subject has had no evidence of episodic jaundice.     

Proteinase inhibitors I and II in fruit of wild tomato species: Transient components of a mechanism for defense and seed dispersal

The juice of unripe fruit from a wild species of tomato, Lycopersicon peruvianum (L.) Mill., LA 107, contains over 50% of its soluble proteins as the sum of two proteinase inhibitors. These are the highest levels of proteinase inhibitors and highest percentage of soluble proteins as proteinase inhibitors of any plant or animal tissue found to date. Fruit of the modern tomato, L. esculentum Mill., contains only negligible quantities of the two inhibitors. The two proteinase inhibitors in the fruit of L. peruvianum are members of the Inhibitor I and II families previously found in potato tubers and in leaves of wounded potato and tomato plants. The levels of the two inhibitors in the unripe fruit decrease significantly during ripening. Unripe fruit from other wild Lycopersicon species such as L. parviflorum Rick, Kesicki, Fobes et Holle, L. hirsutum Humb. et Bonpe., L. pimpinellifolium Mill., and other lines of L. peruvianum contain moderate levels of the inhibitors that also decrease during ripening. Another wild tomato species, L. pennellii Corr., is similar to L. esculentum in not containing the two proteinase inhibitors in either unripe or ripe fruit. The transient levels of the inhibitors in fruit of wild species indicate that they are present in unripe fruit as defensive chemicals against insects, birds or small mammals and their disappearance during ripening may render them edible to facilitate seed dispersal. High levels of mRNAs coding for Inhibitors I and II in unripe fruit of L. peruvianum, LA 107, indicate that strong promoters may regulate the developmentally expressed proteinase-inhibitor genes in tomato fruit that may have a substantial potential for use in genetic-engineering experiments to enhance the production of large quantities of proteinase inhibitors or other proteins in field tomatoes.Abbreviations poly(A)⁺ mRNA polyadenylated mRNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide electrophoresis Project 1791, College of Agriculture and Home Economics Research Center, Washington, State University     

Structural polymorphism of the human platelet Fc gamma receptor

A variable T lymphocyte proliferative response to murine IgG1 anti-T3 monoclonal antibodies, in which most North American Caucasians respond whereas a minority do not, is well established. This is most likely the result of a genetic polymorphism manifested by 1) the inability of the monocyte 40-kDa IgG FcR of some individuals to bind murine IgG1, and 2) a distinctive trimorphic pattern on IEF of the monocyte 40-kDa FcR, one form being seen in all individuals who do not respond and another form (or a combination of both forms) being seen in those who do respond. We have evaluated the IEF patterns of the platelet 40-kDa FcR and find that in every individual tested the pattern for platelet FcR correlates with that seen for the monocyte 40-kDa FcR pattern. Furthermore, the platelets of those individuals whose "nonresponder" monocyte 40-kDa FcR did not mediate a murine IgG1 anti-T3 response did not respond with an aggregation reaction to murine IgG1 immune complexes (opsonized E). In contrast, platelets from donors possessing "responder" monocytes displayed positive "aggregation" responses to E coated with murine IgG1 antibody. However, the platelet FcR structural polymorphism described earlier did not correlate with the donor-specific variability in capacity of platelets to respond functionally to aggregated human IgG described in an earlier paper. Rather, the variation in capacity of platelets from individual donors to respond functionally to aggregated human IgG was related to the quantitative expression of platelet FcR. These data indicate that the molecular mechanisms responsible for the platelet 40-kDa FcR structural polymorphism are quite different from the mechanisms governing the variation in quantitative expression of the receptor.     

Identification and characterization of a novel postlarval hemolymph protein from Manduca sexta

The identification, purification and characterization of a new postlarval specific hemolymph protein from Manduca sexta is described. Incorporation of [³⁵S]methionine into Manduca sexta hemolymph proteins in vivo was investigated as a function of development. A major protein band of M_r ≈ 50,000 was highly labeled during the prepupal and adult stage but not in feeding larvae. This postlarval protein (PLP) was isolated from adult male hemolymph and its chemical and immunological properties determined. PLP is a basic protein (pI ～8.6). Electrophoresis under denaturing conditions reveals a subunit M_r ≈ 50,000 while the native protein has an apparent M_r ～ 85,000 by gel permeation chromatography. Anti-PLP serum recognized PLP but not other hemolymph proteins on immunoblots. In vitro translation of fat body mRNA followed by immunoprecipitation revealed that fat body is the site of PLP synthesis. Quantitation of PLP levels in hemolymph throughout development was performed and suggests PLP may play a role in adult development of M. sexta.     

Integration of a barcode reader with a commercial flow cytometer.

This report describes the application and installation of a barcode reader on a standard EPICS Elite flow cytometer. The barcode reader system eliminates keyboard entry of sample information on the cytometer. The system automates the transfer of sample information already present in our laboratory database to the cytometer at run time. The system uses a standard "off-the-shelf" bar code wand with a personal computer keyboard interface and requires no additional software at run time. No typing of sample information is required by the operator at any stage of normal sample operation at the cytometer. All operations are automatically coded into the cytometry software using the macro functions of the software. Tubes are inserted into the tube reader and sample information is transferred automatically into the cytometer. We have found that the system allows rapid and continuous operation of routine clinical and research samples. This automated data entry also reduces the possibility of data input errors.     

Extraction and measurement of myocardial nucleotides, nucleosides, and purine bases by high-performance liquid chromatography

Nucleotides, nucleosides, and purine bases were extracted from human endomyocardial biopsies, freeze-clamped rat hearts, and porcine coronary sinus plasma. Perchloric acid extracts were neutralized with Freon-trioctylamine and analyzed at 250 nm by reverse-phase ion-pairing high-performance liquid chromatography. To achieve the sensitivity necessary for analyzing small (1-3 mg wet wt) tissue samples, a small-bore, 2.1-mm-internal-diameter, C18, 5-micron reverse-phase column and a flow rate of 0.2 ml/min were used. All of the myocardial nucleotides and AMP degradation products were resolved in a total separation time of 27 min with 30 mM KH2PO4, 7.5 mM tetrabutylammonium phosphate buffers, and binary pH and acetonitrile gradients.     

Evidence for concerted kinetic oxidation of progesterone by purified rat hepatic cytochrome P-450g

Purified cytochrome P-450g, a male-specific rat hepatic isozyme, was observed to metabolize progesterone to two primary metabolites (6 beta-hydroxyprogesterone and 16 alpha-hydroxyprogesterone), two secondary metabolites (6 beta,16 alpha-dihydroxyprogesterone and 6-ketoprogesterone), and one tertiary metabolite (6-keto-16 alpha-hydroxyprogesterone). The Km,app for the formation of these products from progesterone was determined to be approximately 0.5 microM, while the Km,app for metabolism of 6 beta- and 16 alpha-hydroxyprogesterone was found to be 5-10 microM. The ratio of primary to secondary metabolites did not change significantly at progesterone concentrations from 6 to 150 microM, and a lag in formation of secondary metabolites was not observed in 1-min incubations. Concerted oxidation of progesterone to secondary products without the intermediate products leaving the active site was suggested by these results and confirmed by isotopic dilution experiments in which little or no dilution of metabolically formed 6 beta,16 alpha-dihydroxyprogesterone and 6-keto-16 alpha-hydroxyprogesterone was observed in incubations containing a mixture of radiolabeled progesterone and unlabeled 6 beta-hydroxyprogesterone or 16 alpha-hydroxyprogesterone. Incubation of 6 beta-hydroxyprogesterone with a reconstituted system in an atmosphere of 18O2 resulted in greater than 90% incorporation of 18O in the 16 alpha-position of 6 beta,16 alpha-dihydroxyprogesterone but no incorporation of 18O into 6-ketoprogesterone, even though the reaction was dependent upon enzyme and O2, and not inhibited by mannitol, catalase, or superoxide dismutase. Factors which characterize the metabolism of progesterone by cytochrome P-450g in terms of active-site constraints and the catalytic competence of the enzyme in microsomes were also explored.     

Regulation of phospholipid biosynthesis in Saccharomyces cerevisiae by inositol. Inositol is an inhibitor of phosphatidylserine synthase activity

The addition of inositol to the growth medium of Saccharomyces cerevisiae resulted in rapid changes in the rates of phospholipid biosynthesis. The partitioning of the phospholipid intermediate CDP-diacylglycerol was shifted to phosphatidylinositol at the expense of phosphatidylserine and its derivatives phosphatidylethanolamine and phosphatidylcholine. Serine at 133-fold greater concentrations than that of inositol shifted the partitioning of CDP-diacylglycerol to phosphatidylserine at the expense of phosphatidylinositol but to a much lesser degree. Kinetic experiments with pure phosphatidylserine synthase and phosphatidylinositol synthase indicated that the partitioning of CDP-diacylglycerol between phosphatidylserine and phosphatidylinositol was not governed by the affinities both enzymes have for their common substrate CDP-diacylglycerol. Instead, the main regulation of phosphatidylinositol and phosphatidylserine synthesis was through the exogenous supply of inositol. The Km of inositol (0.21 mM) for phosphatidylinositol synthase was 9-fold higher than cytosolic concentration of inositol (24 microM). The Km of serine (0.83 mM) for phosphatidylserine synthase was 3-fold below the cytosolic concentration of serine (2.6 mM). Therefore, inositol supplementation resulted in a dramatic increase in the rate of phosphatidylinositol synthesis, whereas serine supplementation resulted in little affect on the rate of phosphatidylserine synthesis. Inositol also contributed to the regulation of phosphatidylinositol and phosphatidylserine synthesis by having a direct affect on phosphatidylserine synthase activity. Kinetic experiments with pure phosphatidylserine synthase showed that inositol was a noncompetitive inhibitor of the enzyme with a Ki of 65 microM.