Characterization of the subunit composition of HGPRTase from human erythrocytes and cultured fibroblasts

Hypoxanthine-guanine phosphoribosyltransferase is a ubiquitous human enzyme, the inherited deficiency of which leads to a specific metabolic-neurological syndrome. Native acrylamide isoelectric focusing revealed that the human enzyme consists of different numbers of isoenzymes depending on the tissue of origin. The erythrocytic enzyme has the most isoenzymes while the enzyme from cultured fibroblasts has only a single isoenzyme. The isoenzyme pattern of the erythrocytic enzyme changes on storage of the crude hemolysate at 4 C. Treatment of the stored crude hemolysate with 4.5 m urea and 0.35 mm -mercaptoethanol results in an isoenzyme pattern similar to that of the fresh crude extract. Thus the additional isoenzymes are generated on storage not by covalent modification of the enzyme but probably by binding of small molecules to the enzyme or to association of the enzyme molecules. Hypoxanthine-guanine phosphoribosyltransferase has been purified to 80% homogeneity in three steps, DEAE Sephadex chromatography, heat treatment at 85 C for 5 min, and hydroxylapatite chromatography. Denaturing two-dimensional gel electrophoresis of the erythrocytic enzyme revealed that the erythrocytic enzyme is composed of three major types of subunits (1–3) with the same molecular weight but different isoelectric points. In contrast, the fibroblast enzyme is composed of only a single type of subunit, which comigrates with subunit 1 of the erythrocytic enzyme. Since there is a single genetic locus in humans for HGPRTase (the enzyme is X linked) (Nyhan et al., 1967), the observed subunit modification of the erythrocyte enzyme appears to be the result of posttranslational modification. These findings provide a simple explanation for the observed electrophoretic properties of human HGPRTase. A patient with 0.5% of HGPRTase activity in his erythrocytes was found to have small amounts (> 0.5% but < 5% of normal) of the erythrocytic HGPRTase subunits.This work was supported by a grant from NIAMDD, National Institutes of Health, United States Public Health Service. L. J. G. was supported by a fellowship from the National Institute of Child Health and Human Development. D. W. M. is an Investigator, Howard Hughes Medical Institute.     

Characterization of the subunits of purine nucleoside phosphorylase from cultured normal human fibroblasts

In previous communications we have demonstrated that the subunits of normal human erythrocyte purine nucleoside phosphorylase can be resolved into four major (1–4) and two minor (1p and 2p) components with the same molecular weight but different apparent isoelectric points (and net ionic charge). The existence of subunits with different charge results in a complex isoelectric focusing pattern of the native erythrocytic enzyme. In contrast, the isoelectric focusing pattern of the native enzyme obtained from cultured human fibroblasts is simpler. The multiple native isoenzymes obtained from human erythrocytes and human brain have isoelectric points ranging from 5.0 to 6.4 and from 5.2 to 5.8, respectively, whereas cultured human fibroblasts have two major native isoenzymes with apparent isoelectric points of 5.1 and 5.6.Purine nucleoside phosphorylase has been purified at least a hundredfold from ³⁵S-labeled cultured human fibroblasts. A two-dimensional electrophoretic analysis of the denatured purified normal fibroblast enzyme revealed that it consists mainly of subunit 1 (90%) with small amounts of subunits 2 (10%) and 3 (1%). This accounts for the observed differences between the native isoelectric focusing and the electrophoretic patterns of the erythrocyte and fibroblast enzymes. The purine nucleoside phosphorylase subunit 1 is detectable in the autoradiogram from a two-dimensional electrophoretic analysis of a crude, unpurified extract of ³⁵S-labeled cultured normal human fibroblasts. The fibroblast phosphorylase coincides with the erythrocytic subunit 1 of the same enzyme, and the cultured fibroblasts of a purine nucleoside phosphorylase deficient patient (patient I) lack this protein component, genetically confirming the identity of the purine nucleoside phosphorylase subunit in cultured fibroblasts.This work was supported by a grant from the National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, United States Public Health Service. L. J. G. is supported by a fellowship from the National Institute of Child Health and Human Development. D. W. M. is an Investigator, Howard Hughes Medical Institute.     

H(Tla) system: Identification of two new loci,H-31 andH-32, and alleles

Skin and tumor grafting experiments performed on F₁ hybrid mice indicate that theH(Tla) histocompatibility system is composed of at least two genetic loci,H(Tla-1) andH(Tla-2), and that one of these loci,H(Tla-1), has at least three alleles. We suggest thatH(Tla-1) andH(Tla-2) be renamedH-31 andH-32, respectively.     

Inhibitors of lipoxygenase products improve survival in traumatic shock

We have used three selective inhibitors of arachidonic acid metabolism in order to investigate the role of lipoxygenase metabolites in the pathogenesis of traumatic shock (LD₉₀). The following inhibitors were used: CGS-5391B (2.5 mg/kg), a cyclooxygenase and lipoxygenase inhibitor, CGS-5677 (2.0 mg/kg), a selective lipoxygenase inhibitor, and U-60, 257 (0.3 mg/kg), a putative inhibitor of glutathione-s-transferase. These inhibitors did not alter arterial blood pressure or heart rate when given to sham shock rats. The traumatic shock model was characterized by a 4.5-fold increase in plasma cathepsin D activity, a 4-fold increase in plasma myocardial depressant factor (MDF) activity, and a mean survival time of 1.5 ± 0.2 h. Only the dual inhibitor significantly blunted the accumulation of cathepsin D in the plasma (7.5 ± 0.8 vs 11.3 ± 0.8 U/ml, p<0.01). However, all three inhibitors significantly suppressed plasma MDF accumulation by 50–60%: CGS-5391B, CGS-5677, and U-60,2257 (p<0.01). Moreover, these three agents significantly improved survival time in traumatic shock. The increased survival time and reduced MDF activity afforded by these inhibitors suggest a significant role for lipoxygenase metabolites, particularly LTC₄ and LTD₄, in the pathogenesis of traumatic shock.     

Portrayals of Canine Obesity in English-Language Newspapers and in Leading Veterinary Journals, 2000–2009: Implications for Animal Welfare Organizations and Veterinarians as Public Educators

In industrialized societies, more than 1 in 3 dogs and people currently qualify as overweight or obese. Experts in public health expect both these figures to rise. Although clinical treatment remains important, so are public perceptions and social norms. This article presents a thematic analysis of English-language mass media coverage on canine obesity from 2000 through 2009 and compares these results with a thematic analysis of articles on canine obesity in leading veterinary journals during the same time period. Drawing on Giddens's theory of structuration, this study identified articles that emphasized individual agency, environmental structure, or both as contributors to canine obesity. Comparisons with weight-related health problems in human populations were virtually absent from the veterinary sample. Although such comparisons were almost always present in the media sample, quotations from veterinarians and other spokespeople for the welfare of nonhuman animals emphasized the agency of individual caregivers (owners) over structural influences. Now that weight gain and obesity have been established as a pressing animal welfare problem, these results suggest a need for research and for interventions, such as media advocacy, that emphasize intersections between animal-owner agency, socioenvironmental determinants, and connections between animal welfare and human health.     

RNAi‐mediated Ultra‐low gossypol cottonseed trait: performance of transgenic lines under field conditions

Cottonseed remains a low‐value by‐product of lint production mainly due to the presence of toxic gossypol that makes it unfit for monogastrics. Ultra‐low gossypol cottonseed (ULGCS) lines were developed using RNAi knockdown of δ‐cadinene synthase gene(s) in Gossypium hirsutum. The purpose of the current study was to assess the stability and specificity of the ULGCS trait and evaluate the agronomic performance of the transgenic lines. Trials conducted over a period of 3 years show that the ULGCS trait was stable under field conditions and the foliage/floral organs of transgenic lines contained wild‐type levels of gossypol and related terpenoids. Although it was a relatively small‐scale study, we did not observe any negative effects on either the yield or quality of the fibre and seed in the transgenic lines compared with the nontransgenic parental plants. Compositional analysis was performed on the seeds obtained from plants grown in the field during 2009. As expected, the major difference between the ULGCS and wild‐type cottonseeds was in terms of their gossypol levels. With the exception of oil content, the composition of ULGCS was similar to that of nontransgenic cottonseeds. Interestingly, the ULGCS had significantly higher (4%–8%) oil content compared with the seeds from the nontransgenic parent. Field trial results confirmed the stability and specificity of the ULGCS trait suggesting that this RNAi‐based product has the potential to be commercially viable. Thus, it may be possible to enhance and expand the nutritional utility of the annual cottonseed output to fulfil the ever‐increasing needs of humanity.     

TNFα Altered Inflammatory Responses,Impaired Health and Productivity,but Did Not Affect Glucose or Lipid Metabolism in Early-Lactation Dairy Cows

Inflammation may be a major contributing factor to peripartum metabolic disorders in dairy cattle. We tested whether administering an inflammatory cytokine, recombinant bovine tumor necrosis factor-α (rbTNFα), affects milk production, metabolism, and health during this period. Thirty-three Holstein cows (9 primiparous and 24 multiparous) were randomly assigned to 1 of 3 treatments at parturition. Treatments were 0 (Control), 1.5, or 3.0 µg/kg body weight rbTNFα, which were administered once daily by subcutaneous injection for the first 7 days of lactation. Statistical contrasts were used to evaluate the treatment and dose effects of rbTNFα administration. Plasma TNFα concentrations at 16 h post-administration tended to be increased (P<0.10) by rbTNFα administration, but no dose effect (P>0.10) was detected; rbTNFα treatments increased (P<0.01) concentrations of plasma haptoglobin. Most plasma eicosanoids were not affected (P>0.10) by rbTNFα administration, but 6 out of 16 measured eicosanoids changed (P<0.05) over the first week of lactation, reflecting elevated inflammatory mediators in the days immediately following parturition. Dry matter and water intake, milk yield, and milk fat and protein yields were all decreased (P<0.05) by rbTNFα treatments by 15 to 18%. Concentrations of plasma glucose, insulin, β-hydroxybutyrate, non-esterified fatty acids, triglyceride, 3-methylhistidine, and liver triglyceride were unaffected (P>0.10) by rbTNFα treatment. Glucose turnover rate was unaffected (P = 0.18) by rbTNFα administration. The higher dose of rbTNFα tended to increase the risk of cows developing one or more health disorders (P = 0.08). Taken together, these results indicate that administration of rbTNFα daily for the first 7 days of lactation altered inflammatory responses, impaired milk production and health, but did not significantly affect liver triglyceride accumulation or nutrient metabolism in dairy cows.