A study of human leukocyte D locus related antigens in Graves' disease.

An association between Graves' disease and the human leukocyte antigen (HLA) system has previously been reported. The disease was more strongly associated with the HLA D locus antigen Dw3 than with HLA B8. Products of the HLA D locus are determined by the interaction of test cells with standard typing lymphocytes, a technically difficult procedure. Recently, it has been possible to type serologically for D locus related (DRw) specificities on peripheral bone marrow-derived (B) lymphocytes. Blood B lymphocytes from 50 unrelated controls and 41 patients with Graves' disease were typed for seven HLA DRw specificities. 28 patients with Graves' disease (68%) were positive for DRw3, in contrast to 14 controls (28%); whereas only 21 patients (50%) were HLA B8 positive, compared with 13 (26%) controls. Thus, positivity for DRw3 afforded a relative risk for Graves' disease of 5.5, whereas that for HLA B8 amounted to 3.0. Additionally, a family with multiple cases of Graves' disease in which the disease was previously shown to be inherited with the haplotype, was linked to DRw2, which suggests that the susceptibility to the disease was inherited in association with that antigen. Two HLA B/glyoxalase recombination events were observed in this family; in both instances HLA DRw followed HLA B. This study thus demonstrates that the disease susceptibility gene for Graves' disease is in strong linkage disequilibrium with DRw3; however, it may be associated with other DRw specificities and inherited within family units in association with them.

AMP-kinase alpha2 subunit gene PRKAA2 variants are associated with total cholesterol, low-density lipoprotein-cholesterol and high-density lipoprotein-cholesterol in normal women.

BACKGROUND: 5'-AMP-activated protein kinase (AMPK) inactivates critial ensymes in fatty acid and cholesterol synthesis. We hypothesised that the serum lipid profile may be influenced by genetic variation in the AMPK catalytic alpha2 subunit. METHOD: We examined association of 5 tagging SNPs (tSNPs) in the PRKAA2 gene with serum lipids in 2777 normal Caucasian females (mean age 47.4+/-12.5 years). RESULTS: All tSNPs were associated with total- and LDL-cholesterol, (p<0.001 to 0.034), explaining variances of 0.13-0.59% and 0.11-0.55% respectively. One haplotype (frequency 34.7%) showed lower total- and LDL-cholesterol compared with the most common haplotype (frequency 45.7%) (p< or =0.001), explaining 0.78% of total- and 0.75% of LDL-cholesterol. Another haplotype (frequency 10.5%) was significantly associated with lower HDL-cholesterol (p = 0.005), explaining 0.59% of variance. CONCLUSIONS: PRKAA2 gene variants are significantly associated with serum lipoproteins in a large sample of normal female Caucasians.

Role of C-terminal B-chain residues in insulin assembly: the structure of hexameric LysB28ProB29-human insulin.

BACKGROUND: LysB28ProB29-human insulin (Humalog), a fully potent insulin analog in which the prolyl, lysyl sequence at the C-terminal end of the B-chain is inverted, exhibits a decreased association of monomers to dimers leading to rapid in vivo absorption. This provides important benefits for the insulin-requiring diabetic. In spite of its monomeric nature, LysB28ProB29-human insulin can exist as a discrete hexameric structure in the presence of both zinc and phenol. Studies of the crystal structure of LysB28ProB29-human insulin in a hexameric complex were initiated to gain a molecular understanding of the effect of the sequence inversion on the analog's self-association properties and, consequently, its in vivo efficacy. RESULTS: Under the conditions reported, LysB28ProB29-human insulin crystallized as a T3Rf3 hexamer that is isomorphous with the uncomplexed T3Rf3 native human insulin hexamer previously known as '4Zn insulin'. The three-dimensional structure of the T3Rf3 hexamer was determined by X-ray crystallographic methods to a resolution of 2.3 A. The prolyl, lysyl sequence inversion leads to local conformational changes at the C termini of the B-chains which eliminate two critical hydrophobic interactions and weaken two terminal beta-sheet hydrogen bonds that stabilize the dimer. CONCLUSIONS: The loss of these native dimer interactions weakens the hexameric LysB28ProB29-human insulin complex formed in the presence of phenolic ligands. Thus, it is hypothesized that the diffusion of the phenolic ligands from the site of injection results in the dissociation of hexamers directly to monomers, thereby maintaining the rapid time-action of the monomeric analog in spite of the hexameric conformation in therapeutic formulations.

Expression of hepatic mitochondrial carbonic anhydrase V.

We have raised specific (rabbit anti-rat) polyclonal antibodies to hepatic mitochondrial carbonic anhydrase V (CA V) and used them to assay the amounts of protein expressed in liver mitochondria isolated from term-foetal, control or diabetic adult rats and in perivenous and periportal rat hepatocytes. The levels of CA V expressed in mitochondria isolated from the livers of adult male and female rats are similar and increase (about 2-fold) in mitochondria from adult diabetic rats when compared to those isolated from the livers of control rats. The level of enzyme in adult liver was higher than in the livers of term-foetal rats. CA V is expressed in both perivenous and periportal hepatocytes, but the level of expression is greater (approx. 40%) in perivenous cells. The implications and significance of these findings are discussed with reference to the roles and properties of the other carbonic anhydrase isoenzymes and the metabolic function of the mitochondrial isoenzyme.

Sexual differentiation of rat liver carbonic anhydrase III.

Using radioimmunoassay, the concentration of carbonic anhydrase III in the livers of adult male rats was found to be approx. 30-times greater than that observed in mature females. Castration of male rats led to a marked reduction in liver carbonic anhydrase III concentrations which could be partially restored to control levels by testosterone replacement. Administration of testosterone to ovariectomised female rats induced about a 5-fold increase in liver carbonic anhydrase III concentration. Immunoprecipitation analysis of the products of liver mRNA translation in vitro with antiserum specific for carbonic anhydrase III showed that hormonal control of the levels of carbonic anhydrase III in liver is mediated by changes in the amount of translatable carbonic anhydrase III mRNA. Marked changes in liver carbonic anhydrase III concentrations were also observed in developing and ageing male rats.

Membrane specific carbonic anhydrase (CAIV) expression in human tissues.

Membrane-bound carbonic anhydrase IV (CAIV) expression has been evaluated in a range of fetal and adult human tissues and in cell culture. All tissues tested showed expression of CAIV, assessed by Western blotting, with a single immunodetected band at 55 kDa. The levels varied in fetal lung and liver during development and in various zones of the fetal brain. CAIV was clearly expressed in lung, pancreatic tumour and skin cell cultures.

Leptin expression in the fetus and placenta during mouse pregnancy.

During pregnancy, leptin concentrations in the maternal circulation are elevated in both humans and rodents but decrease to pre-pregnancy levels at birth, suggesting a role for leptin in the maintenance of pregnancy. Synthesis of leptin by the human placenta is established but whether the murine placenta synthesizes leptin remains controversial. The aims of this study were to determine (a) if the mouse wild-type placenta expresses the ob gene using Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and (b) whether the mouse fetus and placenta contribute to the significant increase of leptin in the maternal circulation during pregnancy. The mouse placenta did not express the ob gene at a level that could be readily detected using RT-PCR. Moreover, both maternal gain in weight and undetectable concentrations of leptin in sera in leptin-deficient ob/ob mothers bearing heterozygote (ob/+) fetuses suggested that the mouse fetus and placenta do not make a significant contribution to the dramatic increase in maternal plasma concentrations of leptin during late gestation. It is therefore concluded that neither fetal- nor placental-derived leptin modulates maternal weight gain during pregnancy.

Studies of cardiopulmonary bypass in children: implications for the regulation of brain natriuretic peptide.

OBJECTIVE: The aim was to examine the influence of cardiopulmonary bypass on brain natriuretic peptide (BNP) and on hormones of importance in the control of sodium and water balance and blood volume. METHODS: Nine patients (mean age 4 years, range 2-9) undergoing cardiac surgery were studied. Blood samples were taken before, during, and up to 24 h after bypass. Plasma levels of BNP, atrial natriuretic peptide (ANP), arginine vasopressin (AVP), plasma renin activity, aldosterone, and catecholamines were measured. RESULTS: Preoperative concentrations of plasma BNP [573(SEM 68) pg.ml-1] and ANP [332(74) pg.ml-1] were greatly increased (p < 0.05) before bypass in all patients when compared to normal levels in children [BNP = 31(4) pg.ml-1; ANP = 27(3) pg.ml-1, n = 28]. With general anaesthetic and sternotomy, there were large reductions (p < 0.05) in both plasma BNP [180(62) pg.ml-1] and plasma ANP [163(59) pg.ml-1]. During bypass, there were no further significant decreases in plasma ANP or BNP concentrations compared with preoperative levels. Postoperatively, plasma BNP gradually increased for 12 h, to 170(28) pg.ml-1, whereas plasma ANP showed a further small decrease, to 107(20) pg.ml-1. However, postoperative plasma levels of both ANP and BNP remained well below preoperative values (p < 0.01). Plasma AVP increased rapidly within 15 min of the onset of bypass, reaching a peak value of 153(5) pg.ml-1 after 45 min. Off bypass, plasma AVP decreased slowly and was still almost 10-fold above preoperative levels 12 h after end of bypass [137(11) pg.ml-1]. Mean central venous pressure decreased during the onset of bypass, from 4.3(1.9) to 0.4(1.1) mm Hg (p < 0.05), and increased again at the end of bypass, to 9.0(3.3) mm Hg (p < 0.05); there was little further change during the postoperative period. CONCLUSIONS: The major source of plasma BNP in patients with congenital heart disease is the cardiac ventricle. The lower plasma ANP and BNP levels and the narrow band of change in central venous pressure following surgical repair of cardiac abnormalities may be a response to improved cardiac function.

Ventricular expression of brain natriuretic peptide gene following orthotopic cardiac transplantation in children--a three year follow up.

OBJECTIVE: The aim was to examine ventricular brain natriuretic peptide (B-type natriuretic peptide, BNP) gene expression and to determine its relationship with ventricular BNP and circulating BNP levels in paediatric cardiac transplant recipients, over a three year period after transplantation. METHODS: Total RNA extracted from endomyocardial right ventricular biopsy tissues (n = 26) of 13 cardiac transplant recipients (age range 5-17 years) and 10 normal hearts obtained at necropsy (age range 19-76 years) as controls was analysed by northern and slot blot hybridisations. Specific radioimmunoassay techniques were used to determine levels of BNP and atrial natriuretic peptide (A-type natriuretic peptide, ANP) in plasma (n = 26) and ventricular biopsy (n = 26) samples. RESULTS: Ventricular BNP messenger ribonucleic acid (mRNA) levels from slot blot hybridisations in the transplanted heart [122(3) arbitrary units, range 97-143] were significantly higher (p < 0.01) than in the normal heart [63(5) arbitrary units, range 37-98]. Northern blot hybridisations confirmed this result and gave a major BNP mRNA transcript of approximately 900 nucleotides. There was no significant relationship between ventricular BNP mRNA levels and ventricular BNP (r = 0.15, p = 0.5, n = 26) or plasma BNP levels (r = 0.16, p = 0.4, n = 26). There was also no significant relationship between ventricular BNP mRNA levels and any of the haemodynamic variables, or immunosuppressive drugs. A ventricular ANP RNA transcript of approximately 900 nucleotides was detected in the transplanted heart but was below the limit of detection in the normal heart. For the long term study, increased levels of BNP and ANP in both plasma and ventricular samples were observed in the first year after transplantation, with a significant reduction (p < 0.01) in levels three years later. CONCLUSIONS: Ventricular BNP gene expression is increased at the mRNA level after heart transplantation in children. Expression of both ventricular BNP mRNA and ANP mRNA in the transplanted heart may be an important response in the modulation of cardiac function after transplantation.

Leptin requirement for conception, implantation, and gestation in the mouse.

The ob/ob mouse has a complete absence of circulating leptin, resulting in obesity and infertility. Using the minimum daily dose of leptin required to maintain normal body weight and sexual maturation (5 mg/kg, ip), leptin-treated ob/ob females were mated with either wild-type (+/+) or leptin-treated ob/ob males. The leptin treatment continued throughout pregnancy until weaning or was withdrawn at 0.5, 3.5, 6.5, or 14.5 d post coitum (dpc). Normal pregnancy and parturition with pups of normal weight resulted when ob/ob females were mated with +/+ males and leptin treatment was continued throughout pregnancy (6 of 8 pregnancies), to 14.5 dpc (6 of 8 pregnancies), or to 6.5 dpc (9 of 12 pregnancies). Pregnancy did not result when treatment was stopped at 3.5 dpc (1 of 7 pregnancies) or 0.5 dpc (0 of 6 pregnancies). Similar results were obtained when leptin-treated ob/ob females were mated with leptin-treated ob/ob males. The newborn pups failed to survive after birth in groups treated with leptin up to 14.5 and 6.5 dpc despite reinstating leptin at birth. This appeared to be due to a lack of development of the mammary glands. In conclusion, we have shown that leptin is essential for normal preimplantation and/or implantation processes. It is also essential for normal development of the mammary glands, but is not required for pregnancy and parturition once implantation is established.

A new restriction endonuclease, TspEI, from the genus Thermus that generates cohesive termini compatible with those of EcoRI.

The detection, isolation and properties of the restriction endonuclease TspEI are described. The canonical recognition sequence (AATT) is the same as the 4-bp core of the 6-bp sequence (GAATTC) of EcoRI. Hydrolysis occurs 5' to the palindromic tetramer so that TspEI produces the same cohesive termini as EcoRI. TspEI therefore has an obvious application in producing partial digests of DNA for ligation to EcoRI-digested cloning vectors.

Hypophysectomy abolishes sexual dimorphism of liver carbonic anhydrase III.

Hypophysectomy was found to have no effect on the concentration of carbonic anhydrase III (CAIII) in male rat liver, whereas in the female, CAIII was elevated 10-fold, to male levels.

Novel inhibition of carbonic anhydrase isozymes I, II and III by carbamoyl phosphate.

Carbamoyl phosphate has been shown to inhibit carbonic anhydrase (CA) isozymes CA I, CA II and CA III. This physiologically important molecule is the most potent, naturally occurring inhibitor of carbonic anhydrase yet found. It is also unique, among carbonic anhydrase inhibitors discovered hitherto, in that it inhibits the 3 isozymes with equal effect, despite their strikingly different properties. The results imply the participation of carbonic anhydrase in the regulation of substrate availability for the urea cycle.

Atrial natriuretic peptide gene G664A polymorphism and the risk of ischemic cerebrovascular disease.

The atrial natriuretic peptide (ANP) gene may underlie stroke susceptibility and sensitivity to cerebral ischemia in an animal model of stroke. The authors investigate its role in humans by genotyping a polymorphism (G664A) in 436 patients with ischemic cerebrovascular disease and 295 community control subjects. The frequency of this variant was similar in both groups and across the different stroke subtypes. The ANP gene G664A polymorphism is therefore unlikely to be an important risk factor for ischemic stroke in this population.

Angiotensin converting enzyme insertion/deletion polymorphism: association with ethnic origin.

OBJECTIVE: To determine the distribution of the insertion/deletion (I/D) polymorphism of the angiotensin converting enzyme (ACE) gene in several ethnic groups: Caucasian Europeans, Black Nigerians, Samoan Polynesians and Yanomami Indians. RESULTS: The ratio of the frequencies of the II, ID and DD genotypes were 1:2:1 in the Europeans, but there was a tendency towards a higher frequency of the D allele in the Nigerians. In contrast, the Samoans and the Yanomami Indians displayed a much higher frequency of the I allele than of the D allele. CONCLUSION: The relationship between ACE genotype and disease in these latter groups is still not known, but the present results clearly suggest that ethnic origin should be carefully considered in the increasing number of studies on the association between I/D ACE genotype and disease aetiology.

Renin and atrial natriuretic peptide restriction fragment length polymorphisms: association with ethnicity and blood pressure.

An investigation of restriction fragment length polymorphisms (RFLPs) and association with blood pressure was carried out for the candidate genes coding for renin (REN) and atrial natriuretic peptide (ANP). The relationship between blood pressure, REN and ANP gene RFLPs was tested in a population of black Afro-Caribbean and white European subjects sampled randomly from family practice registers. Upper and lower quintiles for diastolic blood pressure were selected for analysis. Digests of DNA were prepared from leucocytes and RFLPs were determined using Southern blotting analysis with REN and ANP gene probes. There were highly significant ethnic differences found for BglI, Bg/lI and TaqI polymorphisms with 5'REN probe and for BglI polymorphism with an ANP probe. There was also an association between blood pressure and the BglI/REN polymorphism in Afro-Caribbeans but not with any other polymorphisms studied in either ethnic group. The findings show significant ethnic RFLP differences at the gene loci for both renin and ANP and provide evidence for a possible link between variations within or close to the renin gene and elevated blood pressure in Afro-Caribbeans.

Immunocytochemical localization of carbonic anhydrase isozymes I, II, and III in rat skeletal muscle.

Specific antisera were raised against the three carbonic anhydrase (CA) isozymes, CAI, CAII, and CAIII, and were used to determine the fiber distribution of these isozymes in skeletal muscle. Fiber types were determined by ATPase staining, and the CA isozymes were detected using a peroxidase-anti-peroxidase (PAP) technique. All three isozymes were present in type I fibers; CAII and CAIII were exclusive to these fibers, and CAI were also present in some small type 2A fibers.

Thyroidectomy significantly alters carbonic anhydrase III concentration and fiber distribution in rat muscle.

Thyroidectomy has a dramatic effect on rat muscle, greatly increasing the number of Type I fibers and the concentrations of carbonic anhydrase III (CAIII) in the muscle. Carbonic anhydrase III is not confined to the Type I fibers, as was previously believed, but also occurs in fibers that exhibit a level of ATPase staining less than that of 2A fibers but greater than 2B. These fibers are rare in normal muscle but become numerous after thyroidectomy, when they stain heavily for CAIII.

Localization of human muscle carbonic anhydrase isozymes using immunofluorescence.

Human muscle sections were examined for the presence of carbonic anhydrase using fluorescent second antibody and antisera specific for the three isozymes, carbonic anhydrase I (CAI), carbonic anhydrase II (CAII), and carbonic anhydrase (CAIII). CAIII was present in all fibers, CAII only in the connective tissue, and CAI showed a weak association with the sarcolemma.

Immunocytochemical localization of mitochondrial carbonic anhydrase in rat tissues.

We used a monospecific polyclonal antiserum against mitochondrial carbonic anhydrase (CA V) from rat liver to study tissue localization of this new member of the carbonic anhydrase gene family. Strong granular immunostaining reaction of CA V was observed in hepatocytes, myocardium, and in certain populations of skeletal muscle fibers. This is the first time that mitochondrial carbonic anhydrase is described in cardiac tissue of rat or any other species. Different epithelial cells revealed very heterogeneous staining reaction, suggesting that mitochondria are a heterogeneous population with respect to their CA V content. Many cells in different glandular epithelia did not show any CA V, whereas some cells, such as gastric parietal cells, were intensely stained with CA V antibodies. No systematic co-expression of CA V with CA I, CA II, or CA III was observed, although the distribution of CA V in skeletal muscle was somewhat similar to that of CA III. Connective tissue cells such as fibroblasts, chondroblasts, and osteoblasts were negative.