Evaluation of photo-crosslinked fibrinogen as a rapid and strong tissue adhesive.

Tissue adhesives and sealants are commonly used in surgery either as an adjunct to, or replacement for, sutures. Previously, we have shown that fibrinogen can be crosslinked rapidly to give a high-strength bond in the presence of a ruthenium(II) complex, a persulfate and irradiation with visible light, and that the crosslinked fibrinogen is nontoxic to cells in vitro. This approach addresses limitations to current fibrin sealants that typically have relatively slow curing times and low bond strengths. In the present study, we have evaluated the efficacy and safety of this new biological scaffold sealant in various animal models. When placed as solid implants into rats, the crosslinked fibrinogen persisted for at least 8 weeks but was fully resorbed by 18 weeks with minimal inflammatory responses. When used as a tissue adhesive for repair of skin incisions in rats or as an arterial haemostat in pig, the photo-crosslinked fibrinogen sealed tissue or arrested bleeding within 20 s of application. For the skin incisions, the fibrinogen sealant promoted rapid tissue vascularization and cellular infiltration with no adverse foreign body cell generation. New collagen deposition occurred and with time the matrix had remodelled to acquire large mature collagen fiber bundles which were accompanied by maximum regenerated tensile strength. This biomaterial system may find useful applications in surgical procedures where rapid curing and/or high strength tissue sealing is required.

Molecular cloning and expression of the dihydrofolate reductase (DHFR) gene from adult buffalo fly (Haematobia irritans exigua): effects of antifolates.

The folate analogues methotrexate, aminopterin and pyrimethamine were toxic when fed in a blood meal to adult buffalo flies (Haematobia irritans exigua), but aminopterin caused greater mortality than methotrexate, while trimethoprim was not toxic to adult flies. This is the first recorded instance of mortality in adult insects caused by ingestion of folate analogues. In order to investigate the mechanism of this toxicity, the dihydrofolate reductase (DHFR) gene was cloned from adult buffalo fly cDNA using a PCR-based approach. The full-length DHFR coding sequence (BF-DHFR) was 887 bp and contained an open reading frame encoding a protein of 188 amino acids. The deduced protein sequence identities between BF-DHFR and the other known insect DHFR sequences were: Drosophila melanogaster, 75%; Aedes albopictus, 54%; Heliothis virescens, 43%. The BF-DHFR gene has a single 52 bp intron, an organization more similar to Dipteran species (Drosophila and Aedes). The cDNA encoding BF-DHFR was inserted into an Escherichia coli expression vector and the recombinant protein was expressed to levels representing about 25% of total cell protein. The active enzyme was purified by affinity chromatography on methotrexate-agarose and displayed a relatively low affinity (IC50 = 30 nm) for methotrexate.

Angiotensinogen genotype, plasma protein and mRNA concentration in isolated systolic hypertension.

Isolated systolic hypertension (ISH) occurs predominantly in the elderly, with a considerable morbidity and mortality. Its etiology is unknown but is likely to involve a significant genetic component. The aim of this study was to examine the angiotensinogen gene in ISH. The M235T and G(- 6)A polymorphisms were genotyped by polymerase chain reaction (PCR) in 86 ISH patients and 120 normotensive controls. Plasma angiotensinogen concentration was determined in 198 subjects by an indirect radioimmunoassay technique. Angiotensinogen mRNA concentration was determined by quantitative competitive reverse transcription (RT)-PCR in subcutaneous adipose tissue from a subset of these patients (n = 8) and controls (n = 6). Both the M235T (p = 0.0015) and G(- 6)A (p = 0.029) polymorphisms were associated with ISH. Plasma angiotensinogen concentration was higher in patients than controls (p < 0.0001), but was not associated with genotype. Angiotensinogen mRNA concentration in adipose tissue from ISH subjects was significantly lower than in adipose tissue from normotensive subjects (p = 0.033). The association of angiotensinogen gene variants with ISH and the elevation of plasma angiotensinogen concentration in these patients suggests a role of the angiotensinogen gene in this form of hypertension. Angiotensinogen gene expression may be altered in ISH, but this requires further examination.

Isolation and characterisation of a novel rabbit sulfotransferase isoform belonging to the SULT1A subfamily.

Sulfotransferases (SULTs) catalyse the sulfonation of both endogenous and exogenous compounds including hormones, catecholamines, drugs and xenobiotics. While in most occasions, sulfonation is a detoxication pathway, in the case of certain drugs and carcinogens, it leads to metabolic activation. Since, the rabbit has been extensively used for both pharmacological and toxicological studies, the purpose of this study was to further characterise the sulfotransferase system of this animal. In the present study, a novel sulfotransferase isoform (GenBank Accession no. AF360872) was isolated from a rabbit liver cDNA lambdaZAP II library. The full-length sequence of the clone was 1138 bp long and contained a coding region of 888 bp encoding a cytosolic protein of 295 amino acids (deduced molecular weight 34,193 Da). The amino acid sequence of this novel SULT isoform showed >70% identity with members of the SULT1A subfamily of sulfotransferases from other species. Upon expression of the encoded rabbit sulfotransferase in Escherchia coli (E. coli), it was shown that the enzyme was capable of sulfonating both p-nitrophenol (K(m) and Vmax values of 0.15 microM and 897.5 nmol/min/mg protein, respectively) and dopamine (K(m) and V(max) values of 175.3 microM and 151.1 nmol/min/mg protein, respectively). Based on the sequence data obtained and substrate specificity, this new rabbit sulfotransferase was named rabSULT1A1. Immunoblotting was used to demonstrate that rabSULT1A1 protein is expressed in liver, duodenum, jejunum, ileum, colon and rectum.

Localisation and functional studies on the 5'-nucleotidase of the cattle tick Boophilus microplus.

The ecto-5'-nucleotidase from the cattle tick Boophilus microplus is an unusual enzyme, hydrolysing a variety of nucleoside mono-, di- and triphosphates to release the free nucleoside. The gene has been sequenced and the recombinant protein expressed as a functional, active enzyme. Nevertheless, the function of the enzyme in the tick remains obscure. The enzyme is present throughout the life cycle, but in largest amounts in unfed larvae and adult ticks. The tissue location has been studied in adult female ticks by Western blotting, RT-PCR and immunofluorescence. All methods show the enzyme to be principally in the Malpighian tubules, though significant amounts are also present on the surface of ovaries and in detectable amounts in other tissues. This, together with the known specificity of the enzyme, suggests a role in purine salvage pathways. Sensitivity of ticks to allopurinol, an inhibitor of hypoxanthine-guanine-phosphoribosyltransferase, supports the importance of purine salvage in this tick and the potential role of nucleotidase in this pathway.

Site-directed mutagenesis of the substrate-binding cleft of human estrogen sulfotransferase.

The sulfonation of estrogens by human estrogen sulfotransferase (humSULT1E1) plays a vital role in controlling the active levels of these hormones in the body. To understand more fully the structural and functional characteristics of humSULT1E1, we have carried out site-directed mutagenesis of critical amino acids found in the substrate-binding cleft. Three single amino acid mutations of humSULT1E1 (V145E, H107A, and K85A) were created in this study. Kinetic studies were used to provide information about the importance of these residues in substrate specificity and catalysis, using a variety of substrates. Lysine at position 85 has been proposed to be within hydrogen bonding distance to the 3alpha-phenol group of beta-estradiol, thereby stabilising the substrate in the active site. However, substitution to a neutral alanine at this position improved substrate specificity of humSULT1E1 for beta-estradiol, estrone, and dehydroepiandrosterone (DHEA). The exchange of valine 145 for negatively charged glutamic acid markedly improved the ability of humSULT1E1 to sulfonate dopamine, but caused a reduction in specificity constants toward steroids tested, in particular DHEA. The presence of a histidine residue at position 107 was shown to be essential for the production of a functional protein, as substitution of this amino acid to alanine resulted in complete loss of activity of humSULT1E1 towards all substrates tested.

The 460Trp polymorphism of the human alpha-adducin gene is not associated with isolated systolic hypertension in elderly Australian Caucasians.

The study was undertaken to determine whether polymorphic variants of the alpha-adducin gene are associated with isolated systolic hypertension (ISH) in elderly Australian Caucasians. Participants were classified with ISH (n = 87, systolic blood pressure (SBP) > or =160 mm Hg and diastolic blood pressure (DBP) < or =90 mm Hg) or normotension (n = 124, SBP <140 mm Hg and DBP <90 mm Hg with no family history of hypertension). To collect demographic data, a structured questionnaire was used. DNA was extracted using standard techniques from 211 subjects (age range 61-89, mean age 73 +/- 6.6 years, male: female ratio 1.1:1). Genotypes (gly/gly, trp/gly and trp/trp) were assigned in triplicate by polymerase chain reaction (PCR) followed by electrophoresis, using a laser scanning electrophoresis unit. The validity of the method was confirmed by sequencing. Frequencies of allele distribution in ISH or control groups were determined by Chi-square tests and a stepwise logistic regression model, which controlled for potential confounders, was used to examine any independent association between alpha-adducin genotypes or alleles with ISH and normotensive controls. Mean BP (+/- s.d.) was: 170/79.8 +/- 14.9/8.3 mm Hg and 122.1/ 73.4 +/- 8. 8/7.6 mm Hg in the ISH and normotension groups respectively. The unadjusted allele and genotypes frequencies were not significantly different in the ISH patients groups compared with normotensive controls (chi2 = 1.59, P = 0.45 and chi2 = 1.23, P = 0.28 respectively). In this elderly cohort, after adjustment for potential confounders, no statistically significant association was found between alpha-adducin genotype and SBP (P = 0.65 for homozygotes, P = 0.59, for heterozygotes), DBP (P = 0.49 homozygotes, for heterozygotes P = 0.45) pulse pressure (P = 0.87 homozygotes, for heterozygotes P = 0.95) diagnosis of ISH (P = 0.72 for homozygotes, P = 0.68 for heterozygotes). However age and renal disease predicted the diagnosis of ISH (P = 0.001, P = 0.459, respectively), a large pulse pressure (P < 0.0001, P = 0.033, respectively) and a higher SBP (P < 0.0001, P = 0.025, respectively) in this large cohort of elderly Australian Caucasian volunteers. Journal of Human Hypertension (2000) 14, 199-203.

Molecular cloning and expression in Escherichia coli of an aquaporin-like gene from adult buffalo fly (Haematobia irritans exigua).

A gene fragment encoding a putative member of the aquaporin gene family was amplified using cDNA prepared from unfed adult buffalo fly poly(A)+ RNA and degenerate PCR primers designed from highly conserved regions of amino acids found in all members of the aquaporin gene family. This PCR product was labelled with digoxigenin-dUTP and used as a probe to screen a lambdagt-11 cDNA library constructed from unfed adult buffalo fly. One positively hybridizing clone (AqpBF1), contained an insert of 1878 bp, and DNA sequence analysis revealed an open reading frame of 753 bp encoding a polypeptide of predicted Mr = 26 163 Da. Comparison of the AqpBF1 deduced protein sequence with the GenBank database revealed significant homology to many aquaporin genes, including 72% identity with a partial DNA sequence encoding a member (DRIP) of the MIP protein family isolated from Drosophila melanogaster. The most closely related, full-length, GenBank sequence was an aquaporin gene isolated from the digestive tract of the sap-sucking insect Cicadella viridis, which was 53% identical to the buffalo fly AqpBF1 protein sequence. The full-length coding sequence of AqpBF1 was cloned into the (His)6-fusion vector, pQE10, and the recombinant protein was expressed in Escherichia coli following induction by IPTG. The recombinant (His)6-fusion protein was localized predominantly in the membrane fraction of E. coli. The protein was solubilized from E. coli membranes with n-octyl beta-D-glucopyranoside and purified by affinity chromatography on a Ni++-sepharose column in the presence of detergent.

The A1166C mutation in the angiotensin II type I receptor and hypertension in the elderly.

1. Using a nested case-control study of 661 non-institutionalized elderly (> or = 60 years) residents of Dubbo, New South Wales, Australia, the aim of this study is to determine whether the A1166C polymorphism of the angiotensin II type I (AT1) receptor gene is associated with hypertension in the elderly. 2. Individuals were classified as isolated systolic hypertension (ISH, n = 146), systolic diastolic hypertension (SDH, n = 188), or normotensive, age- and sex-matched controls (n = 327). AA, CC and AC genotypes were determined using restriction fragment length polymorphism analysis of DNA generated by nested polymerase chain reaction. 3. A univariate analysis (chi 2) was complemented by a logistic regression analysis, facilitating adjustment for potential confounders. The unadjusted genotype and allele frequencies in ISH or SDH subjects did not differ significantly from the control subjects (chi 2 = 3.0, P = 0.55, 4 d.f.; chi 2 = 3.0, P = 0.23, 2 d.f., respectively). After adjustment for potential confounders neither genotype nor allele predicted ISH or SDH in this cohort. 4. From this study we conclude that the A1166C polymorphism of the AT1 receptor gene is not a marker for ISH nor for SDH in this large, elderly community sample.

Cloning and expression of ecto 5-nucleotidase from the cattle tick Boophilus microplus.

Although 5'-nucleotidases are ubiquitous in higher vertebrates, the arthropod enzymes have been little studied. The cDNA sequence of the mature 5'-nucleotidase from the tick Boophilus microplus was therefore determined (GENBANK accession number: U80634). The enzyme has 39-41% sequence identity with the vertebrate 5'-nucleotidases and contains binuclear metal ion binding sites. There are no significant introns within the coding region of the genomic sequence. Southern blot analysis indicates the presence of multiple related genes encoding 5'-nucleotidases. Recombinant tick 5'-nucleotidase was expressed in both Escherichia coli and in baculovirus-infected insect cells. The E. coli recombinant protein was truncated, inactive and produced in abundance. The enzyme was expressed in baculovirus-infected insect cells as a secreted, soluble, glycosylated and enzymatically active protein. This represents the first successful expression and characterization of enzymatically active recombinant 5'-nucleotidase from any organism. Supplementation of the culture medium with 25 microM zinc resulted in a twofold increase in the activity of the expressed protein. The enzyme was purified to homogeneity. It exists under non-denaturing conditions as a homodimer, with an apparent molecular mass of 135 kDa. The Km for the hydrolysis of AMP was 0.37 microM and the k(cat) = 11.5/s, in agreement with data for the native enzyme.

Variations in the monoamine oxidase B (MAOB) gene are associated with Parkinson's disease.

The monoamine oxidase B gene (MAOB; Xp15.21-4) is a candidate gene for Parkinson's disease (PD) given its role in dopamine metabolism and its possible role in the activation of neurotoxins. The association of MAOB polymorphisms (a [GT] repeat allelic variation in intron 2 and an A-G transition in intron 13) with Parkinson's disease (PD) was studied in an Australian cohort of 204 (male:female ratio 1.60) people with PD and 285 (male:female ratio 1.64) age- and gender-matched control subjects. Genomic DNA was extracted from venous blood and polymerase chain reaction was used to amplify the appropriate regions of the MAOB gene. The length of each (GT) repeat sequence was determined by 5% polyacrylamide denaturing gel electrophoresis and a DNA fragment analyzer, while the G-A genotype was determined using 2% agarose gel electrophoresis. The G-A polymorphism showed no association with PD (odds ratio [OR] = 0.80; p = 0.51; 95% confidence interval [CI] = 0.42-1.53). There was a significant difference in allele frequencies of the (GT) repeat allelic variation between patients and control subjects (chi2 = 20.09; p<0.01). After statistical adjustment for potential confounders using a logistic regression analysis, the (GT) repeat alleles > or =188 base pairs in the intron 2 marker of the MAOB gene were significantly associated with PD (OR = 4.60; p<0.00005; 95% CI = 1.97-10.77). The 186 base pair allele was also significantly associated with PD (OR = 1.85; p = 0.048; 95% CI = 1.01-3.42). The GT repeat in intron 2 of the MAOB gene is a powerful marker for PD in this large Australian cohort.

Endocytosis by digest cells of the cattle tick Boophilus microplus: regulation by protein kinase C.

Endocytosis of fluorescently-labeled bovine serum albumin by digest cells of the gut of the cattle tick Boophilus microplus is inhibited by approx 60% in the presence of the tumour promoter 12-O-tetradecanoylphorbol 13-acetate. The results are consistent with a role for protein kinase C in regulating the uptake of blood meal by digest cells. Protein kinase C activity has been measured in the digest cell and the amount of enzyme has also been determined using a phorbol ester binding assay. The presence of a small number of specific protein kinase C substrates in the plasma membrane of the digest cell has been demonstrated. Preliminary experiments indicate that one of these substrates, a protein of approximately 30 kDa, is an integral membrane protein, part of which is exposed on the extracellular surface of the digest cell.