ARH missense polymorphisms and plasma cholesterol levels.

Mutations in a putative low-density lipoprotein (LDL) receptor adaptor protein called ARH have been recently described in patients with autosomal recessive hypercholesterolemia (ARH). ARH plays a tissue-specific role in determination of LDL receptor function. In the ARH gene three mismatched polymorphisms have been detected: Pro202Ser, Pro202His and Arg238Trp. These are of putative interest in plasma cholesterol level determination. To evaluate the effect of polymorphisms on plasma cholesterol levels, all polymorphisms were analyzed by PCR and restriction enzyme analysis by MnII, HpyCH4IV and SacII in 100 Caucasian males with high (>90%, 6.29 +/- 0.89 mmol/l), and 100 males with low (<10%, 3.60 +/- 0.57 mmol/l), total plasma cholesterol levels. No significant differences were observed in frequencies of ARH genotypes or alleles between these two extreme groups. These results suggest that ARH polymorphisms are unlikely to be important genetic determinants of plasma cholesterol levels.     

Hereditary Hypertriglyceridemic Rat: A New Animal Model of Metabolic Alterations in Hypertension

Hereditary hypertriglyceridemic rats (hHTg) were developed as a new genetic model for the study of relationships between blood pressure (BP) and metabolic abnormalities. This strain has been produced by selective inbreeding from Wistar rats according to the rise of plasma triglycerides induced by a high-sucrose diet. Though hHTg rats display hypertriglyceridemia, impaired glucose tolerrance, hyperinsulinemia, insulin resistance and increased BP even without nutritional stimuli, high sucrose feeding further aggravates these symptoms. High plasma triglycerides levels in hHTg rats seem to be a consequence of their hyperproduction. Impaired insulin action is responsible for the defective glucoregulation in this strain. The loss of insulin responsiveness might be due to a reduction in the number of glucose transporters. Highly significant relationships among plasma triglycerides, ouabain-resistant Na⁺ transport and BP were demonstrated in the hHTg rats. Segregating populations (F2 hybrids) should be used for genetic analysis of the primary role of lipid and/or ion transport abnormalities in the pathogenesis of this form of genetic hypertension.     

Indirect detection of anti-acetylcholinesterase compounds in microcolumn liquid chromatography using packed bed reactor with immobilized human red blood cell acetylcholinesterase and choline oxidase

The inhibiting compounds were separated by micro-column liquid chromatography in the mobile phase containing the natural substrate acetylcholine. A home-made packed bed microbioreactor system containing immobilized enzyme acetylcholinesterase (ACHE) in human red blood cell membrane and choline oxidase (CHO) from alcaligenes was used for the post-column conversion of acetylcholine to hydrogen peroxide which was detected by an electrochemical detector. The inhibition effect of the solutes caused a decrease in the acetylcholinesterase activity, a decrease in the formation of hydrogen peroxide and also a decrease in the response corresponding to the concentration of the solutes. The rate of the enzyme regeneration was also recorded. The micro-system was compared with a conventional LC system comprising commercially prepared enzyme reactor. The stability of the enzymes is at least 3 weeks at ambient temperature. The limit of detection depends on biological activity of inhibition and for galanthamine was 1 pmol.     

Antihypertensive mechanisms of chronic captopril or N-acetylcysteine treatment in L-NAME hypertensive rats.

Hypertension due to chronic inhibition of NO synthase (NOS) by Nomega-nitro-L-arginine methyl ester (L-NAME) administration is characterized by both impaired NO-dependent vasodilation and enhanced sympathetic vasoconstriction. The aim of our study was to evaluate changes in the participation of major vasoactive systems in L-NAME-treated rats which were subjected to simultaneous antihypertensive (captopril) or antioxidant (N-acetylcysteine, NAC) treatment. Three-month-old Wistar males treated with L-NAME (60 mg/kg/day) for 5 weeks were compared to rats in which L-NAME treatment was combined with simultaneous chronic administration of captopril or NAC. Basal blood pressure (BP) and its acute responses to consecutive i.v. injections of captopril (10 mg/kg), pentolinium (5 mg/kg), L-NAME (30 mg/kg), tetraethylammonium (TEA, 16 mg/kg) and nitroprusside (NP, 20 microg/kg) were determined in conscious rats at the end of the study. The development of L-NAME hypertension was prevented by captopril treatment, whereas NAC treatment caused only a moderate BP reduction. Captopril treatment normalized the sympathetic BP component and significantly reduced residual BP (measured at full NP-induced vasodilation). In contrast, chronic NAC treatment did not modify the sympathetic BP component or residual BP, but significantly enhanced NO-dependent vasodilation. Neither captopril nor NAC treatment influenced the compensatory increase of TEA-sensitive vasodilation mediated by endothelium-derived hyperpolarizing factor in L-NAME-treated rats. Chronic captopril treatment prevented L-NAME hypertension by lowering of sympathetic tone, whereas chronic NAC treatment attenuated L-NAME hypertension by reduction in the vasodilator deficit due to enhanced NO-dependent vasodilation.     

1H and 13C NMR determination of the hydroxy end-groups in poly(oxypropylene)s

¹H and ¹³C NMR signals of tri(propylene glycol) and of its benzoate and phenyl urethane were assigned by means of 1D and 2D NMR methods such as super-selective long-range INEPT, H-H COSY, H-C COSY and H-H-C RCT spectra. Both ¹H and ¹³C NMR were used as methods for the determination of primary and secondary hydroxy end-groups and of their degree of conversion. ¹³C NMR was found to be a reliable and universal method of end-group determination even for cases where ¹H NMR fails due to the presence of water in the sample or to the nature of the reactant. The results are applied to the analysis of poly(oxypropylene)s with two or three hydroxy end-groups and their derivatives.     

Antibody response to a synthetic peptide derived from the human papillomavirus type 6/11 L2 protein in recurrent respiratory papillomatosis: Correlation between southern blot hybridization,polymerase chain reaction,and serology

Recurrent respiratory papillomatosis (RRP) is the most common benign tumour of the larynx, affecting both children and adults. We present a series of 25 patients, including 10 cases of juvenile multiple, 8 cases of adult solitary, and 7 cases of adult multiple RRP. Biopsy tissue from each patient was screened by Southern blot hybridization and polymerase chain reaction for the presence of human papillomavirus (HPV) DNA. Sera from patients and age- and sex-matched controls were tested for the presence of HPV-specific antibodies using a synthetic pep-tide derived from the minor capsid protein (L2) of HPV 6/11. By Southern blot hybridization and/or polymerase chain reaction, biopsies from all patients were positive for HPV 6/11 DNA. There was no difference in antibody response between cases and controls. Female cases and controls had significantly higher antibody titers than male subjects. A correlation was observed between the HPV-specific antibody level and the number of surgery-necessitating recurrences. © 1994 Wiiey-Liss, Inc.     

Acrocentric prophasing in bromodeoxyuridine-incorporated chromosomes

Chromosomes that appear to be incompletely condensed [pulverized, prematurely condensed chromosomes (PCCs), prophasing] are known to occur in metaphase spreads of cells from normal individuals and more frequently in cells from individuals with malignant disease or in cells exposed in vitro or in vivo to various agents such as viruses, chemicals, and radiation. In this study involving bromodeoxyuridine (BrdU)-treated lymphocytes, a selective prophasing of acrocentrics appeared to be occurring. The acrocentrics involved were generally in interconnected groups.     

Optimization of nonviral transfection: variables influencing liposome-mediated gene transfer in proliferating vs. quiescent cells in culture and in vivo using a porcine restenosis model

Cationic liposomes/DNA complexes are widely used vectors for delivering genes in clinical and experimental trials. Relatively low transfer efficiencies in vivo compared with viral gene transfer may be improved using local application. In addition, markedly increased transfer efficiency may be achieved in vitro and in vivo via optimization of known variables influencing liposomal transfection. Lipofection under different conditions was performed in various cell lines and primary porcine smooth muscle cells. Optimized conditions found in vitro were verified in vivo using a porcine restenosis model. Toxicity was monitored analyzing cell metabolism. Transfer efficiency and safety were determined using morphometry, histology, galactosidase assays, PCR, and RT-PCR. The most important variables enabling maximum transfer efficiency were firstly the appropriate selection of cationic lipids for the cell type to be transfected, secondly the DNA/liposome ratio chosen, which depended on the cell type and cationic lipids used, and thirdly the state of proliferation of the targeted cells. Transfection in vivo demonstrated two- to fivefold higher transfer efficiencies when transfer conditions were extrapolated from optimization experiments in stationary cells compared with the use of conditions established in proliferating cells. Application of the therapeutic gene for cecropin using optimized transfer conditions resulted in a significantly reduced neointima formation compared with the transfection using a control gene for ss-galactosidase. Thus, in this vascular model, initial optimization of lipofection in stationary cells in culture followed by local delivery in vivo and with selection of a suitable therapeutic gene led to markedly improved transfer efficiencies, gene expression, and biological effect. Stationary cell cultures simulate more realistically the in vivo situation and may therefore represent a better model for future in vivo experiments. In addition, the advantages of liposomes are easy handling, low toxicity, and the lack of carcinogenicity or immunogenic reactions.     

Polymerization of lactams, 55. Cyclic dimer of 6-caprolactam and its polymerization

Polymerization of the cyclic dimer of 6-caprolactam was investigated in N-methyl-2-pyrroli-done solution at 260°C under initiation with 6-aminocaproic acid or anhydrous phosphoric acid on the one hand, and in the solid phase at 230, 260, and 280°C in the absence of any other component or under initiation with anhydrous phosphoric acid, on the other. Monomeric 6-caprolactam was formed in all cases already at the beginning of reaction. It has been suggested that, in addition to the direct incorporation of the cyclic dimer into a polymer chain, the dimer is alos incorporated via the monomer, which is formed from the dimer by an intermolecular transamidation reaction.