Comparison of two HLA-DRB high resolution microtiter plate reverse hybridization typing methods: advantage of a codon-86 valine or glycine PCR segregation.

Two rapid, nonisotopic, high-resolution HLA-DRB typing methods have been developed for DRB1, DRB3, DRB4 and DRB5 alleles. These methods are based on a single procedure consisting of the reverse hybridization of biotinylated amplicons to oligonucleotide probes that are covalently attached to a microtiter plate. Detection is by an enzymatic reaction with a fluorescent substrate. The 1 Generic Amplification (1GA) method amplifies all HLA-DRB alleles in the same reaction mix. The 2 Allelic Subset Amplification (2SA) method uses two distinct amplification reactions that distributes all DRB alleles into two equal-size subsets, according to the codon 86 Gly or Val polymorphism; this adds an extra discrimination level to the typing. 108 samples were typed using the 1GA and the 2SA methods and no discrepancies were found. Typing indeterminations due to overlapping probe combinations were compared; it was found that the 2SA method, with the extra discrimination level at the PCR step, greatly improved resolution.

Polymerase chain reaction assay for the detection of Helicobacter pylori in gastric biopsy specimens: comparison with culture, rapid urease test, and histopathological tests.

Ulcer recurrence is probably related to residual Helicobacter pylori (H pylori). Histological examination and culture are considered to be the most specific tests. CLO test is a rapid but less specific test, which is usually used as an alternative test to culture. The aim of this study was to investigate the efficiency of a simplified polymerase chain reaction (PCR) assay as a procedure for the diagnosis of gastric H pylori infection of patients. Biopsy specimens were obtained from antral mucosa of 58 patients at endoscopy and submitted to four tests for detection of H pylori. The bacteria were found in 53%, 43%, 48%, and 50% of patients according to the results of PCR, CLO test, culture, and histological examination. Twenty three patients had both negative histology and negative culture and PCR was negative in all of these. Thirteen patients were not classified because only histology or culture was positive and 10 of these had a positive PCR test. When the diagnosis of H pylori was established by agreement with both histology and culture or three positive tests out of four, 29 patients were H pylori positive (28 having had three positive tests and one displaying positive histology and culture), and 26 were negative, and three undetermined. PCR proved the most sensitive and specific test. These results suggest the simplified PCR assay may be a valuable test for the detection of H pylori.

Vitamin A deficiency and glucocorticoid receptor activity in rat liver.

The influence of vitamin A on the binding properties of hepatic glucocorticoid receptors (GR) was studied in young rats 7 weeks after they had been given a diet with or without vitamin A. Scatchard analysis showed an increased capacity of cytosolic GRs to bind dexamethasone in vitamin A deficiency. In these rats, an increase in tyrosine aminotransferase also occurred, and this could be related to the increased formation of hormone-GR complexes. Measurement of protein kinase C activity showed an increase which might be related to the functional activity of GRs.

c-erb-A mRNA content and triiodothyronine nuclear receptor binding capacity in rat liver according to vitamin A status.

Vitamin A is required for normal growth and development and synergistically acts with thyroid hormones in these processes. Effects of retinol dietary intake (0, 5, 50 U retinol/g food) on the triiodothyronine (T3) nuclear receptor were examined in rat liver. Properties of this receptor have been investigated by an in vitro binding method. The amount of c-erb-A mRNA has been deduced by reverse transcription and amplification methods (PCR). Results show that both retinol deficiency and excess leads to a reduced amount of c-erb-A mRNA and to a decreased T3 binding capacity, suggesting that retinol, or retinoic acid which is its natural metabolite, contributes to the regulation of the T3 nuclear receptor properties.

Effect of retinoids on protein kinase C activity and on the binding characteristics of the tri-iodothyronine nuclear receptor.

Retinoids and thyroid hormones exert profound effects on the development, growth and homeostasis of vertebrates. The receptor proteins which bind retinoic acid, tri-iodothyronine (T3) or steroid hormones and, as a result of this binding, interact with DNA to stimulate expression of specific genes, belong to the same recently discovered superfamily. The functionality of thyroid and steroid hormone receptors is thought to be related to a phosphorylation-dephosphorylation cycle. In the present work, the action of two retinoids (retinol and retinoic acid) was studied on the properties of T3-nuclear receptors and on protein kinase C (PKC) activity in the rat liver (PKC is known to be a phosphorylating enzyme for various proteins). The influence of 12-O-tetradecanoyl phorbol-13-acetate (TPA; known to enhance PKC activity) on the properties of T3-nuclear receptors was also investigated. Measurements of binding characteristics and enzyme activity were performed 4 or 12 h after a single i.p. injection of retinol or retinoic acid (6 mg/kg body weight) or 1 h after a single i.p. injection of TPA (0.7 mg/kg). The activity of PKC was increased 4 h after administration of the retinoids, and the affinity of the T3-nuclear receptor protein was increased markedly after 12 h. The activity of PKC and the affinity of nuclear T3 receptor were both increased 1 h after administration of TPA. These observations provide indirect evidence that retinoids, particularly retinoic acid, induce an increase in PKC activity and a subsequent increase in the affinity of the T3-nuclear receptor protein.