Cystic fibrosis mutation detection by hybridization to light-generated DNA probe arrays

We have combined photochemistry and photolithography with solid-phase DNA synthesis chemistry to form a new technology that makes high density oligonucleotide probe array synthesis possible. Hybridization to these two-dimensional arrays containing hundreds or thousands of oligonucleotide probes provides a powerful DNA sequence analysis tool. Two types of light-generated DNA probe arrays have been used to test for a variety of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. One array, made up of 428 probes, was designed to scan through the length of CFTR exon 11 and identify differences from the wild type reference sequence. The second type of array contained 1480 probes chosen to detect known deletions, insertions, or base substitution mutations. The validity of the probe arrays was established by hybridizing them with fluorescently labeled control oligonucleotide targets. Characterized mutant CFTR genomic DNA samples were then used to further test probe array hybridization specificity. Finally, ten unknown patient samples were genotyped using the CFTR probe array assay. The genotype assignments were identical to those obtained by PCR product restriction fragment analysis. Our results show that light-generated DNA probe arrays are highly effective in analyzing complex mutation and polymorphism patterns in a relatively large gene such as CFTR. © 1996 Wiley-Liss, Inc.     

Influence of Common and Specific HLA-DRB1/DQB1 Haplotypes on Genetic Susceptibilities of Three Distinct Arab Populations to Type 1 Diabetes

The contribution of HLA DRB-DQB to type 1 diabetes (T1D) in Bahrainis, Lebanese, and Tunisians was investigated. DRB1*030101-DQB1*0201 was a locus that conferred susceptibility in three populations, while DRB1*040101-DQB1*0302 was a locus that conferred susceptibility only in Tunisians and Bahrainis. The DRB1*100101-DQB1*050101 (Bahrainis) and DRB1*150101-DQB1*060101 (Lebanese) loci were largely protective. The contribution of HLA to T1D must be evaluated with regard to ethnic background.     

Genotype-phenotype relationship in human ATP6i-dependent autosomal recessive osteopetrosis

Autosomal-recessive osteopetrosis is a severe genetic disease caused by osteoclast failure. Approximately 50% of the patients harbor mutations of the ATP6i gene, encoding for the osteoclast-specific a3 subunit of V-ATPase. We found inactivating ATP6i mutations in four patients, and three of these were novel. Patients shared macrocephaly, growth retardation and optic nerve alteration, osteosclerotic and endobone patterns, and high alkaline phosphatase and parathyroid hormone levels. Bone biopsies revealed primary spongiosa lined with active osteoblasts and high numbers of tartrate-resistant acid phosphatase (TRAP)-positive, a3 subunit-negative, morphologically unremarkable osteoclasts, some of which located in shallow Howship lacunae. Scarce hematopoietic cells and abundant fibrous tissue containing TRAP-positive putative osteoclast precursors were noted. In vitro osteoclasts were a3-negative, morphologically normal, with prominent clear zones and actin rings, and TRAP activity more elevated than in control patients. Podosomes, alphaVbeta3 receptor, c-Src, and PYK2 were unremarkable. Consistent with the finding in the bone biopsies, these cells excavated pits faintly stained with toluidine blue, indicating inefficient bone resorption. Bone marrow transplantation was successful in all patients, and posttransplant osteoclasts showed rescue of a3 subunit immunoreactivity.     

Immunohistochemical localization of nNOS in the skin and nerve fibers of the earthworm Lumbricus terrestris L. (Annelida Oligochaeta)

The epidermis of the earthworm Lumbricus terrestris is a multifunctional tissue. It is composed of supporting, mucous, neuroendocrine-like, sensory and basal cells. NO is considered to be a molecule that regulates numerous functional activities (also in non-neuronal cells) in vertebrates. In the earthworm epidermis, we found neuronal NO synthase immunopositivity in orthochromatic and metachromatic mucous cells, neuroendocrine-like cells and in epidermal and subepidermal nerve fibers and striated muscle fibers. It is suggested that NO has a multitude of biological actions, affecting functional activities of the epidermis such as tissue homeostasis, control of secretion, proliferation, respiration, defense, water-salt balance, as well as regulation of tonus in vascular and striated muscles.     

Comparative study of nasopharyngeal aspirate and nasal swab specimens for diagnosis of acute viral respiratory infection

Paired nasopharyngeal aspirate (NPA) and nasal swab (NS) samples from 475 children hospitalized for acute respiratory infection were studied for the detection of influenza virus, parainfluenza virus, respiratory syncytial virus, and adenovirus by immunofluorescence test, viral culture, and multiplex PCR assay. The overall sensitivity of viral detection with NPA specimens was higher than that obtained with NS specimens.     

Untersuchungen zur Regulation der Ammoniumassimilation von Candida maltosa

Activities of ammonia-metabolizing enzymes from Candida maltosa were measured. The synthesis of glutamate and glutamine involves three primary enzymes: glutamate dehydrogenase (GDH), glutamine synthetase (GS), and glutamate synthase (GOGAT). This yeast has two distinct GDH, the first responsible for glutamate catabolism, and the second solely biosynthetic. The GS is derepressed during growth on low ammonia or on a variety of alternative nitrogen sources, whereas the catabolic GDH (NAD) is repressed under this conditions. Ammonia limitation did not significantly affect GOGAT as well as biosynthetic GDH (NADP) levels. The relatively low K_m-value for ammonia suggest that the GS is catalytically active during cell growth on low concentrations of ammonia. It seems that under N-limitation ammonia assimilation is achieved via GS/GOGAT and under N-excess via GDH/GS.     

Activation of immunological memory cells in vitro

Peritoneal cell populations from NMRI mice injected three times intraperitoneally with sheep red blood cells do not contain a significant number of plaque-forming cells to sheep red blood cells. However, plaque-forming cells do arise in such populations 3–5 days after transfer into tissue culture. The in vitro activation of these cells into antibody-producing cells occurs without further exposure to the antigen. The culture system contained, in addition to the peritoneal cells, irradiated spleen cells from non-immunized animals which were shown not to respond when cultured alone. The peritoneal cells as well as non-producing spleen cells could be activated in vitro at varying times after the last stimulation of the donor animal up to a period of several weeks. These observations suggest that there is a compartment of resting memory cells whose activation is not dependent on the addition of antigen.     

5-Year control and treatment of edema and increased capillary filtration in venous hypertension and diabetic microangiopathy using O-(beta-hydroxyethyl)-rutosides: a prospective comparative clinical registry

This independent prospective controlled trial evaluates the efficacy of O-(beta-hydroxyethyl)-rutosides (HR) during 5 years of administration against signs and symptoms and further degeneration of microcirculatory disturbances. The protective effect of HR in preventing end-point complications such as venous ulceration is evaluated. This study is based on evaluation of edema and the capillary filtration rate (CFR) in association with a clinical score scale. Patients having a severe degree of chronic venous insufficiency (CVI) and venous microangiopathy and completing at least 5 years of treatment are included. The following 4 groups are considered: group A (patients with CVI but without diabetes mellitus, receiving 1500 mg/d of HR), group B (patients with CVI and diabetes mellitus, receiving 2 g/d of HR), group C (control subjects receiving no pharmacologic or compression treatment), and group D (patients using elastic compression stockings only). All patients received the "best" available treatment. No adverse effects or intolerance is noted, with good compliance (>85%). In group A, there is a statistically significant decrease in the CFR during 5 years of follow-up. In group B, the decrease in the CFR is greater than that in group A. Reductions in edema, swelling, and the CFR during 5 years are notable, and values approach normal levels. During 5 years, HR is effective in treating venous edema and hypertension and in preventing deterioration of the distal venous system. The prevention of ulcerations with HR is another important observation. The effects of HR seem to be partially dose related, and tolerability and compliance are good.     

Serological Diagnosis of Tropical Canine Pancytopenia by Indirect Immunofluorescence

An indirect fluorescent-antibody test for detection and titration of antibodies to Ehrlichia canis, the causative agent of tropical canine pancytopenia, has been described. The organism propagated by an in vitro technique in canine blood monocytes served as an antigen in the test. The specificity of the test was revealed by absence of cross-reactivity between the antigen and sera from dogs infected with various common pathogens and specific sera against eight rickettsial species. The accuracy of the test was ascertained by isolation of the organism from reactor dogs located in and outside the United States. Histopathological examination of nine reactor dogs revealed plasmacytosis of meninges and kidneys in eight of them.