Restriction fragment length polymorphism analysis reveals different allele frequency and a linkage disequilibrium at locus D1S94 in neuroblastoma patients

Deletion of chromosome 1p and MYCN amplification have been reported as frequent abnormalities in human neuroblastoma. We studied loss of heterozygosity (LOH) in 50 (48 informative) Italian neuroblastoma patients by restriction fragment length polymorphisms (RFLPs) analysis using anonymous and hypervariable region (HVR) sequences. Twelve cases (25%) showed LOH at one or more loci. Locus D1S94 was the most frequently involved in LOH events (8/12) of deleted cases (66.6%). MYCN amplification was observed in 20% of patients which showed a significantly lower event-free survival probability (EFSp) (P = 0.004). We also studied the allelic distribution in the constitutional DNA of neuroblastoma patients (n = 44) and a matched group of healthy Italian subjects (n = 79) for loci D1S112 and D1S94. A significantly (P = 0.01) different allele frequency was detected for the two groups at locus D1S94, but not at D1S112. Moreover, the neuroblastoma population did not confirm the Hardy-Weinberg expectations at the former locus. This observation suggests the existence of an allelotype associated with neuroblastoma susceptibility.     

Global patterns of linkage disequilibrium at the CD4 locus and modern human origins

Haplotypes consisting of alleles at a short tandem repeat polymorphism (STRP) and an Alu deletion polymorphism at the CD4 locus on chromosome 12 were analyzed in more than 1600 individuals sampled from 42 geographically dispersed populations (13 African, 2 Middle Eastern, 7 European, 9 Asian, 3 Pacific, and 8 Amerindian). Sub-Saharan African populations had more haplotypes and exhibited more variability in frequencies of haplotypes than the Northeast African or non-African populations. The Alu deletion was nearly always associated with a single STRP allele in non-African and Northeast African populations but was associated with a wide range of STRP alleles in the sub-Saharan African populations. This global pattern of haplotype variation and linkage disequilibrium suggests a common and recent African origin for all non-African human populations.     

Inhibition of Trimeresurus flavoviridis phospholipase A2 by lung surfactant protein A (SP-A)

A marked sequence homology has been noted between lung surfactant protein A (SP-A) and an inhibitor of phospholipase A2 (PLA2) isolated from the serum of Trimeresurus flavoviridis (Habu snake). This study evaluated the effect of SP-A on PLA2 activity from several sources. SP-A was isolated from bovine or rat lung surfactant by extraction with 1-butanol and octyl beta-D-glucopyranoside. The addition of SP-A produced a concentration-dependent inhibition of T. flavoviridis PLA2 that indicated non-competitive kinetics with Ki 5 micrograms/ml. Inhibition was reversed by heat inactivation, disulfide bond reduction or alkylation of SP-A, or by the presence of anti-SP-A antibody. Treatment of SP-A with endoglycosidase F or the presence of variation monosaccharides or lectins did not alter SP-A inhibition. Binding of PLA2 to SP-A was shown by ultrafiltration and was abolished by SP-A alkylation or the presence of SDS. The SP-A/PLA2 complex recovered from the ultrafilter had essentially no enzymatic activity, but activity was restored by treatment with mercaptoethanol. SP-A had no effect on activity of PLA2 from Naja naja, Crotalus atrox, or bovine pancreas. These results indicate that surfactant protein A selectively inhibits Trimeresurus phospholipase A2 activity and suggest that binding to the enzyme is the mechanism for inhibition.     

Calcium-triggered selective intermembrane exchange of phospholipids by the lung surfactant protein SP-A

It is shown that human lung surfactant protein (SP-A) mediates selective exchange of phospholipid probes with unlabeled phospholipid in excess vesicles in the presence of calcium and NaCl. The exchange occurs without leakage of vesicle contents, or transbilayer movement (flip-flop) of the phospholipid probes, or fusion of vesicles. Individual steps preceding the exchange are dissected by a combination of protocols, and the results are operationally interpreted in terms of a model where a calcium-dependent change in SP-A triggers aggregation of vesicles followed by probe exchange between the vesicles in contact through SP-A. The contacts remain stable in the presence of calcium; i.e., the vesicles in contact do not change their partners on the time scale of several minutes. The binding of SP-A to vesicles and the aggregation of vesicles are rapid, and the aggregation is rapidly reversed by EGTA; i.e., both the forward and reverse aggregation reactions are complete in about 1 min. The exchange rate of the various probes between aggregated vesicles below 1 mM calcium in the presence of NaCl shows selectivity, i.e., a modest dependence on the net anionic charge on vesicles and for the headgroup of the probe. Exchange with lower selectivity is seen at >2 mM Ca in the absence of NaCl. SP-A binding to vesicles does not show an absolute specificity for the phospholipid structure, but the time course of the subsequent changes does. The results suggest that SP-A contacts between phospholipid interfaces could mediate the exchange of phospholipid species (trafficking and sorting) between lung surfactant pools in the hypophase and all accessible phospholipid interfaces of the alveolar space.     

Absence of linkage disequilibrium at amyloid precursor protein gene locus in Japanese familial Alzheimer's disease with 717Val-->Ile mutation

To date, eleven independent early onset familial Alzheimer's disease (EOFAD) pedigrees with the 717Val-->Ile mutation of amyloid precursor protein (APP) gene have been identified. Interestingly, five pedigrees have been of Japanese origin. The apparent ethnic prediction of this mutation raises the possibility that there is a founder effect in Japan. If the hypothesis holds true, we can expect the presence of linkage disequilibrium at the APP locus. We did not, however, observe any significant linkage disequilibrium at any locus of APP or the adjacent GT12 locus in the five Japanese EOFAD probands with the 717Val-->Ile mutation. The result indicates that a founder effect would probably not be present in Japanese EOFAD pedigrees with the 717Val-->Ile mutation.     

A haplotype and linkage disequilibrium analysis of the hereditary hemochromatosis gene region

Monoclonal antibodies were generated against serotonin (5-HT) and the C-terminal portion of the neuronal form of nitric oxide synthase (nNOS), the enzyme producing nitric oxide in neurons. These antibodies were used to compare the distribution of 5-HT- and nNOS-containing neurons in the raphe nuclei of four animal species (rat, mouse, guinea pig, and cat). It was found that the rat was the only species in which the raphe nuclei contain a substantial number of nNOS-immunoreactive (IR) cell bodies. In this species and as observed by other authors, all mesencephalic raphe nuclei contained nNOS-IR cells, the largest group being located in the nucleus raphe dorsalis. The coexistence of nNOS and 5-HT immunoreactivities in these nuclei was visualized by double labeling. In the medulla, the nuclei raphe magnus and obscurus displayed a rather low number of nNOS-IR neurons. In the other species, nNOS-IR cell bodies were found in very low numbers, whatever raphe nucleus was considered. The rostral pole of the nucleus raphe dorsalis and the nuclei raphe magnus and obscurus contained a few nNOS-IR neurons which did not show any coincidence with the 5-HT neurons. In addition, nNOS-IR axons were rare. It is concluded that in the mouse, guinea pig, and cat the involvement of nitric oxide in functions subserved by 5-HT within the raphe nuclei might be minimal.     

Human lung surfactant protein A exists in several different oligomeric states: oligomer size distribution varies between patient groups

BACKGROUND: Lung surfactant protein A (SP-A) is a complex molecule composed of up to 18 polypeptide chains. In vivo, SP-A probably binds to a wide range of inhaled materials via the interaction of surface carbohydrates with the lectin domains of SP-A and mediates their interaction with cells as part of a natural defense system. Multiplicity of lectin domains gives high-affinity binding to carbohydrate-bearing surfaces. MATERIALS AND METHODS: Gel filtration analyses were performed on bronchoalveolar lavage (BAL) fluid samples from three patient groups: pulmonary alveolar proteinosis (n = 12), birch pollen allergy (n = 11), and healthy volunteers (n = 4). Sucrose density gradient centrifugation was employed to determine molecular weights of SP-A oligomers. SP-A was solubilized from the lipid phase to compare oligomeric state with that of water soluble SP-A. RESULTS: SP-A exists as fully assembled complexes with 18 polypeptide chains, but it is also consistently found in smaller oligomeric forms. This is true for both the water- and lipid-soluble fractions of SP-A. CONCLUSION: The three patient groups analyzed show a shift towards lower oligomeric forms of SP-A in the following sequence: healthy-pulmonary alveolar proteinosis-pollen allergy. Depolymerization would be expected to lead to loss of binding affinity for carbohydrate-rich surfaces, with loss or alteration of biological function. While there are many complex factors involved in the establishment of an allergy, it is possible that reduced participation of SP-A in clearing a potential allergen from the lungs could be an early step in the chain of events.     

Developmental changes of neutral glycosphingolipids as receptors for pulmonary surfactant protein SP-A in the alveolar epithelium of murine lung

A dramatic change in the glycosphingolipid composition in murine lung occurred between 1 day and 1 week after birth. GlcCer and LacCer were the predominant neutral glycosphingolipids prenatally and 1 day after birth, and the concentrations of globo- and ganglio-series glycosphingolipids increased abruptly from 1 week after birth, reaching maxima at 2-3 weeks. To explore the functional significance of the change, we examined the role of neutral glycosphingolipids as receptors for the murine pulmonary surfactant protein, SP-A, and found that SP-A bound to LacCer, Gg3Cer, and Gg1Cer, but not to Gb3Cer, Gb4Cer, IV3GalNAc alpha-Gb4Cer, sulfatide, or several gangliosides. On TLC-blotting with 125I-labeled SP-A, the binding of SP-A to Gg3Cer and Gg4Cer was 5 times higher than that to LacCer, and on immunohistochemical staining Gg4Cer and Gg3Cer was mainly observed in the alveolar epithelium. Thus, the capacity to retain SP-A of glycolipid receptors per gram dry weight of lung at 1 week after birth was 1.6 times higher than that at 1 day after birth, and reached a maximum 3 weeks after birth. These findings suggest that the enhanced synthesis of the ganglio-series neutral glycosphingolipids 1 week after birth results in an increase in the binding capacity for SP-A on the epithelial cell surface of alveoli.     

A new allele at the short tandem repeat locus HumF13A01

We investigated the (AAAG)n short tandem repeat (STR) polymorphism HumF13A01 an Austrian Caucasoid population sample (n = 674). PCR amplified fragments were detected on an automatic A.L.F. DNA sequencer using laser-induced fluorescence. A total of 14 alleles could be identified including a new 179 bp allele which was designated allele 3. Sequence determination of allele 3 confirmed the typing results by revealing three continuous copies of the core repeat, whereas in sequencing of 54 additional alleles no further variants or microheterogeneities could be observed. The population data showed no significant deviation from Hardy-Weinberg equilibrium.     

Evidence for genetic linkage between the ure and trh genes in Vibrio parahaemolyticus

BACKGROUND: Many factors have been demonstrated to influence flexure of rigid contact lenses, but the contributions of surface tension and eyelid forces to flexure are not well understood. METHODS: We placed lenses on a model eye consisting of a polymethyl methacrylate (PMMA) base which could be flexed, and measured resultant flexure with a videokeratoscope. We varied the base toricity, sequence of measurement, and lens base curve. The effects of evaporation of the postlens fluid were also observed. RESULTS: Clinically significant flexure (> 0.50 D) occurred when two conditions were met: (1) the volume of the postlens space would increase if the lens unflexed, and (2) there was a paucity of fluid available to fill that space. Flexure was minimal (< or = 0.50 D) when ample fluid was present. CONCLUSION: Surface tension forces serve more to maintain rather than create rigid lens flexure. Our model helps to explain why steep-fitting lenses flex more and leads to several predictions for flexure, which appear generally to be obeyed.     

Cloning of two loci for synapse protein Snap25 in zebrafish: comparison of paralogous linkage groups suggests loss of one locus in the mammalian lineage

Recreational and subsistence hunters and anglers consume a wide range of species, including birds, mammals, fish and shellfish, some of which represent significant exposure pathways for environmental toxic agents. This study focuses on the Department of Energy's (DOE's) Savannah River Site (SRS), a former nuclear weapons production facility in South Carolina. The potential risk of contaminant intake from consuming mourning doves (Zenaida macroura), the most popular United States game bird, was examined under various risk scenarios. For all of these scenarios we used the mean tissue concentration of six metals (lead, mercury, cadmium, selenium, chromium, manganese) and radiocesium, in doves collected on and near SRS. We also estimated risk to a child consuming doves that had the maximum contaminant level. We used the cancer slope factor for radiocesium, the Environmental Protection Agencies Uptake/Biokinetic model for lead, and published reference doses for the other metals. As a result of our risk assessments we recommend management of water levels in contaminated reservoirs so that lake bed sediments are not exposed to use by gamebirds and other terrestrial wildlife. Particularly, measures should be taken to insure that the hunting public does not have access to such a site. Our data also indicate that doves on popular hunting areas are exposed to excess lead, suggesting that banning lead shot for doves, as has been done for waterfowl, is desirable.     

Genetic susceptibility factors in type 1 diabetes: linkage, disequilibrium and functional analyses

Rehabilitation after soft-tissue autograft reconstructions is controversial because there is indirect evidence that some grafts fail by creeping over time. The vulnerability of soft-tissue grafts to creep over healing time and the effects of the load environment during healing on this vulnerability have never been studied specifically. We hypothesized that immobilization would decrease the magnitude of the vulnerability of ligament grafts to creep. Thirty-nine skeletally mature New Zealand White rabbits underwent a standardized medial collateral ligament autograft procedure to the right hindlimb, and 19 of the rabbits also had the limb rigidly pinned into flexion. Subgroups were killed at 3 or 8 weeks, and all isolated tibia/medial collateral ligament/femur complexes were tested for creep at 4.1 MPa under a standardized protocol. Eight normal medial collateral ligament controls were tested similarly. Results showed that all grafts were quantitatively more susceptible to cyclic and static creep than were normal medial collateral ligament controls (p < 0.05). By 3 weeks of healing, immobilization significantly increased the magnitude of the vulnerability of the grafts to cyclic, static, and total creep (all: p < 0.05). Furthermore, the grafts had more unrecovered creep strain than did the controls following a 20-minute recovery period. Contrary to our hypothesis, immobilization resulted in increased vulnerability of these ligament autografts to creep even with this relatively nonprovocative test of short duration and low stress. We postulate that following immobilization, this increase in the magnitude of susceptibility of the grafts to creep will result in functionally significant elongation of the graft if it is exposed to higher loads and over longer periods of time in vivo.     

Assembly of an active enzyme by the linkage of two protein modules

BACKGROUND & AIMS: Oxidative stress mediates activation and stimulates collagen production of cultured hepatic stellate (Ito) cells. The aim of this study was to assess whether oxidative stress contributes to hepatic fibrogenesis in chronic hepatitis C. METHODS: In liver biopsy specimens of patients with chronic hepatitis C, the following fibrogenesis cascade was analyzed: (1) oxidative stress, determined by the presence of malondialdehyde protein adducts; (2) activation of stellate cells as indicated by their expression of alpha-smooth muscle actin; (3) stimulation of c-myb expression in stellate cells, a critical step in the activation of these cells; and (4) induction of collagen gene expression as detected by in situ hybridization. RESULTS: Treatment with d-alpha-tocopherol (1200 IU/day for 8 weeks) in 6 of these patients, who were refractory to interferon therapy, prevented the fibrogenesis cascade observed before antioxidant treatment. In addition, d-alpha-tocopherol treatment significantly decreased the carbonyl modifications of plasma proteins, a sensitive index of oxidative stress. However, 8 weeks of d-alpha-tocopherol treatment did not significantly affect serum alanine aminotransferase levels, hepatitis C virus titers, or histological degree of hepatocellular inflammation or fibrosis. CONCLUSIONS: These data suggest that enhanced oxidative stress initiates a fibrogenesis cascade in the liver of patients with chronic hepatitis C.     

Comparing the power of linkage detection by the transmission disequilibrium test and the identity-by-descent test

The purpose of this study was to identify new trends in the changing indications for penetrating keratoplasty. We retrospectively reviewed the clinical and pathologic diagnoses of 1,104 corneal buttons that had been submitted to the Estelle Doheny Eye Pathology Laboratory, Los Angeles, during the 5-year period 1989-1993. The leading indications, in order of decreasing frequency, were pseudophakic corneal edema (24.8%), regrafts (21.3%), scarring with or without chronic inflammation (11.1%), keratoconus (7.1%), aphakic corneal edema (6.4%), and ulcerative conditions (5.8%). The incidence of pseudophakic corneal edema remained stable over the study period and was actually surpassed by regraft in the last year of the study. Although pseudophakic corneal edema remains the predominant indication for penetrating keratoplasty, our findings suggest that its occurrence rate has begun to level off.