Candidate genes colocalized to linkage regions in inflammatory bowel disease.

BACKGROUND/AIMS: The genes encoding for tumor necrosis factor-alpha (TNF-alpha), epidermal growth factor receptor (EGFR) and the vitamin D receptor (VDR) are colocalized to inflammatory bowel disease-associated linkage regions on chromosomes 6, 7 and 12. An association study of these gene polymorphisms with ulcerative colitis or Crohn's disease and a stratification according to disease phenotypes was performed in order to identify genetically homogenous subgroups. PATIENTS AND METHODS: 119 healthy, unrelated controls, 95 patients with Crohn's disease and 93 patients with ulcerative colitis were genotyped for the (G to A) -308 TNF-alpha promoter polymorphism on chromosome 6, the codon 497 EGFR polymorphism on chromosome 7 and the TaqI polymorphism of the VDR gene on chromosome 12. After genotyping, patients were stratified according to the respective disease phenotype. RESULTS: A disequilibrium in the distribution of the VDR genotypes was found in patients with ulcerative colitis compared to controls (p = 0.024). In fistulizing and fibrostenotic Crohn's disease the 'TT' genotype was significantly reduced compared with other phenotypes (p = 0.006), whereas the 'tt' genotype was found more frequently (p = 0.04). The frequency of the WT allele of the EGFR gene was significantly higher in ulcerative colitis (p = 0.04) than in controls. Further significant differences, concerning the associations of the different polymorphisms and disease susceptibility or clinical phenotypes, were not observed. CONCLUSIONS: Regardless of the disease phenotype, the associations between the polymorphisms and inflammatory bowel disease investigated herein are modest, even after stratification for the disease phenotypes. Hence, these polymorphisms are unlikely to confer the reported linkage between inflammatory bowel disease and chromosomes 6, 7 and 12.

K-ras mutations in sera of patients with colorectal neoplasias and long-standing inflammatory bowel disease.

BACKGROUND: About 50% of colorectal carcinomas and adenomas display K-ras mutations, which have also been described in stool or colonic lavage fluid. Moreover, the presence of K-ras mutations in plasma samples originating from patients with colorectal cancer has been reported recently. METHODS: DNA was extracted from sera of 16 patients with colorectal carcinomas, 6 with large adenomas, 3 with Crohn disease and 4 with ulcerative colitis. Sera of 20 healthy blood donors served as negative controls. K-ras mutations at the first or second position of codon 12 were detected by an enriched RFLP-PCR method and confirmed by sequencing. RESULTS: Mutations were found in sera of 5 patients with colorectal carcinomas (31%) and 2 patients with long-standing ulcerative pancolitis (50%), but not in patients with adenomas, Crohn disease or the controls. CONCLUSIONS: K-ras mutations can be detected in serum samples from patients with manifest colorectal cancer and in patients who display an increased risk for malignant transformation of the colonic mucosa. This observation may have clinical application concerning noninvasive surveillance of these patients. Because of the low sensitivity of this approach it may be useful to combine it with other molecular markers.

Delta 32 mutation of the chemokine-receptor 5 gene in inflammatory bowel disease.

The gene encoding chemokine receptor 5 (CCR5) is colocalized to the microsatellite marker D3S1573, which was linked with inflammatory bowel disease. Genetic heterogeneity in inflammatory bowel disease might be defined by a combination of the p-ANCA status and immunoregulatory genes. One hundred and twenty healthy unrelated controls, 101 patients with Crohn's disease, and 99 patients with ulcerative colitis were genotyped for the Delta 32 mutation of the CCR5 gene. The presence of p-ANCA was determined by the use of indirect immunofluorescence. After genotyping, patients were stratified according to p-ANCA status. The frequency of the Delta 32 mutation was not significantly different in controls and patients with Crohn's disease or ulcerative colitis (P 0.207 or more). Moreover, the frequency of the mutation was not significantly different in patients with inflammatory bowel disease after stratification for the p-ANCA status (P 0.482). Regardless of the p-ANCA status, Crohn's disease and ulcerative colitis are not associated with the Delta 32 mutation of the CCR5 gene.

The position dependent 15N enrichment of nitrous oxide in the stratosphere.

The position dependent 15N fractionation of nitrous oxide (N2O), which cannot be obtained from mass spectrometric analysis on molecular N2O itself, can be determined with high precision using isotope ratio mass spectrometry on the NO+ fragment that is formed on electron impact in the source of an isotope ratio mass spectrometer. Laboratory UV photolysis experiments show that strong position dependent 15N fractionations occur in the photolysis of N2O in the stratosphere, its major atmospheric sink. Measurements on the isotopic composition of stratospheric N2O indeed confirm the presence of strong isotope enrichments, in particular the difference in the fractionation constants for 15N14NO and 14N15NO. The absolute magnitudes of the fractionation constants found in the stratosphere are much smaller, however, than those found in the lab experiments, demonstrating the importance of dynamical and also additional chemical processes like the reaction of N2O with O(1D).

Design and pilot testing of the DVA/Seattle Footwear System for diabetic patients with foot insensitivity.

Clinical epidemiology studies suggest the majority of lower limb amputations were preceded by a minor traumatic event, often footwear-related, and lower limb ulcers. To reduce foot trauma and ulcers, the diabetic patient with foot insensitivity has unique footwear needs. To address these needs for patients not requiring custom shoes, the DVA/Seattle Footwear System was developed. The six components of this system include: 1) a specially designed shoe last based on the geometry of the diabetic foot and research findings on foot regions at highest risk of ulceration, 2) a depth-inlay shoe, "Custom Stride by PRS," designed to be paired with either a custom-fabricated cork insole or a preformed polyurethane insole, 3) a laser digitizing system that captures 3-D plantar foot contours, 4) DVA/Seattle ShapeMaker software adaptation for modifying plantar surface contours and applying free-form and template modifications to increase or relieve loading, 5) software that translates files into code used by a milling machine to define the cutting path and carve cork blockers into custom insoles, and 6) a preformed polyurethane insole thicker than a typical insole to accommodate the extra volume and the interior dimensions of the shoe. A 6-month pilot cross-over trial of 24 diabetic male veterans without prior foot ulcers was conducted to determine the feasibility of producing, and the safety of wearing, these depthinlay shoes and both types of insoles. During the first 4 weeks, patients were assigned to the study shoes and one type of insole. During the next 4 weeks, they wore the other type of insole, and during the final 4 months, they chose which pair of insoles to wear with the study shoes. Over 150 person-months of footwear observation revealed no breaks in the cutaneous barrier with use of either cork or polyurethane insoles and the study shoes. Patient compliance with the footwear was 88%. Patients were highly satisfied with the appearance, stability, and comfort of the shoes and the comfort of both types of insole. However, 75% of the patients noted that the study shoes felt heavier than their customary shoes. Further research is needed to determine the long-term effectiveness of footwear in prevention of foot ulcers in the population at highest risk for diabetic reulceration and amputation.

Effects of selected thermal variables on the mechanical properties of trabecular bone.

Osteoarticular allografts are commonly used in the treatment of segmental bone loss due to a wide resection of tumour. While the use of such grafts has met with considerable clinical success, fractures are a recognized complication of allograft use. Although trabecular bone can play an important structural role in the function of segmental allografts, few data exist on the effects of common storage and sterilization procedures on the mechanical properties of trabecular bone. To this end, we investigated with these experiments the effects of freezing at -20 degrees C, freezing at -70 degrees C, eight freeze-thaw cycles at -20 degrees C, freeze-drying, boiling and autoclaving on the compressive modulus and strength of bovine trabecular bone. Of these treatments, boiling and autoclaving were the only treatments to alter the properties of bovine trabecular bone, resulting in 26 and 58% reductions in strength, respectively. Autoclaving also significantly reduced the compressive modulus by 59%. From these data, freezing at temperatures between -20 and -70 degrees C does not appear to compromise the structural integrity of trabecular bone.

Aluminium production as a source of atmospheric carbonyl sulfide (COS).

Carbonyl sulfide is found as a major sulfur compound in anodic gases of commercial aluminium electrolysis. Recent spectroscopic measurements on industrial aluminium smelters found typical CO/COS ratios between 80 and 200. This results in specific COS emissions of between 1 and 7 kg/t(Al) if all COS is released into the atmosphere. In 1993 aluminium production would have been responsible for between 0.02 and 0.14 Tg of COS emissions. Currently, aluminium production does not seem to influence the total atmospheric COS budget to an extent beyond its natural variability. If recent growth rates of global aluminium production are sustained, however, COS emissions would quadruple until 2030. Together with increasing aircraft emissions into the stratosphere, an increase of the sulfate background aerosol is to be expected that could significantly enhance ozone depletion. The use of inert anodes is recommended to reduce aluminium production emissions of COS and CF4, C2F6, CO2, and CO at the same time.

COS, CS2 and SO 2 in aluminium smelter exhaust : The contribution of aluminium production to the global COS budget.

Measurements of carbonyl sulfide (COS) and carbondisulfide (CS2) were carried out on samples drawn from a smoke stack of an aluminium smelter. Volume mixing ratios of 6 ppm COS and 0.1 ppm CS2 were measured for gases from the electrolysis unit that had previously passed an Al2O3 fluid bed reactor and electrostatic precipitators. Specific emissions of 1.6 kg COS and 0.03 kg CS2 per ton of primary aluminium were found. Extrapolating from this particular smelter's conditions to a world mix specific COS emissions of about 4 kg/t(Al) are calculated resulting in emissions of annually 0.08 Tg COS into the atmosphere due to electrolytic aluminium production in 1995. Besides the photochemical conversion of anthropogenic CS2 aluminium production is established to be the second major industrial source of COS probably exceeding automotive tire wear's and coal combustion's contributions.

Trabecular bone modulus and strength can depend on specimen geometry.

We performed a series of uniaxial compression tests on wet bovine trabecular bone to compare both modulus and strength when measured using 2:1 aspect ratio (10 mm long, 5 mm diameter) cylinders (n = 30) and 5 mm cubes (n = 29). We also compared the correlation coefficients in the resulting modulus-density and strength-density regressions and the standard errors of the estimate. When comparing the mean values of modulus and strength for each group, the confounding variations in apparent density were accounted for with an analysis of covariance. The Fisher's Z transformation was used to compare the correlation coefficients statistically. Results from the analysis of covariance indicated that the modulus and strength of the cubes were higher by 36% (p < 0.01) and 18% (p < 0.05), respectively, with respect to the 2:1 cylinder values. The correlation coefficients in the modulus-density and strength-density regressions were not sensitive to the regression model (linear versus power law). However, correlation coefficients for both modulus-density and strength-density regressions were higher (p < 0.05) for the 2:1 cylinders (r = 0.90, modulus; r = 0.94, strength) than for the cubes (r = 0.57, modulus; r = 0.82, strength). In addition, the standard errors of the estimate in both modulus and strength were substantially lower for the 2:1 cylinders. These data indicate that both modulus and strength can depend on the specimen geometry when using conventional compression testing techniques. We conclude, therefore, that inter-study comparisons of modulus and strength may be invalid if these confounding effects of different specimen geometries are not addressed. Our data also indicate that density can better explain the observed variance in modulus and strength when 2:1 cylinders are used as opposed to cubes. Using this phenomenon as a rationale for choosing a standard specimen gometry, we recommend that the 2:1 cylinder be used as a standard specimen in studies designed to determine the effects of various treatments on the uniaxial compressive modulus and strength of trabecular bone.

Theoretical analysis of the experimental artifact in trabecular bone compressive modulus.

A theoretical analysis was performed to characterize potential experimental artifacts in conventional compression testing of trabecular bone, where strains are based on the relative displacements of the two loading platens. We assumed that the total experimental artifact for modulus was the sum of a damage and friction artifact and derived equations to describe these artifacts. The two unknown constants in these equations were found using a combination of data derived from linear finite element analyses and in vitro uniaxial compression tests. Subsequent finite element analyses allowed estimation of the artifacts for a wide range of specimens (cube, 1:4-3:1 aspect ratio cylinders). If friction is completely eliminated at the specimen-platen interface, the Young's modulus of a 5 mm sized (1:1 aspect ratio dimension) specimen which has a damage artifact due to machining may be underestimated by at least 45% regardless of specimen geometry; otherwise, the platens modulus may vary from less than 30 to over 175% of the Young's modulus, depending upon the specimen geometry and Poisson's ratio of the bone. Increasing the specimen size reduces the artifact only slightly. Since Poisson's ratio can be large for trabecular bone and is rarely known a priori, the precision of the conventional compression test will, therefore, be poor unless friction is completely eliminated at the specimen-platen interface. However, without friction at the interface, the platens modulus will always underestimate Young's modulus, thereby reducing the accuracy of this test. There was also evidence that the strength may be affected by these artifacts.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of 4-amino-1-hydroxybutylidene bisphosphonate on bone biomechanics in rats.

Bisphosphonates inhibit osteoclast-mediated bone resorption, but their effects on the mechanical behavior of bone remain uncertain. This study investigated the effects of 4-amino-1-hydroxybutylidene bisphosphonate (AHBuBP) on the biomechanical and morphologic properties of bone in ovariectomized rats. Sprague-Dawley rats (four groups, n = 6) were ovariectomized at 3 months of age. From 7 to 13 months, the groups received vehicle or 0.28, 2.8, or 28 micrograms/kg of AHBuBP twice weekly through subcutaneous injection. An additional group of control animals (n = 6) received neither surgery nor drug. We determined the stiffness, yield, and ultimate loads of the femoral midshaft, the sixth lumbar (L6) vertebra, and the femoral neck. Geometric properties of the cortical bone were measured from digitized images of the tibial diaphysis at the level of the synostosis. The area fraction of trabecular bone was determined through the midsagittal plane of the fifth lumbar (L5) vertebra. There were no significant differences in the structural properties of the femoral neck and midshaft, with the exception that the medium-dose group had a greater ultimate load than the vehicle group for the femoral midshaft in bending. Cross-sectional analysis of the tibia did not show significant differences in the inertial properties or area. Ovariectomy caused a significant reduction in the stiffness and ultimate load of L6 and in the area fraction of trabecular bone of L5.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduced nicotinamide adenine dinucleotide-nitrate reductase of Chlorella vulgaris. Purification, prosthetic groups, and molecular properties.

NADH:nitrate reductase (EC 1.6.6.1) from Chlorella vulgaris has been purified 640-fold with an over-all yield of 26% by a combination of protamine sulfate fractionation, ammonium sulfate fractionation, gel chromatography, density gradient centrifugation, and DEAE-chromatography. The purified enzyme is stable for more than 2 months when stored at minus 20 degrees in phosphate buffer (pH 6.9) containing 40% (v/v) glycerol. After the initial steps of the purification, a constant ratio of NADH:nitrate reductase activity to NADH:cytochrome c reductase and reduced methyl viologen:nitrate reductase activities was observed. One band of protein was detected after polyacrylamide gel electrophoresis of the purified enzyme. This band also gave a positive stain for heme, NADH dehydrogenase, and reduced methyl viologen:nitrate reductase. One band, corresponding to a molecular weight of 100, 000, was detected after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme contains FAD, heme, and molybdenum in a 1:1:0.8 ratio. One "cyanide binding site" per molybdenum was found. No non-heme-iron or labile sulfide was detected. From a dry weight determination of the purified enzyme, a minimal molecular weight of 152, 000 per molecule of heme or FAD was calculated. An s20, w of 9.7 S for nitrate reductase was found by the use of sucrose density gradient centrifugation and a Stokes radius of 89 A was estimated by gel filtration techniques. From these values, and the assumption that the partial specific volume is 0.725 cc/g, a molecular weight of 356, 000 was estimated for the native enzyme. These data suggest that the native enzyme contains a minimum of 2 molecules each of FAD, heme, and molybdenum and is composed of at least three subunits.