The oxygenase domain of inducible NO synthase (residues 1−498, iNOSox) is the enzyme's catalytic center. Its active form is a homodimer that contains heme and tetrahydrobiopterin (H4biopterin) and ...binds l-arginine Ghosh, D. K., & Stuehr, D. J. (1995) Biochemistry 34, 801. To help identify protein residues involved in prosthetic group and dimeric interaction, we expressed H4biopterin-free iNOSox in Escherichia coli. The iNOSox was 80% dimeric but contained a low-spin heme iron that bound DTT as a sixth ligand. The iNOSox bound H4biopterin or l-arginine with high affinity, which displaced DTT from the heme and caused spectral changes consistent with a closing up of the heme pocket. The H4biopterin-replete iNOSox could catalyze conversion of N ω-hydroxyarginine to citrulline and NO in a H2O2-supported reaction. Limited trypsinolysis of the H4biopterin-free iNOSox dimer cut the protein at a single site in its N-terminal region (K117). H4biopterin protected against the cleavage whereas l-arginine did not. The resulting 40 kDa protein contained thiol-ligated low-spin heme, was monomeric, catalytically inactive, showed no capacity to bind H4biopterin or l-arginine, and did not dimerize when provided with these molecules, indicating that residues 1−117 were important for iNOSox dimerization and H4biopterin/l-arginine interaction. A deletion mutant missing residues 1−114 was partially dimeric but otherwise identical to the 40 kDa protein regarding its spectral and catalytic properties and inability to respond to l-arginine and H4biopterin, whereas a deletion mutant missing residues 1−65 was equivalent to wild-type iNOSox, narrowing the region of importance to amino acids 66−114. Mutation of a conserved cysteine in this region (C109A) decreased H4biopterin affinity without compromising iNOSox dimeric structure, l-arginine binding, or catalytic function. These results suggest that residues 66−114 of iNOSox are involved in productive H4biopterin interaction and subunit dimerization. H4biopterin binding appears to stabilize the protein structure in this region, and through doing so activates iNOS for NO synthesis.
To examine the effect of a modification of a typically Irish dairy calf-to-beef production system, Charolais×Friesian steers were offered a finishing ration of grass silage ad libitum and 5.6kg ...concentrates daily for 174days prior to slaughter at 25months of age or grass silage ad libitum for 174days, followed by pasture for 167days and slaughter at 30months of age. Finishing at pasture increased carcass weight (376 vs. 342kg) but did not affect intra-muscular lipid concentration (28 vs. 24g/kg). Finishing at pasture decreased Longissimus thoracis et lumborum lightness (35.6 vs. 36.9) and increased shear force of muscle at 2 (8.54 vs 4.32) and 7days (5.21 vs 3.64kg) post-mortem but not at 14days post-mortem (4.45 vs. 3.42kg). Finishing at pasture did not affect the sensory characteristics of tenderness, juiciness, firmness or chewiness and tended (P<0.1) to decrease texture and acceptability. It is concluded that modification of this beef production system as described, had minor effects on beef quality which are unlikely to be of commercial significance.
Backgound
Abdominal aortic aneurysm (AAA) surgery provides a unique opportunity to study the impact of arterial stiffness on central haemodynamics, reflected in augmentation index (AI). The ...aneurysmal aorta is significantly stiffer than undilated age-matched aorta.
Aim
We investigated whether replacement of an aneurysmal aorta with a compliant graft would result in a decrease in AI, which would thus decrease myocardial workload parameters.
Methods
Patients undergoing elective open or endovascular AAA repair were assessed with applanation tonometry and laser fluximetry pre-operatively, immediately and long-term post-operatively.
Results
Replacement of a small segment of abnormal conduit vessel resulted in improvements in AI, demonstrating that arterial stiffness can be surgically manipulated.
Conclusions
These results reflect a decreased myocardial workload post-aortic grafting. This decrease in AI is important from a risk factor management perspective, and arterial stiffness should become a further recognised and screened for risk factor in patients with known aneurysmal disease.
We investigate theoretically the master oscillator power amplifier using a semiconductor laser model that is fully time and space (laterally and longitudinally) resolved. We numerically examine the ...stability of the device and identify the nature of the different instabilities. These can arise from undamped relaxation oscillations, beating between the longitudinal modes of any of the cavities that comprise the device, or lateral filamentation.
To quantify the risk of radiation-induced leukemia and provide further information on the nature of the relationship between dose and response, a case-control study was undertaken in a cohort of over ...150,000 women with invasive cancer of the uterine cervix. The cases either were reported to one of 17 population-based cancer registries or were treated in any of 16 oncologic clinics in Canada, Europe, and the United States. Four controls were individually matched to each of 195 cases of leukemia on the basis of age and calendar year when diagnosed with cervical cancer and survival time. Leukemia diagnoses were verified by one hematologist. Radiation dose to active bone marrow was estimated by medical physicists on the basis of the original radiotherapy records of study subjects. The risk of chronic lymphocytic leukemia, one of the few malignancies without evidence for an association with ionizing radiation, was not increased relative risk (RR) = 1.03; n = 52. However, for all other forms of leukemia taken together (n = 143), a twofold risk was evident (RR = 2.0; 90% confidence interval = 1.0-4.2). Risk increased with increasing radiation dose until average doses of about 400 rad (4 Gy) were reached and then decreased at higher doses. This pattern is consistent with experimental data for which the down-turn in risk at high doses has been interpreted as due to killing of potentially leukemic cells. The dose-response information was modeled with various RR functions, accounting for the nonhomogeneous distribution of radiation dose during radiotherapy. The local radiation doses to each of 14 bone marrow compartments for each patient were incorporated in the models, and the corresponding risks were summed. A good fit to the observed data was obtained with a linear-exponential function, which included a positive linear induction term and a negative exponential term. The estimate of the excess RR per rad was 0.9%, and the estimated RR at 100 rad (1 Gy) was 1.7. The model proposed in this study of risk proportional to mass exposed and of risk to an individual given by the sum of incremental risks to anatomic sites appears to be applicable to a wide range of dose distributions. Furthermore, the pattern of leukemia incidence associated with different levels of radiation dose is consistent with a model postulating increasing risk with increasing exposure, modified at high doses by increased frequency of cell death, which reduces risk.
The objective of this experiment was to quantify variation in bovine M. longissimus dorsi tenderness and determine the extent such variation is explained by variation in the ultrastructure of muscles ...after different postmortem treatments. Eight muscles were hot-boned and aged for 2 d at 2 degrees C (T1) to achieve very contracted actomyosin crossover and tough beef. Eight carcass sides were tenderstretched for 10 h at 10 degrees C and a further 38 h at 2 degrees C (T2) to achieve lengthened actomyosin crossover and tender beef. Both T1 and T2 were compared with conventionally hung carcasses, which underwent similar chilling regimes, C1 (n = 8) and C2 (n = 8), respectively. Measurements of sarcomere length, pH, Warner Bratzler shear force (WBSF), and sensory tenderness were taken, and transmission electron microscopy images analyzed. Variances of attributes were analyzed on Bartlett's test. Variances of the 4 groups were homogenous for all attributes except for pH after 24 h postmortem (with T1 0.00 having lower variances than C1 = 0.04, T2 = 0.06, and C2 = 0.05) and WBSF after 2 d aging (with T2 74.33 having lower variances than T1 = 236.76, C1 = 398.82, and C2 = 856.74). The variation in the tenderness of beef was quantified through ultrastructural variation in bovine muscle, with tenderstretched moderately chilled beef having the least variable tenderness as a result of more uniform overlap between actin and myosin filaments. Variation in the eating quality of beef was not reduced by hot-boning with fast chilling or conventional hanging with fast or moderate chilling. The development of the uniformity within filaments of tenderstretched muscle requires further analysis as residual variation remains.
Radiotherapy has been linked infrequently to secondary leukemia despite extensive exposure of the active bone marrow to ionizing radiation. Few studies include substantial numbers of elderly ...patients.
We evaluated women with cancer of the uterine corpus, the majority of whom were treated at older ages, to gain additional information on cancer risk following partial-body radiotherapy and to examine differences in risk between external-beam therapy and brachytherapy.
A cohort of 110,000 women with invasive cancer of the uterine corpus who survived at least 1 year following their initial cancer was assembled from nine population-based cancer registries. Cancer diagnoses occurred from 1935 through 1985, and most patients were diagnosed during the 1960s and 1970s. Radiation doses were computed to 17 sections of the active bone marrow for 218 women who developed leukemia and for 775 matched control subjects.
Radiotherapy did not increase the risk of chronic lymphocytic leukemia (CLL) (relative risk RR = 0.90; 95% confidence interval CI = 0.4-1.9). However, for all leukemias except CLL, a significant risk was identified (RR = 1.92; 95% CI = 1.3-2.9). Overall, the pattern of risk in relation to dose was erratic and was most consistent with a constant increased risk across the entire dose range. The risk following continuous exposures from brachytherapy at comparatively low doses and low dose rates (RR = 1.80; 95% CI = 1.1-2.8; mean dose = 1.72 Gy) was similar to that after fractionated exposures at much higher doses and higher dose rates from external-beam treatment (RR = 2.29; 95% CI = 1.4-3.7; mean dose = 9.88 Gy), indicating a large difference in the estimated risk per unit dose. Risk did not vary by age at first exposure; increased risks were apparent for irradiated patients aged 65 years or older (RR = 1.77; 95% CI = 0.9-3.5).
The leukemia risk associated with partial-body radiotherapy for uterine corpus cancer was small; about 14 excess leukemia cases were due to radiation per 10,000 women followed for 10 years. Women aged 65 years or older had a radiation risk comparable with that found in younger women. The relationship of leukemia risk to radiation dose was found to be complex due to the competing processes of cell killing, transformation, and repair. At very high doses delivered at high rates, destruction of cells likely dominates, and the risk per unit dose is low. In the low dose range, where dose was protracted and delivered at relatively low dose rates, the leukemia risk appears lower than that projected from risk estimates derived from the instantaneous whole-body exposures of atomic bomb survivors.
The relationship between exposure to sparsely ionizing radiation and mortality due to cancers of hematopoietic and lymphopoietic tissues was studied among 12,955 women treated for benign ...gynecological disorders at any of 17 hospitals in New England or New York State and followed for an average of 25 years; 9770 women were treated by radiation (intracavitary 226 Ra, external-beam X rays), while 3185 were treated by other methods, including curettage, surgery, and hormones. The average age at treatment was 46.5 years, and the mean dose to active bone marrow among irradiated women was 119 cGy. Forty deaths due to acute, myelocytic, or monocytic leukemia were observed among irradiated women. This number was 70% higher than expected based on U.S. mortality rates standardized mortality ratio (SMR) = 1.7; 90% confidence interval (CI) 1.3-2.3. A deficit was recorded among nonirradiated women, based on three observed deaths (SMR = 0.5; 90% CI 0.1-1.2). A well-defined gradient in the SMR with dose among exposed women was not detected. The SMR was highest within 5 years after irradiation but remained elevated even after 30 years. The temporal pattern differed by subtype of leukemia: excess mortality due to chronic myelocytic leukemia occurred almost exclusively within the first 15 years, whereas the SMR for acute leukemia, though also elevated, varied little over time. Cancers of lymphoreticular tissue occurred more often than expected based on U.S. mortality rates, but not appreciably differently for irradiated and nonirradiated women. There was little or no evidence of effects attributable to radiotherapy for chronic lymphocytic leukemia relative risk (RR) = 1.1; 90% CI 0.5-3.0, Hodgkin's disease (RR = 0.9; 90% CI 0.3-3.2), non-Hodgkin's lymphoma (RR = 0.9; 90% CI 0.6-1.6), or multiple myeloma (RR = 0.6; 90% CI 0.3-1.4). These results corroborate previous findings indicating that acute and myelocytic leukemias are the most prominent malignancies after exposure to sparsely ionizing radiation, occurring in excess shortly after irradiation, and that lymphomas are either not caused by radiation or are induced only rarely.
O-Linked fucose is an unusual form of glycosylation recently shown to modify the hydroxyls of serine or threonine residues at a strict consensus site within epidermal growth factor-like domains of ...several serum proteins. Here we demonstrate that Chinese hamster ovary cells modify numerous proteins with O-linked fucose and that the fucose is elongated on specific proteins. We have identified at least two forms of O-linked fucose elongation in Chinese hamster ovary cells: a disaccharide (Glcbeta1,3Fuc) and a larger oligosaccharide of indeterminate structure. Interestingly, it appears that the level of monosaccharide accumulates in the cells over time whereas the disaccharide does not. Analysis of the O-linked fucose-containing saccharides on individual proteins revealed that some proteins are modified with the monosaccharide only, whereas others are modified with monosaccharide and disaccharide, or monosaccharide and oligosaccharide. These results suggest that elongation of the O-linked fucose monosaccharide is a protein-specific phenomena. The presence of elongated O-linked fucose moieties suggests that a novel glycosylation pathway exists in mammalian cells with O-linked fucose as the core.
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