Chemical-Shift Perturbations Reflect Bile Acid Binding to Norovirus Coat Protein: Recognition Comes in Different Flavors

Bile acids have been reported as important cofactors promoting human and murine norovirus (NoV) infections in cell culture. The underlying mechanisms are not resolved. Through the use of chemical shift perturbation (CSP) NMR experiments, we identified a low-affinity bile acid binding site of a human GII.4 NoV strain. Long-timescale MD simulations reveal the formation of a ligand-accessible binding pocket of flexible shape, allowing the formation of stable viral coat protein–bile acid complexes in agreement with experimental CSP data. CSP NMR experiments also show that this mode of bile acid binding has a minor influence on the binding of histo-blood group antigens and vice versa. STD NMR experiments probing the binding of bile acids to virus-like particles of seven different strains suggest that low-affinity bile acid binding is a common feature of human NoV and should therefore be important for understanding the role of bile acids as cofactors in NoV infection.     

Nutrient digestibility and endogenous protein losses in the foregut and small intestine of weaned dairy calves fed calf starters with conventional or enzyme-treated soybean meal

The aims of this experiment were (1) to compare the effects of a soybean meal with an enzymatic treatment (ESBM) to reduce the concentration of antinutritional factors versus a standard soybean meal (SBM) on foregut and small intestine digestion in weaned dairy calves and (2) to estimate the endogenous losses of crude protein (CP) in the small intestine. Our hypothesis was that a diet containing ESBM instead of SBM would improve ruminal and small intestine digestion and absorption of nutrients. A T-cannula was placed in the duodenum, and a second T-cannula was installed in the distal ileum of 12 Holstein calves at approximately 3 wk of age. Calves were weaned on d 42, and on d 50 they were assigned randomly to a quadruplicated 3 × 3 Latin square with 10-d periods. Digesta samples were collected on d 7 and 8 from the ileum and d 9 and 10 from the duodenum. The diets were fed for ad libitum intake and consisted of a calf starter (CS) of 20% CP with SBM as the main source of protein (CTRL), and an isonitrogenous CS with an ESBM instead of SBM (ENZT). A third diet with a low content of CP (10%) and no soy protein was fed to estimate endogenous N losses and digestibilities of test ingredients. Flows and digestibilities of nutrients were compared between CTRL and ENZT and their test ingredients (SBM vs. ESBM, respectively). Duodenal net flows of CP and total AA as well as ruminal microbial protein synthesis per kilogram of digested CP were greater, and flow of nonprotein N and CP true (corrected by endogenous and microbial flows) foregut digestibility were lower with ENZT than CTRL. The apparent small intestine digestibilities of CP and total AA were greater for ESBM than SBM, but there were no differences between the CTRL and ENZT diets. We observed no differences in digestibilities at the duodenum or ileum of starch or NDF, but true small intestine digestibilities of CP and all AA were greater with ENZT than CTRL. Total endogenous protein losses in the small intestine estimated from calves fed the low-CP with no soy protein diet were 37 ± 1.5 g of CP and 29 ± 1.4 g of AA/kg of DMI. These values may be considered the basal endogenous losses as they are similar to values obtained with the regression method, which estimates N losses when dietary N is null. Our results indicated that the inclusion of an ESBM improved the efficiency of ruminal microbial protein synthesis per digested kilogram of organic matter and CP, and increased CP and AA absorption in the small intestine despite a greater proportion of undigested dietary protein entering the duodenum.     

Measurements of single chain form factors by small-angle neutron scattering from polystyrene blends containing high concentrations of labelled molecules

A series of small angle neutron scattering measurements on blends of normal polystyrene (PSH) and labelled (deuterated) polystyrene (PSD) have been made with concentrations of PSD from 5 to 50 mol %. It is shown that the single chain form factor of the polymer in bulk can be obtained from a single concentration measurement for any concentration of labelled molecules, providing the molecular weights of the parent and labelled molecules are the same and the molecular weight distributions are narrow.     

Treatment of radiation-induced nervous system injury with heparin and warfarin

When radiation is used to treat nervous system cancer, exposure of adjacent normal nervous system tissue is unavoidable, and radiation-induced injury may occur. Acute injury is usually mild and transient, but late forms of radiation-induced nervous system injury are usually progressive and debilitating. Treatment with corticosteroids, surgery, and antioxidants is often ineffective. We treated 11 patients with late radiation-induced nervous system injuries (eight with cerebral radionecrosis, one with a myelopathy, and two with plexopathies, all unresponsive to dexamethasone and prednisone) with full anticoagulation. Some recovery of function occurred in five of the eight patients with cerebral radionecrosis, and all the patients with myelopathy or plexopathy. Anticoagulation was continued for 3 to 6 months. In one patient with cerebral radionecrosis, symptoms recurred after discontinuation of anticoagulation and disappeared again after reinstitution of treatment. We hypothesize that anticoagulation may arrest and reverse small-vessel endothelial injury--the fundamental lesion of radiation necrosis--and produce clinical improvement in some patients.     

Effects of single doses of irradiation on the expression of resistance-related proteins in murine NIH 3T3 and human lung carcinoma cells

In this report the effects of single doses of ionizing radiation on the mRNA expression of several proteins involved in multiple drug resistance were analyzed. Murine NIH 3T3 cells treated with single doses of 5, 10 and 20 Gy during the time interval from 1.5 to 72 h after irradiation were compared with their corresponding controls at the same points of time. The glutathione S-transferase-pi (GST pi) level was elevated in cells treated with 10 or 20 Gy from 24 to 72 h after irradiation compared with the control. Topoisomerase II alpha and thymidylate synthase were decreased in irradiated cells 24-72 h after exposure. These down-regulations were associated with cellular proliferation, determined by mRNA expression of the proliferation marker histone 3. Irradiated cells exhibited no alteration in the P-glycoprotein or glutathione peroxidase mRNA content. The finding that GST pi mRNA was overexpressed after irradiation was validated by investigations on a human lung carcinoma cell line (LXF 289) on the mRNA and protein level. Thus, our results indicate that irradiation alters the expression of proteins involved in multidrug resistance and may, therefore, play a role in clinical drug response.     

Bladder injury model induced in rats by exposure to protamine sulfate followed by bacterial endotoxin

PURPOSE: A bladder injury model was developed using protamine sulfate (PS) and endotoxin lipopolysaccharide (LPS) administered intravesically to female Sprague-Dawley rats. MATERIALS AND METHODS: Experimental and control animals were catheterized and intravesically exposed to PS-LPS, PS, LPS, or phosphate buffered saline. After 4, 24 or 72 hours, rats were sacrificed. Urines and bladder tissues were then obtained. Bladder mucosal permeability was evaluated by measuring 14C-urea uptake 24 hours after injury. Repeated instillations of PS/LPS were also made in another group of rats over a period of 5 weeks to attempt to establish a more serious mucosal injury, possibly reflected by altered staining of the collagen IV component of the urinary basement membrane (UBM). RESULTS: Histological examination of the tissues indicated a maximal inflammatory response in the mucosa 4 hours after instillation of PS/LPS. Neutrophils and macrophages in close proximity to the UBM and intraepithelially could be demonstrated. Bladder permeability was significantly altered (26.9% 14C-urea uptake) in rats assayed 24 hours after the PS/LPS treatment, but not after exposure to PS or LPS alone (11.9 and 17.5%, respectively). Protease activity detected in urines from experimental, but not control, animals coincided with the appearance of inflammatory cells in the lamina propria. Inflammatory injury did not appear to alter the collagen IV staining of the UBM. CONCLUSIONS: This rat bladder injury model is useful for examining controversial issues regarding bladder wall structure-function alterations induced by inflammation and possibly important in the pathobiological mechanisms involved in some patients with interstitial cystitis.     

Alfalfa yield response to inoculation with recombinant strains of Rhizobium meliloti with an extra copy of dctABD and/or modified nifA expression

The construction of rhizobial strains which increase plant biomass under controlled conditions has been previously reported. However, there is no evidence that these newly constructed strains increase legume yield under agricultural conditions. This work tested the hypothesis that carefully manipulating expression of additional copies of nifA and dctABD in strains of Rhizobium meliloti would increase alfalfa yield in the field. The rationale for this hypothesis is based on the positive regulatory role that nifA plays in the expression of the nif regulon and the fact that a supply of dicarboxylic acids from the plant is required as a carbon and energy source for nitrogen fixation by the Rhizobium bacteroids in the nodule. These recombinant strains, as well as the wild-type strains from which they were derived, are ideal tools to examine the effects of modifying or increasing the expression of these genes on alfalfa biomass. The experimental design comprised seven recombinant strains, two wild-type strains, and an uninoculated control. Each treatment was replicated eight times and was conducted at four field sites in Wisconsin. Recombinant strain RMBPC-2, which has an additional copy of both nifA and dctABD, increased alfalfa biomass by 12.9% compared with the yield with the wild-type strain RMBPC and 17.9% over that in the uninoculated control plot at the site where soil nitrogen and organic matter content was lowest. These increases were statistically significant at the 5% confidence interval for each of the three harvests made during the growing season. Strain RMBPC-2 did increase alfalfa biomass at the Hancock site; however, no other significant increases or decreases in alfalfa biomass were observed with the seven other recombinant strains at that site. At three sites where this experiment was conducted, either native rhizobial populations or soil nitrogen concentrations were high. At these sites, none of the recombinant strains affected yield. We conclude that RMBPC -2 can increase alfalfa yields under field conditions of nitrogen limitation, low endogenous rhizobial competitors, and sufficient moisture.     

Inclusion body fibromatosis of the breast. Two cases with immunohistochemical and ultrastructural findings

Two cases of fibromatosis of the breast, characterized by a proliferation of spindle cells containing intracytoplasmic, spherical, eosinophilic inclusion bodies, are reported. The light and electron microscopic features, as well as the immunohistochemical features, are indistinguishable from those found in infantile digital fibromatosis. The proliferating spindle cells are characterized as myofibroblasts, whereas the inclusion bodies show an immunohistochemically nonreactive, hollow-like pattern with peripheral reactivity for actin filaments. This lesion, observed for the first time in the breast, expands the number of extradigital inclusion body fibromatoses.