Charged surface area of maurocalcine determines its interaction with the skeletal ryanodine receptor

The 33 amino acid scorpion toxin maurocalcine (MCa) has been shown to modify the gating of the skeletal-type ryanodine receptor (RyR1). Here we explored the effects of MCa and its mutants ([Ala⁸]MCa, [Ala¹⁹]MCa, [Ala²⁰]MCa, [Ala²²]MCa, [Ala²³]MCa, and [Ala²⁴]MCa) on RyR1 incorporated into artificial lipid bilayers and on elementary calcium release events (ECRE) in rat and frog skeletal muscle fibers. The peptides induced long-lasting subconductance states (LLSS) on RyR1 that lasted for several seconds. However, their average length and frequency were decreased if the mutation was placed farther away in the 3D structure from the critical ²⁴Arg residue. The effect was strongly dependent on the direction of the current through the channel. If the direction was similar to that followed by calcium during release, the peptides were 8- to 10-fold less effective. In fibers long-lasting calcium release events were observed after the addition of the peptides. The average length of these events correlated well with the duration of LLSS. These data suggest that the effect of the peptide is governed by the large charged surface formed by residues Lys²⁰, Lys²², Arg²³, Arg²⁴, and Lys⁸. Our observations also indicate that the results from bilayer experiments mimic the in situ effects of MCa on RyR1.     

Cubicle Refusal and Rearing Accommodation as Possible Mastitis Risk Factors in Cubicle-Housed Dairy Heifers

Fifty-nine of the 65 dairy farms with cubicle sheds in the Norwegian county of Oppland in 1990 were included in a study of rearing accommodation, cubicle refusal and mastitis incidence. The farmers recorded the favoured resting location of the individual cows and heifers throughout the final week of pregnancy as well as during calving. The observations were matched with individual records of mastitis cases during the first 30 days after calving. Mastitis incidence in the heifers was analysed by logistic regression using rearing accommodation and cubicle refusal as independent variables, controlling for herd as a clustering factor. Cubicle refusal was found in 29% of the heifers, but in only 3% of older cows. The results of the analysis indicated a tendency for cubicle refusal to be associated with an increased mastitis incidence among the heifers (OR = 2.2, c.i._95%OR = 0.9–5.4, P = 0.08). Cubicle refusal accounted for 21% (0–32%) of the mastitis cases in the study population (PAF = 0.21).     

Cubicle Refusal in Norwegian Dairy Herds

In order to survey the behaviour of choosing the alley area instead of a cubicle as a lying place (cubicle refusal), a questionnaire was sent to the 273 dairy farms in Norway known to keep cows in cubicle housing systems. Sixty-six percent of the farmers contacted were included in the study. The median herd size was 18 cows (range 7–118). More than 85% of the herds had sheds providing one or more cubicles per cow. The mean herd occurrence of cubicle refusal was 6%, but showed great variation (range 0–55%). Regression analysis showed a significant association between rearing heifers in slatted floor pens and an increased cubicle refusal occurrence (p = 0.02, R² = 0.05), while herd size, use of litter, or cubicle-to-animal ratio were not found to be associated with cubicle refusal. The practice of rearing heifers in slatted floor pens accounted for about one half of the observed cubicle refusal (etiologic fraction = 0.51).     

ABE production from corn: a recent economic evaluation

This article details an economic assessment of butanol production from corn using the newly developed hyper-butanol-producing strain of Clostridium beijerinckii BA101. Butanol is produced in batch reactors and recovered by distillation. For a plant with 153,000 metric tons of acetone, butanol, and ethanol (ABE) production capacity, the production equipment cost and total working capital cost is US$33.47×10⁶ and US$110.46×10⁶, respectively. Based on a corn price (C _p) of US$79.23 ton⁻¹ (US$2.01 bushel⁻¹), an ABE yield of 0.42 (g ABE/g glucose) butanol price is projected to be US$0.34 kg⁻¹. An improved yield of 0.50 will reduce this price to US$0.29 kg⁻¹. Assumptions, such as by-product credit for gases and complete conversion of corn steep liquor (CSL) to fermentation by-products, have been taken into consideration. An increased price of corn to US$197.10 ton⁻¹ would result in a butanol price of US$0.47 kg⁻¹. A grass-rooted plant would result in a butanol price of US$0.73 kg⁻¹ (C _p US$79.23 ton⁻¹). In a worst case scenario, the price of butanol would increase to US$1.07 kg⁻¹ (C _p 197.10 ton⁻¹ for a grass-rooted plant and assuming no credit for gases). This is based on the assumption that corn price would not increase to more than US$197.10 ton⁻¹. Journal of Industrial Microbiology & Biotechnology (2001) 27, 292–297. Received 12 September 2000/ Accepted in revised form 12 January 2001     

Isolation, Biochemical Characterization, Long-Term Culture, and Phenotype Modulation of Oval Cells from Carcinogen-Fed Rats

Oval cells are liver epithelial cells that proliferate during hepatocarcinogenesis and chemically induced severe liver injury. It has been suggested that these cells represent hepatic stem cells which might play an important role in the histogenesis of cholangiocellular as well as hepatocellular carcinomas. In order to test this hypothesis highly purified oval cell preparations and propagable oval cell lines are needed. In the present study the isolation, biochemical characterization, and longterm culture of oval cells from rats fed a choline-deficient/DL-ethionine-supplemented diet for 6, 14, or 22 weeks are described. The freshly isolated oval cells were γ-glutamyltranspeptidase-positive, cytokeratin 7-, 8-, 18-, and 19-positive, albumin-positive, peroxidase-negative, and α-fetoprotein-negative and expressed lactate dehydrogenase isoenzymes 1-5. In addition, low but clearly measurable glucose-6-phosphatase and high γ-glutamyltranspeptidase and alkaline phosphatase activities (when compared to activities in untreated liver parenchymal cells) were measured in oval cells. Three oval cell lines, OC/CDE 6, OC/CDE 14, and OC/CDE 22, were established. They contained small and large epithelial cells replicating to form uniform monolayers with a cobblestone appearance; furthermore, a very low number of mononucleated giant cells were also present in the three cell lines. OC/CDE 6, OC/CDE 14, and OC/CDE 22 cells were γ-glutamyltranspeptidase-negative, were transiently albumin-positive, maintained the glucose-6-phosphatase activity levels measured in freshly isolated oval cells, and expressed lactate dehydrogenase isoenzymes 2-5. After exposure of the cultured oval cells to dimethyl sulfoxide or sodium butyrate, 35-40% of the cells reexpressed albumin, and glucose-6-phosphatase activity was enhanced; in addition, sodium butyrate strongly increased γ-glutamyltranspeptidase and alkaline phosphatase activities. In conclusion, oval cells express phenotypic markers of liver parenchymal as well as bile duct epithelial cells and possess a certain intrinsic plasticity. In order to test if the oval cells indeed represent an intermediate step in the differentiation of certain cells within the bile duct and ductular epithelial cell compartment to parenchymal cells, the three cell lines described herein will be transformed in vitro and their potential to give rise to cholangiocellular and/or hepatocellular carcinomas will be verified in vivo.     

Permanent Stained Agar Preparation of Mycoplasma and of L Forms of Bacteria

A procedure is described that permits the preparation of permanent stained mounts of Mycoplasma and of bacterial L forms grown on the surface of and within agar media. These preparations are especially useful for making representative photographs. The cultures are fixed with Formalin vapor. Thin slices of agar are stained at elevated temperature between 50 and 60 C and at a low pH, are dried rapidly, and are mounted in Canada balsam. The results with this staining procedure are illustrated by photographs of various strains of Mycoplasma and of bacterial L forms.     

Crystalloid lysozyme inclusions in Paneth cells of vitamin A-deficient rats

Summary The effect of vitamin A-deficiency on jejunal Paneth cells in rats was investigated. Crystalloid particles were observed in secretion granules of Paneth cells from 6 out of 8 rats with vitamin A-deficiency. The particles were similar to those found in Paneth cells under other experimental conditions. Using an immuno-electron-microscopic technique we demonstrated a clear lysozyme immunoreactivity of these particles. In 2 vitamin A-deficient rats tubular structures have been detected in addition to the crystalloid particles. Crystalloid particles or tubular structures were not detectable in a control group of 8 vitamin A-supplemented rats. The morphological alterations of Paneth cells may be correlated to an impaired local immunity of the intestine during vitamin A-deficiency.This work represents a portion of a doctoral thesis presented by M.J. Koch in partial fullfillment for the degree of medical doctor     

Lymphocytes from hepatic inflammatory infiltrate kill rat hepatocytes in primary culture

In the last few years it has become possible in the liver to isolate lymphocytes from inflammatory infiltrates and to culture them in vitro. Most of the lymphocyte clones obtained are CD 8 + cytotoxic cells, but interactions between these lymphocytes and hepatocytes in primary culture have not been analysed previously. In this study, cloned human T lymphocytes from liver biopsies and from the peripheral blood of patients with chronic hepatitis B or primary biliary cirrhosis, after phenotypical and functional characterization into CD 8+ or CD 4+ cytotoxic lymphocytes, were activated in an antigen-independent fashion by adding either anti CD 3 or anti CD 2/R-3 monoclonal antibodies to the cell suspension. The activated cells were then coincubated with rat hepatocytes in primary culture. The killing capacity of the activated lymphocytes was monitored by light and electron microscopy and by measurement of lactic dehydrogenase (LDH)-release into the culture medium. It was found that cytotoxic CD 8 +, but not CD 4 + helper lymphocytes very effectively killed hepatocytes. The killing effect was dependent on the time of cocultivation and on effector-target (E/T) ratio. Total breakdown of the hepatocyte monolayer was achieved after 10–20 h coculture and at an E/T ratio of 10 to 1. As LDH-release in the culture medium reached about 80% of the total LDH-content, most of the hepatocytes were lysed by activated lymphocytes. Cytotoxic activity of clones obtained from different biopsies was comparable with that of clones from peripheral blood. Hepatocytes in primary culture seem to be very sensitive to the killing capacity of activated cytotoxic lymphocytes. Supported by DFG grants Ra 362/5-2 and SFB 311 A7 (G.R.) and A5 (H.P.D.)     

Lewis(y) antigen (CD174) and apoptosis in gastric and colorectal carcinomas: correlations with clinical and prognostic parameters

Lewis(y) (Le(y)), also designated CD174, represents a carbohydrate blood group antigen which is strongly expressed in neoplastic gastrointestinal tissues. Previous reports indicated an association between Le(y) expression and apoptosis. Therefore, we tried to elucidate its clinicopathological relevance in a series of 160 gastric and 215 colorectal carcinomas by immunohistochemical detection of Le(y) and visualization of apoptotic cells applying the in-situ-end labelling (ISEL) method, followed by semiquantitative scoring of the specimens. In both gastric as well as colorectal carcinomas, between 40 and 50% of the cases were Le(y) reactive. Signet-ring cell carcinomas of the stomach exhibited a significantly stronger Le(y) expression compared to other tumor types. In colorectal cancers, Le(y) was associated with increased tumor staging, showing the strongest positivity in stage IV. Further correlations with clinicopathological variables or prognosis were not observed. On the other hand, the amount of apoptotic cells was significantly reduced in mucinous adenocarcinomas of the colorectum compared to non-mucinous carcinomas. Scoring of apoptotic cells did not result in any other clinicopathologically relevant correlations. In addition, a significant association between Le(y) antigen expression and apoptosis score could not be established. Therefore, the hypothesis of a functional relationship between these two aspects of gastrointestinal tumor biology is not confirmed by our data.