Role of gastrin/pentagastrin in regulation of intestinal cytochrome P-450

1. In the absence of intraluminal inducers, low "basal" levels of cytochrome P-450 and its dependent MFO activities are detected in the rat intestinal mucosa, and may be regulated by endogenous hormones. 2. Rats were nutritionally maintained by either short term (48 hr) intravenous glucose infusion or chronic (8 days) intravenous hyperalimentation, and were treated with various doses of pentagastrin in the infusate. 3. Regardless of the dose (6-90 micrograms/kg/hr) or duration of infusion (2-8 days), pentagastrin had no effect on small intestinal cytochrome P-450, its dependent MFO activity, or the activity of delta-aminolevulinic acid synthetase. 4. The intestinal trophic peptide hormone, gastrin, apparently does not regulate the cytochrome P-450-dependent MFO system of the small intestine.     

血管内皮舒张因子在氧自由基所致慢性缺氧大鼠肺内动脉收缩中的作用

以黄嘌岭(X)-黄嘌呤氧化酶(XO)系统产生氧自由基,应用微量生物测定法观察慢性缺氧(5000m,10d)对大鼠氧自由基所致肺内动脉收缩的影响及内皮舒张因子(EDRF)在其中的作用。慢性缺氧大鼠有内皮的肺内动脉环对氧自由基的收缩反应较正常环境中的对照动物明显增强,加入EDRF灭活剂还原型血红蛋白(RHb)后更加显著;而加入超氧化物歧化酶(铜锌SOD)后则减弱,甚至消除。反之,不论加入RHb或SOD对氧自由基所致去内皮肺内动脉环的收缩反应均无明显影响。上述结果表明慢性缺氧引起肺内动脉收缩增强与EDRF有密切关系:慢性缺氧可能使EDRF的作用减弱,肺内动脉对氧自由基的反应性增强。表示EDRF及其与氧自由基的关系在慢性缺氧性肺动脉高压的形成中可能具有十分重要的意义。     

The use of a simple haematoxylin and eosin staining procedure to demonstrate micronuclei within rodent bone marrow

In the pharmaceutical industry, the majority of drug-safety evaluation studies are carried out preferentially in the rat. Consequently, drug absorption, distribution, metabolism and excretion profiles are available for this species. Such data usually have to be generated independently in the mouse, to allow validation of any micronucleus tests carried out in this species. Unfortunately, at the present time, the rat is not ideal for use in the micronucleus test due to the presence of large numbers of contaminating mast cell granules. Such granules are stained blue by the most commonly accepted staining procedure (May-Grunwald-Giemsa), and can be erroneously scored as micronuclei when they overlay erythrocytes. A simple haematoxylin and eosin staining procedure was evaluated in the micronucleus test using rats and mice. With this procedure, micronuclei stained blue-black and were readily distinguishable from cell inclusions resembling micronuclei such as mast cell granules, which remained unstained. Essentially similar quantitative data for micronucleus incidence and erythrocyte distribution were obtained in mice using this staining technique when compared to the use of the more established May-Grunwald-Giemsa staining procedure. However, unlike the use of the May-Grunwald-Giemsa procedure, the use of the haematoxylin and eosin stains allowed the accurate estimation of micronucleus incidence within the marrows of treated rats in the presence of contaminating mast-cell granules. Furthermore, unlike alternative procedures using fluorescent stains, the haematoxylin and eosin stained preparations are stable, constitute a permanent record of the experiment, and can be analysed at the convenience of the investigator. Therefore, this staining procedure may offer a useful alternative, for example, when evaluating rat bone-marrow smears within which considerable mast cell contamination can occur.     

Rat liver alcohol dehydrogenase. Purification and properties

Alcohol dehydrogenase (EC 1.1.1.1) from the rat liver supernatant fraction has been purified 200-fold and partially characterized. The isolation procedure involved ammonium sulphate fractionation, DEAE-Sephadex chromatography and gel filtration. The purified enzyme behaved as a homogeneous preparation as evaluated by cellulose acetate and polyacrylamide-gel disc electrophoresis. Sulphoethyl-Sephadex chromatography and immunoelectrophoresis with rabbit antiserum indicated the presence of a minor component. Rat liver alcohol dehydrogenase appears to contain 4mol of zinc/mol, has an estimated molecular weight of 65000 and consists of two subunits of similar molecular weight. Heavy-metal ions, thiol-blocking reagents, urea at concentrations below 8m, low pH (5.5) and chelating agents deactivate the enzyme but do not dissociate it into subunits. Deactivated enzyme could not be reactivated. The enzyme is strictly specific for NAD(+) and has a broad specificity for alcohols, which are bound at a hydrophobic site. Inhibition occurred with the enzyme equilibrated with Zn(2+) at concentrations above 0.1mm.     

Muscle glycogenolysis during differing intensities of weight-resistance exercise

Skeletal muscle glycogen metabolism was investigated in eight male subjects during and after six sets of 70% one repetition maximum (1 RM, I-70) and 35% 1 RM (I-35) intensity weight-resistance leg extension exercise. Total force application to the machine lever arm was determined via a strain gauge and computer interfaced system and was equated between trials. Compared with the I-70 trial, the I-35 trial was characterized by almost double the repetitions (13 +/- 1 vs. 6 +/- 0) and half the peak concentric torque for each repetition (12.4 +/- 0.5 vs. 24.2 +/- 1.0 Nm). After the sixth set, muscle glycogen degradation was similar between I-70 and I-35 trials (47.0 +/- 6.6 and 46.6 +/- 6.0 mmol/kg wet wt, respectively), as was muscle lactate accumulation (13.8 +/- 0.7 and 16.7 +/- 4.2 mmol/kg wet wt, respectively). After 2 h of passive recovery without caloric intake, muscle glycogen increased by 22.2 +/- 6.8 and 14.2 +/- 2.5 mmol/kg wet wt in the I-70 and I-35 trials, respectively. Optical absorbance measurement of periodic acid-Schiff-stained muscle sections after the 2 h of recovery revealed larger absorbance increases in fast-twitch than in slow-twitch fibers (0.119 +/- 0.024 and 0.055 +/- 0.024, P = 0.02). Data indicated that when external work was constant, the absolute amount of muscle glycogenolysis was the same regardless of the intensity of resistance exercise. Nevertheless the rate of glycogenolysis during the I-70 trial was approximately double that of the I-35 trial.     

A novel immunohistochemical technique for demonstration of specific binding of human monoclonal antibodies to human cryostat tissue sections

We describe a novel immunohistochemical technique which permits the detection of specific binding of human monoclonal antibodies (MAb) to cryostat sections of human tissues. The technique overcomes the problem of background staining caused by the presence of endogenous immunoglobulins in tissue sections. This is achieved by the formation of a molecular complex of the primary antibody (a human MAb), horseradish peroxidase-conjugated goat anti-human immunoglobulin, and normal human serum. This complex is then incubated with cryostat sections of human tissue, and binding of the complex is demonstrated using diaminobenzidine/hydrogen peroxide. The method is suitable for immunohistochemical screening of small samples of tissue culture supernatant for the presence of human MAb of potential interest, and for determining the pattern of binding of such MAb to a wide range of normal and pathological human tissues.     

Hepatic sarcocystosis in a horse

Hepatic sarcocystosis was diagnosed in a horse in association with refractory bacterial osteomyelitis and plasma cell tumor of the maxilla and hepatic salmonellosis. Gross lesions included pleural, pericardial, and peritoneal effusions, hepatomegaly, gastric ulceration, colonic edema, and proliferative tissues filling 2 maxillary dental alveoli. Histologically, liver was characterized by severe suppurative, necrotizing, periportal hepatitis, and severe periacinar necrosis. Hepatocytes frequently contained protozoal schizonts in various stages of development. In mature schizonts, merozoites were often arranged radially around a central residual body, consistent with asexual division by endopolygeny. Ultrastructural features of merozoites included an apical conoid and polar ring, anterior micronemes, central nuclei, and absence of rhoptries. These protozoa did not react to antisera raised against Neospora caninum, Sarcocystis neurona, Toxoplasma gondii, or Hammondia hammondi. The microscopic and ultrastructural characteristics and immunoreactivity of this organism are consistent with a Sarcocystis sp. other than S. neurona. This is the first report of Sarcocystis-associated hepatitis in a horse. The life cycle of this organism and source of infection are unknown.     

Carboxy terminus of glucose transporter 3 contains an apical membrane targeting domain

We previously demonstrated that distinct facilitative glucose transporter isoforms display differential sorting in polarized epithelial cells. In Madin-Darby canine kidney (MDCK) cells, glucose transporter 1 and 2 (GLUT1 and GLUT2) are localized to the basolateral cell surface whereas GLUTs 3 and 5 are targeted to the apical membrane. To explore the molecular mechanisms underlying this asymmetric distribution, we analyzed the targeting of chimeric glucose transporter proteins in MDCK cells. Replacement of the carboxy-terminal cytosolic tail of GLUT1, GLUT2, or GLUT4 with that from GLUT3 resulted in apical targeting. Conversely, a GLUT3 chimera containing the cytosolic carboxy terminus of GLUT2 was sorted to the basolateral membrane. These findings are not attributable to the presence of a basolateral signal in the tails of GLUTs 1, 2, and 4 because the basolateral targeting of GLUT1 was retained in a GLUT1 chimera containing the carboxy terminus of GLUT5. In addition, we were unable to demonstrate the presence of an autonomous basolateral sorting signal in the GLUT1 tail using the low-density lipoprotein receptor as a reporter. By examining the targeting of a series of more defined GLUT1/3 chimeras, we found evidence of an apical targeting signal involving residues 473-484 (DRSGKDGVMEMN) in the carboxy tail. We conclude that the targeting of GLUT3 to the apical cell surface in MDCK cells is regulated by a unique cytosolic sorting motif.     

Optimized conjugation of a fluorescent label to proteins via intein-mediated activation and ligation

Intein-mediated ligation provides a site-specific method for the attachment of molecular probes to proteins. The method is inherently flexible with regard to either the protein sequence or the attached probe, but practical difficulties have limited the widespread use of this valuable labeling system for the attachment of small- to medium-sized molecules. We report herein studies to improve the efficiency and practical application of these reactions, including the assembly of plasmids for the expression of target-intein fusion proteins and the analysis of their reaction with a fluorescent cysteine derivative under a range of conditions. Optimal ligation of the fluorophore to the target protein is critically dependent on the degree of oxidation of the fluorescent cysteine derivative. Efficient ligation has been achieved with freshly prepared fluorescent cysteine derivative under rigorously anaerobic conditions. Similar ligation yields have also been achieved using more practically convenient conditions including anaerobic reaction with addition of thiophenol, or aerobic reaction with the further addition of tricarboxyethylphosphine.