Effect of Diet on the Activity of Several Enzymes in Extracts of Rumen Microorganisms

The use of enzymatic techniques to characterize rumen metabolism was investigated. Assays were developed to estimate the activities of 14 enzymes in cell-free extracts of microorganisms collected from rumen contents of cows fed two diets, selected to produce widely different proportions of fermentation end products. The results reflected the differences between the two diets in metabolic potential, fermentation patterns, and microbial populations. The differences between the diets in the relative activities of succinic dehydrogenase and fumaric reductase, for example, indicated a shift in the microbial population favoring organisms of the Viellonella alcalescens type on the concentrate diet. The data presented indicate that, if employed carefully, enzymatic criteria can be utilized effectively in studies of rumen metabolism.     

Mechanical, morphological and biochemical adaptations of bone and muscle to hindlimb suspension and exercise

The influences of weightbearing forces on the structural remodeling, matrix biochemistry, and mechanical characteristics of the rat tibia and femur and surrounding musculature were examined by means of a hindlimb suspension protocol and highly intensive treadmill running. Female, young adult, Sprague-Dawley rats were designated as either normal control, sedentary suspended, or exercise suspended rats. For 4 weeks, sedentary suspended rats were deprived of hindlimb-to-ground contact forces, while the exercise suspended rats experienced hindlimb ground reaction forces only during daily intensive treadmill training sessions. The suspension produced generalized atrophy of hindlimb skeletal muscles, with greater atrophy occurring in predominantly slow-twitch extensors and adductors, as compared with the mixed fiber-type extensors and flexors. Region-specific cortical thinning and endosteal resorption in tibial and femoral diaphyses occurred in conjunction with decrements in bone mechanical properties. Tibial and femoral regional remodeling was related to both the absence of cyclic bending strains due to normal weightbearing forces and the decrease in forces applied to bone by antigravity muscles. To a moderate extent, the superimposed strenuous running counteracted muscular atrophy during the suspension, particularly in the predominantly slow-twitch extensor and adductor muscles. The exercise did not, however, mitigate changes in bone mechanical properties and cross-sectional morphologies, and in some cases exacerbated the changes. Suspension with or without exercise did not alter the normal concentrations of collagen, phosphorus, and calcium in either tibia or femur.     

Fourier transform infrared spectroscopic study of the structure and conformational changes of the human erythrocyte glucose transporter

Fourier transform infrared spectroscopy has been used to study the secondary structure of the human erythrocyte glucose transporter after purification and reconstitution in erythrocyte lipids. The spectra indicate that the glucose transporter contains, in addition to the predominant alpha-helical structure, an appreciable amount of beta-structure and random coil conformation. A study of the time dependency of H-2H exchange revealed that more than 80% of the polypeptide backbone is readily accessible to the solvent. This result indicates that a portion of the intramembrane-spanning region of the membrane protein is exposed to the solvent, suggesting the existence of an intraprotein aqueous channel. The residual (10-20%) portion of the protein which exchanges slowly includes some alpha-helical structure, probably situated in a hydrophobic environment inside the membrane. The infrared spectra of transporter preparations were also examined after incubation with substrate and substrate analogues. Compared with the spectra recorded under conditions in which the "inward-facing" form predominates, a small but reproducible shift in the bands assigned to alpha-helical and beta-strand structures is observed after incubation with 4,6-O-ethylidene-D-glucose, which largely fixes the transporter in the "outward-facing" conformation. An increase of temperature, which is known to increase the proportion of transporter in the outward-facing conformation, results in a similar shift in this alpha-helical absorption band.     

Molybdenum hydroxylases in Drosophila. III. Further characterization of the low xanthine dehydrogenase gene

The biochemical effects of several newly induced low xanthine dehydrogenase (lxd) mutations in Drosophila melanogaster were investigated. When homozygous, all lxd alleles simultaneously interrupt each of the molybdoenzyme activities to approximately the same levels: xanthine dehydrogenase, 25%; aldehyde oxidase, 12%; pyridoxal oxidase, 0%; and sulfite oxidase, 2% as compared to the wild type. In order to evaluate potentially small complementation or dosage effects, mutant stains were made coisogenic for 3R. These enzymes require a molybdenum cofactor, and lxd cofactor levels are also reduced to less than 10% of the wild type. These low levels of molybdoenzyme activities and cofactor activity are maintained throughout development from late larval to adult stages. The lxd alleles exhibit a dosage-dependent effect on molybdoenzyme activities, indicating that these mutants are leaky for wild-type function. In addition, cofactor activity is dependent upon the number of lxd ⁺ genes present. The lxd mutation results in the production of more thermolabile XDH and AO enzyme activities, but this thermolability is not transferred with the cofactor to a reconstituted Neurospora molybdoenzyme. The lxd gene is localized to salivary region 68 A4-9, 0.1 map unit distal to the superoxide dismutase (Sod) gene.     

Endocytosis of a monoclonal antibody recognising a cell surface glycoprotein antigen visualised using fluorescent conjugates

The endocytosis of a monoclonal antibody recognising a cell surface glycoprotein antigen has been investigated using several different fluorescent conjugates. These conjugates have been employed for both fluorescence microscopy to show the qualitative changes in distribution of antibody conjugates during endocytosis, and also flow cytofluorimetry to show the quantitative changes in fluorescence intensity associated with this redistribution. Using an antibody directly labelled with fluorescein it was difficult to demonstrate endocytosis due to the inability to distinguish clearly between internal and external fluorescence. However, a fluorescein-HSA-antibody conjugate which was heavily quenched at the cell surface was endocytosed and degraded during incubation at 37 degrees C for 4 h and was then visualised in a perinuclear distribution by the addition of agents to modify intracellular pH. This experiment demonstrated that such conjugates became localised within an acidic internal compartment. A tetramethylrhodamine-HSA-conjugate also demonstrated a similar perinuclear distribution but without the addition of endosomal pH modifiers. When used in conjunction with a fluorescein rabbit anti-HSA second label this conjugate also showed that not all conjugate was endocytosed during a 4-h period; some conjugate remained bound to the cell surface. These experiments suggested that endocytosis in this system differs from receptor-mediated endocytosis via coated pits which is reported to be more rapid and complete.     

Capacity of tumor necrosis factor to bind and penetrate membranes is pH-dependent

Studies with human U937 cells as targets established that a 15-min exposure to rTNF at pH 5.3 caused a significant increase in TNF-mediated cytolysis when compared to cells exposed to TNF at pH 7.4. A detailed examination of TNF-membrane interactions revealed that although TNF bound avidly to model membrane targets, no damage was generated under any condition tested. Binding of TNF, monitored with 125I-labeled as well as unlabeled protein, was enhanced at low pH. In the pH range tested (i.e., 4 to 8), target membrane permeability actually decreased in the presence of TNF. This membrane stabilization may be a consequence of TNF insertion into the target bilayer, a process we detected through use of an intramembranous photolabeling assay; interestingly, the efficiency of TNF insertion into membranes increased dramatically with decreasing pH. We conclude that native TNF does not cause pore formation directly and that its ability to induce cell lysis, as monitored by 51Cr release, is a consequence of some as yet obscure signaling event or intracellular activity. Parallel studies were carried out with diphtheria toxin, a protein with a more thoroughly characterized pH-dependent intoxification pathway. This toxin displayed acid-enhanced activities with both biologic and artificial targets.     

HIV inhibits the early steps of lymphocyte activation, including initiation of inositol phospholipid metabolism

Mechanisms accounting for HIV-associated suppression of lymphocyte proliferation were investigated. In previous work we demonstrated that purified and inactivated HIV-suppressed lymphoid cell proliferation. In this report we used an inactivated preparation of HIV obtained from infected CEM cells grown in serum free media and demonstrated that this HIV-associated suppression acted in the early steps of activation to inhibit the incorporation of radiolabeled phosphorus into phosphatidylinositol 4,5-bisphosphate and phosphatidic acid. Initially we showed that both purified CD4 and CD8 T lymphocyte subsets were affected and HIV-associated inhibition did not require the CD4 molecule. Impaired lymphocyte blastogenesis (decreased size and granularity and decreased expression of receptors to IL-2 and transferrin) in response to PHA indicated an effect of inactivated HIV on the early steps of activation. This was confirmed by time studies where 1) a 2 min HIV-pretreatment followed by washing before stimulation was sufficient to inhibit PHA induced proliferation of normal lymphocytes, and 2) addition of HIV to PHA prestimulated lymphocytes failed to inhibit proliferation, e.g., there was no effect on preactivated lymphocytes. HIV was mainly inhibitory of lymphocyte proliferation induced by PHA or mAb to the CD3 receptor. In contrast to the effect on the CD3/TiR, responses via the CD2 receptor were not suppressed, e.g., stimulation with the monoclonal antibodies T11(2) + T11(3). Inasmuch as responses by direct A23187 + PMA stimulation of intracellular pathways were also inhibited, it appears that the HIV-induced defect was not (or not only) membrane receptor mediated. The earliest (min) measurable event after stimulation was the initial increase in intracellular Ca2+ which was unaffected by HIV pretreatment. The next measurable event (min to h) of stimulation is a sustained increase in inositol phospholipid turnover. Pretreatment of mononuclear cells with inactivated HIV resulted in a decreased inositol phospholipid turnover as judged from decreased 32P incorporation into phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. This led to decreased generation of DAG as reflected in the reduced radiolabeling of its metabolite PA. Reduced availability of DAG presumably interferes with pkC activation and leads to decreased expression of receptors for IL-2 and transferrin and impaired proliferation.     

Photoaffinity labelling of isopenicillin N synthetase by laser-flash photolysis.

Isopenicillin N synthetase (IPNS) from Acremonium chrysogenum was photolabelled by laser-flash photolysis in the presence of a diazirinyl-containing substrate, 2-[3-(3-trifluoromethyl-3H-diazirin-3-yl)-phenoxy]acetyl-S- methyloxycarbonylsulphenyl-L-cysteinyl-D-valine (DCV). Labelling of IPNS by DCV is partially inhibited in the presence of an excess of L-alpha-aminoadipoyl-L-cysteinyl-D-valine (ACV), the natural substrate. In the absence of light, DCV is converted into the corresponding penicillin with comparable Km but significantly depressed Vmax relative to ACV. Selective incorporation of [14C]DCV into IPNS has been demonstrated by fluorography of IPNS analysed by SDS/polyacrylamide-gel electrophoresis. Scintillation counting of labelled IPNS purified on an ion-exchange f.p.l.c. column confirms this result. This methodology may be applicable for studies aimed at investigating the binding of substrates to IPNS.     

Cellular stress induces a redistribution of the glucose transporter

The mechanism by which cells increase their rate of glucose uptake in response to stress is unclear. Using an immunofluorescence technique to localize the glucose transporter protein in BHK cells, we found that hyperthermia, treatment with arsenite, infection with vesicular stomatitis virus or Semliki Forest virus, and treatment with insulin cause the transporter to move from an intracellular site in the perinuclear region to the plasma membrane; the degree of translocation correlates approximately with the increase in glucose uptake. We conclude that stress induces an insulin-like distribution of certain membrane proteins.