Christopher C. Widnell

Superinfect on exclusion by vesicular stomatitis virus

Abstract The infection of baby hamster kidney (BHK 21 ) cells by the Indiana strain of vesicular stomatitis virus (VSV) causes a rapid loss of the ability of the cells to be superinfected by VSV visions or defective-interfering particles. This exclusion phenomenon is at the level of virus penetration and requires viral gene expression and a functional VSV transmembrane glycoprotein G. Infection with the New Jersey serotype of VSV also inhibits the uptake of the Indiana serotype. However, infection of BHK 21 cells with either encepbalomyocarditis, Newcastle disease, or influenza viruses does not inhibit superinfection by VSV.

Cellular mechanisms in the superinfection exclusion of vesicular stomatitis virus

The superinfection exclusion of VSV has been studied and found to be caused by a combination of three distinct effects on endocytosis by VSV-infected cells: first, a decreased rate of formation of endocytic vesicles as judged by an inhibition of fluid-phase uptake at 2 hr postinfection; second, a decreased rate of internalization of receptor-bound ligands, which was detected at 4 hr postinfection; and third, a competition with newly synthesized virus for occupancy of coated pits, as indicated by electron microscopy of infected cells. At the same time that fluid-phase uptake decreased, numerous uncoated invaginations were observed at the cell surface.

The demonstration of a specific 5′-nucleotidase activity in rat tissues☆

Abstract 5′-Nucleotidase has been partially purified from rat liver, spleen, kidney, heart, lung, brain and skeletal muscle. The majority of the enzyme activity in each of these tissues was insoluble in 1% of Triton X-100, solubilized in 2% Triton X-100,1% sodium deoxycholate, and stable to incubation at 50 °C for 5 min. The partially purified enzyme from each tissue exhibited the same pH optimum, was inhibited by concanavalin A, and was inhibited in an identical manner by antibody to highly purified 5′-nucleotidase from liver. Since the enzyme is usually concentrated in the plasma membrane (De Pierre, J. W. and Karnovsky, M. L. (1973) J. Cell Biol. , 56 , 275–303), the results indicate that the enzyme may represent a convenient and general marker for this organelle in rat tissues.

Sequential disassembly of the cytoskeleton in BHK21 cells infected with vesicular stomatitis virus.

The cytopathic effects of vesicular stomatitis virus (VSV) that result in the rounding of BHK21 cells have been studied. The results indicate that they are mediated by a sequential alteration in the distribution of the components of the cytoskeleton, an effect that requires the expression of the viral L protein. The constituents of the cytoskeleton of BHK21 cells were analyzed by fluorescence microscopy. Actin filaments were the first component to become disorganized, so that disassembly of stress fibers were detected 1 hr after infection. The distribution of microtubules and intermediate filaments was unchanged at 2 hr after infection; however, both these cytoskeletal elements exhibited an altered distribution at 3-4 hr after infection. Actinomycin D and cycloheximide did not cause the same effects as infection with VSV, suggesting that inhibition of host-cell gene expression was not responsible. However, viral gene expression was required, since cells infected with uv-irradiated VSV showed the same distribution of cytoskeletal constituents as mock-infected controls. Cells infected at 39.5 degrees (the nonpermissive temperature) with mutants of VSV temperature sensitive in the viral NS (ts G22), N(ts G41), M(ts 0 23), and G(ts 0 45) proteins showed the same changes in the cytoskeleton as those detected with wild-type virus. In contrast, cells infected with ts G11 (L-) showed the characteristic effect of VSV on the cytoskeleton when incubated at 34 degrees (the permissive temperature), but not when incubated at 39.5 degrees. The T-1026 R1 mutant of VSV, which has a much less dramatic effect on cell morphology than wild-type virus, also caused a less marked disruption of the cytoskeleton.

Reversal of age-related changes in microsomal enzyme activities following the administration of triamcinolone, triiodothyronine and phenobarbital

Abstract 1. 1. Microsomes were isolated from the livers of 3- and 24-month-old Fischer rats, and specific activities were determined for glucose-6-phosphatase, NADPH cytochrome c reductase, NADPH diaphorase, NADH cytochrome c reductase, NADH diaphorase and NADH neotetrazolium reductase. 2. 2. In agreement with other reports the specific activities of glucose-6-phosphatase and NADPH cytochrome c reductase decreased in the old animals, and NADH cytochrome c reductase increased. 3. 3. Chronic administration of low doses of triamcinolone almost doubled the specific activity of glucose-6-phosphatase in old animals, but caused essentially no change in young animals. In contrast, treatment with phenobarbital halved this activity in young animals, but was essentially without effect in old animals. Treatment with triiodothyronine and caused a marked stimulation of this activity in both young and old animals. 4. 4. There was very little change in the specific activity of NADPH cytochrome c reductase or NADPH diaphorase following these treatments. 5. 5. NADH cytochrome c reductase, NADH neotetrazolium reductase and NADH diaphorase activities were greatly decreased in both young and old animals by administration of all three compounds. 6. 6. Age-related differences in the activity of these enzymes were either diminished or reversed following each treatment. Old animals were as responsive to these stimuli as young animals, and the highest and lowest specific activities observed for each enzyme during the course of all experiments were very similar for young and old animals of the same sex. These observations suggest that age-related differences in these enzyme activities are probably caused by a different metabolic or endocrine state in young and old animals or their hepatocytes, and not by a deterioration of the integrity or control of the synthesis of these proteins.

Cellular Stress Causes Accumulation of the Glucose Transporter at the Surface of Cells Independently of their Insulin Sensitivity

Abstract. The stimulation of glucose transport in response to various types of stress has been studied. There is no relationship between effects of stress-inducing agents on glucose transport and their effects on cellular protein synthesis. Although the effect of stress on glucose transport appears analogous to its stimulation by insulin, cells that are slightly insulin-sensitive in terms of glucose transport (BHK cells) show a similar degree of stimulation as highly insulin-sensitive cells (differentiated 3T3-L1 cells). External labeling of the transporter protein with a photoactivatable derivative of mannose, 2-N-4-(1-azi-2,2,2-trifluoroethyl) benzoyl-1, 3-bis-(D-mannos-4-yloxy)-propylamine, shows that most of the increased glucose transport activity correlates with an increase in the amount of the transporter on the cell surface. Cells subjected to K+-depletion, which inhibits endocytosis and results in an accumulation of receptors at the cell surface, show the same increase in glucose transport as cells exposed to stress; stressed cells show no further increase in glucose transport when subjected to K+ depletion. These results support the view (Widnell, C.C., Baldwin, S.A., Davies, A., Martin, S., Pasternak, C.A. 1990. FASEB J4:1634–1637) that cellular stress increases glucose transport by promoting the accumulation of glucose transporter molecules at the cell surface.

Stress-induced increase of hexose transport as a novel index of cytopathic effects in virus-infected cells: Role of the L protein in the action of vesicular stomatitis virus

The VSV-specific increase in hexose transport by BHK cells has been measured by assay of the [3H]dGlc/[14C]AIB uptake ratio. The effect was abolished by uv-irradiation of the virus, indicating that viral gene expression is required. Cells infected with the T1026 R1 mutant of VSV, which causes only slight cytopathic changes, exhibited only a slight increase in hexose uptake. Cells infected with temperature-sensitive (ts) mutants of VSV that are defective in the function of the viral N, NS, G, or M proteins at the restrictive temperature (39.5 degrees) exhibited increased [3H]dGLC/[14C]AIB uptake ratios typical of wild-type virus at either restrictive (39.5 degrees) or permissive temperature (34 degrees). Cells infected with a mutant defective in the function of the viral L protein exhibited an increased [3H]dGlc/[14C]AIB uptake ratio at permissive temperature (34 degrees) only; at restrictive temperature (39.5 degrees) the uptake ratio was essentially the same as that of mock-infected cells. Temperature-shift experiments indicated that the effect on hexose transport persisted for at least 6 hr in cells which no longer expressed function L protein, and that when expression of L was restricted to the first 2 hr of infection, an almost complete stimulation of hexose transport was observed 4 hr later. These results indicate that expression of the L gene is a necessary factor for inducing an increased hexose uptake in VSV-infected BHK cells. They also suggest that the action of the L protein on hexose transport is indirect, and is presumably mediated by other cellular constituents. The studies support the concept that an increased dGlc uptake may be a useful index of the cytopathic consequences of virus infection.

Effects of hormones on the activity of glucose-6-phosphatase in primary cultures of rat hepatocytes☆

Although the activity of glucose-6-phosphatase in rat liver is altered markedly following the administration of a variety of hormones in vivo, it is not certain whether the hormones act directly on the hepatocyte. To study this problem hepatocytes were isolated by a collagenase-perfusion technique and cultured on collagen gel/nylon mesh membranes. The activity of glucose 6-phosphatase in cells cultured with fetal calf serum and with Dulbeccos modified Eagles medium or Leibovitz L-15 medium decreased to less than 10-30% of the activity in freshly isolated cells by 96 h. However, when L-15 plus newborn or fetal calf serum was supplemented with glucagon (10(-6)M), epinephrine (10(-6)M), triiodothyronine (10(-6)M), and dexamethasone (10(-5)M) (L-15-GETD), the activity of glucose-6-phosphatase was maintained so that, after 144 h, the activity was at least 80% of that detected in freshly isolated cells. In cells cultured in L-15 plus serum for 72 or 96 h and then in L-15-GETD, glucose-6-phosphatase increased 30-50% over that in control cultures after 24 h. Insulin, which decreases glucose-6-phosphatase activity when administered to intact animals, also decreased the glucose-6-phosphatase activity in cultured hepatocytes to 20-50% of that in controls.

Control of glucose transport by GLUT1: Regulated secretion in an unexpected environment

Studies designed to elucidate the mechanism of regulation of the GLUT1 isoform of the glucose transporter in response to a variety of cellular stresses are reviewed. Using ts mutants of vesicular stomatitis virus, it was shown that the viral L gene was responsible for the stimulation of glucose transport in infected cells. Immunofluorescence of GLUT1 demonstrated that the increase in glucose transport was the consequence of a translocation of the transporter from a reservoir in cytoplasmic vesicles to the plasma membrane. When cells were cycled between deficient and standard medium, the change in glucose transport rates was paralleled by a cycling of the transporter between the plasma membrane and the cytoplasmic vesicles. The redistribution of GLUT1 was not a consequence of a general redistribution of recycling plasma membrane proteins. Instead, the findings focus attention on the regulated exocytosis of specific membrane constituents in cells that, until recently, were not thought to exhibit this capacity.

Cell biology and its relationship to reproductive endocrinology

I feel very honored by this invitation to deliver the Joseph Price Oration and approach my task with some trepidation, since I believe that this is the most distinguished group I have addressed on this topic. Although my field is cell biology, I come from what was once an anatomy department. Thirty years ago most medical schools considered their anatomy departments to be slightly dull places, performing a necessary service function in the first year of the curriculum. However, the last decade or so has led to dramatic changes, not only in name-with cell biology included frequently in the place of honor-but also in the number of people working enthusiastically at night and on weekends, as well as in the department’s contribution to the research budget of the medical school. Suddenly these departments (or perhaps more accurately, some of them) have become lively places, where young investigators enjoy themselves in an atmosphere of excitement. What is cell biology, and perhaps more importantly, what can it or should it be doing for our academic colleagues in the clinical departments? To try to answer this, I would like you to accompany me in a brief journey first through history and then through the cell.