Isoform Specific Reductions in Na + ,K + -ATPase Catalytic (Α) Subunits in the Nerve of Rats with Streptozotocin-Induced Diabetes

Na + ,K + -ATPase activity in nerve is reduced in rats with streptozotocin-induced diabetes; three different isoforms of the Α (catalytic) subunit of the enzyme are present in nerve. Using western blot to determine subunit isoform polypeptide levels in sciatic nerve, we found a substantial reduction in Α1-isoform polypeptide (88% at 3 weeks, 94% at 8 weeks) after induction of diabetes by streptozotocin. Reductions in Α2 and Α3 polypeptide were smaller and not statistically significant. The reduction in amount of all three isoform polypeptides in the nerve of 3-week diabetic animals was corrected by administration of insulin. Accumulation of Α1 polypeptide at a nerve ligature indicated that rapid transport of that polypeptide in nerve occurs with normal kinetics. The results implicate a specific marked deficit in Α1, much more than Α2 or Α3, catalytic subunit isoform of Na + ,K + -ATPase in the pathogenesis of diabetic neuropathy.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66384/1/j.1471-4159.1994.63051782.x.pd

Stimulation of liver growth and DNA synthesis by glucosylceramide

The nature of the growth‐stimulating effect of glucosylceramide was studied. Mice were injected intraperitoneally with emulsified glucosylceramide and conduritol B epoxide, an inhibitor of cerebroside glucosidase. Within one or two days, the liver grew 18–24%, as reported. Two enzymes involved in DNA synthesis also increased more than the weight. The total liver activity of thymidine kinase increased 46–73%, and the total activity of ornithine decarboxylase increased as much as 101%. It is suggested that elevated liver levels of glucocerebroside stimulate cell proliferation through a relatively direct mechanism.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141613/1/lipd0508.pd

Normalization of liver glucosylceramide levels in the "Gaucher" mouse by phosphatidylserine injection

A model of the human genetic disorder, Gaucher disease, can be rapidly generated in mice by the injection of emulsified glucosylceramide and an inhibitor of the lipid's hydrolase, conduritol B epoxide. The liver grows rapidly as it absorbs the load of lipid but the effect disappears as new glucosidase is formed and the load is hydrolyzed. This normalization process is accelerated by treatment with phosphatidylserine, which is a known stimulator of the enzyme. It is possible that injecting the phospholipid into Gaucher patients would have a therapeutic effect since it might help them utilize their residual glucosidase to destroy stored glycolipid.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27332/1/0000357.pd

Uptake by neuroblastoma cells of glucosylceramide, glucosylceramide glucosidase, its stimulator protein, and phosphatidylserine

Serum-free cultured neuroblastoma cells (clone NIE-115) have been shown to absorb emulsified glucosylceramide, glucosylceramide glucosidase, an activator protein for the enzyme, and phosphatidylserine from a synthetic medium. Uptake of the enzyme was augmented by phosphatidylserine, and vice versa. Uptake of the enzyme-lipid complex was further augmented by the activator protein. It appears likely that the activator forms a complex only with the enzyme-lipid complex, not with the individual components. Two uptake mechanisms for the enzyme seem to be involved, one of which (the complex with activator proteins and acidic lipid) is sensitive to mannosyl phosphate groups. Hydrolysis of absorbed glucosylceramide was slow unless the medium was supplemented with the acidic phospholipid or glucosidase. The most rapid disappearance of stored glycolipid took place when the ternary mixture was added to the cell medium, enzyme + activator protein + phosphatidylserine. These findings may be relevant to enzyme replacement therapy for Gaucher disease.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26097/1/0000173.pd

Early Events Associated with Infection of Epstein-Barr Virus Infection of Primary B-Cells

Epstein Barr virus (EBV) is closely associated with the development of a vast number of human cancers. To develop a system for monitoring early cellular and viral events associated with EBV infection a self-recombining BAC containing 172-kb of the Epstein Barr virus genome BAC-EBV designated as MD1 BAC (Chen et al., 2005, J.Virology) was used to introduce an expression cassette of green fluorescent protein (GFP) by homologous recombination, and the resultant BAC clone, BAC-GFP-EBV was transfected into the HEK 293T epithelial cell line. The resulting recombinant GFP EBV was induced to produce progeny virus by chemical inducer from the stable HEK 293T BAC GFP EBV cell line and the virus was used to immortalize human primary B-cell as monitored by green fluorescence and outgrowth of the primary B cells. The infection, B-cell activation and cell proliferation due to GFP EBV was monitored by the expression of the B-cell surface antigens CD5, CD10, CD19, CD23, CD39, CD40 , CD44 and the intercellular proliferation marker Ki-67 using Flow cytometry. The results show a dramatic increase in Ki-67 which continues to increase by 6–7 days post-infection. Likewise, CD40 signals showed a gradual increase, whereas CD23 signals were increased by 6–12 hours, maximally by 3 days and then decreased. Monitoring the viral gene expression pattern showed an early burst of lytic gene expression. This up-regulation of lytic gene expression prior to latent genes during early infection strongly suggests that EBV infects primary B-cell with an initial burst of lytic gene expression and the resulting progeny virus is competent for infecting new primary B-cells. This process may be critical for establishment of latency prior to cellular transformation. The newly infected primary B-cells can be further analyzed for investigating B cell activation due to EBV infection

Glucosylceramide and the level of the glucosidase‐stimulating proteins

The concentration of β‐glucosidase‐stimulating proteins (called cohydrolase here) was measured in mouse liver and brain by immunoassay. Factors that might influence the levels of cohydrolase were examined. Injecting mice with an inactivator of glucosidase (conduritol B epoxide) rapidly produced elevations in liver glucosylceramide (the enzyme’s substrate) and in liver and brain cohydrolase. Injection of glucosylceramide emulsified with Myrj 52 produced the same two effects in liver but not in brain. The increases in cohydrolase level induced by the enzyme inhibitor persisted in both organs for at least seven days, reaching 61–70% above the normal level. Injection of emulsified galactocerebroside, sphingomyelin and mixed glucosphingolipids but not of ceramide also produced rises in cohydrolase level. An increase in cohydrolase level resulted from injection of phenylhydrazine, which produces hemolysis and consequently an increased workload for the glucosidase of liver. When the enzyme inhibitor and/or larger amounts of glucosylceramide emulsion were injected (750 mg/kg body weight), increases in liver weight of 13 to 37% appeared within one day. The increased weight was characterized by increases in the weights of protein, total lipid and DNA and a very high increase in glucosylceramide level. These procedures have produced a rapidly developing model version of Gaucher disease in mice. Injected glucocerebroside also induced an elevated level of glucosidase activity.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141328/1/lipd0702.pd

Allelopathy in two species of Chenopodium -inhibition of germination and seedling growth of certain weeds

The activity of washed leaf and inflorescence material of Chenopodium ambrosioides and C. murale, decaying leaves and inflorescences, and field soils collected beneath Chenopodium plants were examined in terms of the inhibition of seed germination and seedling growth of five weeds, viz. Abutilon indicum, Cassia sophera var. purpurea, C. tora, Evolvulus numularius and Tephrosia hamiltonii. The allelopathic pattern varied in each of the two test species and this depended on the type of test matter. However, the germination as well as the root and hypocotyl growth of A. indicum and E. nummularius were more hampered by phytotoxins or inhibitors from Chenopodium than were the other weeds. Since the leaf and inflorescence of Chenopodium formed the source of inhibitors, the respective plant-parts from the two species were chemically analysed and the presence of three terpenes (p-cymene, ascaridole and aritazone) from C. ambrosioides and an organic acid (oxalic acid) from C. murale were implicated in the allelopathic effect

Glutamate-Stimulated, Guanine Nucleotide-Mediated Phosphoinositide Turnover in Astrocytes Is Inhibited by Cyclic AMP

The potential for cross-talk between the adenyl cy-clase and phosphoinositide (PPI) lipid second messenger system was investigated in astrocytes cultured from neonatal rat brain. Glutamate-stimulated PPI turnover, measured by the formation of total inositol phosphates from myo -[ 3 H]inositoI-labeled lipids, was inhibited in a concentration-dependent manner by the elevation of intracellular cyclic AMP levels produced either by stimulation of the isoproter-enol receptor linked to adenyl cyclase or by its direct activation by forskolin. N 6 ,2′- O -Dibutyryl cyclic AMP, an analogue that can also activate cyclic AMP-dependent kinase, inhibited glutamate-stimulated PPI turnover in a concentration-dependent manner as well, a result suggesting that cyclic AMP-dependent kinase is involved in mediating the inhibition. Inclusion of an inhibitor of cyclic AMP-dependent kinase, l-(5-isoquinolinesulfonyl)-2 methylpiperazine dihy-drochloride or N -(2-guanidinoethyl)-5-isoquinolinesulfon-amide hydrochloride, blocked the cyclic AMP-mediated inhibition in a concentration-dependent manner, a finding further supporting this hypothesis. The site of inhibition of the phosphoinositol lipid pathway by cyclic AMP was probed using a digitonin-permeabilized cell system. Guanosine 5′- O -(3-thiotriphosphate), a nonhydrolyzable analogue of GTP, stimulated PPI turnover and potentiated glutamate-stimulated PPI turnover, and guanosine 5′- O -(3-thiodiphosphate) inhibited glutamate-stimulated PPI turnover in these cells, results providing evidence that glutamate receptors are coupled to phospholipase C by a guanine nucleotide binding protein in astrocytes. N 6 ,2′- O -Dibutyryl cyclic AMP and agents that elevate cyclic AMP levels inhibited the PPI turnover stimulated by guanosine 5′- O -(3-thiotriphosphate), as well as that potentiated by guanosine 5′- O -(3-thiotriphos-phate) in the presence of glutamate, results suggesting that the cyclic AMP-dependent inhibition occurs at or distal to the putative guanine nucleotide binding protein. Because basal PPI turnover was not altered by elevation of cyclic AMP levels, direct inhibition of phospholipase C is unlikely.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65289/1/j.1471-4159.1990.tb04962.x.pd