Donna Ullrey

An HSV‐1 Vector Containing the Rat Tyrosine Hydroxylase Promoter Enhances Both Long‐Term and Cell Type‐Specific Expression in the Midbrain

Abstract: A defective herpes simplex virus type one (HSV‐1) vector that contains a 6.8‐kb fragment of the rat tyrosine hydroxylase promoter (pTHlac‐7kb) was examined for its capability to target catecholaminergic cell type‐specific expression in the CNS. Cell type‐specific expression was assessed by comparison with a control vector (pHSVlac) that uses the HSV‐1 immediate early 4/5 promoter to support expression in multiple cell types. In initial experiments comparing expression in catecholaminergic and noncatecholaminergic cell lines, pTHlac‐7kb supported a seven‐ to 20‐fold increase in reporter gene expression in catecholaminergic cell lines. Four days after stereotactic injection into the midbrain of adult rats, pTHlac‐7kb supported a 10‐fold targeting of β‐galactosidase expression to tyrosine hydroxylase‐expressing neurons in the substantia nigra pars compacta compared with pHSVlac. Expression from pTHlac‐7kb was stably maintained for 6 weeks with no significant changes in the pattern of expression. Long‐term expression from pTHlac‐7kb was confirmed by RNA and DNA analysis. In contrast, reporter gene expression in the midbrain from pHSVlac decreased ∼30‐fold between 4 days and 6 weeks after gene transfer. Thus, within the context of this HSV‐1 vector system, the tyrosine hydroxylase promoter enhanced cell type‐specific expression and contributed to stable, long‐term expression of a recombinant gene product in neurons. The capability to target recombinant gene expression to catecholaminergic neurons in specific brain areas may be useful for studies on the roles of these neurons in brain physiology and behavior.

Uptake patterns and transport enhancements in cultures of hamster cells deprived of carbohydrates.

Abstract Dense cell cultures of the hamster lines, NIL, and polyoma transformed NIL were exposed to culture media containing various sugars (or no sugar). Various responses to these culture conditions were observed as changes in the uptake of galactose and its subsequent metabolism. Cells deprived of sugar have higher uptake rates for galactose and markedly different accumulation products from identical cells treated with sugar. A persistent increase in the transport of the amino acid, cycloleucine, was also observed as a response to culture conditions devoid of sugar

Studies on the surface and cytoplasmic membranes of Ehrlich ascites-carcinoma cells: I. The hydrolysis of ATP and related nucleotides by microsomal membranes

Abstract The hydrolysis of ATP and related nucleotides by Ehrlich ascites-carcinoma microsomes has been investigated. It has been shown that ATP hydrolysis is due to the membrane fraction of the microsomes and that the membrane fraction is composed of several groups of membrane fragments differing in density and biochemical composition. ATP hydrolysis requires activation by divalent cations, which produce different pH optima of ATP hydrolysis. ATP hydrolysis was found to be sensitive to ionic strength, but no specific effects of Na + or K + could be shown, nor was it sensitive to ouabain. In addition to ATP, ITP, GTP, UTP and CTP are also hydrolyzed by the microsomal membranes, the rats depending on the activating ion used and on pH. ADP is cleaved slowly but its presence inhibits ATP hydrolysis. Evidence is presented to suggest that much of the microsomal. ATPase is due to the presence of surfacep-membrane fragments in the microsomal membranes.

Studies on the surface and cytoplasmic membranes of Ehrlich ascites carcinoma cells: II. Alkali-cation-activated adenosine triphosphate hydrolysis in a microsomal membrane fraction

Abstract A membrane fraction isolated from Ehrlich ascites carcinoma microsomes contians an ATP phosphohydrolas which is strongly stimulated by K+ in the presence of Na+. The enzyme system has an absolute requirement for Na+ and Mg2+, but a number of monovalent cations can simulate K+. Ca2+, ADP and ouabain are inhibitory. The enzyme is believed to be associated with plasma membrane fragments. The quantitative relationships between enzyme activity and reactant concentration is presented and the probable relationship between ion transport in the whole cell and the alkali-cation-sensitive ATP phosphohydrolase is discussed.

Expression of the calcium-binding protein, parvalbumin, in cultured cortical neurons using a HSV-1 vector system enhances NMDA neurotoxicity

Calcium-binding proteins (CaBPs) are a family of proteins having a unique distribution in the brain and are thought to be important in buffering intracellular calcium. Glutamate neurotoxicity is a process by which the over-activation of glutamate receptors can cause the influx of excessive extracellular calcium and neuronal cell death. It has been proposed that neurons containing CaBP may be more resistant to glutamate neurotoxicity due to their increased ability to buffer calcium. Using a herpes simplex virus-1 (HSV-1) vector system we packaged the CaBP gene, parvalbumin, or the marker gene, beta-galactosidase (beta-gal), correctly in viron particles, which were found upon infection to express mRNA specific to these vectors. PC12 and neocortical cultures showed strong immunohistochemical staining for either beta-gal or parv. The cortical cultures stained positively for endogenous glutamate decarboxylase, a marker for GABAergic neurons, but not for endogenous parvalbumin, indicating that parvalbumin was being expressed ectopically from the HSV-1 vector. Interestingly, the expression of parvalbumin increased cortical cultures susceptibility to N-methyl-D-aspartate-induced neurotoxicity. This increase in neurotoxicity was not due to the wild-type virus or the helper virus which accompanies the packaging of these vectors. We speculate that the ectopic expression of parvalbumin in cortical cultures may be increasing glutamate release which in turn increases cell death.

A herpes simplex virus-1 vector containing the rat tyrosine hydroxylase promoter directs cell type-specific expression of beta-galactosidase in cultured rat peripheral neurons.

A defective herpes simplex virus-1 (HSV-1) vector system was used to study cell type-specific expression of the tyrosine hydroxylase (TH) gene. HSV-1 particles containing 663 bp (pTHlac 663), 278 bp (pTHlac 278), or 181 bp (pTHlac 181) of the rat TH promoter driving E. coli LacZ were used to infect superior cervical ganglia (SCG: TH-expressing tissue) and dorsal root ganglia (DRG:non-TH-expressing tissue) cultures. One day after infection, expression of beta-galactosidase was visualized by X-gal cytochemistry. Following viral transduction with pTHlac 663 at a multiplicity of infection of 0.2, 14.4% of the SCG neurons were X-gal positive whereas only about 0.9% of DRG neurons were X-gal positive. Infection with either pTHlac278 or 181 resulted in 3-fold more X-gal-positive DRG neurons. These results suggest that (i) the defective HSV-1 vector system may be useful in defining regulatory promoter motifs; (ii) 663 bp of the rat TH promoter contains sufficient information for cell type-specific expression in peripheral nervous system neurons; and (iii) sequences between -278 and -663 contain an element(s) that represses gene expression in non-catecholamingeric neurons.

Methods for specific characterization of trace amounts of uridine nucleotides in animal cell cultures.

Abstract The use of specific indicator enzymes to characterize the nature of radioactive peaks, obtained from paper chromatograms of protein-free cell filtrates from cell cultures briefly incubated with [ 14 C]galactose, has been described. The use of an internal standard ([ 3 H]UDP-galactose) added to the filtrates before their exposure to the indicator enzymes illustrates the specificity of the method used, as well as its usefulness in quantitative analysis of the abundance of nucleotide sugars in cell cultures under different culture conditions.