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Biomedical subjects

A I Geller

Publications and source records attributed to A I Geller.

At least 19 recordsLinked to original sources
PubMed2004

Receptors specific for the carboxyl-terminal region of parathyroid hormone on bone-derived cells: determinants of ligand binding and bioactivity.

PTH comprises 84 amino acids of which the first 34 are sufficient for full activation of the classical PTH/PTHrP receptor, the type 1 PTH receptor. It is known that multiple carboxyl (C)-terminal fragments of PTH are present in the blood and that they comprise the majority of circulating PTH. C-PTH fragments, previously regarded as by-products of PTH metabolism, are directly secreted by the parathyroid glands or arise from the peripheral cleavage of the intact hormone. Compelling evidence now strongly suggests that these C-PTH fragments mediate biological effects via activation of a receptor that specifically recognizes the C-terminal portion of intact PTH, and this receptor is therefore named the carboxyl-terminal PTH receptor (CPTHR). We have previously reported that osteocytes abundantly express this novel receptor and that its activation is involved in cell survival and communication. Here we report the characterization of determinants of PTH that are required for high-affinity binding to the CPTHR. Using synthetic PTH peptides harboring alanine substitution or truncations, we showed the existence of discrete binding domains and critical residues within the intact hormone. We have furthermore identified eight amino acids within the PTH sequence that play key roles in optimizing the binding affinity of C-PTH fragments to CPTHRs. These include the tripeptide sequence Arg(25)-Lys(26)-Lys(27), the dibasic sequence Lys(53)-Lys(54), and three additional residues within the PTH (55-84) sequence, Asn(57), Lys(65), and Lys(72). Functional analysis of these residues demonstrated a strong correlation between binding affinity and biological effect and points to a potential role of CPTHR activation in regulating bone cell survival.

Amino Acid Sequence↗
PubMed2001

Enhanced auditory reversal learning by genetic activation of protein kinase C in small groups of rat hippocampal neurons.

The hippocampus has a central role in specific types of learning, but there is only limited evidence identifying the requisite molecular changes in ensembles of hippocampal neurons. To investigate the role of protein kinase C (PKC) pathways in hippocampal mediated learning, a constitutively active, catalytic domain of rat PKC betaII was delivered into hippocampal dentate granule neurons using a Herpes Simplex Virus (HSV-1) vector. This PKC causes a long-lasting, activation-dependent increase in neurotransmitter release from cultured cells. Activation of PKC pathways in a small percentage (< or =0.26%) of dentate granule neurons was sufficient to enhance rat auditory discrimination reversal learning. The affected neurons altered hippocampal physiology as revealed by elevated NMDA receptor densities in specific hippocampal areas. Thus, these results directly suggest that activation of PKC pathways in a specific hippocampal area alters rat auditory discrimination reversal learning. Because each rat may contain a unique pattern of affected neurons, there appears to be considerable flexibility and/or redundancy in the groups of neurons that can modify learning.

Animals↗
PubMed2001

Enhanced reporter gene expression in the rat brain from helper virus-free HSV-1 vectors packaged in the presence of specific mutated HSV-1 proteins that affect the virion.

Herpes simplex virus (HSV-1) gene expression is hypothesized to shut off promoters in HSV-1 vectors, but in a helper virus-free HSV-1 vector system, a number of promoters support only short-term expression. Thus, recombinant gene expression remains short-term in the absence of approximately 99% of the HSV-1 genome. To resolve this paradox, we hypothesized that specific HSV-1 proteins that affect the virion can shut off recombinant gene expression. This study evaluated expression from HSV-1 vectors, containing neuronal-specific promoters, that were packaged in the presence of specific mutated HSV-1 proteins that affect the virion. The mutated HSV-1 proteins that were examined included two protein kinases (U(L)13 and U(S)3), the virion host shut-off factor (vhs), the transactivator of immediate early promoters (VP16), and a virion protein that affects RNA metabolism (U(S)11). Helper virus-free packaging could occur in the presence of each mutated protein alone or specific combinations of two or three mutated proteins. In BHK and PC12 cells, vectors packaged in the presence of each mutated protein increased ( approximately 2-fold) the level of expression per cell, and vectors packaged in the presence of specific combinations of mutated proteins supported larger (4-7-fold) increases. In the rat striatum, vectors packaged in the presence of a mutated U(S)3 displayed enhanced gene transfer (13-18-fold increases in the number of cells at 4 days), and vectors packaged in the presence of mutated U(L)13 or VP16 enhanced long-term expression (2 months). Vectors packaged in the presence of mutated vhs or U(S)11 displayed minimal changes in expression.

Animals↗
PubMed2001

Helper virus-free HSV-1 vectors packaged both in the presence of VSV G protein and in the absence of HSV-1 glycoprotein B support gene transfer into neurons in the rat striatum.

Herpes simplex virus (HSV-1) vectors have potential for gene transfer into quiescent cells, but the gene transfer process could be more efficient. In other vector systems, both the titers and the efficiency of gene transfer have been enhanced by pseudotyping the vector particles with vesicular stomatitis virus (VSV) G protein. In this report, we pseudotyped helper virus-free HSV-1 plasmid vectors with VSV G protein. Packaging was performed in the presence of both VSV G protein and a deletion in an essential HSV-1 glycoprotein, gB. The resulting vector stocks supported gene transfer into both fibroblast and neuronal cell lines. VSV G protein was required for gene transfer because preincubation of these vector stocks with antibodies directed against either VSV G protein or VSV reduced the titer to undetectable levels. Although the titers were lower than those obtained using the unmodified vector system, the titers were not increased by use of chimeric proteins that contain the extracellular domain of VSV G protein and the transmembrane and/or cytoplasmic domains of specific HSV-1 glycoproteins. Also, the titers were not increased by performing the packaging in the presence of deletions in multiple HSV-1 glycoproteins. Nonetheless, pHSVlac pseudotyped with VSV G protein supported gene transfer into striatal neurons in the rat brain. Thus, HSV-1 vectors pseudotyped with VSV G protein may be useful for specific gene transfer studies.

Animals↗
PubMed2001

General strategy for constructing large HSV-1 plasmid vectors that co-express multiple genes.

Herpes simplex virus type 1 (HSV-1) plasmid vectors have a number of attractive features for gene transfer into neurons. In particular, the large size of the HSV-1 genome suggests that HSV-1 vectors might be designed to co-express multiple genes. Here, we report a general strategy for constructing large HSV-1 plasmid vectors that co-express multiple genes. Each transcription unit is linked to an antibiotic resistance gene, and genetic selections are used to assemble large vectors. Using this strategy, we constructed large (26 or 31 kb) HSV-1 vectors that contain two transcription units and two or three genes. These vectors were efficiently packaged into HSV-1 particles using a helper virus-free packaging system. The resulting vector stocks supported the expression of two or three genes in both cultured cells and the rat brain. Potential applications of HSV-1 vectors that co-express multiple genes are discussed.

Gene Expression Regulation, Viral↗
PubMed2000

A tyrosine hydroxylase-neurofilament chimeric promoter enhances long-term expression in rat forebrain neurons from helper virus-free HSV-1 vectors.

Helper virus-free herpes simplex virus (HSV-1) plasmid vectors are attractive for neural gene transfer, but a promoter that supports neuronal-specific, long-term expression is required. Although expression from many promoters is unstable, a 6.8-kb, but not a 766-bp, fragment of the tyrosine hydroxylase (TH) promoter supports long-term expression. Thus, 5' upstream sequences in this promoter may enhance expression. In this study, we evaluated expression from vectors that contain 5' upstream sequences from this promoter (-0.5 to -6.8 kb) inserted at the 5' end of either a neurofilament heavy subunit (NF-H) promoter or the cytomegalovirus (CMV) immediate early promoter. The TH-NFH promoter supported expression for 6 months in the striatum, 2 months in the hippocampus, and for 1 month in both perirhinal and postrhinal cortex (the longest time points examined). Expression was targeted to neurons. The enhanced expression may require specific sequences in the TH promoter fragment because replacing this fragment with a similar sized fragment of bacteriophage lambda DNA did not enhance expression. The reverse orientation of the TH promoter fragment also enhanced expression. Insertion of insulators from the chicken beta-globin locus between the TH-NFHlac transcription unit and the vector backbone may support a modest additional enhancement in expression. Other eucaryotic sequences may also enhance expression; a S. cerevisiae (40-kb fragment)-NFH promoter enhanced expression. In contrast, the TH-CMV promoter did not enhance expression. Thus, the TH-NFH promoter may support some physiological studies that require long-term expression in forebrain neurons.

Animals↗
PubMed2000

Fifty-one kilobase HSV-1 plasmid vector can be packaged using a helper virus-free system and supports expression in the rat brain.

Herpes simplex virus type 1 (HSV-1) plasmid vectors have a number of attractive features for gene transfer into neurons. In particular, the large size of the HSV-1 genome suggests that HSV-1 vectors might be designed to accommodate large inserts. We now report the construction and characterization of a 51 kb HSV-1 plasmid vector. This vector was efficiently packaged into HSV-1 particles using a helper virus-free packaging system. The structure of the packaged vector DNA was verified by both Southern blot and PCR analyses. A vector stock was microinjected into the rat striatum, the rats were sacrificed at 4 days after gene transfer, and numerous X-gal positive striatal cells were observed. This 51 kb vector was constructed using general principles that may support the routine construction of large vectors. Potential applications of such HSV-1 vectors include characterizing large promoter fragments or genomic clones and co-expressing multiple genes.

Animals↗
PubMed2000

Modest increases in the titers of helper virus-free herpes simplex virus 1 (HSV-1) vectors by packaging in a cell line with inducible expression of HSV-1 VP16 or by treatment with N,N'-hexamethylene-bis-acetamide.

A recently developed helper virus-free HSV-1 vector system has potential for both gene therapy and physiological studies, but relatively low vector titers have complicated use of this system. In this report, we explored improving the vector titers by isolating a Vero cell line that uses the tetracycline-regulated promoter system to induce HSV-1 VP16 at the beginning of the packaging procedure. We isolated the required cell line and demonstrated that it supports enhanced expression from a HSV-1 immediate early (IE) promoter. However, use of this cell line in helper virus-free vector packaging results in only a modest, approximately 2-fold increase in the vector titers. Additionally, treating the packaging cells with N,N'-hexamethylene-bis-acetamide (HMBA), which is known to induce HSV-1 IE promoters, also supports only modest increases in the vector titers. These results suggest that the lack of VP16 is not the rate limiting factor in the packaging procedure.

Acetamides↗
PubMed1999

Improved titers for helper virus-free herpes simplex virus type 1 plasmid vectors by optimization of the packaging protocol and addition of noninfectious herpes simplex virus-related particles (previral DNA replication enveloped particles) to the packaging procedure.

A helper virus-free herpes simplex virus type 1 (HSV-1) plasmid vector system has potential for both gene therapy and physiological studies, but relatively low titers have complicated use of this system. In this article, the packaging efficiency was improved by optimizing the packaging protocol and by adding noninfectious HSV-1-related particles, i.e., previral DNA replication enveloped particles (PREPs), during the packaging procedure. PREPs contain many of the tegument proteins that are thought to enhance an HSV-1 infection. Use of both the optimized packaging protocol and the PREPs resulted in an approximately 50-fold increase in the titer, and five different HSV-1 vectors were packaged using this procedure. A purified vector stock (7.8x10(8) infectious vector particles/ml) was microinjected into the striatum, the rats were sacrificed 4 days after gene transfer, and the brains were found to contain an average of approximately 6740 X-Gal-positive striatal cells. This improved packaging procedure may augment use of this vector system.

Animals↗
PubMed1999

Diverse stabilities of expression in the rat brain from different cellular promoters in a helper virus-free herpes simplex virus type 1 vector system.

Many neural gene transfer studies require both long-term and cell type-specific expression. We have reported a helper virus-free HSV-1 plasmid vector system (Fraefel et al., 1996), and this system supports at least some long-term expression from herpesvirus immediate-early promoters. In this study, we constructed vectors that placed the lacZ reporter gene under the regulation of five different cellular promoters. Vector stocks were microinjected into the midbrain, striatum, or hippocampus; the rats were sacrificed at 4 days to 2 months after gene transfer; and the numbers of X-Gal-positive cells were determined. A 6.8-kb fragment of the rat tyrosine hydroxylase (TH) promoter supported relatively stable expression for up to 2 months and targeted expression to TH-immunoreactive neurons in the substantia nigra pars compacta. The other promoters that were examined were chosen with the goal of obtaining long-term, neuronal-specific expression. At 4 days after gene transfer, a 766-bp fragment of the TH promoter supported expression in cells with neuronal morphology in the midbrain and striatum, consistent with results in transgenic mice. However, expression was absent by 2 weeks. Similarly, at 4 days after gene transfer, a mouse neurofilament heavy subunit promoter supported expression in cells with neuronal morphology in the midbrain, striatum, and hippocampus, but expression was absent by 2 weeks. A rat neuron-specific enolase promoter supported only a low level of expression in cultured neuronal cells, and expression was not detected in the brain. A rat voltage-gated sodium channel promoter supported only a low level of expression in PC12 cells and expression could not be detected in cultured cortical cells. These results demonstrate that different promoters support a wide range of levels and stabilities of expression in this vector system, and the results suggest approaches to improving the stability of long-term expression.

Animals↗
PubMed1999

An efficient in vivo recombination cloning procedure for modifying and combining HSV-1 cosmids.

A helper virus-free herpes simplex virus type-1 (HSV-1) plasmid vector system developed recently may have applications in gene therapy and basic physiological studies. This system might be improved by mutating specific HSV-1 genes in the packaging system and by creating large vectors. An in vivo recombination cloning procedure is reported that supports the routine manipulation of relatively large DNAs such as the five cosmids that comprise this helper virus-free HSV-1 packaging system. In vivo recombination cloning is carried out by transforming overlapping DNA fragments into a specific RecA+ Escherichia coli, BJ5183. The cloning efficiency was improved by using a modified version of the Hanahan transformation procedure, and the background was lowered by either using vectors with different combinations of ends (5' overhangs, 3' overhangs, blunt ends) or by treating the vector with calf intestinal phosphatase. The range of usable overlap sizes is from 251 bp to 18 kb with 500 bp to 5 kb preferred. This procedure supports the routine construction and mutation of HSV-1 cosmids, by use of up to six different DNA fragments, and the construction of plasmids up to 65 kb in size. This procedure may also have applications to other vector systems and to studies on large viruses.

Cloning, Molecular↗
PubMed1999

Genetic analysis of the role of protein kinase C signaling pathways in behaviors by direct gene transfer with HSV-1 vectors.

A genetic intervention strategy is described to elucidate the specific biochemical pathways in identified types of neurons that underlie behavioral adaptations. This strategy contains three parts: A Herpes simplex virus (HSV-1) vector is used to obtain localized gene transfer, a cell type-specific promoter is used to target expression to a particular type of neuron, and a constitutively active signal transduction enzyme is expressed to alter neuronal physiology. To enable this approach, a constitutively active protein kinase C (PKC) was developed which causes a long-lasting, activation-dependent increase in neurotransmitter release from cultured sympathetic neurons. This genetic intervention strategy was tested using the nigrostriatal system: Microinjection of HSV-1 vectors that contain the tyrosine hydroxylase promoter targeted expression to dopaminergic nigrostriatal neurons. Expression of the constitutively active PKC in a small percentage of nigrostriatal neurons (approximately 0.1-2%) produced a long-term (> or = 1 month) change in apomorphine-induced rotational behavior, the amount of rotational behavior correlated with the number of affected nigrostriatal neurons, and D2-like dopamine receptor levels were elevated in the striatal regions innervated by the affected nigrostriatal neurons. The strengths and limitations of this genetic intervention strategy are discussed.

Animals↗
PubMed1998

Modulation of rat rotational behavior by direct gene transfer of constitutively active protein kinase C into nigrostriatal neurons.

The modulation of motor behavior by protein kinase C (PKC) signaling pathways in nigrostriatal neurons was examined by using a genetic intervention approach. Herpes simplex virus type 1 (HSV-1) vectors that encode a catalytic domain of rat PKCbetaII (PkcDelta) were developed. PkcDelta exhibited a constitutively active protein kinase activity with a substrate specificity similar to that of rat brain PKC. As demonstrated in cultured sympathetic neurons, PkcDelta caused a long-lasting, activation-dependent increase in neurotransmitter release. In the rat brain, microinjection of HSV-1 vectors that contain the tyrosine hydroxylase promoter targeted expression to dopaminergic nigrostriatal neurons. Expression of pkcDelta in a small percentage of nigrostriatal neurons (approximately 0.1-2%) was sufficient to produce a long-term (>/=1 month) change in apomorphine-induced rotational behavior. Nigrostriatal neurons were the only catecholaminergic neurons that contained PkcDelta, and the amount of rotational behavior was correlated with the number of affected nigrostriatal neurons. The change in apomorphine-induced rotational behavior was blocked by a dopamine receptor antagonist (fluphenazine). D2-like dopamine receptor density was increased in those regions of the striatum innervated by the affected nigrostriatal neurons. Therefore, this strategy enabled the demonstration that a PKC pathway or PKC pathways in nigrostriatal neurons modulate apomorphine-induced rotational behavior, and altered dopaminergic transmission from nigrostriatal neurons appears to be the affected neuronal physiology responsible for the change in rotational behavior.

Animals↗
PubMed1997

Helper virus-free herpes simplex virus-1 plasmid vectors for gene therapy of Parkinson's disease and other neurological disorders.

Vectors based on herpes simplex virus type 1 (HSV-1) have potential for gene therapy of neurological disorders. HSV-1 plasmid vectors (amplicons) contain only approximately 1% of the 150-kb HSV-1 genome and have been packaged into virus particles by using a helper virus. We have demonstrated that HSV-1 plasmid vectors which express tyrosine hydroxylase can cause long-term biochemical and behavioral recovery in the 6-hydroxydopamine rat model of Parkinson's disease. Furthermore, we and others have used HSV-1 plasmid vectors which express a wide range of genes that affect neuronal physiology. Because of the pathogenicity of the HSV-1 helper virus, however, the use of this vector system has been limited to studies in animal models or primary cultures of neural cells. Thus, to increase the safety of HSV-1 plasmid vectors, we recently developed a helper virus-free packaging system that may facilitate studies on neuronal physiology and potential therapeutic applications.

Animals↗
PubMed1997

An HSV-1 vector containing the rat tyrosine hydroxylase promoter enhances both long-term and cell type-specific expression in the midbrain.

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 beta-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 approximately 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.

Animals↗