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Biomedical subjects

J L Rubenstein

Publications and source records attributed to J L Rubenstein.

108 records · Page 6Linked to original sources
PubMed1989

Developmental expression of proenkephalin mRNA and peptides in rat striatum.

The development of proenkephalin (PE) gene expression in the rat striatum was examined at the mRNA and peptide levels. Immunocytochemistry was performed with antisera generated to the PE-specific peptide product Met-enkephalin-Arg-Gly-Leu (MERGL). The distribution of immunostaining was compared with the distribution of PE mRNA, determined by in situ hybridization with an oligonucleotide probe. PE mRNA first appeared at E16 in the caudal ventrolateral striatum, followed at E17-18 by the appearance of MERGL immunoreactivity in a similar distribution. The anatomical gradients of PE gene expression were similar to the pattern of histogenesis of striatal neurons, suggesting that the timing of PE gene expression is related to the time of neuronal withdrawal from the mitotic cycle. The relation of the development of PE gene expression to the known patterns of striatal histogenesis, neurochemical compartmentalization and dopaminergic innervation is discussed.

Animals↗
PubMed1989

Suicidal behavior in "normal" adolescents: risk and protective factors.

Risk and protective factors were examined in suicidal and nonsuicidal public high school students. With life stress and depression as independent risk factors, family cohesion was found to offset the effect of stress, and friendships to have a more indirect effect. Differential effects of ten sources of stress were analyzed from a developmental perspective, and the probability of suicidal behavior associated with clusters of factors was estimated for the general population.

Achievement↗
PubMed1988

Retroviral vectors.

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Cloning, Molecular↗
PubMed1986

Use of a recombinant retrovirus to study post-implantation cell lineage in mouse embryos.

We show that a gene introduced into cells of mouse embryos by a retrovirus can serve as a heritable marker for the study of cell lineage in vivo. We constructed a defective recombinant retrovirus in which the Escherichia coli beta-galactosidase (lacZ) gene is inserted in the genome of a Muloney murine leukemia virus (M-MuLV). Expression of lacZ was detected with a histochemical stain that can be applied to cultured cells and embryonic tissue. Infection of cultured cells showed that lacZ has no detectable deleterious effects on cell viability or growth, that the enzyme is stably expressed in the progeny of infected cells for many generations in the absence of selective pressure, and that the virus can induce lacZ in a variety of cell types. Following injection of the virus into mid-gestation mouse embryos, clones of lacZ-positive cells were detected in skin, skull, meninges, brain, visceral yolk sac, and amnion. We identified the cell types comprising a series of lacZ-positive clones in the visceral yolk sac and skin to learn the lineage relationships of the labelled cells. In each tissue, we obtained evidence that several cell types have a pluripotential ancestor and that cell fate is progressively restricted as development proceeds.

Animals↗
PubMed1986

Introduction of genes into preimplantation mouse embryos by use of a defective recombinant retrovirus.

Two-cell and four-cell preimplantation mouse embryos were cocultured in vitro with fibroblasts producing the recombinant retrovirus M-MuLV-neo. No wildtype helper virus was detected in these cultures. Of the embryos that survived the in vitro cultivation and the reimplantation into foster mothers, 2 of 15 that were tested contained the proviral genome. The provirus integrated as a single copy at a unique site. We estimate that approximately equal to 20% of the cells in each of the two transgenic fetuses contained the provirus.

Animals↗
PubMed1984

Construction of a retrovirus capable of transducing and expressing genes in multipotential embryonic cells.

Retroviral gene expression is inhibited in embryonal carcinoma (EC) cells. We have constructed a recombinant retroviral vector that is capable of expressing the neomycin-resistance (neo) gene in EC cells. The critical modification that permits expression of the neo gene is the insertion of a composite simian virus 40 early gene-herpes simplex virus type 1 thymidine kinase gene (SVtk) promoter 3' to the viral first intron and 5' to the neo gene. When the SVtk promoter is deleted, the recombinant retrovirus is either unable or extremely inefficient at expressing the neo gene in EC cells.

Drug Resistance↗
PubMed1984

[Nonsense RNA: a tool for specifically inhibiting the expression of a gene in vivo].

We describe a general technique to inhibit gene expression in eukaryotic cells. The gene we chose to inhibit was the E. coli LacZ gene (encoding beta-galactosidase), which has previously been cloned into a eukaryotic expression vector [1]. This plasmid is called pCH110. We constructed a variant of pCH110 in which we flipped a 2566 base pair 5' fragment of the LacZ gene into the antiparallel orientation. The plasmid containing this mutated LacZ gene is called pNSLacZ (NS signifies non-sense coding sequence). When equal amounts of pCH110 and pNSLacZ are co-transfected into 3T6 mouse fibroblasts, the beta-galactosidase activity is decreased by approximately a factor of ten. Increasing the ratio of pNSLacZ to pCH110 above 1:1 does not appreciably increase the level of inhibition. Next, we prove the specificity of the inhibition by adding a third gene to the transfection mixture. For this purpose, we used pSVneo beta, a plasmid which expresses a phosphotransferase. We found that even when the beta-galactosidase activity was diminished by a factor of 10, the phosphotransferase activity was unaffected. Therefore, we have demonstrated that: the presence of an antiparallel copy of the LacZ gene results in a significant and specific diminution of the LacZ gene's expression; only a fraction of the LacZ gene needs to be in the antiparallel orientation in order to observe this effect. These results suggest that this technique can serve as a tool to decrease the level of gene expression in order to study the function of specific genes, or as a therapeutic manoeuvre in the treatment of disorders of abnormal gene expression.

Animals↗
PubMed1983

Construction of a synthetic messenger RNA encoding a membrane protein.

We have synthesized microgram quantities of a functional eucaryotic mRNA by in vitro transcription. For this purpose, we constructed a plasmid in which the Escherichia coli lactose promoter was 5' to the vesicular stomatitis virus (VSV) G protein gene (Rose, J. K., and C. J. Gallione, 1981, J. Virol., 39:519-528). This DNA served as the template in an in vitro transcription reaction utilizing E. coli RNA polymerase. The RNA product was capped using the vaccinia guanylyltransferase. A typical preparation of the synthetic G mRNA was equivalent to the amount of G mRNA that can be isolated from approximately 10(8) VSV-infected cells. This synthetic mRNA was translated by a wheat germ extract in the presence of microsomes, producing a polypeptide that was indistinguishable from G protein in its size, antigenicity, degree of glycosylation, and its membrane insertion. This technique should aid in identifying features needed by proteins for insertion into membranes.

Animals↗
PubMed1981

Purification of coated vesicles by agarose gel electrophoresis.

We have applied agarose gel electrophoresis as a novel step in the purification of clathrin-coated vesicles. Preparations of coated vesicles obtained by sedimentation velocity and isopycnic centrifugation are resolved into two distinct fractions upon electrophoresis. The slower migrating fraction contains smooth vesicles, whereas the faster contains only coated vesicles and empty clathrin coats. The faster mobility of the coated vesicles is primarily caused by the acidic nature of clathrin. Coated vesicles from three different cell types have different mobilities. In each case, however, all of the major polypeptides previously attributed to coated vesicles comigrate with the now homogeneous particles, even though a powerful ATPase activity is completely removed.

Adenosine Triphosphatases↗
PubMed1980

Lateral diffusion of M-13 coat protein in mixtures of phosphatidylcholine and cholesterol.

The translational diffusion of fluorescent-labeled M-13 phage coat protein (FITC-M-13), an integral membrane protein, has been measured in mixtures of phosphatidylcholines and cholesterol, using a pattern photobleaching technique. At temperatures below the chain-melting transition temperature of dimyristoylphosphatidylcholine (DMPC) (23.8 degrees C), the lateral diffusion coefficient of M-13 shows a marked increase when the cholesterol concentration is increased above 20 mol %. A similar marked increase in the lateral diffusion coefficient of a fluorescent phospholipid is also observed. At temperatures above the chain-melting transition temperature of DMPC, a minimum is observed in the lateral diffusion coefficient of FITC-M-13 for cholesterol concentrations in the vicinity of 25 mol %. This minimum in the diffusion coefficient of FITC-M-13 is also observed at 25 mol % cholesterol in egg phosphatidylcholine at 15 degrees C. No such minimum is observed for the lateral diffusion coefficient of the fluorescent lipid. The lateral diffusion coefficient of FITC-M-13 is large (greater than 10(-9) cm2/s) at all cholesterol concentrations for temperatures above the chain-melting transition temperature of the phosphatidylcholine. Several other proteins contain hydrophobic regions similar to that of the M-13 coat protein. We anticipate that a number of proteins of this type will show similar diffusional behavior, in particular exhibiting rapid diffusion throughout a wide range of lipid composition.

Cholesterol↗
PubMed1980

Dynamic properties of binary mixtures of phosphatidylcholines and cholesterol.

We have observed the paramagnetic resonance spectra of a head group spin-labeled phosphatidylethanolamine (L-alpha-dipalmitoylphosphatidyl-N-ethanolamine) as a function of temperature and cholesterol concentration in binary mixtures of cholesterol and dimyristoylphosphatidylcholine. These spectra bear on two interrelated topics involving mixtures of phosphatidylcholine and cholesterol: (1) lipid phase equilibria and the lateral ordering and diffusion of lipid molecules and (2) model membrane immunochemistry using spin-label lipid haptens.

Cholesterol↗
PubMed1979

Lateral diffusion in binary mixtures of cholesterol and phosphatidylcholines.

The lateral diffusion of a fluorescent-labeled phospholipid, phosphatidyl-N-(4-nitrobenzo-2-oxa-1,3,-diazole)ethanolamine, has been measured in binary mixtures of cholesterol and dimyristoyl phosphatidylcholine at temperatures both above and below 23.8 degrees C, the chain-melting transition temperature of this phosphatidylcholine. There is a temperature-composition region, approximately temperature less than 23 degrees C and mole fraction of cholesterol (X) less than 0.20, in which the lateral diffusion coefficient of the fluorescent probe is at least an order of magnitude smaller than it is at points outside of this temperature-composition region. At temperatures above approximately 23 degrees C there is a significant increasing cholesterol concentration, for X greater than 0.2.

Cholesterol↗
PubMed1979

Infant development in father-absent families.

In a sample of 55 black infants (age 5-6 months) living in the inner city in lower socioeconomic circumstances, 27 infants were being reared by their mothers in single-parent families. Male infants who had experienced minimal interaction with their fathers were significantly lower on the Bayley Mental Developmental Index and in measures of social responsiveness, secondary circular reactions, and preferences for novel stimuli. Female infants in this sample appeared unaffected by the father's presence or absence. After weighing alternative interpretations of the findings, we conclude that the father is a significant component in the early environment and that both observational studies and conceptual development of the role of the father in infancy are needed.

Child Development↗
PubMed1977

The mitochondrial DNA of Drosophila melanogaster exists in two distinct and stable superhelical forms.

We have studied the structure and replication of mitochondrial DNA from Drosophila melanogaster embryos, larvae, adult flies and two established tissue culture lines. The most striking observation is that the organism maintains at least two stable, distinct closed circular forms of mitochondrial DNA throughout development of the early embryo and in the adult fly. The major closed circular monomeric form comprises approximately 75% of the population and has a normal number of superhelical turns. In contrast, closed circular mitochondrial DNA isolated from Drosophila tissue culture cells is comprised almost entirely of molecules with the low superhelix density. We have been unable to detect the D loop form of mitochondrial DNA present in other eucaryotic systems, and find by electron microscope and pulse-chase labeling techniques that the time required to replicate Drosophila mitochondrial DNA is very short (less than 15 min) compared to the mouse L cell system (greater than 1 hr). We conclude that Drosophila mitochondrial DNA utilizes a replication mechanism different from that of other higher eucaryotes. We postulate that the maintenance of markedly different topological forms of mitochondrial DNA is most probably related to different demands for replication and transcription of the genome in these sources.

Cell Line↗
PubMed1977

Structure of hepatitis B Dane particle DNA before and after the Dane particle DNA polymerase reaction.

DNA isolated from the hepatitis B antigen form known as the Dane particle was examined by electron microscopy before and after the endogenous Dane particle DNA polymerase reaction. The most frequently occurring form was an untwisted circular double-stranded DNA molecule approximately 1 mum in length. Less frequently occurring forms included circular DNA of approximately unit length and having one or more small single-stranded regions, similar circular molecules with one or more tails either shorter or longer than 1 mum in length, and very small circular molecules with tails. There was no increase in frequency or length of tails after a DNA polymerase reaction, suggesting that tails were not formed during this reaction. The mean length of circular molecules increased by 23% when DNA was spread in formamide compared with aqueous spreading, suggesting that single-stranded regions are present in most of the molecules. The mean length of circular molecules obtained from aqueous spreading increased by 27% after a Dane particle DNA polymerase reaction. This indicates that single-stranded regions were converted to double-stranded DNA during the reaction.

DNA, Circular↗
PubMedDate not supplied

Anti-VEGF antibody treatment of glioblastoma prolongs survival but results in increased vascular cooption.

Vascular endothelial growth factor (VEGF) is an important mediator of the intense angiogenesis which is characteristic of glioblastoma. While genetic manipulation of VEGF/VEGF receptor expression has previously been shown to inhibit glioblastoma growth, to date, no study has examined the efficacy of pharmacologic blockade of VEGF activity as a means to inhibit intracranial growth of human glioblastoma. Using intraperitoneal administration of a neutralizing anti-VEGF antibody, we demonstrate that inhibition of VEGF significantly prolongs survival in athymic rats inoculated in the basal ganglia with G55 human glioblastoma cells. Systemic anti-VEGF inhibition causes decreased tumor vascularity as well as a marked increase in tumor cell apoptosis in intracranial tumors. Although intracranial glioblastoma tumors grow more slowly as a consequence of anti-VEGF treatment, the histologic pattern of growth suggests that these tumors adapt to inhibition of angiogenesis by increased infiltration and cooption of the host vasculature.

Animals↗