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Biomedical subjects

J L Rubenstein

Publications and source records attributed to J L Rubenstein.

At least 19 recordsLinked to original sources
PubMed1992

Synaptic vesicle-associated Ca2+/calmodulin-dependent protein kinase II is a binding protein for synapsin I.

Synapsin I is a synaptic vesicle-associated phosphoprotein that is involved in the modulation of neurotransmitter release. Ca2+/calmodulin-dependent protein kinase II, which phosphorylates two sites in the carboxy-terminal region of synapsin I, causes synapsin I to dissociate from synaptic vesicles and increases neurotransmitter release. Conversely, the dephosphorylated form of synapsin I, but not the form phosphorylated by Ca2+/calmodulin-dependent protein kinase II, inhibits neurotransmitter release. The amino-terminal region of synapsin I interacts with membrane phospholipids, whereas the C-terminal region binds to a protein component of synaptic vesicles. Here we demonstrate that the binding of the C-terminal region of synapsin I involves the regulatory domain of a synaptic vesicle-associated form of Ca2+/calmodulin-dependent protein kinase II. Our results indicate that this form of the kinase functions both as a binding protein for synapsin I, and as an enzyme that phosphorylates synapsin I and promotes its dissociation from the vesicles.

Amino Acid Sequence↗
PubMed1992

Isolation and characterization of a library of cDNA clones that are preferentially expressed in the embryonic telencephalon.

In order to isolate genes involved in development of the mammalian telencephalon we employed an efficient cDNA library procedure. By subtracting an adult mouse telencephalic cDNA library from an embryonic day 15 (E15) mouse telencephalic cDNA library we generated two subtracted libraries (ES1 and ES2). We estimate that ES1 contains between 200 and 600 different cDNA clones, which approximates the number of genes that are preferentially expressed in the E15 telencephalon, compared to the adult telencephalon. Northern analysis of 20 different cDNA clones shows that 14 of these are expressed at least 5-fold more in the E15 telencephalon than the adult telencephalon. Limited sequencing of the 14 differentially expressed clones reveals that 10 have no significant identity to sequences in GenBank and EMBL databases, whereas the other 4 have significant sequence identity to vimentin, histone 3.3, topoisomerase I and the B2 repeat element. In situ hybridization using one of the differentially expressed cDNAs, TES-1, demonstrates that it is transiently expressed in the anlage of the basal ganglia. In situ hybridization with another differentially expressed cDNA clone, TES-4, shows that it is specifically expressed in differentiating cells of the neural axis with a distinctive rostral-caudal temporal pattern. These findings, and the methods that we have developed, provide a framework for future investigations of the genetic control of telencephalon development.

Animals↗
PubMed1992

DLX2 (TES1), a homeobox gene of the Distal-less family, assigned to conserved regions on human and mouse chromosomes 2.

Dlx-2 (also called Tes-1), a mammalian member of the Distal-less family of homeobox genes, is expressed during murine fetal development in spatially restricted domains of the forebrain. Searching for a candidate neurological mutation that might involve this gene, we have assigned the human and mouse loci to regions of conserved synteny on human chromosome 2, region cen--q33, and mouse chromosome 2 by Southern analysis of somatic cell hybrid lines. An EcoRI dimorphism, discovered in common inbred laboratory strains, was used for recombinant inbred strain mapping. The results place Dlx-2/Tes-1 near the Hox-4 cluster on mouse chromosome 2.

Animals↗
PubMed1992

Depressive affect in "normal" adolescents: relationship to life stress, family, and friends.

Self-reported depressive affect was examined in high school students in relation to stress and the quality of relationships with family and friends. Higher levels of depressive affect were connected with stress around sexuality and achievement, lower levels of family cohesion, and more problematic peer relationships. The effects of high stress were buffered for boys by positive peer relationships, and for girls by cohesive family relationships.

Achievement↗
PubMed1991

Isolation and characterization of a novel cDNA clone encoding a homeodomain that is developmentally regulated in the ventral forebrain.

A complementary DNA, Tes-1, of a novel homeodomain protein has been cloned, and its pattern of expression has been characterized. It is a structural homolog of Distal-less, a homeodomain-encoding gene in D. melanogaster. Its expression is developmentally regulated and is limited to structures in the head. Within the central nervous system of the midgestation mouse embryo, it is expressed exclusively in the ventral forebrain. It is likely that Tes-1 plays a regulatory role in the development of this complex neural structure.

Amino Acid Sequence↗
PubMed1990

Subtractive hybridization system using single-stranded phagemids with directional inserts.

We describe a subtractive hybridization protocol which is designed to permit subtractions between cDNA libraries. The method uses single-stranded phagemids with directional inserts as both the driver and the target. We modified the M13 phagemid vector pBluescript for the directional cDNA cloning and subtractive hybridization. Two simplified methods for efficient construction of directional cDNA libraries are also described. Using a model system, we found that one round of subtractive hybridization results in a 5,000-fold specific subtraction of abundant molecules. We used two methods to quantify the efficiency and verify the specificity of the subtraction. In order to obtain these subtraction efficiencies, it was necessary to develop a method to purify the single-stranded DNA to homogeneity. The single-stranded purification involved using potassium iodide (KI) density centrifugation, restriction endonuclease digestion and phenol extraction in the presence of magnesium. We describe the several advantages of using directional inserts for the subtraction procedure.

Bacteriophages↗
PubMed1990

Depression, episodic behavioral dyscontrol, and polydipsia following right temporal lobe damage.

A patient referred to the authors for evaluation and treatment of depression, behavioral dyscontrol syndrome, and polydipsia is described. The authors reviewed his medical status and, finding damage to his right temporal lobe, conceptualized his symptom constellation as representing interictal syndrome and treated him with carbamazepine. His affective symptoms, but not the polydipsia, improved following treatment with carbamazepine.

Adult↗
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↗