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Biomedical subjects

M Rubinstein

Publications and source records attributed to M Rubinstein.

At least 109 records · Page 6Linked to original sources
PubMed1993

Post-translational processing of proopiomelanocortin (POMC) in mouse pituitary melanotroph tumors induced by a POMC-simian virus 40 large T antigen transgene.

Mice harboring a transgene composed of proopiomelanocortin (POMC) gene promoter sequences (nucleotides -706 to +64) ligated to the simian virus (SV) 40 early gene encoding large T antigen developed large POMC-expressing pituitary tumors. Histologically the tumors arose from the intermediate lobe, contained nuclear SV40 T antigen and POMC peptides, but did not express other pituitary hormones. POMC processing in the pituitary tumors was indistinguishable from normal mouse intermediate lobe melanotrophs and was characterized by high proportions of acetylated and carboxyl-terminal shortened beta-endorphins, and amino-terminal acetylated alpha-melanocyte-stimulating hormone, and virtually no adrenocorticotropic hormone (ACTH)(1-39), beta-lipotropin, or POMC. The tumors contained abundant levels of mRNA for the prohormone convertase PC2 and undetectable levels of PC1. Normal mouse neurointermediate lobe also has a high ratio of PC2/PC1 expression that is distinct from the relative abundance of PC1 in anterior lobe and AtT-20 corticotroph cells. In contrast, extracts from tumors transplanted subcutaneously in nude mice contained predominantly nonacetylated forms of beta-endorphin(1-31) and -(1-27), very little ACTH(1-39), almost no corticotropin-like intermediate peptide or alpha-melanocyte-stimulating hormone, and higher proportions of intact POMC. Surprisingly, despite the less efficient proteolytic cleavage, a transplanted tumor expressed both PC1 and PC2. These studies are the first biochemical documentation of a melanotroph pituitary tumor in a rodent species and provide a new model for the investigation of pituitary oncogenesis and the molecular basis of tissue-specific prohormone post-translational processing.

Adrenocorticotropic Hormone↗
PubMed1993

An antiviral soluble form of the LDL receptor induced by interferon.

Interferons, which induce several intracellular antiviral proteins, also induce an extracellular soluble protein that inhibits vesicular stomatitis virus (VSV) infection. This 28-kilodalton soluble protein was purified to homogeneity and identified by protein sequencing as the ligand-binding domain of the human 160-kilodalton low density lipoprotein receptor (LDLR). The existence of an antiviral soluble LDLR was confirmed by immunoaffinity chromatography with monoclonal antibody to LDLR. This soluble receptor mediates most of the interferon-triggered antiviral activity against VSV, apparently by interfering with virus assembly or budding, and not by inhibiting virus attachment to cells.

Amino Acid Sequence↗
PubMed1993

Real-time measurements of kinetics of EGF binding to soluble EGF receptor monomers and dimers support the dimerization model for receptor activation.

We have tested one aspect of the allosteric dimerization model for the activation of EGF receptor (EGFR) by EGF: whether EGF binding favors dimerization of the receptor. For this to be true, EGF molecules must bind with higher affinity to dimeric receptors than to monomeric receptors. We have tested this directly in a defined system using the soluble, extracellular ligand binding domain of EGFR monomers (sEGFR) and sEGFR dimers stabilized by treatment with a covalent cross-linking agent. We describe real-time kinetic measurements of EGF binding to receptor monomers and dimers employing the method of total internal reflection (surface plasmon resonance). Our data show that sEGFR dimers bound EGF with 30-40-fold higher affinity [KD = (2-3) x 10(-8) M] than did sEGFR monomers. The enhanced binding affinity of sEGFR dimers resulted mainly from a reduced off-rate with k(off) = 0.001 s-1 for sEGFR dimers as compared to k(off) = 0.06 s-1 for sEGFR monomers. These measurements indicate that dimerization of sEGFR increases its affinity for EGF by prolonging the amount of time that EGF remains bound to the receptor. This provides evidence that EGF binding stabilizes receptor dimerization and provides further support for the allosteric dimerization model as a mechanism for ligand induced receptor activation.

Animals↗
PubMed1993

Introduction of a point mutation into the mouse genome by homologous recombination in embryonic stem cells using a replacement type vector with a selectable marker.

The introduction of small mutations instead of null alleles into the mouse genome has broad applications to the study of protein structure-function relationships and the creation of animal models of human genetic diseases. To test a simple mutational strategy we designed a targeting vector for the mouse proopiomelanocortin (POMC) gene containing a single nucleotide insertion that converts the initial tyrosine codon of beta-endorphin 1-31 to a premature translational termination codon and introduces a unique Hpal endonuclease restriction site. The targeting vector also contains a neo cassette immediately 3' to the last POMC exon and a herpes simplex virus thymidine kinase cassette to allow positive and negative selection. Homologous recombination occurred at a frequency of 1/30 clones of electroporated embryonic stem cells selected in G418 and gancyclovir. 10/11 clones identified initially by a polymerase chain reaction (PCR) strategy had the predicted structure without evidence of concatemer formation by Southern blot analysis. We used a combination of Hpa I digestion of PCR amplified fragments and direct nucleotide sequencing to further confirm that the point mutation was retained in 9/10 clones. The POMC gene was transcriptionally silent in embryonic stem cells and the targeted allele was not activated by the downstream phosphoglycerate kinase-1 promoter that transcribed the neo gene. Under the electroporation conditions used, we have demonstrated that a point mutation can be introduced with high efficiency and precision into the POMC gene using a replacement type vector containing a retained selectable marker without affecting expression of the allele in the embryonic stem cells. A similar strategy may be useful for a wide range of genes.

Alleles↗
PubMed1993

Rat and mouse proopiomelanocortin gene sequences target tissue-specific expression to the pituitary gland but not to the hypothalamus of transgenic mice.

The proopiomelanocortin (POMC) gene is expressed predominantly in corticotrophs of the pituitary anterior lobe, melanotrophs of the intermediate lobe and neurons of the arcuate nucleus of the hypothalamus. The different ontogeny of POMC mRNA as well as the complicated hormonal regulation of POMC gene expression in the three different cell types suggests a concerted interaction between several cis-acting elements in the POMC gene and transcription factors located in each of the three cell types. To investigate cell-specific elements in the POMC gene we tested two different constructs in transgenic mice. The construct -4000rPOMCLacZ, carrying 4 kb of the rat POMC promoter fused to the Escherichia coli beta-galactosidase gene, showed appropriate expression in melanotrophs in 50% of the mice analyzed. beta-Galactosidase activity was less evident in corticotrophs under basal environmental conditions. In brain, 7 out of 15 independently derived transgenic founders had ectopic expression of the transgene in different areas; however, none of the animals analyzed expressed beta-galactosidase in neurons of the arcuate nucleus. The construct HAL*, a 'tagged' 10.2-kb mouse genomic fragment, was more efficiently targeted to the pituitary. Using in situ hybridization, we detected uniform expression of HAL* in melanotrophs in 100% of the 6 pedigrees analyzed and transgenic mRNA levels paralleled those of the endogenous POMC mRNA. In corticotrophs, basal expression was low but after adrenalectomy HAL* mRNA levels were comparable to those of POMC. None of the 6 pedigrees had appropriate expression of HAL* in the brain; however, 2 lines had ectopic expression in the dentate gyrus of the hippocampus.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Sexually assaultive male juveniles: a follow-up.

OBJECTIVE: The purpose of this study was to investigate the adult outcome of a group of male juveniles who committed sexual assault. METHOD: Nineteen sexually assaultive male juveniles and a comparison group of 58 violent juveniles were studied over an 8-year period through use of criminal records and clinical interviews. RESULTS: Although in adolescence the two groups were similarly violent, on follow-up those who had committed sexual assault were significantly more likely to commit adult sexual offenses. They also committed significantly more violent nonsexual offenses. Childhood sexual abuse, especially by females, was associated with adult sexual offenses. CONCLUSIONS: Sexually assaultive delinquents are at particularly high risk for subsequent violence. Hence, special efforts must be made to treat these delinquents in adolescence. Prevention of violent sexual behavior must include improved methods of detecting sexual abuse, especially that perpetrated by older females.

Adolescent↗
PubMed1993

Normative data for 40-Hz event-related potentials to 500-Hz tonal stimuli in young and elderly subjects.

Recordings of 40-Hz event-related potentials (ERPs) were obtained in 81 young subjects (18-40 years) and in 20 elderly subjects (60-77 years) with hearing normal for their age. The stimuli were 500-Hz logons. The first negative peak (N1) of the response was analyzed. With increasing intensity the amplitude of the responses increased and the latency decreased for all subjects. The 40-Hz ERP was obtained within 10 dB of the behavioral threshold for more than 80% of the population under study. The N1 was prolonged in latency and enhanced in amplitude in the older subjects. No differences were found between young and elderly subjects regarding binaural interaction, or in responses elicited by ipsi- and contralateral stimulation. From the clinical point of view, the 40-Hz ERP recordings of young and elderly subjects are similar.

Acoustic Stimulation↗
PubMed1992

Soluble interferon-alpha receptor molecules are present in body fluids.

Soluble forms of the interferon-alpha receptor (sIFN-alpha R) were identified in human serum and urine by Western blotting with monoclonal antibodies (MAb) directed against IFN-alpha R, and by immunoprecipitation (Iptn) of a covalently cross-linked complex of IFN-alpha R and [125I]IFN-alpha with anti IFN-alpha MAb. Elevated levels of sIFN-alpha R were found in sera of hairy cell leukemia patients. The soluble receptor from serum migrated as a 55 kDa protein in SDS-PAGE, and, as expected, the cross-linked product migrated as a 75 kDa protein. The soluble receptor from urine was found to be a protein of mol. wt. 45 kDa and its cross-linked complex migrated as a 65 kDa protein. The calculated mol. wt. of the entire extracellular domain of the IFN-alpha R prior to post-translational modifications is 47,000. Since there are 12 potential glycosylation points in this extracellular domain, its actual mol. wt. may be as high as 70,000 Da. It is therefore concluded that sIFN-alpha R molecules, corresponding to truncated forms of the extracellular domain of the cell surface IFN-alpha R, are present in human serum and in normal human urine.

Animals↗
PubMed1992

An inducer of NKCF (NK cytotoxic factor) release: localization on target-cell membrane and initial characterization.

Natural killer (NK) cells are probably involved in the elimination of virus-infected cells and of certain tumor cells. NK cell-mediated cytotoxicity (NK-CMC) was extensively studied and was found to consist of several steps. Following recognition and conjugation between the effector and the target cell, the latter one induces release of NK cytotoxic factor (NKCF) from the effector cells. The NKCF binds to the target cell which is subsequently killed. None of the molecules involved in these steps was completely characterized. In the present study it is demonstrated that isolated membranes of target cells can effectively induce the release of NKCF. Furthermore, the activity of such isolated membranes was found to be modulated by interferon (IFN) treatment of the cells prior to membrane isolation. It was therefore concluded that an NKCF-inducing structure (NKIS) is present on plasma membranes and is distinct from the NK-recognition structure. Similarly, the sensitivity to NK-CMC could be transferred from sensitive cells to IFN-gamma-treated (NK-resistant) cells by membrane fusion with the aid of Sendai virus envelope glycoproteins. It is proposed that transfer of NKIS is responsible for the acquired sensitivity to NK-CMC. In addition, it is shown that NKIS activity was recovered following membrane solubilization and reconstitution. Its level on cell surface was modulated by treatment of cells with tunicamycin, thus indicating that NKIS was probably a cell surface glycoprotein.

Cell Membrane↗
PubMed1992

Expression of active human interferon-beta in transgenic plants.

Tobacco plants were transformed with the human gene for interferon-beta (IFN-beta). Transformation was determined by the polymerase chain reaction (PCR), and expression was determined by Western blot analysis, by purifying the IFN from the transgenic plants, and by bioassays indicating its activity in human cells. Plants expressing IFN-beta were self-pollinated. IFN-beta-expressing progeny plants were selected and produced active IFN-beta, indicating stable transformation.

Base Sequence↗
PubMed1992

Identification of DNA elements cooperatively activating proopiomelanocortin gene expression in the pituitary glands of transgenic mice.

The proopiomelanocortin (POMC) gene is highly expressed in adult mouse pituitary anterior lobe corticotrophs and intermediate lobe melanotrophs. To identify the DNA elements important for this tissue-specific expression, we analyzed a series of POMC reporter genes in transgenic mice. A DNA fragment containing rat POMC 5'-flanking sequences from -323 to -34 recapitulated both basal pituitary cell-specific and hormonally stimulated expression in adult mice when fused to a heterologous thymidine kinase promoter. Developmental onset of the reporter gene expression lagged by 1 day but otherwise closely paralleled the normal ontogeny of murine POMC gene expression, including corticotroph activation at embryonic day 14.5 (E14.5) followed by melanotroph activation at E15.5 to E16.5. AtT20 corticotroph nuclear protein extracts interacted with three specific regions of the functional POMC promoter in DNase I protection assays. The positions of these protected sites were -107 to -160 (site 1), -182 to -218 (site 2), and -249 to -281 (site 3). Individual deletions of these footprinted sites did not alter transgene expression; however, the simultaneous deletion of sites 2 and 3 prevented transgene expression in both corticotrophs and melanotrophs. Electrophoretic mobility shift and Southwestern (DNA-protein) assays demonstrated that multiple AtT20 nuclear proteins bound to these footprinted sites. We conclude that the sequences between -323 and -34 of the rat POMC gene promoter are both necessary and sufficient for correct spatial, temporal, and hormonally regulated expression in the pituitary gland. Our data suggest that the three footprinted sites within the promoter are functionally interchangeable and act in combination with promoter elements between -114 and -34. The inability of any reporter gene construction to dissociate basal and hormonally stimulated expression suggests that these DNA elements are involved in both of these two characteristics of POMC gene expression in vivo.

Animals↗
PubMed1992

Interleukin-6 protects ductal breast carcinoma cells from MHC-unrestricted cell-mediated cytotoxicity.

Interleukin-6 (IL-6) has various biological activities including growth stimulation and maturation of B cells into antibody-producing cells, growth stimulation of murine hybridoma and plasmacytoma cells, induction of acute phase proteins, activation of T cell functions, triggering differentiation of various hematopoietic cells, and inhibition of growth of the human ductal breast carcinoma cell line T-47. Recently it was found that IL-6 also has the ability to enhance natural killer (NK) cell activity. In the present study the possible role of IL-6 as a regulator of NK cell-mediated cytotoxicity (NK-CMC) was evaluated. It was found that IL-6 reduced the sensitivity of T-47 cells (a ductal breast carcinoma cell line) to NK-CMC. The mechanism of IL-6-induced protection was studied. IL-6 had no effect on the level of conjugate formation between T-47 cells and NK cells. However, IL-6 reduced the number of dead conjugated T-47 cells. IL-6-treated T-47 cells were also found to be as sensitive as the nontreated cells to the lytic effect of NK cytotoxic factor (NKCF). However, IL-6 appeared to reduce the ability of T-47 cells to induce release of NKCF from NK cells following conjugate formation. Therefore, it is suggested that IL-6 protects T-47 cells from natural killing by the same mechanism as interferon.

Breast Neoplasms↗
PubMed1991

Epidermal growth factor (EGF) induces oligomerization of soluble, extracellular, ligand-binding domain of EGF receptor. A low resolution projection structure of the ligand-binding domain.

Ligand-induced oligomerization is a universal phenomenon among growth factor receptors. Although the mechanism involved is yet to be defined, much evidence indicates that receptor oligomerization plays a crucial role in receptor activation and signal transduction. Here we show that epidermal growth factor (EGF) is able to stimulate the oligomerization of a recombinant, soluble, extracellular ligand-binding domain of EGF receptor. Covalent cross-linking experiments, analysis by sodium dodecyl sulfate-gel electrophoresis, size exclusion chromatography, and electron microscopy demonstrate that receptor dimers, trimers and larger multimers are formed in response to EGF. This establishes that receptor oligomerization is an intrinsic property of the extracellular ligand-binding domain of EGF receptor. Ligand-induced conformational change in the extracellular domain will stimulate receptor-receptor interactions. This may bring about the allosteric change involved in signal transduction from the extracellular domain across the plasma membrane, resulting in the activation of the cytoplasmic kinase domain. Electron microscopic images of individual extracellular ligand-binding domains appear as clusters of four similarly-sized stain-excluding areas arranged around a central, relatively less stain-excluded area. This suggests that the extracellular ligand-binding domain is structurally composed of four separate domains.

Base Sequence↗