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Biomedical subjects

M Rubinstein

Publications and source records attributed to M Rubinstein.

At least 37 records · Page 2Linked to original sources
PubMed2000

Functional uncoupling of adenosine A(2A) receptors and reduced responseto caffeine in mice lacking dopamine D2 receptors.

Dopamine D(2) receptors (Rs) and adenosine A(2A)Rs are coexpressed on striatopallidal neurons, where they mediate opposing actions. In agreement with the idea that D(2)Rs tonically inhibit GABA release from these neurons, stimulation-evoked GABA release was significantly greater from striatal/pallidal slices from D(2)R null mutant (D(2)R(-/-)) than from wild-type (D(2)R(+/+)) mice. Release from heterozygous (D(2)R(+/-)) slices was intermediate. However, contrary to predictions that A(2A)R effects would be enhanced in D(2)R-deficient mice, the A(2A)R agonist CGS 21680 significantly increased GABA release only from D(2)R(+/+) slices. CGS 21680 modulation was observed when D(2)Rs were antagonized by raclopride, suggesting that an acute absence of D(2)Rs cannot explain the results. The lack of CGS 21680 modulation in the D(2)R-deficient mice was also not caused by a compensatory downregulation of A(2A)Rs in the striatum or globus pallidus. However, CGS 21680 significantly stimulated cAMP production only in D(2)R(+/+) striatal/pallidal slices. This functional uncoupling of A(2A)Rs in the D(2)R-deficient mice was not explained by reduced expression of G(s), G(olf), or type VI adenylyl cyclase. Locomotor activity induced by the adenosine receptor antagonist caffeine was significantly less pronounced in D(2)R(-/-) mice than in D(2)R(+/+) and D(2)R(+/-) mice, further supporting the idea that D(2)Rs are required for caffeine activation. Caffeine increased c-fos only in D(2)R(-/-) globus pallidus. The present results show that a targeted disruption of the D(2)R reduces coupling of A(2A)Rs on striatopallidal neurons and thereby responses to drugs that act on adenosine receptors. They also reinforce the ideas that D(2)Rs and A(2A)Rs are functionally opposed and that D(2)R-mediated effects normally predominate.

Animals↗
PubMed2000

Selective increase of Nurr1 mRNA expression in mesencephalic dopaminergic neurons of D2 dopamine receptor-deficient mice.

The orphan nuclear receptor Nurr1 is critical for the survival of mesencephalic dopaminergic precursor neurons. Little is known about the mechanisms that regulate Nurr1 expression in vivo. Other members of this receptor family have been shown to be activated by dopamine. We sought to determine if Nurr1 expression is also regulated by endogenous dopamine through dopamine receptors. Consequently, we investigated the expression of Nurr1 mRNA in genetically modified mice lacking both functional copies of the D2 dopamine receptor gene and in their congenic siblings. Quantitative in situ hybridization demonstrated a significant increased expression of Nurr1 mRNA in the substantia nigra pars compacta and the ventral tegmental area of D2 dopamine receptor -/- mice. No change in Nurr1 expression was detected in other brain regions, such as the habenular nuclei and temporal cortex. Among the cell groups studied, mesencephalic dopaminergic neurons are unique in that they express both Nurr1 and the D2 dopamine receptor, and synthesize dopamine. Thus, it seems plausible that the selective increase in Nurr1 expression observed in D2 receptor-deficient mice is the consequence of an impaired dopamine autoreceptor function.

Animals↗
PubMed2000

Adsorption of polyelectrolytes at an oppositely charged surface

We develop a scaling theory of polyelectrolyte adsorption at an oppositely charged surface. At low surface charge densities, the thickness of the adsorbed layer is determined by the balance between electrostatic attraction to the charged surface and chain entropy. At high surface charge densities, it is determined by the balance between electrostatic attraction and short-range monomer-monomer repulsion. These different stabilizing mechanisms result in the nonmonotonic dependence of the layer thickness on the surface charge density.

Journal Article↗
PubMed2000

Unexpected scenario of glass transition in polymer globules: an exactly enumerable model.

We introduce a lattice model of glass transition in polymer globules. This model exhibits ergodicity breaking in which the disjoint regions of phase space do not arise uniformly, but as small chambers whose number increases exponentially with polymer density. Chamber sizes obey power law distribution, making phase space similar to a fractal foam. This clearly demonstrates the importance of the phase space geometry and topology in describing any glass-forming system, such as semicompact polymers during protein folding.

Glass↗
PubMed2000

IL-18 binding protein increases spontaneous and IL-1-induced prostaglandin production via inhibition of IFN-gamma.

IL-18 shares with IL-1 the same family of receptors and several identical signal transduction pathways. Because of these similarities, IL-18 was investigated for its ability to induce prostaglandin E(2) (PGE(2)) synthesis in human peripheral blood mononuclear cells (PBMC), a prominent, proinflammatory property of IL-1. IL-18 was highly active in PBMC by inducing the synthesis of the chemokine IL-8; however, no induction of PGE(2) synthesis nor cyclooxygenase type-2 gene expression was observed in PBMC stimulated with IL-18. In the same cultures, IL-1beta induced a 12-fold increase in PGE(2). Although IL-1beta-induced IL-8 synthesis was augmented 3-fold by IL-18, IL-18 suppressed IL-1beta-induced PGE(2) production by 40%. The suppressive effect of IL-18 on PGE(2) production was mediated by interferon (IFN)-gamma because anti-human IFN-gamma-antibody prevented IL-18-induced reduction in PGE(2). Consistent with these observations, IL-12, a known inducer of IFN-gamma, augmented IL-1beta-induced IFN-gamma but suppressed IL-1beta-induced PGE(2) by 75%. IL-18 binding protein (IL-18BP) is a naturally occurring and specific inhibitor of IL-18. When recombinant IL-18BP was added to PBMC cultures, unexpectedly, spontaneous PGE(2) production increased. PGE(2) production was also increased by the addition of IL-18BP to PBMC stimulated with either IL-1beta or IL-12 and also in whole blood cultures stimulated with Staphylococcus epidermidis. These studies demonstrate that IL-18BP decreases endogenous IL-18 activity by reducing IFN-gamma-mediated responses.

Cells, Cultured↗
PubMed2000

Structural requirements of six naturally occurring isoforms of the IL-18 binding protein to inhibit IL-18.

A novel, constitutively expressed and secreted IL-18 binding protein (IL-18BP) neutralizes IL-18 and thereby suppresses the production of IFN-gamma, resulting in reduced T-helper type 1 immune responses. In the present study, four human and two mouse isoforms, resulting from mRNA splicing and found in various cDNA libraries, were expressed, purified, and assessed for binding and neutralization of IL-18 biological activities. Human IL-18BP isoform a (IL-18BPa) exhibited the greatest affinity for IL-18 with a rapid on-rate, a slow off-rate, and a dissociation constant (K(d)) of 399 pM. IL-18BPc shares the Ig domain of IL-18BPa except for the 29 C-terminal amino acids; the K(d) of IL-18BPc is 10-fold less (2.94 nM). Nevertheless, IL-18BPa and IL-18BPc neutralize IL-18 >95% at a molar excess of two. IL-18BPb and IL-18BPd isoforms lack a complete Ig domain and lack the ability to bind or neutralize IL-18. Murine IL-18BPc and IL-18BPd isoforms, possessing the identical Ig domain, also neutralize >95% murine IL-18 at a molar excess of two. However, murine IL-18BPd, which shares a common C-terminal motif with human IL-18BPa, also neutralizes human IL-18. Molecular modeling identified a large mixed electrostatic and hydrophobic binding site in the Ig domain of IL-18BP, which could account for its high affinity binding to the ligand. It is likely that preferential secretion of functional and nonfunctional isoforms of IL-18BP affect the immune response.

Amino Acid Sequence↗
PubMed2000

IL-18 regulates IL-1beta-dependent hepatic melanoma metastasis via vascular cell adhesion molecule-1.

Proinflammatory cytokines, including IL-1beta and tumor necrosis factor-alpha (TNF-alpha), promote cancer cell adhesion and liver metastases by up-regulating the expression of vascular cell adhesion molecule-1 (VCAM-1) on hepatic sinusoidal endothelium (HSE). In this study, hepatic metastasis after intrasplenically injected mouse B16 melanoma (B16M) cells was reduced 84-95% in mice with null mutations for either IL-1beta or the IL-1beta-converting enzyme (ICE, caspase-1) compared with wild-type mice. On day 12, 47% of wild-type mice were dead compared with 19% of either IL-1beta or ICE-deficient mice. In vitro, conditioned medium from B16M cells (B16M-CM) induced the release of TNF-alpha and IL-1beta from cultures of primary murine HSE. The effect of B16M-CM on HSE resulted in increased numbers of B16M cells adhering to HSE, which was completely abrogated by a specific inhibitor of ICE, anti-IL-18 or IL-18-binding protein. Exogenous IL-18 added to HSE also increased the number of adhering melanoma cells; however, this was not affected by IL-1 receptor blockade or TNF neutralization but rather by anti-VCAM-1. These results demonstrate a role for IL-1beta and IL-18 in the development of hepatic metastases of B16M in vivo. In vitro, soluble products from B16M cells stimulate HSE to sequentially release TNF-alpha, IL-1beta, and IL-18. The IL-18 cytokine increases expression of VCAM-1 and the adherence of melanoma cells.

Animals↗
PubMed2000

Quantitative analysis of the dopamine D4 receptor in the mouse brain.

The D4 receptor (D4R), a member of the dopamine D2-like receptor family, has been implicated in the pathophysiology of several diseases and has been the target of various investigations regarding its distribution and quantification. The brain distribution of the D4R has been well described in various species, but the quantification is still an issue of controversy, because no specific ligand is commercially available. To circumvent this difficulty we have performed a biochemical and autoradiographical study in brain samples obtained from mice lacking D4Rs and their wild-type siblings; comparison of their binding parameters allows a more accurate quantification of the members of the D2-like receptor family (D2, D3, and D4 receptors). We found that the distribution of D2-like receptors in mouse brain is similar to that of rat brain, i.e., caudate putamen, nucleus accumbens, olfactory tubercle, and hippocampus. The contribution of the D4R to the overall population of D2-like receptors is 17% in nucleus accumbens, 21% in caudate putamen and olfactory tubercle, and 40% in hippocampus. Based on our study we conclude that nemonapride probably binds to nondopaminergic sites that if not properly blocked may lead to overestimations of D4R levels. We observed that the experimental condition that better estimates the density of D4 receptors is the displacement of D2 and D3 [3H]nemonapride binding sites with cold raclopride.

Animals↗
PubMed2000

Production of a biologically active human interleukin 18 requires its prior synthesis as PRO-IL-18.

Interleukin (IL-)18 is an activator of NK cells and a co-inducer of Th(1)cytokines, sharing structural features with the IL-1 family of proteins. Unlike most other cytokines, IL-18 and IL-1beta lack a signal peptide, have an all beta-pleated sheet structure and are synthesized as biologically inactive precursors (pro-IL-18 and pro-IL-1beta). These precursors are cleaved by caspase-1 (IL-1beta-converting enzyme, ICE) to form the biologically active mature cytokines. Direct expression of mature recombinant human IL-18 in E. coli resulted in a partially active cytokine. We tested the possibility that correct folding of huIL-18 requires its prior synthesis as pro-IL-18. Because caspase-1 is not readily available, we constructed an expression vector encoding human pro-IL-18 in which the caspase-1 cleavage site was mutated into a factor Xa site. To facilitate purification, the mutated pro-IL-18 cDNA was fused in frame to a glutathione-S-transferase (GST) coding sequence. The GST-pro-IL-18 fusion protein was expressed in E. coli, captured on glutathione agarose and mature human IL-18, exhibiting high biological activity was released upon cleavage with factor Xa. This result indicates that correct folding of huIL-18 occurs at the level of pro-IL-18 and provides a practical way to produce biologically active huIL-18.

Amino Acid Sequence↗
PubMed2000

Lack of operant ethanol self-administration in dopamine D2 receptor knockout mice.

RATIONALE: Dopamine D2 receptors are postulated to play an important role in modulating the reinforcing effects of abused drugs including ethanol. OBJECTIVES: This experiment examined operant ethanol self-administration in dopamine D2 receptor knockout (KO) mice and wild-type (WT) mice using a continuous access procedure. METHODS: Adult male KO and WT mice were trained in 30-min sessions to perform a lever press response for access to 10% v/v ethanol. After training, the mice were placed in test chambers on a continuous (23 h/day) basis with access to food (one lever press, i.e., FR1), 10% v/v ethanol (four lever presses, i.e., FR4), and water from a sipper tube (phase 1). After 30 consecutive sessions, response patterns were determined for 0, 5, 10, 20 and 30% v/v ethanol (phase 2). Saccharin (0.2% w/v) was subsequently added to the ethanol mixture and responding was examined for 0, 5, 10 and 20% ethanol (phase 3). RESULTS: During phase 1, WT mice displayed higher ethanol-lever responding compared to KO mice. Food lever responding and water intake was the same in both genotypes. During phase 2, WT mice displayed concentration-dependent ethanol lever responding, whereas KO mice responded at low rates regardless of ethanol concentration. WT mice also responded more for food compared to KO mice. Each genotype showed similar water intakes except at the 20% ethanol concentration, where WT mice had lower intakes. During phase 3, WT mice continued to show higher responding for all concentrations including saccharin alone. WT mice also continued to respond more for food compared to KO mice, but drank less water. In each phase, WT mice displayed episodic (bout) responding on the ethanol lever. KO mice did not respond for ethanol in bouts. CONCLUSIONS: Reduced responding in the KO mice for several reinforcers including ethanol indicates a more general role for dopamine D2 receptors in motivated responding rather than a specific role in ethanol reinforcement.

Animals↗
PubMed2000

Ethanol-conditioned place preference is reduced in dopamine D2 receptor-deficient mice.

Pharmacological blockade studies have supported a role of the dopamine system in ethanol reward for many years, but receptor subtype specificity has been difficult to establish. Recently, genetically engineered mice lacking functional dopamine D2 receptors have been shown to drink less ethanol in a two-bottle choice task. To determine whether reduced ethanol intake reflects a reduction in ethanol reward, D2 receptor-deficient [knockout (KO)] mice were compared to heterozygous (HET) and wild-type (WT; C57BL/6xDBA/2 F2 hybrid) mice in a place conditioning task. Under conditions that produced reliable place preference in both WT and HET mice, KO mice showed no evidence of place conditioning, suggesting that D2 receptor gene inactivation reduced ethanol reward or the ability to learn about ethanol reward. Consistent with previous findings, this mutation also produced a gene dose-related reduction in basal activity levels. Moreover, KO and HET mice showed enhancement of ethanol-stimulated activity relative to WT mice. However, differences in basal and ethanol-stimulated activity did not explain the differences in place conditioning. Overall, this study strongly supports the conclusion that dopamine D2 receptors normally influence ethanol reward in mice.

Animals↗
PubMed2000

The indirect basal ganglia pathway in dopamine D(2) receptor-deficient mice.

Recent pathophysiological models of basal ganglia function in Parkinson's disease predict that specific neurochemical changes in the indirect pathway would follow the lack of stimulation of D(2) dopamine receptors. Post mortem studies of the basal ganglia in genetically modified mice lacking functional copies of the D(2) dopamine receptor gene allowed us to test these predictions. When compared with their congenic N(5) wild-type siblings, mice lacking D(2) receptors show an increased expression of enkephalin messenger RNA in the striatum, and an increased activity and expression of cytochrome oxidase I in the subthalamic nucleus, as expected. In addition, D(2) receptor-deficient mice display a reduced expression of glutamate decarboxylase-67 messenger RNA in the globus pallidus, as the basal ganglia model predicts. This reduction contrasts with the lack of change or increase in glutamate decarboxylase-67 messenger RNA expression found in animals depleted of dopamine after lesions of the mesostriatal dopaminergic system. Furthermore, D(2) receptor-deficient mice show a significant decrease in substance P messenger RNA expression in the striatonigral neurons which form the direct pathway. Finally, glutamate decarboxylase-67 messenger RNA expression in the basal ganglia output nuclei was not affected by mutations in the D(2) receptor gene, a fact that could probably be related to the absence of a parkinsonian locomotor phenotype in D(2) receptor-deficient mice. In summary, these findings provide compelling evidence demonstrating that the lack of endogenous stimulation of D(2) receptors is sufficient to produce subthalamic nucleus hyperactivity, as assessed by cytochrome oxidase I histochemistry and messenger RNA expression, and strongly suggest the existence of interactions between the basal ganglia direct and indirect pathways.

Animals↗
PubMed2000

Disparate spinal and supraspinal opioid antinociceptive responses in beta-endorphin-deficient mutant mice.

The role of endogenous opioid systems in the analgesic response to exogenous opiates remains controversial. We previously reported that mice lacking the peptide neurotransmitter beta-endorphin, although unable to produce opioid-mediated stress-induced antinociception, nevertheless displayed intact antinociception after systemic administration of the exogenous opiate morphine. Morphine administered by a peripheral route can activate opioid receptors in both the spinal cord and brain. However, beta-endorphin neuronal projections are confined predominantly to supraspinal nociceptive nuclei. Therefore, we questioned whether the absence of beta-endorphin would differentially affect antinociceptive responses depending on the route of opiate administration. Time- and dose-response curves were obtained in beta-endorphin-deficient and matched wild-type C57BL/6 congenic control mice using the tail-immersion/withdrawal assay. Null mutant mice were found to be more sensitive to supraspinal (i.c.v.) injection of the micro-opioid receptor-selective agonists, morphine and D-Ala(2)-MePhe(4)-Gly-ol(5) enkephalin. In contrast, the mutant mice were less sensitive to spinal (i.t.) injection of these same drugs. Quantitative receptor autoradiography revealed no differences between genotypes in the density of mu, delta, or kappa opioid receptor binding sites in either the spinal cord or pain-relevant supraspinal areas. Thus we report that the absence of a putative endogenous ligand for the mu-opioid receptor results in opposite changes in morphine sensitivity between discrete areas of the nervous system, which are not simply caused by changes in opioid receptor expression.

Analgesics, Opioid↗
PubMed2000

The neutralization of type I IFN biologic actions by anti-IFNAR-2 monoclonal antibodies is not entirely due to inhibition of Jak-Stat tyrosine phosphorylation.

A panel of monoclonal antibodies (mAb) derived against human interferon-alpha/beta receptor-2 (IFNAR-2) was evaluated for their ability to antagonize the biologic effects of type 1 interferons (IFN-alpha1, IFN-alpha2a, and IFN-beta). Anti-IFNAR-2 mAb 117.7, 35.9, 53.2, and 51.44 neutralized type I IFN-mediated antiviral, antiproliferative, and major histocompatibility complex (MHC) class I upregulation functions. However, only mAb 51.44 neutralized IFN-alpha2a and IFN-beta-mediated natural killer (NK) cell cytotoxicity. In BIAcore and cell binding studies, only mAb 51.44 and 234.28 inhibited IFN-alpha2a and IFN-beta binding to its receptor. The receptor blockade by mAb 51.44 and 234.28 resulted in the inhibition of IFN-alpha2a and IFN-beta-induced tyrosine phosphorylation of Jak1, Tyk2, Stat1/2/3, and IFNAR-1/2 and inhibition of IFN-stimulated gene factor 3 (ISGF3) formation. mAb 117.7, 35.9, and 53.2, although antagonists of IFN's biologic activities, did not block the binding of IFN-alpha/beta to its receptor. The 117.7 mAb, representative of this class of receptor nonblocking mAb, induced hyper-tyrosine phosphorylation of IFNAR-2 in the presence of IFN-alpha/beta but did not inhibit IFN-alpha/beta-induced Jak-Stat tyrosine phosphorylation and ISGF3 complex formation. These results show that the neutralization of type I IFN biologic actions by anti-IFNAR-2 mAb cannot be entirely explained by inhibition of Jak-Stat tyrosine phosphorylation.

Antibodies, Monoclonal↗
PubMed2000

Harvesting the human genome: the Israeli perspective.

The post-genome era is at our door, and soon the complete human genome sequence will be available for the next set of goals. Israel is well equipped and skilled to join the worldwide harvest of the human genome, but additional massive government investment is required. This will affect various domains of activity, including the fields of diagnostics and therapeutics. The technologies and know-how described above constitute the basis for future human genome applications in Israel.

Computational Biology↗
PubMed1999

D3 receptor knockdown through antisense oligonucleotide administration supports its inhibitory role in locomotion.

To study the specific contribution of the D3 dopamine receptor in the generation of locomotor activity, total or partially dopamine-depleted rats were pretreated with an antisense oligodeoxynucleotide for the D3 receptor (D3R-as) and locomotor activity induced by apomorphine was measured. A 35.7% increase in locomotor activity was seen in the totally dopamine-depleted rats pretreated with the D3R-as, whereas the same antisense, caused a significantly greater increase in the locomotor response (95%) in the partially dopamine-depleted rats compared with control groups (pretreated with a control oligodeoxynucleotide or vehicle). In situ autoradiography for D3 receptors showed a 27% fall in the density of D3 receptors in the islands of Calleja compared with control animals. Our results seem to confirm that D3 receptors exert an inhibitory effect on locomotor activity, through the stimulation of both pre- and postsynaptic components.

Adrenergic Uptake Inhibitors↗