Biomedical subjects

D A Greenberg

Publications and source records attributed to D A Greenberg.

At least 19 recordsLinked to original sources

PubMed2001

Evidence for association of polycystic ovary syndrome in caucasian women with a marker at the insulin receptor gene locus.

The polycystic ovary syndrome (PCOS) is one of the commonest female endocrinopathies affecting 5-10% of women of reproductive age. The disorder, characterized by chronic anovulation and signs of hyperandrogenism, results from a complex interaction between genetic predisposing factors and environmental triggers. We have studied 85 Caucasian PCOS patients and 87 age-matched Caucasian control women for associations with four candidate genes: follistatin, CYP19 (aromatase), CYP17a, and the insulin receptor (INSR). These genes were analyzed using microsatellite markers located near or inside the genes. We found that only the insulin receptor gene marker D19S884 was significantly associated with PCOS (p=0.006 and even after a conservative correction p=0.042). The INSR gene region was then fine mapped with an additional panel of 9 markers but only marker D19S884, located 1 cM telomeric to the INSR gene, was again associated with PCOS. In conclusion, our results suggested that a susceptibility gene for PCOS was located on chromosome 19p13.3 in the insulin receptor gene region. It remains to be determined if this susceptibility gene is the insulin receptor gene itself or a closely located gene. Since insulin stimulates androgen secretion by the ovarian stroma it is likely that INSR function in the ovary is involved in the genetic susceptibility ot PCOS.

Adolescent↗

PubMed2001

Genome scan of idiopathic generalized epilepsy: evidence for major susceptibility gene and modifying genes influencing the seizure type.

Idiopathic generalized epilepsy (IGE) is a common, complex disease with an almost exclusively genetic etiology but with variable phenotypes. Clinically, IGE can be divided into different syndromes. Varying lines of evidence point to the involvement of several interacting genes in the etiology of IGE. We performed a genome scan in 91 families ascertained through a proband with adolescent-onset IGE. The IGEs included juvenile myoclonic epilepsy (JME), juvenile absence epilepsy (JAE), and epilepsy with generalized tonic clonic seizures (EGTCS). Our linkage results support an oligogenic model for IGE, with strong evidence for a locus common to most IGEs on chromosome 18 (lod score 4.4/5.2 multipoint/two-point) and other loci that may influence specific seizure phenotypes for different IGEs: a previously identified locus on chromosome 6 for JME (lod score 2.5/4.2), a locus on chromosome 8 influencing non-JME forms of IGE (lod score 3.8/2.5), and, more tentatively, two newly discovered loci for absence seizures on chromosome 5 (lod scores 3.8/2.8 and 3.4/1.9). Our data also suggest that the genetic classification of different forms of IGE is likely to cut across the clinical classification of these subforms of IGE. We hypothesize that interactions of different combinations of these loci produce the related heterogeneous phenotypes seen in IGE families.

Adolescent↗

PubMed2000

Erythropoietin protects cultured cortical neurons, but not astroglia, from hypoxia and AMPA toxicity.

In addition to its better-known hemopoietic action, erythropoietin (Epo) has neurotrophic properties and neuroprotective effects in some models of hypoxic-ischemic injury. To define further the cellular mechanisms underlying neuroprotection by Epo, we studied the effects of Epo on hypoxia with glucose deprivation in cultured rat cortical neurons and astroglia and on exposure to excitotoxins in cultured rat cortical neurons. Epo (30 pM) reduced neuronal, but not astroglial, cell death from hypoxia with glucose deprivation, and also attenuated the neurotoxic effect of (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), but not other excitotoxins. Epo appears to protect against cerebral ischemia through a direct effect on neurons that may be mediated in part by AMPA receptors.

Animals↗

PubMed2000

Vascular endothelial growth factor: direct neuroprotective effect in in vitro ischemia.

Vascular endothelial growth factor (VEGF) is a hypoxia-inducible angiogenic peptide with recently identified neurotrophic effects. Because some neurotrophic factors can protect neurons from hypoxic or ischemic injury, we investigated the possibility that VEGF has similar neuroprotective properties. In HN33, an immortalized hippocampal neuronal cell line, VEGF reduced cell death associated with an in vitro model of cerebral ischemia: at a maximally effective concentration of 50 ng/ml, VEGF approximately doubled the number of cells surviving after 24 h of hypoxia and glucose deprivation. To investigate the mechanism of neuroprotection by VEGF, the expression of known target receptors for VEGF was measured by Western blotting, which showed that HN33 cells expressed VEGFR-2 receptors and neuropilin-1, but not VEGFR-1 receptors. The neuropilin-1 ligand placenta growth factor-2 failed to reproduce the protective effect of VEGF, pointing to VEGFR-2 as the site of VEGF's neuroprotective action. Two phosphatidylinositol 3'-kinase inhibitors, wortmannin and LY294002, reversed the neuroprotective effect of VEGF, implicating the phosphatidylinositol 3'-kinase/Akt signal transduction system in VEGF-mediated neuroprotection. VEGF also protected primary cultures of rat cerebral cortical neurons from hypoxia and glucose deprivation. We conclude that in addition to its known role as an angiogenic factor, VEGF may exert a direct neuroprotective effect in hypoxic-ischemic injury.

Androstadienes↗

PubMed2000

Expression of the RNA-binding protein TIAR is increased in neurons after ischemic cerebral injury.

T-cell restricted intracellular antigen-related protein (TIAR) is an RNA recognition motif-type RNA-binding protein that has been implicated in the apoptotic death of T-lymphocytes and retinal pigment epithelial cells. Western blots prepared with a monoclonal antibody against TIAR showed expression in normal rat hippocampus, and induction by 15 min of global cerebral ischemia. This increased expression was evident at 8 hr after ischemia and maximal at 24 hr, whereas expression at 72 hr was reduced below basal levels. Expression of TIAR protein was also increased in parietal cortex 6 and 24 hr after 90 min of focal cerebral ischemia induced by middle cerebral artery (MCA) occlusion, as well as in cultured cortical neurons and astroglia after exposure to hypoxia in vitro. Immunocytochemistry showed that increased expression of TIAR occurred mainly in the CA1 sector of hippocampus 24 hr after global ischemia, and in cortical and striatal neurons 24 hr after 20 or 90 min of focal ischemia. Double-labeling studies showed that TIAR protein expression was co-localized with DNA damage in neuronal cells. The findings suggest that TIAR may be involved in neuronal cell death after cerebral ischemic injury.

Animals↗

PubMed2000

No evidence for a major susceptibility locus for juvenile myoclonic epilepsy on chromosome 15q.

Juvenile myoclonic epilepsy (JME) is a distinct epileptic syndrome with a complex mode of inheritance. Several studies found evidence for a locus involved in JME on chromosome 6 near the HLA region. Recently, Elmslie et al. [1997] reported evidence of linkage in JME to chromosome 15q14 assuming a recessive mode of inheritance with 50% penetrance and 65% linked families. The area on chromosome 15q14 encompasses the location of the gene for the alpha-7 subunit of the nicotinic acetylcholine receptor. This could fit the hypothesis that there are two interacting loci, one on chromosome 6 and on chromosome 15 or that there is genetic heterogeneity in JME. In an independent dataset of JME families, we tested for linkage to chromosome 15 but found little evidence for linkage. Moreover, families with more than one family member affected with JME provide a lodscore of 3.4 for the HLA-DR/DQ haplotype on chromosome 6. The lodscore for these same families on chromosome 15q14 is <-2 assuming homogeneity and the maximum lodscore is 0.2 assuming alpha =.25. Only one of these families has a negative lodscore on chromosome 6 and a positive lodscore of 0.5 on chromosome 15q14. Our results indicate that this possible gene on chromosome 15 plays at most a minor role in our JME families. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:49-52, 2000.

Chromosome Mapping↗

PubMed2000

Endocannabinoids protect cerebral cortical neurons from in vitro ischemia in rats.

The endogenous cannabinoids (endocannabinoids) anandamide and 2-arachidonylglycerol increased cell viability in cerebral cortical neuron cultures subjected to 8 h of hypoxia and glucose deprivation. This effect was observed at nanomolar concentrations, was reproduced by a non-hydrolyzable analog of anandamide, and was unaltered by CB1 or CB2 cannabinoid receptor antagonists. Like synthetic cannabinoids, endocannabinoids can protect neurons from hypoxic injury, and may represent endogenous neuroprotective molecules in cerebral ischemia.

Animals↗

PubMed2000

Induction of vascular endothelial growth factor and hypoxia-inducible factor-1alpha by global ischemia in rat brain.

Vascular endothelial growth factor is an angiogenic and neurotrophic peptide whose expression is transcriptionally induced in hypoxic tissues through the action of hypoxia-inducible factor-1alpha. To determine if this signaling pathway is activated in the ischemic brain, and might therefore participate in adaptive processes such as angiogenesis and neuroprotection, we examined the expression of vascular endothelial growth factor and hypoxia-inducible factor-1alpha in cerebral cortex and hippocampus following transient global cerebral ischemia in the rat. Northern analysis showed ischemia-inducible expression of multiple vascular endothelial growth factor messenger ribonucleic acid splice variants between 4 and 24h. Western analysis and immunocytochemistry demonstrated the concerted induction of vascular endothelial growth factor and hypoxia-inducible factor-1alpha in the same, apparently neuronal, cells in vulnerable regions of cortex and hippocampus after 15min of ischemia, which persisted for as long as 4 to 72h of reperfusion. These findings demonstrate that hypoxia-sensitive vascular endothelial growth factor signaling can be induced in neurons in global cerebral ischemia in vivo, and are consistent with the hypothesis that ischemic insults trigger hypoxia-sensing and adaptive downstream molecular responses in central neurons.

Alternative Splicing↗

PubMed2000

Induction of vascular endothelial growth factor receptors and phosphatidylinositol 3'-kinase/Akt signaling by global cerebral ischemia in the rat.

Vascular endothelial growth factor is an angiogenic peptide that binds to tyrosine kinase receptors on target cells to activate signal transduction pathways involving phosphatidylinositol 3'-kinase and the serine-threonine protein kinase, Akt. To determine whether this signaling pathway is activated in cerebral ischemia, we examined the expression of vascular endothelial growth factor receptors 1 and 2, and phosphatidylinositol 3'-kinase-activated phospho-Akt, in the cerebral cortex and hippocampus following transient global cerebral ischemia in the rat. Western blot analysis and immunocytochemistry demonstrated induction of vascular endothelial growth factor receptor 1 and 2 expression, and of anti-phosphatidylinositol 3'-kinase-immunoprecipitated phospho-Akt, in vulnerable regions of the cortex and hippocampus after 15 min of global ischemia and 4-72 h of reperfusion. These findings demonstrate that vascular endothelial growth factor receptors and receptor-coupled signal transduction pathways are induced in ischemic brain in vivo, and could therefore participate in endogenous neuroprotective responses to ischemia.

Animals↗

PubMed2000

Increased expression of apoptosis-linked gene 2 (ALG2) in the rat brain after temporary focal cerebral ischemia.

Calcium is an important mediator of programmed cell death induced by transient cerebral ischemia, and calcium-binding proteins have been implicated in calcium-regulated signal transduction. Apoptosis-linked gene 2 is a calcium-binding protein required for cell death induced by different apoptotic stimuli. By Western blot analysis, we found that apoptosis-linked gene 2 protein was expressed in normal brains, and that expression increased in ischemic brains after 20 or 90 min of transient focal cerebral ischemia. Immunocytochemistry showed increased apoptosis-linked gene 2 protein expression in frontal cortex, a region where neurons underwent ischemic stress but still survived, after 20 or 90 min of focal cerebral ischemia. Apoptosis-linked gene 2 protein was also up-regulated in the ischemic border-zone of parietal cortex 24h after 20 min of focal ischemia, and was remarkably over-expressed in the caudate-putamen and parietal cortex, (where cells are destined to die) 24h after 90 min of ischemia. The expression pattern of apoptosis-linked gene 2 protein was similar to that of deoxyribonucleic acid damage detected by Klenow labeling assay. Our results suggest that apoptosis-linked gene 2 may be involved in the regulation of cell death after transient focal cerebral ischemia.

Animals↗

PubMed2000

Reproducibility and complications in gene searches: linkage on chromosome 6, heterogeneity, association, and maternal inheritance in juvenile myoclonic epilepsy.

Evidence for genetic influences in epilepsy is strong, but reports identifying specific chromosomal origins of those influences conflict. One early study reported that human leukocyte antigen (HLA) markers were genetically linked to juvenile myoclonic epilepsy (JME); this was confirmed in a later study. Other reports did not find linkage to HLA markers. One found evidence of linkage to markers on chromosome 15, another to markers on chromosome 6, centromeric to HLA. We identified families through a patient with JME and genotyped markers throughout chromosome 6. Linkage analysis assuming equal male-female recombination probabilities showed evidence for linkage (LOD score 2.5), but at a high recombination fraction (theta), suggesting heterogeneity. When linkage analysis was redone to allow independent male-female thetas, the LOD score was significantly higher (4.2) at a male-female theta of.5,.01. Although the overall pattern of LOD scores with respect to male-female theta could not be explained solely by heterogeneity, the presence of heterogeneity and predominantly maternal inheritance of JME might explain it. By analyzing loci between HLA-DP and HLA-DR and stratifying the families on the basis of evidence for or against linkage, we were able to show evidence of heterogeneity within JME and to propose a marker associated with the linked form. These data also suggest that JME may be predominantly maternally inherited and that the HLA-linked form is more likely to occur in families of European origin.

Alleles↗

PubMed2000

Limited genetic susceptibility to severe Graves' ophthalmopathy: no role for CTLA-4 but evidence for an environmental etiology.

Graves' disease (GD) is an autoimmune thyroid disease (AITD) characterized by hyperthyroidism and by the occurrence of a distinctive ophthalmopathy (orbitopathy), which presents with varying degrees of severity. Graves' disease clusters in families but the importance of heredity in the pathogenesis of the associated ophthalmopathy is unclear. We have studied the family history of 114 consecutive, ethnically mixed patients with severe Graves' ophthalmopathy (GO). Patients were selected by unambiguous single ascertainment. Seventy-seven percent of patients were female and 59% smoked. The mean age at onset of their GD was 43 years (range 17-78 years). Forty-one patients (36%) had a family history of AITD, defined as a first- and/or a second-degree relative affected with either Graves' disease (GD) or Hashimoto's thyroiditis (HT). The segregation ratio for AITD in nuclear families in our ascertained Graves' ophthalmopathy families was 0.07 (0.12 in Caucasians only). Hence, the higher prevalence of AITD among relatives of Graves' ophthalmopathy patients agreed with the known genetic predisposition to AITD and this predisposition was stronger in women than in men. However, only 3 of the 114 patients had a family history of severe Graves' ophthalmopathy (all second-degree relatives) and the segregation ratio for GO was 0. These data did not support a major role for familial factors in the development of severe Graves' ophthalmopathy distinct from Graves' disease itself. In addition, we tested 4 candidate genes, human leukocyte antigen (HLA), tumor necrosis factor-beta (TNF-beta), CTLA-4 and the thyrotropin receptor (TSHR), for association with Graves' ophthalmopathy. These were negative except for the HLA and CTLA-4 genes, which were found to be weakly associated with GO giving similar relative risk (RR) as in GD patients without ophthalmopathy. These data suggested that environmental factors, rather than major genes, were likely to predispose certain individuals with AITD to severe Graves' ophthalmopathy. Smoking remains one example of such potential external insults.

Abatacept↗

PubMed2000

Vascular endothelial growth factor rescues HN33 neural cells from death induced by serum withdrawal.

Vascular endothelial growth factor (VEGF) is an angiogenic factor with neurotrophic effects in the peripheral nervous system. To determine if VEGF can also promote the survival of central neurons, we examined its effect on HN33 (mouse hippocampal neuron x neuroblastoma) cells deprived of serum. Serum-deprived HN33 cells expressed VEGFR-2 receptors, which, in the presence of VEGF, interacted with the downstream signaling molecules phosphatidylinositol 3'-kinase and phospho-Akt, as demonstrated by immunoprecipitation and Western blotting. Treatment of serum-deprived HN33 cells with VEGF also stimulated the phosphorylation of IkappaB-alpha and nuclear translocation of the transcription factor NF-kappaB. Withdrawal of serum for 24 h reduced HN33 cell viability by approximately 50% in the absence of VEGF, but by only approximately 20% in the presence of 100 ng/mL of VEGF. These findings support a neurotrophic role for VEGF in the central nervous system, which may be mediated through VEGFR-2 receptors, the protein kinases phosphatidylinositol 3'-kinase and Akt, and the transcription factor NK-kappaB. Thus, VEGF, like other neurotrophic factors, could exert protective effects in acute or chronic neurodegenerative disorders.

Animals↗

PubMed2000

CB1 cannabinoid receptor induction in experimental stroke.

Cannabinoids protect cortical neurons from ischemic injury by interacting with CB1 receptors. Because a variety of neuroprotective genes are induced in cerebral ischemia, we examined the effect of experimental stroke, produced by 20 minutes of middle cerebral artery occlusion in rats, on CB1 receptor expression. Western blotting and immunohistochemistry showed that CB1 expression on neurons was increased in the arterial boundary zone of the cortical mantle, beginning by 2 hours and persisting for 72 hours or more after ischemia These findings are consistent with a neuroprotective role for endogenous cannabinoid signaling pathways and with a potential therapeutic role in stroke for drugs that activate CB1 receptors.

Animals↗

PubMed1999

Cannabinoids and neuroprotection in global and focal cerebral ischemia and in neuronal cultures.

Marijuana and related drugs (cannabinoids) have been proposed as treatments for a widening spectrum of medical disorders. R(+)-[2, 3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1, 4-benzoxazin-yl]-(1-naphthalenyl)methanone mesylate (R(+)-WIN 55212-2), a synthetic cannabinoid agonist, decreased hippocampal neuronal loss after transient global cerebral ischemia and reduced infarct volume after permanent focal cerebral ischemia induced by middle cerebral artery occlusion in rats. The less active enantiomer S(-)-WIN 55212-3 was ineffective, and the protective effect of R(+)-WIN 55212-2 was blocked by the specific central cannabinoid (CB1) cannabinoid receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide-hydrochloride. R(+)-WIN 55212-2 also protected cultured cerebral cortical neurons from in vitro hypoxia and glucose deprivation, but in contrast to the receptor-mediated neuroprotection observed in vivo, this in vitro effect was not stereoselective and was insensitive to CB1 and CB2 receptor antagonists. Cannabinoids may have therapeutic potential in disorders resulting from cerebral ischemia, including stroke, and may protect neurons from injury through a variety of mechanisms.

Animals↗

PubMed1999

Neuromuscular disease and calcium channels.

Calcium channels that are regulated by voltage have an important role in linking cellular stimulation to physiological responses in the nervous system. In addition, a number of autoimmune or genetic disorders that affect calcium channels (channelopathies) have been identified, including several that affect neuromuscular function. These include the Lambert-Eaton myasthenic syndrome, which is associated with autoantibodies directed against neuronal calcium channels, as well as at least two inherited neuromuscular diseases-hypokalemic periodic paralysis and some varieties of malignant hyperthermia-that affect calcium channels in skeletal muscle. Preliminary progress has been achieved in understanding the relationship between these immunological or genetic abnormalities and the alterations in channel function that they produce. A major challenge that remains is to determine how calcium channelopathies lead to the curious assortment of paroxysmal and progressive disorders that are observed clinically.

Animals↗

PubMed1999

Summary of analyses of problem 2 simulated data for GAW11.

The simulated data for Problem 2 of the 11th Genetic Analysis Workshop (GAW11) consisted of family linkage data and disease data from three populations, each population with different genetic parameters. The disease was simulated such that there were two genetically distinct diseases, one caused by a single locus with three alleles and the other by two epistatically interacting loci. The two diseases were clinically identical. Each of the diseases had a severe and mild form. One of the diseases was influenced by an environmental factor and one by an allele at one of the marker loci (association). Linkage data consisted of 300 polymorphic markers. Investigators analyzed these data in a myriad of ways, using most currently available linkage techniques and association analysis methods. In addition, investigators also tested newly developed methods, some of which took advantage of the existence of the environmental component. Other types of analyses included meta-analyses, methods of combining data from different studies, questions of replication, how much information is available in a single data set, and analyses for gene x gene and gene x environment interaction.

Computer Simulation↗

PubMed1999

Simulated data for a complex genetic trait (problem 2 for GAW11): how the model was developed, and why.

This paper describes a simulated data set created as Problem 2 for GAW11. The generating model for Problem 2 involved two different genetic diseases, or "types," in three separate populations. The two-locus (2L) type results from the epistatic interaction of two genetic loci, and the three-allele type, from a single locus with two disease-causing alleles and one normal allele. Each type has two phenotypic forms: Mild and Severe. Both forms are subject to both genetic and environmental influences. The disease occurs in three different hypothetical populations, each with different disease allele frequencies and penetrances. In two populations there is also a fourth locus with an allele that is associated with the 2L type. Misdiagnosis can occur, but only after a family has already been ascertained through > or = 2 "genetically" affected offspring. Finally, the three different populations are studied by four different hypothetical research groups. These groups each have their own ideas about how the disease is inherited and have therefore devised different ascertainment schemes based on those beliefs. Each research group collected 100-family data sets, including data on 300 markers on six chromosomes and measurements on disease status and on the proposed two environmental factors. GAW participants were supplied with 25 random replicates of each data set.

Alleles↗