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BackgroundFamilial neurohypophyseal diabetes insipidus (FNDI), usually an autosomal dominant disorder, is caused by mutations in the arginine vasopressin (AVP)–neurophysin II preprohormone leading to aberrant preprohormone processing and gradual destruction of AVP-secreting cells. Patients typically present between 1 and 6 years of age with polyuria and polydipsia.ObjectiveClinical, biochemical, and genetic studies of three new cases of autosomal recessive FNDI presenting in early infancy.PatientsThree Palestinian cousins presented with failure to thrive, vomiting, irritability, and fever. The parents were asymptomatic. Patients developed hypernatremia (154–163 mmol/l) and serum hyperosmolality (>320 mOsm/kg), while urine osmolality remained between 73 and 229 mOsm/kg. Plasma AVP levels were low, and the posterior pituitary bright spot was absent on magnetic resonance imaging (MRI). All patients responded to desmopressin.ResultsPatients were homozygous and parents were heterozygous for microsatellite markers flanking the AVP gene. All patients were homozygous for the P26L (proline to leucine) substitution affecting mature AVP. A founder effect with the single original kindred carrying the P26L mutation was confirmed by microsatellite analysis, but patients in that family presented only at 2 years of age. In microsatellite analysis, the new kindred patients were not homozygous and did not share a single allele at the aquaporin 2 and vasopressin receptor-2 genes locuses.ConclusionThis is the first description of autosomal recessive FNDI presenting in the neonatal period. The unusual early clinical and radiological (MRI) presentation argues against gradual destruction of AVP-secreting neurons as the pathophysiological mechanism. Factors beside allelism of AVP-related genes must influence the age of FNDI presentation given the founder effect demonstrated for the P26L mutation.

Vibrio vulnificus is a waterborne pathogen that was responsible for an outbreak of severe soft-tissue infections among fish farmers and fish consumers in Israel. Several factors have been shown to be associated with virulence. However, the transcriptome profile of the pathogen during human infection has not been determined yet. We compared the transcriptome profile, using RNA sequencing, of a human-pathogenic strain harvested directly from tissue of a patient suffering from severe soft-tissue infection with necrotizing fasciitis, with the same strain and three other environmental strains grown in vitro. The five sequenced libraries were aligned to the reference genomes of V. vulnificus strains CMCP6 and YJ016. Approximately 47.8 to 62.3 million paired-end raw reads were generated from the five runs. Nearly 84 % of the genome was covered by reads from at least one of the five runs, suggesting that nearly 16 % of the genome is not transcribed or is transcribed at low levels. We identified 123 genes that were differentially expressed during the acute phase of infection. Sixty-three genes were mapped to the large chromosome, 47 genes mapped to the small chromosome and 13 genes mapped to the YJ016 plasmid. The 123 genes fell into a variety of functional categories including transcription, signal transduction, cell motility, carbohydrate metabolism, intracellular trafficking and cell envelope biogenesis. Among the genes differentially expressed during human infection we identified genes encoding bacterial toxin (RtxA1) and genes involved in flagellar components, Flp-coding region, GGDEF family protein, iron acquisition system and sialic acid metabolism.

Hemangiomas are localized tumors of vascular cells which appear frequently in humans and animals, and their mode of induction is unknown. Recently, a new field strain of avian retrovirus, avian hemangioma virus (AHV), was isolated from spontaneous hemangiomas in layer hens. Sequence analysis of the AHV genome revealed the presence of three prototypic retroviral genes, gag, pol, and env, but no oncogenes. AHV was capable of inducing hemangiomas in hens in vivo, but it induced a strong cytopathic effect in cultured endothelial cells. The AHV envelope glycoprotein, gp85, was found to be responsible for the cell-killing effect. Four independent lines of experimental evidence indicated that AHV induces a cytopathic effect through a typical programmed cell death, apoptosis: (i) morphological changes in cells visualized by light microscopy, (ii) nuclear condensation and fragmentation indicated by 4′,6-diamidino-2-phenylindole staining, (iii) intranucleosomal degradation of DNA demonstrated by terminal deoxynucleotidyl transferase-mediated dUTP–biotin nick end-labeling staining, and (iv) flow cytometry analysis of the DNA content of the infected cells. Quiescent endothelial G 0 /G 1 cells were much more sensitive to AHV-induced apoptosis than actively dividing cells, suggesting that the AHV ability to induce apoptosis is dependent on the proliferative state of the infected cells.

Human immunodeficiency virus (HIV) infection of the brain leads to massive neuronal damage, resulting in the AIDS (acquired immunodeficiency syndrome) dementia complex (ADC). A recent study using transgenic mice indicates that neurons possess transcription factors capable of activating the HIV promoter. To identify these, we transfected two types of primary cultures of rat neurons with HIV promoter-reporter gene constructs. The two kappa B regulatory sites in the HIV long terminal repeat (LTR) are shown to be essential for strong promoter activity. Two proteins present in neurons, BETA and an NF-kappa B-like protein, can bind the kappa B sites. These proteins are shown to belong to distinct families of transcription factors. Mutation analysis and transfection of a dominant negative NF-kappa B mutant, indicate that the neuronal NF-kappa B-like activity mediates HIV promoter activation. cDNA cloning, biochemical and immunological analyses indicate that neuronal NF-kappa B is similar to NF-kappa B of other tissues. Transfections of primary neuron cultures with an HIV promoter-beta-galactosidase construct show that within these cultures, neurons are indeed the cells that highly activate the HIV promoter. Thus, analogous to the situation in T-lymphocytes and macrophages, NF-kappa B is an activator of HIV transcription in neurons.

RGS2 (regulator of G-protein signaling 2) modulates dopamine receptor signal transduction. Functional variants in the gene may influence susceptibility to extrapyramidal symptoms (EPS) induced by antipsychotic drugs. To further investigate our previous report of association of the RGS2 gene with susceptibility to antipsychotic-induced EPS, we performed a replication study. EPS were rated in 184 US patients with schizophrenia (115 African Americans, 69 Caucasian) treated for at least a month with typical antipsychotic drugs (n=45), risperidone (n=46), olanzapine (n=50) or clozapine (n=43). Six single nucleotide polymorphisms (SNPs) within or flanking RGS2 were genotyped (rs1933695, rs2179652, rs2746073, rs4606, rs1819741 and rs1152746). Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by logistic regression. Our results indicate association of SNP rs4606 with antipsychotic-induced parkinsonism (AIP), as measured by the Simpson Angus scale, in the overall sample and in the African-American subsample, the G (minor) allele having a protective effect. ORs for AIP among rs4606 G-allele carriers were 0.23 (95% CI 0.10–0.54, P=0.001) in the overall sample, and 0.20 (0.07–0.57, P=0.003) in the African-American subsample. In the previously studied Israeli sample the OR was 0.31 (0.11–0.84, P=0.02). We completely sequenced the RGS2 gene in nine patients with AIP and nine patients without, from the Israeli sample. No common coding polymorphisms or additional regulatory variants were revealed, suggesting that association of the rs4606 C/G polymorphism with AIP is biologically meaningful and not a consequence of linkage disequilibrium with another functional variant. Taken together, the findings of the current study support the association of RGS2 with AIP and focus on a possible protective effect of the minor G allele of SNP rs4606. This SNP is located in the 3′-regulatory region of the gene, and is known to influence RGS2 mRNA levels and protein expression.

Linkage and association studies in schizophrenia have repeatedly drawn attention to several chromosomal regions and to genes within them. Conflicting patterns of association and the lack of a clear functional significance of the associated variants limit the interpretation of these results. The use of rare pedigrees, where genes with a major effect cause the disorder, has been proven beneficial in studies of other complex disorders. Our objective was to use this advantage by performing a genome wide linkage analysis for schizophrenia in a large, multiplex Israeli Arab pedigree. We genotyped 346 microsatellite markers in 24 pedigree members affected with schizophrenia spectrum disorders and 32 unaffected relatives. Two-point linkage analysis with SUPERLINK demonstrated a LOD score of 2.47 for D20S116 on chromosome 20p13 under an autosomal dominant mode of inheritance. Further fine mapping yielded a two-point LOD score of 2.56 for the adjacent marker D20S193 and narrowed down the linked region to 2–5 cM. A haplotype containing the markers D20S193, D20S889, and D20S116, 0.7 Mb in length, was found to be shared by most affected pedigree members. Genotyping of 43 SNPs in the interval supported these results with a multipoint LOD score of 2.7 around D20S193. We were also able to better define the boundaries of the shared haplotype which contains strong candidate genes for schizophrenia. Our study exemplifies the power of rare and unique pedigrees in drawing attention to novel regions for genetic studies of schizophrenia. © 2007 Wiley-Liss, Inc.

We previously reported an autosomal scan for schizophrenia susceptibility loci in a systematically recruited sample of Arab Israeli families. The scan detected significant evidence for linkage at chromosome 6q23 with a nonparametric LOD score (NPL) of 4.60 (P=0.000004) and a multipoint parametric LOD score of 4.16. In order to refine this finding we typed 42 additional microsatellite markers on chromosome 6q between D6S1570 (99.01 cM from the pter) and D6S281 (190.14 from the pter) in the same sample (average intermarker distance approximately 1.7 cM). In the 23 cM region between D6S1715 and D6S311, markers were more closely spaced ( approximately 1.1 cM). Multipoint nonparametric and parametric and single point linkage analyses were performed. The peak NPL rose to 4.98 (P=0.00000058) at D6S1626 (136.97 cM), immediately adjacent to D6S292 (NPL 4.98, P=0.00000068), the marker that gave the highest NPL in the original genome scan, under the broad diagnostic category. The putative susceptibility region (NPL-1) was reduced from 12.0 to 4.96 cM. The peak multipoint parametric LOD score was 4.63 at D6S1626 under a dominant genetic model, core diagnostic category and the LOD-1 interval was 2.10 cM. The maximum single point LOD score (3.55, theta=0.01) was also at D6S1626 (dominant model, core diagnostic category). Increased evidence for linkage in the same sample as in the original genome scan and consistent localization of the linkage peak add further support for the presence of a schizophrenia susceptibility locus at chromosome 6q23. Moreover, the markedly reduced linkage interval greatly improves prospects for identifying a schizophrenia susceptibility gene within the implicated region.

Several genes have been reported recently to be associated with schizophrenia and bipolar disorder. Because of the complexity of the inheritance of these disorders, there is an urgent need to replicate these findings and to search for additional candidate genes. The study of genetic isolates is a powerful technique that may overcome some of the obstacles caused by genetic heterogeneity and ambiguity of phenotype definition. Identity by descent (IBD) haplotype sharing analysis in these populations may be used to detect mutations within shared haplotypes in smaller samples of affected individuals. In this study, we used IBD haplotype sharing analysis to replicate positive linkage and association findings in psychotic disorders, and to identify other regions of interest. Fifty-two patients with major psychiatric disorders from a genetically isolated village in Israel were studied. By studying eight Y chromosome markers, we were able to confirm the oral tradition of members of this isolate regarding a common paternal origin. Three hundred fifty nine microsatellite markers on 9 candidate chromosomes were genotyped, and haplotypes were reconstructed using information from family members. Two highly significant (P < 0.0001) peaks of haplotype sharing were found. One was for psychotic patients with any diagnosis at the location of dysbindin, a gene previously associated with schizophrenia. The other peak was for patients with schizophrenia on chromosome 1p36. Thus, this study both replicates an earlier finding and points to a novel region of interest, which might be unique to this population.

Anxiety involves complex, incompletely understood interactions of genomic, environmental, and experience-derived factors, and is currently being measured by psychological criteria. Here, we report previously nonperceived interrelationships between expression variations and nucleotide polymorphisms of the chromosome 7q21–22 acetylcholinesterase-paraoxonase 1 ( ACHE-PON1 ) locus with the trait- and state-anxiety measures of 461 healthy subjects from the Health, Risk Factors, Exercise Training, and Genetics Family Study. The AChE protein controls the termination of the stress-enhanced acetylcholine signaling, whereas the PON protein displays peroxidase-like activity, thus protecting blood proteins from oxidative stress damages. Serum AChE and PON enzyme activities were both found to be affected by demographic parameters, and showed inverse, reciprocal associations with anxiety measures. Moreover, the transient scores of state anxiety and the susceptibility score of trait anxiety both appeared to be linked to enzyme activities. This finding supported the notion of corresponding gene expression relationships. Parallel polymorphisms in the ACHE and PON1 genes displayed apparent associations with both trait- and state-anxiety scores. Our findings indicate that a significant source of anxiety feelings involves inherited and acquired parameters of acetylcholine regulation that can be readily quantified, which can help explaining part of the human variance for state and trait anxiety.

Schizophrenia is a complex neuropsychiatric disorder to which an as-yet-unknown number of genes contribute, interacting with each other and the environment. Linkage analyses have implicated several chromosomal regions as harboring schizophrenia susceptibility loci although rarely at levels commensurate with proposed thresholds for genome-wide significance. We systematically recruited Arab Israeli families multiply affected with schizophrenia from the catchment area of a Regional Mental Health Center. Clinical diagnoses were established by semistructured interviews and all other available sources of information under narrow, core and broad categories. Using 350 microsatellite markers, spaced at an average of 10.3 cM, we performed an autosomal scan in 155 subjects from 21 families. Linkage analysis employed affects only, multipoint, nonparametric (model-free) and also parametric (dominant and recessive) approaches. We detected significant evidence for a schizophrenia susceptibility gene at chromosome 6q23 with a nonparametric LOD score (NPL) of 4.60 (P=0.000004) under the broad diagnostic category and a parametric LOD score of 3.33 (dominant model). Under the core diagnostic category the NPL was 4.29 (P=0.00001) and the LOD score 4.16 (dominant model). We also detected suggestive evidence for linkage at chromosome 10q24 under the broad diagnostic category (NPL 3.24, P=0.0008; heterogeneity LOD score, dominant model 2.65, alpha=0.82). Additionally, NPL scores2.0 were observed at chromosome 2q37, 4p15-16, 7p22, 9q21-22 and 14q11.1-11.2. The linkage we detected at chromosome 6q23 fulfills the criteria for genome-wide significance and is located approximately midway between loci suggested by a previous significant report at chromosome 6q25 and findings located more centromerically at 6q21-22.

Hereditary Inclusion Body Myopathy (HIBM) is a unique group of neuromuscular disorders characterized by adult onset and a typical muscle pathology. We have recently identified the gene encoding for a bifunctional enzyme, UDP-N-acetylglucosamine 2 epimerase/N-acetylmannosamine kinase (GNE), as the mutated gene in the prototype form of the disease presenting quadriceps sparing, particularly common in Middle Eastern Jews. Interestingly, we have identified the homozygous M712T Middle Eastern Jewish mutation also in two unrelated Middle Eastern Moslem families. We have also evaluated the involvement of GNE in several families from worldwide non-Jewish ethnic origins presenting symptoms similar to the Middle Eastern HIBM prototype. A total of 14 GNE mutations were identified (one nonsense and 13 missense), of which six are novel: an homozygous missense mutation in a consanguineous family from Italy and in a non consanguineous family from USA, and distinct compound heterozygotes in families from Germany, Italy, Ireland, Bahamas, USA and East India. This study brings to 17 the number of reported GNE mutations in quadriceps sparing myopathy, occurring either in the epimerase or the kinase domain of the enzyme. The mechanism leading to this unique phenotype still remains to be elucidated.

In search of a genetic basis for adverse responses to anticholinesterases, we selected, gentotyped and sequenced five G, C-rich clusters in the 35 kb human (h) acetylcholinesterase (ACHE) gene locus of individuals with a clinical history of anti-AChE hypersensitivity. One exceedingly anti-ACHE sensitive proband and one of her parents, out of 30 patients and ca. 100 controls, carried a 4 nucleotide deletion within a 200 bp region 17 kb upstream to the transcription start site. The deletion disrupted one out of the 2 motifs in this region for binding the transcription factor HNF3A, expressed in hepatocytes, lung and the AChE-rich small intestine. When placed upstream of an hACHE promoter-reporter vector, neither the native nor the variant 200 bp region modified AChE production by this vector in transfected COS cells. In contrast, co-transfection of the constructed hAChE expression vectors with a rat HNF3A encoding vector increased AChE production by over 50%, demonstrating HNF3A-dependent enhancer properties for this domain. Moreover, the mutant construct with only one intact HNF3 A site was 2-fold more effective an enhancer than the construct carrying the native two HNF3A sites (P

Upon activation, T lymphocytes accumulate high levels of the neuropeptide enkephalin which correlate with high levels of proenkephalin mRNA in the cells. Here we investigated the transcriptional basis for these changes. The proenkephalin promoter contains a sequence GGGGACGTCCCC, named B2, which is similar to the kappa B sequence GGGGACTTTCC, the binding site of the transcription factor nuclear factor (NF)-kappa B. Activation of T lymphocytes induces an NF-kappa B-like binding activity to the B2 site, concomitant with activation of the proenkephalin promoter. Mutations at the B2 site abolish this transcriptional activation. The purified homodimer (two p50s) of the DNA-binding subunit of NF-kappa B binds the B2 site of proenkephalin relatively better than does the heterotetramer (two p65s plus two p50s) form of the factor. Thus, it appears that the T-cell-specific activation of the proenkephalin promoter is mediated by NF-kappa B. However, as NF-kappa B is ubiquitous and the transcriptional activation through the B2 site is T cell specific, yet another T-cell-specific factor which synergizes with NF-kappa B should be considered.

To suppress the hyperglycemic effect of malathion in rats, a smaller amount of atropine was required when the drug was injected by intraventricular (i. vent.) than s.c. Pentobarbital, but not diazepam, blocked the hyperglycemic response. The results suggest that central accumulation of acetylcholine was the mediator of the response. Since hyperglycemia was not abolished by adrenalectomy and/or hypophysectomy, a hypothesis is presented to explain how central accumulation of acetylcholine might cause hyperglycemia.

Isoproterenol and other agonists readily dissociate from the beta-adrenergic receptor in turkey erythrocyte membranes. However, when a low concentration of deoxycholate is added, the receptor locks the prebound agonist; i.e., the rate of dissociation of the prebound agonist decreases drastically. The dissociation of prebound antagonists is slightly increased by deoxycholate. Locking, which is thus agonist specific, occurs in the cold, is reversed when detergent is removed from the membranes, and appears not to require the guanyl nucleotide binding protein of the adenylate cyclase system. It is suggested that this induced fit of a receptor to an agonist represents the specific conformational response that normally propagates in the receptor molecule in its interaction with the next component along the pathway of signal transmission.

A diverse panel of monoclonal antibodies was obtained from BALB/c mice immunized with two haptens structurally related to spiroperidol (SPD). Bromoacetyl derivatives of aminospiroperidol (NH2SPD) and N-amino-phenethylspiroperidol (NAPS) were synthesized to couple the haptens covalently to a protein carrier for immunization, thereby maintaining the butyrophenone portion of the immunogen. Hybridomas were selected based on their ability to secrete antibody that binds [3H]SPD with high affinity. Equilibrium dissociation constants for these antibodies ranged from 0.2 to greater than 100 nM. The antigen binding sites of the anti-NH2SPD and anti-NAPS antibodies were characterized in studies of the inhibition of the binding of [3H]-SPD by a series of ligands that are either (a) structurally related to SPD or (b) structurally unrelated to the butyrophenones but known to be selective antagonists of the D2 subtype of dopamine receptor. Based on the patterns of inhibition of the binding of [3H]SPD by these compounds, 12 classes of antibody combining sites were identified. Most of these antibodies bound butyrophenones with high affinity. One anti-NH2SPD and four anti-NAPS antibodies also bound domperidone, a nonbutyrophenone that has a high affinity for D2 receptors. None of the antibodies bound clebopride or sulpiride, D2-selective antagonists of the benzamide class, or the agonist dopamine.

Adenylate cyclase in intact celfecan be inactivated by N-ethylmaleimide or by heat without apparent damage to the coupled hormone receptor. Subsequently the receptor can be transferred to the adenylate cyclase of another type of cell, simply by fusing the two cell types with each other by Sendai virus. Coupling occurs in the fused cell membrane between the preexisting components and requires, at most a few minutes. Coupling is measured by hormonal activation of adenylate cyclase in cell ghosts prepared after fusion (Proc. Nat. Acad. Sci. 73 , 4410, 1976). By this procedure the s-adrenergic receptor in turkey erythrocytes has been transferred to the adenylate cyclase of mouse erythroleukemia cells (F cells) and also to the enzyme in Y-1 rat adrenal tumor cells. The experiments show that the hormone receptor can be dissociated in a functional state from its original, inactivated, adenylate cyclase system and that the receptor is compatible with the enzyme in a different cell. The apparent dissociation constants for epinephrine and norepinephrine, measured through adenylate cyclase activation, remained unchanged when the s-adrenergic receptor was transferred to the foreign adenylate cyclase system. Thus the dissociation constant is an intrinsic property of the receptor which seems not to be much influenced by the other components of the membrane. Cyclic AMP levels in the intact fused cell have also been measured. Within 3 min after onset of fusion the newly transferred s-adrenergic receptor was already able to cause an increase in intracellular cyclic AMP in response to hormone addition. A new hormone responsiveness can therefore indeed be conferred on intact cells. The s-adrenergic receptor has also been transferred from C6 glioma cells to F cells and to Y-1 cells. A different receptor, specific for prostaglandin E1, has been successfully transferred from the F cells to Y-1 and C6 cells. Thus the wedding of hormone receptors to heterologous adenylate cyclase systems can be performed among cells of different species and differentiation characteristics. It is therefore suggested that the various hormone receptors which are coupled to adenylate cyclase are all of similar structure, differing mainly in the confined area which binds the specific hormone. The newly developed experimental approach of heterologous coupling could be used to analyze biological defective systems by transfer of the receptor or the enzyme to their counterparts in a normally functioning cell. Such studies in biochemical compatibility might be extended to study other multicomponent systems in biological membranes.

Cultured bovine adrenal chromaffin cells contain a pool of ATP sequestered within the chromaffin vesicles and an extravesicular pool of ATP. In a previous study it was shown that the turnover of ATP in the extravesicular pool was biphasic. One phase occurred with a t1/2 of 3.5–4.5 h whereas the second phase occurred with a t1/2 of several days. The studies described here were undertaken to characterize further the vesicular and extravesicular pools of ATP by examining the effects of metabolic inhibitors, adenosine, and digitonin on ATP utilization and subcellular localization immediately after and 48 h after labeling with [3H]adenosine and 32Pi. Immediately after labeling a combination of cyanide, 2-deoxy-D-glucose, the β-glucono-l,5-lactone resulted in a 90–95% depletion of the labeled ATP but only a 25% depletion of the endogenous ATP within 30 min. Forty-eight hours after labeling, addition of the inhibitors resulted in a 70% depletion of the [3H]ATP but only a 25% depletion of the [32P]ATP and endogenous ATP. Addition of 10 μM adenosine to the media resulted in a similar loss of [3HJATP in cells examined immediately after or 48 h after labeling. Adenosine increased the amounts of [32P]ATP when added immediately after labeling but had no effect on the [32P]ATP content when added 48 h after labeling. Permeabilization of the plasma membrane by treatment with digitonin resulted in a loss of 90–95% of the labeled ATP but only 20% of the endogenous ATP in cells examined immediately after labeling and in a 79% loss of [3H]ATP, a 32% loss of [32P]ATP, and a 23% loss of endogenous ATP in cells examined 48 h after labeling. These studies show that ATP contained within chromaffin vesicles is metabolically inert and does not exchange readily with cytosolic ATP, but they do not differentiate extravesicular pools of labeled ATP. Calculation of ATP disappearance after abrupt cessation of ATP production indicates that cultured adrenal chromaffin cells utilize ATP at a rate of 0.24 nmol/min/106 cells, corresponding to a turnover time of 12.5 min for the extravesicular pool.

In the last few years considerable progress has been made in the study of receptor-response systems which are located on cell membranes. Yet the unravelling of the underlying mechanisms at the molecular level has been rather slow. Biochemical characterisation of the components required solubilisation of the membrane molecules in detergents, which resulted almost invariably in the loss of biological activity of the receptor-response system. Reconstitution of receptor activity was rarely obtained and reproducibility of such experiments was poor (Heidman and Changeux, 1978; Eimerl et al., 1980). It seemed that tenacious binding of detergents to components of the system was a major problem. Obviously, if even one component of a receptor-response system failed to function the entire reconstituted system would fail to produce the expected response. Another problem in reconstitution was to create conditions of concentration, polarity, etc., under which the solubilised components would associate properly to produce the functional complex. Thus, crude and purified preparations of receptors were obtained which demonstrated specific binding of antagonists or agonists but failed to demonstrate biological activity. The binding data obtained with such preparations are therefore difficult to evaluate. Several years ago, because of the difficulties described above, we developed a different experimental approach to the study of membrane receptors and other hydrophobic membrane molecules (Orly and Schramm, 1976).

We have identified a DNA binding protein, named BETA, that interacts with the same (B) transcriptional regulatory sequence as the known transcription factor NF-kappa B. BETA is found only in gray matter throughout the brain, and not in a variety of other rat tissues. Two binding sites for BETA are present adjacent to the promoter of the rat proenkephalin gene. Transfection of primary brain cultures that express BETA, with a reporter gene driven by the SV40 promoter linked to BETA DNA binding sites, results in transcriptional activation. We infer that BETA is a brain-specific transcription activator.

A new hormone responsiveness can be conferred on an adenylate cyclase system by coupling it to a foreign hormone receptor. Receptor and enzyme from cells of different species can be compatible with each other.

Intraventricular injection of carbachol produces hyperglycemia in rats at doses which are ineffective when given subcutaneously. This effect is suppressed by intraventricular administration of small amounts of atropine, further supporting the suggestion that the effect of carbachol is due to its action on central cholinergic receptors. Carbachol-induced hyperglycemia is not abolished by adrenalectomy and hypophysectomy, or pretreatment with reserpine.

Adenylate cyclase can be inactivated in the intact cell by N-ethylmaleimide or by heat while the hormone receptor remains functional. The hormone receptor in the treated cell can then be coupled to adenylate cyclase in another type of cell by fusing the two cells with each other (J. Orly and M. Schramm, Proc. Nat. Acad. Sci. U.S.A. 73 , 4410, 1976; M. Schramm, J. Orly, S. Eimerl and M. Korner, Nature 268 , 310, 1977). Protein synthesis is not required and it is therefore concluded that the newly formed system is constructed exclusively from the preexisting components. Thus, fusion of two different cell membranes produces a functional biochemical hybrid system in which the hormone receptor originating from one cell activates the adenylate cyclase originating from the other cell. Development of the concepts of receptor transfer and some very recent findings are also discussed. In the earlier experiments, both the β-adrenergic receptor and the prostaglandin E1 receptor have been successfully coupled with adenylate cyclase systems of various cultured cells. Studying the properties of the β-adrenergic receptor, it has been found that the dissociation constants for epinephrine, norepinephrine and propranolol do not change upon transfer of the receptor to a membrane of different properties. It is therefore thought that the dissociation constant is an intrinsic property of the receptor molecule which is not much influenced by the other natural components of the cell membrane. While the above studies involved measurement of adenylate cyclase activation in cell membranes prepared after fusion, function of the transferred receptor in the intact fused cell has also been demonstrated. Within 3 min. after onset of fusion the newly transferred β-adrenergic receptor is already able to cause an increase in intracellular cyclic AMP in response to hormone addition. The experiments indicate that the characteristic biological response of a cell to cyclic AMP can be induced by a foreign hormone receptor implanted in the membrane of that cell. Exploitation of the full potential of biochemical hybridization of membrane components requires that membranes of cells from any tissue could be readily fused with each other. Unfortunately, cell fusion by Sendai virus as used in the above noted work is limited to certain tissue-culture cells and erythrocytes. A new biochemical fusion method (non-viral) has therefore been developed. With the aid of this method the glucagon receptor of purified liver membranes has been successfully coupled to the adenylate cyclase of Friend erythroleukemia cells. It is thus demonstrated that even receptors for a peptide hormone in isolated membranes of a highly differentiated mamalian tissue can be transferred to the adenylate cyclase of a different cell. It is anticipated that hybridization of hormone receptors with adenylate cyclase of different cells will be used to analyze the function of these components in mutants and in diseased states. In addition, hormone receptors discovered by ligand binding could be tested for function by hybridization with a well characterized response system in the cell membrane of another type of cell. Obviously, this novel experimental approach need not be limited to hormone receptors coupled to the adenylate cyclase system. Other receptor-response systems could be analyzed by the same approach. In principle, it seems applicable to any dissociable multicomponent system residing in biological membranes.

Hypersensitivity to acetylcholinesterase inhibitors (anti-AChEs) causes severe nervous system symptoms under low dose exposure. In search of direct genetic origin(s) for this sensitivity, we studied six regions in the extended 22 kb promoter of the ACHE gene in individuals who presented adverse responses to anti-AChEs and in randomly chosen controls. Two contiguous mutations, a T-->A substitution, disrupting a putative glucocorticoid response element, and a 4-bp deletion, abolishing one of two adjacent HNF3 binding sites, were identified 17 kb upstream of the transcription start site. Allele frequencies for these mutations were 0.006 and 0.012, respectively, in 333 individuals of various ethnic origins, with a strong linkage between the deletion and the biochemically neutral H322N mutation in the coding region of ACHE. Heterozygous carriers of the deletion included a proband who presented with acute hypersensitivity to the anti-AChE pyridostigmine and another with unexplained excessive vomiting during a fourth pregnancy following three spontaneous abortions. Electromobility shift assays, transfection studies and measurements of AChE levels in immortalized lymphocytes as well as in peripheral blood from both carriers and non-carriers, revealed functional relevance for this mutation both in vitro and in vivo and showed it to increase AChE expression, probably by alleviating competition between the two hepatocyte nuclear factor 3 binding sites. Moreover, AChE-overexpressing transgenic mice, unlike normal FVB/N mice, displayed anti-AChE hypersensitivity and failed to transcriptionally induce AChE production following exposure to anti-AChEs. Our findings point to promoter polymorphism(s) in the ACHE gene as the dominant susceptibility factor(s) for adverse responses to exposure or to treatment with anti-AChEs.