The role of alpha-tocopherol in preventing disease: from epidemiology to molecular events.

The function of vitamin E has been attributed to its capacity to protect the organism against the attack of free radicals by acting as a lipid based radical chain breaking molecule. More recently, alternative non-antioxidant functions of vitamin E have been proposed and in particular that of a "gene regulator". Effects of vitamin E have been observed at the level of mRNA or protein and could be consequent to regulation of gene transcription, mRNA stability, protein translation, protein stability and post-translational events. Given the high priority functions assigned to vitamin E, it can be speculated that it would be inefficient to consume it as a radical scavenger. Rather, it would be important to protect vitamin E through a network of cellular antioxidant defences, similarly to what occurs with proteins, nucleic acids and lipids.

Ex vivo gene therapy with stromal cells transduced with a retroviral vector containing the BMP4 gene completely heals critical size calvarial defect in rats.

In order to develop a successful gene therapy system for the healing of bone defects, we developed a murine leukemia virus (MLV)-based retroviral system expressing the human bone morphogenetic protein (BMP) 4 transgene with high transduction efficiency. The bone formation potential of BMP4 transduced cells was tested by embedding 2.5 x 10(6) transduced stromal cells in a gelatin matrix that was then placed in a critical size defect in calvariae of syngenic rats. Gelatin matrix without cells or with untransduced stromal cells were the two control groups. The defect area was completely filled with new bone in experimental rats after 4 weeks, while limited bone formation occurred in either control group. Bone mineral density (BMD) of the defect in the gene therapy group was 67.8 +/- 5.7 mg/cm(2) (mean +/- s.d., n = 4), which was 119 +/- 10% of the control BMD of bone surrounding the defect (57.2 +/- 1.5 mg/cm(2)). In contrast, BMD of rats implanted with untransduced stromal cells was five-fold lower (13.8 +/- 7.4 mg/cm(2), P < 0.001). Time course studies revealed that there was a linear increase in BMD between 2-4 weeks after inoculation of the critical size defect with 2.5 x 10(6) implanted BMP4 cells. In conclusion, the retroviral-based BMP4 gene therapy system that we have developed has the potential for regeneration of large skeletal defects.

Dopamine D2 receptor (DRD2) gene and susceptibility to posttraumatic stress disorder: a study and replication.

Subjects on an addiction treatment unit who had been exposed to severe combat conditions in Vietnam were screened for posttraumatic stress disorder (PTSD). Of 24 with PTSD, 58.3% carried the D2A1 allele. Of the remaining eight who did not meet PTSD criteria, 12.5% carried the D2A1 allele (p = 0.04). In a replication study of 13 with PTSD, 61.5% carried the D2A1 allele. Of the remaining 11 who did not meet criteria for PTSD, 0% carried the D2A1 allele (p = 0.002). For the combined group 59.5% of those with PTSD carried the D2A1 allele versus 5.3% of those who did not have PTSD (p = 0.0001). These results suggest that a DRD2 variant in linkage disequilibrium with the D2A1 allele confers an increased risk to PTSD, and the absence of the variant confers a relative resistance to PTSD.

The dopamine D2 receptor gene: a genetic risk factor in substance abuse.

Drug abuse has grown to epidemic proportions. Dopaminergic reward pathways have frequently been implicated in the etiology of drug addiction. To examine the possible role of genetic variants of the dopamine D2 (DRD2) gene in susceptibility to drug abuse we determined the prevalence of the TaqI A1 variant of the DRD2 gene in 200 white patients hospitalized in the Addiction Treatment Unit of a Veterans Administration Hospital. While the prevalence of the D2A1 allele was not significantly increased over controls, it did increase from 21% in subjects with alcohol abuse only to 32% in subjects with alcohol dependence only, consistent with other studies showing an association with the severity of alcoholism. By contrast, of 104 subjects with a discharge diagnosis of drug and alcohol abuse/dependence, 42.3% carried the D2A1 allele versus 29.0% of the 763 white controls (representing all white controls published to date) (P = 0.006). Of those who spent more than $25/week on two or more substances, 56.9% carried the D2A1 allele versus 28.2% of those abusing a single substance (P < 0.0005). Multiple logistic regression analysis showed a highly significant association between multiple substance abuse based on money spent and the presence of the D2A1 allele (P = 0.0003) and age of onset of abuse (P < 0.0001). D2A1 carriers exceeded D2A2A2 subjects for a history of being expelled from school for fighting (P = 0.001), and of those ever jailed for violent crimes, 53.1% carried the D2A1 allele versus 28.8% of those jailed for non-violent crimes (P = 0.011).(ABSTRACT TRUNCATED AT 250 WORDS)

The dopamine D2 receptor (DRD2) as a major gene in obesity and height.

Dopamine plays a major role in the regulation of appetite and growth hormone. Dopaminergic agonists suppress appetite and dopamine D2 receptor antagonists enhance it. We examined the hypothesis that allelic variants of the DRD2 locus may be associated with weight and height. Sarkar and Sommer described two DRD2 polymorphisms that could be haplotyped by PCR. For weight, the mean Z score (National Center for Health Statistics) for 208 subjects without haplotype 4 was 0.086 versus 0.557 for 280 subjects with haplotype 4, P = 0.0003. Two separate sets of subjects were studied and these results were significant for both, providing an internal replication. For height, the mean Z score for 164 subjects without haplotype 4 was 0.1677 versus 0.6885 for 219 subjects with haplotype 4, P < 0.00001. These and other data suggest that the 4 haplotype is in linkage disequilibrium with allelic variants of the DRD2 gene that play a major role in the regulation of weight (obesity) and height, and may serve as a risk factor in late-onset non-insulin-dependent diabetes mellitus (NIDDM).

Dopamine D2 receptor gene Taq I 'A' locus map including 'A4' variant: relevance for alcoholism and drug abuse.

The D2 dopamine receptor gene (DRD2) displays Taq I restriction fragment length polymorphisms (RFLPs) at two different loci, termed A and B. One of the three different Taq I A 'alleles' described at this site, the A1 allele (size = 6.6 kb), has been found to be associated with alcoholism and with drug abuse in the majority of studies reported to date. A complete map of the Taq I A RFLP site has been constructed, through hybridization with different fragments of the 3' flanking region and polymerase chain reaction (PCR). When screening 432 unrelated individuals to establish possible A1 allelic association with drug abuse or dependence, we have encountered a novel Taq I A RFLP, which we have named 'A4' (size = 8.6 kb). This sequence variant displays a frequency of approximately 1% in our sample and shows a Mendelizing genetic pattern in an Italian nuclear family. Primers suitable for detecting A4 using PCR have been designed. The A4, but not the A3 'allele', displays substantial overlap with the A1. In particular, A2 and A3 share the presence of a Taq I restriction site, whose absence in A1 and A4 is apparently associated with substance abuse vulnerability. Therefore, in association studies it is proper to contrast individuals displaying A1 and A4 RFLP patterns, with individuals displaying A2 and A3 RFLPs.

Alkylation of free sulfhydryls fortifies electroplax subsynaptic structures.

The cysteine-rich 43,000-dalton peripheral membrane protein, nu 1, is localized at the cytoplasmic face of electroplax and muscle cholinergic synapses, where it is thought to play an important role in the endplate supramolecular structure. The peripheral membrane protein properties of nu 1 are inferred by its removal from nicotinic cholinergic membranes by the action of mild alkali or lithium diiodosalicylate. An interesting property of nu 1 is its high concentration of free sulfhydryl groups, whose exact role in synaptic structure is still largely unknown. Alkylation of free sulfhydryls with N-ethylmaleimide (3 mM) has a profound effect on the association of nu 1 with synaptic membranes, rendering nu 1 unextractable by pH 11 treatment or by lithium diiodosalicylate and, concomitantly, decreasing nu 1's electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels. Iodoacetamide and iodoacetate have similar effects, but at concentrations 10- to 100-fold higher than required for N-ethylmaleimide. Furthermore, sulfhydryl modification also stabilizes the association of nicotinic receptor subunits with the detergent-insoluble cytoskeleton. N-Ethylmaleimide treatment increases the fraction of insoluble receptor molecules on extraction with Triton X-100, sodium cholate, or octylglucoside. These results suggest an important role of sulfhydryl groups in the structural stability of the postsynaptic cholinergic membrane.

Creatine kinase isoenzymes in Torpedo californica: absence of the major brain isoenzyme from nicotinic acetylcholine receptor membranes.

Creatine kinase, actin, and nu 1 are three proteins of Mr 43 000 associated with membranes from electric organ highly enriched in nicotinic acetylcholine receptor. High levels of creatine kinase are required to maintain adequate ATP levels, while actin may play a role in maintaining the synaptic cytoskeleton. Previous investigations have prompted the conclusion that postsynaptic specializations at the receptor-enriched membrane domains in electroplax contain the brain form of creatine kinase rather than the form of creatine kinase predominantly found in muscle. We have examined this conclusion by purifying Torpedo brain creatine kinase to virtual homogeneity in order to examine its immunochemical, molecular, and electrophoretic properties. On the basis of immunological cross-reactivity and isozyme analysis, the receptor-associated creatine kinase is identified to be of the muscle type. When the molecular characteristics of Torpedo brain and muscle creatine kinase are compared, the brain enzyme is positioned at a more basic pH during chromatofocusing and on two-dimensional gel electrophoresis (pI = 7.5-7.9). Furthermore, electrophoretic mobilities of the brain and muscle forms of creatine kinase differ in sodium dodecyl sulfate electrophoresis: the brain isozyme of creatine kinase has lower apparent molecular weight (Mr 41 000) when compared with the muscle enzyme (Mr 43 000). On the basis of the results of our current investigations, the hypothesis that the brain isozyme of creatine kinase is a component of the postsynaptic specializations of the Torpedo californica electroplax must be abandoned. Recent sequence data have established close homology between Torpedo and mammalian muscle creatine kinases. On the basis of electrophoretic criteria, our results indicate that a lower degree of homology exists between the brain isozymes.

Immunochemical and molecular differentiation of 43 000 molecular weight proteins associated with Torpedo neuroelectrocyte synapses.

Synaptic membranes, highly enriched in nicotinic receptor, contain three 43 000 molecular weight (Mr) peripheral proteins (distinctive in their peptide mapping profiles and earlier designated v1, v2, and v 3) as well as the receptor alpha 2 beta gamma delta integral membrane subunits. Of the three proteins, only v1 is copurified with the membrane-bound receptor, while v2 and v3 are prominent cytosolic proteins, which are retained at significant levels in receptor-rich membranes during multistep centrifugation and affinity partitioning purification procedures [Gysin, R., Wirth, M., & Flanagan, S. D. (1981) J. Biol. Chem. 256, 11373-11376]. Peptide mapping analysis of Torpedo v3 and rabbit skeletal actin indicates that the two proteins are closely related. The enzymatic activity, creatine phosphokinase (EC 2.7.3.2), copurifies with v2 during chromatofocusing fractionation of the cytosol. The Torpedo electroplax form of creatine phosphokinase has an electrophoretic mobility identical with that of the mammalian skeletal muscle form of the enzyme. Upon release of the membrane-bound forms of v1, creatine phosphokinase, and actin by the action of mild alkali, v1 remains in a high molecular weight form. Dissociation of v1 into lower molecular weight species requires urea or sodium dodecyl sulfate (NaDodSO4). Preparation of essentially pure v1 was achieved by eluting the v1 protein spots directly from naDodSO4-isoelectric focusing gels loaded with alkali extracts derived from membranes highly enriched in nicotinic receptor. Amino acid compositions of the purified fractions indicate that v1 and Torpedo creatine phosphokinase have distinct amino acid compositions from each other and from that of actin.(ABSTRACT TRUNCATED AT 250 WORDS)

Cross-correlation analysis of interneuronal connectivity in the motor cortex of the monkey.

Pairs of neurones were recorded within the hand region of the precentral motor cortex of monkeys trained to squeeze a force transducer between the thumb and index finger. Neuronal discharges of a pair of cells, recorded through the same electrode, were first examined for their co-variation with force. The discharges of those pairs with a consistent co-variation were cross-correlated during periods of constant isometric force. Cross-correlograms in the majority of cases had either a sharp peak or trough at latencies suggesting monosynaptic causal relations between the cell discharges. A peak was associated with pairs of cells whose discharges co-varied similarly with force and when a trough was observed, the cell discharges of the pair generally differed in their force co-variation. It is concluded that the action of motor cortical neurones is linked by common and recurrent collateral inputs during isometric co-contraction of finger muscles.

Structural heterogeneity and subcellular distribution of nicotinic synapse-associated proteins.

Peripheral membrane proteins (Mr = 43,000) are associated with Torpedo membranes highly enriched in nicotinic receptor. These 43,000-dalton proteins are not required for ion translocation or other known receptor functions, but they have been implicated in constraint of the nicotinic receptor within the plane of the membrane bilayer. Sodium dodecyl sulfate-polyacrylamide electrophoresis allows partial resolution of the 43,000-dalton band into a doublet. We have carried out further analysis using two-dimensional gel electrophoresis, which reveals the existence of at least seven Coomassie blue-staining spots in the isoelectric focusing dimension. Peptide maps of the individual spots serve to elucidate the observed electrophoretic complexity. Three different membrane-bound proteins, designated v1, v2, and v3, were identified on the basis of their characteristic peptide maps which show no apparent homology in amino acid sequence. Two of these proteins, v1 and v2, are resolved into multiple spots in the isoelectric focusing dimension, but each group of isoelectric focusing variants has nearly identical peptide fingerprints. Of relevance to the putative role of these proteins in synaptic or receptor supramolecular structures is the observation that only v1 is exclusively membrane bound, and co-purifies with receptor whereas both v2 and v3 are also prominent proteins of the cytoplasm and are depleted from membrane fractions most enriched in receptor. These proteins may interact in the formation or maintenance of synaptic and nicotinic receptor supramolecular structures.

Affinity partitioning of membranes. Evidence for discrete membrane domains containing cholinergic receptor.

Subsynaptic membrane domains from Torpedo californica electroplax contain nicotinic cholinergic receptor molecules at densities as high as 20,000 micrometers-2. Intense homogenization of the electroplax releases membrane fragments enriched in nicotinic receptor from basal lamina and other synaptic cleft and presynaptic elements. Ideally, preparations of membrane fragments, highly enriched in nicotinic receptor, should approach 125I-alpha-bungarotoxin-specific binding activities near the levels observed after receptor dispersal in detergents and subsequent affinity chromatography. We report the application of affinity partitioning, combined with multiple extraction techniques, to yield preparations of virtually homogeneous membranes enriched in nicotinic receptor alpha, beta, gamma, and delta subunits as well as the 43,000-dalton peripheral protein subunit. The countercurrent distribution technique serves to resolve three populations of receptor-containing membranes. One fraction is refractory to affinity partitioning and may represent aggregates of receptor-rich membranes with fragments derived from nonsynaptic membranes. The second and third fractions contain membrane fragments derived from the subsynaptic membrane and are highly enriched in nicotinic receptor (5.1 to 7.8 nmol of alpha-bungarotoxin binding sites/mg of protein). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of all three fractions indicates that alpha, beta, and gamma subunits are present in stable stoichiometric ratios, while the 43,000-dalton peripheral protein content varies by 33% between the fractions. However, removal of 90% of the 43,000-dalton component by mild alkali treatment does not result in conversion of one fraction into the other. The combination of affinity partitioning and counter-current distribution techniques utilized in this study should prove useful in the resolution of a variety of subcellular particles that contain specific binding sites.

Regulation of the synthesis of S-100 protein in rat glial cells.

The influence of serum on the synthesis of the nervous system specific S-100 protein has been investigated in the rat glial cell clone C61a. In sparse cells, where S-100 synthesis is low, removal of serum leads to a 2- to 3-fold increase of the rate of S-100 synthesis relative to total soluble cell protein. Measurements of S-100 content by radioimmunoassay showed that sparse cells are accumulating S-100 much more rapidly in the absence of serum. Our findings suggest that serum components inhibit the expression of S-100 synthesis in sparse cells by a concentration-dependent mechanism. This effect does not correlate directly with the reduced cellular growth rate in low serum or serum-free medium. Lipid-free medium, which has little growth-promoting activity, maintains the capacity to lower the rate of S-100 synthesis. A partially purified extract of platelets can substitute for serum in repressing the synthesis of the S-100 protein.

[Optimation and simplification of clinical tumor cytodiagnosis by means of cytocentrifugation]. / Optimierung und Vereinfachung der klinischen Tumorzytodiagnostik mittels Zytozentrifugation

Since the introduction of cytocentrifugation, the methods of clinical cytology have become more refined. The following advantages should be stressed: 1. speed and ease of processing, 2. highest possible cellular yield, 3. good preservation of cellular characteristics. In the light of predominantly CSF analyses in the field of pediatric oncology the above mentioned findings are clearly illustrated (ALL, AML, reticulum cell sarcomatosis, Ewing sarcoma, leptomeninx sarcoma, retinoblastoma, Letterer-Siwe's disease, familial erythrophagocytic lymphohistiocytosis, plexus papilloma). Malignancies are clearly distinguishable from benign conditions (toxic glial reactions, virus meningitides, CNS hemorrhages etc.). The higher cellular yield permits very early diagnosis of meningeal leukemia. This qualitative improvement of cytological diagnostic methods may considerably influence the choice of therapeutic procedures in the treatment of malignancies.

Glia-induced morphological differentiation in neuroblastoma cells.

Glial cells release a factor into their culture medium that induces a high degree of morphological differentiation in neuroblastoma cells under normal growth conditions. This phenomenon is not correlated with a change in intracellular adenosine 3':5'-cyclic monophosphate or in the rate of cell growth. Media from other cell lines tested induce less morphological differentiation or have no effect.