Catecholamines and vitiligo.

Catecholamines and vitiligo. The levels of some catecholamine metabolites, namely homovanillic acid (HVA), vanilmandelic acid (VMA), 3-methoxytyramine (MT), normetanephrine (NMN), metanephrine (MN), 3,4-dihydroxy mandelic acid (DOMAC), and 3,4-dihydroxy phenylacetic acid (DOPAC), have been evaluated in the 24 hr urines of 150 patients affected with different types of vitiligo and in 50 healthy age-matched individuals. The patients were divided into three groups according to the different phases of the disease. The first group included subjects affected either with the early active phase or with progressive increase in both number and/or the size of previous lesions. The second group included patients in whom no new lesions had appeared for between 4-8 months. In the third group the white areas had been stable for 1-5 years. The first and second groups showed values of HVA and VMA from 4 to 10 times and from 1/2 to 3 times higher respectively than those of controls, while no significant differences were found between the third group and controls. Our results clearly show that a significant increase of urinary levels of HVA and VMA, deriving respectively from dopamine and from norepinephrine and epinephrine characterizes the onset and the progressive active phases of vitiligo, irrespective of the type of distribution. The increased release of catecholamines from the autonomic nerve endings in the microenvironment of melanocytes in the affected skin areas might be involved in the etiopathogenesis of vitiligo through two main mechanisms: (1) a direct cytotoxic action of catecholamines and/or their o-diphenol catabolites; (2) an indirect action.(ABSTRACT TRUNCATED AT 250 WORDS)


Biochemical properties of the kringle 2 and protease domains are maintained in the refolded t-PA deletion variant BM 06.022. BM 06.022 is a t-PA deletion variant which comprises the kringle 2 and the protease domain. Production of BM 06.022 in Escherichia coli leads to the formation of inactive inclusion bodies, which have to be refolded by an in vitro refolding process to achieve activity and proper structure of the domains. We analysed the biochemical properties of BM 06.022 to obtain some information about the structure of kringle 2 and the protease as compared with the structure of these domains in the intact t-PA molecule. The kinetic analysis of the amidolytic activity of BM 06.022 and CHO-t-PA yielded similar values for kcat (13.9 s-1 and 11.4 s-1 for the single chain forms and 33.9 s-1 and 27.1 s-1 for the two chain forms of BM 06.022 and CHO-t-PA, respectively) and for Km (2.5 mM and 2.1 mM for the single chains forms and 0.5 mM and 0.3 mM for the two chain forms of BM 06.022 and CHO-t-PA, respectively). BM 06.022 and CHO-t-PA have the same plasminogenolytic activity in the absence of CNBr fragments of fibrinogen. However, BM 06.022 has a lower plasminogenolytic activity in the presence of CNBr fragments of fibrinogen and a lower affinity to fibrin as compared with CHO-t-PA. The affinity of BM 06.022 for fibrin is completely suppressed by 0.3 mM epsilon-aminocaproic acid, while the intact t-PA has a residual affinity of approximately 30%. The dissociation constants for the interaction with the lysine analogue epsilon-aminocaproic acid are 0.10 mM and 0.09 mM for BM 06.022 and the intact t-PA, respectively. Furthermore, BM 06.022 and CHO-t-PA are inhibited by PAI-1 in a similar manner.


Intracellular amplification and expression of a synthetic analog of rotavirus genomic RNA bearing a foreign marker gene: mapping cis-acting nucleotides in the 3'-noncoding region. cDNAs were constructed to encode plus- or minus-sense analogs of gene 9 RNA of porcine rotavirus strain OSU in which the bacterial chloramphenicol acetyltransferase (CAT) reporter gene was flanked by the 5'-terminal 44 nucleotides (nt) and 3'-terminal 35 nt of the authentic rotavirus gene. Transfection of plus-sense gene-9-CAT RNA into rotavirus-infected cells resulted in its amplification and in the efficient expression of CAT; this was greatly enhanced by the presence of a 5' cap structure. Amplification was ablated by omitting the rotavirus superinfection or by removing the 3'-terminal 35-nt rotavirus sequence from the RNA. This result indicated that amplification depended both on rotavirus proteins supplied in trans and on cis-acting rotavirus sequences. Minus-sense or double-stranded gene-9-CAT RNA was essentially inactive, indicating that synthetic RNAs can be introduced into the rotavirus replicative cycle in vivo only when provided in the plus sense. However, incorporation of the CAT-bearing RNA into infectious rotavirus was not detected. Two heterologous rotaviruses, the simian RRV and chicken Ch2 strains, efficiently complemented the OSU-based gene-9-CAT RNA, even though the Ch2 strain was only 50%-66% related in the noncoding regions. Mutational analysis of the 35-nt 3'-noncoding region showed that the 3'-terminal 12 or 17 nt were sufficient for reduced (12% or 23%, respectively) levels of amplification, whereas inclusion of the 3'-terminal 19 nt fully restored amplification. Thus, the 3'-terminal cis-acting signals required for amplification include the 7-nt-terminal consensus sequence together with 12 nt of adjoining, less-well-conserved sequence.


Cloned human neuropeptide Y receptor couples to two different second messenger systems. Neuropeptide Y (NPY) is one of the most abundant neuropeptides in the mammalian nervous system and exhibits a diverse range of important physiological activities, including effects on psychomotor activity, food intake, regulation of central endocrine secretion, and potent vasoactive effects on the cardiovascular system. Two major subtypes of NPY receptor (Y1 and Y2) have been defined by pharmacological criteria. We report here the molecular cloning of a cDNA sequence encoding a human NPY receptor and the corrected sequence for a rat homologue. Analysis of this sequence confirms that the receptor is a member of the G protein-coupled receptor superfamily. When expressed in Chinese hamster ovary (CHO) or human embryonic kidney (293) cells, the receptor exhibits the characteristic ligand specificity of a Y1 type of NPY receptor. In the 293 cell line, the receptor is coupled to a pertussis toxin-sensitive G protein that mediates the inhibition of cyclic AMP accumulation. In the CHO cell line, the receptor is coupled not to the inhibition of adenylate cyclase but rather to the elevation of intracellular calcium. These results demonstrate that second messenger coupling of the NPY-Y1 receptor is cell type specific, depending on the specific repertoire of G proteins and effector systems present in any cell type.


Viral E1 and E2 proteins support replication of homologous and heterologous papillomaviral origins. We have shown that E1 and E2 proteins of human papillomavirus type 11 (HPV-11) were essential to support the replication of the homologous viral origin (ori) in a transient replication assay, similar to reports on bovine papillomavirus type 1 (BPV-1). Unexpectedly, matched or even mixed combinations of E1 and E2 proteins from HPV-11 or BPV-1 replicated either ori in human, monkey, and rodent cell lines of epithelial or fibroblastic lineage, albeit with varied efficiencies. Either set of viral proteins was also able to initiate replication of ori-containing plasmids from many other human and animal papillomaviruses. Thus the interactions among the cis elements and trans factors of papillomaviruses are more conserved than expected from the other members of the papovavirus family, simian virus 40 and polyomavirus, for which large tumor antigen does not replicate a heterologous ori in either permissive or nonpermissive cells. We infer that the stringent species and tissue specificities observed for papillomaviruses in vivo are not entirely due to direct restrictions on viral DNA replication. Rather, transcriptional control of viral gene expression must play a dominant role.


Functional binding of the "TATA" box binding component of transcription factor TFIID to the -30 region of TATA-less promoters. Many viral and cellular promoters transcribed in higher eukaryotes by RNA polymerase II lack obvious A+T-rich sequences, called "TATA" boxes, that bind the transcription factor TFIID. One such TATA-less promoter, the simian virus 40 major late promoter, contains a genetically important sequence element 30 base pairs upstream of its transcription initiation site that has no obvious sequence similarity to a TATA box. We show here that the cloned human TATA box-binding protein, hTFIID tau, functionally binds to this upstream sequence element, although with an affinity one-sixth of that to which it binds the TATA box of the adenovirus type 2 major late promoter. Analysis of point mutations in the -30 element of the simian virus 40 major late promoter shows that the affinity of binding correlates with the efficiency of transcription from this promoter. Furthermore, this element has genetic properties similar to those of a TATA box. (i) It directs RNA polymerase II to initiate transcription approximately 30 base pairs downstream of its location, and (ii) inactivation of this element results in increased heterogeneity in the sites of transcription initiation. All of five other TATA-less promoters tested were found to contain a sequence approximately 30 base pairs upstream of their major transcription initiation sites to which hTFIID tau binds. We conclude that many, if not all, TATA-less promoters differ from TATA box-containing promoters simply in the affinity of their -30 regions for binding of TFIID, with functional binding of TFIID supported in part by other nearby sequence elements of the promoter.


Human chromosomal localization of genes encoding the gamma 1 and gamma 2 subunits of the gamma-aminobutyric acid receptor indicates that members of this gene family are often clustered in the genome. The gamma-aminobutyric acid (GABA) receptors are the major inhibitory neurotransmitter receptors in the brain and the site of action of a number of important pharmacological agents including barbiturates, benzodiazepines, and ethanol. The gamma 1 and gamma 2 subunits have been shown to be important in mediating responses to benzodiazepines, and a splicing variant of the gamma 2 subunit, gamma 2L, has been shown to be necessary for ethanol actions on the receptor, raising the possibility that the gamma 2 gene may be involved in human genetic predisposition to the development of alcoholism. We have assigned the human genes encoding the gamma 1 and gamma 2 subunits of the GABAA receptor to chromosomes 4 and 5, respectively, by PCR amplification of human-specific products from human-hamster somatic cell hybrid DNAs. Using panels of chromosome-specific natural deletion hybrids, we have further localized the gamma 1 gene (GABRG1) to 4p14-q21.1 and the gamma 2 gene (GABRG2) to 5q31.1-q33.2. These data indicate that the gamma 1 gene may be clustered together with the previously mapped alpha 2 and beta 1 genes on chromosome 4 and that the gamma 2 gene may be close to the previously localized alpha 1 gene on chromosome 5. To further examine the latter possibility the alpha 1 gene was mapped using the chromosome 5 deletion hybrids and shown to be within the same region as the gamma 2 gene, 5q31.1-q33.2. A PCR-based screening strategy was used to isolate a 450-kilobase human genomic yeast artificial chromosome clone containing both the alpha 1 and gamma 2 genes. Pulsed-field gel restriction mapping of the yeast artificial chromosome indicates that the two genes are within 200 kilobases of each other. The data presented here provide further evidence for the nonrandom organization of the human genome by demonstrating that members of the GABAA receptor gene family often occur in small gene clusters widely distributed in the genome.


Recognition of the Epstein-Barr virus-encoded nuclear antigens EBNA-4 and EBNA-6 by HLA-A11-restricted cytotoxic T lymphocytes: implications for down-regulation of HLA-A11 in Burkitt lymphoma. Evasion from cytotoxic T-lymphocyte (CTL) surveillance may be an important step in the pathogenesis of Epstein-Barr virus (EBV)-carrying Burkitt lymphoma (BL) as suggested by the consistent down-regulation of all transformation-associated viral antigens, except EBV nuclear antigen 1 (EBNA-1), and of certain HLA class I alleles in BL biopsies and cell lines that maintain the tumor cell phenotype in vitro. The most common HLA class I defect recorded in BL lines is a selective down-regulation of HLA-A11. To gain some insight into the role of HLA-A11 down-regulation in pathogenesis of BL, we have investigated the target specificity of HLA-A11-restricted CTLs derived by stimulation of lymphocytes from three EBV-seropositive individuals with autologous EBV-transformed lymphoblastoid cell lines. Recombinant vaccinia viruses carrying the coding sequences for EBNA-1, -2A, -2B, -5, -3, -4, and -6 (also known as EBNA-1, -2A, -2B, -LP, -3a, -3b, and -3c, respectively) and EBV latent membrane protein 1 were used to induce high levels of expression of the relevant EBV antigen in fibroblasts derived from HLA class I-matched individuals. EBNA-4-expressing fibroblasts were the predominant target of HLA-A11-restricted CTLs in all three donors. A less pronounced and less regular EBNA-6-specific cytotoxic component was found in two of the donors.


Membrane-bound Dictyostelium myosin heavy chain kinase: a developmentally regulated substrate-specific member of the protein kinase C family. A cDNA clone corresponding to the Dictyostelium myosin heavy chain kinase (MHCK) gene was isolated using antibodies specific to the purified enzyme. Sequence analysis of the cDNA revealed that the Dictyostelium MHCK possesses all of the domains characteristic of members of the protein kinase C family. The amino-terminal region of the MHCK contains the cysteine-rich motif with an internal duplication that is present in all known protein kinase C species. This domain precedes sequences that are highly homologous to protein kinase catalytic domains. The carboxyl-terminal region contains a cluster of 23 serine and threonine residues that may represent the autophosphorylation domain of the Dictyostelium MHCK. These results, along with previous studies that indicate that this enzyme has very restrictive substrate specificity, incorporates approximately 20 mol of phosphate per mol of kinase through an autophosphorylation reaction, and is expressed only during development, suggest that the Dictyostelium MHCK is a distinct member of the protein kinase C family and imply that this kinase family, which may include members with very specific cellular functions, may be even more heterogeneous than previously thought.


Hormonal regulation of the thyrotropin beta-subunit gene by phosphorylation of the pituitary-specific transcription factor Pit-1. The pituitary-specific transcription factor Pit-1 is a cell-specific activator of prolactin and growth hormone gene transcription in the anterior pituitary. Pit-1 has also been shown to mediate both thyrotropin-releasing hormone (TRH) and cAMP stimulation of the prolactin and thyrotropin beta-subunit (TSH beta) genes. The molecular mechanism by which Pit-1 mediates these stimulatory effects remains unclear. At least three Pit-1-binding elements within the TSH beta gene mediate responsiveness to TRH and cAMP. The present studies were designed to test the hypothesis that phosphorylation is an important modulator of Pit-1 interaction with the TSH beta gene. TSH beta elements bind less well to nonphosphorylated Pit-1 than to phosphorylated Pit-1 and are weak activators of gene expression, unlike high-affinity Pit-1 binding sites in the prolactin and growth hormone genes. Phosphorylation by protein kinase A or C enhances Pit-1 binding to TSH beta elements 3- to 8-fold. Conversely, phosphorylation generally reduces binding of Pit-1 to elements within the prolactin and growth hormone genes. A variation within the consensus sequence for Pit-1 binding in TSH beta gene elements [A(A/T)(A/T)AATNCAT in the TSH beta gene versus A(A/T)(A/T)TATNCAT in the prolactin and growth hormone genes] could explain these differences. These elements may limit basal activation of the TSH beta gene by binding less well to nonphosphorylated Pit-1 while conferring hormonal stimulation through enhanced binding of phosphorylated Pit-1.


Transient immunoglobulin M antibody response to hepatitis C virus capsid antigen in posttransfusion hepatitis C: putative serological marker for acute viral infection. The development of serological assays for hepatitis C virus (HCV) has made specific diagnosis possible. However, markers useful in indicating acute-phase HCV infection have not been identified. By an immunoblotting method, we characterized the IgM and IgG antibody response against HCV capsid antigen in patients with HCV infection. Among 88% of patients with acute posttransfusion hepatitis C recruited in a prospective study, there was a transient IgM antibody response. The IgM antibody appeared shortly after onset of hepatitis (average 3.7 weeks), persisted for several months (average 18 weeks), and then disappeared. In contrast, the IgG antibody persisted long-term once it appeared. Among patients with chronic hepatitis C with milder disease activities (serum aminotransferase increase above normal levels of less than 4-fold), the IgM antibody was negative in the majority (72%). In those with acute exacerbations (aminotransferase increase of greater than 10-fold), about 55% were negative for the IgM antibody. The reactivity of the IgM antibody in the rest was weaker or became negative upon further dilution of serum. The results suggest that IgM anti-capsid antibody may serve as a marker indicating acute or active HCV infection.


Oxytocin receptor distribution reflects social organization in monogamous and polygamous voles. The neuropeptide oxytocin has been implicated in the mediation of several forms of affiliative behavior including parental care, grooming, and sex behavior. Here we demonstrate that species from the genus Microtus (voles) selected for differences in social affiliation show contrasting patterns of oxytocin receptor expression in brain. By in vitro receptor autoradiography with an iodinated oxytocin analogue, specific binding to brain oxytocin receptors was observed in both the monogamous prairie vole (Microtus ochrogaster) and the polygamous montane vole (Microtus montanus). In the prairie vole, oxytocin receptor density was highest in the prelimbic cortex, bed nucleus of the stria terminalis, nucleus accumbens, midline nuclei of the thalamus, and the lateral aspects of the amygdala. These brain areas showed little binding in the montane vole, in which oxytocin receptors were localized to the lateral septum, ventromedial nucleus of the hypothalamus, and cortical nucleus of the amygdala. Similar differences in brain oxytocin receptor distribution were observed in two additional species, the monogamous pine vole (Microtus pinetorum) and the polygamous meadow vole (Microtus pennsylvanicus). Receptor distributions for two other neurotransmitter systems implicated in the mediation of social behavior, benzodiazepines, and mu opioids did not show comparable species differences. Furthermore, in the montane vole, which shows little affiliative behavior except during the postpartum period, brain oxytocin receptor distribution changed within 24 hr of parturition, concurrent with the onset of maternal behavior. We suggest that variable expression of the oxytocin receptor in brain may be an important mechanism in evolution of species-typical differences in social bonding and affiliative behavior.


A dominant mutation in the Wilms tumor gene WT1 cooperates with the viral oncogene E1A in transformation of primary kidney cells. Wilms tumor is a pediatric kidney cancer that has been linked to the inactivation of a tumor-suppressor gene at chromosome locus 11p13. The WT1 gene, mapping to this locus, is developmentally regulated in the kidney and encodes a putative transcription factor that has been shown to be mutated in Wilms tumor specimens. We have suggested that one such altered product of the WT1 gene may be capable of trans-dominant suppression, since the mutated allele was found to be coexpressed with the wild-type allele in a sporadic Wilms tumor. We therefore tested the ability of this mutant WT1 allele, containing an in-frame deletion within the DNA-binding domain, to transform primary baby rat kidney cells. The mutant WT1 gene was found to cooperate with the adenoviral E1A gene in transforming baby rat kidney cells, as demonstrated by growth in soft agar and tumorigenicity in nude mice. The wild-type WT1 gene in all of its alternatively spliced forms neither suppressed E1A-induced focus formation nor cooperated with E1A. Our results indicate that impairment of DNA binding of the WT1 tumor-suppressor gene product can result in a dominant negative mutation.


Rapid fitness losses in mammalian RNA virus clones due to Muller's ratchet. Muller's ratchet is an important concept in population genetics. It predicts that when mutation rates are high and a significant proportion of mutations are deleterious, a kind of irreversible ratchet mechanism will gradually decrease the mean fitness of small populations of asexual organisms. In contrast, sexual recombination may stop or reverse this mutational ratchet by recombinational repair of genetic damage. Experimental support for Muller's ratchet has previously been obtained in protozoa and in a tripartite RNA bacteriophage. We now show clear evidence that Muller's ratchet can operate on a nonsegmented nonrecombining pathogenic RNA virus of animals and humans. We did genetic bottleneck passages (plaque-to-plaque transfers) of vesicular somatitis virus (VSV) and then quantitated relative fitness of the bottleneck clones by allowing direct replication competition in mixed infections in cell culture. We document variable fitness drops (some severe) following only 20 plaque-to-plaque transfers of VSV. In some clones no fitness changes (or only insignificant changes) were observed. Surprisingly, the most regular and severe fitness losses occurred during virus passages on a new host cell type. These results again demonstrate the extreme genetic and biological variability of RNA virus populations. Muller's ratchet could have significant implications for variability of disease severity during virus outbreaks, since genetic bottlenecks must often occur during respiratory droplet transmissions and during spread of low-yield RNA viruses from one body site to another (as with human immunodeficiency virus). Likewise, the lower-probability generation of increased-fitness clones during repeated genetic bottleneck transfers of RNA viruses in nature might also affect disease pathogenesis in infected individuals and in host populations. Whenever genetic bottlenecks of RNA viruses occur, enhanced biological differences among viral subpopulations may result.


Molecular cloning of a Drosophila diacylglycerol kinase gene that is expressed in the nervous system and muscle. We have isolated a Drosophila melanogaster diacylglycerol kinase (DGK, EC 2.7.1.107) homologue by using a porcine DGK cDNA probe and we have characterized its structure and expression. The DGK cDNA has a single open reading frame that encodes 791 amino acids. The Drosophila and porcine DGKs share a similar carboxyl-terminal region, a putative catalytic domain, which is divided into two separate domains in Drosophila. The DGK gene was mapped to the cytogenetic position 43F1, and its DGK mRNA is abundant both in embryo and in adult fly. By in situ hybridization to sections of adult flies, we demonstrated that the mRNA is present predominantly in the nervous system and muscles, including compound eyes, brain cortex, fibrillar muscle, and tubular muscle. In a 10- to 11-hr embryo, the DGK gene is expressed abundantly in a limited number of cells in the procephalic region and in the ventral nerve cord. The pattern of temporal and spatial expression suggests that the DGK protein has an important function in the adult nervous system and muscle and during the development of the embryonic nervous system.


High rates of Ac/Ds germinal transposition in Arabidopsis suitable for gene isolation by insertional mutagenesis. Overexpression of the Activator (Ac) transposase gene in Arabidopsis thaliana resulted in a minimal germinal transposition frequency of 27% in which independent Dissociation (Ds) transposition events were observed. Molecular analysis of 45 F1 generation Ac/Ds plants indicated that high rates of somatic excision had occurred, and independent germinal insertions were identified in F2 generation progeny plants. A tandem cauliflower mosaic virus (CaMV) promoter fused to two different Ac coding sequences significantly increased the rate of Ds transposition. The CaMV-Ac fusions activated single and multiple copies of two different Ds elements, DsDHFR and Ds35S-1, and reciprocal crosses resulted in similar transposition frequencies. The improved rate of independent germinal transposition observed makes Arabidopsis an ideal system for insertional mutagenesis.


Vitamin D-responsive protein-DNA interactions at multiple promoter regulatory elements that contribute to the level of rat osteocalcin gene expression. The observation that vitamin D-mediated enhancement of osteocalcin (OC) gene expression is dependent on and reciprocally related to the level of basal gene expression suggests that an interaction of the vitamin D responsive element (VDRE) with basal regulatory elements of the OC gene promoter contributes to both basal and vitamin D-enhanced transcription. Protein-DNA interactions at the VDRE of the rat OC gene (nucleotides -466 to -437) are reflected by direct sequence-specific and antibody-sensitive binding of the endogenous vitamin D receptor present in ROS 17/2.8 osteosarcoma nuclear protein extracts. In addition, a vitamin D-responsive increase in OC gene transcription is accompanied by enhanced non-vitamin D receptor-mediated protein-DNA interactions in the "TATA" box region (nucleotides -44 to +23), which also contains a potential glucocorticoid responsive element. Evidence for proximity of the VDRE with the basal regulatory elements is provided by two features of nuclear architecture. (i) Nuclear matrix attachment elements in the rat OC gene promoter that bind nuclear matrix proteins with sequence specificity may impose structural constraints on promoter conformation. (ii) Limited micrococcal nuclease digestion and Southern blot analysis indicate that three nucleosomes can be accommodated in the sequence spanning the OC gene VDRE, the OC/CCAAT box (nucleotides -99 to -76), and the TATA/glucocorticoid responsive element, and thereby the potential distance between the VDRE and the basal regulatory elements can be reduced. A model is presented for the contribution of both the VDRE and proximal promoter elements to the enhancement of OC gene transcription in response to vitamin D. The vitamin D receptor plus accessory proteins may function cooperatively with basal regulatory factors to modulate the extent to which the OC gene is transcribed.


Protein-induced bending and DNA cyclization. We have applied T4 ligase-mediated DNA cyclization kinetics to protein-induced bending in DNA. The presence and direction of a static bend can be inferred from J factors for cyclization of 150- to 160-base-pair minicircles, which include a catabolite activator protein binding site phased against a sequence-directed bend. We demonstrate a quasi-thermodynamic linkage between cyclization and protein binding; we find that properly phased DNAs bind catabolite activator protein approximately 200-fold more tightly as circles than as linear molecules. The results unambiguously distinguish DNA bends from isotropically flexible sites and can explain cooperative binding by proteins that need not contact each other.


Stable expression of mammalian type A gamma-aminobutyric acid receptors in mouse cells: demonstration of functional assembly of benzodiazepine-responsive sites. The differential sensitivity of type A gamma-aminobutyric acid (GABAA) receptors to benzodiazepine ligands seen in the mammalian nervous system is thought to be generated by the existence of a number of different receptor subtypes, assembled from a range of closely related subunits (alpha 1-6, beta 1-3, gamma 1-3, and delta) encoded by discrete genes. The characteristics of a given subtype can be determined by the coexpression of cloned cDNAs encoding the subunits of interest. Two transient expression systems have so far been employed in the study of the ligand-binding characteristics and chloride channel properties of such GABAA receptors--Xenopus oocytes and transfected mammalian cells. Here we report on the use of a steroid-inducible promoter expression system for the production of a permanently transfected clonal cell line expressing the alpha 1 beta 1 gamma 2L GABAA receptor subtype. Using both immunoprecipitation by subunit-specific antisera and gel-exclusion chromatography, we have shown that the alpha 1, beta 1, and gamma 2L subunits coassemble to form receptor macromolecules that are of the same size as native GABAA receptors. Additionally, the recombinant receptors have the same benzodiazepine pharmacology as native alpha 1-containing GABAA receptors and function as GABA-gated chloride channels. Such cell lines expressing individual GABAA receptor subtypes will prove important tools in the study of the structure, function, and pharmacology of GABAA receptors and in the development of subtype-specific drugs.


Unmutated proto-src coding region is tumorigenic if expressed from the promoter of Rous sarcoma virus: implications for the gene-mutation hypothesis of cancer. The transforming (onc) genes of oncogenic retroviruses share most or all of their coding sequences with normal cellular genes termed proto-onc genes. The viral genes differ from proto-onc genes in virus-specific promoters and in various point mutations and substitutions of cell-derived coding regions. In view of the structural similarities between viral oncogenes and cellular proto-onc genes, the hypothesis has been advanced that proto-onc genes become cellular cancer genes if they have suffered mutations. Indeed, point mutations and substitutions have been observed in the proto-onc genes of some cancers. However, the hypothesis has been difficult to prove because mutated proto-onc genes from tumors do not transform diploid cells. Moreover, owing to the popularity of this hypothesis, even viral oncogenes are thought to derive transforming function from mutations of this cell-derived coding region. A competing hypothesis proposes that enhanced expression from retroviral promoters is necessary and sufficient for oncogenic function of proto-onc genes. To distinguish between these hypotheses we have tested tumorigenicity of RpSV, a synthetic retrovirus with the normal proto-src coding region in a vector derived from Rous sarcoma virus (RSV). In addition, we have tested the role of RSV-specific src point mutations on the tumorigenicity of RpSV. It was found that RpSV with an unmutated proto-src coding region is tumorigenic in chickens and that tumorigenicity is enhanced by RSV-specific src point mutations. It is concluded that retroviral promoters are essential for the transforming function of viral oncogenes and that certain point mutations merely supplement their transforming function. Thus retroviral onc genes are not models for the hypothesis that mutated, but transcriptionally normal, proto-onc genes of certain tumors are cancer genes.


ATP is a coupling modulator of parallel Na,K-ATPase-K-channel activity in the renal proximal tubule. A fundamental and essential property of nearly all salt-transporting epithelia is the tight parallel coupling between the magnitude of the K-conductive pathway at the basolateral membrane and the activity of the Na,K-dependent ATPase (Na,K-ATPase). In the present study, we demonstrate that the coupling response in the renal proximal tubule is governed, at least in part, through the interaction between ATP-sensitive K channels and Na,K-ATPase-mediated changes in intracellular ATP levels. First, we identified a K-selective channel at the basolateral membrane, which is inhibited by the cytosolic addition of ATP. Second, conventional microelectrode analysis in the isolated perfused proximal straight tubule revealed that these channels are the major determinant of the macroscopic K conductance so that ATP-mediated changes in the open probability of the K channel could alter the extent of K recycling. Indeed, the increase in the macroscopic K conductance upon stimulation of transcellular Na transport and pump activity was found to be paralleled by a decrease in intracellular ATP. Finally, a causal link between parallel Na,K-ATPase-K-channel activity and ATP was established by the finding that intracellular ATP loading uncoupled the response. With our recent observations that similar ATP-sensitive K channels are expressed abundantly in other epithelia, we postulate that ATP may act as a universal coupling modulator of parallel Na,K-ATPase-K-channel activity.


Long-chain fatty acids activate calcium channels in ventricular myocytes. Nonesterified fatty acids accumulate at sites of tissue injury and necrosis. In cardiac tissue the concentrations of oleic acid, arachidonic acid, leukotrienes, and other fatty acids increase greatly during ischemia due to receptor or nonreceptor-mediated activation of phospholipases and/or diminished reacylation. In ischemic myocardium, the time course of increase in fatty acids and tissue calcium closely parallels irreversible cardiac damage. We postulated that fatty acids released from membrane phospholipids may be involved in the increase of intracellular calcium. We report here that low concentrations (3-30 microM) of each long-chain unsaturated (oleic, linoleic, linolenic, and arachidonic) and saturated (palmitic, stearic, and arachidic) fatty acid tested induced multifold increases in voltage-dependent calcium currents (ICa) in cardiac myocytes. In contrast, neither short-chain fatty acids (less than 12 carbons) or fatty acid esters (oleic and palmitic methyl esters) had any effect on ICa, indicating that activation of calcium channels depended on chain length and required a free carboxyl group. Inhibition of protein kinases C and A, G proteins, eicosanoid production, or nonenzymatic oxidation did not block the fatty acid-induced increase in ICa. Thus, long-chain fatty acids appear to directly activate ICa, possibly by acting at some lipid sites near the channels or directly on the channel protein itself. We suggest that the combined effects of fatty acids released during ischemia on ICa may contribute to ischemia-induced pathogenic events on the heart that involve calcium, such as arrhythmias, conduction disturbances, and myocardial damage due to cytotoxic calcium overload.


Regulation of exoprotein expression in Staphylococcus aureus by a locus (sar) distinct from agr. A single insertion of transposon Tn917LTV1 into the chromosome of a Staphylococcus aureus clinical isolate, strain DB, resulted in a pleiotropic effect on the expression of a number of extracellular and cell-wall-associated proteins. Detailed comparison of phenotypes associated with the mutant, 11D2, and the parent, DB, indicated that the chromosomal locus inactivated as a result of transposon mutagenesis differs from the S. aureus accessory gene regulator locus (agr). In particular, the expression of alpha-hemolysin, which is not detectable in Agr- mutants, was enhanced in mutant 11D2, while it remained at a low level in strain DB. Likewise, protease activity was significantly enhanced in 11D2 compared with DB. In addition, most of the cell-bound proteins were expressed at lower levels in the mutant than the parent strain. This pattern is contrary to that found in switching from Agr+ to Agr- phenotypes. Southern blot hybridization with an agr probe indicated that the inactivated chromosomal locus is distinct from agr. Transduction experiments demonstrated that the phenotypes associated with mutant 11D2 could be transferred to the parental strain DB as well as to RN450, an S. aureus strain with a genetic background similar to strain 8325-4. This locus on the S. aureus chromosome, possibly regulatory in nature, has been designated sar for staphylococcal accessory regulator.


Photoinduced electron transfer from tris(2,2'-bipyridyl)ruthenium to cytochrome c oxidase. Flash photolysis has been used to effect electron transfer from tris(2,2'-bipyridyl)ruthenium(II) to cytochrome c oxidase in the presence of a sacrificial electron donor, aniline. The observation that photoreduction occurs only at low ionic strength and high pH indicates that an electrostatic complex between the ruthenium compound and the enzyme is the reactive species. The reaction was followed at 830, 605, and 445 nm. The initial absorbance changes observed suggest that the copper ion CuA is the preferred electron acceptor and that electron transfer from the excited ruthenium complex takes place in less than 1 microsecond. Some rapid cytochrome a reduction is also observed. Absorbance changes after the initial transients suggest that the reduced CuA then equilibrates with cytochrome a with a rate constant of 2 x 10(4) s-1. Comparison of the absorbance changes at 605 and 445 nm and the kinetic difference spectrum in the Soret region indicate that no reduction of cytochrome a3 takes place. With the oxidized enzyme, no further reactions were detected, whereas, in the peroxide and ferryl intermediates, cytochrome a reoxidizes on a millisecond time scale. The reaction appears biphasic in both intermediates, with rate constants in the range 2 x 10(2) to 4 x 10(3) s-1. This is considerably slower than the maximal rates observed for electron transfer between cytochrome a and the bimetallic site found in earlier work and suggests rate limitation by other processes. The rates obtained for the slower phases are close to the rate for catalysis of cytochrome c oxidation.


Coinfection with multiple strains of the Epstein-Barr virus in human immunodeficiency virus-associated hairy leukoplakia. Epstein-Barr virus DNA was analyzed from specimens of hairy leukoplakia, an oral lesion that occurs in patients infected with the human immunodeficiency virus. The simultaneous presence of both type 1 and type 2 Epstein-Barr virus was demonstrated by Southern blot analysis and polymerase chain reaction assay. Restriction fragment length polymorphisms in the BamHI WYH region and in clones of the EcoRI C region suggested the presence of multiple strains of type 1 and type 2 viruses. The demonstration of multiple variably sized BamHI H fragments on Southern blot analysis and cloning of the EBNA-2 gene coding region also suggested the presence of multiple viral strains or variants coinfecting hairy leukoplakia. Recombination of the viral genome in and around the EBNA-2 gene apparently generated viral variants that replicated efficiently, one of which appeared to increase in abundance in a lesion over time. These data indicate that hairy leukoplakia involves coinfection with multiple strains of replicating Epstein-Barr virus and the endogenous generation of viral variants, some of which have mutations of the EBNA-2 gene.


Phosphorylation by cellular casein kinase II is essential for transcriptional activity of vesicular stomatitis virus phosphoprotein P. We have previously shown that phosphorylation of vesicular stomatitis virus (VSV) phosphoprotein P by cellular protein kinase activity is an essential prerequisite for its transcriptional function. We have now purified this protein kinase by monitoring its ability to phosphorylate bacterially expressed, unphosphorylated P protein. Biochemical studies showed that the kinase is indistinguishable from casein kinase II, a ubiquitous cyclic AMP-independent protein kinase present in a wide variety of eukaryotic cells and tissues. Functional VSV transcription could be reconstituted with viral L protein, N-RNA template, and P protein phosphorylated by either purified cellular protein kinase or purified casein kinase II. The unusual role of casein kinase II in the transcription process of a nonsegmented negative-strand RNA virus would have important implications in host-virus interactions and antiviral therapy.


Drug binding to higher ordered DNA structures: netropsin complexation with a nucleic acid triple helix. We have used a combination of spectroscopic and calorimetric techniques to characterize how netropsin, a ligand that binds in the minor groove of DNA, influences the properties of a DNA triple helix. Specifically, our data allow us to reach the following conclusions: (i) netropsin binds to the triplex without displacing the major-groove-bound third strand; (ii) netropsin binding to the triplex exhibits a lower saturation binding density (7.0 base triplets per netropsin bound) than netropsin binding to the corresponding duplex (5.5 base pairs per netropsin bound); (iii) the netropsin-free and the netropsin-bound triplexes each melt in two well-resolved transitions, initial conversion of the triplex to the duplex state followed by duplex melting to the component single-stranded states; (iv) netropsin remains bound to DNA as the triplex melts to the duplex state; (v) netropsin binding thermally destabilizes the triplex in equilibrium with duplex equilibrium dramatically, while thermally stabilizing the duplex to single-strand equilibrium; (vi) netropsin binding to the triplex is enthalpically 4 times more favorable (more exothermic) than netropsin binding to the corresponding duplex; (vii) netropsin binding to the triplex decreases the cooperativity of the triplex----duplex melting event. These results demonstrate that occupancy of the minor groove of a triplex by a ligand such as netropsin can exert a profound impact on the properties of the host triplex, particularly with regard to the equilibrium in which the third strand is expelled from the major groove. Thus, our results reveal considerable major groove/minor groove crosstalk. Such knowledge may prove of practical importance by providing an approach for modulating the affinity and specificity of major-groove-binding third strands in triplex-forming protocols designed to target specific duplex domains. Fundamentally, our results provide insights into the crosstalk that can result when ligands bind to the two major receptor sites of duplex DNA--namely, the major and minor grooves.


The existence of distinct classes of prostaglandin E2 receptors mediating adenylate cyclase and phospholipase C pathways in osteoblastic clone MC3T3-E1. We have reported previously that PGE2 evoked an increase in intracellular calcium level ([Ca2+]i) in mouse osteoblastic cells (1). Here, we investigated the effects of PGE1 and PGF2 alpha on cAMP production and [Ca2+]i in comparison with those of PGE2. In osteoblastic clone, MC3T3-E1 cells, PGE1 stimulated cAMP production, but had no effect on [Ca2+]i, whereas PGF2 alpha evoked only [Ca2+]i increase. In contrast, PGE2 not only stimulated cAMP production, but also increased [Ca2+]i. From the Scatchard plot analysis of PGE2 it was confirmed that there were two classes of PGE2 binding sites (Kd value, 9.2 nM; binding site, 29 fmole/mg protein, and Kd value, 134 nM; binding site, 148 fmole/mg protein). As the increase in [Ca2+]i was caused by PGF2 alpha and PGE2, but not by PGE1, we investigated the displacement of [3H]-PGF2 alpha binding. The displacement capacity of unlabeled PGE2 was about 110 of that of PGF2 alpha, while that of PGE1 was very low even at 500-fold excess. These data indicate the possibility that the dual action of PGE2 is mediated by distinct receptor systems.


Use of gamma-linolenic acid in the treatment of schizophrenia and tardive dyskinesia. There is good background evidence to suggest that essential fatty acids and their eicosanoid derivatives may play a role in schizophrenia and in with tardive dyskinesia. Trials involving treatment with essential fatty acids, or eicosanoids or drugs which stimulate eicosanoid synthesis have shown modestly promising results. Particularly favourable outcomes in both schizophrenia and tardive dyskinesia were associated with combined treatment using essential fatty acids and nutritional supplements.


The relationship between schizophrenia and essential fatty acid and eicosanoid metabolism. Essential fatty acids (EFAs) and their eicosanoid derivatives are important constituents of the brain and regulators of neuronal function. There is direct and indirect evidence of impaired metabolism of prostaglandin (PG)E1 in schizophrenia. There is also direct evidence of abnormal EFA biochemistry with plasma phospholipids from five populations and brain phospholipids from another all showing reduced levels of linoleic acid and elevated levels of 22-carbon EFAs of both n-6 and n-3 series. Clinical trials of PGE1 and of the PGE1 precursors, gamma-linolenic acid (GLA) and dihomo-gamma-linolenic acid (DGLA) have shown modest therapeutic effects. In view of lack of therapeutic process involving drugs based on the dopamine concept of schizophrenia, it is time for new approaches based on the EFA/PG concept to be evaluated thoroughly.


Comparison of behavioral and radioprotective effects of WR-2721 and WR-3689. The behavioral effects of the radioprotectant agents ethiofos, S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721) and S-2-(3-methylaminopropyl)aminoethylphosphorothioic acid (WR-3689) were evaluated in rats trained to respond under a multiple fixed-interval 120-s, fixed-ratio 50-response (mult FI FR) schedule of milk reinforcement. Each compound produced dose-dependent reductions in responding under both schedules over the same dose range (100-180 mg/kg, IP); ED50s indicated that WR-3689 was slightly more potent than WR-2721. On several performance measures, WR-3689 produced greater decrements during a second dose-effect determination, whereas WR-2721 had more pronounced effects during the initial one. In a second series of studies, low (56 mg/kg) and high (180 mg/kg) doses of both drugs were tested for radioprotective effects in rats responding under an FR-50 schedule of milk reinforcement and exposed to a nonlethal (5 gray, Gy) or lethal (10 Gy) dose of ionizing radiation (60Co gamma rays). Neither dose of radiation altered FR response rates on the day of exposure (day 1). Five Gy of gamma radiation produced a 25-40% reduction in response rates on days 2-5 (24-72 h) after exposure. Neither dose of WR-2721 or WR-3689 provided significant protection against these performance decrements. All groups exposed to 10 Gy experienced a progressive decline in FR responding on days 2-5 after exposure. Performance of groups that received pretreatment with the 180-mg/kg dose of either drug or the 56-mg/kg dose of WR-3689 was maintained at significantly higher levels than saline-treated controls on days 4-5 after exposure to 10 Gy; however, even at these higher levels of performance response rates remained below 50% of preirradiation control levels. Subsequently, 56 and 180 mg/kg WR-3689 and 180 mg/kg WR-2721 were found to provide protection against the lethal consequences of the 10-Gy exposure. Thus, neither WR-2721 nor WR-3689 afforded any significant short-term protection against radiation-induced performance decrements when these drugs were administered at either behaviorally ineffective or behaviorally disruptive doses. Rather, the beneficial effects of these drugs paralleled their ability to antagonize radiation-induced lethality.


Acute effects of delta-9-tetrahydrocannabinol on dopaminergic activity in several rat brain areas. In this work, we examined the acute effects of two doses of delta-9-tetrahydrocannabinol (THC) on several pre- and postsynaptic biochemical measures of dopaminergic activity in the striatum, limbic forebrain, and hypothalamic-anterior pituitary area of adult male rats. The exposure to a low dose of THC (0.5 mg/kg bw) decreased the number of striatal D2 dopaminergic binding sites, but did not affect their affinity. Treatment with a higher dose of THC was ineffective. In addition, both doses decreased the number of D1 dopaminergic binding sites in the limbic forebrain without changing their affinity. We did not find any changes in the dopamine (DA) or L-3,4-dihydroxyphenylacetic acid (DOPAC) content, or in the DOPAC/DA ratio, in either the striatum or limbic forebrain. THC treatment produced a dose-related decline in plasma prolactin (PRL) levels. Furthermore, both the basal and DA-inhibited in vitro release of PRL were reduced in animals exposed to THC in a dose-dependent manner. This inhibitory effect of THC on PRL release was accompanied by a decreased DOPAC/DA ratio in medial basal hypothalamus that, in turn, may be a result of the fall in PRL levels rather than a direct action of the drug. These data show that acute exposure to THC can alter brain dopaminergic neurotransmission. Our results suggest that the reduction of PRL release following THC exposure, both in vivo and in vitro, might be elicited by a direct action of THC on the pituitary.


Hippocampal phosphoinositide turnover is altered by hippocampal sympathetic ingrowth and cholinergic denervation. Cholinergic denervation of the hippocampus, by medial septal (MS) lesions, results in an unusual neuronal rearrangement in which peripheral sympathetic nerves, which originate from the superior cervical ganglia, grow into the hippocampal formation. To assess the functional significance of hippocampal sympathetic ingrowth (HSI), hydrolysis of phosphoinositides was examined in three groups: control, MS lesions + sham ganglionectomy (HSI group); and MS lesions + ganglionectomy (MSGx; no ingrowth). Four months after surgery, both norepinephrine (NE) and carbachol were found to produce a dose-dependent increase in the hydrolysis of hippocampal phosphoinositides in all groups. However, the presence of HSI, when compared to control and MSGx groups, significantly enhanced the turnover of phosphoinositides when stimulated by carbachol, but not NE. In further studies, the time course of this effect was studied. One week after surgery, carbachol-stimulated phosphoinositide turnover was equivalent among all groups; by 2 weeks, phosphoinositide turnover was enhanced in the HSI and MSGx group; by 4 weeks, PI turnover was markedly diminished in the MSGx group when compared to both the HSI and control groups, which were equivalent to each other. To ensure that the ganglionectomy alone did not alter phosphoinositide turnover, a ganglionectomy-alone group was studied at the 4-week time point. In this group, phosphoinositide turnover was equivalent to controls, suggesting no influence of the superior cervical ganglia on this response. In all groups, atropine inhibited carbachol-stimulated phosphoinositide turnover. These results suggest that both cholinergic denervation (i.e., MSGx group) and HSI produce marked functional alterations in hippocampal metabolic activity.


Experimental hydroxyapatite cement cranioplasty. Hydroxyapatite cement is a calcium phosphate-based material that when mixed with water forms a dense paste that sets within 15 minutes and isothermically converts in vivo to a microporous hydroxyapatite implant. This cement was used to reconstruct bilateral 2.5-cm-diameter full-thickness critical-sized parietal skull defects in six cats. One side was reconstructed with 100 percent hydroxyapatite cement, and the other with a mixture of 50 percent hydroxyapatite cement and 50 percent ground autogenous bone by weight. These animals were sacrificed at 6 and 12 months after implantation. Positive and negative controls also were prepared. The anatomic contour of the soft tissue overlying all hydroxyapatite cement implants was well maintained, there were no wound infections or structural failures, and the implants were well tolerated histologically. None of the negative (unreconstructed) control defects was completely filled with repair bone, and all positive (methyl methacrylate) controls demonstrated foreign-body giant-cell formation and fibrous encapsulation of the implants. Examination of decalcified and undecalcified sections revealed progressive but variable replacement of the cement by new bone and soft tissue without a change in the shape or volume of the hydroxyapatite cement-reconstructed areas. New bone comprised 77.3 and 64.7 percent of the tissue replacing the hydroxyapatite cement and hydroxyapatite cement-bone implants, respectively. Replacement of the hydroxyapatite cement implants by new bone is postulated to occur by a combination of osteoconduction and implant resorption. These results indicate that further experimental research leading to the possible application of hydroxyapatite cement for full-thickness calvarial defect reconstruction in humans is warranted.


Inferior alveolar nerve reconstruction with a polyglycolic acid bioabsorbable nerve conduit. A custom-designed polyglycolic acid (PGA) bioabsorbable nerve conduit was used to reconstruct a 25-mm defect in the right inferior alveolar nerve. The initial nerve injury, following a dental extraction, resulted in loss of lower lip sensation and severe facial pain. Sixteen months after tooth extraction, with no improvement in symptomatology, the alveolar canal was enlarged in diameter by means of mandibular osteotomy to accommodate a 2-mm-diameter polyglycolic acid tube. The proximal end of the inferior alveolar nerve was sutured into the polyglycolic acid tube. The mental nerve was sutured into the distal end of the tube. Pain of neural origin was relieved in the early postoperative period. Two years following nerve reconstruction, pain relief remains excellent and perception of pressure and vibration is similar to the thresholds for these perceptions on the contralateral lip.


Glycogen storage disease diagnosed in adults. Glycogen storage diseases are usually identified in childhood. We present the clinical, biochemical and histological features of 10 patients first diagnosed in adult life. Five had glycogen storage disease type 1a, one type 1c, two type IX, and in two patients there were previously unreported abnormalities of hepatic glucose-6-phosphatase system activity. Of the latter, one patient had an inhibitor of liver glucose-6-phosphatase (pseudo-1b glycogen storage disease) the other having abnormal glucose-6-phosphatase activity and microsomal pyrophosphate transport. A glucagon test is suggested as a useful screening procedure. Glycogen storage disease should be considered in adults with symptoms suggesting hypoglycaemia.


The neurological features of HIV-positive patients in Glasgow--a retrospective study of 90 cases. A retrospective study of the neurological problems arising in HIV-I seropositive patients in a single defined geographical area was undertaken. Ninety patients were referred for a neurological opinion from a total known HIV-I seropositive population of 436. Minor problems were frequently encountered early in the course of disease (20 at CDC stage II, 12 at CDC stage III), including seizures related to drug abuse in six. The most frequent neurological problem in those patients in CDC group IV (58 patients) were the AIDS dementia complex (14 patients), an axonal sensorimotor neuropathy (12), toxoplasmosis (nine) and cryptococcal meningitis (three). All patients with a structural lesion had appropriate focal signs on examination. The value and role of CT cranial scanning in the diagnosis of toxoplasmosis is discussed and the importance of recognizing potentially treatable causes of both intellectual impairment and cytomegalovirus-related neuropathies is stressed. This is the first report of an unselected series of patients at all stages of HIV-I related neurological disease from a single UK centre.


Effects of treatments by calcium and sex hormones on vertebral fracturing in osteoporosis. Lateral radiographs of the thoracic and lumbar spine were taken periodically in 49 patients with osteoporosis. Thirty patients were postmenopausal, and 19 nonmenopausal with osteoporosis due to steroids, male hypogonadism, alcoholism, thyrotoxicosis or unknown cause. Patients were studied before, during and after treatment with high calcium alone, or with combined calcium and sex steroids. Calcium was given as effervescent calcium lactate gluconate, and sex hormones as oestradiol valerate, testosterone oenanthate, or methenolone oenanthate. A total of 964 films covering 409 patient-years were available for measurement. On each vertebra, deformity due to loss of anterior height was measured and assigned to one of four grades. For the time interval between each consecutive pair of films, a patient's vertebral fracture rate score was calculated and expressed per thousand patient-years. In comparison with the corresponding pretreatment fracture rate score, both the postmenopausal and the nonmenopausal groups who had not received sex hormones previously, failed to show significant changes (p = 0.144; p = 0.017) on high calcium alone during mean periods of 4.3 and 2.8 years respectively. If the first 2 years on high calcium were excluded for the postmenopausal group, they still failed to show a reduction in fracture rate score (observed for a mean period of 5.0 years; p = 0.04). When treated with combined calcium and sex hormones, both postmenopausal and nonmenopausal groups showed a lower fracture rate score of 20 and 207 respectively when compared with the pretreatment levels of 1500 and 1697 (in mean treatment periods of 3.2 and 4.4 years; p less than 0.001 in each case). When given high-dose calcium alone, but after treatment with sex hormones as well, the postmenopausal group showed no change in fracture rate score from pretreatment (in a mean of 3.1 years; p = 0.069); however the nonmenopausal group still showed a significant reduction in fracture rate score from 1697 to 42 over a mean period of 2.3 years (p = 0.001). The postmenopausal group, after stopping all treatment, showed a higher fracture rate score of 1286 (in a mean of 2.6 years) than did those on combined calcium and sex hormones, in whom the fracture rate score was 20 (in a mean of 3.2 years; p = 0.008). A subgroup of 11 patients with osteoporosis of both the menopausal and nonmenopausal types, had data both before (in a mean of 5.5 years) and during (for a mean of 2.5 years) treatment with calcium alone; the fracture rate scores were 1473 and 918 (p = 0.247).(ABSTRACT TRUNCATED AT 400 WORDS)


The organization of GABAergic neurons in the mammalian superior colliculus. GABA is an important inhibitory neurotransmitter in the mammalian superior colliculus. As in the lateral geniculate nucleus, GABA immunoreactive neurons in SC are almost all small and are distributed throughout the structure in all mammalian species studied to date. Unlike the LGN, GABA-labeled neurons in SC have a variety of morphologies. These cells have been best characterized in cat, where horizontal and two granule cell morphologies have been identified. Horizontal cells give rise to one class of presynaptic dendrite while granule C cells give rise to another class of spine-like presynaptic dendrite. Granule A cells may be the origin of some GABAergic axon terminals. GABA containing synaptic profiles form serial synapses, providing a possible substrate for disinhibition. The distribution of GABAA and GABAB receptor subtypes appears similar to that of GABA neurons, with the densest distribution found within the superficial gray layer. However, antibody immunocytochemistry of the beta 2 and beta 3 subunits of the GABAA receptor reveals that it is located at both synaptic and non-synaptic sites, and may be associated with membrane adjacent to terminals with either flattened or round vesicles. A few GABA containing neurons in SC colocalize the pentapeptide leucine enkephalin or the calcium binding protein calbindin. However, none appear to co-localize parvalbumin, a situation different from GABA containing interneurons in the LGN and visual cortex. The diversity of GABA neurons in SC rivals that found in visual cortex, although unlike visual cortex, the pattern of co-occurrence does not distinguish GABA cell types in SC. The superior colliculus also differs from both LGN and visual cortex in that GABA and calbindin immunoreactivity is not altered by either long-term occlusion and/or short-term enucleation in adult Rhesus monkeys. No consistent differences have been found in the optical density of GABA labeling in either cells or neuropil. To conclude, GABA neurons in the superior colliculus share some properties like those in LGN and others like those in visual cortex. In other properties, they differ from GABA neurons in both the LGN and visual cortex. The GABA systems in the superior colliculus are similar in all mammalian species studied, suggesting that they are phylogenetically conserved systems which are not amenable to plastic alterations, a situation different to that in the geniculostriate system.


Organization and plasticity of GABA neurons and receptors in monkey visual cortex. The GABA neurons of monkey area 17 are a morphologically and chemically heterogeneous population of interneurons that are normally distributed most densely within the geniculocortical recipient zones of the visual cortex. In adult monkeys deprived of visual input from one eye, the levels of immunoreactivity for GABA and GAD within neurons of these geniculocortical zones is reduced. Similar changes are seen in the levels of proteins that make up the GABAA receptor sub-type. The effects of monocular deprivation on other substances suggest that specific types of GABA neurons, such as those in which the tachykinin neuropeptide family and parvalbumin coexist with GABA, are greatly influenced by changes in visual input. That some proteins remain normal within deprived-eye neurons and that other proteins are increased indicates the changes in the GABA cells of the cortex are not the result of a general reduction in protein synthesis. Comparisons of what is known about the morphological and synaptic features of GABA cells in area 17 and the characteristics of cells affected by monocular deprivation suggests that certain classes, such as the clutch cell, may be preferential targets of deprivation. Such a selective loss of certain GABA neurons would have broad implications for the possible physiological plasticity of cortical cells, for if ongoing studies determine that specific receptive field properties are affected by monocular deprivation in adults, the correlation of functional properties and classes of GABA cells would be possible.


Evidence that the prostaglandin E2 receptor and the anhydrolevuglandin E2 receptor in the rat uterus are the same receptor. Anhydrolevuglandin E2 (AnLGE2) is closely related to prostaglandin E2 (PGE2) and has been found to inhibit the uterotonic activity of PGE2. The binding of PGE2 and its inhibition by AnLGE2 has been determined in rat uterine membrane fractions. AnLGE2 inhibited the binding of 3HPGE2 in a dose related fashion. 3HAnLGE2 also binds to rat uterine membrane fractions and its binding is inhibited by PGE2 in a dose related fashion. These data support previous physiological observations that AnLGE2 inhibits the actions of PGE2 by acting at the PGE2 receptor. Thus, AnLGE2 appears to be a specific inhibitor of PGE2 actions at its uterine receptors.


Inhibitory effect of 17 beta -estradiol on prostaglandin E2-induced phosphoinositide hydrolysis in osteoblast-like cells. We examined the effect of estradiol on PGE2-induced phosphoinositide hydrolysis and cAMP production in cloned osteoblast-like MC3T3-E1 cells. 17 beta -Estradiol pretreatment significantly inhibited the formation of inositol phosphates induced by 10 microM PGE2 in a dose-dependent manner between 1 pM and 10 nM. This effect of 17 beta -estradiol was dependent on the time of pretreatment and submaximum inhibition was observed at 4 h. However, 17 beta -estradiol had little effect on the formation of inositol phosphates induced by 20 mM NaF, a GTP-binding protein activator. The cAMP production induced by PGE2 was not influenced by 17 beta -estradiol. These results suggest that 17 beta -estradiol modulates the signal transduction by PGE2 and that the effect seems to be exerted between PGE2 receptor and the GTP-binding protein coupled to phospholipase C in osteoblast-like MC3T3-E1 cells.


Strategy for studying health effects of pesticides/fertilizer mixtures in groundwater. This paper is a "historical account" of the entire process leading to the final implementation of a toxicology research program on the health-effect studies of pesticide and fertilizer contaminants in groundwater conducted at the National Institute of Environmental Health Sciences/National Toxicology Program (NIEHS/NTP). Starting with the perception of a potential problem area, the program evolved through several stages, including planning and information-gathering, deliberation on experimental designs, formulation of a draft protocol, peer-review and public comments on the protocol, finalization of the protocol, announcement of open bidding to the NTP master agreement laboratories (i.e., a group of laboratories that has been previously evaluated and approved to conduct NTP toxicology contract work), implementation of chemistry developmental work, selection of toxicology laboratories and awarding of contracts, final implementation of toxicology studies, and cultivation of special collaborative efforts among NIEHS in-house scientists as well as colleagues at the neighbouring laboratories in Research Triangle Park. To put things in perspective, the author's present approach, a coupling of physiologically based pharmacokinetics/pharmacodynamics and computer modeling with the toxicology of chemical mixtures at Colorado State University, is used to address the issues of toxicology and risk assessment of chemical mixtures with respect to rationale and reality.


Arterial blood gases during cardiac arrest: markers of blood flow in a canine model. Measures of CO2 have been shown to correlate with coronary perfusion pressure and cardiac output during cardiac arrest. We evaluated arterial pH (pHa) relative to blood flow during cardiac arrest in a canine electromechanical dissociation (EMD) model of cardiac arrest using different resuscitation techniques. Following 15 min of cardiac arrest, 24 mongrel dogs received epinephrine with continued CPR or closed-chest cardiopulmonary bypass. Central arterial blood gases, end-tidal carbon dioxide (PetCO2), coronary perfusion pressure and cardiac output were measured. During CPR, prior to epinephrine or bypass, there was no correlation of pHa, PACO2 and PetCO2, with cardiac output or coronary perfusion pressure. Immediately after instituting the resuscitation techniques, both pHa and PaCO2 showed a significant correlation with cardiac output (pHa; R = -0.78, P less than 0.001 and PaCO2; R = 0.87, P less than 0.001) and with coronary perfusion pressure (pHa; R = -0.75, P less than 0.001 and PaCO2; R = 0.75, P less than 0.001). Eventual survivors (n = 15) had an early significant decrease in pHa, base excess and a significant increase in PaCO2 which was not present in non-survivors (n = 9). Neither pHa nor PaCO2 correlate with blood flow under low flow conditions of CPR. However, with effective circulatory assistance, pHa and PaCO2 reflect systemic blood flow and reperfusion washout.


Influence of ACTH-(1-24) on metabolic acidosis and hypoxemia induced by massive hemorrhage in rats. In anesthetized rats, step-wise bleeding to a severe condition of hemorrhagic shock causes a decrease in arterial and venous pH and in venous PO2 and SO2 and an increase in arterial PO2 and in venous PCO2 and lactic acid. The intravenous bolus injection of ACTH-(1-24) (160 micrograms/kg)--which causes a rapid and sustained reversal of the shock condition--produces a gradual and almost complete recovery (within 60 min) of venous PO2, PCO2 and SO2; on the other hand, the normalization of blood pH and lactate is preceded by a further worsening during the first minutes after treatment. On the whole, these data are compatible with the ACTH-(1-24)-induced mobilization of the residual blood--which is pooled in poorly oxygenated tissues--and with the improved circulatory and respiratory functions.


The effects of convective cooling and rewarming on systemic and central nervous system physiology in isoflurane-anesthetized dogs. Recent studies have demonstrated that small (i.e., 2-5 degrees C) reductions in temperature may protect the brain and spinal cord from ischemic injury. The present study evaluated the physiologic response of anesthetized animals to convective-based cooling and warming. Six shaved, isoflurane-anesthetized (1.50% end-expired; 1 MAC), pancuronium-paralyzed dogs were subjected to temperature manipulation. The flow of cool (13-14 degrees C) or warm (39-41 degrees C) air was uniformly applied to the the dorsal and lateral surfaces of the dog using an inflatable blanket with perforations in the interior surface. Convective cooling reduced pulmonary artery temperature (Tpa) from 37.0 +/- 0.2 degrees C (Mean +/- S.D.) to 33.0 +/- 0.0 degrees C over a 93 +/- 18 min period. Thereafter, the active cooling was discontinued and passive cooling resulted in a further reduction in Tpa to 32.4 +/- 0.3 degrees C over the next 60 min. Institution of convective warming resulted in an increase in Tpa from 32.4 +/- 0.3 to 33.0 +/- 0.0 degrees C in 23 +/- 14 min and from 33.0 to 37.0 +/- 0.0 in an additional 137 +/- 26 min. During the periods of active cooling, passive cooling and active warming, there were strong correlations between Tpa and temperature within the brain, cisterna magna, parietal epidural space, lumbar subarachnoid space and other commonly used temperature measurement sites non-invasively monitored (e.g. tympanic membrane, esophagus, rectum) r greater than or equal to 0.97; P less than 0.0001). The combination of isoflurane anesthesia (a potent EEG-suppressor) plus mild hypothermia (less than 34 degrees C) resulted in an EEG attenuation in five dogs, two of which progressed to burst suppression. The magnitude of EEG changes correlated with the degree of temperature reduction. Upon rewarming to 37 degrees C, all dogs had normal EEG activity and normal brain concentrations of high energy phosphates, glucose and lactate. Blood pressure and cardiac output did not change during the study and no dog exhibited acid-based anomalies or blood lactate accumulation. Whole body oxygen consumption and heart rate decreased in a temperature-dependent fashion. Cardiac rhythm disturbances were rare. The authors conclude that convection-based corporeal cooling and rewarming are efficacious methods for non-invasively and uniformly altering CNS temperatures without adversely affecting cerebral or systemic physiology.


Blood rheology in the endotoxin-administered rabbits. The effects of endotoxin on blood rheology were studied in rabbits. Non-treated six rabbits (Group A) were used to obtain control data. Thirteen rabbits who were administered 0.1 mg of endotoxin three times at intervals of 3 days were divided into two groups; seven rabbits (Group B) were injected with 1 ml saline solution as the vehicle for endotoxin at 7 days after the final administration of endotoxin, while the remaining six rabbits (Group C) were administered endotoxin at 0.2 mg/kg on the same day. Blood was sampled from the femoral artery 120 min after the final treatment. Blood viscosity was measured at a shear rate of 150 s-1 at 37 degrees C using a cone-plate viscometer. The passage time for a 5% red blood cell suspension and that for plasma were determined by filtration; the former represents erythrocyte deformability while the latter is related to plasma fluidity. The hematocrit, whole blood viscosity and erythrocyte deformability did not show significant differences among these three groups. The ratio of hematocrit to whole blood viscosity is considered to be an index of oxygen delivery from the hemorheologic point. This index did not show significant difference either. A good correlation was observed between whole blood viscosity and hematocrit in Group A, but not in the endotoxin-treated groups. The plasma fluidity was lower in Groups B and C than in Group A. These data indicate that plasma fluidity and the hematocrit-viscosity relationship are affected in endotoxin-treated rabbits, although blood viscosity, erythrocyte deformability and oxygen delivery hardly changed.


Amino acids and glucose in human cerebrospinal fluid after acute ischaemic brain damage. In the search for potential biochemical markers of value for prognosis after acute hypoxic brain damage, amino acids and glucose were assessed in the cerebrospinal fluid (CSF) and glucose in blood. Samples were taken by lumbar puncture 4, 28, 76 and 172 h after resuscitation from 20 patients and once from 10 control patients. Eight of the resuscitated patients recovered neurologically but 12 remained comatose. The concentrations of alanine (P less than 0.001) and phenylalanine (P less than 0.035) differed most in 4-h samples between the groups. The concentration of alanine was higher in all patient groups with hypoxic brain damage as compared to the controls, the concentrations in patients dying within 76 h (disabled-s group) being higher than in the recovered patients. Phenylalanine in the disabled-s group was significantly higher than the control value. Furthermore, there were significant differences between various patient groups in the concentrations of glutamine, isoleucine, leucine, lysine, serine, tyrosine and valine. When taking into account the permeability of the BBB to these amino acids, alanine, valine and isoleucine most clearly represent brain amino acid metabolism. CSF glucose in the control group and in the recovered patients was lower than in patients dying within 76 h.


Stroke models: strengths and pitfalls.

Stroke models: strengths and pitfalls. Recent editorials and reviews express disillusionment and sharp criticism with the contribution of animal experimental studies to stroke prevention and treatment. The basis for these comments appears to be a frustration with the absence of pharmacologic agents to effectively treat stroke patients despite considerable research efforts. In response to these nihilistic views, Zivin and Grotta (Stroke, 21 (1990) 981-983) have written a poignant rebuttle and Goldstein (Stroke, 21 (1990) 373-374) has outlined the considerable progress which has been achieved in experimental cerebral stroke research. It is our goal in the present discussion to highlight the strengths and to examine the potential pitfalls of stroke models, some of which may have contributed to the equivocal results obtained in testing pharmacologic agents. In addition, based on our own experience with a model of middle cerebral artery (MCA) occlusion in cats, we will describe the benefits of large versus small animal models for experimental stroke studies.


The ethics of resuscitation; differences between Europe and the USA--Europe should not adopt American guidelines without debate. There are differences between Europe and the USA in the style of medical decision-making for patients who are critically ill or requiring CPR. These differences are both legal and philosophical. They concern principally the degree of influence the patient and next of kin should have on critical medical decisions. Currently American physicians transfer more of the decision-making to patients and relatives than do their European counterparts. The current state of the art in cardio-pulmonary resuscitation (CPR) from cardiac arrest occurring out-of-hospital requires public education programmes. These heighten public awareness of CPR-related questions. There has been a wide acceptance in Europe of the American guidelines for CPR. Cultural and legal differences, however, should encourage the acceptance of specific European guidelines. The author believes that it is important to introduce in the European CPR programmes discussions on the ethical dilemmas that may occur. This may help to conserve the relatively high level of public trust that facilitates the patient-doctor relationship in Europe, compared with the USA. Arguments are also put forward for a heightened sensitivity in the European medical profession concerning communication with the patients and their next of kin and with the mass media, in view of the increasing public sophistication and interest that the citizen CPR programmes are generating.


Pathogenesis and pharmacorrection of early postresuscitation cardiac arrhythmia. Effect of acute lethal blood loss on character and frequency of cardiac arrhythmias in postresuscitation period has been studied. Experiments were carried out on mongrel male rats resuscitated after 4- and 6-min clinical death caused by acute blood loss. Electric cardiac instability was found in early postresuscitation period. Pacemaker migration, paroxysmal ventricular tachycardia, blockades and extrasystole that lead to ventricular fibrillation were observed in 20 percent of cases. Supported by correlative analysis it has been established that the main arrhythmogenic factors are abundance of catecholamines, free fatty acids, dienic conjugates, lactate and inhibition of Ca dependent ATPase. Antiarrhythmogenic effects of antihypoxant gutimin, the beta-adrenoreceptor blocker inderal, antioxidant oxypiridin-6 were noticed after their separate administration before clinical death. The same effect of carnosine and phosphocreatine administered during resuscitation also was noticed.


Comparison of two methods of transporting paramedics to cardiac arrests outside hospital. To compare the deployment of paramedics in a separate rapid response unit with their deployment in a standard emergency ambulance. A one year period of each deployment. Throughout the community in some parts of West Yorkshire. All patients receiving resuscitation for cardiac arrest by paramedics. Using the same group of paramedics and central control, 12 months with the paramedics deployed in separate cars in addition to the standard ambulances (period 1) were followed by another 12 months when they were deployed as one crew member of a standard emergency ambulance (period 2). Number of arrests attended, number of patients in ventricular fibrillation at paramedic arrival, response times, survival to leave hospital. In period 1, 580 arrests were attended with 31 survivors. In period 2, 462 arrests resulted in 25 survivors. The mean response time was shorter in period 1 (6.24 versus 6.60 min, Cl--0.01-0.73 min). In period 1, 217 patients were found in ventricular fibrillation (23 survivors): In period 2, 141 patients were found in ventricular fibrillation (11 survivors). Separating paramedics from the standard emergency ambulances increases the number of survivors of cardiac arrest but the difference may not be sufficiently large to justify the additional expenditure.


[Autologous bone marrow graft in solid tumors in childhood]. Curability in children suffering from malignant solid tumor is 50%. Thus, high dose chemotherapy with or without total body irradiation followed by autologous bone marrow transplantation (ABMT) has been proposed to patients suffering from cancer either at initial diagnosis (poor prognosis tumor) or at relapse. Thanks to these studies, drugs having dose effects properties have been selected. In some tumors, ABMT has significantly improved patients median survival. It remains to be determined if: 1. high dose chemotherapy protocols with ABMT are superior to new aggressive chemotherapeutic protocol without ABMT. 2. ABMT increases the curability of high risk patients.


Regional flow during experimental hemorrhage and crystalloid resuscitation: persistence of low flow to the splanchnic organs. BACKGROUND AND METHODS. Rapid changes in cardiac output (CO) and organ perfusion occur with hemorrhagic shock and fluid resuscitation. To assess regional alterations of flow, 40 Sprague-Dawley male rats were subjected to hemorrhagic shock and crystalloid resuscitation under halothane anesthesia. Polyethylene microspheres were injected before and after hemorrhage and after resuscitation. At sacrifice, brain, lungs, heart, liver, intestine, spleen and kidneys were harvested, weighed and radioactivity counted. Changes in mean arterial pressure, oxygen consumption, organ flow and CO were also measured. Cardiac output decreased during hemorrhage (P less than 0.01), it increased with resuscitation but did not return to baseline even with infusion of fluid volumes of three times the blood loss. Flow decreased during hemorrhage in all organs, but the difference was not statistically significant in the liver (P greater than 0.05), since a larger percentage of CO was maintained as hepatic perfusion. During resuscitation, flow to brain and kidneys increased over the percentage values expected by increased CO (P less than 0.01), but flow to the liver did not increase significantly. Flow to small bowel remained depressed (P less than 0.005). Following hemorrhage there is hypoperfusion of all splanchnic organs; however, flow to the liver decreases least. Crystalloid resuscitation in our model failed to return CO to baseline. Blood supply to intestine remained depressed in disproportion to CO both after hemorrhage and resuscitation and hepatic blood flow remained decreased after resuscitation.


Epstein-Barr virus (EBV) genomes and c-myc oncogene in oral Burkitt's lymphomas. In addition to Burkitt's lymphomas, tentative evidence suggests the involvement of Epstein-Barr virus (EBV) in malignant lymphomas of T-cell origin. The c-myc proto-oncogene is strongly associated with the development of lymphoid neoplasias. In the present study, a series of 38 biopsies of oral lymphomas (29 Burkitt's lymphomas, 9 malignant lymphomas of other type) obtained from patients in Tanzania were studied using in situ hybridization (ISH) and polymerase chain reaction (PCR) for detection of EBV DNA and c-myc oncogene. In ISH applied on formalin-fixed, paraffin wax-embedded biopsies, the Bam HI W fragment of EBV DNA was used as the probe. Amplification of c-myc oncogene was studied by PCR with a primer set from Exon II area. As an internal standard beta-globin gene was simultaneously amplified. EBV DNA was disclosed by ISH in five Burkitt's lymphomas only. Using the PCR, 20 of the 29 cases (70%) of Burkitt's lymphomas showed amplification for EBV DNA. Of the other EBV-positive lymphomas, two were of the lymphocytic type (large non-cleaved cell), one histiocytic and one Burkitt's-like lymphoma. All EBV-positive cases found on the agarose gel were positive also with the dot blot, when hybridized with the 32P-labeled EBV Bam HI W-fragment probe. All lymphomas showed similar bands on the gel for c-myc and beta-globin indicating that no amplification of c-myc was present.


Severe head injury: should expected outcome influence resuscitation and first-day decisions? An early 'prognosis' based on initial findings can influence clinical decisions. To evaluate the quality of first-day outcome prediction based on either clinical or neuroradiological information, we prospectively examined 100 consecutive severely head-injured patients from the surgical intensive care unit. The prognoses were always made by the same experienced neurosurgeon and neuroradiologist according to a contracted Glasgow Outcome Scale (GOS). Every patient's outcome was predicted according to a three-class GOS within 24 h after injury. Correct 'first-day' prognoses were made in 59 and 56% of the cases by the neuroradiologist and neurosurgeon, respectively. In those instances where the prognoses made by the clinician and the radiologist coincided, the prognoses were correct for 73% of the patients. Based on accepted criteria, the neuroradiologist tended to overpredict favorable outcomes and missed many of the unfavorable outcomes (dead or vegetative) whereas the clinician overestimated unfavorable outcomes. Our study showed that outcome prognosis of patients with severe head injury has limited accuracy when made within 24 h after the injury, although an improvement in accuracy occurred when there was agreement between clinical and radiological predictions. Even with sophisticated clinical and radiological technologies, it is not possible to predict outcome on the first day after the accident with sufficient accuracy to guide early management.


Anti-hepatitis C virus screening will reduce the incidence of post-transfusion hepatitis C also in low-risk areas. The incidence of post-transfusion hepatitis non-A, non-B (PTH-NANB) was prospectively assessed in two areas in the southeast region of Sweden. Patients undergoing hip arthroplasty were studied with blood sampling for alanine aminotransferase analysis before and at 2, 3, and 4 months after transfusion. Of the patients 97% and 82% were transfused and received a mean of 5.5 and 3.4 units in Linköping and Oskarshamn, respectively. None of 38 patients in Oskarshamn but 4 of 144 patients (2.8%) in Linköping contracted PTH-NANB. Two of these four patients developed antibodies against hepatitis C virus (HCV) by the first-generation anti-HCV enzyme-linked immunosorbent assay (ELISA) (C100). The other two patients remained negative by this test. HCV infection was, however, indicated in all four patients by positive second-generation anti-HCV ELISA confirmed by positive second-generation recombinant immunoblot assay (4-RIBA). Three of the patients were positive by polymerase chain reaction (PCR). Serum from one blood donor to the four hepatitis patients (altogether three donors) was found positive by first- and second-generation anti-HCV ELISA and 4-RIBA and was also PCR-positive. Three other blood donors, who did not transmit hepatitis, were anti-HCV ELISA (C100)-positive. This study shows that if anti-HCV ELISA had been available at the start of the trial, all cases of PTH would have been avoided at the expense of only 0.7% transfusion units discarded. Routine anti-HCV ELISA testing of all transfusion units will reduce the incidence of PTH-C even in low-risk areas.


Evaluation of the central nervous function in resuscitated comatose patients by multilevel evoked potentials. Multilevel evoked potentials were examined in 17 patients who became comatose after cardiac arrest and resuscitation. In 4 patients, the P1 through N3 components of the somatosensory evoked cerebral potential (SECP) were present altogether within 100 ms after the ischemic insults. They all subsequently regained consciousness, though three of them developed intelligence and motor disturbances to some extent. In 11 patients who regained consciousness, or remained in a vegetative state, the evoked potentials which reflect brainstem functions, such as the auditory evoked brainstem potential, the R1 wave of the orbicularis oculi reflex and the slow positive wave of the somatosensory evoked brainstem potential, were recognized. The somatosensory evoked spinal potential and spinal monosynaptic reflex showed normal appearances in the state of vegetation and even after the determination of brain death. The measures of SECP could be useful in predicting restoration of consciousness.


Interaction between heparin and acetylsalicylic acid on gastric mucosal and skin bleeding in humans. The haemorrhagic effect of unfractionated heparin and of the low molecular weight heparin, enoxaparin, on gastric mucosal bleeding induced by acetylsalicylic acid (ASA) and on skin bleeding induced by the Simplate technique was investigated in healthy human volunteers. Endoscopic estimation of gastric bleeding by visual analogue scores was more sensitive than biochemical quantitation of blood in the gastric washing by a modified HaemoQuant method. Contrary to what was expected, the ASA-induced gastric mucosal bleeding was not increased by heparin pretreatment, whereas heparin in combination with ASA, but not ASA alone, significantly increased the skin bleeding time. In the interaction with ASA, enoxaparin and unfractionated heparin appeared to act similarly.


Shock index: a re-evaluation in acute circulatory failure. To evaluate the relationship between the shock index SI (ratio of heart rate to systolic arterial pressure) and cardiac function and oxygen transport in an experimental model of hemorrhage and clinical septic shock. This study was conducted in a hypovolemic circulatory failure model; 40% hemorrhage in the anesthetized pig and normovolemic hyperdynamic septic patients in the intensive care unit (ICU). Hemodynamic and oxygen transport variables were measured and their relationships to SI was examined. SI was inversely related to blood loss, cardiac index (CI), stroke volume (SV), mean arterial pressure (MAP) and left ventricular stroke work (LVSW) (r = -0.73, -0.75, -0.89 and -0.75, respectively P less than 0.01) following hemorrhage in the anesthetized pig. Oxygen transport variables, i.e. oxygen delivery (DO2) and mixed venous oxygen saturation (SvO2P) (r = -0.68 and -0.74, respectively, P less than 0.01) were also inversely related to the SI. Oxygen consumption (VO2) increased initially with increasing SI and fell when SI was greater than 3.0. In clinical septic shock and following blood volume expansion, the SI was not correlated to CI, SVI, MAP or systemic vascular resistance (SVR) (r = -0.01, -0.47, -0.34 and -0.14, respectively, P-value NS) but was inversely related to LVSWI (r = -0.68, P less than 0.01). There were no relationships between the SI and oxygen transport variables (DO2, SvO2) (r = -0.02 and -0.17, P-value NS) in septic shock. SI provides a non-invasive means to monitor deterioration or recovery of LVSW during acute hypovolemic and normovolemic circulatory failure and its therapy. SI may be of limited value in the assessment of systemic oxygen transport and response to therapy in clinical shock.


An audit of cardiac arrest management by extended trained ambulance crew. To audit the use of extended skills by South Glamorgan Ambulance crew in attempted resuscitations from out-of-hospital cardio-respiratory arrest, in terms of successful discharge of patients from hospital and the accuracy with which agreed protocols were applied. Design-Retrospective analysis of ambulance report forms, electrocardiograph rhythm strips, casualty cards and discharge summaries during 26 months (1st May 1987-30th June 1989). A mixed urban and semi-rural area of 187 square miles with a population of 396,000. There were 274 attempted resuscitations. Seven patients (2.5%) were managed for primary respiratory arrest and 3 were discharged. In 98 patients (35.8%) the initial resuscitation protocol was for ventricular fibrillation: 26 were admitted and 17 were discharged. In 169 patients (61.7%) the initial resuscitation protocol was for asystole or electromechanical dissociation: 11 were admitted and 1 discharged. The majority of patients who were successfully discharged from hospital were those in ventricular fibrillation who responded to defibrillation alone (13 survivors). Drug administration may have played a role in the successful resuscitation of the remainder. Endotracheal intubation was successful in 94.7% and vein cannulation in 87.7% of attempts. There were deviations from the ventricular fibrillation protocol in 27 cases (27.5%) and from the asystole protocol in 27 cases (16.0%). Survival rates for ventricular fibrillation managed by these personnel were satisfactory with early defibrillation. Defibrillation alone was responsible for the majority of successful resuscitations. The additional benefit of drug administration appears small, though potentially important. The majority of patients were in asystole by the time the ambulance arrived. Extended trained crews use their skills effectively. The most important skill is defibrillation. Further studies are required to explain the high proportion of patients found in asystole. The performance of individual ambulance personnel should be assessed prospectively, because agreed resuscitation protocols are not always followed.


[Lyme borreliosis: significance of the serological diagnosis of an infection with Borrelia burgdorferi in neurological diseases with inflammatory cerebrospinal fluid syndrome]. To look for a correlation between positive antibody-response against Borrelia burgdorferi (Bb) and an inflammatory CSF-syndrome, from May 1988 to May 1989 333 patients from the Neurological Department of the University of Bern underwent lumbar puncture with cell count, quantitative and qualitative protein analysis and antibody determination against Bb in serum and CSF. 6 patients with active syphilis were excluded. The results of the 333 remaining patients were analyzed using chi 2 or Fisher's exact test. The antibody determination was performed using an immunoperoxidase assay (IPA). Our results are calculated for three cut-off points: Bb-IgG 1:64, 1:128, 1:256 and/or Bb-IgM 1:16, 1:32, 1:64. We found 11.7% patients to be seropositive (Bb-IgG 1: greater than or equal to 256 and/or Bb-IgM 1: greater than or equal to 64). We demonstrated the following correlations: elevated cell count (greater than 10/mm3 cells CSF) versus elevated Bb-titer (1: greater than or equal to 256), elevated total protein of CSF (greater than 48 mg%) versus elevated Bb-titer, blood-brain-barrier dysfunction versus elevated Bb-titer. In diagnostic subgroups, the same correlations were only demonstrated for PNS disorders (n = 134), and especially PNS-disorders without compression. 8 cases showed the high risk constellation inflammatory CSF syndrome and highly positive titer (Bb-IgG 1: greater than or equal to 256). Only 2 had typical neuroborreliosis, while in 2 cases the possibility of neuroborreliosis was open. Patients with MS did not show a special risk for Bb-infection.(ABSTRACT TRUNCATED AT 250 WORDS)


Successful CPCR in two patients.

Successful CPCR in two patients. Authors have salvaged two cases suffering from respiratory and cardiac arrest with active effective cardiopulmonary cerebral resuscitation (CPCR). One was a 53-year-old woman with myasthenia gravis whose tracheostomy tube was dislodged on the way to being transferred to the ICU. Another case, a 56-year-old farmer, the victim of an anesthesia accident which occurred in the cystoscopic examination room where equipment for CPR was unavailable. The patients were discharged with complete recovery of brain function after 64 days of unconsciousness in case 1 and weaning off after 74 days of mechanical ventilation in case 2. It is very important for the success of CPR to understand and practice the technique of CPR in the order: A (airway), B (breathing) and C (circulation). Early intubation and defibrillation is effective measurements for successful CPR. Training programs of CPR must be held not only for medical personnel but also for citizens in the developing counties.


[The beta-mimetic effects following the enoral injection of epinephrine with articaine]. A randomized, double-blind study was conducted to assess the influence of 1:200,000 or 1:100,000 epinephrine in a four percent articaine-HCl solution on the concentration-time profiles of epinephrine in the serum of patients during removal of an impacted lower wisdom tooth. In order to estimate the amount of endogenous epinephrine secretion during operation, volunteers were injected without subsequent operation. The heart rate (HR), the concentration of cAMP and norepinephrine were determined as indicators of beta-adrenoceptor stimulation and effective signal transduction. These parameters were correlated with the epinephrine serum concentration. HR measurements were done automatically using the Hewlett Packard monitor, cAMP and the catecholamins were determined immuno- and radioenzymatically. The results showed a dose-dependent absorption of epinephrine from the intraoral depot. The peak concentration and the velocity at which 50% of the epinephrine had been eliminated were affected by the operative manipulation that had induced an increase in endogenous epinephrine secretion. HR, cAMP and norepinephrine were linearly correlated with serum epinephrine.


Impaired spatial learning in alpha-calcium-calmodulin kinase II mutant mice. Although long-term potentiation (LTP) has been studied as the mechanism for hippocampus-dependent learning and memory, evidence for this hypothesis is still incomplete. The mice with a mutation in the alpha-calcium-calmodulin-dependent kinase II (alpha-CaMKII), a synaptic protein enriched in the hippocampus, are appropriate for addressing this issue because the hippocampus of these mice is deficient in LTP but maintains intact postsynaptic mechanisms. These mutant mice exhibit specific learning impairments, an indication that alpha-CaMKII has a prominent role in spatial learning, but that it is not essential for some types of non-spatial learning. The data considerably strengthen the contention that the synaptic changes exhibited in LTP are the basis for spatial memory.


Modulation of DNA binding specificity by alternative splicing of the Wilms tumor wt1 gene transcript. The technique of whole-genome polymerase chain reaction was used to study the DNA binding properties of the product of the wt1 gene. The zinc finger region of this gene is alternatively spliced such that the major transcript encodes a protein with three extra amino acids between the third and fourth fingers. The minor form of the protein binds specifically to DNA. It is now shown that the major form of wt1 messenger RNA encodes a protein that binds to DNA with a specificity that differs from that of the minor form. Therefore, alternative splicing within the DNA binding domain of a transcription factor can generate proteins with distinct DNA binding specificities and probably different physiological targets.


Phosphorylation-independent modulation of L-type calcium channels by magnesium-nucleotide complexes. Free magnesium ions and magnesium-nucleotide complexes can exert opposite effects on many fundamental cellular processes. Although increases in the intracellular concentration of magnesium ions inhibit the L-type calcium current in heart cells, magnesium-adenosine triphosphate complexes (MgATP) would be expected to increase the current by promoting channel phosphorylation. Rapid increases in the intracellular concentration of MgATP induced by flash photolysis of caged magnesium or caged ATP resulted in enhanced calcium current. The increase in calcium current was not prevented by blocking phosphorylation, revealing a previously unrecognized direct regulatory action of the magnesium-nucleotide complex.