Showing papers by "Karolinska Institutet published in 1988"

Topogenic signals in integral membrane proteins.

Abstract: Integral membrane proteins are characterized by long apolar segments that cross the lipid bilayer. Polar domains flanking these apolar segments have a more balanced amino acid composition, typical for soluble proteins. We show that the apolar segments from three different kinds of membrane-assembly signals do not differ significantly in amino acid content, but that the inside/outside location of the polar domains correlates strongly with their content of arginyl and lysyl residues, not only for bacterial inner-membrane proteins, but also for eukaryotic.proteins from the endoplasmic reticulum, the plasma membrane, the inner mitochondrial membrane, and the chloroplast thylakoid membrane. A positive-inside rule thus seems to apply universally to all integral membrane proteins, with apolar regions targeting for membrane integration and charged residues providing the topological information.

Central D2-Dopamine Receptor Occupancy in Schizophrenic Patients Treated With Antipsychotic Drugs

Abstract: • Using positron emission tomography and the carbon 11—labeled ligand raclopride, central D2-dopamine receptor occupancy in the putamen was determined in psychiatric patients treated with clinical doses of psychoactive drugs. Receptor occupancy in drug-treated patients was defined as the percent reduction of specific carbon 11—raclopride binding in relation to the expected binding in the absence of drug treatment. Clinical treatment of schizophrenic patients with 11 chemically distinct antipsychotic drugs (including both classic and atypical neuroleptics such as clozapine) resulted in a 65% to 85% occupancy of D2-dopamine receptors. In a depressed patient treated with the tricyclic antidepressant nortriptyline, no occupancy was found. The time course for receptor occupancy and drug levels was followed after withdrawal of sulpiride or haloperidol. D2-dopamine receptor occupancy remained above 65% for many hours despite a substantial reduction of serum drug concentrations. In a sulpiride-treated patient, the dosage was reduced in four steps over a nine-week period and a curvilinear relationship was demonstrated between central D2-dopamine receptor occupancy and serum drug concentrations. The results demonstrate that clinical doses of all the currently used classes of antipsychotic drugs cause a substantial blockade of central D2-dopamine receptors in humans. This effect appears to be selective for the antipsychotics, since it was not induced by the antidepressant nortriptyline.

Clonal Genomic Alterations in Glioma Malignancy Stages

Abstract: Comparison of constitutional and tumor genotypes at chromosomal loci defined by restriction fragment length alleles has proven useful in determining the genomic position and tissue specificity of recessive mutations that predispose to cancer (Hansen, M. F., and Cavenee, W. K. Cancer Res., 47: 5518–5527, 1987). Here we have applied this approach to 53 unrelated patients with glial tumors of varying histological malignancy grade. Loss of constitutional heterozygosity for loci on chromosome 10 was observed in 28 of 29 tumors histologically classified as glioblastoma (malignancy grade IV) whereas no similar losses were observed in any of 22 gliomas of lower malignancy grade. Examination of restriction fragment length alleles on other chromosomes revealed that loss of sequences on chromosomes 13, 17, or 22 had occurred at nonrandom frequencies and in at least one instance of each malignancy grade of adult glioma. The tumors in which loss of constitutional heterozygosity was observed were composed of one or a mixture of glial cell subtypes displaying astrocytic, oligodendrocytic, and/or ependymal differentiation. These results demonstrate a close association of the loss of chromosome 10 sequences with the most malignant histological stage of glioma and that glioblastoma arises as the clonal expansion of an earlier staged precursor. Furthermore they suggest that glioblastoma is a common phenotypic and malignancy terminus for glial tumors of various cellular subtypes which is reached through a common molecular pathway. This approach which involves the identification of malignancy stage specific somatic losses of heterozygosity provides a genotypic, rather than phenotypic, analysis of tumor progression.

Distinct replicative and cytopathic characteristics of human immunodeficiency virus isolates.

Abstract: According to their capacity to replicate in vitro, human immunodeficiency virus (HIV) isolates can be divided into two major groups, rapid/high and slow/low. Rapid/high viruses can easily be transmitted to a variety of cell lines of T-lymphoid (CEM, H9, and Jurkat) and monocytoid (U937) origin. In contrast, slow/low viruses replicate transiently, if at all, in these cell lines. Except for a few isolates, the great majority of slow/low viruses replicate in peripheral blood mononuclear cells and Jurkat-tatIII cells constitutively expressing the tatIII gene of HIV-1. The viruses able to replicate efficiently cause syncytium formation and are regularly isolated from immunodeficient patients. Poorly replicating HIV isolates, often obtained from individuals with no or mild disease, show syncytium formation and single-cell killing simultaneously or, with some isolates, cell killing only.

2,3,7,8-Tetrachlorodibenzo-p-dioxin kills immature thymocytes by Ca2+-mediated endonuclease activation.

Abstract: Suspensions of thymocytes from young rats were incubated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which resulted in a sustained increase in cytosolic free Ca2+ concentration followed by DNA fragmentation and loss of cell viability. Both the Ca2+ increase and DNA fragmentation were prevented in cells treated with the inhibitor of protein synthesis, cycloheximide, and DNA fragmentation and cell killing were not detected when cells were incubated in a "Ca2+-free" medium or pretreated with high concentrations of the calcium probe, quin-2 tetraacetoxymethyl ester. These results indicate that TCDD can kill immature thymocytes by initiating a suicide process similar to that previously described for glucocorticoid hormones.

Biological monitoring of toxic metals.

Abstract: The biological monitoring of metals, when used properly, allows total exposure to a particular metal to be measured from various media. It takes into consideration inter- and intraindividual variations in uptake due to differences in metabolism and physical work load and can be used to identify individuals, or groups of individuals, with high exposure or at high risk. As many metals are retained for long periods, biological monitoring may not only provide information on recent exposure, but also on exposure which occurred a long time ago. Under optimal conditions, the concentration of a metal in biological media can be used to assess exposure, the concentration of the metal in the target or critical organ (ie, the organ where the adverse effects are first observed) and the risks for adverse effects. This paper gives an overview of several important aspects of biological monitoring but does not provide detailed information on particular metals.

Expression of Epstein-Barr virus-encoded proteins in nasopharyngeal carcinoma.

Abstract: Expression of the Epstein-Barr virus (EBV) encoded nuclear antigens (EBNA 1 to 6) and membrane-associated protein (LMP) was investigated by immunoblotting in 83 nasopharyngeal carcinoma (NPC) biopsies and 25 other tumor and normal tissue specimens from the head and neck region. Fifty-eight of the 83 NPC biopsies were large enough to yield parallel data on virus DNA and viral expression. All 16 cases of clinically diagnosed and histologically confirmed NPCs from North Africa contained EBV DNA and expressed EBNA-1. Of 31 clinically diagnosed NPCs from China, 29 contained EBV DNA and 25 of these expressed EBNA-1. One control tissue biopsy from the oropharynx of NPC patients contained EBV DNA, but none expressed EBNA-1. The latent membrane protein (LMP) was detected in 22/31 of the Chinese and in 10/16 of the North African NPC biopsies. None of the NPC biopsies or control tissues expressed detectable amounts of EBNA 2 or any of the other 4 nuclear antigens which are invariably expressed in EBV-transformed B cells. A smaller number of tumors from Malaysia and East Africa exhibited a similar pattern of expression. EBV was rescued from a nude-mouse-passaged North African NPC tumor by co-cultivation of the tumor cells with umbilical cord blood lymphocytes. The tumor expressed EBNA 1 and LMP, but not EBNA 2 or the other 4 EBNAs. The resulting LCLs expressed all 6 nuclear antigens, EBNA 1 to 6 and LMP. Our data suggest that expression of the EBV genome is regulated in a tissue-specific fashion.

Diverse effects of fibronectin and laminin on phenotypic properties of cultured arterial smooth muscle cells.

Abstract: Plasma fibronectin promotes modulation of rat arterial smooth muscle cells from a contractile to a synthetic phenotype during the first few days in primary culture. This process includes cell adhesion and spreading, loss of myofilaments, and formation of a widespread rough endoplasmic reticulum and a prominent Golgi complex. The structural reorganization is accompanied by activation of overall RNA and protein synthesis. Moreover, the cells gain the ability to replicate their DNA and divide in response to platelet-derived growth factor. Here, it is demonstrated that the power of fibronectin to bring about this change in the differentiated properties of the smooth muscle cells resides in a 105-kD cell-binding fragment, whereas a 70-kD collagen-binding fragment and a 31-kD heparin-binding fragment are inactive in this respect. Laminin, another adhesive glycoprotein and a component of the basement membrane that normally surrounds arterial smooth muscle, was contrarily found to maintain the cells in a contractile phenotype. However, with increasing time more and more cells went through the modulation into a synthetic phenotype. This "catch-up" was counteracted by a peptide that contained the cell-attachment sequence of fibronectin (Arg-Gly-Asp-Ser). Hence, it is possible that the delayed modulation on laminin was due to production of fibronectin by the cells themselves. In support of this notion, fibronectin isolated from smooth muscle cultures was found to be as effective as plasma fibronectin in stimulating the phenotypic modulation. Moreover, using a combination of chemical, immunochemical, and immunocytochemical methods, it was demonstrated that the cells secreted fibronectin as well as laminin at an increasing rate during the first 4 d in primary culture and, notably, cells cultured on laminin produced more fibronectin than cells cultured on fibronectin. Newly synthesized fibronectin was incorporated into a network of pericellular and intercellular fibrils, whereas laminin formed a more diffuse layer covering the cells in a basement membrane-like manner. Taken together, the findings suggest diverse roles for fibronectin and laminin in the control of the differentiated properties of arterial smooth muscle cells. They further indicate that the ability of arterial smooth muscle cells to produce fibronectin and laminin early in primary culture is not directly related to the phenotypic state as determined morphologically and by measurement of overall rates of RNA and protein synthesis. This may be due to the cells being able to sense the macromolecular composition of the pericellular matrix and to modify their secretory activity accordingly.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence against linkage of schizophrenia to markers on chromosome 5 in a northern Swedish pedigree.

Abstract: Schizophrenia is a severe mental illness with a typically chronic course affecting nearly 1% of the human population. It is generally accepted that genetic factors have an important pathogenic role in a substantial portion of schizophrenia cases; however, despite decades of family studies, there is no agreed-upon mode of inheritance. The discovery of genetic aetiologic factors and resolution of the inheritance pattern(s) will undoubtably emerge from genetic linkage studies. With these objectives in mind, we undertook a linkage project, starting in 1985, in a previously well-documented kindred from north Sweden. Multipoint linkage analyses were used to screen the proximal long arm of chromosome 5 using restriction fragment length polymorphism (RFLP) markers at five loci and the distal long arm using RFLPs at two loci, one of which was the locus for the glucocorticoid receptor. We found strong evidence against linkage between schizophrenia and the seven loci. These results, together with the positive evidence for linkage of schizophrenia with markers in the proximal long arm of chromosome 5 lead us to conclude that the genetic factors underlying schizophrenia are heterogeneous.

Expression of the beta-nerve growth factor gene in hippocampal neurons

Abstract: In situ hybridization with complementary DNA probes for nerve growth factor (NGF) was used to identify cells containing NGF messenger RNA in rat and mouse brain. The most intense labeling occurred in hippocampus, where hybridizing neurons were found in the dentate gyrus and the pyramidal cell layer. The neuronal identity of NGF mRNA-containing cells was further assessed by a loss of NGF-hybridizing mRNA in hippocampal areas where neurons had been destroyed by kainic acid or colchicine. RNA blot analysis also revealed a considerable decrease in the level of NGF mRNA in rat dentate gyrus after a lesion was produced by colchicine. This lesion also caused a decrease in the level of Thy-1 mRNA and an increase in the level of glial fibrillary acidic protein mRNA. Neuronal death was thus associated with the disappearance of NGF mRNA. These results suggest a synthesis of NGF by neurons in the brain and imply that, in hippocampus, NGF influences NGF-sensitive neurons through neuron-to-neuron interactions.

Histopathological classification of nonantral gastric endocrine growths in man.

Abstract: Recently, the gastric endocrine system has been recognized as the origin of benign and malignant tumors in pernicious anemia. It has also been found that the gastric endocrine cells respond to permanent elevation of serum gastrin levels induced by changes in acid secretion in response to surgical procedures, drug therapy and age. Therefore, a definition of nonantral gastric endocrine hyperplasia (simple or diffuse, linear or chain-forming, micronodular, adenomatoid), dysplasia (enlarging or fusing micronodules, microinvasion, nodular growth) and neoplasia (intramucosal carcinoid, invasive carcinoid) is presented. The individual entities are illustrated, together with the literature discussed and the techniques for their identification presented.

Regulation of glucocorticoid receptor expression: evidence for transcriptional and posttranslational mechanisms.

Abstract: The mechanism of ligand-induced (homologous) down-regulation of the glucocorticoid receptor (GR) has been studied. Dexamethasone caused a down-regulation of the levels of GR mRNA and protein both in hepatoma tissue culture cells and rat liver in vivo. The decrease in the level of rat liver GR mRNA was due to a reduced transcription rate of the GR gene, as assessed by nuclear run-on transcription experiments. The half-life of GR mRNA in hepatoma tissue culture cells was determined to be approximately 4.5 h and was unaffected by dexamethasone. In addition to the transcriptional regulation of GR gene expression, a dexamethasone-dependent posttranslational modification in the rate of GR protein turnover was observed. In the absence of dexamethasone, GR protein half life was approximately 25 h whereas it decreased to approximately 11 h in the presence of hormone. Down-regulation of GR protein occurred with a 6- to 24-h delay as compared to the decline in GR mRNA. This is most likely due to the differences in h...

Angiogenic properties of Kaposi's sarcoma-derived cells after long-term culture in vitro

Abstract: Cells derived from lung biopsies and pleural effusions from AIDS patients with Kaposi's sarcoma (KS) of the lungs were established in long-term culture with the aid of conditioned medium from HTLV-II-transformed T cells (HTLV-II CM). These AIDS-KS cells were similar to the so-called spindle cells in KS lesions and had some of their features. They produced factors that supported their own growth (autocrine) and the growth of other cells (paracrine), including umbilical vein endothelium and fibroblasts. That the AIDS-KS cells also expressed potent angiogenic activity was demonstrated by the chorioallantoic membrane assay and by subcutaneous inoculation of AIDS-KS cells into nude mice, which resulted in the development of angiogenic lesions composed of mouse cells and showing histological features similar to those of human KS lesions. These data suggest that AIDS-associated KS and possibly other types of KS may be initiated by signals that induce the growth of particular cells (spindle cells of lymphatic or vascular origin) and the expression of autocrine and paracrine activities.

Ethanol-, fasting-, and acetone-inducible cytochromes P-450 in rat liver: regulation and characteristics of enzymes belonging to the IIB and IIE gene subfamilies.

Abstract: Two major forms of hepatic microsomal cytochrome P-450 were purified from starved and acetone-treated rats. On the basis of amino acid sequence analysis, they were identified as P-450j and P-450b. Ethanol or acetone treatment of rats caused a 9-fold increase in the amount of P-450j in liver microsomes accompanied by similar increases in the rate of NADPH-dependent metabolism of carbon tetrachloride, acetone, and benzene. Immunological experiments indicated that P-450j constitutes the major catalyst of the microsomal metabolism of the latter agents and contributes by about 50% to microsomal P-450-dependent ethanol oxidation under the conditions used. The P-450j-dependent catalytic activities had a high rate of turnover. In contrast, this was not the case for the immunodetectable P-450j, indicating the occurrence of inactive forms of this protein in microsomes. Starvation or ethanol or acetone treatment caused 10-30-fold increases in the amount of both mRNA and apoprotein of P-450b,e compared to control. Run-on experiments and the concomitant increases of the P-450b,e gene products at the mRNA and protein levels indicated the appearance of mainly a transcriptional activation by acetone, ethanol, or starvation. Fasting exerted, in addition, a pronounced synergistic effect on acetone-dependent induction of P-450b,e mRNA (3-fold), apo-P-450b,e (4.3-fold), P-450j mRNA (2-fold), and apo-P-450j (2-fold). No increase of mRNA coding for P-450j, compared to control, was seen after acetone or ethanol treatment alone. The results indicate that effects of ethanol, acetone, and/or starvation on drug and xenobiotic metabolism are caused by the induction of P-450 forms belonging to at least two gene subfamilies.

Central distribution of trigeminal and upper cervical primary afferents in the rat studied by anterograde transport of horseradish peroxidase conjugated to wheat germ agglutinin.

Abstract: Injections of WGA-HRP were made in the rat trigeminal ganglion and C1-3 dorsal root ganglia (DRGs) to study the central projection patterns and their relations to each other. Trigeminal ganglion injections resulted in heavy terminal labeling in all trigeminal sensory nuclei. Prominent labeling was also observed in the solitary tract nucleus and in the medial parts of the dorsal horn at C1-3 levels, but labeling could be followed caudally to the C7 segment. Contralateral trigeminal projections were found in the nucleus caudalis and in the dorsal horn at C1-3 levels. The C1 DRG was found to be inconstant in the rat. When it was present, small amounts of terminal labeling were found in the external cuneate nucleus (ECN) and the central cervical nucleus (CCN). No dorsal horn projections were seen from the C1 DRG. Injections in the C2 DRG resulted in heavy labeling in the ECN, nucleus X, CCN, and dorsal horn, where it was mainly located in lateral areas. Labeling could be followed caudally to the Th 7 segment. C2 DRG projections also appeared in the cuneate nucleus (Cun), in all the trigeminal sensory nuclei, and in the spinal, medial, and lateral vestibular nuclei. A small C2 DRG projection was observed in the ventral cochlear nucleus. C3 DRG injections resulted in heavy labeling in both medial middle and lateral parts of the dorsal horn, in the ECN, and in nucleus X, whereas the labeling in the CCN was somewhat weaker. Smaller projections were seen to trigeminal nuclei, Cun, and the column of Clarke. Comparisons of the central projection fields of trigeminal and upper cervical primary afferents indicated a somatotopic organization but with a certain degree of overlap.

Specific glucocorticoid receptor binding to DNA reconstituted in a nucleosome.

Abstract: We have reconstituted a nucleosome with core histones from rat liver using a restriction fragment containing a sequence from the mouse mammary tumour virus (MTV) long terminal repeat (LTR). This sequence harbours glucocorticoid responsive elements (GREs) which mediate glucocorticoid hormone induction of transcription from the MTV promoter via glucocorticoid receptor (GR) binding. Exonuclease III and DNase I footprinting demonstrated that the reconstituted nucleosome was specifically located between positions -219 and -76. A nucleosome was previously shown to be located at a similar or identical position in the MTV promoter in situ and to be structurally altered upon glucocorticoid hormone induction. We demonstrated, by DNase I footprinting, that GR is able to bind sequence specifically to the DNA in the in vitro assembled nucleosome. No evidence for unfolding of the nucleosome was obtained, but the DNase I footprinting pattern demonstrated GR induced local alterations in the DNA.

Human fetal dopamine neurons grafted in a rat model of Parkinson's disease: immunological aspects, spontaneous and drug-induced behaviour, and dopamine release.

Abstract: We have used a rat model of Parkinson's disease (PD) to address issues of importance for a future clinical application of dopamine (DA) neuron grafting in patients with PD Human mesencephalic DA neurons, obtained from 65-8 week old fetuses, were found to survive intracerebral cell suspension xenografting to the striatum of rats immunosuppressed with Cyclosporin A The grafts produced an extensive new DA-containing terminal network in the previously denervated caudate-putamen, and they normalized amphetamine-induced, apomorphine-induced and spontaneous motor asymmetry in rats with unilateral lesions of the mesostriatal DA pathway Grafts from an 115-week old donor exhibited a lower survival rate and smaller functional effects As assessed with the intracerebral dialysis technique the grafted DA neurons were found to restore spontaneous DA release in the reinnervated host striatum to normal levels The neurons responded with large increases in extracellular striatal DA levels after the intrastriatal administration of the DA-releasing agent d-amphetamine and the DA-reuptake blocker nomifensine, although not to the same extent as seen in striata with an intact mesostriatal DA system DA fiber outgrowth from the grafts was dependent on the localization of the graft tissue Thus, grafts located within the striatum gave rise to an extensive axonal network throughout the whole host striatum, whereas grafted DA neurons localized in the neocortex had their outgrowing fibers confined within the grafts themselves In contrast to the good graft survival and behavioural effects obtained in immunosuppressed rats, there was no survival, or behavioural effects, of human DA neurons implanted in rats that did not receive immunosuppression In addition, we found that all the graft recipients were immunized, having formed antibodies against antigens present on human T-cells This supports the notion that the human neurons grafted to the non-immunosuppressed rats underwent immunological rejection Based on an estimation of the survival rate and extent of fiber outgrowth from the grafted human fetal DA neurons, we suggest that DA neurons that can be obtained from one fetus may be sufficient to restore significant DA neurotransmission unilaterally, in one putamen, in an immunosuppressed PD patient

Skeletal muscle adaptations consequent to long-term heavy resistance exercise.

Abstract: Heavy resistance training is associated with increased body weight, lean body mass, and muscle cross-sectional area The increased muscle cross-sectional area is mainly brought about by hypertrophy of individual muscle fibers There is a greater increase in the area of fast twitch fibers compared to slow twitch fibers In addition, long-term heavy resistance training may produce fiber proliferation Mitochondrial volume density decreases in proportion to muscle hypertrophy in response to training Typically, no capillary neoformation occurs during strength training Therefore, capillary density decreases consequent to heavy resistance training It appears, though, that bodybuilders, relying on a high repetition training system, in contrast to Olympic weight- and power lifters, display a small increase in number of capillaries per fiber Enzyme activities, reflecting oxidative potential; decrease during long-term heavy resistance training, resulting in muscle hypertrophy Although glycogen storage capacity is enhanced in strength trained athletes, enzyme activities reflecting anaerobic metabolism do not increase in response to heavy resistance exercise

Localization of glucocorticoid receptor mRNA in the male rat brain by in situ hybridization

Abstract: The localization and distribution of mRNA encoding the glucocorticoid receptor (GR) was investigated in tissue sections of the adult male rat brain by in situ hybridization and RNA blot analysis. GR mRNA levels were measured by quantitative autoradiography with 35S- and 32P-labeled RNA probes, respectively. Strong labeling was observed within the pyramidal nerve cells of the CA1 and CA2 areas of the hippocampal formation, in the granular cells of the dentate gyrus, in the parvocellular nerve cells of the paraventricular hypothalamic nucleus, and in the cells of the arcuate nucleus, especially the parvocellular part. Moderate labeling of a large number of nerve cells was observed within layers II, III, and VI of the neocortex and in many thalamic nuclei, especially the anterior and ventral nuclear groups as well as several midline nuclei. Within the cerebellar cortex, strong labeling was observed all over the granular layer. In the lower brainstem, strong labeling was found within the entire locus coeruleus and within the mesencephalic raphe nuclei rich in noradrenaline and 5-hydroxytryptamine cell bodies, respectively. A close correlation was found between the distribution of GR mRNA and the distribution of previously described GR immunoreactivity. These studies open the possibility of obtaining additional information on in vivo regulation of GR synthesis and how the brain may alter its sensitivity to circulating glucocorticoids.

Changes in plasma concentrations of aromatic and branched-chain amino acids during sustained exercise in man and their possible role in fatigue

Abstract: The plasma concentrations of branched-chain and aromatic amino acids have been measured in two different types of sustained dynamic exercise. Twenty-two subjects participated in the 1986 Stockholm Marathon and eight subjects took part in an army training programme of approximately 1.5-h duration. Both types of exercise caused a significant decrease in the plasma concentration of branched-chain amino acids, while there was no change in the concentration of total (free plus bound to albumin) tryptophan. The plasma concentration of free tryptophan, which was measured in the marathon runners, was found to increase 2.4-fold during the race. This increase is probably caused by a pronounced elevation in the concentration of plasma free fatty acids during exercise, since these are known to displace tryptophan from albumin. The observed increase in plasma free tryptophan concentration, together with the decrease in plasma concentration of branched-chain amino acids, gives rise to a marked increase in the plasma concentration ratio of free tryptophan/branched-chain amino acids. This should lead to an increase in the rate of transport of tryptophan across the blood-brain barrier and hence to an increase in the rate of synthesis of 5-hydroxytryptamine (5-HT) in the brain. An elevated concentration of 5-HT in specific areas of the brain may be responsible, at least in part, for the development of physical, and/or mental fatigue during prolonged exercise.