Biomedical subjects
Publications and source records attributed to C A Ross.
Seven cDNA fragments containing polymorphic (AAT)n trinucleotide repeats were isolated from a human brain cDNA library and mapped by linkage to specific loci. These repeats may serve as gene markers or as candidates for diseases caused by expansion mutation.
We studied the interaction between Trypanosoma congolense and bovine aorta endothelial (BAE) cell monolayers. Our findings suggest that trypanosomes adhere predominantly to the flattened, peripheral cell surface domains as well as to filamentous endothelial outgrowths that are present during in vitro cultivation in non-confluent monolayers. Adhesion is mediated exclusively by the flagellum in a distinct geometrical order with respect to the flagellar cytoskeleton. Thus, it is possible to define exactly the trypanosomal cell surface domain involved in the attachment process. After 24-48 h of cultivation on monolayers, trypanosomes start to develop short, filopodia-like flagellar protrusions, which serve as additional elements in assisting parasite attachment. Small filaments (3-5 nm) also serve as cross-links between flagellar and endothelial cell surface membranes. Lectin-gold labeling shows that these cross-links contain sialic acid residues. In vitro assays confirm that sialic acid is involved in the adhesion process, whereas the extracellular matrix (ECM) proteins fibronectin, collagen, laminin and vitronectin are not. The presence of T. congolense exhibits a mitogenic effect on BAE cells.
Expansion mutation is the cause of eight neuropsychiatric disorders. Thus far each disease is the result of expansion of a C-G rich trinucleotide repeat that is polymorphic for length in the general population. We now report the identification of seven novel cDNA clones with CCA or equivalent trinucleotide repeats obtained by screening a human frontal cortex cDNA library. The repeat lengths of two clones, CCA11 (linked to D20S101, expressed in human brain as a 3.2 kb message) and CCA38 (linked to D5S404), are highly polymorphic in a normal human population. CCA54, mapped to chromosome 19, appears to correspond to a portion of the human gene encoding the alpha 1 subunit of a P-type calcium channel. Expansion mutations at these loci should be considered as possible candidates in evaluating the genetic etiologies of diseases linked to chromosomes 5, 19, and 20.
The Inositol 1,4,5-trisphosphate receptor (IP3R) is an intracellular calcium channel involved in coupling cell membrane receptors to calcium signal transduction pathways within the cell. We have cloned a cDNA for the human type 1 inositol 1,4,5-trisphosphate receptor. The sequence contains the S2 splice site which appears to be the region most divergent between rat and human. We now report an additional alternatively spliced region in the coupling domain, that is 9 amino acids long, which we term S3. Alternatively spliced forms are found in both human and rat. PCR analysis of brain and peripheral tissues from human and rat shows both transcripts of the type 1 inositol 1,4,5-trisphosphate receptor in all tissues. The long form predominates in most brain regions (except the cerebellum) while the short form predominates in peripheral tissues. The sequence of the longer form in human appears to create an additional consensus protein kinase C phosphorylation site.
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STUDY OBJECTIVE: Tricyclic antidepressants (TCAs) are a leading cause of death from intentional drug overdose. Available therapies are often unsatisfactory. In this study we evaluated the use of a high-affinity drug-specific polyclonal Fab fragment (TFab) as an antidote to desipramine toxicity. DESIGN: We gave anaesthetized rats under mechanical ventilation IV desipramine so that we might study the effect of TFab on survival or IP desipramine to facilitate study of the interaction of TFab and hypertonic sodium bicarbonate (NaHCO3), the standard clinical treatment for TCA overdose. INTERVENTIONS: For the study of the effects of TFab and NaHCO3 on survival, each rat was given a constant IV infusion of desipramine until it died, together with TFab 2 g/kg, bovine serum albumin, or .9% NaCl starting 5 minutes after the desipramine infusion. In the study of the interaction of TFab and NaHCO3, each rat received 30 mg/kg IP desipramine followed by TFab (molar TFab:desipramine ratio, .11), NaHCO3, TFab+NaHCO3, or NaCl at the time of maximal toxicity (15 minutes). RESULTS: In the survival protocol, QRS-interval duration, systolic blood pressure, and heart rate were significantly improved by TFab, and survival was prolonged by 58% compared with that in the albumin and NaCl groups (P < .001). The molar ratio of TFab to administered desipramine was .21. The unbound fraction of desipramine in serum at the time of death was reduced by TFab, but the unbound desipramine concentration was not, suggesting that TFab prolonged survival by delaying the increase in the unbound serum desipramine concentration. In the interaction protocol, neither TFab nor NaHCO3 was effective alone, but the combination significantly reduced QRS-interval prolongation (P = .001). CONCLUSION: These data demonstrate the efficacy of TFab in reducing desipramine-induced cardiovascular toxicity and prolonging survival. The pharmacokinetic effects of TFab in rats with severe desipramine toxicity were similar to those observed in sublethal desipramine toxicity. Therapeutic benefit is enhanced by the concurrent use of NaHCO3 and may be achieved despite binding only a fraction of the desipramine dose.
Microsatellite DNA sequences are rapidly becoming the dominant source of nuclear genetic markers for a wide range of applications, from genome mapping to forensic testing to population studies. If misinterpretation is to be avoided, it is vital that we understand fully the way in which microsatellite sequences evolve. We have therefore compared allele length distributions for 42 microsatellites in humans with their homologues in a range of related primates. We find a highly significant trend for the loci to be longer in humans, showing that microsatellites can evolve directionally and at different rates in closely related species.
Huntington's disease (HD) is an inherited neurodegenerative disorder expressed when a trinucleotide repeat in the gene IT-15 is expanded. The mechanism by which the expanded repeat causes the expression of the disease is unknown. Possible mechanisms include alterations in the amount of the mRNA, potentially resulting from changes in gene transcription or abnormal mRNA stability. In order to determine whether the expanded IT-15 allele is present in mRNA, we isolated total RNA from the cortex and striatum of patients and controls. To distinguish the two alleles of the IT-15 transcript in HD patients, we amplified across a region containing a dimorphic single triplet deletion observed on some chromosomes and found that the relative intensity of the two PCR bands amplified from genomic DNA and those amplified from first strand cDNA from brain tissue were essentially equal. In order to determine whether the exon containing the expanded CAG repeat is present in IT-15 mRNA from HD patients, we amplified across this region and demonstrated the presence of the expanded repeat in cDNA from both striatum and cortex. Based on this evidence, we suggest that the mechanism of disease expression does not occur during transcription or in the stability of the RNA, but rather occurs during translation or postranslationally.
Huntington's disease (HD) is an inherited, neurodegenerative disorder caused by expansion of a CAG repeat in the IT15 gene, leading to an expanded glutamine repeat in the HD protein. The mechanism by which the expanded repeat causes expression of the disease is not known, though there do not appear to be changes in the mRNA levels. We have conducted quantitative Western blot analyses of HD patients and controls. Expression of the IT15 protein is essentially equal in control and HD frontal cortex. In caudate from HD patients, IT15 protein is decreased in parallel with the decrease in a neuronal marker, suggesting that loss of IT15 protein is secondary to neuronal loss. In order to determine expression of the two alleles of the IT15 protein we used Western blots of 4% polyacrylamide gels. Both alleles of the IT15 protein were expressed at similar levels in HD lymphoblastoid cell lines and HD post-mortem hippocampus and cerebellum (regions relatively spared in HD), indicating that even very long CAG repeats can be translated into polyglutamine. In contrast, in cerebral cortex and caudate (regions severely affected in HD), in the longer expanded repeat cases the expanded allele of the IT15 protein was present at a significantly lower level (compared with the normal length allele), often with a smear of more slowly migrating reactivity above it. These data suggest the possibility of altered structure, abnormal processing or abnormality of protein-protein interactions involving the IT15 protein with the expanded glutamine repeat.
A substantial number of patients with dissociative identity disorder have had previous diagnoses of schizophrenia, due to the presence of positive symptoms of schizophrenia. The authors investigated the pattern of positive and negative symptoms in patients with dissociative identity disorder, and compared it with norms in schizophrenia. A total of 108 patients with a clinical diagnosis of dissociative identity disorder were administered the Positive and Negative Syndrome Scale. The positive symptom and general psychopathology scores were significantly more severe in the dissociative identity group than the norms for schizophrenia, while the negative symptoms were significantly more severe in schizophrenia. Since patients with dissociative identity disorder report more positive symptoms of schizophrenia than do schizophrenics, while schizophrenics report more negative symptoms, a primary emphasis on positive symptoms may result in false-positive diagnoses of schizophrenia and false-negative diagnoses of dissociative identity disorder.
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Hereditary factors play a major role in the etiology of idiopathic generalized epilepsies (IGEs). The pivotal function of glutamate receptors (GluRs) in excitatory neurotransmission implicates their involvement in epileptogenesis and genetic susceptibility to IGEs. A trinucleotide repeat polymorphism detected in the 3' untranslated region of the kainate-selective GluR6 receptor gene (GRIK2) on chromosome 6 makes it possible to perform linkage and association studies with this high-ranking candidate gene. The present study tested the hypothesis that allelic variants of GRIK2 contribute to the genetic susceptibility to the common IGEs. Linkage and association analyses were conducted in 63 families ascertained through IGE patients with juvenile myoclonic epilepsy, juvenile absence epilepsy, or childhood absence epilepsy. Our linkage and association results suggest that allelic variants of GRIK2 are not involved in the expression of the common familial IGEs, and radiation hybrid mapping assigns GRIK2 to the chromosomal region 6q16.3-q21. This localization excludes GRIK2 as a candidate for the putative IGE susceptibility locus "EJM1" on the short arm of chromosome 6.
96 patients with a clinical diagnosis of Dissociative Identity Disorder were administered the Million Clinical Multiaxial Inventory-II. The most elevated personality disorder scales were Avoidant, Self-defeating, Borderline, and Passive-Aggressive personality disorders. Elevated Axis I scales included Dysthymia, Major Depression, Thought Disorder, and Anxiety Disorder. Million-II profiles of a patient before and after integration showed normalization of a previously pathological profile.
In a previous study, three in vitro methods for the assessment of drug sensitivity among Trypanosoma evansi isolates were compared--a direct counting method, pyruvate production method and uptake of radiolabelled hypoxanthine. The pyruvate assay system, which measures the amount of pyruvate in the supernatant of growing populations of trypanosomes by a spectrophotometric method, was selected for further investigation with regard to its suitability for field studies. The effect of initial seeding density and incubation time on the growth of three stocks of T. evansi--TREU 1840 and TREU 1981 (suramin sensitive) and TREU 2136 (suramin resistant)--and drug sensitivities revealed by the pyruvate assay and direct counting were examined to optimise assay conditions. Maximum densities and pyruvate production achieved were not affected by varying the initial seeding densities in the range of 5 x 10(4)-5 x 10(5)/ml and had been reached after 48 hours incubation with one exception: Pyruvate levels continued to increase up to 72 hours in the suramin resistant stock. However, inhibition curves were affected by initial seeding density and incubation period. Results suggested that an initial seeding density of 1 x 10(5)/ml and an incubation time of 48 hours are optimal for the assay. Using these assay conditions, the isolates were screened against suramin, quinapyramine sulphate and Cymelarsan, the trypanocides used most commonly against T. evansi. This assay proved to be a relatively simple and cheap technique applicable to screening large numbers of isolates of differing sensitivities to trypanocidal drugs.
In an earlier subcellular fractionation study of epithelial tissue (liver and pancreas), we demonstrated that the inositol 1,4,5-trisphosphate receptor (IP3R) is found in association with biochemically distinct cellular membranes, including the endoplasmic reticulum (ER) and plasma membrane (Sharp, A. H., Snyder, S. H., and Nigam, S. K. (1992) J. Biol. Chem. 267, 7444-7449). To further characterize epithelial IP3Rs, we have now employed cultured Madin-Darby canine kidney (MDCK) cells, a well studied tight polarized epithelial cell type. Indirect immunofluorescence with an antiserum which specifically recognizes IP3R in MDCK cells by immunoblotting and immunoprecipitation gave an ER-like staining pattern as well as a basolateral plasma membrane-like staining pattern, the latter being particularly evident in highly confluent monolayers. In sections of adult rat kidney tubules a similar staining pattern was observed. Interestingly, whereas known basolateral proteins (Na+,K(+)-ATPase and the facilitated glucose transporter) gave a continuous basolateral staining pattern, that seen for IP3R was discontinuous (punctate). A highly similar staining pattern was observed for the caveolar protein, caveolin, suggesting that the punctate basolateral plasma membrane-like staining pattern observed for IP3R reflects its localization to basolateral caveolae. Biotinylation of non-permeabilized and permeabilized MDCK cells, followed by immunoprecipitation of IP3R and detection with streptavidin, indicated that while most IP3R is localized to biotin-inaccessible compartments (i.e. ER), a fraction (10-20%) of IP3R was accessible to externally added biotin primarily from the basolateral side. This result is compatible with the dual ER and basolateral caveolar localization suggested by immunocytochemistry, although it does not exclude the presence of some IP3R in the basolateral plasma membrane as well. Solubility studies revealed IP3R to be considerably more insoluble than the basolateral proteins, Na+,K(+)-ATPase and the liver cell adhesion molecule, as well as the cytoskeletal proteins, ankyrin and fodrin. In the most insoluble fraction, IP3R was found along with caveolin, further supporting the notion that part of the cellular IP3R pool associates with caveolae. Since multiple localizations of IP3R within a cell might reflect the existence of multiple isoforms, polymerase chain reaction amplification of first strand cDNA with primers specific for the three isotypes of IP3R was performed. All three isoforms of IP3R were expressed in the homogeneous population of MDCK cells. The existence of distinct membrane localizations and multiple isoforms of IP3R within the same cell type suggests an explanation for the complex spatiotemporal patterns of Ca2+ release from inositol 1,4,5-trisphosphate-sensitive Ca2+ pools in epithelial and other cells.
Drug-specific antibody fragments can enhance the elimination of some drugs by redistributing drug from tissues into serum and allowing renal excretion of the drug-antibody complex. This approach could potentially be used to enhance the elimination of compounds such as polychlorinated biphenyls that have very long elimination half-lives. As a first step in testing this hypothesis, the effects of 2,2',4,4',5,5'-hexachlorobiphenyl (HCB)-specific antibodies and their corresponding Fab fragments on HCB disposition were studied in rats. Antibodies to HCB were produced in chickens, and the corresponding Fab fragments were produced by digestion with papain. To study antibody effects on HCB distribution, [14C]HCB (0.1 mg) was administered i.v. to rats. Two weeks later, after distribution to tissues was complete, anti-HCB IgG or control IgG was administered i.v. The serum radiolabel concentration 2 hr after IgG administration increased 185 +/- 64% in animals treated with specific antibody vs 51 +/- 19% in control animals (P < 0.001). The increase in serum radiolabel concentration was apparent within 30 min and maximal at 2 hr. To study effects on HCB excretion, anti-HCB or control Fab fragment was administered 2 weeks after [14C]HCB. Urinary HCB excretion over the next 24 hr, measured by gas chromatography, was 10-fold greater in the group treated with anti-HCB Fab (P < 0.01). These data demonstrate that anti-HCB IgG can redistribute HCB rapidly from tissues into serum and that anti-HCB Fab can enhance urinary HCB excretion. While the magnitude of these changes was small, the data suggest that increasing HCB excretion using drug-specific antibody fragments is feasible, and can serve as a model for enhancing the excretion of compounds that have very long elimination half-lives.