Role of myelin basic protein and proteolipid protein genes in multiple sclerosis: single strand conformation polymorphism analysis of the human sequences

Susceptibility to multiple sclerosis (MS) is widely held to have a strong genetic component. While the identities of genes conferring susceptibility are currently unknown, possible candidates include those genes coding for proteins which function in central nervous system (CNS) myelin. Two such genes are the human myelin basic protein (MBP) and proteolipid protein (PLP) genes, whose products make up approximately 80% of the total protein in CNS myelin. The association of a variable number tandem repeat (VNTR) 5' to the human MBP gene with MS has been the subject of conflicting reports. Here we test the hypothesis that mutations in the human MBP and PLP genes might be associated with MS by examining the entire expressed sequence of both genes by single strand conformation polymorphism (SSCP) analysis, using a panel of 71 MS patients and 71 controls. We have also re-examined the VNTR region in patients and controls. Three base changes were found in the human PLP gene and nine base changes in the human MBP gene; these were essentially equally distributed between patients and controls. No preferential distribution of various alleles of the VNTR between patients and controls was found. Although intronic and regulatory regions have not been examined, it would appear unlikely that mutations in these genes coding for the two major CNS myelin proteins contribute significantly to genetic susceptibility to MS.     

Computer-assisted measurement of lumbar spine radiographs

RATIONALE AND OBJECTIVES: The authors evaluated a method for obtaining reproducible, reliable measurements from standard lumbar spine radiographs for determining the degree of spondylolisthesis, vertebral body height, intervertebral disk space height, disk space angle, and degree of vertebral body wedging. MATERIALS AND METHODS: Four to six easily defined points were identified on each vertebral body on anteroposterior and lateral plain radiographs of the lumbosacral spine of patients. From these points, the degree of spondylolisthesis, the vertebral body height, the intervertebral disk space height, the disk space angle, and the degree of vertebral body wedging were easily calculated by using well-known geometric relationships. This method requires the use of a personal computer and a standard spreadsheet program but does not require the use of any other specialized radiographic equipment, computer hardware, or custom software. RESULTS: Calculations of intra- and interobserver variability for the measurement of spondylolisthesis, disk space height, disk space angle, and vertebral body height measurement showed that the technique is extremely reproducible. CONCLUSION: This technique may prove useful in the prospective evaluation of potential candidates for lumbar spinal stenosis surgery.     

BIACORE analysis of histidine-tagged proteins using a chelating NTA sensor chip

While BIACORE instruments are routinely used for kinetic measurements and for the determination of binding constants, the immobilization of a ligand onto the sensor chip surface has to be individually optimized for every system. We show here that the histidine (His) tag, routinely used in protein purification and in detection is an ideal tag for immobilization, despite the intrinsically low affinity between an immobilized metal ion and the His tag. This is due to strong rebinding effects caused by the high surface density of immobilized Ni2+-nitrilotriacetic acid (NTA) on the chips used here. The immobilization of the ligand can be adjusted to a low level using the same chip, such that mass transport limitation and rebinding of the analyte to the immobilized ligand is minimal. Nine different proteins with different numbers of His tags were tested for stable binding to the Ni2+-NTA surface. Most proteins with one His tag dissociate very rapidly from the Ni2+-NTA surface, and the KD for the interaction between His tag and Ni2+-NTA was estimated to about 10(-6) m at neutral pH. In contrast, two His tags are usually found to be sufficient for stable binding. The kinetics of the chaperonin system of Escherichia coli GroEL and GroES were analyzed as a model using this system and found to be very similar to those obtained with covalently immobilized ligands. The sensor chip can be reused many times, because of the powerful regeneration methods. The ligand can be freshly immobilized after each cycle, thus eliminating potential denaturation upon regeneration as a source of error.     

The effect of alterations to action potential duration on beta-adrenoceptor-mediated aftercontractions in human and guinea-pig ventricular myocytes

Aftercontractions induced by beta-adrenoceptor stimulation in human and guinea-pig cardiomyocytes may be related to changes in action potential duration (APD). We investigated the effects of altering APD during the occurrence of isoproterenol-induced aftercontractions, using the KATP channel openers cromakalim and lemakalim or the action potential voltage clamp technique, in guinea-pig and human ventricular cardiomyocytes. Contractile responses were measured at 32 degrees C using a video-based edge-detection system. In guinea-pig myocytes, action potentials, Indo-1 fluorescence and contraction were measured at 22 degrees C. Isoproterenol (< or = 12 nM) had variable effects on APD but induced aftercontractions, the majority (14/19 cells) of which occurred during the action potential. Short action potentials were produced using K+ channel openers. These compounds reduced or completely abolished the isoproterenol-induced aftercontractions. Increasing isoproterenol in the presence of K+ channel opener restored the main contraction to a level similar to or above those with isoproterenol alone, but without the reappearance of aftercontractions. When cells were stimulated to contract under action potential voltage clamp, isoproterenol-induced aftercontractions were abolished by voltage clamping with action potentials of short duration. It was possible to induce aftercontractions in some cells without application of isoproterenol if voltage clamp-imposed action potentials of very long duration were used. These aftercontractions were also abolished by shortening action potential duration. We conclude that K+ channel openers or the imposition of action potentials of short duration can dissociate positively inotropic beta-adrenoceptor stimulation from aftercontraction formation and that action potentials of long duration can be pro-arrhythmic.     

Neutralizing capacity of ++antiophidic sera against the venom of Bothrops moojeni (lance-headed viper)

Human oncostatin M (OM) is a M(r) 28,000 glycoprotein that has been shown to regulate cell proliferation and differentiation. The biological activities of OM can be mediated by two different heterodimeric receptor complexes, the leukemia inhibitory factor (LIF)/OM shared receptor and the OM-specific receptor. In this study, we have examined the growth-regulatory effect of OM on 10 breast cancer cell lines derived from human tumors. The cellular proliferation of seven of these breast cancer cell lines was inhibited by OM. The three cell lines that did not respond to OM treatment lacked the expression of OM receptors. The growth-inhibitory activity of OM is examined further in the H3922 breast cancer cell line, which expresses the high-affinity OM receptor at a relatively higher level. We found that the cellular proliferation of H3922 cells was induced strongly by extrogenous epidermal growth factor (EGF), EGF-like factor, and basic fibroblast growth factor. The proliferative activities of these growth factors can be abolished totally by cotreatment of H3922 cells with OM. Treatment of H3922 cells with OM for 24 h did not block EGF binding or the induction of EGF receptor tyrosine phosphorylation. This finding suggests that OM interferes with the mitogenic signal at steps distal to the EGF receptor. Examination of proto-oncogene expression demonstrated that OM down-regulates the c-myc gene in H3922 cells. The biological effects reported herein are not shared by the OM-related cytokines interleukin 6 or LIF, as demonstrated by the inability of these proteins to inhibit cell growth or modulate c-myc gene expression in breast cancer cells. Additionally, the high-affinity binding of labeled OM cannot be displaced by LIF. Together, these data suggest that OM is a growth inhibitor for breast cancer cells. The inhibitory activity is mediated predominantly through the OM-specific receptor, and activation of this receptor abrogates growth factor stimulation and down-regulates the c-myc proto-oncogene.     

Mutational analysis of the Drosophila sister-chromatid cohesion protein ORD and its role in the maintenance of centromeric cohesion

The ord gene is required for proper segregation of all chromosomes in both male and female Drosophila meiosis. Here we describe the isolation of a null ord allele and examine the consequences of ablating ord function. Cytologically, meiotic sister-chromatid cohesion is severely disrupted in flies lacking ORD protein. Moreover, the frequency of missegregation in genetic tests is consistent with random segregation of chromosomes through both meiotic divisions, suggesting that sister cohesion may be completely abolished. However, only a slight decrease in viability is observed for ord null flies, indicating that ORD function is not essential for cohesion during somatic mitosis. In addition, we do not observe perturbation of germ-line mitotic divisions in flies lacking ORD activity. Our analysis of weaker ord alleles suggests that ORD is required for proper centromeric cohesion after arm cohesion is released at the metaphase I/anaphase I transition. Finally, although meiotic cohesion is abolished in the ord null fly, chromosome loss is not appreciable. Therefore, ORD activity appears to promote centromeric cohesion during meiosis II but is not essential for kinetochore function during anaphase.