Inherited deficiency of the sixth component of complement: a silent or null gene

Four families have been studied, some members of which have inherited deficiency of the sixth component of complement. The genetically determined electrophoretic variants of C6 were evaluated in all family members. Seven individuals were found who did not have the variant found in the serum of the parent from whom they inherited the deficiency. It is inferred that the isolated low levels of C6 in these individuals results from the heterozygous state of a normal C6 variant gene and a silent or null C6 gene; the genes determining electrophoretic variants and the low serum levels of C6 are allelic.     

The influence of potassium content on the kinetics of potassium influx into excised ryegrass and barley roots

The influx of K⁺ into excised roots of barley (Hordeum vulgare L.) and ryegrass (Lolium multiflorum L.) previously grown with or without K⁺ was measured in K⁺ solutions ranging in concentration from 0.01 to 50 mM. In both species the K⁺ influx was lower in the roots with high K⁺ content. The extent of reduction by high internal [K⁺] decreased with external concentration above 1 mM. These results support the contention that at high external concentrations passive diffusion makes significant contributions to observed fluxes.     

Value of stool examination in patients with diarrhoea.

Findings of stool examinations in 1593 patients with diarrhoea due to a single enteric pathogen--enterotoxigenic Escherichia coli rotavirus, Shigella, Campylobacter jejuni, Vibrio cholerae 0:1, Entamoeba histolytica, or Giardia lamblia--were reviewed to determine how well they predicted the agent associated with the diarrhoea. Specimens were examined visually for blood and mucus, tested for pH, and examined under a microscope for the presence of red and white blood cells, parasites, and stool fat. Although visible blood was more common in specimens from patients infected with Shigella (51%) and Ent histolytica (39%) than in those from patients infected with other agents (6%; p less than 0.01), patients infected with Shigella were most likely to have numerous faecal leucocytes (greater than 50/high power field: 39% v 8% of all patients and 7% of patients infected with Ent histolytica, p less than 0.01 in both cases). Patients infected with enterotoxigenic E coli, rotavirus, V cholerae 0:1, or C jejuni had loose stools with fewer red or white cells. Patients infected with rotavirus and C jejuni were more likely to have acid stools with 3 to 4+ fat, but these findings were related to young age and breast feeding. Stool examination is most useful in establishing a diagnosis of dysentery and in helping to distinguish between patients infected with Shigella and Ent histolytica; it is of limited usefulness in discriminating between pathogens causing watery diarrhoea.     

CENP-B is a highly conserved mammalian centromere protein with homology to the helix-loop-helix family of proteins

CENP-B is a centromere associated protein originally identified in human cells as an 80 kDa autoantigen recognized by sera from patients with anti-centromere antibodies (ACA). Recent evidence indicates that CENP-B interacts with centromeric heterochromatin in human chromosomes and may bind to a specific subset of human alphoid satellite DNA. CENP-B has not been unambiguously identified in non-primates and could, in principal, be a primate-specific alphoid DNA binding protein. In this work, a human genomic DNA segment containing the CENP-B gene was isolated and subjected to DNA sequence analysis. In vitro expression identified the site for translation initiation of CENP-B, demonstrating that it is encoded by an intronless open reading frame (ORF) in human DNA. A homologous mouse gene was also isolated and characterized. It was found to possess a high degree of homology with the human gene, containing an intronless ORF coding for a 599 residue polypeptide with 96% sequence similarity to human CENP-B. 5 and 3 flanking and untranslated sequences were conserved at a level of 94.6% and 82.7%, respectively, suggesting that the regulatory properties of CENP-B may be conserved as well. CENP-B mRNA was detected in mouse cells and tissues and an immunoreactive nuclear protein identical in size to human CENP-B was detected in mouse 3T3 cells using human ACA. Analysis of the sequence of CENP-B revealed a segment of significant similarity to a DNA binding motif identified for the helix-loop-helix (HLH) family of DNA binding proteins. These data demonstrate that CENP-B is a highly conserved mammalian protein that may be a member of the HLH protein family and suggest that it plays a role in a conserved aspect of centromere structure or function.     

Isolation of Neurospora Crassa a Mating Type Mutants by Repeat Induced Point (Rip) Mutation

In the filamentous fungus, Neurospora crassa, mating type is regulated by a single locus with alternate alleles, termed A and a. The mating type alleles control entry into the sexual cycle, but during vegetative growth they function to elicit heterokaryon incompatibility, such that fusion of A and a hypha results in death of cells along the fusion point. Previous studies have shown that the A allele consists of 5301 bp and has no similarity to the a allele; it is found as a single copy and only within the A genome. The a allele is 3235 bp in length and it, too, is found as a single copy within the a genome. Within the A sequence, a single open reading frame (ORF) of 288 amino acids (mt A-1) is thought to confer fertility and heterokaryon incompatibility. In this study, we have used repeat induced point (RIP) mutation to identify functional regions of the A idiomorph. RIP mutations in mt A-1 resulted in the isolation of sterile, heterokaryon-compatible mutants, while RIP mutations generated in a region outside of mt A-1 resulted in the isolation of mutants capable of mating, but deficient in ascospore formation.     

Effects of nitrite, chlorate, and chlorite on nitrate uptake and nitrate reductase activity

Effects of NO₂⁻, ClO₃⁻, and ClO₂⁻ on the induction of nitrate transport and nitrate reductase activity (NRA) as well as their effects on NO₃⁻ influx into roots of intact barley (Hordeum vulgare cv Klondike) seedlings were investigated. A 24-h pretreatment with 0.1 mol m⁻³ NO₂⁻ fully induced NO₃⁻ transport but failed to induce NRA. Similar pretreatments with ClO₃⁻ and ClO₂⁻ induced neither NO₃⁻ transport nor NRA. Net ClO₃⁻ uptake was induced by NO₃⁻ but not by ClO₃⁻ itself, indicating that NO₃⁻ and ClO₃⁻ transport occur via the NO₃⁻ carrier. At the uptake step, NO₂⁻ and ClO₂⁻ strongly inhibited NO₃⁻ influx; the former exhibited classical competitive kinetics, whereas the latter exhibited complex mixed-type kinetics. ClO₃⁻ proved to be a weak inhibitor of NO₃⁻ influx (K_i = 16 mol m⁻³) in a noncompetitive manner. The implications of these findings are discussed in the context of the suitability of these NO₃⁻ analogs as screening agents for the isolation of mutants defective in NO₃⁻ transport.     

Use of lectins for detection of electrophoretically separated glycoproteins transferred onto nitrocellulose sheets

A method of rapidly identifying lectin-binding glycoproteins separated by polyacrylamide gel electrophoresis is described. The method is particularly useful for comparing the glycoprotein content of different cell types and fractions. Normal rat liver, Novikoff hepatoma, and rat mammary tumor cell line 13762 MAT-B were fractionated to give purified nuclei and other fractions defined by their sedimentation properties in low ionic strength buffer. The subcellular fractions were separated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate, transferred to nitrocellulose sheets, and localized by an immunochemical method to identify lectin-binding activities. The localization pattern of concanavalin A and wheat germ agglutinin-binding activities in the fractions from the three cell types showed the greatest similarities between the glycoprotein contents of normal liver and Novikoff hepatoma fractions. On a per-cell basis the purified nuclei from each of the cell types contained less activity overall than did other particulate cell fractions. Washing the nuclei from normal liver and Novikoff hepatoma, but not MAT-B cells, in nonionic detergent removed or depressed most of the lectin-binding activities. However, two major bands were unaffected by the detergent. One of these localized with wheat germ agglutinin at an apparent molecular weight of 62,000 in the nuclei of all three cell types. The other localized with concanavalin A at an apparent molecular weight of 200,000 in normal liver and Novikoff hepatoma nuclei.     

Activation of Retinal Tyrosine Hydroxylase In Vitro by Cyclic AMP-Dependent Protein Kinase: Characterization and Comparison to Activation In Vivo by Photic Stimulation

Abstract: Tyrosine hydroxylase in rat retina is activated in vivo as a consequence of photic stimulation. Tyrosine hydroxylase in crude extracts of dark-adapted retinas is activated in vitro by incubation under conditions that stimulate protein phosphorylation by cyclic AMP-dependent protein kinase. Comparison of the activations of the enzyme by photic stimulation in vivo and protein phosphorylation in vitro demonstrated several similarities. Both treatments decreased the apparent K _m of the enzyme for the synthetic pterin cofactor 6MPH₄. Both treatments also produced the same change in the relationships of tyrosine hydroxylase activity to assay pH. When retinal extracts containing tyrosine hydroxylase activated either in vivo by photic stimulation or in vitro by protein phosphorylation were incubated at 25°C, the enzyme was inactivated in a time-dependent manner. The inactivation of the enzyme following both activation in vivo and activation in vitro was partially inhibited by sodium pyrophosphate, an inhibitor of phosphoprotein phosphatase. In addition to these similarities, the activation of tyrosine hydroxylase in vivo by photic stimulation was not additive to the activation in vitro by protein phosphorylation. These data indicate that the mechanism for the activation of tyrosine hydroxylase that occurs as a consequence of light-induced increases of neuronal activity is similar to the mechanism for activation of the enzyme in vitro by protein phosphorylation. This observation suggests that the activation of retinal tyrosine hydroxylase in vivo may be mediated by phosphorylation of tyrosine hydroxylase or some effector molecule associated with the enzyme.     

Vitellin and vitellogenin incorporation by isolated oöcytes of Locusta migratoria migratorioides (R.F.)

Vitellin and vitellogenin labelled in vitro with ¹²⁵I and in vivo with ³H were incorporated into yolk by locust oöcytes incubated in an in vitro system. This incorporation was specific and linear with the duration of incubation. Uptake of vitellin by oöcytes was 3–4 times higher than ¹²⁵I-bovine serum albumin in 2.1-mm oöcytes and 20 times higher than ¹²⁵I-bovine serum albumin in 4.0-mm long oöcytes. The uptake of the albumin was enhanced by the presence of vitellin in the incubation medium. ³H-labelled yolk protein was incorporated at higher rates than that labelled with ¹²⁵I. The addition of the juvenile hormone analogue ZR 515, caused the incorporation rates of vitellogenin to be increased. The amount of vitellin or vitellogenin taken up by the oöcytes increased with their length, and the rate of incorporation per unit surface area was highest in 3–4-mm long oöcytes. These results corroborate previously reported in vivo patterns of incorporation rates of developing oöcytes.