Apolipoprotein B gene variants are involved in the determination of serum cholesterol levels: a study in normo- and hyperlipidaemic individuals

We have investigated the frequencies of 3 restriction fragment length polymorphisms (RFLPs) of the apolipoprotein B (apo B) gene in normo- and hyperlipidaemic individuals. In individuals with type III hyperlipidaemia, the allele frequency for the RFLP detected with XbaI was significantly different from the allele frequency in normolipidaemic individuals and in those with other types of hyperlipidaemia. No significant difference in allele frequency was found among these groups for the RFLPs detected with MspI or EcoRI. Within a sample of 62 normolipidaemic individuals, homozygotes for the X2 allele (cutting site) of the XbaI RFLP had a significantly higher serum cholesterol level than homozygotes for the XI allele, with individuals of the genotype X1X2 having an intermediate value (X2X2 mean 5.71 mmol/l, X1X1 mean 4.81 mmol/l, X1X2 mean 5.30 mmol/l). There were also significant differences in serum triglyceride levels in individuals with different XbaI genotypes. In these normolipidaemic individuals there was no correlation between the EcoRI and MspI RFLP genotypes and levels of any serum lipid variable. Information from the XbaI and EcoRI RFLPs was used in conjunction to define apo B haplotypes. These haplotypes are a more precise measure of the genotypic variation, and they explain a greater fraction of the serum cholesterol and triglyceride levels than the single-site polymorphisms considered separately. This study suggests that variations in the gene for apo B are associated with the determination of serum cholesterol and triglyceride levels both in patients with type III hyperlipidaemia and in the normal population.     

Intramolecular phenotypic capacitance in a modular RNA molecule

Phenotypic capacitance refers to the ability of a genome to accumulate mutations that are conditionally hidden and only reveal phenotype-altering effects after certain environmental or genetic changes. Capacitance has important implications for the evolution of novel forms and functions, but experimentally studied mechanisms behind capacitance are mostly limited to complex, multicomponent systems often involving several interacting protein molecules. Here we demonstrate phenotypic capacitance within a much simpler system, an individual RNA molecule with catalytic activity (ribozyme). This naturally occurring RNA molecule has a modular structure, where a scaffold module acts as an intramolecular chaperone that facilitates folding of a second catalytic module. Previous studies have shown that the scaffold module is not absolutely required for activity, but dramatically decreases the concentration of magnesium ions required for the formation of an active site. Here, we use an experimental perturbation of magnesium ion concentration that disrupts the folding of certain genetic variants of this ribozyme and use in vitro selection followed by deep sequencing to identify genotypes with altered phenotypes (catalytic activity). We identify multiple conditional mutations that alter the wild-type ribozyme phenotype under a stressful environmental condition of low magnesium ion concentration, but preserve the phenotype under more relaxed conditions. This conditional buffering is confined to the scaffold module, but controls the catalytic phenotype, demonstrating how modularity can enable phenotypic capacitance within a single macromolecule. RNA’s ancient role in life suggests that phenotypic capacitance may have influenced evolution since life’s origins.Phenotypic capacitance requires mutations that reversibly alternate between hidden and revealed states in response to environmental or genetic perturbations (1, 2). In the hidden or “cryptic” state the mutations can survive selection because they do not change the phenotype, and multiple such mutations can accumulate within individual genomes in a population. Perturbation after such accumulation can reveal the combined effects of multiple mutations. By exposing the phenotypic effects of mutations that may not have been beneficial individually, capacitance provides a mechanism of generating new phenotypes—from macromolecules to morphological traits—with novel functions (1–4). The term “phenotypic capacitance” is appropriate for situations where altered phenotypes with a genetic basis can be hidden and revealed, even when no adaptive potential of the revealed phenotypes is demonstrated (2). “Evolutionary capacitance,” on the other hand, is a term reserved for instances when an adaptive role is demonstrated. Phenotypic capacitance has been known in fruit flies since the 1950s (5), but demonstrations of adaptive potential (4, 6), and the various molecular mechanisms behind it (7–11) have been reported only relatively recently.A module in a biological system is a group of system parts that interact more with each other than with parts outside of the module (12). In RNA molecules, the parts are nucleotides, and modules are units of tertiary structure that fold independently, and often perform different functions, such as binding to different proteins, RNAs, or small molecules. Modularity has been described in the telomerase RNA component (13), ribosomal RNA (14), long noncoding RNA (e.g., Xist, Hotair) (15), riboswitches (16), and self-splicing introns (17, 18). A link between modularity and phenotypic capacitance could provide a mechanism for the evolution of novel functions involving modules of RNA structure, and especially for functional innovations that require multiple simultaneous mutations. It is not known how modularity might change the potential to hide and reveal the effects of mutations in RNA structures, which is a prerequisite for phenotypic capacitance.To investigate a possible link between modularity and phenotypic capacitance in RNA, we chose to study the Azoarcus group I RNA enzyme (ribozyme). Group I ribozymes such as this have two structural modules that are functionally distinct (Fig. 1). The first of them is a scaffold module (Fig. 1, yellow) that folds rapidly, and forms a nearly identical structure even when removed from the context of the rest of the ribozyme (19, 20). The scaffolding it provides facilitates the folding of the less thermodynamically stable catalytic module (Fig. 1, blue) (21). Biochemical studies have shown that the catalytic phenotype (protein-free splicing) resides in the catalytic module, which can maintain activity if the scaffold module is deleted, but only under conditions of very high magnesium (80 mM) and extended incubation times (16 h) (22). This instability caused by deleting the scaffold supports the idea that this module acts as an intramolecular chaperone.Open in a separate windowFig. 1.The modular structure of the Azoarcus group I ribozyme. (A) The scaffold module (yellow) and the catalytic module (blue) are shown in the context of the ribozyme secondary structure. The regions that show base pairing (P) are numbered sequentially from the 5′ to the 3′ end, according to group I intron standards. The substrate for the ribozyme is written in lowercase letters. The modules are also shown in the context of the 3D crystal structure (PDB ID: 1ZZN), from a “top view” (B) and “side view” (C), with respect to the scaffold module.The diversification of RNA functions has played an important role in the evolution of extant organisms, and may have been even more important at life’s beginnings when RNA enzymes (ribozymes) played a central role as catalysts in the RNA world scenario (23). In a previous publication using the Azoarcus ribozyme, we demonstrated an adaptive role for accumulated cryptic variation that was revealed when altered enzymatic activity was required (24). Here, we focus on how the functionally distinct modules of this structure might facilitate the occurrence of environmentally conditional mutations that enable phenotypic capacitance.We aimed to identify mutations that maintain the catalytic phenotype under normal conditions but alter this phenotype under stressful conditions. The ideal stressful condition for our RNA system is a low concentration of magnesium ions. The concentration of magnesium ions inside of cells is maintained at a higher concentration than any other divalent ion due to its role in many cellular functions. Importantly for our current experiments, it is known that magnesium ions are critical for stabilizing the native structure of RNA molecules (25). Low concentrations of magnesium only allow folding of the most stable RNA structures and can lead to misfolding of less stable structures. In addition, many ribozymes, including group I introns, use highly coordinated magnesium ions in their active site (26). Based on previous reports on the magnesium dependence of the Azoarcus ribozyme (19), we here studied a stressful environment with low (2 mM) MgCl₂, a relaxed environment with high (25 mM) MgCl₂, and an intermediate environment (10 mM MgCl₂). We note that magnesium availability is often limited in natural environments, a stressful condition that organisms have evolved adaptive responses to. For example, magnesium homeostasis in bacteria is maintained by the expression of ion transporters that are sometimes regulated by cis-acting RNA regulatory elements. These “magnesium riboswitch” elements control downstream mRNA expression by conformational changes induced by altered magnesium concentrations (27). This example and others (28) highlight the widespread importance of our chosen experimental stressor.     

Inhibition of experimental autoimmune encephalomyelitis in Lewis rats by nasal administration of encephalitogenic MBP peptides: synergistic effects of MBP 68-86 and 87-99

Induction of mucosal tolerance by inhalation of soluble peptides with defined T cell epitopes is receiving much attention as a means of specifically down-regulating pathogenic T cell reactivities in autoimmune and allergic disorders. Experimental autoimmune encephalomyelitis (EAE) induced in the Lewis rat by immunization with myelin basic protein (MBP) and Freund's adjuvant (CFA) is mediated by CD4+ T cells specific for the MBP amino acid sequences 68-86 and 87-99. To further define the principles of nasal tolerance induction, we generated three different MBP peptides (MBP 68-86, 87-99 and the non- encephalitogenic peptide 110-128), and evaluated whether their nasal administration on day -11, -10, -9, -8 and -7 prior to immunization with guinea pig MBP (gp-MBP) + CFA confers protection to Lewis rat EAE. Protection was achieved with the encephalitogenic peptides MBP 68-86 and 87-99, MBP 68-86 being more potent, but not with MBP 110-128. Neither MBP 68-86 nor 87-99 at doses used conferred complete protection to gp-MBP-induced EAE. In contrast, nasal administration of a mixture of MBP 68-86 and 87-99 completely blocked gp-MBP-induced EAE even at lower dosage compared to that being used for individual peptides. Rats tolerized with MBP 68-86 + 87-99 nasally showed decreased T cell responses to MBP reflected by lymphocyte proliferation and IFN-gamma ELISPOT assays. Rats tolerized with MBP 68-86 + 87-99 also had abrogated MBP-reactive IFN-gamma and tumor necrosis factor-alpha mRNA expression in lymph node cells compared to rats receiving MBP 110-128 nasally, while similar low levels of MBP-reactive transforming growth factor-beta and IL-4 mRNA expressing cells were observed in the two groups. Nasal administration of MBP 68-86 + 87-99 only slightly inhibited guinea pig spinal cord homogenate-induced EAE, and passive transfer of spleen mononuclear cells from MBP 68-86 + 87-99-tolerized rats did not protect naive rats from EAE. Finally, we show that nasal administration of MBP 68-86 + 87-99 can reverse ongoing EAE induced with gp-MBP, although higher doses are required compared to the dosage needed for prevention. In conclusion, nasal administration of encephalitogenic MBP peptides can induce antigen-specific T cell tolerance and confer incomplete protection to gp-MBP-induced EAE, and MBP 68-86 and 87-99 have synergistic effects. Non-regulatory mechanisms are proposed to be responsible for tolerance development after nasal peptide administration.      

Are relational style and neuropsychological performance predictors of social attributions in chronic schizophrenia?

Attributional style is defined as the pervasive tendency to explain the cause of social actions in terms of oneself, or others, or the context of the event. While the clinical correlates of this aspect of social cognition have been widely researched, its links with relationship style and neuropsychological performance, although hypothesised, have received less attention. This study investigated whether attributional style is predicted by variance in either relationship style or neuropsychological performance in schizophrenia. We assessed attributional style (using the Internal, Personal and Situational Attributions Questionnaire [IPSAQ]), relationship style (using Bartholomew and Horowitz's Relationship Questionnaire), and neuropsychological function (using the Wechsler Abbreviated Scale of Intelligence, the Wechsler Memory Test, and the Cambridge Automated Test Battery) in 73 stabilised outpatients with chronic schizophrenia and 78 controls matched for age and gender. 'Externalising bias' (attributing positive rather than negative events to oneself) was predicted by verbal ability in both patients and controls. 'Personalising bias' (attributing negative events to others rather than to situational factors) was predicted by higher secure relationship style ratings, but only in the patient group. This study highlights the importance of relationship style and neuropsychological performance for different aspects of attributional style in schizophrenia.     

Sensitivity and specificity of four assays to detect human T− lymphotropic virus type I or type I/II antibodies

BACKGROUND: Assays that detect human T-lymphotropic virus type I and type II antibody (HTLV-I/II) are widely used in the routine screening of blood donors. STUDY DESIGN AND METHODS: Four commercially available anti-HTLV-I (Fujirebio and Organon Teknika) or -HTLV-I/II assays (Murex and Ortho) were evaluated in various serum panels: A) HTLV-I-positive specimens (n = 41), confirmed by Western blot and polymerase chain reaction; B) a commercially available anti-HTLV-I/II panel; C) serial dilutions of sera from HTLV-I-positive individuals (n = 30), confirmed by immunofluorescence assay and Western blot: D) serial dilutions of HTLV-II-positive blood donors (n = 20), confirmed by Western blot and polymerase chain reaction, and E) sera from first-time blood donors (n = 1055). RESULTS: All four assays elicited reactions in all 82 HTLV-I- positive samples in Panels A, B, and C. Of 32 HTLV-II-positive specimens in Panels B and D, 31 (96.9%) reacted in the Organon Teknika assay and all 32 reacted in the remaining tests. Probit analysis of test results in Panels C and D indicated that the Fujirebio test was the most sensitive assay, followed by Organon Teknika, Ortho, and Murex. The specificities of Fujirebio, Murex, Organon Teknika, and Ortho tests in 1055 first-time blood donors were 99.9, 100, 99.6, and 99.9 percent, respectively. CONCLUSION: All four studied assays for detecting HTLV-I or HTLV-I/II antibodies are appropriate as screening tests.     

Human marrow erythropoiesis in culture. I. Characterization of methylcellulose colony assay

We examined the morphological and functional characteristics of human marrow erythrocytes cultured with a recently developed methylcellulose colony assay technique. Erythrocytic cells in various stages of development were observed, and a significant degree of maturational synchrony within individual colonies was noted. By light microscopy, colonies consisting of late normoblasts appeared compact, had an orange hue attributable to their hemoglobin, and demonstrated pseudoperoxidase activity, whereas colonies composed of early erythroblasts grew less compact or in clusters of smaller cell aggregates and showed no reddish tinge. Colonies possessing intermediate features were also observed. Maturational synchrony of individual colonies was confirmed using ransmission and scanning electron microscopy. The ultrastructure and cytochemistry of most immature cells were normal. The mature erythrocytes, however, were severely microcytic and hypochromic and contained one to several Heinz bodies. These defects in the cytoplasmic maturation of erythrocytes corresponded with impaired granulocytic maturation in culture, which we observed previously, and suggest environmental or nutritional defects in culture. Linearity of the method was confirmed using five normal bone marrows. Erythropoietin dose-responses observed in ten normal marrows were comparable to the previously reported results and revealed significant variation in individual plating efficiencies.     

A strabismus susceptibility locus on chromosome 7p

Strabismus has been known to have a significant genetic component, but the mode of inheritance and the identity of the relevant genes have been enigmatic. This paper reports linkage analysis of nonsyndromic strabismus. The principal results of this study are: (i) the demonstrated feasibility of identifying and recruiting large families in which multiple members have (or had) strabismus; (ii) the linkage in one large family of a presumptive strabismus susceptibility locus to 7p22.1 with a multipoint logarithm of odds score of 4.51 under a model of recessive inheritance; and (iii) the failure to observe significant linkage to 7p in six other multiplex families, consistent with genetic heterogeneity among families. These findings suggest that it will be possible to localize and ultimately identify strabismus susceptibility genes by linkage analysis and mutation screening of candidate genes.     

Activity of 2-(3,4-dichlorophenoxy)-5-nitrobenzonitrile (MDL-860) against picornaviruses in vitro.

The newly synthesized compound 2-(3,4-dichlorophenoxy)-5-nitrobenzonitrile (MDL-860) has been found to inhibit picornavirus replication. In multiple growth cycle experiments, 1 microgram of MDL-860 per ml caused a reduction in virus yield of at least 1.0 log10 50% tissue culture infectious doses per 0.2 ml for 8 of 10 enteroviruses and 72 of 90 rhinovirus serotypes. This antiviral activity was dependent on both compound concentration and virus inoculum size. At concentrations that had no toxic effects on cell cultures, MDL-860 did not inhibit cytopathic effect or hemadsorption activity due to coronavirus 229-E, vesicular stomatitis virus, herpes simplex virus type 1, adenovirus, influenza virus A, or parainfluenza virus 1. Compound concentrations up to 25 micrograms/ml did not cause cytopathic effect in short-term cultures of rhesus monkey, WI-38, or HeLa cells; 10 micrograms/ml did not inhibit the replication of HeLa cells. The mechanism of action of MDL-860 has not been defined, although it was not directly virucidal and appeared to inhibit picornaviruses specifically at an early step in the virus-host cell interaction.