Adi D. Bharucha

Geographic distribution and genealogy of mutation 207 of the lipoprotein lipase gene in the French Canadian population of Québec

SummaryMutations in the lipoprotein lipase (LPL) gene, leading to partial or total inactivation of the enzyme, result in a hereditary clinical syndrome called familial LPL deficiency. The French Canadian population, which is primarily and historically located in the province of Québec, has the highest worldwide frequency of LPL-deficient patients. We have analyzed the prevalence, spatial distribution, and genealogy in the Québec population of a LPL gene mutation, M-207 (P207L in conventional notation), which changes the amino acid proline to leucine in position 207 of the LPL protein and inactivates the enzyme. Our results show that M-207 is the most prevalent LPL gene mutation among French Canadians and accounts for the largest proportion of LPL-deficient patients in this population. Genealogical reconstruction of French Canadian LPL-deficient patients point to 16 founders of M-207, all of whom migrated to Québec in the early seventeenth century from the north-western part of France, especially from the region of Perche. Most of the carriers of M-207 are, at present, found in the Charlevoix, Saguenay-Lac-St-Jean regions of eastern Québec. On the basis of the number of homozygote M-207 LPL-deficient patients so far identified, we estimate that there are at least 31,000 carriers of this mutation in the province of Québec. This constitutes a large pool of individuals at risk for atherosclerosis and other lipid-related diseases, since LPL deficiency is considered to be a significant contributing factor in the etiology and development of these diseases.

Effects of trichothecenes (T-2-toxin) on protein synthesis in vitro by brain polysomes and messenger RNA

The effects of T-2 toxin on protein synthesis were tested in two reticulocyte lysate in vitro systems pretreated with micrococcal nuclease. One of the test systems contained purified globin mRNA and was initiation dependent. The other contained rat brain polysomes and incorporated amino acids by an elongation dependent process. T-2 toxin inhibited the translation of globin mRNA at all concentrations tested, from 10(-8) M to 10(-4) M. Rat brain polysomes were much less sensitive to T-2 toxin than globin mRNA. While high concentrations of the toxin (10(-4) M) led to partial inhibition of protein synthesis by polysomes, low concentrations (10(-8) M and 10(-6) M) stimulated protein synthesis. Comparison of the above results with those obtained by other workers suggest that the T-2 toxin may inhibit not only the initiation step of translation, but also elongation and termination, depending upon the concentration of the toxin and the nature of the translation system. A similar mechanism may operate for all the trichothecene toxins that exert their effect through binding to ribosomal peptidyl transferase.

MOLECULAR BIOLOGICAL MODELS IN GERIATRIC NEUROBIOLOGY

Abstract 1 Some of the theoretical and experimental approaches based on concepts of molecular biology that have been developed recently for the study of the aging phenomenon have been summarized. The importance of investigation of the aging nervous system in this context has been discussed. 2 Experimental results have been presented showing that chromatins from cerebral cortices of young and old rats differ from each other in a number of qualitative and quantitative parameters, for example, in the relative proportion of DNA, RNA, histone and nonhistone proteins, in the stability of DNA secondary structure to thermal denaturation and in the amounts of single-stranded DNA, free double-stranded DNA and protein-covered DNA. 3 Cerebral cortex from the young rat appears to possess a larger portion of DNA which is free or attached to acidic protein and a smaller portion of DNA attached to histories, as compared to the old tissue. This correlates with the greater metabolic activity of the nervous system of the young as compared to the old animal. 4 The data suggest that the stabilization of DNA in the aged tissue may be due to intercalation with protein and that both the unique and reiterated sequences may be stabilized in this manner.

Biochemical manipulations of minute quantities of mRNAs and cDNAs immobilized on cellulose paper discs

Publisher Summary This chapter discusses the biochemical manipulations of minute quantities of messenger RNA (mRNAs) and complementary DNA (cDNA) immobilized on cellulose paper discs. In principle, cDNA is prepared directly from small amounts of mRNA trapped on oligo (dT) cellulose paper disks, using the oligo (dT) of the paper as primer for reverse transcription. The newly formed cDNA, covalently linked to the disk, is then employed in further reactions. This procedure: (1) permits manipulations of very small amounts of mRNA and cDNA, (2) reduces the processing time of serial reactions considerably, because the bound nucleic acid may be purified by rinsing in an appropriate buffer rather than by precipitation and centrifugation, and (3) reduces losses of nucleic acid products that might otherwise occur during each of the recovery steps. A target mRNA of interest in a given population of mRNA can be detected or even quantitated by the dot-blot procedure, using an appropriate probe. But the extreme susceptibility of mRNA to degradation by chance contamination with RNase is a practical drawback in the storage and manipulations of RNA samples. However, once reverse transcribed and immobilized on cellulose disk, the cDNA act as stable and durable records of their corresponding mRNAs and can be repeatedly screened by the same or different probes, as needed.

Structure and biological activity of polysomes stained with Coomassie blue.

Polysomes prestained with Coomassie blue were fractionated on sucrose density gradients giving rise to visible bands corresponding to different size classes of aggregates. Coomassie blue staining enhanced the capacities of brain and liver polysomes to synthesize proteins in vitro, including the synthesis of neuron‐specific enolase. This positive action of the dye was restricted to polysomes and was not manifested when mRNAs isolated from prestained polysomes were tested in in vitro translation or reverse transcription, indicating that the action of the dye consists in stabilization of polysomal structure.

MOLECULAR CORRELATES OF BRAIN DEVELOPMENT: tRNA AS A POSSIBLE INTERMEDIATE IN THE FORMATION OF NEUROACTIVE SUBSTANCES

Abstract The following considerations suggest that tRNA may serve as an intermediate in the formation of compounds having neuropharmacologies or neurophysiological activities: 1 The aminoacyl moieties of glutaminyl and asparaginyl-tRNAs undergo simultaneous nonenzymatic cyclization and deesterification under very mild reaction conditions giving rise to α-aminoglutarimide and α-aminosuccinimide. Derivatives of these two compounds are known to be active neuropharmacologically. 2 N-Formylmethionine and N-Formylmethionyl oligopeptides which could be synthesized through participation of mitochondrial initiator tRNA are known to have strong chemotactic properties. 3 tRNAs containing isopentenyl side chain release, in the course of catabolism, modified bases or nucleoside derivatives having cytokinin or cytotoxic activities.

[4] – Biochemical Manipulations of Minute Quantities of mRNAs and cDNAs Immobilized on Cellulose Paper Discs

Publisher Summary Immobilization of the critical reactants on insoluble matrices is the means through which secondary products may be removed from a given reaction. This may enhance the rates and yields of the reaction. The oligonucleotide linked to cellulose powder is extensively used for the separation and purification of messenger RNAs (mRNAs) from other nucleic acids and proteins. Poly (U) covalently bound to diazothiophenyl paper is also used for the isolation of mRNA from total cytoplasmic RNA. In principle, complementary DNA (cDNA) is prepared directly from small amounts of mRNA trapped on oligo cellulose paper disks, using the oligo of the paper as primer for reverse transcription. The newly formed cDNA is then employed in further reactions. This procedure permits manipulation of small amounts of mRNA and cDNA; reduces the processing time of serial reactions considerably because the bound nucleic acid may be purified by rinsing in an appropriate buffer rather than by precipitation and centrifugation; and reduces losses of nucleic acid products that might otherwise occur during each of the recovery steps. The mRNA profiles of a cell may undergo significant changes in response to a variety of genetic, physiological, pathological, chemical, or environmental signals. This may involve the transcription of a large number of genes or may result in the induction or suppression of a limited number of unique genes. The method of hybridization subtraction is often used to enrich the transcripts of the affected genes for eventual cloning and characterization.

Influence of Dye Binding on the Integrity and Function of Nucleic Acids and Nucleoproteins: Complexation of Polysomes with Coomassie Blue

Polysomes prestained with low concentrations of Coomassie blue gave rise to visible bands representing aggregates of different sizes when centrifuged in a sucrose density gradient and showed profiles similar to unstained polysomes. The visibility of the bands rendered them easy to recuperate and analyze. The stained polysomes were more active than the unstained specimens in protein synthesis, including the synthesis of tissue specific proteins in vitro. This enhanced activity was abolished when messenger RNAs were isolated from the prestained polysomes and translated in vitro. It is suggested that Coomassie blue may exert its influence by acting on one or more of the protein components involved in stabilizing polysomal structure and function.