Winston Salser

DOSE-DEPENDENT SUPPRESSION OF SERUM CHOLESTEROL BY TOCOTRIENOL-RICH FRACTION (TRF25) OF RICE BRAN IN HYPERCHOLESTEROLEMIC HUMANS

Tocotrienols are effective in lowering serum total and LDL-cholesterol levels by inhibiting the hepatic enzymic activity of beta-hydroxy-beta-methylglutaryl coenzymeA (HMG-CoA) reductase through the post-transcriptional mechanism. alpha-Tocopherol, however, has an opposite effect (induces) on this enzyme activity. Since tocotrienols are also converted to tocopherols in vivo, it is necessary not to exceed a certain dose, as this would be counter-productive. The present study demonstrates the effects of various doses of a tocotrienol-rich fraction (TRF25) of stabilized and heated rice bran in hypercholesterolemic human subjects on serum lipid parameters. Ninety (18/group) hypercholesterolemic human subjects participated in this study, which comprised three phases of 35 days each. The subjects were initially placed on the American Heart Association (AHA) Step-1 diet and the effects noted. They were then administered 25, 50, 100, and 200 mg/day of TRF25 while on the restricted (AHA) diet. The results show that a dose of 100 mg/day of TRF25 produce maximum decreases of 20, 25, 14 (P<0.05) and 12%, respectively, in serum total cholesterol, LDL-cholesterol, apolipoprotein B and triglycerides compared with the baseline values, suggesting that a dose of 100 mg/day TRF25 plus AHA Step-1 diet may be the optimal dose for controlling the risk of coronary heart disease in hypercholesterolemic human subjects.

Novel tocotrienols of rice bran modulate cardiovascular disease risk parameters of hypercholesterolemic humans

Tocotrienols inhibit cholesterol synthesis by post-transcriptionally suppressing β-hydroxy-β-methylglutaryl-coenzyme A reductase activity. A double blind, 12-week study was performed to investigate the effect of a novel tocotrienol-rich fraction (TRF25; obtained by molecular distillation from specially processed rice bran oil) on cardiovascular disease risk factors of hypercholesterolemic human subjects (serum total cholesterol >5.69 mmol/L). After acclimation to an alcohol-free regimen (baseline) participants were assigned to the National Cholesterol Education Program (NCEP) Step-1 diet (saturated fat <19%, total fat <30% of total calories and cholesterol <7.76 mmol/L). The participants were evaluated after 4 weeks of exposure to the NCEP Step-1 diet; one group of 21 participants was continued on the NCEP Step-1 diet for 4 weeks receiving an additional 1.2 gm corn oil (placebo group) and a second group of 20 received 200 mg TRF25 dissolved in 1.0 gm corn oil (TRF25 group). Serum total cholesterol and LDL-cholesterol levels of all the participants, stable during the baseline phase of the study, decreased 5% and 8%, respectively, during the 4-week NCEP Step-1 diet. Placebo continuing on the NCEP Step-1 diet for an additional 4 weeks experienced additional but modest decreases in serum total cholesterol (2%) and LDL-cholesterol (3%), yielding significant (P < 0.05) decreases when compared with the baseline values. These responses confirm the cholesterol-lowering action of a low fat, low cholesterol diet. Participants receiving TRF25 had 12% and 16% reductions (P < 0.05) in total cholesterol and LDL-cholesterol levels during the 4-week experimental phase; during the two phases (NCEP Step-1 diet plus treatment) the serum total cholesterol and LDL-cholesterol levels of these participants were decreased (P < 0.05) by 17% and 24%, respectively. TRF25-mediated decreases in Apo B, Lp(a), platelet factor 4 and thromboxane B2 (15%, 17%, 14%, and 31%, respectively) were significant (P < 0.05). There was no change in the levels of HDL-cholesterol and apolipoprotein A-I by this treatment. The treatments also resulted in remarkable increases in the levels of LDL-bound antioxidants, especially tocotrienols, which have substantially greater antioxidant activity than vitamin E.

The primary sequence of rabbit α-globin mRNA

Abstract The rabbit α-globin DNA insertion in the chimeric plasmid pHb72 (Liu et al., 1977) has been sequenced by the method of Maxam and Gilbert (1977). This has enabled us to determine the messenger RNA (mRNA) sequence beginning in the 5′ untranslated region 9 nucleotides before the initiation codon and extending through the first 361 nucleotides of the translated region. The data reported here overlap and are in complete agreement with sequences determined by Baralle (1977) for the 5′ end of the mRNA and by Proudfoot et al. (1977) for the 3′ end. Our sequence is also in agreement with the partial complementary RNA (cRNA) sequencing data which we reported previously (Paddock et al., 1977) and with the published amino acid sequence. Save for three uncertain nucleotides in the 3′ sequence determined by Proudfoot et al. (1977), this work marks the completion of the primary sequence of the rabbit α-globin mRNA. These observations reaffirm the high fidelity with which gene copies can be synthesized in vitro, cloned in a bacterial plasmid and maintained in the host. The general features of the mRNA nucleotide sequence are discussed with particular attention given to the base composition and codon preferences observed and to comparison of this sequence with other completed mRNA gene sequences. A new computer program has been used to search for the most stable base-pairing arrangement of the completed mRNA.

Synergistic effect of tocotrienol-rich fraction (TRF25) of rice bran and lovastatin on lipid parameters in hypercholesterolemic humans

Tocotrienols exert hypocholesterolemic action in humans and animals. Lovastatin is widely used for that purpose. Both agents work by suppressing the activity of beta-hydroxy-beta-methylglutaryl coenzyme A reductase through different mechanisms, post-transcriptional vs competitive inhibition. A human study with 28 hypercholesterolemic subjects was carried out in 5 phases of 35 days each, to check the efficacy of tocotrienol-rich fraction (TRF(25)) of rice bran alone and in combination with lovastatin. After placing subjects on the American Heart Association (AHA) Step-1 diet (phase II), the subjects were divided into two groups, A and B. The AHA Step-1 diet was continued in combination with other treatments during phases III to V. Group A subjects were given 10 mg lovastatin, 10 mg lovastatin plus 50 mg TRF(25), 10 mg lovastatin plus 50 mg alpha-tocopherol per day, in the third, fourth, and fifth phases, respectively. Group B subjects were treated exactly to the same protocol except that in the third phase, they were given 50 mg TRF(25) instead of lovastatin.The TRF(25) or lovastatin plus AHA Step-1 diet effectively lower serum total cholesterol (14%, 13%) and LDL-cholesterol (18%, 15% P < 0.001), respectively, in hypercholesterolemic subjects. The combination of TRF(25) and lovastatin plus AHA Step-1 diet significantly reduces of these lipid parameters of 20% and 25% (P < 0.001) in these subjects. Substitution of TRF(25) with alpha-tocopherol produces insignificant changes when given with lovastatin. Especially significant is the increase in the HDL/LDL ratio to 46% in group (A) and 53% (P < 0.002) in group (B). These results are consistent with the synergistic effect of these two agents. None of the subjects reported any side-effects throughout the study of 25-weeks. In the present study, the increased effectiveness of low doses of tocotrienols (TRF(25)) as hypocholesterolemic agents might be due to a minimum conversion to alpha-tocopherol. The report also describes in vivo the conversion of gamma-[4-3H]-, and [14C]-desmethyl (d-P(21)-T3) tocotrienols to alpha-tocopherol.

Structure and expression of ferritin genes in a human promyelocytic cell line that differentiates in vitro.

HL-60 is a human promyelocytic cell line with the capability of differentiating in vitro to give neutrophils, macrophages, or eosinophils. We screened libraries of HL-60 cDNA clones representing different time points during these differentiation processes to isolate clones corresponding to mRNAs whose expression is regulated during terminal differentiation. Upon sequencing this group of regulated clones, one clone encoding the heavy subunit and two clones encoding the light subunit of human ferritin were identified by reference to published amino acid sequences. Southern blot analyses showed that these clones are encoded by distinct multigene families. These clones identify two mRNAs whose ratios vary in a complex manner during both neutrophil and macrophage differentiation.

Human ribosomal RNA gene spacer sequences are found interpersed elsewhere in the genome

A cloned EcoRI fragment containing human 18 S rRNA gene sequences was used to screen a gene library to obtain a set of 8 overlapping cloned DNA segments extending into the non-transcribed spacer region of the human ribosomal RNA gene cluster. 19.4 kb of the approx. 43-kb rDNA repeat was obtained in cloned form and mapped with restriction endonucleases. None of the clones obtained extended into 28 S rRNA sequences. A 7-kb region of non-transcribed spacer DNA shared in common between five independent clones was subjected to comparative restriction digests. It was estimated that sequences among the five different spacer isolated varied by not more than 1.0%, if all the observed differences are assumed due to point mutation. HaeII-restriction fragments from within this same 7-kb region contain sequences carried not only within the tandem repeats of the gene cluster but interspersed elsewhere in the genome. Some of these sequences correspond to the Alu family of highly repeated interspersed sequences.

mRNA species regulated during the differentiation of HL-60 cells to macrophages and neutrophils☆

Using cDNA clone banks from differentiated and undifferentiated HL-60 promyelocytic leukemia cells, we have selected clones for genes which are regulated during this differentiation. Regulation of the corresponding mRNAs in HL-60 cells during both monocytic and neutrophilic differentiation was measured for 21 of these clones. The levels of mRNA hybridizing to some of these clones changed by more than 100-fold during differentiation. Unlike erythropoiesis or myogenesis, in which the synthesis of a few new proteins is synchronously regulated, mRNAs in differentiating HL-60 cells are asynchronously regulated, suggesting a complex series of regulatory events. About half of these regulation-selected clones contained repeat sequences, including both Alu and novel repeat families. Most of the regulated genes are members of extensive gene families.

Secondary structures formed by random RNA sequences

Summary Three techniques (optical melting curves, sedimentation in the presence and absence of dimethylsulfoxide, and resistance to RNase Tl) are used to investigate different aspects of the structure of random RNA sequences. The hypochromicity of melting curves indicate that these sequences have about 60% base-pairing, but the low T m and large σ T suggest that the base-pairing is much less stable than that observed in rRNA. Both the small hydrodynamic radius revealed in sedimentation studies and the relatively low resistance to RNase are interpreted as confirming the presence of base-pairing but base-pairing which is disorderly .

DNA polymorphism in the human Thy-1 gene

Thy-1 is a membrane glycoprotein expressed predominantly in brain tissue and occasionally in lymphoid tissue. The human Thy-1 gene is located on chromosome 11q22.3. Although two allelic forms of Thy-1 exist in mice (Thy-1.1 and Thy-1.2), no allelic forms have been described for the human Thy-1 gene. We describe a polymorphic MspI site within the human Thy-1 gene that distinguishes two alleles, 8 and 9, which are represented in a northern European population at frequencies of 0.7 and 0.3, respectively. Thy-1, therefore, provides a potentially useful marker to identify linkages with human disease genes located near 11q22.

Mutation rates in globin genes: the genetic load and Haldane's dilemma.

Publisher Summary This chapter discusses the mutation rates for mammalian genomes that have often been computed from rates of amino-acid substitution. The fact that rates differing by manyfold are obtained for different proteins emphasizes that in some proteins, such as the histones, most amino-acid substitutions are sufficiently deleterious to be eliminated during the course of evolution. It reviews that by measuring acceptance rates of silent mutations through direct nucleotide sequence comparisons between species, it can remove one of the most important limitations of previous estimates of the mutation rate, which have been based on amino-acid substitution data. The chapter also discusses that Haldane computed the number of genetic deaths required for a gene substitution to occur according to strict Darwinian principles. With reasonable assumptions about permissive levels of mortality associated with Darwinian evolution, he estimated that there could be about one gene substitution every 300 generations. To account for the observed maintenance of mammalian genomes in the face of Haldanes dilemma, it proposes two solutions—that is, by Truncation selection and Neutral mutations. Truncation selection provides a useful way of explaining accurate maintenance of the repetitive gene clusters and satellite DNAs, but it is difficult to imagine that truncation selection could effectively maintain the accuracy of the single-copy DNA coding for most proteins. It is suggested that only a small fraction of the genome, perhaps less than 2%, is kept accurate as single-copy DNA. Since most mammalian genomes contain large amounts of single-copy DNA, the implication is that most such sequences must be genetically drifting.