Search

We are studying novel tocotrienols, which have a number of activities that might interfere with the formation of atherosclerotic plaques, including hypocholesterolemic, antioxidant, anti-inflammatory and antiproliferation effects. This study compared the effects of alpha-tocopherol, the tocotrienol-rich fraction (TRF(25)) and didesmethyl tocotrienol (d-P(25)-T3) of rice bran on the pathogenesis of atherosclerotic lesions in C57BL/6 apolipoprotein (apo)E-deficient (-/-) mice. These mice are an excellent model because they become hyperlipidemic even when they consume a low fat diet and they develop complex atherosclerotic lesions similar to those of humans. These compounds were also tested in wild-type C57BL/6 apoE (+/+) and (+/-) mice fed low or high fat diets. When a high fat diet was supplemented with alpha-tocopherol, TRF(25) or d-P(25)-T3 and fed to mice (+/+) for 24 wk, atherosclerotic lesion size was reduced 23% (P = 0.33), 36% (P = 0.14) and 57% (P < 0.02), respectively, and in mice (+/-) fed for 18 wk, lesions were reduced by 19% (P = 0.15), 28% (P < 0.01) and 33% (P < 0.005), respectively, compared with mice fed a control diet. A low fat diet did not cause atherosclerotic lesions in these mice. The low fat diet supplemented with TRF(25) or d-P(25)-T3 fed to apoE-deficient (-/-) mice for 14 wk decreased atherosclerotic lesion size by 42% (P < 0.04) and 47% (P < 0.01), respectively, whereas alpha-tocopherol supplementation resulted in only an 11% (P = 0.62) reduction. These results demonstrate the superior efficacy of tocotrienols compared with alpha-tocopherol. Although tocotrienols decreased serum triglycerides, total and LDL cholesterol levels, the decreases in atherosclerotic lesions seem to be due to the other activities. Serum tocol concentrations in various groups are also described. This is the first report of a significant reduction in the atherosclerotic lesion size in all three genotypes of apoE mice fed a novel tocotrienol (d-P(25)-T3) of rice bran. Dietary tocotrienol supplements may provide a unique approach to promoting cardiovascular health.

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.

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

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

(1975). Recent Advances in DNA Sequence Analysi. CRC Critical Reviews in Biochemistry: Vol. 2, No. 4, pp. 455-512.

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.

Measurements of the sedimentation velocity under completely denaturing conditions in dimethylsulfoxide were used to obtain the molecular weights of some T4-specific mRNAs including the biologically active lysozyme mRNA. The amount of folding was determined by comparing their sedimentation in sucrose gradients in the presence and absence of this reagent. T4-specific mRNAs sediment much more slowly in the presence of dimethylsulfoxide than in its absence. This difference is linearly dependent on molecular weight; the dependence was similar to that observed for Escherichia coli stable RNA controls and very different from that of random coils (E. coli stable RNA treated with formaldehyde so that no base-pairing is possible). We conclude that the T4-specific mRNAs are not random coils but resemble much more closely the highly base-paired E. coli stable RNAs.

Rabbit alpha globin gene copies have been made, using reverse transcriptase and DNA polymerase I, and cloned in bacterial plasmids. Plasmid pHb72 has been shown to contain the alpha gene sequence by restriction enzyme analysis and nucleotide sequencing studies, and therefore has been approved for propagation under P2 plus EK1 conditions by the National REcombinant DNA Committee.

DNA synthesized in vitro from a phage M13 template has been cleaved at either guanine, adenine, or cytosine residues by ribosubstitution techniques. Fingerprints of the fragments obtained suggest that DNA sequencing will be possible with this technique.

A 169-nucleotide region from the rabbit beta globin gene has been sequenced by analysis of complementary DNA's cloned in bacterial plasmids. Comparison of these sequences with those established for this gene by other techniques provides evidence of a high degree of fidelity and allows the unambiguous establishment that these plasmids do not contain harmful sequences.

Complementary DNA transcribed in vitro from purified rabbit globin messenger RNA and made double-stranded has been inserted into E. coli plasmid pSC 101 by the poly-dT/poly-dA “tailing” and annealing technique. E. coli transformants given by this DNA preparation have been shown to contain globin sequences by the hybridization of globin RNA to DNA from clones grown and lysed in situ on nitrocellulose filters. Some measure of the amount and identity of inserted globin sequences has been determined by fingerprinting fragments of 32p-labeled globin RNA hybridized to the purified plasmid chimeras.

A mouse MOPC21 cDNA previously cloned in plasmid pMB9(Higuchi et al. , Proc. Natl. Acad. Sci. 73 (1976) 2136–2140; Wall et al. , Nucleic Acid Res. 5 (1978) 3113–3128) and is designated pL21-3 has been extensively characterized. Cleavage of pL21-3 with Hpall has shown the insert to be 910 basepairs long, consistent with the length of the entire variable and constant regions and the untranslated regions. Digestion of pL21-3 with various restriction endonucleases has established that the insert sequence starts from parts of the 5′ leader region and extends downstream to include the untranslated 3′ terminus. 131 nucleotides in the variable region corresponding to amino acids 49–91 have been determined.

Dna from human sperm cells can be isolated with size and purity sufficient for use in recombinant DNA research. The DNA averages 100 000 base pairs (bp) in size (about 70.10(6) daltons) and is free of somatic cell, bacterial, and viral DNA. It can therefore be cloned under P2 + EK2 conditions as stipulated in the 1978 NIH Guidelines for recombinant DNA research.

Rabbit globin complementary DNA made with RNA-dependent DNA polymerase (reverse transcriptase) was used as template for in vitro synthesis of 32 P-labeled RNA. The sequences of the nucleotides in most of the fragments resulting from combined ribonuclease T 1 and alkaline phosphatase digestion have been determined. Several fragments were long enough to fit uniquely with the α or β globin amino-acid sequences. These data demonstrate that the cDNA was copied from globin mRNA and contained no detectable contaminants.

The existence of non-random base pairing in T4-specific mRNA is supported by the nuclease digestion patterns of the messenger for phage T4 lysozyme. Under conditions which denature RNA, the nuclease nicked lysozyme mRNA recovers its activity. This behaviour can be explained by a model in which secondary structure controls initiation at existing ribosomal attachment sites.

Complementary DNA, transcribed in vitro from purified rabbit globin messenger RNA and made double-stranded, has been inserted into Escherichia coli plasmids pSC101 and pMB9 by the poly(dT)/poly(dA) "tailing" and annealing technique. E. coli transformants given by this DNA preparation have been shown to contain globin sequences by the hybridization of globin RNA to DNA from clones grown and lysed in situ on nitrocellulose filters. An estimate of the amount of inserted globin sequences has been provided by fingerprint analysis of globin mRNA sequences hybridized to the purified plasmid chimeras. Inserted sequences so far subjected to detailed analysis have been ascribed to the rabbit beta globin chain. The susceptibility of inserted beta globin, sequences to the restriction endonuclease EcoRI confirms the existence of a site already found through previous nucleotide sequence analysis.

Previous studies have shown that 60–70% of the nucleotides in rRNA, tRNA and the RNA of the RNA bacteriophages are involved in base pairing. To determine whether this is also true of mRNAs coded from double-stranded DNA, T4-specific mRNAs were purified using two different techniques: hybridization to T4 DNA or chromatography on benzoylated DEAE-cellulose columns followed by sucrose gradients. We have determined the optical melting curves of the purified mRNAs. The hypochromicities calculated from these melting curves were similar to those of rRNA, indicating comparable amounts of base pairing in rRNA and T4-specific mRNA. If this hypochromicity were due to random base pairing, we would have expected a less sharp melting curve and a lower T m than for the rRNA controls. On the contrary, the mRNA samples had very high cooperativities and T m values.

Deoxynucleotides have been incorporated into RNA synthesized in vitro by RNA polymerase with either double-stranded or single-stranded DNA as a template. By use of this technique to block or promote cleavage at a particular phosphodiester bond, a variety of specific cleavages may be obtained with the available ribonucleases and deoxyribonuclease I. These methods should greatly increase the ease and rapidity of nucleotide sequence determinations.

DNA containing the herpes simplex virus thymidine kinase (HSVtk) gene was used to transform wild-type tk+ mouse L cells to a tk++ status in vitro using methotrexate as a selective agent. HSVtk DNA was also used to transform mouse bone marrow cells in vitro. Transformed marrow cells injected into irradiated and methotrexate-treated recipient mice gave rise to proliferating cells which in some cases dominated the marrow population and which contained HSVtk gene sequences.

Publisher Summary This chapter reviews that cDNA serves as the template for synthesis of a variety of products used in various phases. The synthesis products include 32 P-labeled RNA for sequence analysis of fragments resulting from a cleavage at G residues, 32 P-labeled dC- or dT-substituted RNAs for specific cleavage at U or at C residues, and duplex gene copies for insertion into bacterial plasmids. It reviews that molecular cloning approach is applied to a wide variety of eukaryotic mRNAs. One of the advantages of the technique that is developed for the insertion of eukaryotic mRNA copies into bacterial plasmids is that a rigorous purification is not necessary; the act of cloning such material definitively eliminates all contaminating eukaryotic nucleic acid sequences. This should be especially useful to the researcher who, for instance, has a mixture of 10 different mRNA species that are difficult to separate. To obtain the corresponding sequences in pure form, it suffices to make cDNA copies of the mixture of mRNAs, convert this mixture of cDNAs to duplex gene copies, insert the DNA into bacterial plasmids and introduce the mixture of plasmids thus obtained into bacteria. Since each bacterial transformant can receive only one of the eukaryotic gene inserts, analysis of a few dozen bacterial clones should suffice to provide one with a clone for each of the interesting sequences. With such clones, it is possible to produce milligram quantities of these sequences in duplex form, sufficient for the most rapid sequencing techniques.

Publisher Summary Recent developments indicate that in vitro synthesis of ribosubstituted DNA may allow the relatively rapid determination of DNA sequences of small to moderate complexity. In vitro synthesis provides an advantage as one can synthesize the same ribosubstituted DNA with the 32 P labeling a different one of the four bases each time, so that nearest-neighbor labeling information will greatly simplify the subsequent analysis. The ready availability of small molecules such as the tRNAs and 5 S ribosomal RNA was extremely important in permitting the rapid development of RNA sequencing methods. In order to obtain very simple sequences for working out DNA sequencing techniques we have turned to the highly iterated satellite DNA fractions of higher organisms. This chapter describes the methods for synthesis and cleavage of ribosubstituted DNA and discusses the analysis of the sequences of individual fragments and the use of micrococcal nuclease to determine the sequence of larger fragments.

The sequence of the highly repetitive satellite HS-β DNA fraction from kangaroo rat Dipodomys ordii was determined independently by RNA and DNA sequencing techniques. A basic iterated sequence of 10 nucleotides with several mutational variations was found. Base-composition data are consistent with the proposed sequence and revealed a high content of 5-methylcytosine. DNA and RNA sequencing techniques used gave identical results, showing that the fidelity of synthesis of riboguanidine-substituted DNA under our conditions is adequate for nucleotide sequence studies.

PROTEIN synthesis requires initiation of translation at the correct site on mRNA. Two initiation factors, A and C (ref. 1), or F1 and F2 (ref. 2), promote the ApUpG-dependent attachment of formylmethionyl-tRNA (fmet-tRNAf) to Escherichia coli ribosomes2–4, and account for initiation AUG triplets5,6. A mechanism must exist, however, for the discrimination between internal and initial methionine codons. In order to study the possible role of the third initiation factor, B, in this selection mechanism, we have examined the role of the three factors in the synthesis of lysozyme promoted by T4 mRNA7. We assumed that polypeptide synthesis as measured by amino-acid incorporation should not necessarily require correct initiation, while active enzyme formation should be highly dependent on this process. We have used the ratio of enzyme formed to valine incorporated as a measure of the proportion of polypeptide molecules correctly initiated.

A new technique for measuring the rate of turnover and quantity of unstable RNA in exponentially growing cells has been developed which escapes the interpretive difficulties of earlier methods. It depends upon the fact that an unstable RNA fraction has easily measurable effects upon the kinetics of labelling of GTP; before the GTP pool can become fully labelled, unlabelled nucleotides present as GTP or in unstable RNA must be washed into stable RNA. (There is no equilibration with guanine in the medium.) Consequently, if the uptake of label into GTP is rapid, with only the delay predicted from the experimentally measured GTP pool size, then it is possible to conclude that there is little or no unstable RNA. In Bacillus subtilis, however, the GTP pool is labelled very slowly which, with other data, indicates a large amount of unstable RNA (9% of the guanine residues of the cell in unstable RNA). Similar measurements of the rapidity of GTP labelling in Escherichia coli indicate that there is much less unstable RNA (about 3%) in this organism. The curve expressing GTP specific activity as a function of time is shown to be the sum of two exponential terms with very different time constants. From the relative sizes of the two exponential terms, it is possible to estimate the rate of turnover of the unstable RNA. The result is weighted most heavily for the slow components of the decaying messenger, and thus it sets an upper limit to the true average decay time for the unstable RNA. This decay time (upper limit) was three minutes for the unstable RNA in B. subtilis grown at 37 °C with a doubling time of 115 minutes and six minutes when the cells were grown at 30 °C with a doubling time of 225 minutes. For E. coli (growing at 30 °C with a doubling time of 66 minutes) the decay time measured in this way was about four minutes. Thus the decay times as measured in these experiments are the same order of magnitude as have been observed by measuring the decay of pulse-labelled RNA and the decay of protein synthetic capacity after the addition of actinomycin. The various estimates of the decay rate of messenger RNA in bacteria are discussed in terms of the possible theoretical ambiguities in the interpretation of each.