Lifestyle modification interventions differing in intensity and dietary stringency improve insulin resistance through changes in lipoprotein profiles.

OBJECTIVE: Metabolic dysfunction characterized by insulin resistance (IR) is an important risk factor for type-2 diabetes and coronary artery disease (CAD). The aim of this study was to determine if clinical lifestyle interventions differing in scope and intensity improve IR, defined by the lipoprotein IR (LPIR) score, in individuals differing in the severity of metabolic dysfunction. METHODS: Subjects with diagnosed type-2 diabetes, CAD or significant risk factors participated in one of two clinical lifestyle modification interventions: (i) intensive non-randomized programme with a strict vegetarian diet (n = 90 participants, 90 matched controls) or (ii) moderate randomized trial following a Mediterranean-style diet (n = 89 subjects, 58 controls). On-treatment and intention-to-treat analyses assessed changes over 1 year in LPIR, lipoprotein profiles and metabolic risk factors in intervention participants and controls in both programmes. RESULTS: In the on-treatment analysis, both interventions led to weight loss: [-8.9% (95% CI, -10.3 to -7.4), intensive programme; -2.8% (95% CI, -3.8 to -1.9), moderate programme; adjusted P < 0.001] and a decrease in the LPIR score [-13.3% (95% CI, -18.2 to -8.3), intensive; -8.8% (95% CI, -12.9 to -4.7), moderate; adjusted P < 0.01] compared with respective controls. Of the six lipoprotein parameters comprising LPIR, only large very-low-density lipoprotein particle concentrations decreased significantly in participants compared with controls in both programmes [-26.3% (95% CI, -43.0 to -9.6), intensive; -14.2% (95% CI, -27.4 to -1.0), moderate; P < 0.05]. Intention-to-treat analysis confirmed and strengthened the primary results. CONCLUSION: A stringent lifestyle modification intervention with a vegetarian diet and a moderate lifestyle modification intervention following a Mediterranean diet were both effective for improving IR defined by the LPIR score.

Sequential assignments and secondary structure of the RNA-binding transcriptional regulator NusB.

The NusB protein is involved in transcriptional regulation in bacteriophage lambda. NusB binds to the RNA form of the nut site and along with N, NusA, NusE and NusG, stabilizes the RNA polymerase transcription complex and allows stable, persistent antitermination. NusB contains a 10 residue Arg-rich RNA-binding motif (ARM) at the N-terminus but is not sequentially homologous to any other proteins. In contrast to other known ARM-containing proteins, NusB forms a stable structure in solution in the absence of RNA. NMR spectroscopy was used to determine that NusB contains six alpha-helices: R10-Q21, 127-F34, V45-L65, Q79-S93, Y100-F114 and D118-L127. The structure of NusB makes it a member of a newly emerging class of alpha-helical RNA-binding proteins.

The alpha subunit of RNA polymerase and transcription antitermination.

The N gene product of coliphage gamma, with a number of host proteins (Nus factors), regulates phage gene expression by modifying RNA polymerase to a form that overrides transcription-termination signals. Mutations in host nus genes diminish this N-mediated antitermination. Here, we report the isolation and characterization of the rpoAD305E mutation, a single amino acid change in the carboxy terminal domain (CTD) of the alpha subunit of RNA polymerase, that enhances N-mediated antitermination. A deletion of the 3' terminus of rpoA, resulting in the expression of an alpha subunit missing the CTD, also enhances N-mediated antitermination and, similar to rpoAD305E, suppresses the effect of nus mutations. Thus, the N-Nus complex may be affected through contacts with the CTD of the alpha subunit of RNA polymerase, as is a group of regulatory proteins that influences initiation of transcription. What distinguishes our findings on the N-Nus complex from those of previous studies with transcription proteins is that all of the regulators characterized in those studies bind DNA and influence transcription initiation; whereas the N-Nus complex binds RNA and affects transcription elongation. A screen of some previously identified rpoA mutations that influence transcription activators revealed only one other amino acid change, L290H, in the CTD of the alpha subunit, that influences antitermination. Although our results provide evidence that interactions of the alpha subunit of RNA polymerase must be considered in forming models of transcription antitermination, they do not provide information as to whether the interactions of alpha that ultimately influence antitermination occur during initiation or during elongation of transcription.

Structural and functional analyses of the transcription-translation proteins NusB and NusE.

The NusB and NusE (ribosomal protein S10) proteins function in transcription and translation. The two proteins form a complex that binds to the boxA sequence found in the leader RNA of rrn operons; boxA is required for transcription antitermination in rrn operons. Although binding of these two proteins to the boxA RNA of the bacteriophage lambda nut site has not been observed, both NusB and NusE as well as the RNA boxA sequence are required for lambda N-mediated antitermination. Studies identifying the amino acid changes caused by mutations in nusB and nusE and relating these changes to altered function are reported. It is concluded that boxA is essential for an effective NusB contribution to N-mediated antitermination and that by mutation NusB may be changed to allow more-effective binding to boxA variants.

Improved bacterial hosts for regulated expression of genes from lambda pL plasmid vectors.

The construction and use of a set of Escherichia coli strains with defective lambda prophages that facilitate expression of genes cloned in lambda pL-plasmid vectors is described. These bacteria allow high and regulated expression of such genes, whereas a kanamycin-resistance marker (KmR) on the prophage allows easy identification and genetic transfer from strain to strain. Optimal conditions for examining gene expression with the pL-vector systems using these strains are discussed.

Analysis of mutations in the ninR region of bacteriophage lambda that bypass a requirement for lambda N antitermination.

Two mutations in the ninR region of bacteriophage lambda that bypass a requirement for antitermination have been studied. One mutation, byp, has been cloned and mapped by marker rescue to a 417-base-pair segment in the ninR region of the genome. Analysis of the byp mutation by using promoter detection vectors, DNA sequencing, and S1 nuclease analysis showed that the byp mutation created a new promoter that transcribed gene Q. The second mutation analyzed was the deletion nin3. Sequence analysis revealed that 2,485 base pairs of the ninR region were removed, beginning within the ren gene and ending in an open reading frame termed ninG. The tR2 and tR3 terminators, and probably others, were removed by the nin3 deletion, thereby allowing the phage to be N independent and to grow in hosts defective for Nus antitermination factors.

Strain Improvement of Rhodotorula graminis for Production of a Novel l-Phenylalanine Ammonia-Lyase.

l-Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) from Rhodotorula rubra has been used in the commercial manufacture of l-phenylalanine from trans-cinnamic acid. In this study, R. graminis PAL was investigated. Mutant strain GX6000 was isolated after ethyl methanesulfonate mutagenesis of wild-type R. graminis GX5007 by selecting for resistance to phenylpropiolic acid, an analog of trans-cinnamic acid. Mutant strain GX6000 produced inducible PAL at levels four- to fivefold higher than had wild-type R. graminis. Furthermore, this strain had several other physiological traits that make it more commercially useful than R. rubra. For example, during fermentation, the PAL half-life was three- to fivefold longer, PAL specific activity was six to seven times higher, and PAL synthesis was significantly less inhibited by temperatures above 30 degrees C. Induction of PAL in strain GX6000 appeared to be less tightly regulated; l-leucine acted synergistically with l-phenylalanine, the physiological inducer, to increase the PAL specific activity and titer to 165 U/g (dry weight) and 3,000 U/liter, respectively, a 40% increase over the effect of l-phenylalanine alone. Strain GX6000 PAL showed significantly greater stability in bioreactors for the synthesis of l-phenylalanine, a finding that is consistent with the stability properties observed during fermentation.

[Dynamic electrocardiographic evaluation of the circadian rhythms of the ectopic ventricular beats in healthy and cardiopathic subjects]. / Valutazione mediante elettrocardiografia dinamica dei ritmi circadiani dei battiti ectopici ventricolari in soggetti sani e cardiopatici.

The 24 hour study was carried out on 34 subjects, 17 cardiopathy 13 male and 4 female, mean age 56 +/- 14) and 17 normal (10 male and 7 female, mean age 41 +/- 18) with, on average, over 30 PVC/h in the dynamic ECG: no statistically significant difference in mean PVC/h was recorded between the two groups. Single cosinor analysis demonstrated a statistically significant circadian rhythm (p less than 0.05) in 11 cardiopathic (64.7%) and 13 normal subjects (76.4%). Population mean cosinor failed to demonstrate a significant rhythm in either of the two groups. Results suggest that personal circadian rhythms should be studied to identify the most appropriate antiarrhythmic treatment.

Chromatographic and fluorescence spectroscopic studies of individual 7,12-dimethylbenz[a]anthracene--deoxyribonucleoside adducts.

Compared with standard Sephadex LH-20 column chromatography, a newly developed high pressure liquid chromatographic separation of hydrocarbon deoxyribonucleoside adducts derived from the DNA of mouse embryo cell cultures exposed to 7,12-dimethylbenz[a]anthracene (DMBA) provides markedly superior resolution. Once resolved, the fluorescence spectroscopic properties of the three major DMBA--DNA adducts indicate that the fluorescence exhibited by adducts derived from a bay region syn dihydrodiol epoxide of DMBA differs subtly from that exhibited by adducts derived from the isomeric anti dihydrodiol epoxide.

Evidence that binding of 7,12-dimethylbenz(a)anthracene to DNA in mouse embryo cell cultures results in extensive substitution of both adenine and guanine residues.

Primary mouse embryo cell cultures were grown in the presence of [14C]guanine, labeling primarily deoxyguanosine residues in the cellular DNA, or in the presence of [14C]adenine, labeling both deoxyguanosine and deoxyadenosine residues in the cellular DNA. These cultures were subsequently exposed to 7,12-[3H]dimethylbenz(a)anthracene for 24 hr. The DNA was isolated and hydrolyzed to deoxyribonucleosides, and the 7,12-dimethylbenz(a)anthracene:deoxyribonucleoside adducts were separated chromatographically allowing the three major adducts found to be identified as bay-region anti-dihydrodiol-epoxide:deoxyguanosine and :deoxyadenosine adducts and a bay-region syn-dihydrodiol-epoxide:deoxyadenosine adduct. Therefore, in contrast to what is known for benzo(a)pyrene, substantial amounts of deoxyadenosine adducts are formed with the more potent carcinogen, 7,12-dimethylbenz(a)anthracene.