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The inability of current recommendations to control the epidemic of diabetes, the specific failure of the prevailing low-fat diets to improve obesity, cardiovascular risk, or general health and the persistent reports of some serious side effects of commonly prescribed diabetic medications, in combination with the continued success of low-carbohydrate diets in the treatment of diabetes and metabolic syndrome without significant side effects, point to the need for a reappraisal of dietary guidelines. The benefits of carbohydrate restriction in diabetes are immediate and well documented. Concerns about the efficacy and safety are long term and conjectural rather than data driven. Dietary carbohydrate restriction reliably reduces high blood glucose, does not require weight loss (although is still best for weight loss), and leads to the reduction or elimination of medication. It has never shown side effects comparable with those seen in many drugs. Here we present 12 points of evidence supporting the use of low-carbohydrate diets as the first approach to treating type 2 diabetes and as the most effective adjunct to pharmacology in type 1. They represent the best-documented, least controversial results. The insistence on long-term randomized controlled trials as the only kind of data that will be accepted is without precedent in science. The seriousness of diabetes requires that we evaluate all of the evidence that is available. The 12 points are sufficiently compelling that we feel that the burden of proof rests with those who are opposed.

Despite extensive study, there is little experimental information available as to which of the deoxyribose hydrogen atoms of duplex DNA reacts most with the hydroxyl radical. To investigate this question, we prepared a set of double-stranded DNA molecules in which deuterium had been incorporated specifically at each position in the deoxyribose of one of the four nucleotides. We then measured deuterium kinetic isotope effects on the rate of cleavage of DNA by the hydroxyl radical. These experiments demonstrate that the hydroxyl radical reacts with the various hydrogen atoms of the deoxyribose in the order 5 H > 4 H > 3 H Ϸ 2 H Ϸ 1 H. This order of reactivity parallels the exposure to solvent of the deoxyribose hydrogens. Our work therefore reveals the structural basis of the reaction of the hydroxyl radical with DNA. These results also provide information on the mechanism of DNA damage caused by ionizing radiation as well as atomic-level detail for the interpretation of hydroxyl radical footprints of DNA-protein complexes and chemical probe experiments on the structure of RNA and DNA in solution.The hydroxyl radical (⅐OH), the quintessential reactive oxygen species, is the mediator of much of the DNA damage caused by ionizing radiation (1). This damage includes strand breaks, which are initiated by abstraction of a deoxyribose hydrogen atom by the hydroxyl radical. DNA strand breaks induced by the hydroxyl radical also form the basis of a widely used method for making footprints of DNA-protein complexes (2, 3) and for studying the structure of DNA (4) and RNA (5) in solution. The key experimental advantage of the hydroxyl radical as a chemical probe is that it effects DNA cleavage with no base-or sequence-specificity (6-8). The hydroxyl radical produces highly detailed footprints that yield information about DNA structure (4, 7) and protein-DNA interactions (3, 8, 9) at single-nucleotide resolution.Mechanistic information on the reaction of the hydroxyl radical with nucleic acids will benefit our understanding of radiation damage to DNA as well as the interpretation of chemical probe experiments. The extensive literature on the radiation chemistry of DNA (1) is a rich source of mechanistic possibilities. Not surprisingly, because of the high reactivity of the hydroxyl radical, a wide spectrum of products has been detected on treatment of the constituents of DNA (nucleic bases, nucleosides, nucleotides, or simple-sequence singlestranded DNA, for example) with ionizing radiation (1). It has been more difficult to conduct similarly detailed experiments on the biologically relevant duplex form of DNA. It is not hard to conceive, though, that the hydroxyl radical might react in a different manner with double-stranded DNA compared with simpler nucleic acid systems because the shape of the double helix would strongly influence the accessibility of the various COH bonds in DNA.Until now, the extent of cleavage at a particular nucleotide in a hydroxyl radical footprinting experiment only could be interpreted at th...

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A computer program, GelExplorer, which uses a new methodology for obtaining quantitative information about electrophoresis has been developed. It provides a straightforward, easy-to-use graphical interface, and includes a number of features which offer significant advantages over existing methods for quantitative gel analysis. The method uses curve fitting with a nonlinear least-squares optimization to deconvolute overlapping bands. Unlike most curve fitting approaches, the data is treated in two dimensions, fitting all the data across the entire width of the lane. This allows for accurate determination of the intensities of individual, overlapping bands, and in particular allows imperfectly shaped bands to be accurately modeled. Experiments described in this paper demonstrate empirically that the Lorentzian lineshape reproduces the contours of an individual gel band and provides a better model than the Gaussian function for curve fitting of electrophoresis bands. Results from several fitting applications are presented and a discussion of the sources and magnitudes of uncertainties in the results is included. Finally, the method is applied to the quantitative analysis of a hydroxyl radical footprint titration experiment to obtain the free energy of binding of the lambda repressor protein to the OR1 operator DNA sequence.

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Xenomitochondrial mice harboring trans-species mitochondria on a Mus musculus domesticus (MD) nuclear background were produced. We created xenomitochondrial ES cell cybrids by fusing Mus spretus (MS), Mus caroli (MC), Mus dunni (Mdu), or Mus pahari (MP) mitochondrial donor cytoplasts and rhodamine 6-G treated CC9.3.1 or PC4 ES cells. The selected donor backgrounds reflected increasing evolutionary divergence from MD mice and the resultant mitochondrial-nuclear mismatch targeted a graded respiratory chain defect. Homoplasmic (MS, MC, Mdu, and MP) and heteroplasmic (MC) cell lines were injected into MD ova, and liveborn chimeric mice were obtained (MS/MD 18 of 87, MC/MD 6 of 46, Mdu/MD 31 of 140, and MP/MD l of 9 founder chimeras, respectively). Seven MS/MD, 1 MC/MD, and 11 Mdu/MD chimeric founder females were mated with wild-type MD males, and 18 of 19 (95%) were fertile. Of fertile females, only one chimeric MS/MD (1% coat color chimerism) and four chimeric Mdu/MD females (80-90% coat color chimerism) produced homoplasmic offspring with low efficiency (7 of 135; 5%). Four male and three female offspring were homoplasmic for the introduced mitochondrial backgrounds. Three male and one female offspring proved viable. Generation of mouse lines using additional female ES cell lineages is underway. We hypothesize that these mice, when crossbred with neurodegenerative-disease mouse models, will show accelerated age-related neuronal loss, because of their suboptimal capacity for oxidative phosphorylation and putatively increased oxidative stress.

One of the challenges in teaching biochemistry is facilitating students' interest in and mastery of metabolism. The many pathways and modes of regulation can be overwhelming for students to learn and difficult for professors to teach in an engaging manner. We have found it useful to take advantage of prevailing interest in popular yet controversial weight-loss methods, particularly low-carbohydrate diets. The metabolic rationale behind these eating plans can be linked to glycolysis, the citric acid cycle, lipolysis, gluconeogenesis, ketosis, glycogen metabolism, fatty acid oxidation, and hormonal regulation. When this approach was used in undergraduate biochemistry classes at the State University of New York at Geneseo, students were highly motivated to learn the biochemical principles behind these diets. The following provides information about low-carbohydrate diet plans that will enable professors to speak authoritatively on the subject. History and studies regarding efficacy as well as biochemical metabolic effects are included.