Nutrition programs enhance exercise effects on body composition and resting blood pressure.

The purpose of our study was to examine the effects of exercise alone and exercise combined with specific nutrition programs on body composition and resting blood pressure rate. Adult participants (99 women, 22 men; aged 20-86 years) completed a combined strength and endurance exercise program (Exercise Only), or in conjunction with 1 of 2 nutrition plans (Exercise/Protein; Exercise/Protein/Diet). The Exercise-Only group performed 1 set of 9 resistance machines regimens interspersed with 3 bouts of recumbent cycling (5 minutes each). The Exercise/Protein group performed the same exercise program as Exercise-Only group, plus consumed 1.5 g of protein per kg of ideal body weight on a daily basis. The Exercise/Protein/Diet group followed an identical Exercise/Protein protocol along with a restricted daily caloric intake (1200-1500 cals/day for women; 1500-1800 cals/day for men). After 10 weeks of training, the Exercise/Protein group attained greater increases (P < 0.05) in lean weight and greater decreases (P < 0.05) in diastolic blood pressure (DBP) rate than the Exercise-Only group. The Exercise/Protein/Diet group experienced greater reductions (P < 0.05) in body weight, body mass index (BMI), percent fat, fat weight, waist circumference (WC), systolic blood pressure (SBP) rate, and DBP rate than the Exercise-Only group, as well as greater reductions (P < 0.05) in body weight, BMI, percent fat, fat weight, and WC than the Exercise/Protein group. Our findings suggest that a higher protein nutrition plan may enhance the effects of exercise for increasing subject lean weight and decreasing DBP rate. The findings further indicate that a higher protein and lower calorie nutrition plan may enhance the effects of exercise for decreasing subject body weight, BMI, percent fat, fat weight, WC, SBP rate, and DBP rate, while attaining similar gains in lean body mass.

Biochemical and mutagenic analysis of I-CreII reveals distinct but important roles for both the H-N-H and GIY-YIG motifs.

Homing endonucleases typically contain one of four conserved catalytic motifs, and other elements that confer tight DNA binding. I-CreII, which catalyzes homing of the Cr.psbA4 intron, is unusual in containing two potential catalytic motifs, H-N-H and GIY-YIG. Previously, we showed that cleavage by I-CreII leaves ends (2-nt 3' overhangs) that are characteristic of GIY-YIG endonucleases, yet it has a relaxed metal requirement like H-N-H enzymes. Here we show that I-CreII can bind DNA without an added metal ion, and that it binds as a monomer, akin to GIY-YIG enzymes. Moreover, cleavage of supercoiled DNA, and estimates of strand-specific cleavage rates, suggest that I-CreII uses a sequential cleavage mechanism. Alanine substitution of a number of residues in the GIY-YIG motif, however, did not block cleavage activity, although DNA binding was substantially reduced in several variants. Substitution of conserved histidines in the H-N-H motif resulted in variants that did not promote DNA cleavage, but retained high-affinity DNA binding-thus identifying it as the catalytic motif. Unlike the non-specific H-N-H colicins, however; substitution of the conserved asparagine substantially reduced DNA binding (though not the ability to promote cleavage). These results indicate that, in I-CreII, two catalytic motifs have evolved to play important roles in specific DNA binding. The data also indicate that only the H-N-H motif has retained catalytic ability.

Expression, purification, and biochemical characterization of the intron-encoded endonuclease, I-CreII.

The ORF of the Cr.psbA4 intron of Chlamydomonas reinhardtii mediates efficient intron homing, and contains an H-N-H and possibly a GIY-YIG motif. The ORF was over-expressed in Escherichia coli without non-native amino acids, but was mostly insoluble. However, co-over-expression of E. coli chaperonins GroEL/GroES solubilized approximately 50% of the protein, which was purified by ion-exchange and heparin-affinity chromatography. Biochemical characterization showed that the protein is a double-strand-specific endonuclease that cleaves fused psbA exon 4-exon 5 DNA, and was named I-CreII. I-CreII has a relatively relaxed divalent metal ion requirement (Mg(2+), Mn(2+), Ca(2+), and Fe(2+) supported cleavage), is insensitive to salt <350 mM, and is stabilized by DNA. Cleavage of target DNA occurs close (4 nt on the top strand) to the intron-insertion site, and leaves 2-nt 3'-OH overhangs, similar to GIY-YIG endonucleases. The boundaries of the recognition sequence span approximately 30 bp, and encompass the cleavage and intron-insertion sites. Cleavage of heterologous psbA DNAs indicates the enzyme can tolerate multiple, but not all, substitutions in the recognition site. This work will facilitate further study of this novel endonuclease, which may also find use in site-specific manipulation of chloroplast DNA.