Electrostatic interactions between charged amino acid residues and the bacteriochlorophyll dimer in reaction centers from Rhodobacter sphaeroides.

The extent of electrostatic contributions from the protein environment was assessed by the introduction of ionizable residues near the bacteriochlorophyll dimer in reaction centers from Rhodobacter sphaeroides. Two mutations at symmetry-related sites, M199 Asn to Asp and L170 Asn to Asp, resulted in a 48 and 44 mV lowering of the midpoint potential, respectively, compared to the wild type at pH 8, while a 75 mV decrease in the midpoint potential was observed for the mutation L168 His to Glu. The decrease relative to wild type was found to be approximately additive, up to 147 mV, for various combinations of the mutations. As the pH was lowered from 9.5 to 6.0, the relative decrease in the midpoint potential became smaller for each of these three mutations. Titration of the pH dependence of the change in midpoint potential of the M199 Asn to Asp mutant compared to wild type yielded a pK(a) value of 7.9 and a change in midpoint potential from low to high pH of 59 mV. The major effect of the mutation on the midpoint potential of the dimer is interpreted as stemming from a negative charge on the residue. An average dielectric constant of approximately 20 was estimated for the local protein environment, consistent with a relatively hydrophobic environment for residue M199. The rate of charge recombination between the primary quinone acceptor and the bacteriochlorophyll dimer decreased in the M199 Asn to Asp mutant at high pH, reflecting the decrease in midpoint potential.

Blue light drives B-side electron transfer in bacterial photosynthetic reaction centers.

The core of the photosynthetic reaction center from the purple non-sulfur bacterium Rhodobacter sphaeroides is a quasi-symmetric heterodimer, providing two potential pathways for transmembrane electron transfer. Past measurements have demonstrated that only one of the two pathways (the A-side) is used to any significant extent upon excitation with red or near-infrared light. Here, it is shown that excitation with blue light into the Soret band of the reaction center gives rise to electron transfer along the alternate or B-side pathway, resulting in a charge-separated state involving the anion of the B-side bacteriopheophytin. This electron transfer is much faster than normal A-side transfer, apparently occurring within a few hundred femtoseconds. At low temperatures, the B-side charge-separated state is stable for at least 1 ns, but at room temperature, the B-side bacteriopheophytin anion is short-lived, decaying within approximately 15 ps. One possible physiological role for B-side electron transfer is photoprotection, rapidly quenching higher excited states of the reaction center.

Dietary content may prevent secondary hyperparathyroidism in female rhesus monkeys (Macaca mulatta).

Macaque laboratory chows provide relatively more calcium (Ca) and vitamin D (D) than human diets; this may influence skeletal aging. To evaluate this possibility, parameters of skeletal relevance in premenopausal and naturally postmenopausal rhesus monkeys were measured in a cross-sectional study. Serum osteocalcin (Oc) was elevated in the postmenopausal group (P < 0.01), but levels of parathyroid hormone (PTH) and 25-hydroxyvitamin D (25OHD) were not different. Subsequently, in premenopausal animals, dietary Ca and/or D intake was reduced to optimal human levels for 8 weeks prior to the evaluation of the skeletal parameters. Serum 25OHD concentration was reduced (P < 0.01) and a trend (P=0.10) towards increased PTH was observed in both low D groups. In addition, serum alkaline phosphatase (ALP) levels were increased in the low Ca group (P < 0.01). In conclusion, skeletal turnover, as measured by serum Oc, was increased in naturally postmenopausal rhesus monkeys in the absence of hyperparathyroidism. Dietary D reduction causes a decline in serum 25OHD and an upward trend in PTH.

Diet- or warfarin-induced vitamin K insufficiency elevates circulating undercarboxylated osteocalcin without altering skeletal status in growing female rats.

To further characterize the skeletal role of vitamin K (K), markers of bone turnover, density, and strength were evaluated in rats with diet- or warfarin (W)-induced K insufficiency. One hundred two, 7-week-old, female rats were randomly assigned to low K (phylloquinone [K1], 20 microg/kg diet), control K (K1, 1300 microg/kg diet), low-dose W (W, 1.5 mg/kg control diet), or high-dose W plus K (W/K1, 10/100 mg/kg diet). Femur bone mineral content (BMC) and bone mineral density (BMD), plasma prothrombin time (PT) and prothrombin concentration (PC), and serum total alkaline phosphatase (ALP) and skeletal alkaline phosphatase (sALP) were measured at baseline and days 20, 40, 60, and 80. Serum total osteocalcin (OC) and undercarboxylated osteocalcin (ucOC) and femur length (FL) were measured at baseline and day 80. Left femur OC was measured and biomechanical testing of the right femur and third lumbar vertebral body was performed at day 80. Low dietary K elevated circulating ucOC (17% higher than control; p < 0.0001) at day 80. Furthermore, in both W groups, essentially all circulating OC was undercarboxylated and femur OC was lower than control (p < 0.0001). However, there was no change in femur percent ucOC, suggesting deposition of less newly synthesized OC. No between group differences were observed in PT, ALP, sALP, FL, BMC, BMD, or bone strength. In conclusion, skeletal K insufficiency can be induced by W or diet manipulation. This does not hinder peak bone mass attainment in female rats; however, W causes less newly synthesized OC to be deposited in bone.

Serum dehydroepiandrosterone sulfate concentrations across the life span of laboratory-housed rhesus monkeys.

Cross-sectional studies of humans have shown that dehydroepiandrosterone sulfate (DHEAS) peaks shortly after sexual maturation and declines thereafter, suggesting that the progressive reduction in DHEAS may play a role in the aging process and in the development of age-related morbidity. The present study examines changes in DHEAS concentrations across the life span of rhesus monkeys as part of the development of this primate model for studies of aging. Serum concentrations of DHEAS were measured in 792 laboratory-housed rhesus monkeys (Macaca mulatta) aged 0.5-36 years (527 females, 265 males). DHEAS concentrations in all monkeys were used to formulate an equation that describes two levels of decline of DHEAS with age. The most rapid decline occurs from infancy until approximately 5 years of age. The decline then occurs gradually with increasing age. There were no signs of an andrenarche just prior to sexual maturation, as is seen in humans or the great apes. This equation can be used to predict the expected mean serum DHEAS concentration and normal ranges of male or female rhesus monkeys at any age greater than 5 months.

Central region-of-interest analysis of lumbar spine densitometry demonstrates lower bone mass in older rhesus monkeys.

Osteoarthritis (OA) spuriously elevates spine bone mineral density (BMD) as measured by dual-energy X-ray absorptiometry (DXA). This study documents spinal OA prevalence in adult female rhesus monkeys, and evaluates a custom central region-of-interest (CROI) analysis technique designed to minimize OA effects on BMD measurement. Lumbar spine radiographs were obtained on 71 animals, age 10-37 years. OA degree was blindly scored as none, minimal, or moderate/severe. Moderate/severe OA was not observed before age 19, but was present in 66% of older animals. Subsequently, lumbar spine (L2-4) BMD was determined by standard DXA analysis and manual placement of 0.92 cm2 CROIs in two groups of female rhesus monkeys. One group (eight control, eight postovariectomy, ages 10-19 years) was assessed longitudinally, the second (n = 90, ages 10-37) cross-sectionally. Measured bone loss following ovariectomy (8.1% standard analysis, 11.5 % CROI) was comparable with both techniques. By contrast, CROI demonstrated lower bone mass with age (p < 0.0001), whereas only a trend (p = 0.06) was observed with standard analysis. When World Health Organization criteria were applied, 42% of animals > or = age 19 years were classified as osteopenic/osteoporotic by standard analysis compared with 67% by CROI. All "normal" animals reclassified as osteopenic/osteoporotic by CROI had OA. In conclusion, female rhesus monkeys often develop spinal OA with advancing age. CROI analysis demonstrated lower bone density in older monkeys and was as sensitive to estrogen-depletion bone loss as standard methodology. This suggests that alternative analysis techniques, such as CROI, may be more appropriate to evaluate bone density in nonhuman primates, and potentially in people.

Zoledronate prevents the development of absolute osteopenia following ovariectomy in adult rhesus monkeys.

This study assessed effects of the bisphosphonate zoledronate (ZLN) on bone density and biochemical markers of bone turnover in ovariectomized (OVX) adult female rhesus monkeys. Forty monkeys were randomly assigned to one control or four OVX groups. The control and one OVX group received saline, and the other three OVX groups received ZLN (0.5, 2.5, or 12.5 microg/kg) by a single weekly subcutaneous injection for 69 weeks. Bone mass of the total body (TB), lumbar spine (LS), distal and central radius (dual-energy X-ray absorptiometry), and skeletal turnover markers were measured at baseline and at 13, 26, 39, 52, and 69 weeks of treatment. Increased skeletal turnover and decreased bone mass (LS and TB) were demonstrable by 13 weeks post-OVX. Maximal bone loss (7-8%) at these sites occurred by 39 weeks after OVX and persisted for the study duration. Long-term ZLN treatment was well tolerated and prevented increased skeletal turnover and bone loss in a dose-dependent fashion. Progressive turnover suppression was not observed with any ZLN dose. In conclusion, after OVX, adult rhesus monkeys develop persistent increased bone turnover and absolute osteopenia of the LS and TB, making them an outstanding model of skeletal behavior in perimenopausal women. These OVX-related skeletal changes are dose-dependently blocked by ZLN.

The effect of high fat diet and dehydroepiandrosterone (DHEA) administration in the rhesus monkey.

Dehydroepiandrosterone (DHEA) lowers serum cholesterol, particularly the low density lipoprotein (LDL) fraction, in rhesus monkeys on a commercial diet (12% calories from fat, 0.0083% cholesterol). We fed rhesus monkeys a diet of 30% calories from fat and 0.1% cholesterol for 12 weeks, then commercial chow for 7 weeks. Six monkeys each received DHEA or placebo, orally for 17 weeks. Food intake increased the first 6 weeks, but decreased thereafter. Monkeys had a 22% mean weight gain while on high fat diet. DHEA monkeys had higher T4 levels than placebo monkeys at weeks 8 and 16. After 12 weeks on high fat diet, all monkeys had elevated serum cholesterol concentrations, an increase in amount and percentage of intermediate density lipoprotein and LDL cholesterol, and an increase in amount, but a decrease in percentage of high density lipoprotein cholesterol. There were no significant differences in serum cholesterol or plasma lipoprotein concentrations between DHEA and placebo monkeys while on high fat diet (a trend toward lower levels was noted in the DHEA group). On commercial chow, plasma lipoprotein concentrations decreased for all monkeys, and DHEA monkeys had significantly lower total and LDL cholesterol than placebo monkeys. We conclude that a high fat diet (30% fat) masks any cholesterol-lowering effects of DHEA.

Hypocholesterolemic effect of exogenous dehydroepiandrosterone administration in the rhesus monkey.

The effect(s) of exogenous dehydroepiandrosterone (DHEA) was studied in healthy non-obese rhesus monkeys. Six monkeys were given 60 mg/kg/day DHEA for 4 weeks. The dose was then increased to 75 mg/kg/day for an additional 4 weeks. Another six monkeys were given placebo, daily for 8 weeks. Body weight, activity level, average daily food intake, and plasma T4, insulin, total androgen and cortisol concentrations remained unchanged for all 12 monkeys throughout the 8 weeks. Both groups had a significant (p < 0.05) decrease in total plasma cholesterol, however the DHEA treated monkeys were significantly lower than the placebo monkeys. The change in cholesterol in the DHEA treated group primarily affected the fraction containing the low density lipoprotein cholesterol which remained overall unchanged for the placebo monkeys. Both groups had a decrease in high density lipoprotein cholesterol, however there was no difference between the treatment groups. We conclude that in the rhesus monkey, exogenous administration of DHEA acutely reduces cholesterol concentration, particularly the lipoprotein fraction containing low density lipoprotein, without changing food intake, activity level, or body weight.

Reduction in body weight and cholesterol in spontaneously obese dogs by dehydroepiandrosterone.

We studied the effect(s) of exogenous dehydroepiandrosterone (DHEA) in spontaneously obese dogs. Nineteen euthyroid obese and six non-obese normal dogs were evaluated. Dogs received DHEA for three months at an escalating dose of 30-75 mg/kg p.o. daily. We found a 3 percent reduction in total body weight/month in 68 percent of the obese dogs, without reduction in food intake. The normal dogs did not lose weight or reduce food intake. Serum cholesterol in obese dogs went from 226 to 173 mg/dl post-treatment and in normal dogs from 128 to 89 mg/dl. Analysis of lipoproteins in four normal dogs revealed that the marked reduction in cholesterol most significantly affected the LDL-HDL1 fraction.