Defining the structure of the substrate-free state of the DnaK molecular chaperone.

Members of the Hsp70 (heat-shock protein of 70 kDa) family of molecular chaperones bind to exposed hydrophobic stretches on substrate proteins in order to dissociate molecular complexes and prevent aggregation in the cell. Substrate affinity for the C-terminal domain of the Hsp70 is regulated by ATP binding to the N-terminal domain utilizing an allosteric mechanism. Our multi-dimensional NMR studies of a substrate-binding domain fragment (amino acids 387-552) from an Escherichia coli Hsp70, DnaK(387-552), have uncovered a pH-dependent conformational change, which we propose to be relevant for the full-length protein also. At pH 7, the C-terminus of DnaK(387-552) mimics substrate by binding to its own substrate-binding site, as has been observed previously for truncated Hsp70 constructs. At pH 5, the C-terminus is released from the binding site, such that DnaK is in the substrate-free state 10-20% of the time. We propose that the mechanism for the release of the tail is a loss of affinity for substrate at low pH. The pH-dependent fluorescence changes at a tryptophan residue near the substrate-binding pocket in full-length DnaK lead us to extend these conclusions to the full-length DnaK as well. In the context of the DnaK substrate-binding domain fragment, the release of the C-terminus from the substrate-binding site provides our first glimpse of the empty conformation of an Hsp70 substrate-binding domain containing a portion of the helical subdomain.

Effects on blood lipids of a blood pressure-lowering diet: the Dietary Approaches to Stop Hypertension (DASH) Trial.

BACKGROUND: Effects of diet on blood lipids are best known in white men, and effects of type of carbohydrate on triacylglycerol concentrations are not well defined. OBJECTIVE: Our goal was to determine the effects of diet on plasma lipids, focusing on subgroups by sex, race, and baseline lipid concentrations. DESIGN: This was a randomized controlled outpatient feeding trial conducted in 4 field centers. The subjects were 436 participants of the Dietary Approaches to Stop Hypertension (DASH) Trial [mean age: 44.6 y; 60% African American; baseline total cholesterol: < or = 6.7 mmol/L (< or = 260 mg/dL)]. The intervention consisted of 8 wk of a control diet, a diet increased in fruit and vegetables, or a diet increased in fruit, vegetables, and low-fat dairy products and reduced in saturated fat, total fat, and cholesterol (DASH diet), during which time subjects remained weight stable. The main outcome measures were fasting total cholesterol, LDL cholesterol, HDL cholesterol, and triacylglycerol. RESULTS: Relative to the control diet, the DASH diet resulted in lower total (-0.35 mmol/L, or -13.7 mg/dL), LDL- (-0.28 mmol/L, or -10.7 mg/dL), and HDL- (-0.09 mmol/L, or -3.7 mg/dL) cholesterol concentrations (all P < 0.0001), without significant effects on triacylglycerol. The net reductions in total and LDL cholesterol in men were greater than those in women by 0.27 mmol/L, or 10.3 mg/dL (P = 0.052), and by 0.29 mmol/L, or 11.2 mg/dL (P < 0.02), respectively. Changes in lipids did not differ significantly by race or baseline lipid concentrations, except for HDL, which decreased more in participants with higher baseline HDL-cholesterol concentrations than in those with lower baseline HDL-cholesterol concentrations. The fruit and vegetable diet produced few significant lipid changes. CONCLUSIONS: The DASH diet is likely to reduce coronary heart disease risk. The possible opposing effect on coronary heart disease risk of HDL reduction needs further study.

Signal peptides bind and aggregate RNA. An alternative explanation for GTPase inhibition in the signal recognition particle.

N-terminal signal sequences can direct nascent protein chains to the inner membrane of prokaryotes and the endoplasmic reticulum of eukaryotes by interacting with the signal recognition particle. In this study, we show that isolated peptides corresponding to several bacterial signal sequences inhibit the GTPase activity of the Escherichia coli signal recognition particle, as previously reported (Miller, J. D., Bernstein, H. D., and Walter, P. (1994) Nature 367, 657-659), but not by the direct mechanism proposed. Instead, isolated signal peptides bind nonspecifically to the RNA component and aggregate the entire signal recognition particle, leading to a loss of its intrinsic GTPase activity. Surprisingly, only "functional" peptide sequences aggregate RNA; the peptides in general use as "nonfunctional" negative controls (e.g. those with deletions or charged substitutions within the hydrophobic core), are sufficiently different in physical character that they do not aggregate RNA and thus have no effect on the GTPase activity of the signal recognition particle. We propose that the reported effect of functional signal peptides on the GTPase activity of the signal recognition particle is an artifact of the high peptide concentrations and low salt conditions used in these in vitro studies and that signal sequences at the N terminus of nascent chains in vivo do not exhibit this activity.

Dietary composition and physiologic adaptations to energy restriction.

BACKGROUND: The concept of a body weight set point, determined predominantly by genetic mechanisms, has been proposed to explain the poor long-term results of conventional energy-restricted diets in the treatment of obesity. OBJECTIVE: The objective of this study was to examine whether dietary composition affects hormonal and metabolic adaptations to energy restriction. DESIGN: A randomized, crossover design was used to compare the effects of a high-glycemic-index (high-GI) and a low-glycemic-index (low-GI) energy-restricted diet. The macronutrient composition of the high-GI diet was (as percent of energy) 67% carbohydrate, 15% protein, and 18% fat and that of the low-GI diet was 43% carbohydrate, 27% protein, and 30% fat; the diets had similar total energy, energy density, and fiber contents. The subjects, 10 moderately overweight young men, were studied for 9 d on 2 separate occasions. On days -1 to 0, they consumed self-selected foods ad libitum. On days 1-6, they received an energy-restricted high- or low-GI diet. On days 7-8, the high- or low-GI diets were consumed ad libitum. RESULTS: Serum leptin decreased to a lesser extent from day 0 to day 6 with the high-GI diet than with the low-GI diet. Resting energy expenditure declined by 10.5% during the high-GI diet but by only 4.6% during the low-GI diet (7.38 +/- 0.39 and 7.78 +/- 0.36 MJ/d, respectively, on days 5-6; P = 0.04). Nitrogen balance tended to be more negative, and energy intake from snacks on days 7-8 was greater, with the high-GI than the low-GI diet. CONCLUSION: Diets with identical energy contents can have different effects on leptin concentrations, energy expenditure, voluntary food intake, and nitrogen balance, suggesting that the physiologic adaptations to energy restriction can be modified by dietary composition.

Descriptive characteristics of the dietary patterns used in the Dietary Approaches to Stop Hypertension Trial. DASH Collaborative Research Group.

The Dietary Approaches to Stop Hypertension trial was a randomized, multicenter, controlled feeding study to compare the effect on blood pressure of 3 dietary patterns: control, fruits and vegetables, and combination diets. The patterns differed in selected nutrients hypothesized to alter blood pressure. This article examines the food-group structure and nutrient composition of the study diets and reports participant nutrient consumption during intervention. Participants consumed the control dietary pattern during a 3-week run-in period. They were then randomized either to continue on the control diet or to change to the fruits and vegetables or the combination diet for 8 weeks. Sodium intake and body weight were constant during the entire feeding period. Analysis of variance models compared the nutrient content of the 3 diets. Targeting a few nutrients thought to influence blood pressure resulted in diets that were profoundly different in their food-group and nutrient composition. The control and fruits and vegetables diets contained more oils, table fats, salad dressings, and red meats and were higher in saturated fat, total fat, and cholesterol than was the combination diet. The fruits and vegetables and combination diets contained relatively more servings of fruits, juices, vegetables, and nuts/seeds, and were higher in magnesium, potassium, and fiber than was the control diet. Both the fruits and vegetables and combination diets were low in sweets and sugar-containing drinks. The combination diet contained a greater variety of fruits, and its high calcium content was obtained by increasing low-fat dairy products. In addition, the distinct food grouping pattern across the 3 diets resulted in substantial differences in the levels of vitamins A, C, E, folate, B-6, and zinc.

Pre-enrollment diets of Dietary Approaches to Stop Hypertension trial participants. DASH Collaborative Research Group.

A large body of evidence suggests that several nutrients are related to blood pressure. Less is known about the eating patterns of special populations, such as those at risk for hypertension, or how demographic factors affect the diets of these populations. This article characterizes the usual diets of participants before they enrolled in the Dietary Approaches to Stop Hypertension (DASH) trial. During screening for DASH, 380 participants completed the National Cancer Institute food frequency questionnaire. Nutrient and food group intake, the Keys score (a measure of a diet's atherogenicity), and the Diet Quality Index were estimated from the food frequency questionnaire. The effects of age, sex, race, baseline weight, and education on these dietary factors were assessed among DASH participants and compared with similar data from the Third National Health and Nutrition Examination Survey and other published reports. Among DASH participants, African-Americans reported lower intakes of dairy products (P < .001), calcium (P < .001), and magnesium (P < .05) than did whites. Older women reported greater intakes of calcium, magnesium, and potassium (all P < .05) and less fat (P < .05) than did younger women. Older men consumed fewer servings of fruits (P < .03), less vitamin C (P < .05), and had a higher Keys score (P < .05) than did younger men. Heavier (body mass index > or = 25) participants reported lower intakes of protein and potassium, but higher fat and energy intakes (all P < .05). Taken together, these data show that younger, overweight African-American women have the least healthful diets, because they consume more atherogenic foods and fewer of the nutrients related to decreased blood pressure. Overall Diet Quality Index scores did not differ between African-American and white participants. Despite differences in dietary assessment methods between the population samples of DASH and the Third National Health and Nutrition Examination Survey, within each population sample patterns of micronutrient intake were similar between African-American and white participants.

Menu design and selection for multicenter controlled feeding studies: process used in the Dietary Approaches to Stop Hypertension trial. DASH Collaborative Research Group.

Outpatient feeding studies are being used increasingly more often than inpatient studies because they are less expensive to conduct and less disruptive to participants' daily lives. Frequently, however, they are more difficult to implement. Studies involving multiple feeding centers add an additional layer of cooperation, coordination, and standardization to the already complex task of developing and delivering research diets. This was true for the 4-year Dietary Approaches to Stop Hypertension (DASH) trial, one of the first multicenter outpatient controlled feeding studies. This 4-center, randomized clinical trial was designed to compare the effects of 3 dietary patterns on blood pressure. After a year of development, 3 sets of 7-day cycle menus that met the study nutrient criteria and that were appropriate for varying food production routines and staffing patterns at the 4 clinical centers were adopted. The major development tasks were: defining methodologies to guide menu design and food production; selecting a nutrient database and calculating nutrient content of menus; evaluating and selecting the menus; and adjusting the menus for final use. The purpose of this article is to describe the steps and considerations in the design and selection of menus for the DASH trial, a process applicable to all well-controlled feeding studies.

Validation of diet composition for the Dietary Approaches to Stop Hypertension trial. DASH Collaborative Research Group.

The Dietary Approaches to Stop Hypertension trial involved 4 clinical sites at which 459 participants (in 5 cohorts) were fed 3 dietary patterns over 11 weeks per cohort. The 3 patterns were a control diet, a fruits and vegetables diet, and a combination diet. Before the intervention, key nutrient levels in each diet were validated at 2 energy levels (2,100 and 3,100 kcal) by chemical analysis of the prepared menus. During intervention, diets were sampled across all cohorts, sites, and energy levels, and 7-day menu cycle composites were assayed. In general, sodium, potassium, calcium, and magnesium in the validated menus for each diet/energy level met the nutrient targets, though moderate variability was evident among individual menus, particularly for potassium, calcium, and magnesium. However, as intended, there was clear separation and no overlap in mineral levels in individual menus of diets that were designed to differ. During intervention, macronutrient contents met nutrient goals. Sodium, potassium, calcium, and magnesium in the diets generally met target levels, though potassium in the fruits and vegetables diet was 11% to 23% below target. There were no consistent differences in nutrient levels between sites. The mean nutrient levels in the validated menus and diets sampled during intervention were in excellent agreement with each other, though sodium was somewhat higher (approximately 6%) in the diets from intervention vs validation. These results indicate the success of the quality control measures implemented and suggested consistent overall diet composition throughout the 28 months during which the study was conducted.

Dietary adherence in the Dietary Approaches to Stop Hypertension trial. DASH Collaborative Research Group.

Participants in controlled feeding studies must consume all study foods and abstain from all other foods. In outpatient studies in which adherence may be compromised by free-living conditions, promoting, documenting, and monitoring dietary adherence are necessary. In the Dietary Approaches to Stop Hypertension (DASH) trial, a thorough participant screening process, an orientation session, and a run-in feeding period before randomization aided in the selection of participants who would most likely adhere to the demands of the study protocol. Throughout the feeding period, various educational and motivational techniques were used to encourage DASH participants to adhere to the dietary protocol. Both objective and subjective methods documented excellent participant adherence. Daily monitoring of individual adherence was based on meal attendance, body weight measurements, and daily diaries. Urinary sodium, potassium, phosphorus, and urea nitrogen values and an anonymous poststudy survey were used to evaluate adherence at the end of the study. Most DASH participants adhered to the feeding regimen by consuming only study foods and no other foods. When adherence lapsed, participants generally cited the lack of menu variety as a reason. Successful participant adherence to the constraints of an outpatient controlled feeding study is possible with carefully selected participants and a variety of adherence-promoting strategies incorporated into the study protocol.

Translating the Dietary Approaches to Stop Hypertension diet from research to practice: dietary and behavior change techniques. DASH Collaborative Research Group.

The Dietary Approaches to Stop Hypertension (DASH) clinical trial demonstrated that a diet that emphasizes fruits, vegetables, and low-fat dairy products, includes whole grains, nuts, fish, and poultry, and is reduced in fats, red meats, sweets, and sugar-containing beverages can be highly effective in lowering blood pressure. The National High Blood Pressure Education Program now suggests the DASH diet for preventing and managing hypertension. For persons modifying their diets, the DASH diet offers varied choices. However, simultaneously modifying several dimensions of a diet can be challenging, even for knowledgeable and motivated persons. Persons who are uncertain about modifying their diet may become overwhelmed by the needed dietary changes. Dietitians and other health care practitioners can help patients adopt the DASH diet by exploring possible ambivalence, increasing motivation, and strengthening commitment to change; encouraging patients to select dietary modifications that will fit their lifestyle; and, finally, offering information about how to change their eating behavior. In this article, we offer dietary advice and counseling suggestions for tailoring interventions to match patients' readiness for adopting the DASH diet.

The DASH Diet, Sodium Intake and Blood Pressure Trial (DASH-sodium): rationale and design. DASH-Sodium Collaborative Research Group.

The DASH Diet, Sodium Intake and Blood Pressure Trial (DASH-Sodium) is a multicenter, randomized trial comparing the effects of 3 levels of sodium intake and 2 dietary patterns on blood pressure among adults with higher than optimal blood pressure or with stage 1 hypertension (120-159/80-95 mm Hg). The 2 dietary patterns are a control diet typical of what many Americans eat, and the DASH diet, which, by comparison, emphasizes fruits, vegetables, and low-fat dairy foods, includes whole grains, poultry, fish, and nuts, and is reduced in fats, red meat, sweets, and sugar-containing beverages. The 3 sodium levels are defined as higher (typical of current US consumption), intermediate (reflecting the upper limit of current US recommendations), and lower (reflecting potentially optimal levels). Participants are randomly assigned to 1 of the 2 dietary patterns using a parallel group design and are fed each of the 3 sodium levels using a randomized crossover design. The study provides participants with all of their food during a 2-week run-in feeding period and three 30-day intervention feeding periods. Participants attend the clinic for 1 meal per day, 5 days per week, and take home food for other meals. Weight is monitored and individual energy intake adjusted to maintain baseline weight. The primary outcome is systolic blood pressure measured at the end of each intervention feeding period. Systolic blood pressure is compared across the 3 sodium levels within each diet and across the 2 diets within each sodium level. If effects previously observed in clinical trials are additive, sodium reduction and the DASH diet together may lower blood pressure to an extent not as yet demonstrated for nonpharmacologic treatment. The DASH-Sodium results will have important implications for the prevention and treatment of high blood pressure.

Feasibility of outpatient electrolyte balance studies.

Clinical studies requiring controlled electrolyte balance have traditionally been conducted in an inpatient (IP), metabolic ward setting. The purpose of this study was to test the feasibility of performing such studies in an outpatient (OP) clinical research setting. Focusing on sodium (Na) and potassium (K) balance, we retrospectively compared 28 subjects studied as OP vs 25 studied as IP on our metabolic ward. We assessed their adherence to our metabolic diets and their compliance with serial 24-hr urine collections. Dietary compliance was assessed by checksheet and urinary Na excretion; urine collection accuracy was determined by serial 24-hr creatinine excretion. The diets for both studies contained a low Na phase (10 mEq) and a high Na phase (200 mEq for IP and 250 mEq for OP), each lasting 1 week. When in balance on the low Na diet, 24-hr Na excretion was 4.6 +/- 0.7 mEq for OP and 13.4 +/- 2.2 mEq for IP, indicating excellent compliance with the low salt diet. Na excretion on the high Na diet was 184.5 +/- 7.4 mEq for OP and 195.3 +/- 9.6 mEq for IP. These values were not significantly different from each other; however, the OP were significantly less than their diet of 250 mEq Na (p less than 0.05). This difference may have been due to dermal Na losses. K excretion was also similar in the two groups. There was no significant difference in the reproducibility of individual multiple urinary creatinine measurements in OP vs IP.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of the effects of oat bran and low-fiber wheat on serum lipoprotein levels and blood pressure.

Previous studies have shown that supplementation of the diet with oat bran may lower serum cholesterol levels. However, it is not known whether oat-bran diets lower serum cholesterol levels by replacing fatty foods in the diet or by a direct effect of the dietary fiber contained in oat bran. To determine which is the case, we compared the effect of isocaloric supplements of high-fiber oat bran (87 g per day) and a low-fiber refined-wheat product on the serum lipoprotein cholesterol levels of 20 healthy subjects, 23 to 49 years old. After a one-week base-line period during which they consumed their usual diets, the subjects were given each type of supplement for six-week periods in a double-blind, crossover trial. Mean serum cholesterol levels (+/- SD) were not significantly different during the high-fiber and low-fiber periods: total cholesterol, 4.44 +/- 0.73 and 4.46 +/- 0.64 mmol per liter (172 +/- 28 and 172 +/- 25 mg per deciliter); low-density lipoprotein, 2.69 +/- 0.63 and 2.77 +/- 0.59 mmol per liter (104 +/- 24 and 107 +/- 23 mg per deciliter); and high-density lipoprotein, 1.40 +/- 0.39 and 1.32 +/- 0.39 mmol per liter (54.2 +/- 15.0 and 50.9 +/- 15.2 mg per deciliter), respectively. However, both types of supplements lowered the mean base-line serum cholesterol level, 4.80 +/- 0.80 mmol per liter (186 +/- 31 mg per deciliter), by 7 to 8 percent (95 percent confidence interval for high fiber, 11 to 4 percent, and for low fiber, 11 to 3 percent). The subjects ate less saturated fat and cholesterol and more polyunsaturated fat during both periods of supplementation than at base line. Those changes in dietary fats were sufficient to explain all of the reduction in serum cholesterol levels caused by the high-fiber and low-fiber diets. The average blood pressure was 112/68 mm Hg at base line and did not change during either dietary period. We conclude that oat bran has little cholesterol-lowering effect and that high-fiber and low-fiber dietary grain supplements reduce serum cholesterol levels about equally, probably because they replace dietary fats.