Energy extraction from nuts: walnuts, almonds and pistachios.

The bioaccessibility of fat has implications for satiety and postprandial lipidaemia. The prevailing view holds that the integrity of plant cell wall structure is the primary determinant of energy and nutrient extraction from plant cells as they pass through the gastrointestinal (GI) tract. However, comparisons across nuts (walnuts, almonds and pistachios) with varying physical properties do not support this view. In the present study, masticated samples of three nuts from healthy adults were exposed to a static model of gastric digestion followed by simulated intestinal digestion. Primary outcomes were particle size and lipid release at each phase of digestion. Walnuts produced a significantly larger particle size post-mastication compared with almonds. Under gastric and intestinal conditions, the particle size was larger for walnuts compared with pistachios and almonds (P < 0·05). However, the masticated and digesta particle sizes were not related to the integrity of cell walls or lipid release. The total lipid release was comparable between nuts after the in vitro intestinal phase (P > 0·05). Microstructural examination showed ruptured and fissured cell walls that would allow digestion of cellular contents, and this may be governed by internal cellular properties such as oil body state. Furthermore, the cell walls of walnuts tend to rupture rather than separate and as walnut tissue passes through the GI tract, lipids tend to coalesce reducing digestion efficiency.

Effect of a high-fat meal containing conventional or high-oleic peanuts on post-prandial lipopolysaccharide concentrations in overweight/obese men.

BACKGROUND: An increased plasma lipopolysaccharide (LPS) concentration may favour metabolic disorders such as insulin resistance. The meal composition influences plasma LPS concentrations. The present study aimed to investigate the effect of the acute consumption of a high-fat meal (49% of energy from fat) containing conventional or high-oleic peanuts on post-prandial LPS concentrations and its relationship with lipaemia and insulinaemia in overweight and obese men. METHODS: The test meal consisted of a shake containing conventional peanuts (CVP; n = 21), high-oleic peanuts (HOP; n = 23) or a control biscuit (CT; n = 21). Blood samples were collected in the fasting state and 1, 2 and 3 h post-prandially. LPS, insulin, lipids and glucose concentrations were assessed. RESULTS: LPS concentrations were lower in CVP [mean (SE) 0.7 (0.5) EU mL(-1) ] and HOP [1.0 (0.9) EU mL(-1) ] groups compared to CT [1.6 (1.2) EU mL(-1) ] at 3 h post-prandially. Triacylglycerol and insulin concentrations increased in all groups. Triacylglycerol started to increase only after 2 h in the CVP and HOP groups. LPS correlated positively with triacylglycerol. Insulin returned to basal concentrations at 3 h only in the CVP and HOP groups. CONCLUSIONS: The acute consumption of peanuts delayed the increase in serum triacylglycerol and favoured the quicker return of insulin to basal concentrations, especially in the CVP group. Our results suggest that the consumption of conventional or high-oleic peanuts may help to reduce the risk of endotoxaemia and metabolic disorders.

Appetitive, dietary and health effects of almonds consumed with meals or as snacks: a randomized, controlled trial.

BACKGROUND/OBJECTIVES: Snacks contribute toward a significant proportion of human total daily energy intake. This study investigated the effects of almonds, a satiating and nutrient-rich, common snack, on postprandial glycemia, appetite, short-term body weight and fasting blood parameters when consumed with meals or alone as a snack. METHODS: This was a 4-week randomized, parallel-arm study that entailed consuming almonds (43 g/day) with breakfast (BF) or lunch (LN), alone as a morning (MS) or afternoon (AS) snack or no almonds (CL). Participants (N=137) with increased risk for type 2 diabetes completed an oral glucose tolerance test (OGTT) and acute-feeding session at baseline, followed by almond consumption for 4 weeks before repeating the OGTT and acute-feeding trials. Anthropometric, biochemical and appetite responses were assessed. RESULTS: Almonds lowered serum glucose responses postprandially. Effects were most prominent in the snack groups. Almonds, consumed as snacks, also reduced hunger and desire to eat during the acute-feeding session. After 4 weeks, anthropometric measurements and fasting blood biochemistries did not differ from the control group or across intervention groups. Without specific guidance, daily energy intake was reduced to compensate for energy from the provided almonds. Dietary monounsaturated fat and α-tocopherol intakes were significantly increased in all almond groups. CONCLUSION: Almonds provide post-ingestive metabolic and appetitive benefits and did not increase the risk for weight gain. This suggests that almonds may be a healthful snack option.

Effects of fruit and vegetable, consumed in solid vs beverage forms, on acute and chronic appetitive responses in lean and obese adults.

BACKGROUND: The effects of fruits and vegetables in solid vs beverage forms on human appetite and food intake, acutely and chronically, are unclear. METHODS: This 21-week, randomized, crossover study assessed appetitive ratings following the inclusion of fruits and vegetables, in solid and beverage form, into the habitual diet of healthy lean (n=15) and overweight/obese (n=19) adults with low customary consumption. The primary acute outcomes were satiation (amount of challenge meal consumed), satiety (latency of subsequent eating event) and dietary compensation after a 400 kcal fruit preload. Ratings of appetite were also obtained before and after 8 weeks of required increased fruit and vegetable consumption (20% estimated energy requirement). RESULTS: Acutely, overweight/obese participants reported smaller reductions of hunger after consuming the fruit preload in beverage compared with solid form (preload × form × body mass index effects, P=0.03). Participants also consumed significantly less of a challenge meal (in both gram and energy) after the ingestion of the solid fruit preload (P<0.005). However, the subsequent meal latency was not significantly different between the solid and the beverage fruit preloads. Total daily energy intake was significantly higher when the obese participants consumed the beverage fruit preload compared with the solid (P<0.001). Daily energy intake was markedly, but not significantly, higher among the lean with the beverage vs solid food form. Hunger and fullness ratings remained stable when participants consumed fruits and vegetables in solid or beverage form for 8 weeks each. CONCLUSION: Acute post-ingestive appetitive responses were weaker following consumption of fruits in beverage vs solid food forms. Consumption of beverage or solid fruit and vegetable food loads for 8 weeks did not chronically alter appetitive responses.

Flaxseed energy and macronutrients balance.

BACKGROUND/OBJECTIVES: Flaxseed has functional properties in the reduction of the risk of chronic non-communicable diseases such as cardiovascular disease, diabetes and cancer. Regardless of its high energy density, the consumption of flaxseed tends to promote body weight maintenance. The purpose of this study was to evaluate energy and macronutrient balance after flaxseed consumption. SUBJECTS/METHODS: Twenty four healthy volunteers were allocated into 3 experimental groups, when they consumed flaxseed (FS), defatted flaxseed flour (FF), or flaxseed oil (FO). During the control period they were provided a diet without flaxseed products for 7-9 days. Following that diets containing 70 g of one of the flaxseed products were consumed for another 7-9 day- period. Test foods were consumed exclusively in the laboratory and fecal excretion was collected during the study. There was a higher energy excretion (P < 0.05) in the FF and FS groups, compared to their control and FO group. RESULTS: The excretions of total lipid and the PUFA α-linolenic acid were higher in FS group (P < 0.05). CONCLUSIONS: The intake of 70 g/day of FS and FF raised lipid and energy excretion, which may mitigated the effect of flaxseed consumption on body weight.

Appetitive and Dietary Effects of Consuming an Energy-Dense Food (Peanuts) with or between Meals by Snackers and Nonsnackers.

Background. Energy-dense foods are inconsistently implicated in elevated energy intake (EI). This may stem from other food properties and/or differences in dietary incorporation, that is, as snacks or with meals. Objective. Assess intake pattern and food properties on acute appetitive ratings (AR) and EI. Design. 201 normal and overweight adults consuming a standard lunch. Test loads of 1255.2 kJ (300 kcal) were added to the lunch or provided as snack. Loads (peanuts, snack mix, and snack mix with peanuts) were energy, macronutrient, and volumetrically matched with a lunch portion as control. Participants completed meal and snack sessions of their randomly assigned load. Results. No differences were observed in daily EI or AR for meal versus snack or treatment versus control. Consumption of peanuts as a snack tended to strengthen dietary compensation compared to peanuts or other loads with a meal. Conclusions. Inclusion of an energy-dense food as a snack or meal component had comparable influence on AR and EI. Peanuts tended to elicit stronger dietary compensation when consumed as a snack versus with a meal. If substantiated, this latter observation suggests that properties other than those controlled here (energy, macronutrient content, and volume) modify AR and EI.

Nutritively sweetened beverage consumption and body weight: a systematic review and meta-analysis of randomized experiments.

Nutritively sweetened beverages (NSBs) may play a role in the obesity epidemic. We abstracted data from randomized controlled trials (RCTs) and evidence-based reviews through January 2009 concerning effects of consumption of NSBs on changes in body weight and adiposity. Studies included were those (i) conducted in humans; (ii) lasting at least 3 weeks; (iii) incorporating random assignment of subjects to conditions that differed only in the consumption of NSBs and (iv) including an adiposity indicator as an outcome. Twelve studies met the inclusion criteria. Meta-analysis of six studies that added NSBs to persons' diets showed dose-dependent increases in weight. Contrarily, meta-analysis of studies that attempted to reduce NSB consumption consistently showed no effect on body mass index (BMI) when all subjects were considered. Meta-analysis of studies providing access to results separately for subjects overweight at baseline showed a significant effect of a roughly 0.35 standard deviations lesser BMI change (i.e. more weight loss or less weight gain) relative to controls. The current evidence does not demonstrate conclusively that NSB consumption has uniquely contributed to obesity or that reducing NSB consumption will reduce BMI levels in general. We recommend an adequately powered RCT with overweight persons, for whom there is suggestive evidence of an effect.

Short-term dietary compensation in free-living adults.

Evidence suggests that compensatory behaviors operate in infants and pre-school children, such that the high variance characteristic of single eating occasions is much reduced over the day. However, the concept has not been fully explored in adults. The present within-subject, observational study investigated short-term dietary compensation patterns in fifty, weight-stable, normal weight (n=27), overweight (n=14), and obese (n=9) free-living adults (11 M, 39 F; age 30+/-11 y; BMI 26.3+/-5.9). Twenty four-hour diet recalls were obtained for 7 consecutive days, by the multi-pass technique. Each 24-h period was divided into 7 eating occasions. The coefficient of variation for energy intake was calculated for each adult, for each eating occasion, and over each 24-h period. Sub-group variability was assessed by BMI and frequency of consumption of sweetened energy-yielding beverages. The mean coefficient of variation for energy intake for the 7 eating occasions was 110.5%, compared to 28.9% for the day as a whole. Correlations between energy intakes at successive eating events were uniformly negative. No significant differences were noted in the sub-group analyses. Significantly greater variation in energy intake was noted for snacks compared to meals (P<0.0001). These data suggest that adults regulate energy intake over a 24-h period more closely than they do at individual eating occasions, similar to the pattern previously observed in children. Further studies of compensatory responses by larger sub-groups of individuals at risk for weight gain are warranted.

Peanut digestion and energy balance.

OBJECTIVE: To explore the effects of peanut consumption on fecal energy excretion with a balanced, non-vegetarian diet. DESIGN: Four arm parallel group design (that is, whole peanut (P), peanut butter (PB), peanut oil (PO) or peanut flour (PF) consumption) with one crossover (control and intervention). SUBJECTS: In total 63 healthy men and women from Ghana, Brazil and USA (N=15-16 per group) with an average body mass index of 21.8 kg m(-2). MEASUREMENTS: Percent fat of fecal wet weight daily energy excretion during the control and the treatment periods. RESULTS: Compared to control, the percentage of fat in the feces increased significantly for the P group (5.22+/-0.29%) relative to the other three groups ((PO=3.07+/-0.36%, PB=3.11+/-0.31% (P=0.001), and PF=3.75+/-0.40% (P=0.019)). The same findings held for kJ g(-1) of feces excreted. During the P supplementation period, the energy excretion was 21.4+/-1.0 kJ g(-1) versus 18.7+/-1.0 kJ g(-1) for PO (P=0.034), 18.8+/-0.7 kJ g(-1) for PB (P=0.042) and 18.5+/-0.8 kJ g(-1) for PF (P=0.028). CONCLUSION: Fecal fat and energy loss is greater with consumption of whole peanuts compared to peanut butter, oil or flour. This may contribute to the less than predicted change of body weight observed with peanut consumption. There were no cultural differences.

Effects of appetite, BMI, food form and flavor on mastication: almonds as a test food.

OBJECTIVES: To investigate the effects of appetitive sensations, body mass index (BMI) and physical/sensory properties of food (almonds) on masticatory indices and resultant pre-swallowing particle sizes. SUBJECTS/METHODS: Twelve lean (BMI=22.2+/-0.3) and 12 obese (BMI=34.3+/-0.6) adults. After collecting appetitive ratings, electromyographic recordings were used to assess participants' microstructure of eating for five almond products (raw, dry unsalted roasted, natural sliced, roasted salted and honey roasted) under fasted and satiated conditions. Duplicate samples were masticated to the point of deglutition and then were expectorated and size sorted. RESULTS: No statistically significant effects of BMI were detected for any of the mastication measures. Maximum and mean bite forces were greater under the fasted condition. Sliced almonds required lower bite force than did the other almond varieties. The pre-swallowing particle sizes were significantly greater for the sliced almonds than all other varieties. Both the number of chews and mastication time were negatively correlated with particle size. There were no significant effects of almond form or flavor on particle size. CONCLUSIONS: These results do not support differences in masticatory performance between lean and obese individuals, nor effects of sensory properties. Instead, the physical form of foods as well as an individuals' appetitive state may have a greater influence on masticatory behavior. The health implications of these observations warrant further investigation.

Effects of food form on appetite and energy intake in lean and obese young adults.

OBJECTIVE: To investigate the independent effect of food form on appetite and energy intake in lean and obese adults using high carbohydrate, fat or protein food stimuli. DESIGN: Crossover dietary challenge with matched beverage and solid food forms: high carbohydrate (watermelon and watermelon juice); high protein (cheese and milk); high fat (coconut meat and coconut milk). A total of 120 lean (18-23 kg/m(2); N=60) and obese (30-35 kg/m(2); N=60) adults (18-50 years old) with stable body weight. Forty different participants (N=20 lean and 20 obese) were tested with each of the food systems. MEASUREMENTS: Appetitive sensations, food palatability and dietary intake. RESULTS: Regardless of the predominant energy source, the beverage food form elicited a weaker compensatory dietary response than the matched solid food form. Thus, total daily energy intake was significantly higher by 12.4, 19 and 15% on days the beverage forms of the high-carbohydrate, -fat and -protein foods were ingested, respectively. This was due more to a weak effect on satiety than satiation. The obese participants had higher energy intake at the lunch, including the beverage high-protein load, but overall differences between lean and obese participants were small and not systematic. CONCLUSION: Food rheology exerts an independent effect on energy intake. Dietary compensation for beverages is weaker than for solid food forms of comparable nutrient content. Thus, they pose a greater risk for promoting positive energy balance.

Effects of solid versus liquid meal-replacement products of similar energy content on hunger, satiety, and appetite-regulating hormones in older adults.

OBJECTIVE: To examine whether solid versus liquid meal-replacement products differentially affect appetite and appetite-regulating hormones in older adults. METHODS: On two occasions, 9 subjects (age: 61+/-3 years; BMI: 25.6+/-1.3 kg/m (2)) consumed 25% of daily energy needs as solid or liquid meal-replacements of similar energy contents. Blood and appetite ratings were collected over 4 hours. RESULTS: The post-prandial hunger composite (area under the curve) was lower following the solid versus liquid meal-replacement (p<0.005) and remained below baseline over 4 hours (p<0.05). Similar responses were observed with the desire to eat. The insulin and ghrelin composites were lower following the solid trial compared to the liquid [insulin: 5825 (range: 4676-11639) VS. 7170 (4472-14169) uIU/l x 240 min, p<0.01; ghrelin: -92798 (range: -269130-47528) VS. -56152 (range: -390855-30840) pg/ml x 240 min, p<0.05]. Ghrelin also remained below baseline over 4 hours (p<0.05). No differences in cholecystokinin and leptin were observed between products. CONCLUSION: The consumption of comparable meal-replacement products in solid versus liquid versions with similar energy contents led to differential appetitive responses and should not be viewed as dietary equivalents in older adults.

Fluid calories and energy balance: the good, the bad, and the uncertain.

Energy-yielding fluids are a large and growing proportion of daily energy intake. The specific form and nutrient composition of fluids may hold divergent implications for energy balance. Ethanol elicits a weak compensatory dietary response, resulting in positive energy balance. However, its impact on body weight is unclear, possibly due to metabolic inefficiencies. In contrast, the weak dietary compensation for clear beverages containing other energy sources is associated with weight gain. How these beverages elude satiety mechanisms has not been studied. Soups hold higher satiating value, at least in part, due to cognitive factors. Nutrient dense beverages have been used successfully in meal replacement regimens for weight management, but due to their relatively weak satiety value, are widely consumed for weight gain and as nutrient supplements. A better understanding of the role of fluid calories in the diet is needed to improve dietary guidelines.

Crystallization and preliminary X-ray diffraction studies of two thermostable alpha-galactosidases from glycoside hydrolase family 36.

alpha-Galactosidases from thermophilic organisms have gained interest owing to their applications in the sugar industry. The alpha-galactosidases AgaA, AgaB and AgaA A355E mutant from Geobacillus stearothermophilus have been overexpressed in Escherichia coli. Crystals of AgaB and AgaA A355E have been obtained by the vapour-diffusion method and synchrotron data have been collected to 2.0 and 2.8 A resolution, respectively. Crystals of AgaB belong to space group I222 or I2(1)2(1)2(1), with unit-cell parameters a = 87.5, b = 113.3, c = 161.6 A. Crystals of AgaA A355E belong to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 150.1, c = 233.2 A.

Effects of peanut oil consumption on appetite and food choice.

OBJECTIVE: Peanut consumption may improve lipid profiles without promoting weight gain. Both properties have been attributed to their high-unsaturated fat content. Mono and polyunsaturated fatty acids reportedly hold stronger satiety value than saturated fats and may help appetite control. This study investigated the effects of chronic peanut oil consumption on appetite and food choice. RESEARCH METHODS AND PROCEDURES: A total of 129 healthy adults from three countries (Brazil, Ghana and US) were randomly assigned to one of four treatment arms: consumption of peanut oil, olive oil or safflower oil as 30% of individual resting energy expenditure (REE) for 8 weeks or no dietary intervention. Participants received no other dietary guidance. They completed appetite questionnaires eliciting information about hunger, fullness, desire to eat, and prospective consumption during all waking hours for 1 day at weeks 2 and 6 and for 1 or 3 days at weeks 0, 4 and 8. Diet records were completed at weeks 0, 4 and 8. RESULTS: No differences in appetitive ratings were observed over the 8-week trial. There were no significant treatment by time interactions. Total caloric intake was significantly higher at week 8 relative to baseline (F=10.08, P<0.05). The increases for each treatment were: peanut oil=197+/-114; olive oil=237+/-121; safflower oil=274+/-90; control=75+/-71. Free-feeding intake, an index of dietary compensation, was reduced significantly at weeks 4 and 8 compared to baseline (F=9.08, P<0.00). The declines (compensation scores) were (kcals): peanut oil=-208+/-105 (46%); olive oil=-235+/-105 (50%); safflower oil=-186+/-102 (44%). There were no significant differences across countries in appetite ratings. DISCUSSION: A prior intervention with whole peanuts reported a dietary compensation score of 66% over 8 weeks, this compares to a 46% compensation score observed with peanut oil. Our data suggests that the lipid fraction in peanuts elicits a weak effect on satiety.

Effects of chronic peanut consumption on energy balance and hedonics.

OBJECTIVE: To investigate the effects of chronic peanut consumption on energy balance and hedonics. DESIGN: Thirty-week, cross-over, intervention study. Participants were provided 2113+/-494 kJ/day (505+/-118 kcal/day) as peanuts for 8 weeks with no dietary guidance (free feeding-FF), 3 weeks with instructions to add peanuts to their customary diet (addition-ADD) and 8 weeks where peanuts replaced an equal amount of other fats in the diet (substitution-SUB). SUBJECTS: Fifteen, healthy, normal-weight (BMI of 23.3+/-1.8) adults, aged 33+/-9 y. MEASUREMENTS: Dietary intake, appetitive indices, energy expenditure, body weight and hedonics. RESULTS: During FF, peanut consumption elicited a strong compensatory dietary response (ie subjects compensated for 66% of the energy provided by the nuts) and body weight gain (1.0 kg) was significantly lower than predicted (3.6 kg; P<0.01). When customary dietary fat was replaced with the energy from peanuts, energy intake, as well as body weight, were maintained precisely. Participants were unaware that body weight was a research focus. Resting energy expenditure was increased by 11% after regular peanut consumption for 19 weeks (P<0.01). Chronic consumption of peanuts did not lead to a decline in pleasantness or hunger ratings for peanuts nor did it lead to any hedonic shift for selected snack foods with other taste qualities during any of the three treatments. CONCLUSIONS: Despite being energy dense, peanuts have a high satiety value and chronic ingestion evokes strong dietary compensation and little change in energy balance.

The taste of fat elevates postprandial triacylglycerol.

To explore the contributions of taste and smell stimulation by dietary fat on the postprandial rise of serum triacylglycerol (TAG), 19 healthy, overnight-fasted adults received lipid loads (50 g safflower oil in capsules) followed by oral (i.e., taste and smell) or odor-only stimulation with cream cheese on crackers or no load with odor or no stimulation. In eight participants, lipid loading was followed by taste-only stimulation. Stimulation was provided (where applicable) at 3-min intervals for 60 min and 15-min intervals for an additional 60 min. Blood was drawn at stipulated times. Fat loading with oral stimulation led to serum TAG concentrations that were significantly elevated over baseline at 2, 4, and 6 h. Fat loading with odor stimulation led to a significant rise only at 4 h. No significant elevation was observed with either no-load treatment. Taste-only treatment led to a significantly higher TAG response than all other treatments except loading with oral stimulation. These data provide additional evidence supporting a taste component for human fat perception.

Oral stimulation influences postprandial triacylglycerol concentrations in humans: nutrient specificity.

OBJECTIVE: To determined whether the nature of the lipid in an oral stimulus modifies postprandial triacylglycerolemia. METHODS: Sixteen healthy adults (eight male, eight female) participated in six test sessions conducted weekly. The test sessions were administered randomly after overnight fasts and included: ingestion of 50 grams of butter in capsules (to avoid oral stimulation with lipids) and 500 mL of water in 15 minutes followed by oral stimulation with one of the following foods on a cracker: butter, unsaturated fatty acid (UFA) margarine, jelly, UFA margarine + jelly, cracker alone or no oral stimulation. Sensory stimulation entailed masticating and expectorating approximately 5.0 g samples of each stimulus every three minutes for 110 minutes. Blood was drawn immediately after preload ingestion and at minutes 35, 85, 200, 320, and 440 post loading and analyzed for serum triacylglycerol (TAG), insulin and glucose concentrations. RESULTS: Only the oral samples containing the UFA margarine led to significant elevations of serum TAG concentration compared to baseline (p < 0.05). Maximum change of TAG concentrations were greater following orosensory stimulation with UFA margarine compared to orosensory stimulation with butter, jelly or UFA margarine + jelly. No differences were observed relative to vehicle alone or no orosensory stimulation, but this is due to lower nadir values for these treatments. Insulin and glucose concentrations were not different between treatments. CONCLUSION: Oral exposure specifically to an unsaturated dietary lipid augments the postprandial rise of TAG, compared to baseline.

Modulation of the regioselectivity of a Bacillus alpha-galactosidase by directed evolution.

Alpha-galactosidase AgaB of Bacillus stearothermophilus was subjected to directed evolution in an effort to modify its regioselectivity. The wild-type enzyme displays a major 1,6 and minor 1,3 regioselectivity. We used random mutagenesis and staggered extension process (StEP) to obtain mutant enzymes displaying modified regioselectivity. We developed a screening procedure allowing first the elimination of AgaB mutants bearing the 1,6 regioselectivity and secondly the selection of those retaining a 1,3 regioselectivity. Our results show that, among the evolved enzymes that have lost most of their activity towards the 1,6 linkage both in hydrolysis and in synthesis, one (E901) has retained its 1,3 activity. However the transglycosylation level reached by this mutant is quite low versus that of the native enzyme. This work constitutes the first example of modification of glycosylhydrolase regioselectivity by directed evolution.

The production of improved tissue-type plasminogen activator in Escherichia coli.

Tissue-type plasminogen activator (t-PA) is a valuable thrombolytic agent because of its high affinity to fibrin. When produced in mammalian cell lines, it is glycosylated, a modification that is believed to promote its rapid clearance from the circulation. Bacteria such as Escherichia coli have been tested as alternative expression systems but were not able to express the cDNA of t-PA effectively. The coding sequence for t-PA revealed a significant proportion of AGA and AGG codons, which are rarely used in the coding sequences of E. coli. The argU and argW gene products of E. coli proved to be minor tRNA(arg) species, respectively decoding the very rare triplets AGA/AGG and AGG for arginine. Analysis of genomic fragments from E. coli for both tRNA(arg) genes revealed the presence of defective, cryptic prophages integrated within the impaired tRNA genes. Cloning and supplementation of the limiting tRNA genes argU and argW on helper plasmids improved the translation of the rare AGA and AGG codons. This augmentation improved bacterial growth and enhanced t-PA production in the form of inactive inclusion bodies. This dependence on augmentation of tRNA(arg4) or tRNA(arg5) for improved cell growth and expression was also observed for other genes with a high content of these rare arginine codons. Construction and production of nonglycosylated t-PA in inclusion bodies in E. coli along with improvement of the subsequent renaturation and purification procedures resulted in material comparable to that derived from CHO cells. Deletion of domain-encoding segments yielded various "muteins" of t-PA (e.g., reteplase [rPA]) that could be produced in and activated from the purified inclusion bodies analogously. Furthermore, it was shown that rPA has an extended half-life in the circulation because of its lack of glycosylation and impaired receptor binding capability. rPA was successfully used in various clinical studies. It is a new-generation thrombolytic agent with a longer half-life and can thus be administered more conveniently as a double bolus.