A quantitative description of components of in vitro morphometric change in the rat osteoclast model: relationships with cellular function.

We describe the in vitro morphometric changes shown by rat osteoclasts that accompany their functional responses to the application of a range of regulatory agents of known physiological importance. We introduce a cellular motility parameter, mu, which was defined through a quantification of retraction-protrusion behaviour. This was used in conjunction with a net cell retraction, rho, which is derived from the change in total cell area following the application of an agent. These terms were used together for the description of cellular motility changes in response to specific cellular regulatory agents. The definition of retraction-protrusion was normalised against control cell area, to give a dimensionless variable independent of the net cell retraction. Thus, mutual terms present in either descriptor cancelled when the complementary parameter was held constant. Furthermore, the descriptor, mu remained time-invariant for extended intervals (around 20 min) even when rho was varying following cell introduction into culture. Interventions also with substances known to modify osteoclast function, were capable of altering each descriptor, to different extents. Thus elevation of the extracellular Ca2+ concentration ([Ca2+]e) at the osteoclast calcium "receptor" altered rho without changes in mu. In contrast, the polypeptide amylin (250 nM), within 20 minutes of application, elicited a marked change in mu, but only a relatively small change in rho. Finally, human calcitonin treatment (300 pM) influenced both descriptors. When combined together, these morphometric findings accordingly offer complementary descriptions of visible cellular changes in response to added agents of physiological relevance.(ABSTRACT TRUNCATED AT 250 WORDS)


Application of dynamic light scattering to studies of protein folding kinetics.

The applicability of dynamic light scattering to studies of the kinetics of unfolding and refolding reactions of proteins is discussed and demonstrated experimentally. The experimental set-up and the data acquisition and data evaluation schemes that have been optimized for kinetic experiments are described. The relationship of the signal-to-noise ratio to the minimum data acquisition time that is needed to obtain results of sufficiently high precision is discussed. It turns out that the attainable time resolution is of the order of a few seconds for proteins with molar masses of about 50,000 g.mol(-1) and concentrations of 1 g.1(-1). Thus, DLS is too slow to follow conformational changes in the subsecond region, but it is useful for studies of unfolding-refolding reactions of proteins that proceed with time constants in the range of seconds or minutes. This is demonstrated by investigations of the kinetics of the cold denaturation of 3-phosphoglycerate kinase from yeast.


Dietary fat intake and plasma lipid levels in adolescents.

The relationship between dietary fat intake and fasting plasma lipid levels was assessed in 35 female and 19 male adolescents recruited from two local education authority schools in Norwich, UK. Dietary intakes were assessed using a 7-day weighed dietary record method, coupled with the collection of duplicate diets. Fat and energy intakes calculated using food composition tables were compared with values obtained by direct analysis of duplicate diets. Fasting plasma lipid levels (total, HDL and LDL cholesterol and triglycerides) were measured and compared with total dietary lipids and fatty acid intakes. The average proportion of energy consumed as fat was higher than is currently considered desirable but lower than previous studies have reported for adults. Mean serum total cholesterol values were 4.2 (SEM 0.1) mmol for females and 4.5 (SEM 0.2) mmol for males; this difference was not statistically significant. In male subjects the dietary fatty acid profiles were significantly correlated with several parameters of plasma lipid status which are thought to be risk factors for coronary heart disease, and in particular with the ratio of total:HDL cholesterol.


Variability of body density and body impedance at different frequencies.

Body density and body impedance at 15 selected frequencies ranging from 1 to 1350 kHz were determined in 13 subjects on four consecutive days of the week. Bioelectrical impedance at the same frequencies was also determined in 11 subjects on the same day of the week during four consecutive weeks. Day-to-day variability of body density and body impedance and week-to-week variability of body impedance were studied. Statistical analyses did not reveal significant day- or week-effects in variabilities. The mean within-person variation in body density between days was 0.0019 kg/l (CV 0.2%). Mean within-person variation in body impedance between days was 57 ohms (CV 8.7%) at 1 kHz. At 5, 50 and 100 kHz the within-person variation between days was 16 ohms (CV 2.4%), 12 ohms (CV 2.1%) and 11 ohms (CV 2.0%), respectively. Mean within-person variation between weeks was 44 ohms (CV 2.4%) at 1, 5, 50 and 100 kHz, respectively. Overall the within-subject variability in impedance at all frequencies was higher in females. The larger mean within-person variation in impedance at 1 kHz could only partly be explained by variation in body weight and may be a real error in the measurement. Electrodes with a larger surface area reduced the variability to values comparable with those at higher frequencies. The day-to-day variation in impedance at higher frequencies causes an error in calculated FFM, which is about two times larger compared to the estimated error in FFM by body density.


Vitamin A metabolism in plasma of normal and anorectic women.

Plasma retinol and retinyl ester kinetics were determined following an oral bolus of vitamin A in normal women and in women suffering from anorexia nervosa. Circulating levels of vitamin A and alpha-tocopherol were lower in anorectics than in normals with alpha-tocopherol levels close to minimal adequate levels. This suggests the possibility of deficiencies of other dietary factors. Following an oral bolus, plasma retinol increased more in anorectics than in normals, but transport values were not changed. Retinyl ester levels increased sevenfold more in anorectics than normals with a parallel enhancement in transport. Retinyl esters in plasma of normals and anorectics were present mainly in low-density lipoprotein with lesser amounts in very-low-density and high-density lipoprotein, but only minor amounts were found in chylomicrons. These data were used to calculate a relative dose response and indicated that anorectics had higher values than normals. Anorectics appear to have a lower status of vitamins A and E than normals but no defect in vitamin A metabolism was observed.


Diet, body size and micronutrient status in Parkinson's disease.

Nutritional status was assessed in a group of patients with Parkinson's disease. Weight loss since the onset of disease occurred in 52% of the patients and 22% had lost more than 12.8 kg. Although 67% of patients experienced eating difficulties of some kind, dietary intakes of protein and energy were not significantly lower than recommended intakes. Plasma levels of albumin (44.2 g/l vs 45.7 g/l), vitamin A (2.61 vs 2.94 mumol/l), vitamin E (22.0 vs 32.0 mumol/l), iron (15.3 vs 18.3 mumol/l) and zinc (14.2 vs 18.7 mumol/l) were significantly lower (P < 0.05) in the patients than in healthy controls. Levels of ferritin, total iron-binding capacity and copper were similar between groups. The potential significance of low levels of vitamin E and zinc are discussed in relation to oxidative stress in the pathogenesis of this disease.


Nutritional status of children of urban leprosy patients staying at preventoria based on biochemical parameters.

Measurements of nutritionally relevant biochemical and endocrine variables were made on 60 apparently healthy children (group A) whose parents suffered from leprosy and who had been separated at the age of 4 years and brought up in preventoria. Most of the measurements were also made on a comparison group of healthy children from the same poor socio-economic class (group B). In both groups the serum concentrations of cholesterol and triglycerides were well below those found in Western populations. Almost all the children in both groups were anaemic, but serum iron and ferritin levels were satisfactory. Folate and vitamin B12 levels were measured in group A only and were low in a significant proportion. Deficiency of these water-soluble vitamins may be a cause of the anaemia. Low albumin levels were found in 40% of group A children, compared with 2% in group B. The concentrations of calcium and magnesium were lower and that of phosphate higher in group A than in B. In both groups one-third of the children had low levels of serum zinc. Fifteen per cent of group A children had biochemical evidence of vitamin A deficiency, but none were deficient in vitamin E. Levels of total T3 and total T4 were below the lower limit of normal in a substantial proportion of children in both groups. Concentrations of parathyroid hormone were increased in parallel with the low values for serum calcium. Radiological studies of ossification centres in 57 group A children showed delayed maturation in 11 cases. The relevance of these findings to previous studies of the children of lepers in India is discussed.


The effect of a high protein-low calorie diet on the energy expenditure of obese adolescents.

Resting energy expenditure (REE), and body composition, as fat-free mass (FFM) and fat mass, were determined in seven obese adolescents before and after weight loss of a mean 13.5 kg on an approximately 800 kcal/d (3349 kJ), high protein reducing diet regimen. Ideal body weight decreased from 166% to 142% in 8 weeks. There were no significant changes in total body potassium (TBK), extracellular water (ECW), intracellular water (ICW) or total body water (TBW) with weight loss. The REE (kcal/d) fell from 2034 +/- 392 (8514 +/- 1641 kJ) to 1762 +/- 453 (7376 +/- 1896 kJ) with weight loss (P < 0.05). However, when the REE was expressed as kcal/body weight there was no difference between before and after weight loss, 21.4 +/- 2.8 (90 +/- 21 kJ) and 21.6 +/- 4.5 (90 +/- 19 kJ). Similarly, when REE was examined in relation to FFM (kcal/kg) before and after weight loss, there were also no significant differences: 34.6 +/- 5.1 (145 +/- 21 kJ) and 32.1 +/- 7.9 (134 +/- 33 kJ).


Fascial planes within subcutaneous fat in humans.

We have used B-mode (brightness-mode) ultrasound to investigate the fascial planes within subcutaneous fat at the triceps and abdominal sites in a group of 17 women attending a weight control group over a 12 month period. In most subjects there was a single intralipid fascial plane at each site. As the thickness of adipose tissue increased, most of the change at the abdominal site was in the deep rather than the superficial layer of fat. At the triceps site both deep and superficial layers increased. Our findings confirm the presence of two different layers in human subcutaneous fat at the triceps and abdominal sites. These layers have been shown to be functionally different in animals and our study supports this in humans at the abdominal site.


On the use of dimension-less parameters in acid-base theory. I. The buffer capacity of simple ampholyte solutions.

A complete mathematical account of the buffer capacity of bivalent ampholytes is given. The consistent use of dimensionless parameters makes it possible to obtain very neat expressions and equations. Direct proof of the well-known condition K1 > or = 4 K2 is presented. Since water is also an ampholyte, its buffer capacity is considered as well.


Polyhydroxy and polyethyleneglycol (meth)acrylate polymers: physical properties and general studies for their use as electrophoresis matrices.

A new series of materials have been tested for their suitability as electrophoresis matrices. The mechanical and optical properties of gels composed of polyethyleneglycol (meth)acrylate esters or polyhydroxy (meth)acrylate esters in water and in various concentrations of organic solvents are described. Several crosslinkers including polyethyleneglycol and polyhydroxy di(meth)acrylates, piperazine diacrylate, and bisacrylamide were used in these studies. Electrophoretic migration and separation of a series of protein standards through polyethyleneglycol methacrylate (PEGM) 200, PEGM 400, and glyceryl methacrylate is demonstrated. Further, copolymerization of all of the monomers with acrylamide was performed and the distribution of monomer incorporation into the polymer network calculated. All monomers and copolymers that were examined by IR spectroscopy showed greater than 99% polymerization. These results justify their further study for biomolecule separations.


Polyethyleneglycol methacrylate 200 as an electrophoresis matrix in hydroorganic solvents.

The properties of gels composed of poly(polyethyleneglycol methacrylate) and copolymers of polyethyleneglycol methacrylate and acrylamide were studied. These novel electrophoresis matrices are amphigels (swellable in water and organic solvents) that have unprecedented organic solvent compatibility. Hydrophobic proteins which are poorly solubilized in aqueous detergent systems (e.g., zein) are well-resolved in these gels with hydro-organic solvents. This is especially relevant for isoelectric focusing, to avoid using either ionic detergents that may interfere with the focusing, or urea that may cause carbamylation of proteins. Variations of crosslinker, buffer, and solvent systems in these gels were explored.


Investigation of the electroosmotic flow effect on the efficiency of capillary electrophoresis.

The influence of the electroosmotic flow profile on the efficiency and resolution of capillary electrophoresis is studied. The mathematical model is formulated and the set of equations is solved numerically. The results of the analysis are applicable to a wide range of buffer concentrations and capillary diameters. The temperature dependence of electrophoretic mobility, viscosity and thermal conductivity and the dependence of electrical conductivity on temperature and ion concentration in the double layer are taken into account. It is shown that there exists a region of buffer concentrations and capillary diameters where the influence of the electroosmotic flow profile on the efficiency and resolution is much greater than that of the temperature dependence of the electrophoretic mobility. The results are especially essential for small buffer concentrations or capillary diameters comparable with the double electrical layer thickness.


Computer simulation of transient states in capillary zone electrophoresis and isotachophoresis.

Transient states in the evolution of electrophoretic systems comprising aqueous solutions of weak monovalent acids and bases are simulated. The mathematical model is based on the system of nonstationary partial differential equations, expressing the mass and charge conservation laws while assuming local chemical equilibrium. It was implemented using a high resolution finite-difference algorithm, which correctly predicted the behavior of the concentration, pH and conductivity fields at low computational expense. Both the regular and the irregular modes of separation in capillary zone electrophoresis and isotachophoresis are considered. It is shown that the results of separation, particularly zone order, strongly depend on pH distribution. Simulation data as well as simple analytical assessments may help to predict and correctly interpret the experimental results.


Identification of gram-negative bacteria by isoelectric focusing of soluble cellular proteins.

Strains of Escherichia coli, Pseudomonas aeruginosa and Proteus mirabilis, isolated from clinical specimens were disrupted by repeated freezing in liquid nitrogen and thawing at room temperature. The samples were separated by isoelectric focusing using polyacrylamide gels. The resulting protein patterns showed clear differences between the three species and made identification possible.


Two-dimensional gel electrophoresis analysis of Syrian hamster embryo cells: morphological transformation is not cell type specific.

Studies were conducted to investigate the protein phenotype of normal and morphologically transformed Syrian hamster embryo (SHE) cells. Based upon two-dimensional gel protein phenotype analysis, we conclude that (i) SHE cells are a mixture of multiple cell types including mesenchymal and epithelial cells and (ii) several cell types present in the SHE cell population can be morphologically transformed by a variety of genotoxic and non-genotoxic carcinogens.


Quantitative two-dimensional gel electrophoresis analysis of human fibroblasts transformed by ras oncogenes.

Quantitative two-dimensional gel electrophoresis was used to compare the cellular protein patterns of a normal foreskin-derived human fibroblasts cell line (LG1) and three immortal derivatives of LG1. One derivative, designated MSU-1.1 VO, was selected for its ability to grow in the absence of serum and is non-tumorigenic in athymic mice. The other two strains were selected for focus-formation following transfection with either Ha-ras or N-ras oncogenes and form high grade malignant tumors. Correspondence and cluster analysis provided a nonbiased estimate of the relative similarity of the different two-dimensional patterns. These techniques separated the gel patterns into three distinct classes: LG1, MSU-1.1 VO, and the ras transformed cell strains. The MSU-1.1 VO cells were more closely related to the parental LG1 than to the ras-transformed cells. The differences between the three classes were primarily quantitative in nature: 16% of the spots demonstrated statistically significant changes (P < 0.01, T test, mean ratio of intensity > 2) in the rate of incorporation of radioactive amino acids. The patterns from the two ras-transformed cell strains were similar, and variations in the expression of proteins that occurred between the separate experiments obscured consistent differences between the Ha-ras and N-ras transformed cells. However, while only 9 out of 758 spots were classified as different (1%), correspondence analysis could consistently separate the two ras transformants. One of these spots was five times more intense in the Ha-ras transformed cells than the N-ras.(ABSTRACT TRUNCATED AT 250 WORDS)


Superimposing two-dimensional gels to study genetic variation in malaria parasites.

Two-dimensional polyacrylamide gel electrophoresis is a valuable tool for studying genetic variation in the human malaria parasite, Plasmodium falciparum. It involves examining the position of protein spots in gel produced from different isolates. Some spots have been seen to vary, while others have had a constant position in all isolates so far examined. These invariant spots provide a reference frame to compare variations in other spots. This paper discusses the usefulness of digital image handling, warping and superimposition in a personal computer environment. Rather than produce a fully automatic interpretation system, we show how the computer may be used as a tool for manipulating gel images, although interpretation of the gels' features remains with the human expert. Autoradiographs are scanned on a desktop scanner, and the images in digital form can be displayed on a monitor attached to a personal computer. The coordinates of the invariant spots on each of several gels are identified by the user. Each of the gels is then warped so that the invariant spots of all the gels coincide as closely as possible. The variable spots are then examined. We have used both affine warping transformations, which match the invariant spots as closely as possible, and thin plate spline transformations, which match them exactly. Colour superimposition proved a useful way of examining the gels.


The local reciprocal method for sizing DNA fragments: an obscure limitation.

The local reciprocal method, for sizing DNA fragments, has been found to fail when the experimental data produces a linear relationship. A simple solution for computerised systems is to test for this unexpected condition.


A simple and rapid method for HLA-DQA1 genotyping by polymerase chain reaction-single strand conformation polymorphism and restriction enzyme cleavage analysis.

A simple and rapid method for identification of alleles at the human leucocyte antigen (HLA)-DQA1 locus is described. The polymorphic second exon of the HLA-DQA1 locus was amplified by the polymerase chain reaction (PCR) method. The amplified DNA was analyzed by single-strand conformation polymorphism (SSCP) and restriction enzyme cleavage assay. Using this method, the eight known DQA1 alleles could be distinguished from each other. This paper suggests that the method can be used for quick genotyping of DQA1 alleles, but detecting point mutations at various positions in a fragment as well as new HLA-DQA1 genotypes should also be possible.


The terminator, an apparatus for simultaneous homogenization of 96 small seeds individually.

An apparatus was developed to homogenize 96 individual seeds of tomato, pepper, cabbages, and other seeds of similar size in a 96-well tissue culture plate in 45 s. The apparatus, named the terminator, consists of two parts: a base plate that can hold the 96-well tissue culture plate, and a vibrating part that is attached to a variable transformer. The seeds are homogenized by the vibrating action of 96 small homogenizers that are attached to the vibrating part and that fit perfectly into the wells of the tissue culture plate. The seed homogenates made in this way can be used for electrophoretic analysis of genetic variations of seed proteins.


Preincubation with cysteine prevents modification of sulfhydryl groups in proteins by unreacted acrylamide in a gel.

It has been found that the double bond of free, unreacted acrylamide in a gel can react with a free -SH group of proteins, forming a cysteinyl-S-propionamide adduct. When beta-lactoglobulin was incubated at concentration levels lower than those of free acrylamide, left after polymerizing a 5% T, 4% C gel (barely 12 mM), under anaerobic conditions in 0.1 M borate, pH 9.5, for 1 h and then the tryptic digests analyzed by high performance liquid chromatography (HPLC), two new peptides were detected. The two new peaks were recovered and sequenced by the Edman degradation procedure. They correspond to the sequence from Leu-149 to Ile-162. Residue No. 160 was found to be a cysteinyl-S-propionamide reaction product. Interestingly, only this residue, out of a total of 5 Cys residues, had reacted. No other amino acids (including -NH2 terminus and free -NH2 in Lys) reacted with free acrylamide. The addition of free acrylamide to the -SH group could be completely inhibited if: (i) the gel was extensively washed prior to sample application, or (ii) the gel was incubated for 1 h in 100 mM free Cys.


Chicory seed lot variety identification by leucine-aminopeptidase and esterase zymogram analysis.

A method of identifying closely related chicory varieties has been developed using native polyacrylamide gel electrophoresis and subsequent leucine aminopeptidase and esterase staining of bulked seed sample extracts. The variety zymograms are analyzed according to a mobility-density scheme. This quick, easy and low-cost method can be a useful tool for chicory breeders.


Marathon running: physiological and chemical changes accompanying late-race functional deterioration.

Twenty-one experienced runners were studied before, during and immediately after a marathon race to ascertain whether either depletion of energy substrate or rise in body temperature, or both, contribute to late-race slowing of running pace. Seven runners drank a glucose/electrolyte (GE) solution ad libitum (Na+ 21 mmol l-1, K+ 2.5 mmol l-1, Cl- 17 mmol l-1, PO4(2-) 6 mmol l-1, glucose 28 mmol l-1) throughout the race; 6 drank water and 8 drank the GE solution diluted 1:1 with water. Although average running speeds for the three groups were not significantly different during the first two-thirds (29 km) of the race, rectal temperature was significantly higher (P < 0.05) and reduction of plasma volume was greater (P < 0.05) in runners who replaced sweat losses with water. During the last one-third of the race, the average running pace of the water-replacement group slowed by 37.2%; the pace slowed by 27.9% in the 8 runners who replaced their sweat loss with GE diluted 1:1 with water (1/2 GE) and 18.2% in runners who replaced fluid loss with full-strength solution (GE). Eleven runners (5 in the water group, 4 in the 1/2 GE group and 2 in the GE group) lapsed into a walk/run/walk pace during the last 6 miles of the race. Ten of these had a rectal temperature of 39 degrees C or greater after 29 km of running, and plasma volume in these runners was reduced by more than 10%.(ABSTRACT TRUNCATED AT 250 WORDS)


Thermoregulatory responses of young and older men to cold exposure.

Nine young (20-25 years) and ten older (60-71 years) men, matched for body fatness and surface area:mass ratio, underwent cold tests in summer and winter. The cold tests consisted of a 60-min exposure, wearing only swimming trunks, to an air temperature of 17 degrees C (both seasons) and 12 degrees C (winter only). Rectal (Tre) and mean skin (Tsk) temperatures, metabolic heat production (M), systolic (BPs) and diastolic (BPd) blood pressures and heart rate (fc) were measured. During the equilibrium period (28 degrees C air temperature) there were no age-related differences in Tre, Tsk, BPs, BPd, or fc regardless of season, although M of the older men was significantly lower (P < 0.003). The decrease in Tre and Tsk (due to the marked decrease in six of the older men) and the increase in BPs and BPd were significantly greater (P < 0.004) for the older men during all the cold exposures. The rate of increase in M was significantly greater (P < 0.01) for the older group when exposed to 12 degrees C in winter and 17 degrees C in summer (due to the marked increase in four of the older men). This trend was not apparent during the 17 degrees C exposure in winter. There was no age-related difference in fc during the exposures. Significant decreases in Tre and Tsk and increases in M, BPs and BPd during the 12 degrees C exposure were observed for the older group (P < 0.003) compared to their responses during the 17 degrees C exposure in winter. In contrast, Tre, M, BPs in the young group were not affected as much by the colder environment. It was concluded that older men have more variable responses and some appear more or less responsive to mild and moderate cold air than young men.


Influence of body position and pre-exercise activity on cardiac output and oxygen uptake following step changes in exercise intensity.

Parallel measurements of breath-by-breath oxygen uptake, cardiac output (Doppler technique), blood pressure (Finapres technique) and heart rate were performed in nine subjects during cycle ergometer exercise in the upright and supine positions. Transients were monitored during power steps starting from and leading to either rest or lower levels of exercise intensity. Oxygen uptake (VO2) and cardiac output kinetics were markedly faster than in all other conditions when exercise was started from rest. In contrast to exercise-exercise on steps, the computed arteriovenous difference in O2 content increased almost immediately in this situation, indicating that not only the additional energy expenditure due to the acceleration of the flywheel but also an increased venous admixture from non-exercising parts of the body contributed to the early kinetics. The off kinetics generally showed a more uniform pattern and did not simply mirror the on transients. The present findings indicate that transitions from rest should be avoided when muscle VO2 kinetics are to be assessed on the basis of VO2 measurements at the mouth.


Muscle strength according to level of physical exercise and educational background in middle-aged women in Finland.

The aim of this study was to examine the role of socio-economic status and the practice of physical exercise in explaining variation in muscle strength in 50 to 60-year-old women. Consequently, four study groups combining education and physical activity were formed: (1) university education, physically active; (2) university education, sedentary; (3) vocational or lower level of education, physically active; (4) vocational or lower level of education, sedentary. Maximal isometric strength of hand grip, arm flexion, body flexion and extension as well as dynamic power of the abdominal muscles were measured in 112 women. The results of the maximal isometric strength measurements were standardised by body mass index. The means of the maximal isometric strength results and sit-up tests were compared between the groups using two-way analysis of variance. The physically active women performed better than the sedentary in both the isometric and dynamic tests. Greater muscle strength was also found among the more highly educated compared with the less educated. The less educated sedentary women showed the poorest performance in the strength tests.


Effects of carbohydrate feedings on plasma free tryptophan and branched-chain amino acids during prolonged cycling.

Brain serotonin (5-hydroxytryptamine, 5-HT) has been suggested to be involved in central fatigue during prolonged exercise. Changes in the ratio of plasma free tryptophan (free Trp) to branched-chain amino acids (BCAA) are associated with altered brain 5-HT synthesis. The purposes of this study were to describe systematically the effects of prolonged exercise on changes in plasma free Trp and BCAA and to examine the effects of carbohydrate (CHO) feedings on these same variables. Eight well-trained men [VO2max = 57.8 (SE 4.1) ml kg-1 min-1] cycled for up to 255 min at a power output corresponding to VO2 at lactate threshold (approximately 68% VO2max) on three occasions separated by at least 1 week. Subjects drank 5 ml kg-1 body wt-1 of either a water placebo, or a liquid beverage containing a moderate (6% CHO) or high (12% CHO) concentration of carbohydrate beginning at min 14 of exercise and every 30 min thereafter. Exercise time to fatigue was shorter in subjects receiving placebo [190 (SE 4) min] as compared to 6% CHO [235 (SE 10) min] and 12% CHO [234 (SE 9) min] (P < 0.05). Glucose and insulin decreased in the placebo group, and free Trp, free-Trp/BCAA, and free fatty acids increased approximately five- to sevenfold (P < 0.05). These changes were attenuated in a dose-related manner by the carbohydrate drinks.(ABSTRACT TRUNCATED AT 250 WORDS)


Physical activity and energy expenditure in lean and obese adult human subjects.

We compared the physical activity of 11 lean and 11 obese men and women over a 7-day period. There were no significant differences in either the amount of movement recorded with an accelerometer (9.5 (SD 3.9) vs 9.9 (SD 2.6) kcounts.day-1), or in the energy expenditure due to physical activity reflected by the difference between the average daily metabolic rate measured by the doubly labelled water technique and the sleeping metabolic rate measured in a respiration chamber and adjusted for fat-free mass: 112 (SD 33) vs 118 (SD 22) kJ.kg-1.day-1. The obese showed a non-significant loss of body mass of 0.5 (SD 1.1) kg, probably due to reduced intake during the 7-day intake recording period.


Differential mobilization of leucocyte and lymphocyte subpopulations into the circulation during endurance exercise.

A total of 14 healthy subjects [means (SD): 27.6 (3.8) years; body mass 77.8 (6.6) kg; height 183 (6) cm] performed endurance exercise to exhaustion at 100% of the individual anaerobic threshold (Th(an)) on a cycle ergometer (mean workload 207 (55) W; lactate concentrations 3.4 (1.2) mmol.l-1; duration 83.8 (22.2) min, including 5 min at 50% of individual Th(an)). Leucocyte subpopulations were measured by flow cytometry and catecholamines by radioimmunological methods. Blood samples were taken before and several times during exercise. Values were corrected for plasma volume changes and analysed using ANOVA for repeated measures. During the first 10 min of exercise, of all cell subpopulations the natural killer cells (CD3-CD16/CD56+) increased the most (229%). Also CD3+CD16/CD56+ (84%), CD8+CD45RO- (69%) cells, eosinophils (36%) and monocytes (62%) increased rapidly during that time. CD3+, CD3+HLA-DR+, CD4+CD45RO+, CD4+CD45RO-, CD8+CD45RO+ and CD19+ cells either did not increase or increased only slightly during exercise. Adrenaline and noradrenaline increased nearly linearly by 36% and 77% respectively at 10 min exercise. The increase of natural killer cells and heart rates between rest and 10 min of exercise correlated significantly (r = 0.576, P = 0.031). We conclude that natural killer cells, cytotoxic, non-MHC-restricted T-cells, monocytes and eosinophils are mobilized rapidly during the first minutes of endurance exercise. Both catecholamines and increased blood flow are likely to contribute this effect.


Reduced short-term thermic effects of a meal in obese adolescent girls.

Post-meal energy expenditure (TEM) was compared for 14 healthy obese (body fat = 45.3%, body mass index, BMI = 35.9 kg m-2) and 9 healthy nonobese (body fat = 20.7%, BMI = 17.8 kg m-2) adolescent girls. The test meal for both groups was a standard 3348.8-kJ, 0.473-1 chocolate milkshake of 15% protein (casein), 40% fat (polyunsaturated/saturated ratio = 0.05; 75 mg cholesterol) and 45% carbohydrate (lactose and sucrose). Glucose, insulin and resting energy expenditure (RMR) were measured at rest prior to meal consumption and 20, 40, 60, 90, and 120 min after the meal. Cumulative net TEM was calculated as the integrated area under the TEM curve with RMR as baseline. Reliability was assessed by retesting 4 subjects, and a placebo effect was tested by administering a flavored energy-free drink. Results indicated high reliability and no placebo effect. The meal resulted in a greater rise in insulin and glucose for the obese compared to the nonobese subjects (P < or = 0.05), and a significant TEM for both groups (P < or = 0.05). The cumulative TEM (W kg-1) was 61.9% greater for the nonobese (P < 0.01) when expressed relative to body mass, and 33.2% greater for the nonobese (P < or = 0.01) when expressed relative to the fat-free body mass. Expressed relative to the meal, the TEM was 25.5% less for the obese (P < 0.01). The data support an energy conservation hypothesis for obese female adolescents.


Effects of temperature on electromyogram and muscle function.

The effects of 30 min of cooling (15 degrees C water) and warming (40 degrees C water) on arm muscle function were measured. A reference condition (24 degrees C air) was included. Of nine young male subjects the maximal grip force (Fmax), the time to reach 66% of Fmax (rate of force buildup) and the maximal rhythmic grip frequency were determined, together with surface electromyographic activity (EMG) of a forearm muscle (flexor digitorum superficialis). The results showed that in contrast to warming, cooling resulted in a significant decrease of 20% in the Fmax and a significant 50% decrease in force build-up time and the maximal rhythmic grip frequency. The relationship between the root mean square value (rms) of the EMG and the static grip force did not change due to temperature changes. The median power frequency (MPF) in the power spectrum of the EMG signal decreased by 50% due to cooling but remained unchanged with heating. During a sustained contraction at 15% of Fmax (Fmax depending on the temperature) the increase in the rms value with contraction time was 90% larger in the warm condition and 80% smaller in the cold condition compared to the increase in the reference condition. The MPF value remained constant during the warm and reference conditions but in the cold it started at a 50% lower value and increased with contraction time.(ABSTRACT TRUNCATED AT 250 WORDS)


Glucocorticoid response to exercise as measured by serum and salivary cortisol.

Serum and salivary cortisol concentrations were studied in 78 elite athletes engaged in different sports, by subjecting them to high-intensity laboratory exercise. The mean difference in the pre-exercise cortisol concentrations in the seven groups studied were more marked in serum (from 311 to 768 nmol.l-1) than in saliva (from 17.9 to 22.7 nmol.l-1, only one group reaching 40 nmol.l-1). Judging from the correlation coefficients based on total variances, the post-/pre-exercise differences in cortisol concentrations in serum depended chiefly on pre-exercise values, while those in saliva tended to depend more on the postexercise concentrations. The coefficients of correlation between that difference and either the pre- or postexercise values were -0.71 and 0.47, respectively, for serum, and -0.51 and 0.58, respectively, for saliva. This would suggest that salivary cortisol concentration might be a more suitable variable for assessing glucocorticoid activity in exercise than serum cortisol concentration, probably being less sensitive to pre-exercise emotional state.


Thermographic studies on patterns of skin temperature after exercise.

In six subjects thermograms of the thighs and the forearms were taken before, during and after 10 min ergometer exercise at 100 W at an ambient temperature of 23 degrees C. During exercise, an intra-individually constant and reproducible skin temperature pattern with local temperature differences exceeding 3 degrees C evolved. Reactions after external local cooling or after occlusion of blood flow and measurements with a laser Doppler-flowmeter showed dispersed convective heat transport to be the source of this irregular pattern. Temperature differences of 3.6 degrees C and deviations of blood flow in the skin microcirculation of 300% within a distance of a few centimetres reduce the value of single-spot measurements of skin temperature with reference to the whole extremity.


The acute effect of 30 min of moderate exercise on high density lipoprotein cholesterol in untrained middle-aged men.

Fifteen middle-aged, untrained (defined as no regular exercise) men (mean age 49.9 years, range 42-67) cycled on a cycle ergometer at 50 rpm for 30 min at an intensity producing 60% predicted maximum heart rate [(fc,max), where fc,max = 220 - age]. Total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and triglyceride (Tg) concentrations were measured from fasting fingertip capillary blood samples collected at rest, after 15 and 30 min of exercise, and at 15 min post-exercise. The mean HDL-C level increased significantly from the resting level of 0.85 mmol.l-1 to 0.97 mmol.l-1 (P < 0.05) after 15 min of exercise, increased further to 1.08 mmol.l-1 (P < 0.01) after 30 min of exercise and remained elevated at 1.07 mmol.l-1 (P < 0.01) at 15 min post-exercise. These increases represented changes above the mean resting level of 14.1%, 27.1% and 25.9% respectively. The HDL-C/LDL-C ratio increased significantly from a resting ratio of 0.20 to 0.26 after 30 min of exercise (P < 0.01) and to 0.24 at 15 min post-exercise (P < 0.05). The mean Tg level increased significantly from a resting level of 0.88 mmol.l-1 to 1.05 mmol.l-1 after 15 min, and to 1.06 mmol.l-1 after 30 min of exercise (P < 0.05 at each time). The TC/HDL-C ratio decreased significantly (P = 0.05) after 30 min of exercise and at 15 min post-exercise by 18.8% and 14%, respectively. No significant changes were observed in the levels of TC or LDL-C over time.(ABSTRACT TRUNCATED AT 250 WORDS)


Physiological correlates of middle-distance running performance. A comparative study between men and women.

To compare the relative contributions of their functional capacities to performance in relation to sex, two groups of middle-distance runners (24 men and 14 women) were selected on the basis of performances over 1500-m and 3000-m running races. To be selected for the study, the average running velocity (v) in relation to performances had to be superior to a percentage (90% for men and 88% for women) of the best French v achieved during the season by an athlete of the same sex. Maximal O2 consumption (VO2max) and energy cost of running (CR) were measured in the 2 months preceding the track season. This allowed us to calculate the maximal v that could be sustained under aerobic conditions, va,max. A v:va,max ratio derived from 1500-m to 3000-m races was used to calculate the maximal duration of a competitive race for which v = va,max (tva,max). In both groups va,max was correlated to v. The relationships calculated for each distance were similar in both sexes. The CR [0.179 (SD 0.010) ml.kg-1 x m-1 in the women versus 0.177 (SD 0.010) in the men] and tva,max [7.0 (SD 2.0) min versus 8.4 (SD 2.1)] also showed no difference. The relationships between VO2max and body mass (mb) calculated in the men and the women were different. At the same mb the women had a 10% lower CR than the men; their lower mb thus resulted in an identical CR.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of hypnosis on plasma proenkephalin peptide F and perceptual and cardiovascular responses during submaximal exercise.

Little information is available concerning the influence of subconscious mechanisms on neuroendocrine function, more specifically, proenkephalin peptide F release. Ten men [5 middle distance runners (21.6 (SD 0.54 years) and 5 untrained men (24.0 (SD 4.3 years)] consented to be volunteers in this investigation. Submaximal exercise intensities of 25% and 50% of peak oxygen consumption (VO2) (8 min stages) were used for both the control and hypnosis treatments. A traditional hypnotic induction was used, with the suggestion of two higher intensities of exercise stress (50% and 75% peak VO2) previously experienced in familiarization and testing by each subject. Each minute oxygen consumption was measured using open circuit spirometry, heart rate via an ECG, and ratings of perceived exertion (RPE) using the Borg scale. Plasma peptide F immunoreactivity (ir) [preproenkephalin-(107-140)] in blood sampled from an indwelling cannula was measured by radioimmunoassay at 7-8 min of each stage of the exercise test. Expected significant increases were observed for all cardiorespiratory and perceptual variables over the increasing exercise intensities and there were no significant differences between trained and untrained groups for peptide F if response patterns. Hypnosis did not significantly affect peptide F ir concentrations (P > 0.05) and did not significantly alter exercise heart rate, RPE or minute ventilation (P > 0.05). However, hypnosis did significantly increase oxygen consumption during exercise (P = 0.0095) but not of the magnitude needed for the metabolic demands of the higher exercise intensities. Thus, traditional hypnosis was unable to make functionally significant changes in the cardiorespiratory variables.(ABSTRACT TRUNCATED AT 250 WORDS)


Short-chain and medium-chain aliphatic-ester synthesis in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the enzymes which catalyse the synthesis of ethyl acetate, ethyl n-hexanoate and isoamyl acetate were partly resolved from a fraction containing slowly sedimenting lipoproteins released during cell disruption with glass beads. Solubilization with detergents and fractionation by affinity chromatography have demonstrated the presence of at least three, and probably four, ester synthases which differ in their catalytic properties. Isoamyl-acetate synthase was solubilized and extensively purified to apparent homogeneity by successive chromatographies on various columns. On the basis of its specific activity in cell-free extracts, the enzyme was purified 19,000-fold with a 5% activity yield. As judged by SDS/PAGE, it consists of a single polypeptide chain with a molecular mass of 57 +/- 3 kDa and its apparent pI is 5.5. The enzyme acetylates isoamyl alcohol, ethanol and 12-DL-hydroxystearic acid from acetyl-CoA but is unable to use n-hexanoyl-CoA as a cosubstrate. This enzyme, defined as an acetyl-CoA: O-alcohol acetyltransferase, could be the product of one of the anaerobically induced genes in S. cerevisiae.


Identification and isolation of common and tissue-specific geranylgeranylated gamma subunits of guanine-nucleotide-binding regulatory proteins in various tissues.

Heterotrimeric guanine-nucleotide-binding regulatory proteins (G proteins) have been classified into several subtypes on the basis of the properties of their alpha subunits, though a notable multiplicity of gamma subunits has also been demonstrated. To investigate whether each subtype of alpha subunit is associated with a particular gamma subunit, various oligomeric G proteins, purified from bovine tissues, were subjected to gel electrophoresis in a Tricine buffer system. All G proteins examined were shown to have more than two kinds of gamma subunit. Of the brain G proteins, GoA, GoB, and Gi1 contain the same set of three gamma subunits, but Gi2 contains only two of these subunits. Lung Gi1 and Gi2 and spleen Gi2 and Gi3 had similar sets of two gamma subunits, one of which was distinct from the gamma subunits of brain G proteins. These observations indicate that each subtype of alpha subunit is associated with a variety of beta gamma subunits, and that the combinations differ among cells. For analyses of the structural diversity of the gamma subunits, beta gamma subunits were purified from the total G proteins of each tissue and subjected to reverse-phase HPLC under denaturing conditions, where none of the beta subunits were eluted from the column. Three distinct gamma subunits were isolated in this way from brain beta gamma subunits. In contrast, lung and spleen beta gamma subunits contained at least five gamma subunits, the elution positions and electrophoretic mobilities of which were indistinguishable between the two tissues. Among several gamma subunits, two subspecies appeared to be common to the three tissues. In fact, in each case, the partial amino acid sequence of the most abundant gamma subunit in each tissue was identical, and the sequences coincided exactly with that of 'gamma 6' [Robishaw, J. D., Kalman, V. K., Moomaw, C. R. & Slaughter, C. A. (1989) J. Biol. Chem. 264, 15758-15761]. Fast-atom-bombardment mass spectrometry analysis indicated that this abundant gamma subunit in lung and spleen was geranylgeranylated and carboxymethylated at the C-terminus, as was 'gamma 6' from brain. In addition to abundant gamma subunits, other tissue-specific gamma subunits were also shown to be geranylgeranylated by gas-chromatography-coupled mass spectrometry analysis of Raney nickel-treated gamma subunits. These results suggest that most gamma subunits associated with many different subtypes of alpha subunit are geranylgeranylated in a variety of tissues, with the single exception being the retina where the G protein transducin has a farnesylated gamma subunit.


Assignment of the magnetic resonances of the imino protons and methyl protons of Bombyx mori tRNA(GlyGCC) and the effect of ion binding on its structure.

The magnetic resonances in the low-field H-NMR spectra of Bombyx mori tRNA(GlyGCC), corresponding to the hydrogen-bonded imino protons of the helical stems and tertiary base pairs, could be tentatively assigned by means of the sequential nuclear Overhauser effects. While B. mori tRNA(GlyGCC) does not contain the G19C56 tertiary base pair, the D20G57 base pair exists between the D and T loops, which was not found in the X-ray crystal structure of yeast tRNA(Phe). The effects of Mg2+, spermine and temperature on the conformation of this tRNA have also been examined based on the behavior of the assigned resonance signals. Mg2+ stabilize the D and T stems and the tertiary structure between the D and T loops. Spermine affects the resonances of the D and anticodon stems, and A23G9, but does not stabilize them. While the acceptor stem melts sequentially from both ends (G7C66 and G1C72) with increasing temperature, the anticodon stem melts from only one end (G39C31) and the G26C44 base pair is the most stable. In the tertiary structure between the variable loop and D stem, G10G45 melts first and G22G46 last. Yeast tRNA(Phe) has also been examined, and the results were compared with those for B. mori tRNA(Gly).


Cloning and expression of a heat-inducible hsp70 gene in two species of Hydra which differ in their stress response.

A heat-inducible, intron-containing member of the hsp70 gene family has been isolated and characterized in Hydra magnipapillata and Hydra oligactis, two species previously shown [Bosch, T. C. G., Krylow, S. M., Bode, H. R. & Steele, R. E. (1988) Proc. Natl Acad. Sci. USA 85, 7927-7931] to differ in their stress response. The gene, hsp70.1, encodes a 654-amino-acid protein of predicted molecular mass 70 kDa with 78% amino acid identity to Xenopus HSP70. Northern-blot analysis revealed that polyps of H. oligactis accumulate significantly less hsp70.1 mRNA after heat shock than polyps of H. magnipapillata. In nuclear run-off experiments, we found that transcriptional induction of hsp70.1 expression in response to stress is similar in both species. Thus, the previously reported inability of H. oligactis to synthesize heat-shock proteins in response to stress is at least in part due to reduced stability of hsp70.1 mRNA during heat shock.


1H-NMR assignments and local environments of aromatic residues in bovine, human and guinea pig variants of alpha-lactalbumin.

1H-NMR assignments have been defined for the aromatic-ring protons of the bovine, guinea pig and human variants of alpha-lactalbumin. Spin-system networks were identified by means of double-quantum-filtered two-dimensional J-correlated spectroscopy and two-dimensional relayed coherence spectroscopy data. Analysis of two-dimensional nuclear-Overhauser-enhancement spectroscopy data of the proteins indicated that in each case two clusters of aromatic residues exist. The two clusters are also evident in the crystal structure of the human protein, and this evidence, in conjunction with sequence differences between the three proteins, permitted sequence-specific assignments to be made for the majority of aromatic residues. Remaining ambiguities in the assignments could be resolved by analysis of photochemically induced dynamic nuclear polarization (PCIDNP) effects. Comparison of the PCIDNP spectra of the three proteins indicated the presence of only minor differences in the surface exposure of conserved aromatic residues. Taken together, these results indicate that the environments of the conserved aromatic residues in bovine, guinea pig and human alpha-lactalbumin in solution are very similar to each other, and that the solution and the crystal forms of at least the human protein are similar.


Fluorescence study of the ligand stereospecificity for binding to lumazine protein.

6,7-Dimethyllumazine derivatives, substituted at the 8-position with aldityls or monohydroxyalkyl groups, have been examined for their binding ability to lumazine apo-protein from two strains of Photobacterium phosphoreum using fluorescence dynamics techniques. On the protein the lumazine has a nearly monoexponential decay of fluorescence with lifetime 13.8 ns (20 degrees C). In free solution the lifetime is 9.6 ns. The concentration of free and bound lumazine in an equilibrium mixture can be recovered readily by analysis of the fluorescence decay. Only the aldityl derivatives D-xylityl and 3'-deoxy-D-ribityl, having stereoconfigurations at the 2' and 4' positions identical to the natural ligand, 8-(1'-D-ribityl), show comparable dissociation constants (0.3 microM, 20 degrees C, pH 7.0). D-Erythrityl and L-arabityl have dissociation constants of 1-2 microM. All other ligands show no interaction at all or have dissociation constants in the range 6-80 microM, which can still be determined semi-quantitatively using the fluorescence decay technique. In the case of these very weakly bound ligands, unambiguous detection of bound ligand can be shown by a long correlation time (23 ns, 2 degrees C) for the fluorescence anisotropy decay. Examination of the bound D-xylityl compound's fluorescence anisotropy decay at high time resolution (< 100 ps) shows rigid association, i.e. no mobility independent of the macromolecule. All bound ligands appear to be similarly positioned in the binding site. The influence of the stereoconfiguration at the 8-position found for lumazine protein parallels that previously observed for the enzyme riboflavin synthase, where the lumazines are substrates or inhibitors. This is consistent with the finding of significant sequence similarity between these proteins. The binding rigidity may have implications for the mechanism of the enzyme.


Mechanism of the degradation of non-enzymatically glycated proteins under physiological conditions. Studies with the model fructosamine, N epsilon-(1-deoxy-D-fructos-1-yl)hippuryl-lysine.

The degradation of fructosamines, formed from the non-enzymic glycation of proteins under physiological conditions, to advanced glycation end products was investigated by studying the model peptide fructosamine N epsilon-(1-deoxy-D-fructos-1-yl)hippuryl-lysine (DHL). At pH 7.4 and 37 degrees C in aerobic phosphate buffer, DHL degraded to form N epsilon-carboxymethyl-hippuryl-lysine, and hippuryl-lysine over a 29-day incubation period. The expected N epsilon-(3-lactato)hippuryl-lysine and 'hippuryl-lysylpyrraline' derivatives were not found. Superoxide radicals and hydrogen peroxide were formed during the degradation of DHL but were also both consumed during the degradation reaction. Reversal of the Amadori rearrangement was not a major fate of the fructosamine. The formation of N epsilon-carboxymethyl-hippuryl-lysine was decreased by desferrioxamine, catalase, superoxide dismutase, catalase with superoxide dismutase, anaerobic conditions and aminoguanidine. The formation of hippuryl-lysine was decreased by desferrioxamine, catalase and catalase with superoxide dismutase, but was increased by the addition of aminoguanidine. N epsilon-Carboxymethyl-serine and unmodified lysine residues are major peptide-based end products in the degradation of lysyl-fructosamine under physiological conditions. Oxygen, redox-active metal ions, catalase, superoxide dismutase and the pharmacological agent aminoguanidine are expected to be influential on the rate and fate of fructosamine degradation.


Inhibition of cytochrome-P450 reductase by polyols has an electrostatic nature.

The present study was undertaken to examine the nature of the inhibitory action of glycerol on the liver microsomal monooxygenase system. In agreement with earlier observations, glycerol inhibited benzphetamine N-demethylation by liver microsomes of the phenobarbital-treated rabbit. The presence of glycerol in the medium did not affect binding of the substrate to cytochrome P450. Another polyol, ethylene glycol, was equally efficient in inhibiting benzphetamine N-demethylation. Both also inhibited reduction of rabbit cytochrome P450 LM2, cytochrome c and potassium ferricyanide by NADPH-cytochrome-P450 reductase in microsomes. Recently, we showed that the stimulation of electron transfer by increased ionic strength is due to neutralization of electrostatic interaction between NADPH-cytochrome-P450 reductase and its charged redox partners [Voznesensky, A. I. & Schenkman, J. B. (1992) J. Biol. Chem. 267, 14669-14676]. Polyols have an opposite effect to that of salt on ionic properties of a solution. They decrease the dielectric constant, thereby promoting electrostatic interactions between proteins. Addition of polyols decreased the conductivity of the medium. When rates of electron transfer to charged acceptors, cytochrome P450, cytochrome c and potassium ferricyanide, at various salt and polyol concentrations, relative to activities in 200 mM sodium phosphate, were plotted as a function of the conductivity the data for each acceptor fit on the same line. In contrast, neither alteration of ionic strength nor polyol addition affected the rate of electron transfer from NADPH-cytochrome-P450 reductase to an uncharged acceptor 1,4-benzoquinone. The data obtained is consistent with our earlier suggestion that charge repulsion limits redox interactions between rabbit cytochrome P450 LM2 and its reductase at low ionic strength, and suggest that the observed action of polyols is the result of enhancement of electrostatic interactions that inhibits electron transfer between NADPH-cytochrome-P450 reductase and its charged redox partners. In congruence with the hypothesis, the Km of rabbit cytochrome P450 LM2 for NADPH-cytochrome-P450 reductase was increased almost one order of magnitude by elevating the glycerol content from 5% to 25% (by vol.) without a change in Vmax.


A weighting method for predicting protein structural class from amino acid composition.

A protein is generally classified into one of the following four structural classes: all alpha, all beta, alpha+beta and alpha/beta. In this paper, based on the weighting to the 20 constituent amino acids, a new method is proposed for predicting the structural class of a protein according to its amino acid composition. The 20 weighting parameters, which reflect the different properties of the 20 constituent amino acids, have been obtained from a training set of proteins through the linear-programming approach. The rate of correct prediction for a training set of proteins by means of the new method was 100%, whereas the highest rate of previous methods was 82.8%. Furthermore, the results showed that the more numerous training proteins, the more effective the new method.


Catalysis by alpha-chymotrypsin entrapped into surface-modified polymeric nanogranules in organic solvent.

Physicochemical characteristics of previously suggested surface-modified polymeric nanogranules (SMPN) and catalytic and stability properties of alpha-chymotrypsin entrapped into such nanogranules in a nonpolar solvent were investigated in more details. SMPN were obtained by polymerization of an acrylamide/N,N'-methylene-bisacrylamide mixture in a mixed reversed micellar system composed of Aerosol OT [sodium di(2-ethylhexyl)sulfosuccinate] and the polymeric surfactant Pluronic F-108 modified with polymerizable groups, followed by the chromatographic removal of the auxiliary surfactant, Aerosol OT. An optimal solvent system was found providing the required orientation of the polymeric surfactant in starting mixed micelles, i.e. with polar fragments immersed into the micellar interior and apolar fragments protruding into organic solvent. The hydrodynamic diameter of SMPN in benzene solution was estimated by means of quasi-elastic light scattering to be 84 +/- 1 nm. Catalytic and stability properties of alpha-chymotrypsin entrapped into SMPN strongly depended on conditions of preparation of SMPN. The optimal concentration of acrylamide monomers in the micellar interior and hydration degree of starting reversed micelles were found to be 20% by mass and wo = 15, respectively. alpha-Chymotrypsin-containing SMPN were used as a catalyst in the synthesis of N-acetyl-L-tyrosine ethyl ester from N-acetyl-L-tyrosine and ethanol, performed in a membrane reactor.


Studies on the aminopeptidase activity of rat cathepsin H.

Three synthetic substrates H-Arg-NH-Mec, Bz-Arg-NH-Mec and H-Cit-NH-Mec (Bz, Benzoyl; NH-Mec, 4-methylcoumaryl-7-amide; Cit, citrulline) were used to characterize specificity requirements for the P1-S1 interaction of cathepsin H from rat liver. From rapid equilibrium kinetic studies it was shown that Km, kcat and the specificity constants kcat/Km are quite similar for substrates with a free alpha-amino group. In contrast, a 25-fold decrease of kcat/Km was observed for the N-terminal-blocked substrate Bz-Arg-NH-Mec. The activation energies for H-Arg-NH-Mec and Bz-Arg-NH-Mec were determined to be 37 kJ/mol and 55 kJ/mol, respectively, and the incremental binding energy delta delta Gb of the charged alpha-amino group was estimated to -8.1 kJ/mol at pH 6.8. The shown preference of cathepsin H for the unblocked substrates H-Arg-NH-Mec and H-Cit-NH-Mec was further investigated by inspection of the pH dependence of kcat/Km. The curves of the two substrates with a charged alpha-amino group showed identical bell-shaped profiles which both exhibit pKa1 and pKa2 values of 5.5 and 7.4, respectively, at 30 degrees C. The residue with a pKa1 of 5.5 in the acid limb of the activity profile of H-Arg-NH-Mec was identified by its ionization enthalpy delta Hion = 21 kJ/mol as a beta-carboxylate or gamma-carboxylate of the enzyme, whereas the residue with a pKa2 of 7.4 was assigned to the free alpha-amino group of the substrate with a delta Hion of 59 kJ/mol. Bz-Arg-NH-Mec showed a different pH-activity profile with a pKa1 of 5.4 and a pKa2 of 6.6 at 30 degrees C. Cathepsin H exhibits no preference for a basic P1 side chain as has been shown by the similar kinetics of H-Arg-NH-Mec and the uncharged, isosteric substrate H-Cit-NH-Mec. In summary, specific interactions of an anionic cathepsin H active site residue with the charged alpha-amino group of substrates caused transition state stabilization which proves the enzyme to act preferentially as an aminopeptidase.


Metabolic effects of short-chain ceramide and glucosylceramide on sphingolipids and protein kinase C.

Recent studies have identified a potential role for glucosylceramide (GlcCer) in growth promotion and hormonal signalling. In an effort to demonstrate a growth-promoting activity of GlcCer, we prepared a GlcCer having a short-chain acid (octanoyl), in the belief that this glycolipid could be absorbed more readily and more uniformly by cultured cells. By using a mixture of two specific lecithins, dioleoylglycerophosphocholine and 1-stearoyl-2-palmitoylglycerophosphocholine, we were able to prepare dispersions containing a high molar proportion of the GlcCer and the related ceramide, octanoyl sphingosine. Unexpectedly, both sphingolipids inhibited protein and DNA synthesis in Madin-Darby canine kidney cells and produced large increases in the levels of the natural lipids, GlcCer, ceramide, free sphingosine, and an amine that may be glucosylsphingosine (GlcSph). Decreases were seen in the level of sphingomyelin and the proportion of protein kinase C in the cell membranes. The level of lactosylceramide was diminished by octanoyl GlcCer but elevated considerably by octanoyl sphingosine. Diacylglycerols were increased by the lecithins in the liposomes, but the exogenous sphingolipids had no effect. Octanoyl sphingosine labeled in the sphingoid base yielded labeled GlcCer and sphingomyelin labeled in both long-chain and very-long-chain fatty acid families, as well as the octanoyl version. The two families of ceramides, however, had relatively little radioactivity. Some of these changes are attributed to rapid hydrolysis of the added lipids with the formation, particularly from the ceramide, of sphingosine and its anabolic metabolite, GlcSph. Several observations support the idea that the octanoyl sphingosine inhibited the phosphocholinetransferase that synthesizes sphingomyelin while the octanoyl GlcCer inhibited GlcCer beta-glucosidase and GlcCer galactosyltransferase. The use of unnatural short-chain lipids in the study of cell growth and other phenomena may result in unexpected changes in related metabolites and the findings from such experiments should therefore be interpreted cautiously.


Characterisation of the control of respiration in potato tuber mitochondria using the top-down approach of metabolic control analysis.

Control over oxidative phosphorylation by purified potato mitochondria was determined using the top-down approach of metabolic control analysis. The control over the respiration rate, phosphorylation rate, proton-leak rate and proton motive force exerted by the respiratory chain, phosphorylation reactions and the proton leak were measured over a range of phosphorylation rates from resting (state 4) to maximal (state 3). These rates were obtained by adding different amounts of hexokinase in the presence of glucose, or different amounts of oligomycin in the presence of ADP. The respiratory substrate was NADH or succinate, both of which feed electrons directly to ubiquinone. The rate of oxygen consumption by the alternative oxidase pathway was negligible with NADH as substrate but was measurable with succinate and was subtracted. Control over the respiration rate in potato mitochondria was predominantly exerted by the respiratory chain at all rates except close to state 4, where control by the proton leak was equally or more important. For oxidation of NADH, the flux control coefficient over the respiration rate exerted by the respiratory chain in state 3 was between 0.8 and 1.0, while in state 4, control over the respiration rate was shared about equally between the chain and the proton leak. The control over the phosphorylation rate was predominantly exerted by the respiratory chain, although at low rates control by the phosphorylation system was also important. For oxidation of NADH, the flux control coefficient over the phosphorylation rate exerted by the respiratory chain in state 3 was 0.8-1.0, while near state 4 the flux control coefficients over the phosphorylation rate were about 0.8 for the phosphorylation system and 0.25 for the chain. Control over the proton leak rate was shared between the respiratory chain and the proton leak; the phosphorylation system had negative control. For oxidation of NADH, the flux control coefficients over the leak rate in state 3 were 1.0 for the leak, 0.4 for the chain and -0.4 for the phosphorylation system, while in state 4 the flux control coefficients over leak rate were about 0.5 for the leak and 0.5 for the chain. Control over the magnitude of the protonmotive force was small, between -0.2 and +0.2, reflecting the way the system operates to keep the protonmotive force fairly constant; the respiratory chain and the phosphorylation system had equal and opposite control and there was very little control by the proton leak except near state 4.


Purine-cytosine permease of Saccharomyces cerevisiae. Effect of external pH on nucleobase uptake and binding.

The cloned FCY2 gene (strain pAB4) of the purine-cytosine permease (PCP) of Saccharomyces cerevisiae and the cloned allele fcy2-21 (strain pAB25) introduced into an S. cerevisiae strain carrying a chromosomal deletion at the FCY2 locus [Weber, E., Rodriguez, C., Chevallier, M. R. & Jund, R. (1990) Mol. Microbiol. 4, 585-596] were studied. The influence of external pH (varying over 3.5-6) has been analysed on the uptake of adenine, hypoxanthine and cytosine (Ktapp, apparent Michaelis constant and Vm) and on the binding constants of these three solutes (Kdapp, apparent half-saturation constant and Bmax, total binding sites) determined on plasma membranes. For pAB4, the variations of Ktapp and Vm were the same for the three bases, i.e. an increase in Ktapp when the pH increased and a maximum Vm around pH 5. For pAB25, Ktapp values varied in the same way and were significantly higher for the three bases than those found in pAB4. There was almost no variation of Vm for adenine, and there was a continuous decrease when the pH increased in the Vm of hypoxanthine and cytosine. Equilibrium binding measurements were performed for the three bases with plasma membrane isolated from pAB4 and pAB25. One single class of binding sites was detected. For pAB4, the affinity increased when the pH decreased for the three bases. The affinity of PCP for adenine was always greater than for cytosine or hypoxanthine. For pAB25, the same phenomenon was observed. However, the curves showing the variation of Kdapp as a function of pH were shifted towards more acidic pH values. A model was used to fit the experimental binding data obtained with hypoxanthine for the calculation of the dissociation constants of its binding to PCP and to determine the ionization constants of an amino acid involved in ligand binding. For pAB4, at acid pH, the dissociation constant was 1.7 +/- 0.4 microM. An amino acid displaying a pK of 3.8 was determined; this value was shifted to pK 4.8 when hypoxanthine was bound. For pAB25, the main effects of the mutation were a large decrease in the affinity of PCP for hypoxanthine (Kd of 14.4 +/- 4.3 microM) and a shift in the pK of the amino acid towards a more acidic pH (about 2.9). The pK of this group remained similar to the value obtained with pAB4 when hypoxanthine was bound. From these data, it is proposed that the binding of hypoxanthine and H+ is a random process.


Decreased stress inducibility of the HSP68 protein in a rat hepatoma variant clone.

Analysis of the stress response of closely related rat hepatoma clones revealed that the major inducible heat-shock protein 68 (HSP68) was only slightly inducible upon stress in the glucocorticoid-resistant, dedifferentiated clone-2 cells, but strongly activated in the differentiated, glucocorticoid-sensitive Faza 967 cells from which clone 2 was derived. The decreased inducibility of HSP68 in clone-2 cells was not the consequence of altered kinetics of protein synthesis recovery, was not correlated with the deficient inducibility of other major heat-shock proteins and had no effect on the heat sensitivity of the cells. This deficiency was observed after treatment with mild and strong heat and various chemicals. The results of nuclear run-on experiments suggested that the impairment of HSP68 mRNA induction most likely occurs at the transcriptional level and is probably specific for the corresponding gene. In Faza 967 and clone-2 cells, stress activated comparable levels of heat-shock-factor binding to the heat-shock element, and the expression of a reporter gene under the control of murine HSP70.1 promoter was strongly stimulated in both cells. Therefore, our results raise the possibility that the deficient stress inducibility of HSP68 is due to some specific regulation of the endogeneous HSP68 gene, rather than to a deficiency of the heat-shock factor or mutation of the corresponding gene.


A chick skeletal-muscle alpha-actinin gene gives rise to two alternatively spliced isoforms which differ in the EF-hand Ca(2+)-binding domain.

A chick non-muscle alpha-actinin cDNA probe encoding the EF-hand region of molecule was used to screen a lambda gt10 chick brain cDNA library from 14-day embryos. A partial 2.1-kb alpha-actinin cDNA was isolated (8W cDNA) which encoded a protein identical to chick skeletal-muscle alpha-actinin, except in the C-terminal part of the first EF hand. In the variant, the 22 residues found in the skeletal-muscle isoform were replaced by a stretch of 26 unique residues. Analysis of the structure of the skeletal-muscle alpha-actinin gene showed that the region of divergence was encoded by two exons which are alternatively spliced. Quantitative reverse transcriptase/polymerase chain reaction (RT/PCR) was used to investigate the levels of the alpha-actinin transcripts in various tissues. The skeletal-muscle alpha-actinin variant was expressed at low levels in brain, liver and spleen, but could not be detected in skeletal muscle. Surprisingly, skeletal-muscle alpha-actinin mRNA was also expressed in brain, liver and spleen. The RT/PCR products were authenticated by using diagnostic restriction enzyme sites and by sequencing. The splice variant derived from the skeletal-muscle alpha-actinin gene was also detected in a variety of cDNA libraries from both adult and embryonic tissues by PCR. Although a transcript encoding this alpha-actinin splice variant is expressed in non-muscle tissues, neither of the two EF-hands would be predicted to be functional, making it unlikely to be a typical non-muscle isoform which are calcium-sensitive with respect to binding actin. The two vertebrate non-muscle alpha-actinins sequenced to date also have a spacer of five amino acids between the two EF hands, whereas in the variant, the spacer is just four residues in length. Further analysis will be required before this alpha-actinin isoform, which we refer to as SKv, can be classified as muscle or non-muscle alpha-actinin. We propose a new nomenclature to describe the various alpha-actinin genes and their transcripts.


A mutant Kex2 enzyme with a C-terminal HDEL sequence releases correctly folded human insulin-like growth factor-1 from a precursor accumulated in the yeast endoplasmic reticulum.

Mutations in the pro region of the yeast DNA hybrid of prepro-alpha-factor and human insulin-like growth factor-1 (IGF-1) cause the accumulation, in the yeast Saccharomyces cerevisiae, of an unglycosylated precursor protein where the pre sequence is missing. The prepro sequence of the prepro-alpha-factor consists of a pre or signal sequence and a proregion which possesses three sites for N-glycosylation. Isolation of a precursor, where the pro region is still linked to IGF-1 through a pair of dibasic amino acid residues, implies that the polypeptide may have translocated into the endoplasmic reticulum (ER) but has not been processed by the Golgi membrane-bound Kex2 endoprotease. However, the lack of any N-glycosylation in the translocated polypeptide is surprising. The mutated pro region, can be processed, in vitro, by treatment with a soluble form of the Kex2 enzyme. It is also possible to release the pro region, in vivo, by coexpressing a mutant Kex2 protease which is partially retained in the ER with the help of the C-terminal tetrapeptide sequence, HDEL. The mature IGF-1, which is secreted from the intracellular pool of precursor proteins, is predominantly an active, monomeric molecule, corroborating observations that early removal of the pro region before folding in the ER helps to prevent aberrant intermolecular disulfide-bond formation in IGF-1. These results have revealed the utility of the ER-retained Kex2 enzyme as a novel in vivo biochemical tool.


Cardiolipin liposomes sequester a reactivatable partially folded rhodanese intermediate.

The interaction was studied between the mitochondrial enzyme thiosulfate sulfurtransferase and liposomes, in the form of large unilamellar vesicles (LUV), prepared from either cardiolipin (CL), PtdCho or PtdSer. At equivalent concentrations of lipid, more partially folded thiosulfate sulfurtransferase bound to CL/LUV than to PtdSer/LUV, and only traces were bound to PtdCho/LUV. Native thiosulfate sulfurtransferase did not bind to any of these LUV. We show that CL/LUV-sequestered thiosulfate sulfurtransferase is inactive but may be reactivated (approximately 56%) with the aid of detergents, thiosulfate, beta-mercaptoethanol and phosphate buffer. Reactivations in the presence of PtdSer/LUV or PtdCho/LUV was only 9% or 1%, respectively. Analysis of the complex by protease digestion and fluorescence spectroscopy indicated that thiosulfate sulfurtransferase was held by CL/LUV and PtdSer/LUV as a folding intermediate. Data presented here suggest that detergents may not interact directly with the protein, but, rather, their primary role in reactivation is to disrupt the LUV, allowing flexibility to the anchored thiosulfate sulfurtransferase molecule, thereby promoting folding. These studies complement other reports which imply a possible role for CL in protein translocation across the mitochondria, since we find that CL binds to thiosulfate sulfurtransferase and sequesters it in a translocation-competent prefolded conformation, which may readily lead to a correctly folded enzyme.


Amino-acid sequence and cell-adhesion activity of a fibril-forming collagen from the tube worm Riftia pachyptila living at deep sea hydrothermal vents.

We have determined the amino acid sequence of the alpha chain of a fibril-forming collagen from the body wall of the marine invertebrate Riftia pachyptila (vestimentifera) by Edman degradation. The pepsin-solubilized collagen chain consists of a 1011-residue triple-helical domain and short remnants of N- and C-telopeptides. The triple-helical sequence showed one imperfection of the collagen Gly-Xaa-Yaa triplet repeat structure due to a Gly-->Ala substitution. This imperfection is correlated to a prominent kink in the molecule observed by electron microscopy. No strong sequence similarity was found with the fibril-forming vertebrate collagen types I-III, V and XI except for the invariant Gly residues. However, one of the two consensus cross-linking sequences was well conserved. The Riftia collagen shared with the vertebrate collagens many post-translational modifications. About 50% of the Pro and Lys residues are found in the Yaa position and were extensively hydroxylated to 4-hydroxyproline (4Hyp) and hydroxylysine (Hyl). A few proline residues in Xaa position were partially hydroxylated to either 4Hyp or 3Hyp. Despite the low sequence similarity, Riftia collagen was a potent adhesion substrate for two human cell lines. Cell adhesion could be inhibited by antibodies against the integrin beta 1 subunit but not by RGD peptides. This biological activity is apparently conserved in fibril-forming collagens of distantly related species but does not require the two RGD sequences present in Riftia collagen.


Single-stranded DNA binding protein from calf thymus. Purification, properties, and stimulation of the homologous DNA-polymerase-alpha-primase complex.

A binding protein for single-stranded DNA (ssDNA) was purified from calf thymus to near homogeneity by chromatography on DEAE-cellulose, blue-Sepharose, ssDNA-cellulose and FPLC Mono Q. The most purified fraction consisted of four polypeptides with molecular masses of 70, 55, 30, and 11 kDa. The polypeptide with the molecular mass of 55 kDa is most likely a degraded form of the largest polypeptide. The complex migrated as a whole on both glycerol gradient ultracentrifugation (s = 5.1 S) and gel filtration (Stokes' radius approximately 5.1 nm). Combining these data indicates a native molecular mass of about 110 kDa, which is in accord with a 1:1:1 stoichiometry for the 70 + 55/30/11-kDa complex. The ssDNA binding protein (SSB) covered approximately 20-25 nucleotides on M13mp8 ssDNA, as revealed from both band shift experiments and DNase I digestion studies. The homologous DNA-polymerase-alpha-primase complex was stimulated by the ssDNA binding protein 1.2-fold on poly(dA).(dT)14 and 10-13-fold on singly primed M13mp8 DNA. Stimulation was mainly due to facilitated DNA synthesis through stable secondary structures, as demonstrated by the vanishing of many, but not all, pausing sites. Processivity of polymerase-primase was not affected on poly(dA).(dT)14; with poly(dT).(rA)10 an approximately twofold increase in product lengths was observed when SSB was present. The increase was attributed to a facilitated rebinding of polymerase alpha to an already finished DNA fragment rather than to an enhancement of the intrinsic processivity of the polymerase. Similarly, products 300-600 nucleotides long were formed on singly primed M13 DNA in the presence of SSB, in contrast to 20-120 nucleotides when SSB was absent. DNA-primase-initiated DNA replication on M13 DNA was inhibited by SSB in a concentration-dependent manner. However, with less sites available to begin with RNA priming, more homogeneous products were formed.


Effect of actin on the tryptic digestion of myosin subfragment 1 in the weakly attached state.

The structure of myosin subfragment 1 (S1) in the weakly attached complex with actin was studied at three specific sites, at the 50-kDa/20-kDa and 27-kDa/50-kDa junctions, and at the N-terminal region, using tryptic digestion as a structure-exploring tool. The structure of S1 at the vicinity of the 50-kDa/20-kDa junction is pH dependent in the weakly attached state because the tryptic cleavage at this site was fully protected by actin at pH 6.2, but the protection was only partial at pH 8.0. Since the actin protection is complete in rigor at both pH values, the results indicate that the structure of S1 at the 50-kDa/20-kDa junction differs in the two states at pH 8.0, but not at pH 6.2. Actin restores the ADP-suppressed tryptic cleavage after Lys213 at the 27-kDa/50-kDa junction in the strongly attached state, but not in the weakly attached state, which indicates structural difference between the two states at this site. ATP and ADP open a new site for tryptic cleavage in the N-terminal region of the S1 heavy chain between Arg23 and Ile24. Actin was found to suppress this cleavage in both weakly and strongly attached states, which shows that, in the vicinity of this site, the structure of S1 is similar in both states. The results indicate that the binding of S1 to actin induces localized changes in the S1 structure, and the extent of these changes is different in the various actin-S1 complexes.


Determination of the three-dimensional solution structure of the histidine-containing phosphocarrier protein HPr from Escherichia coli using multidimensional NMR spectroscopy.

We recorded several types of heteronuclear three-dimensional (3D) NMR spectra on 15N-enriched and 13C/15N-enriched histidine-containing phosphocarrier protein, HPr, to extend the backbone assignments [van Nuland, N. A. J., van Dijk, A. A., Dijkstra, K., van Hoesel, F. H. J., Scheek, R. M. & Robillard, G. T. (1992) Eur. J. Biochem, 203, 483-491] to the side-chain 1H,15N and 13C resonances. From both 3D heteronuclear 1H-NOE 1H-13C and 1H-NOE 1H-15N multiple-quantum coherence (3D-NOESY-HMQC) and two-dimensional (2D) homonuclear NOE spectra, more than 1200 NOE were identified and used in a step-wise structure refinement process using distance geometry and restrained molecular dynamics involving a number of new features. A cluster of nine structures, each satisfying the set of NOE restraints, resulted from this procedure. The average root-mean-square positional difference for the C alpha atoms is less than 0.12 nm. The secondary structure topology of the molecule is that of an open-face beta sandwich formed by four antiparallel beta strands packed against three alpha helices, resembling the recently published structure of Bacillus subtilis HPr, determined by X-ray crystallography [Herzberg, O., Reddy, P., Sutrina, S., Saier, M. H., Reizer, J. & Kapafia, G. (1992) Proc. Natl, Acad. Sci. USA 89, 2499-2503).


The uncoupling protein from brown adipose tissue mitochondria. The environment of the tryptophan residues as revealed by quenching of the intrinsic fluorescence.

The uncoupling protein from brown adipose tissue is a member of the family of metabolite carriers of the mitochondrial inner membrane. It contains two tryptophan residues which have been characterized by fluorescence spectroscopy. Application of fluorescence-quenching-resolved spectroscopy (FQRS) allowed the determination of the emission maximum for each residue, both of which occur at 332 nm, thus suggesting that they are both located in a non-polar environment. Fluorescence quenching has demonstrated that both residues are accessible to acrylamide and inaccessible to Cs+, while only one of them is accessible to I-. When FQRS is combined with guanidinium hydrochloride denaturation, the unfolding of the regions containing each tryptophan can be monitored separately as they are transferred to the polar medium where the emission maximum appears at 359 nm, revealing also that the iodide-accessible residue is more sensitive to the denaturant. Secondary structure predictions, together with the data presented here, suggest that the iodide-accessible residue could correspond to Trp173 and the denaturant-resistant iodide-inaccessible one to Trp280, located in the center of the sixth transmembrane alpha-helix. Interaction of the protein with GDP (a transport inhibitor) has been studied and has revealed that it partially shields Trp173 from the interaction with I-, as well as reducing the static component of the acrylamide quenching.


Sequence-specific 1H-NMR assignment and determination of the secondary structure of bovine heart fatty-acid-binding protein.

The nearly complete sequence-specific 1H resonance assignment of the pI = 4.9 isoform of cytosolic 15-kDa fatty-acid-binding protein from bovine heart (H-FABPc) by homonuclear two-dimensional NMR spectroscopy is presented. Regular secondary structure elements were identified from NOE spectra and the sequence locations of slowly exchanging backbone amide protons. The molecular structure of the protein was found to consist mainly of ten antiparallel beta-strands and two short alpha-helices. The data presented here for the first time for a hydrophobic molecule transporter of the fatty-acid-binding protein type is the basis for a complete tertiary structure determination currently in progress.


Conformation/activity studies of rationally designed potent anti-adhesive RGD peptides.

The Arg-Gly-Asp (RGD) sequence is a universal cell-recognition site of various extracellular proteins that interact with integrin cell-surface receptors. In order to design low-molecular-mass RGD protein antagonists, the determination of the biologically active conformation is a prerequisite. We present a method that yields detailed insight into the steric factors which govern the binding of the ligands to their receptors by systematically scanning the conformational space accessible for the tripeptide sequence RGD. The investigation is based on the conformationally controlled design of homodetic cyclic oligopeptides and their structural determination, coupled with biological assays. For this purpose, a whole set of cyclic pentapeptides and hexapeptides has been synthesized and their three-dimensional structures in solution analyzed by modern two-dimensional NMR techniques in combination with restrained and free molecular dynamics simulations. Their biological activity was compared with that of linear GRGDS in inhibition assays of tumor cell adhesion to laminin P1 and vitronectin substrates. An up to 100-fold, and in part selective, increase in activity was observed for two cyclic pentapeptides. Most other peptides showed a decreased activity which, however, was useful to correlate activity with rather small variations in conformation. Detailed comparative studies of the systematically designed conformations and the corresponding anti-adhesive activities offer an access to lead structures for a rational indirect drug design of peptide and peptidomimetic pharmaceuticals with strong interfering activity for integrin-mediated cell-cell and cell-matrix interactions.


Molecular analysis of the protein-protein interaction between the E9 immunity protein and colicin E9.

The specificity-determining region of the colicin E9 immunity protein (Im9) for its interaction with its cognate E colicin has been localized to residues 16-43 of the 86-amino-acid protein by the use of gene fusions. A comparison of the alignment of residues in this region of the Im2, Im8 and Im9 proteins have identified nine candidate specificity-determining residues. Using site-directed mutagenesis, we have changed each of these residues in the Im9 protein to the residue found in the same position in the Im8 protein. The immunity phenotype conferred by the mutant immunity protein was then tested. Of the nine residues, only one (Val34 to Asp) showed any evidence of conferring immunity to colicin E8. Changing other residues in the specificity-determining region to the equivalent Im8 residue did not affect the phenotype conferred by the mutant protein, with the exception of the change of Val37 to Glu, which resulted in low-level E8 immunity. While the substitutions at positions 34 and 37 of the Im9 protein introduced immunity towards ColE8, they did not diminish the immunity towards ColE9, suggesting that the two immunity proteins may have a common specificity framework which can be modified by single mutations. In addition, we have used chemical modification of the unique cysteine residue of Im9 (Cys23) in order to probe further this specificity-determining region. Cys23 in the purified Im9 protein is accessible to modification with the thiol-specific reagent 5,5'-dithiobis(2-nitrobenzoic acid) and the stoichiometry of labelling is close to 1:1. This residue, however, cannot be labelled by 5,5'-dithiobis(2-nitrobenzoic acid) when the Im9 protein is complexed to colicin E9. This result is consistent with the Cys23 residue being buried in the complex. However, when the purified Im9 protein modified at Cys23 with a variety of reagents was used in DNase inhibition assays with colicin E9, the modified Im9 proteins still possessed anti-DNase activity but only up to a certain derivative molecular mass. These results are discussed in terms of the proximity of Cys23 to the specificity-determining region.


The primary structure of piscine (Oncorhynchus mykiss) retinol-binding protein and a comparison with the three-dimensional structure of mammalian retinol-binding protein.

1. The primary structures of two variants of rainbow trout (Oncorhynchus mykiss) plasma retinol-binding protein (RBP) were determined and found to be approximately 60% identical with those of both human and Xenopus laevis RBPs. The comparable sequence similarities that we have found agree with the estimate of similar divergence times between bony fishes and mammals and between bony fishes and amphibians. The two piscine RBP variants differ by six amino acid substitutions at positions that are not crucial for the interaction with retinol, on the basis of the human RBP three-dimensional structure [Cowan, S. W., Newcomer, M. E. & Jones, T. A. (1990) Proteins Struct. Func. Genet. 8, 44-61]. 2. Models were developed for the three-dimensional structures of rainbow trout and X. laevis RBPs, based on that of human RBP. The overall three-dimensional structure appears to be very well preserved for RBPs isolated from vertebrate species for which the divergence time is 350-400 million years. At variance with an almost absolute conservation for the residues that participate in the formation of the retinol binding site in mammalian RBPs, several amino acid replacements are found for this part of the RBP molecule when the comparison is extended to piscine and amphibian RBPs. However, the only allowed amino acid replacements are either conservative or more than 0.4 nm distant from retinol. Besides the retinol binding site, a few regions at the protein surface appear to be rather conserved during phylogenetic development of vertebrates and, therefore, might be involved in molecular interactions.


Biochemical characterization of single-chain chimeric plasminogen activators consisting of a single-chain Fv fragment of a fibrin-specific antibody and single-chain urokinase.

K12G0S32 is a 57-kDa recombinant single-chain chimeric plasminogen activator consisting of scFv-K12Go, a single-chain variable-region antigen-binding fragment (Fv) of the monoclonal antibody MA-15C5, which is specific for fragment D-dimer of human cross-linked fibrin, and a low-molecular-mass (33 kDa) urokinase-type plasminogen activator (u-PA-33k) containing amino acids Ala132-Leu411 (Holvoet, P., Laroche, Y., Lijnen, H. R., Van Cauwenberghe, R., Demarsin, E., Brouwers, E., Matthyssens, G. & Collen D. (1991) J. Biol. Chem. 266, 19717-19724). In addition, the Arg156-Phe157 thrombin-cleavage site in the u-PA moiety of K12G0S32 is removed by substitution of Phe157 with Asp. In the present study, the fibrinolytic potency of K12G0S32, determined in a system composed of a 125I-fibrin-labeled human plasma clot submerged in citrated plasma, was found to be only twofold higher than that of intact single-chain u-Pa (rscu-PA), but 17-fold higher than that of rscu-PA(M), a variant of rscu-PA in which the thrombin-cleavage site was removed by substitution of Phe157 with Asp. The fibrinolytic potency of K12G0S32T, with an intact thrombin-cleavage site, was 6-15-fold higher than that of rscu-PA. Conversion of 1 microM single-chain K12G0S32 or rscu-PA(M) into their two-chain derivatives with plasmin occurred at a rate of 1.0 +/- 0.15 nmol.min-1.nmol plasmin-1 and 0.85 +/- 0.074 nmol.min-1.nmol plasmin-1, compared to 14 +/- 2.3 nmol.min-1.nmol plasmin-1 and 18 +/- 2.6 nM.min-1.nmol plasmin-1 for K12G0S32T and rscu-PA, respectively. Purified fragment D-dimer of human cross-linked fibrin inhibited the fibrinolytic potency of single-chain K12G0S32T, but not of two-chain K12G0S32T, in a dose-dependent manner. Furthermore, the fibrinolytic potencies of two-chain K12G0S32 and K12G0S32T were not significantly higher than those of recombinant two-chain u-PA (rtcu-PA) or of rtcu-PA(M). These findings suggest that the 59-fold increase in fibrinolytic potency of K12G0S32T, relative to that of rscu-PA(M), is due both to targeting of the activator to the clot via the single-chain Fv fragment (sixfold increase) and to a more efficient conversion of single-chain K12G0S32T to its two-chain derivative (eightfold increase). Thus, targeting to clots by means of fibrin-specific antibodies results in a significant increase of the fibrinolytic potency of single-chain but not of two-chain u-PA.(ABSTRACT TRUNCATED AT 400 WORDS)


Time-resolved fluorescence study of human recombinant interferon alpha 2. Association state of the protein, spatial proximity of the two tryptophan residues.

Human recombinant interferon alpha 2 belongs a to family of proteins active against a wide range of viruses. It contains two tryptophan residues located at positions 77 and 141 in the peptide sequence. The fluorescence emission spectrum of these tryptophan residues displays a maximum at 335 nm. The fluorescence intensity decay is described by one broad excited-state-lifetime population centered around a value of 1.7 ns (full width at half maximum, 1.5 ns). These observations suggest that in the native protein, both tryptophan residues emit from similar environments, not directly exposed to the surrounding solvent. The anisotropy decay is essentially biexponential. The correlation-time value characterizing the Brownian rotation of the protein varies linearly with the viscosity/temperature ratio. The calculated hydrodynamic volumes are compatible with the existence of a dimer and a tetramer, at pH 5.5 and 9.4, respectively. Addition of urea at pH 5.5 disrupts the dimer and modifies to some extent the excited-state-lifetime distribution which becomes more heterogeneous. Disulfide-bond reduction also dissociates the dimer and leads to a highly heterogeneous fluorescence-intensity decay with four excited-state-lifetime populations. An opening of the local structure in the Trp region of the protein is likely to occur in these conditions. The fast-anisotropy-decay components can be due to either fast rotation or energy transfer between the indoles. Close proximity of the two Trp residues (less than 1 nm) is suggested from steady-state and time-resolved fluorescence-anisotropy measurements in vitrified medium [95% (by mass) glycerol at -38 degrees C]. This suggestion is in agreement with the recently published three-dimensional structure of the homologous protein murine interferon beta [Senda, T., Shimazu, T., Matsuda, S. Kawano, G., Shimizu, H., Nakamura, K. T. & Mitsui, Y. (1992) EMBO J. 11, 3193-3201].


The hemoglobins of Notothenia angustata, a temperate fish belonging to a family largely endemic to the Antarctic Ocean.

The blood of the teleost Notothenia angustata contains a major hemoglobin (Hb 1, over 95% of the total), accompanied by a minor component (Hb 2). The two hemoglobins have identical beta chains and differ in their alpha chains. The primary structure of both hemoglobins has been established through the elucidation of the complete amino acid sequence of the three chains. The study of the oxygen-binding properties shows that Hb 1 displays the Bohr and Root effects and has high affinity for organic phosphates. N. angustata belongs to the family Nototheniidae, suborder Notothenioidei. Unlike the vast majority of nototheniid species, which live in isolation in the Antarctic Ocean and have developed cold adaptation, N. angustata inhabits the waters of southern New Zealand and is not cold adapted. Although some hematological parameters typically favour oxygen transport in a temperate environment, the hemoglobin multiplicity and structural and functional features closely resemble those of the Antarctic species of the same family and suborder. Thus, N. angustata may be considered as a link between temperate and Antarctic habitats. The hypothetical separation history of N. angustata from the Antarctic species of the same family is discussed in the light of the present findings.


Salt dependence, kinetic properties and catalytic mechanism of N-formylmethanofuran:tetrahydromethanopterin formyltransferase from the extreme thermophile Methanopyrus kandleri.

N-Formylmethanofuran(CHO-MFR):tetrahydromethanopterin(H4MPT) formyltransferase (formyltransferase) from the extremely thermophilic Methanopyrus kandleri was purified over 100-fold to apparent homogeneity with a 54% yield. The monomeric enzyme had an apparent molecular mass of 35 kDa. The N-terminal amino acid sequence of the polypeptide was determined. The formyltransferase was found to be absolutely dependent on the presence of phosphate or sulfate salts for activity. The ability of salts to activate the enzyme decreased in the order K2HPO4 > (NH4)2SO4 > K2SO4 > Na2SO4 > Na2HPO4. The salts KCl, NaCl and NH4Cl did not activate the enzyme. The dependence of activity on salt concentration showed a sigmoidal curve. For half-maximal activity, 1 M K2HPO4 and 1.2 M (NH4)2SO4 were required. A detailed kinetic analysis revealed that phosphates and sulfates both affected the Vmax rather than the Km for CHO-MFR and H4MPT. At the optimal salt concentration and at 65 degrees C, the Vmax was 2700 U/mg (1 U = 1 mumol/min), the Km for CHO-MFR was 50 microM and the Km for H4MPT was 100 microM. At 90 degrees C, the temperature optimum of the enzyme, the Vmax was about 2.5-fold higher than at 65 degrees C. Thermostability as well as activity of formyltransferase was dramatically increased in the presence of salts, 1.5 M being required for optimal stabilization. The efficiency of salts in protecting formyltransferase from heat inactivation at 90 degrees C decreased in the order K2HPO4 = (NH4)2SO4 >> KCl = NH4Cl = NaCl >> Na2SO4 > Na2HPO4. The catalytic mechanism of formyltransferase was determined to be of the ternary-complex type. The properties of the enzyme from M. kandleri are compared with those of formyltransferase from Methanobacterium thermoautotrophicum, Methanosarcina barkeri and Archaeoglobus fulgidus.