Roles of the Domain I Segment in Emulsifying Properties of Bovine Serum Albumin

Emulsifying properties of bovine serum albumin (BSA) were compared between intact BSA (molecular mass of 66 kDa) and the domain I-truncated fragment (molecular mass of 45 kDa). The particle size considerably decreased with increasing both intact BSA and the fragment concentration. The intact BSA formed a more homogeneous emulsion and smaller particle distributions than the fragment at all protein concentrations. The relative amount of protein adsorbed at an oil-water interface was much less for intact BSA than for the fragment, and the surface concentration of intact BSA per unit surface area was also much less than that of the fragment, indicating that less protein is required to form a stable film for intact BSA than for the fragment. The particle size distribution in the fragment-stabilized emulsion became more homogeneous during storage time, while that in intact BSA-stabilized BSA had no significant change. These results strongly suggested that the domain I-truncated fragment with a higher hydrophobic value is more favorable toward the oil phase, while the highly charged domain I segment inhibits the droplet association, and consequently is important in stabilizing droplets dispersed in the emulsion.     

Absence of carbohydrate in crystalline Fraction I protein isolated from tobacco leaves

By means of gas—liquid chromatography—mass spectra analyses, three-times recrystallized Fraction I protein from tobacco leaved contains less than 0.0005% carbohydrate and is therefore different from other species of protein which contain up to 5% carbohydrate.     

Study of insulin-like growth factor I in human obesity.

In obesity there is a decrease in basal and stimulated GH secretion. IGF-I, which has negative feedback effects on GH secretion, could be the initial mediator of such alterations. We studied IGF-I levels in obese subjects and their relationship to the obesity level and GH secretion. We determined plasma IGF-I, basal and stimulated GH in 30 normal and 30 obese women and related these variables to obesity indices (body mass index, BMI, and % overweight). Baseline plasma GH values were 1.2 +/- 0.3 and 2.3 +/- 0.6 micrograms/l in obese subjects and controls, respectively (NS). Mean peak GH secretion after stimuli were 11.2 +/- 1.4 and 34.4 +/- 5.6 micrograms/l in obese subjects and controls, respectively (p less than 0.001). Plasma IGF-I were 1.0 +/- 0.1 U/ml and 0.7 +/- 0.1 U/l in obese subjects and controls, respectively (NS). There was a significant negative correlation between plasma IGF-I and age (r = -0.55, p less than 0.001) and a significant negative correlation between mean peak GH secretion and weight (r = -0.60, p less than 0.001), BMI (r = -0.64, p less than 0.001) and percentage of ideal body weight (r = -0.67, p less than 0.001). We did not find any correlation between IGF-I and indices of overweight. These data suggest that the reduced GH secretion found in obesity is not related to a negative feedback inhibition by elevated levels of IGF-I and that adiposity is not associated with a decline in IGF-I levels. We confirm the existence of a negative correlation between GH secretion and obesity indices.     

Increase of IGF I binding to erythrocyte receptors during the TRH-test: correlation between IGF I binding and thyroid hormone values

The effects of TRH on insulin-like growth factor I receptors were investigated on erythrocytes from 7 GH-deficient children having plasma GH levels less than 10 ng/ml during two provocation tests. Intravenous injection of synthetic TRH (0.2 mg/m2) was followed by a marked increase of IGF I binding on erythrocytes, from 3.9% +/- 0.3% to 5.9% +/- 0.3% (P less than 0.005) after 1 hour and 7.3% +/- 0.4% (P less than 0.005) after 2 hours. The IGF I binding variations were due to an increase in both the receptor affinity and the number of sites. The levels of plasma GH, IGF I, T3, T4, free T4, TSH and prolactin having been determined during the TRH test at 0, 1 hour, and 2 hours after the injection, the increase in the IGF I binding to erythrocytes at the same time correlated with the rise of thyroid hormones: triiodothyronine T3 (P less than 0.001) and thyroxine T4 (P less than 0.005) and not with the level of the other hormones. These findings suggest that thyroid hormones play a role in the regulation of insulin-like growth factor I receptors.     

In vitro development of rabbit pronuclear embryos in rabbit peritoneal fluid

The use of rabbit peritoneal fluid (PF) for the culture of rabbit embryos in vitro was evaluated. Development of zygotes cultured in PF and Earle's balanced salts solution (EBSS) + 10% fetal calf serum (EBSS/FCS) was compared. The effects of increasing the concentration of PF in EBSS and of culturing embryos in fractionated PF were also investigated. In addition, embryonic development in PF was compared to that in vivo. Development to hatching blastocysts was enhanced with PF (73%) compared to EBSS/FCS (3%, p less than 0.001). PF manifested greater mitogenic activity than EBSS/FCS, as indicated by higher cell number in embryos at 48, 72, and 96 h post-mating/hCG (p less than 0.001). PF also promoted blastocyst cell proliferation in a dose-dependent manner (r = 0.98, p less than 0.01); however, embryo growth remained slower than in vivo. Culture in the high (greater than 30,000 Da) molecular mass fraction of PF reduced incidence of hatching (56% vs. 92%, p less than 0.001) and mean cell number in Day 4 blastocysts (151 +/- 4 vs. 243 +/- 5, p less than 0.001). Rates of blastocyst hatching (10%) and cell number (110 +/- 3) were further reduced in the low (less than 30,000 Da) molecular mass fraction. When the high molecular mass fraction was dialyzed, embryos did not develop beyond the early morula stage. This suggests that the interaction or the synergy of high and low molecular mass components of PF is necessary for optimum development of rabbit embryos.     

Mass analysis of bacteriophage T4 proheads and mature heads by scanning transmission electron microscopy and hydrodynamic measurements.

Quantitative mass analysis of bacteriophage T4 proheads by scanning transmission electron microscopy (STEM) revealed a mass of 79.5 +/- 0.6 MDa, while hydrodynamic measurements yielded a prohead mass of about 80 MDa. This is 25% less than the prohead mass deduced from its polypeptide composition, and this finding implies that the bacteriophage T4 prohead is built of fewer polypeptide copies than previously reported. In contrast, the mass of mature heads measured by STEM, 194 +/- 2 MDa, is in agreement with previous mass measurements of DNA and protein content, and it is consistent with the previously determined stoichiometry. This good agreement of average STEM values for proheads and mature heads with corresponding hydrodynamic measurements suggests that STEM allows faithful evaluation of the masses of large supramolecular assemblies (i.e., greater than or equal to 200 MDa) such as whole viruses or cellular organelles.     

Effects of Nitrate Application on Amaranthus powellii Wats. : I. Changes in Photosynthesis, Growth Rates, and Leaf Area

Physiological effects of different nitrate applications were studied using the C₄ plant, Amaranthus powellii Wats. Plants were grown in a controlled environment chamber and watered daily with nutrient solutions containing 45, 10, 5, or 1 millimolar nitrate. Chloride and sulfate were used to keep the cation and phosphate concentrations constant. Total leaf nitrogen concentration, chlorophyll concentration, specific leaf mass, leaf area, relative growth rate, relative leaf growth rate, unit leaf rate (increase of dry mass per unit leaf area per day), net photosynthetic rate, and incident quantum yield decreased with decreasing nitrate concentration. The per cent decrease of unit leaf rate was similar to the decrease of light-saturated net photosynthetic rate; however, the decrease in relative growth rate was less than that of unit leaf rate because leaf area ratio (leaf area per unit dry mass) increased with decreasing nitrate concentration. Essential mineral concentrations per unit leaf area were about equal among all treatments. Leaf expansion, determined by stomatal density, decreased except for the 1 millimolar treatment which showed relatively more cell expansion but less cell division. Decreased nitrate application was correlated with higher osmotic potentials and lower pressure potentials (determined by pressure-volume curves), whereas leaf water potentials were equal among treatments. Even though total leaf area and shoot mass decreased with decreasing applied nitrate, the increase of the leaf area ratio may be related to selection for the highest possible growth rate.     

Prepubertal Asians have less limb skeletal muscle.

Skeletal muscle mass in prepubertal Asian children has not been examined previously. The aims of this study were to test the hypotheses that 1) prepubertal Asians have less appendicular skeletal muscle (ASM) mass compared with African-Americans and Caucasians, and 2) ASM is less in prepubertal Asian girls compared with Asian boys. ASM was estimated by using dual-energy X-ray absorptiometry in healthy prepubertal girls (n = 170) and boys (n = 166). The results showed that, after adjusting for age, height, and body weight, 1) Asian girls and boys had less amounts of ASM than African-Americans (P < 0.001); 2) Asian girls had less amounts of ASM than Caucasian girls (P = 0.004); 3) there was a trend towards less ASM in Asian compared with Caucasian boys (P = 0.07); 4) and Asian girls had significantly less ASM than Asian boys (P < 0.001). This study indicates that skeletal muscle mass as a fraction of body weight is smaller in Asian compared with African-American and Caucasian children.     

Subunit stoichiometry of the chloroplast photosystem I complex

A native photosystem I (PS I) complex and a PS I core complex depleted of antenna subunits has been isolated from the uniformly 14C-labeled aquatic higher plant, Lemna. These complexes have been analyzed for their subunit stoichiometry by quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis methods. The results for both preparations indicate that one copy of each high molecular mass subunit is present per PS I complex and that a single copy of most low molecular mass subunits is also present. These results suggest that iron-sulfur center X, an early PS I electron acceptor proposed to bind to the high molecular mass subunits, contains a single [4Fe-4S] cluster which is bound to a dimeric structure of high molecular mass subunits, each providing 2 cysteine residues to coordinate this cluster.     

The PSI-K subunit of photosystem I is involved in the interaction between light-harvesting complex I and the photosystem I reaction center core

PSI-K is a subunit of photosystem I. The function of PSI-K was characterized in Arabidopsis plants transformed with a psaK cDNA in antisense orientation, and several lines without detectable PSI-K protein were identified. Plants without PSI-K have a 19% higher chlorophyll a/b ratio and 19% more P700 than wild-type plants. Thus, plants without PSI-K compensate by making more photosystem I. The photosystem I electron transport in vitro is unaffected in the absence of PSI-K. Light response curves for oxygen evolution indicated that the photosynthetic machinery of PSI-K-deficient plants have less capacity to utilize light energy. Plants without PSI-K have less state 1-state 2 transition. Thus, the redistribution of absorbed excitation energy between the two photosystems is reduced. Low temperature fluorescence emission spectra revealed a 2-nm blue shift in the long wavelength emission in plants lacking PSI-K. Furthermore, thylakoids and isolated PSI without PSI-K had 20-30% less Lhca2 and 30-40% less Lhca3, whereas Lhca1 and Lhca4 were unaffected. During electrophoresis under mildly denaturing conditions, all four Lhca subunits were partially dissociated from photosystem I lacking PSI-K. The observed effects demonstrate that PSI-K has a role in organizing the peripheral light-harvesting complexes on the core antenna of photosystem I.     

3D whole body scanning to determine mass properties of legs.

We describe a novel method of measuring the mass properties of the limbs, specifically legs. We use the method to obtain the mass and centre of mass of the legs which enables us to calculate the leg joint moments from measurements of ground reaction forces using force plates. The data are obtained by using a 3D whole body scanner to obtain a data set representing the surface of both legs. The bones are significantly denser than the soft tissue so their mass is calculated. Textbook values for the densities of bone and soft tissue are used. The actual bones are approximated by stretching appropriate bone shapes to fit the X-ray of the subject. Numerical integration is then used to obtain the mass and centre of mass of the limb. The system is fast and reliable and allows an individual's mass properties to be measured rather than relying upon population surveys which may be biased, particularly, when the subject is atypical by being disabled. Paraplegics can be measured in the scanner using a modified Oswestry standing frame. When compared with a water displacement method, for 10 legs the errors in the total leg volume using this method are less than 1% and in the location of the centre of mass are less than 4%.     

Characterization of dehydropeptidase I in the rat lung

The activity of dehydropeptidase I in rat tissues decreases in the order of lung greater than kidney greater than liver-spleen greater than other tissues, while aminopeptidase activity is high in the kidney, and lower in the lung than in other tissues. Dehydropeptidase I was solubilized from the membrane fraction of rat lung by treatment with papain and purified by DEAE-cellulose column chromatography, affinity chromatography on concanavalin-A-Sepharose and high-performance liquid chromatography gel filtration. The purified preparation was found to be homogeneous on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The relative molecular mass was estimated to be 150,000 by gel filtration, comprising a homodimer of two 80,000-Mr subunits. The enzyme activity was inhibited by cilastatin, o-phenanthroline and ATP. This enzyme catalyzed the hydrolysis of S(substituent)-L-cysteinyl-glycine adducts such as L-cystinyl-bis(glycine) and N-ethylmaleimide-S-L-cysteinyl-glycine, as well as the conversion of leukotriene D4 to E4. Furthermore it catalyzed a hydrolytic splitting of L-Leu-L-Leu, but not S-benzyl-L-cysteine p-nitroanilide, which is a good substrate for aminopeptidase. Our enzyme preparation was immunologically identical to the rat renal dehydropeptidase I. The physiological significance of the pulmonary dehydropeptidase I on the metabolism of glutathione and its adducts is discussed.     

Analytical biotechnology of recombinant peptides and proteins. I. Analysis of the purity, composition and structure of human, porcine and bovine insulins

A method for analysis of the type, purity, and possible structural modifications of insulins of bovine, porcine, and human origin was proposed. It is based on a combination of narrow-bore reversed-phase HPLC and mass spectrometry. The hydrolysis of insulins with highly specific Glu-protease V8 from Staphylococcus aureus followed by peptide mapping of the hydrolysis products and mass spectrometry of the isolated fragments helps rapidly and reliably localize and identify substitutions of amino acid residues in insulin structure by using insulin samples of less than 1 nmol.     

Effects of lower limb unloading on skeletal muscle mass and function in humans

A model to simulate effects of microgravity on skeletal muscle mass and function in humans has been developed. Unilateral lower limb unloading that allowed ankle, knee, and hip joint mobility was conducted in six healthy men by suspending one lower limb and having the subjects walk on crutches. They performed maximal unilateral concentric or eccentric quadriceps actions at different angular velocities before and after 4 wk of suspension and after 4 days and after 7 wk of uncontrolled recovery. Peak torque (PT) and angle-specific torque (AST) were measured. Muscle cross-sectional area (CSA) and radiological density (RD) of the thigh were assessed by means of computerized tomography. Concentric and eccentric PT and AST across speeds decreased (P less than 0.05) by 22 and 16%, respectively, in response to unloading. At 4 days of recovery PT (-11%) and AST (-7%) were still lower (P less than 0.05) than before. Muscle CSA and RD decreased (P less than 0.05) by 7 and 6%, respectively. After 7 wk of recovery PT, AST, CSA, and RD had returned to normal. The control limb showed no changes over the experimental period except for a 6% decrease (P less than 0.05) in RD. It is suggested that this human model of unloading could serve to simulate effects of microgravity on skeletal muscle mass and function because reductions in muscle mass and strength were of similar magnitude to those produced by bed rest.     

Analytical biotechnology of recombinant peptides and proteins: I. determination of the purity, composition, and structure of human, porcine, and bovine insulins

A method for analysis of the type, purity, and possible structural modifications of insulins of bovine, porcine, and human origin was proposed. It is based on a combination of narrow-bore reversed-phase HPLC and mass spectrometry. The hydrolysis of insulins with highly specific Glu-protease V8 fromStaphylococcus aureus followed by peptide mapping of the hydrolysis products and mass spectrometry of the isolated fragments helps rapidly and reliably localize and identify substitutions of amino acid residues in insulin structure by using insulin samples of less than 1 nmol.     

Mathematical modelling of dynamics and control in metabolic networks. I. On Michaelis-Menten kinetics

As a starting point for modeling of metabolic networks this paper considers the simple Michaelis-Menten reaction mechanism. After the elimination of diffusional effects a mathematically intractable mass action kinetic model is obtained. The properties of this model are explored via scaling and linearization. The scaling is carried out such that kinetic properties, concentration parameters and external influences are clearly separated. We then try to obtain reasonable estimates for values of the dimensionless groups and examine the dynamic properties of the model over this part of the parameter space. Linear analysis is found to give excellent insight into reaction dynamics and it also gives a forum for understanding and justifying the two commonly used quasi-stationary and quasi-equilibrium analyses. The first finding is that there are two separate time scales inherent in the model existing over most of the parameter space, and in particular over the regions of importance here. Full modal analysis gives a new interpretation of quasi-stationary analysis, and its extension via singular perturbation theory, and a rationalization of the quasi-equilibrium approximation. The new interpretation of the quasi-steady state assumption is that the applicability is intimately related to dynamic interactions between the concentration variables rather than the traditional notion that a quasi-stationary state is reached, after a short transient period, where the rates of formation and decomposition of the enzyme intermediate are approximately equal. The modal analysis reveals that the generally used criterion for the applicability of quasi-stationary analysis that total enzyme concentration must be much less than total substrate concentration, et much less than St, is incomplete and that the criterion et much less than Km much less than St (Km is the well known Michaelis constant) is the appropriate one. The first inequality (et much less than Km) guarantees agreement over the longer time scale leading to quasi-stationary behavior or the applicability of the zeroth order outer singular perturbation solution but the second half of the criterion (Km much less than St) justifies zeroth order inner singular perturbation solution where the substrate concentration is assumed to be invariant. Furthermore linear analysis shows that when a fast mode representing the binding of substrate to the enzyme is fast it can be relaxed leading to the quasi-equilibrium assumption. The influence of the dimensionless groups is ascertained by integrating the equations numerically, and the predictions made by the linear analysis are found to be accurate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of lincomycin on the chlorophyll protein complex I content and Photosystem I activity of greening leaves

1. 1. Greening barley and pea leaves treated with lincomycin have a reduced chlorophyll content. Lincomycin does not alter the proportion of chlorophyll in chlorophyll-protein complex II (CPII) but greatly reduces that in chlorophyll-protein complex I (CPI).

2. 2. Difference spectra show that chloroplasts from lincomycin-treated leaves are deficient in at least two long wavelength forms of chlorophyll a. These have maxima at 77 K of 683 and 690 nm.

3. 3. The chemically determined P-700/chlorophyll ratio of chloroplasts is unaffected by lincomycin but the photochemical P-700/chlorophyll ratio is less than half of that of the control. It is less affected than the chlorophyll-protein complex I content.

4. 4. Photosystem I activity expressed on a chlorophyll basis is unaffected by lincomycin but the light intensity for half saturation is increased 8-fold.

5. 5. Chlorophyll-protein complex I apoprotein content is reduced by lincomycin. No evidence was found for an accumulation of its precursor(s). The relative abundance of major peptides of 18 000, 15 000 and 12 000 daltons in lincomycin-treated chloroplasts is attributed to a general inhibition of greening and associated membrane formation.

Enhanced cholesteryl ester transfer protein activities and abnormalities of high density lipoproteins in familial hypercholesterolemia.

Cholesteryl ester transfer protein may play a role in the cholesteryl ester metabolism between high density lipoproteins (HDL) and apolipoprotein B-containing lipoproteins. To investigate relationship between HDL and cholesteryl ester transfer protein (CETP) activity in the development of atherosclerosis, the present study has focused on CETP activity in the patients with familial hypercholesterolemia (GH). HDL-C and HDL-C/apo A-I mass ratio in heterozygous FH were lower than those in normolipidemic controls. There was a 2-fold increase in total CETP activity in incubated FH serum compared with normolipidemic controls. Assays for CETP activity in the lipoprotein deficient serum (d greater than 1.215 g/ml) were carried out by measuring the transfer of radioactive cholesteryl ester from HDL (1.125 less than d less than 1.21 g/ml) to LDL (1.019 less than d less than 1.060 g/ml). CETP activities in heterozygous FH (79 +/- 4 nmol/ml/h) was significantly higher than those in normolipidemic controls (54 +/- 6 nmol/ml/h). The increased total cholesteryl ester transfer mainly results from increased CETP activity in the d greater than 1.215 g/ml, possibly reflecting an increase in CETP mass in serum. Increased CETP activity in the d greater than 1.215 g/ml was correlated positively with IDL-cholesterol/triglyceride mass ratio (r = 0.496, p less than 0.01), and negatively with HDL-cholesterol/apo A-I mass ratio (r = -0.334, p less than 0.05). These results indicate that the enhanced CETP activities may contribute to increase risk for developing atherosclerosis in FH by changing the distribution of cholesteryl ester in serum lipoproteins.     

Active oxygen produced during selective excitation of photosystem I is damaging not only to photosystem I, but also to photosystem II

With the aim to specifically study the molecular mechanisms behind photoinhibition of photosystem I, stacked spinach (Spinacia oleracea) thylakoids were irradiated at 4 degrees C with far-red light (>715 nm) exciting photosystem I, but not photosystem II. Selective excitation of photosystem I by far-red light for 130 min resulted in a 40% inactivation of photosystem I. It is surprising that this treatment also caused up to 90% damage to photosystem II. This suggests that active oxygen produced at the reducing side of photosystem I is highly damaging to photosystem II. Only a small pool of the D1-protein was degraded. However, most of the D1-protein was modified to a slightly higher molecular mass, indicative of a damage-induced conformational change. The far-red illumination was also performed using destacked and randomized thylakoids in which the distance between the photosystems is shorter. Upon 130 min of illumination, photosystem I showed an approximate 40% inactivation as in stacked thylakoids. In contrast, photosystem II only showed 40% inactivation in destacked and randomized thylakoids, less than one-half of the inactivation observed using stacked thylakoids. In accordance with this, photosystem II, but not photosystem I is more protected from photoinhibition in destacked thylakoids. Addition of active oxygen scavengers during the far-red photosystem I illumination demonstrated superoxide to be a major cause of damage to photosystem I, whereas photosystem II was damaged mainly by superoxide and hydrogen peroxide.     

Mononuclear phagocyte function in SLE. I. Bipartite Fc- and complement-dependent dysfunction

To determine the relative contributions of Fc- and complement-mediated immune clearance to the overall mononuclear phagocyte system (MPS) dysfunction in SLE, we performed a kinetic analysis of clearance rate data from 32 patients and 49 normal controls. Three rate constants regulating complement-mediated MPS clearance and one rate constant regulating Fc-mediated MPS clearance were evaluated. Mean values for rate constants regulating complement-mediated phagocytosis (k4) and Fc-mediated clearance (k3) were significantly lower for the patient population as a whole when compared with normal controls (p less than 0.01 and p less than 0.001, respectively). Further analysis by clinical subgroups revealed that mean k3 values were significantly low for all but the inactive nonrenal subset of patients, whereas mean k4 values were significantly low for both the active and inactive renal patients but not for nonrenal patients. Rate constant values for Fc-mediated clearance correlated significantly with disease activity scores for the entire SLE group (p less than 0.001) as well as for the subset of patients with active and inactive renal disease (p less than 0.001). Neither k3 nor k4 correlated significantly with anti-DNA antibody, titers, total hemolytic complement levels, or circulating immune complexes. These data indicate that both Fc- and complement-mediated clearance defects occur in SLE. Nonrenal patients have at least one clearance mechanism intact, whereas immune complex glomerulonephritis is associated with dysfunctions in both of these clearance mechanisms.