Showing papers on "Low protein published in 1996"

The determinants of pKas in proteins.

Abstract: Although validation studies show that theoretical models for predicting the pKas of ionizable groups in proteins are increasingly accurate, a number of important questions remain: (1) What factors limit the accuracy of current models? (2) How can conformational flexibility of proteins best be accounted for? (3) Will use of solution structures in the calculations, rather than crystal structures, improve the accuracy of the computed pKas? and (4) Why does accurate prediction of protein pKas seem to require that a high dielectric constant be assigned to the protein interior? This paper addresses these and related issues. Among the conclusions are the following: (1) computed pKas averaged over NMR structure sets are more accurate than those based upon single crystal structures; (2) use of atomic parameters optimized to reproduce hydration energies of small molecules improves agreement with experiment when a low protein dielectric constant is assumed; (3) despite use of NMR structures and optimized atomic pa...

Weanling Rats Exposed to Maternal Low-Protein Diets during Discrete Periods of Gestation Exhibit Differing Severity of Hypertension

Abstract: 1. In the rat, hypertension is induced by fetal exposure to maternal low-protein diets. The effect on blood pressure of undernutrition before conception and during discrete periods in early, mid or late pregnancy was assessed using an 18% casein (control) diet and a 9% casein diet to apply mild protein restriction. 2. The offspring of rats fed 9% casein developed raised blood pressure by weaning age. Feeding a low-protein diet before conception was not a prerequisite for programming of hypertension. 3. Hypertension was observed in rats exposed to low protein during the following gestational periods: days 0–7, days 8–14 and days 15–22. Blood pressure increases elicited by these discrete periods of undernutrition were lower than those induced by feeding a low-protein diet throughout pregnancy. The effect in early gestation was significant only in male animals. Post-natal growth of male rats exposed to low-protein diets was accelerated, but kidneys were small in relation to body weight. 4. Biochemical indices of glucocorticoid action in liver, hippocampus, hypothalamus and lung were elevated in rats exposed to low-protein diets in utero. The apparent hypersensitivity to glucocorticoids was primarily associated with undernutrition in mid to late gestation. 5. Plasma renin activity was elevated in rats exposed to 9% casein over days 15–22 of gestation. Animals undernourished over days 0–7 and 8–14 produced pups with lower plasma angiotensin II concentrations at weaning. 6. Fetal exposure to maternal low-protein diets for any period in gestation may programme hypertension in the rat. Alterations to renal structure, renal hormone action or the hypothalamic—pituitary-adrenal axis may all play a role in the programming phenomenon, either independently or in concert.

Maternal Protein Restriction Influences the Programming of the Rat Hypothalamic-Pituitary-Adrenal Axis

Abstract: The role of glucocorticoids in the intrauterine programming of hypertension was assessed in the progeny of rats fed either 18 g casein/100 g diet (control diet) or 9 g casein/100 g diet (low protein diet), before conception and throughout pregnancy. Rats exposed to the low protein diet had significantly (P < 0.05) higher systolic blood pressures than control animals, when weaned. These rats had elevated brain and liver activities of specific glucocorticoid-inducible marker enzymes, relative to controls. Glycerol 3-phosphate dehydrogenase activity was also higher (377%) in whole brains of newborn rats exposed to low protein diet in utero, but no similar effect of corticosteroids was noted in brains of d 20 fetuses. Weanling rats of the low protein group exhibited a blunted diurnal pattern of adrenocorticotrophin (ACTH) concentrations in plasma. Plasma corticosterone concentrations were unaltered by prenatal dietary experience and exhibited a normal pattern of diurnal variation. Brain regional 11beta-hydroxysteroid dehydrogenase activities were unaltered by prenatal dietary experience, as was binding of 3H-corticosterone to type I glucocorticoid receptors in hippocampus, hypothalamus and liver. Type II glucocorticoid receptor binding capacity and receptor numbers in male rats were apparently elevated in hippocampus of low protein-exposed rats and were significantly lower in liver (P < 0.05), relative to control rats. Programming of the hypothalamic-pituitary-adrenal axis is inferred, and the observation that binding of steroid to type II receptor sites in vascular tissue is increased in low protein exposed rats may provide a direct mechanism for modulation of blood pressure by glucocorticoids in this model.

Targeting of stealth liposomes to erbB-2 (Her/2) receptor : in vitro and in vivo studies

Abstract: Long-circulating (stealth) liposomes coated with polyethylene glycol (PEG), which show reduced uptake by the reticuloendothelial system (RES) and enhanced accumulation in tumours, were used for conjugation to monoclonal antibodies (MAbs) as a drug-targeting device. A MAb (N-12A5) directed against erbB-2 oncoprotein, a functional surface antigen, was used. Amplification and overexpression of the erbB-2 gene product, being unique to malignancy, confer onto this antibody-mediated therapy high tumour specificity. In vitro binding of [3H]cholesteryl ether ([3H]Chol ether) labelled anti-erbB-2 conjugated liposomes to N-87 cells (erbB-2-positive human gastric carcinoma) was compared with the binding of non-targeted liposomes and indicated a 16-fold increase in binding for the targeted liposomes. No difference in binding to OV1063 cells (erbB-2-negative human ovary carcinoma) was observed. These results indicate highly selective binding of antibody-targeted liposomes to erbB-2-overexpressing cells. Despite increased cell binding, doxorubicin (DOX) loaded in anti-erbB-2-conjugated liposomes did not cause increased in vitro cytotoxicity against N-87 cells, suggesting lack of liposome internalisation. In vivo, the critical factor needed to decrease the non-specific RES uptake and prolong the circulation time of antibody-conjugated liposomes is a low protein to phospholipid ratio ( < 60 micrograms mumol-1). Using these optimised liposome preparations loaded with DOX and by monitoring the drug levels and the [3H]Chol ether label, biodistribution studies in nude mice bearing subcutaneous implants of N-87 tumours were carried out. No significant differences in liver and spleen uptake between antibody-conjugated and plain liposomes were observed. Nevertheless, there was no enhancement of tumour liposome levels over plain liposomes. Both liposome preparations considerably enhanced DOX concentration in the tumour compared with free drug administration. Therapeutic experiments with N-87 tumour-bearing nude mice indicated that anti-tumour activity of targeted and non-targeted liposomes was similar, although both preparations had an increased therapeutic efficacy compared with the free drug. These studies suggest that efficacy is dependent on drug delivery to the tumour and that the rate-limiting factor of liposome accumulation in tumours is the liposome extravasation process, irrespective of liposome affinity or targeting to tumour cells.

Protein Adsorption on Cation Exchangers: Comparison of Macroporous and Gel-Composite Media

Abstract: The protein uptake equilibrium and kinetics and the breakthrough behavior of recently developed commercial chromatography media are evaluated using lysozyme as a model solute. One of the adsorbents, known by the trade name POROS 50, is a macroporous matrix based on a styrene-divinylbenzene copolymer. The other adsorbent, known by the trade name HyperD, is a composite obtained by filling the pores of high-porosity polystyrene-coated silica particles with a polyacrylamide-based hydrogel. Both materials are designed for preparative and process ion-exchange chromatography of proteins at high speed and have a strong cation exchange functionality. The equilibrium and transport properties of lysozyme in each adsorbent are studied in batch experiments. The maximum equilibrium uptake capacity in a 10 mM sodium phosphate buffer at pH 6.5 is 160 ± 5 mg/cm 3 of particle volume for the macroporous adsorbent and 260 ± 10 mg/cm 3 for the gel-composite material. With the macroporous adsorbent mass transfer appears to be dominated by macropore diffusion with an effective pore diffusivity of 1.1 x 10 -7 cm 2 /s, except during the initial saturation of the outermost layer of the particle, when external film mass transfer is controlling. An idealized two-step model of this process is found to be consistent with the experimental data. With the gel-composite adsorbent, however, mass transfer appears to be dominated by homogeneous gel diffusion with an effective pseudo-homogeneous diffusivity of 7.5 x 10 -9 cm 2 /s at high protein concentrations and by the external film resistance at low protein concentrations. For both adsorbents, breakthrough profiles obtained experimentally at elevated flow rates in packed columns are in good agreement with predictions based on the batch measurements and external film coefficients predicted from literature correlations. In spite of the fact that it possesses a larger particle size, the gel-composite adsorbent appears to be superior to the macroporous medium, exhibiting a dynamic capacity at 10% breakthrough more than a factor of 2 larger at mobile phase velocities in the range 0-5000 cm/h.

Detritus-Bacteria-Meiofauna interactions in a seagrass bed (Posidonia oceanica) of the NW Mediterranean

Abstract: The biochemical composition of the sediment organic matter, and bacterial and meiofaunal dynamics, were monitored over an annual cycle in aPosidonia oceanica bed of the NW Mediterranean to test the response of the meiofauna assemblage to fluctuations in food availability. Primary production cycles of the seagrass and its epiphytes were responsible for relatively high (compared to other Mediterranean systems) standing stocks of organic carbon in sediments (from 1.98 to 6.16 mg Cg−1 sediment dry weight). The biopolymeric fraction of the organic matter (measured as lipids, carbohydrates, and proteins) accounted for only a small fraction (18%) of the total sedimentary organic carbon. About 25% of the biopolymeric fraction was of microphytobenthic origin. Sedimentary organic carbon was mostly refractory (56 to 84%) and probably largely not utilizable for benthic consumers. The biopolymeric fraction of the organic matter was characterized by high carbohydrate concentrations (from 0.27 to 5.31 mg g−1 sediment dry weight in the top 2 cm) and a very low protein content (from 0.07 to 0.80 mg g−1 sediment dry weight), which may be a limiting factor for heterotrophic metabolism in seagrass sediments. RNA and DNA concentrations of the Sediments varied significantly during the year. High RNA and DNA values occurred during the microphytobenthic bloom and in correspondence with peaks of bacterial abundance. Bacteria accounted for a small fraction of the total organic carbon (0.65%) and of the biopolymeric organic carbon (4.64%), whilst microphytobenthos accounted for 3.79% of total organic carbon and for 25.08% of the biopolymeric carbon. Bacterial abundance (from 0.8 to 5.8 × 108 g−1 sediment dry weight) responded significantly to seasonal changes of organic matter content and composition and was significantly correlated with carbohydrate concentrations. Bacteria might be, in the seagrass system, an important N storage for higher trophic levels as il accounted for 25% of the easily soluble protein. pool and contributed significantly to the total DNA pool (on average 12%). Total meiofaunal density ranged from 236 to 1858 ind. 10 cm−2 and was significantly related, with a time lag, to changes in bacterial standing stocks indicating that microbes might represent an important resource. Bacterial abundance and biomass were also significantly related to nematode abundance. These results indicate that bacteria may play a key role in the benthic trophic

Altered regulation of hepatic glucose output in the male offspring of protein-malnourished rat dams

Abstract: Offspring of protein-malnourished rat dams have permanent alterations in hepatic enzyme activities associated with glucose homeostasis. Hormonal control of hepatic glucose output (HGO) was studied in male offspring of dams fed either a 20% (control) or 8% (low protein) protein diet during pregnancy and lactation. Glucagon (210 pM) stimulated HGO significantly more (P < 0.04) in controls (from 0.72 +/- 0.11 to 3.18 +/- 0.30 mumol.min-1.g liver-1) compared with low-protein animals (from 0.53 +/- 0.11 to 2.05 +/- 0.24 mumol.min-1.g liver-1). Insulin (1 nM) decreased (P < 0.001) HGO in controls to 2.39 +/- 0.37 mumol.min-1.g liver-1 after 10 min but increased HGO (to 2.82 +/- 0.40 mumol.min-1.g liver-1; P < 0.04) in low-protein rats. There were fivefold fewer (P = 0.01) glucagon receptors but a threefold increase (P < 0.05) in hepatic insulin receptor number in the low-protein rats, which was reflected by increased in insulin degradation (P < 0.001). The glucose transporter GLUT-2 was also raised threefold in the low-protein group (P < 0.001). The anomalous response to insulin indicates changes in its metabolic signaling, but normal insulin binding suggests that this alteration is a postreceptor event.

Association of disproportionate growth of fetal rats in late gestation with raised systolic blood pressure in later life.

Abstract: In human populations, patterns of disproportionate fetal growth are associated with cardiovascular disease in later life. Protein restriction of pregnant rats is known to impair fetal growth and is also associated with increased systolic blood pressure in later life. Growth of fetuses exposed to maternal low protein diets was found to be accelerated between day 14 and day 20 of gestation, but this growth appeared to falter in late gestation, resulting in low or normal birthweights. Placental growth was also accelerated by protein restriction. Day 20 fetuses from rats fed low protein diets were heavier but had proportionally smaller brains than did control fetuses. These animals were also longer in proportion to body mass. Between day 20 and full term (day 22), growth of the brain was spared at the expense of the trunk and at birth, pups exposed to low protein were short in relation to body mass. At weaning, rats exposed to low protein diets in utero had significantly higher systolic blood pressure relative to control animals. These data indicate that increased blood pressure in rats is linked to disproportionate patterns of growth in middle and late gestation.

Improved refolding of an immobilized fusion protein

Abstract: Fusion proteins of monomeric α-glucosidase from Saccharomyces cerevisiae containing N- or C-terminal hexa-arginine peptides were expressed in the cytosol of Escherichia coli in soluble form. The polycationic peptide moieties allow noncovalent binding of the denatured fusion proteins to a polyanionic solid support. Upon removal of the denaturant, refolding of the matrix-bound protein can proceed without perturbation by aggregation. However, nonspecific interactions of the denatured polypeptide, or of folding intermediates, with the matrix cause a drastic decrease in renaturation under suboptimal folding conditions. At low salt concentrations, ionic interactions of the refolding polypeptide with the matrix result in lower yields of renaturation. At higher salt concentrations, renaturation is prevented by hydrophobic interactions with the matrix. Apart from ionic strength, renaturation of the denatured matrix-bound fusion protein must be optimized with respect to pH, temperature, cosolvents, and matrix material used. Under optimum conditions, immobilized α-glucosidase can be renatured with a high yield at protein concentrations up to 5 mg/ml, whereas folding of the wild-type enzyme in solution is feasible only at an extremely low protein concentration (15 μg/ml). Thus, folding of the immobilized α-glucosidase allows an extremely high yield of the renaturated model protein. The technology should be applicable to other proteins that tend to aggregate during refolding.

Computing ionization states of proteins with a detailed charge model

Abstract: A convenient computational approach for the calculation of the p Kas of ionizable groups in a protein is described. The method uses detailed models of the charges in both the neutral and ionized form of each ionizable group. A full derivation of the theoretical framework is presented, as are details of its implementation in the UHBD program. Application to four proteins whose crystal structures are known shows that the detailed charge model improves agreement with experimentally determined pKas when a low protein dielectric constant is assumed, relative to the results with a simpler single-site ionization model. It is also found that use of the detailed charge model increases the sensitivity of the computed pKas to the details of proton placement. © 1996 by John Wiley & Sons, Inc.

Association of antithrombotic factor deficiencies and hypofibrinolysis with Legg-Perthes disease.

Abstract: Thirty-three (75 per cent) of forty-four unselected children who had Legg-Perthes disease were found to have coagulation abnormalities. Twenty-three children had thrombophilia (a deficiency in antithrombotic factor C or S, with an increased tendency toward thrombosis); nineteen of the twenty-three children had protein-C deficiency and four had protein-S deficiency. Seven children had a high level (0.25 gram per liter or more) of lipoprotein(a), a thrombogenic, atherogenic lipoprotein associated with osteonecrosis in adults. Three children had hypofibrinolysis (a reduced ability to lyse clots). The mean age of the children when the Legg-Perthes disease was first diagnosed was 5.8 ± 2.7 years, and the mean age at the time of the present study was 10.1 ± 4.4 years. At least one of the first-degree relatives of eleven of the nineteen probands who had a low protein-C level had a low protein-C level as well; all of these low levels represented previously undiagnosed familial protein-C deficiency. The eleven probands who had familial protein-C deficiency were more likely to have early onset of Legg-Perthes disease (at or before the age of five years) than the eleven children who had normal levels of protein C, protein S, and lipoprotein(a) as well as normal fibrinolytic activity (chi-square = 6.6; p = 0.01). At least one first-degree relative of one of the four probands who had a low protein-S level had a low protein-S level and previously undiagnosed familial protein-S deficiency. At least one first-degree relative of six of the seven probands who had a high level of lipoprotein(a) had a familial high level of lipoprotein(a). Six of the seven children who had a high level of lipoprotein(a) also had a low level of stimulated tissue-plasminogen activator activity, the major initiator of fibrinolysis. At least one first-degree relative of one of the three probands who had normal levels of protein C, protein S, and lipoprotein(a) but low stimulated tissue-plasminogen activator activity also had low stimulated tissue-plasminogen activator activity (familial hypofibrinolysis). Legg-Perthes disease, thrombophlebitis, premature myocardial infarction, and stroke, which are ramifications of the familial thrombophilic-hypofibrinolytic disorders, were common in the first and second-degree relatives of the thirty-three children with Legg-Perthes disease who also had thrombophilic-hypofibrinolytic disorders. CLINICAL RELEVANCE: Protein-C or S deficiency, hypofibrinolysis, or a high level of lipoprotein(a) may result in thrombotic venous occlusion of the femur, which leads to the venous hypertension and osteonecrosis of the femoral head characteristic of Legg-Perthes disease. When Legg-Perthes disease develops in a child, the levels of proteins C and S, lipoprotein(a), and stimulated fibrinolysis should be measured. Early diagnosis of protein-C or S deficiency, hypofibrinolysis, or a high level of lipoprotein(a) in such children may open avenues for pharmacological preventive therapy to reduce thrombophilia, stimulate fibrinolysis, or lower the level of lipoprotein(a), potentially ameliorating the Legg-Perthes disease process.

Randomized Controlled Trial of a Low Animal Protein, High Fiber Diet in the Prevention of Recurrent Calcium Oxalate Kidney Stones

Abstract: Low protein diets are commonly prescribed for patients with idiopathic calcium nephrolithiasis, who account for > 80% of new diagnoses of kidney stones. This dietary advice is supported by metabolic studies and epidemiologic observational studies but has not been evaluated in a controlled trial. Using 1983-1985 data from three Northern California Kaiser Permanente Medical Centers, the authors randomly assigned 99 persons who had calcium oxalate stones for the first time to a low animal protein, high fiber diet that contained approximately 56-64 g daily of protein, 75 mg daily of purine (primarily from animal protein and legumes), one-fourth cup of wheat bran supplement, and fruits and vegetables. Intervention subjects were also instructed to drink six to eight glasses of liquid daily and to maintain adequate calcium intake from dairy products or calcium supplements. Control subjects were instructed only on fluid intake and adequate calcium intake. Both groups were followed regularly for up to 4.5 years with food frequency questionnaires, serum and urine chemistry analysis, and abdominal radiography; and they were urged to comply with dietary instructions. In the intervention group of 50 subjects, stones recurred in 12 (7.1 per 100 person-years) compared with two (1.2 per 100 person-years) in the control group; both groups received a mean of 3.4 person-years of follow-up (p = 0.006). After adjustment for possible confounding effects of age, sex, education, and baseline protein and fluid intake, the relative risk of a recurrent stone in the intervention group was 5.6 (95% confidence interval 1.2-26.1) compared with the control group. The authors conclude that advice to follow a low animal protein, high fiber, high fluid diet has no advantage over advice to increase fluid intake alone.

Grafting of biocompatible hydrophilic polymers onto inorganic and metal surfaces

Abstract: Methods for grafting unmodified PEO or any other water-soluble polymers to the surfaces of metals and glasses to form biocompatible surfaces having low protein affinity is provided. One technique includes the steps of: (a) providing a support member having a plurality of hydroxyl or oxide groups attached to a surface of said support member; (b) exposing said surface to a silane coupling agent to cause the silane coupling agent to form a silane layer that is covalently bound to the surface wherein the silane layer comprises a plurality of vinyl groups; and (c) exposing the silane layer to a hydrophilic polymer and causing the silane layer to react with the hydrophilic polymer to covalently bond to the silane layer. Exposure of the silane layer to γ-radiation to induce grafting with low radiation to induce grafting of the hydrophilic polymer to the silane layer. Another technique comprises grafting a silylated hydrophilic polymer or chain having a hydrophobic domain directly to the metal or inorganic surface.

Nutritive quality of plant protein: Sources of variation and insect herbivore responses

Abstract: Protein quality has received comparatively little attention as a factor in host plant suitability for insects. It is argued here that plant protein quality is subject to considerable variation from genetic and environmental influences and thus may significantly impact herbivore performance. Furthermore, other phytochemicals that are ingested with protein may negatively impact protein utilization. There is a wide distribution of alkylating agents found in plants (e.g., quinones, phenolics, aldehydes, pyrrolizidine alkaloids, sesquiterpene lactones, isothiocyanates) that form covalent bonds with nucleophilic side chains of proteins (e.g., -SH, -NH, -NH2) and potentially limit amino acid availability. The behavioral and physiological adaptations of insects to variation in protein quality are also discussed. Finally, preliminary evidence for physiological adaptation to low protein quality in Helicoverpa zea is provided. The potential role of protein quality in host plant specialization is summarized. © 1996 Wiley-Liss, Inc.

Plasma protein adsorption to sulfonated poly(ethylene oxide)-grafted polyurethane surface.

Abstract: Adsorption of proteins (fibrinogen, albumin, and gamma globulin) from plasma onto surface-modified PUs (PU-PEO, PU-SO3, and PU-PEO-SO3) was evaluated. Adsorbed fibrinogen at steady state decreased in the order PU-SO3 > PU > PU-PEO-SO3 > PU-PEO, suggesting that sulfonate groups have specific high affinity to fibrinogen. The intermediate fibrinogen adsorption on PU-PEO-SO3 can be explained by the compensatory effect between the low protein binding affinity of the PEO chain and the high fibrinogen binding affinity of the sulfonate group. In addition, PU-PEO-SO3 showed a very fast fibrinogen adsorption due to the high accessibility of the sulfonate group to fibrinogen by the poly(ethylene oxide) (PEO) spacer. The kinetic profiles of their surfaces showed that as the adsorption time increases, fibrinogen initially adsorbed was decreased and a plateau reached, demonstrating that all the surfaces exhibited the Vroman effect (the fibrinogen displacement phenomenon). PU-PEO showed the least fibrinogen and albumin adsorption among PUs, confirming the known nonadhesive property of PEO chains. It is very interesting that PU-PEO-SO3 exhibited the highest adsorption of albumin and the lowest adsorption of IgG. Therefore, it may be concluded that such adsorption behaviors of proteins to PU-PEO-SO3 contribute to improved blood compatibility.

Metabolic differences between the postbreeding, moulting and migratory periods in feeding and fasting passerine birds

Abstract: 1. After the breeding season, migrant passerine birds moult and subsequently migrate to their winter quarters. Moult and migration involve different physiological processes (replacement of body proteins vs energy storage for endurance flights). This study investigates metabolic responses to three phases of the annual cycle (postbreeding, moulting and migratory periods) in three physiological situations (feeding during the day, overnight fasted, short-term fasted) by examining six plasma metabolite levels of three bird species. 2. In birds feeding during the day, diurnal body mass gain, triglyceride and free fatty-acid plasma levels were higher during the migratory period than during the postbreeding and moulting periods. This reflects hyperphagia and hyperlipogenesis in preparation for migratory flights. 3. No clear effects of moult were found on the metabolite levels examined. 4. Overnight fasting was generally characterized by low protein catabolism (low uric acid levels) and increased fat utilization (high free fatty-acid, glycerol and β-hydroxy-butyrate levels), compared with feeding birds. 5. During the migratory period, however, overnight fasted birds showed no elevated free fatty-acid levels, a more marked drop in triglyceride levels, unchanged glucose levels, less increased β-hydroxy-butyrate levels and a stronger decrease in uric acid levels than birds in the postbreeding and moulting periods. This suggests that the increased fat deposition rate during the migratory period leads to increased hepatic and muscular lipid stores by the end of the day. During the migratory period, a higher utilization of these triglycerides allows a lower utilization of fatty acids from adipose tissues and a more effective sparing of protein and carbohydrates during overnight fasting than during the postbreeding period. 6. These metabolic responses were more pronounced in the Blackcap and Robin than in the Garden Warbler which starts migration when it is in the last stages of moult.

α‐Lactalbumin‐enriched low‐protein infant formulas: a comparison to breast milk feeding

Abstract: Tryptophan (TRP) is the limiting amino acid in low-protein infant formulas. This is mainly due to lower alpha-lactalbumin (alpha LA) content in cow's milk whey as compared with human milk protein. To study the effect of alpha LA-enrichment on the TRP supply, cross-over studies were carried out in 20 healthy infants up to 3 months of age. In this study, two protein-reduced (1.3%) infant formulas (moderate TRP content of 1.88% and higher TRP content of 2.10%) were alternately fed over a 2 week period in two groups of infants. Serum TRP levels of the formula-fed infants with the higher TRP content did not differ significantly from an exclusively breastfed control group of 11 infants (10.5 +/- 4.8 versus 10.9 +/- 4.7 mg l-1, p = 0.841), whereas levels of the formula-fed infants with the moderate TRP content were significantly lower (7.4 +/- 3.9, p = 0.038). The supplementation of alpha LA resulting in a higher TRP supply to low-protein diets is a further step towards the production of infant formulas more closely adapted to human breast milk.

Analysis of heterologous protein production in defined recombinant Aspergillus awamori strains.

Abstract: A study was carried out to obtain more insight into the parameters that determine the secretion of heterologous proteins from filamentous fungi. A strategy was chosen in which the mRNA levels and protein levels of a number of heterologous genes of different origins were compared. All genes were under control of the Aspergillus awamori 1,4-β-endoxylanase A (exlA) expression signals and were integrated in a single copy at the A. awamori pyrG locus. A Northern (RNA) analysis showed that large differences occurred in the steady-state mRNA levels obtained with the various genes; those levels varied from high values for genes of fungal origin (A. awamori 1,4-β- endoxylanase A, Aspergillus niger glucoamylase, and Thermomyces lanuginosa lipase) to low values for genes of nonfungal origin (human interleukin 6 and Cyamopsis tetragonoloba [guar] α-galactosidase). With the C. tetragonoloba α-galactosidase wild-type gene full-length mRNA was even undetectable. Surprisingly, small amounts of full-length mRNA could be detected when a C. tetragonoloba α-galactosidase gene with an optimized Saccharomyces cerevisiae codon preference was expressed. In all cases except human interleukin 6, the protein levels corresponded to the amounts expected on basis of the mRNA levels. For human interleukin 6, very low protein levels were observed, whereas relatively high steady-state mRNA levels were obtained. Our data suggest that intracellular protein degradation is the most likely explanation for the low levels of secreted human interleukin 6.

Chronic Low Protein Intake Reduces Tissue Protein Synthesis in a Pig Model of Protein Malnutrition

Abstract: To determine the effect of severe chronic protein deficiency on protein synthesis in different tissues and total protein in plasma, and on plasma biochemical constituents involved in amino acid metabolism, we fed diets containing either 20 or 3% protein to two groups of four age-matched piglets. After consuming the diets for 8 wk, the pigs received a primed-constant infusion of 2 H 3 -leucine for 8 h to measure the fractional synthesis rates (FSR) of tissue protein and total protein in plasma. Plasma urea and amino acid concentrations, particularly indispensable amino acids, were significantly lower in protein-deficient pigs. Fractional protein synthesis rates were lower in skin by 66% (P < 0.01), in jejunal mucosa by 50% (P < 0.05), in masseter muscle by 40% (P < 0.05), and in liver by 25% (P < 0.02) ; the fractional synthesis rate of the longissimus muscle was not different than controls. Although plasma protein concentration was significantly (P < 0.01) lower in protein-deficient pigs, the fractional synthesis rate of the total intravascular plasma protein pool was not different. We conclude that adaptation to a low protein diet involves a reduction in the rate of protein synthesis in most body tissues, with the most marked changes occurring in skin and intestine, two tissues which frequently exhibit severe functional impairment in protein malnutrition.

Quantitative trait loci influencing grain protein content in tetraploid wheats

Abstract: Seed storage protein content of durum wheat (Triticum turgidum var. durum) has an important effect on nutritional value and pasta-making characteristics. The objective of this study was to determine by association with genetic markers the number, chromosomal location, and magnitude of effect of quantitative trait loci (QTLs) controlling protein concentration in kernels. A set of 65 recombinant inbred lines (RIs) was developed by single seed descent from a cross between cultivated durum wheat cv. ‘Messapia’ (low protein content) and accession MG4343 of the wild tetraploid wheat var. dicoccoides (high protein content). This population was characterized for eight morphological, six storage protein, one isozyme and 124 RFLP loci. Field trials were conducted in one location in 1993 and two locations in 1994. QTLs were mapped by regression analysis on each marker locus for each location and for the average across environments. A total of six putative QTLs were located on chromosome arms 4BS, SAL, 6AS, 6BS and 7BS. The number and size of QTLs detected varied across environments. The marker with the highest r2 value per QTL in each environment and across environments was chosen for a multiple linear regression analysis, which explained 49.2- 56.4% of the phenotypic variation for protein content. Only some of the markers were found to be negatively associated with plant grain yield and/or seed weight in one or two of the environments.

Effect of protein and protein-free energy intake on protein and fat deposition rates in preruminant calves of 80 to 240 kg live weight

Abstract: Two experiments were conducted to quantify the effects of protein intake on protein and fat deposition rates at two protein-free, energy intake levels in 90 preruminant Holstein Friesian x Dutch Friesian calves. The two experiments were similar in design, but were performed in two different weight ranges: 80 to 160 kg BW and 160 to 240 kg BW in Exp. 1 and 2, respectively. In each experiment, calves were allocated to either an initial slaughter group or to one of 12 treatments (three calves per treatment), which consisted of six protein intake levels at each of two protein-free energy intake levels. Calves were slaughtered and analyzed for body composition when they had reached the target weight. A balance study was conducted when calves reached 120 and 200 kg BW in Exp. 1 and 2, respectively. Protein digestibility increased with increasing protein intake in both experiments (P < .001). Average daily gain of the empty body varied between 640 and 1,340 g/d and between 420 and 1,370 g/d in Exp. 1 and 2, respectively, and was affected by protein (P < .001) and protein-free energy intake (P < .001). The calves responded to increased protein intake by increasing their protein (P < .001) and fat (P < .01) deposition rates. Maximum protein deposition was reached in the second experiment at 244 g/d. Extra protein-free energy intake resulted mainly in extra fat deposition (P < .001), but also increased the protein deposition (P < .01), even at low protein intake levels. In both experiments, the response of protein deposition rate to increased protein intakes was low: about 30% of the extra ingested protein was deposited. These results clearly demonstrate a low priority for partitioning dietary protein into protein gain in these calves.

Cutaneous microdialysis. Methodology and validation.

Abstract: This thesis describes the methodology and validation of cutaneous microdialysis for the study of skin penetration of various topically applied substances in experimental dermatological research. Microdialysis is a sampling technique which makes it possible to measure substances in the extracellular water space in human and animal skin in vivo. A microdialysis probe, i.e. a tubular semipermeable membrane connected to afferent and efferent tubings, is placed in the dermis and perfused. Substances from extracellular space may diffuse through the pores of the membrane and be collected in the dialysate for further analysis. Glucose, sodium fusidate, betamethasone 17,21-dipropionate and calcipotriol were chosen as model substances and were investigated by in vitro microdialysis. The perfusion rate, the length of the membrane, stirring rate and temperature influenced recovery of the substances. Lipophilic compounds tend to have low recoveries and differ in recovery and loss. Insertion of the microdialysis probe causes a trauma in the skin. Rat and human skin were studied in vivo. Increase in skin blood flow, erythema and skin thickness were demonstrated by laser Doppler perfusion imaging, Dermaspectrometer colorimetry, Minolta Chromameter colorimetry and ultrasound imaging of cross-sectional skin structure. In addition histamine was released in rat skin due to the needle insertion. An equilibration period of minimum 90 min in human skin and 30 min in rat skin after the insertion is necessary to allow the effects of trauma to diminish. To obtain measurable concentrations in the dialysate in rats treated topically with the lipophilic drug betamethasone 17-valerate, unrealistic high doses and penetration enhancement were required. The highly protein-bound drug fusidic acid was not measurable in the dialysate after topical application, probably due to very low concentrations of free diffusible drug. Measurable concentrations were only observed after high doses of oral administrations of fusidic acid. Calcipotriol could not be detected in the dialysate. The microdialysis technique is probably primarily useful for the study of hydrophilic substances and substances with low protein binding and low molecular weight. However, application of cutaneous microdialysis for the study of lipophilic substances need further methodologically development.

The effect of protein intake on 24-h energy expenditure during energy restriction.

Abstract: OBJECTIVE : To investigate whether protein intake influences the decline in energy expenditure during energy restriction DESIGN : Cross-over study of three diets of 42 MJ/d for 7 days : one diet with 36% energy as protein and two with 15% energy as protein, one high in carbohydrate and the other high in fat SUBJECTS : Two men and six women aged 31-57 y, BMI 278-341 kg/m 2 MEASUREMENTS : 24-h energy expenditure (24-h EE), sleeping metabolic rate (SMR) and body weight on days 0 and 7 of each diet ; 24-h urinary nitrogen excretion (24-h UN) on days 0-7 of each diet RESULTS : 24-h EE and SMR declined on all three diets but the decrease was significantly less on the high protein diet than on the two low protein diets Weight loss was similar on all three diets 24-h UN was less than N intake on the high protein diet but greater than N intake on the two low protein diets CONCLUSIONS : Maintaining protein intake reduces the decrease in energy expenditure during energy restriction