Protein requirement

Polyacrylamide gel electrophoresis, which can effectively resolve a mixture of proteins into individual constituents with a simple apparatus, has been widely and routinely used for analytical purposes. However, its application for preparative purposes is limited owing to the lack of a universal method that permits the recovery of general proteins from the gel in high yields. In early days, the electrophoretically separated protein in gel was recovered by extraction or solubilization of the excised gel, and later by electrophoretic elution. Regarding the electrophoretic recovery of proteins, a number of methods have been reported, which can be divided into two categories: (1) "electroelution", which recovers the protein of interest from excised gel electrophoretically, and (2) "continuous elution" of applied proteins from a preparative-scale gel during electrophoresis, where the electrophoretically separated proteins that migrate through the gel into a buffer stream at the gel bottom are fractionated consecutively. Characteristic features of the electroelution method reside in its simple requirements for the elution apparatus and that microgram amounts of protein can be quantitatively recovered in the concentrated form. The major drawbacks of electroelution are that the method requires manipulations for locating the protein in the gel, followed by the excision and re-electrophoresis of the relevant part of gel for eluting the protein. The continuous elution method, on the other hand, has advantages in its high loading capacity of sample protein and easy monitoring of the elution process. This method, however, requires expensive equipment and gives low concentrations of the recovered protein, the purity of which is sometimes poor. For those who are trying the electrophoretic recovery of protein from polyacrylamide gel for the first time, the electroelution method would be the first choice in view of the easy manipulation, the small amount of protein required for loading and the satisfactory recovery yield with least expense without using of any costly equipment.

Six feeding experiments were conducted to determine energy and protein requirements of different sized yellowtail seriola quinqueradiata (8-280 g) for maximum growth and maintenance of bodyweight under different water temperatures (21.7-27.1°C) in 500 L polycarbonate tanks. For each experiment, fish were fed to satiation or fed at levels between 0 and 80% of the satiation level twice a day. The experimental diets were extruded dry pellets with known digestible energy (DE) and protein (DP) values. The growth rate was highest for the satiation group and decreased proportional to the feeding levels. The DE (kcal/kgBW per day)and DP (g/kgBW per day) requirements for maximum growth, calculated on the basis of the daily feeding rate of the satiation group, were 225 and 21.7, 136 and 14.8, 125 and 11.2, 111 and 10.7, and 92 and 8.2, respectively, for fish with an average initial bodyweight of 8, 63, 160, 237, and 280 g. The requirements for maintenance of bodyweight, based on when the growth rate was zero for the above sizes, were 61 and 5.9, 15 and 1.7, 15 and 1.3, 24 and 2.3, and 12 and 1.0, respectively. The requirements for maintenance of body energy content, estimated on the basis of the relationship between the DE intake and energy retention, were significantly higher than those for maintenance of bodyweight.

Although fish have been kept for more than three centuries as ornamentals, and the development of manufactured feed since 50 years ago has contributed to the tremendous growth of this hobby, nutrition of ornamental fish is based on extrapolation of results derived from food fishes under intensive farming conditions. Some research on nutrient (protein, minerals) requirements of growing freshwater ornamental species (live-bearers) in a production environment has been conducted, mainly in Singapore, with emphasis on the provision of live feed during the early stages of the life cycle. Protein requirements varied from around 30% dietary protein for growing omnivorous goldfish (Carassius auratus) to 50% for the carnivorous discus (Symphysodon aequifasciata). Whereas mineral (phosphorus, iron, magnesium, zinc) requirements have received some attention in the guppy (Poecilia reticulata), few researches have concentrated on vitamin requirements of ornamental species. Requirements for fatty acids have been conducted mainly on marine ornamentals (damselfish, seahorses), and accentuated the need for dietary supplementation of n-3 highly unsaturated fatty acids. Fish kept in public and home aquaria presents the problem of diversity of species in the same enclosure, each with its own specific requirements and needs. Maintenance energy levels of ornamental fish varied from 0.068 kJ per day for small neon tetras (Paracheirodon innesi) to 0.51 kJ per day for moonlight gouramis (Trichogaster microlepsis) kept at a water temperature of 26 °C. Research on nutrient requirements of ornamental fish urges for suitable measurements other than only growth rate in order to determine optimal dietary inclusion levels.Les exigences alimentaires des poissons d'ornementation. Les exigences alimentaires des poissons d'ornementation. L'élevage de poissons ornementaux effectué pendant plus de trois siècles et le développement de la manufacture d'aliment depuis 50 ans ont contribué à l'essor de ce loisir, mais la nutrition des poissons ornementaux se fonde sur une extrapolation de données dérivées de poissons de consommation et en conditions de culture intensive. Quelques études ont été faites sur les besoins alimentaires (en protéines, en éléments minéraux) d'espèces ornementales d'eau douce (des poissons vivipares) dans le cadre de production, à Singapour en particulier, et sur la nécessité d'aliment vivant dans les premiers stades de leur cycle de vie. Les besoins protéiniques varient de 30 % de protéine brute pour le poisson rouge omnivore (Carassius auratus), jusqu'à 50 % pour le discus carnivore (Symphysodon aequifasciata). Les besoins en minéraux (phosphore, fer, magnésium et zinc) ont été étudiés pour le guppy (Poecilia reticulata), mais peu d'investigations ont été faites pour connaître les besoins vitaminiques des espèces ornementales. Les besoins en acides gras ont été étudiés chez les poissons ornementaux marins (demoiselles, hippocampes) et se limitent à la supplémentation diététique en acides gras très insaturés n-3. Que les poissons soient présentés en aquariums publics ou cultivés en aquariums personnels, un problème de diversité d'espèces se pose dans un même bassin ; chaque espèce ayant ses exigences spécifiques. Leur besoin énergétique d'entretien varie de 0,07 kJ/jour pour les petits néons-tétras (Paracheirodon innesi) jusqu'à 0,51 kJ/jour pour les gouramis serpents (Trichogaster microlepsis), élevés dans une eau à 26 °C. Dans la recherche sur les besoins alimentaires des poissons ornementaux, il faut utiliser des paramètres adéquats, autres que le taux de croissance pour obtenir les taux optimaux des nutriments.

Considerable debate has taken place over the safety and validity of increased protein intakes for both weight control and muscle synthesis. The advice to consume diets high in protein by some health professionals, media and popular diet books is given despite a lack of scientific data on the safety of increasing protein consumption. The key issues are the rate at which the gastrointestinal tract can absorb amino acids from dietary proteins (1.3 to 10 g/h) and the liver's capacity to deaminate proteins and produce urea for excretion of excess nitrogen. The accepted level of protein requirement of 0.8g • kg -1 • d -1 is based on structural requirements and ignores the use of protein for energy metabolism. High protein diets on the other hand advocate excessive levels of protein intake on the order of 200 to 400 g/d, which can equate to levels of approximately 5 g • kg -1 • d -1 , which may exceed the liver's capacity to convert excess nitrogen to urea. Dangers of excessive protein, defined as when protein constitutes > 35% of total energy intake, include hyperaminoacidemia, hyperammonemia, hyperinsulinemia nausea, diarrhea, and even death (the "rabbit starvation syndrome"). The three different measures of defining protein intake, which should be viewed together are: absolute intake (g/d), intake related to body weight (g • kg -1 • d -1 ) and intake as a fraction of total energy (percent energy). A suggested maximum protein intake based on bodily needs, weight control evidence, and avoiding protein toxicity would be approximately of 25% of energy requirements at approximately 2 to 2.5 g • kg -1 • d -1 , corresponding to 176 g protein per day for an 80 kg individual on a 12,000kJ/d diet. This is well below the theoretical maximum safe intake range for an 80 kg person (285 to 365 g/d).

La presente revisión se realizó con el objetivo de presentar la importancia y los avances que se han registrado en la nutrición proteica de los bovinos productores de carne en pastoreo. Se inicia con un análisis sobre la composición nutrimental de los forrajes, la utilización y degradación de la proteína, la síntesis de proteína microbiana y la proteína de escape ruminal; también, se analizan los requerimientos de proteína del rumiante, la suplementación proteica y por último se discute sobre la fermentación ruminal y cinética digestiva.

An estimate was made of the optimum protein level in growth diets for bullfrog Rana catesbeiana. A nested design of six treatments with three replicates per treatment was used. Juvenile bullfrogs (8.84 ± 1.42 g average initial weight) were fed in lots at 6% body weight per day with six practical diets containing 20, 28, 34, 42 50 or 58% crude protein from fish meal. Each batch contained 12 frogs placed randomly in plastic cages with an automatic water sprinkler system to keep the animals wet and the cages clean. Cages were installed under a controlled environment: 28°C temperature; 100% relative humidity; and 14L:10D photoperiod. The diets were assigned randomly to the cages. Results were evaluated in terms of survival, growth and feed efficiency. All the treatments showed 83% survival except the 28% protein diet, which had 60.44%. Productive performance tended to improve as protein level increased. The greatest growth was observed in the 58% protein diet, although this was not statistically different from the 42 and 50% protein diets. Feed conversion ratio (FCR) also improved as dietary protein level increased, with the best FCR in the 58% protein diet, although this was not significantly different from the 42 and 50% protein diets. Optimum protein level was estimated using a second-order quadratic regression using weight gain (%) and established as 39.21% crude protein at a protein: energy ratio of 81.34 mg P/kcal. Under the experimental conditions applied, a 40% crude protein level is recommended for growth in juvenile R. catesbeiana.

The physicochemical properties of emulsions play an important role in food systems as they directly contribute to texture, sensory and nutritional properties of foods. Sodium caseinate (NaCas) is a well-used ingredient because of its good solubility and emulsifying properties and its stability during heating. One of most significant aspects of any food emulsion is its stability. Among the methods used to study emulsion stability it may be mentioned visual observation, ultrasound profiling, microscopy, droplet size distribution, small deformation rheometry, measurement of surface concentration to characterize adsorbed protein at the interface, nuclear magnetic resonance, confocal microscopy, diffusing wave spectroscopy, and turbiscan. They have advantages and disadvantages and provide different insights into the destabilization mechanisms. Related to stability, the aspects more deeply investigated were the amount of NaCas used to prepare the emulsion, and specially the oil-toprotein ratio, the mobility of oil droplets and the interactions among emulsion components at the interface. It is known that the amount of protein required to stabilize oil-in-water emulsions depends, not only on the structure of protein at the interface, and the average diameters of the emulsion droplets, but also on the type of oils and the composition of the aqueous phase. Several authors have investigated the effect of a thickening agent or of a surface active molecule. Factors such as pH, temperature, and processing conditions during emulsion preparation are also very relevant to stability. There is a general agreement among authors that the most stable systems are obtained for conditions that produce size reduction of the droplets, an increase in viscosity of the continuous phase and structural changes in emulsions such as gelation. All these conditions decrease the molecular mobility and slow down phase separation.

Structural and biochemical analysis of proteins requires access to purified protein material. Modern molecular biology technologies facilitate straightforward molecular cloning and expression analysis of multiple protein constructs in parallel, and such approaches have proven very efficient to identify samples suitable for further analysis.

Effect of isolipidic (62.7 ± 5.0 g kg−1) diets with protein levels of 204.6 (T20), 302.3 (T30), 424.6 (T40) or 511.0 g kg−1 (T50) on growth and survival in Nile tilapia (Oreochromis niloticus Linnaeus 1758) fry cultured for 70 days at one of four salinities (0, 15, 20 and 25 g L−1) was evaluated. A bifactorial (4 × 4) design was used with 16 treatments run in triplicate and 20 fry (0.25 ± 0.04 g) per replicate under semi-controlled conditions. Four independent, recirculating systems (one per salinity level) were used, each one with 12 circular tanks (70 L capacity), filters and constant aeration. The different salinities had no significant effect on growth. Weight gain improved significantly as dietary protein content increased, although organisms fed the T50 diet had a lower growth rate. Survival was highest (98.33%) in the T50/15 (protein/salinity levels) treatment and lowest (71.0%) in the T20/20 treatment, with no pattern caused by the variables. The T40/25, T40/20 and T50/0 treatments produced the most efficient growth and feed utilization values while the T20 treatments at all the salinities resulted with the lowest performance. With the exception of the T50 treatments, a non-significant tendency to increased weight gain was observed as water salinity increased, suggesting that the salinity of the culture environment does not influence dietary protein requirements in Nile tilapia O. niloticus fry.

This study assessed the protein and energy requirements of Tra catfish (Pangasianodon hypothalamus) using a bio-energetic factorial approach. To examine these parameters, a series of inter-related studies were undertaken to define the various factorial parameters. Studies included controlled laboratory experiments and field-data collection from commercial farms in the Mekong Delta region of Vietnam. Key components of the model include parameters for maintenance energy (39.7 kJ kg )0.844 day )1 at 32°C) and protein (0.467 g kg )0.833 day )1 at 32°C) demands, the effect of animal live-weight and temperature on growth and maintenance energy demands, the efficiencies of energy and protein utilization at various energy and protein intake levels and the variability in whole body composition with varying liveweight. Interestingly, both the energy [0.510AE (Energy intake) ) 11.143] and protein [0.322AE(Protein intake) + 0.665] utilization efficiencies at near satiety feed intake levels were substantially lower than that observed from other fish species. Modelling using these equations was used to iteratively derive optimal diet designs and feeding rations for growing Pangasius catfish.

The Cornell Net Carbohydrate and Protein System (CNCPS), a mechanistic model that predicts nutrient requirements and biological values of feeds for cattle, was modified for use with sheep. Published equations were added for predicting the energy and protein requirements of sheep, with a special emphasis on dairy sheep, whose specific needs are not considered by most sheep-feeding systems. The CNCPS for cattle equations that are used to predict the supply of nutrients from each feed were modified to include new solid and liquid ruminal passage rates for sheep, and revised equations were inserted to predict metabolic fecal N. Equations were added to predict fluxes in body energy and protein reserves from BW and condition score. When evaluated with data from seven published studies (19 treatments), for which the CNCPS for sheep predicted positive ruminal N balance, the CNCPS for sheep predicted OM digestibility, which is used to predict feed ME values, with no mean bias (1.1 g/100 g of OM; P > 0.10) and a low root mean squared prediction error (RMSPE; 3.6 g/100 g of OM). Crude protein digestibility, which is used to predict N excretion, was evaluated with eight published studies (23

Reported here is a native chemical ligation strategy for the total chemical synthesis of the B1 domain of protein L. A synthetic construct of this 76 amino acid protein domain was prepared by the chemoselective ligation of two unprotected polypeptide fragments, one containing an N-terminal cysteine residue and one containing a C-terminal thioester moiety. The polypeptide fragments utilized in the ligation reaction were readily prepared by stepwise solid phase peptide synthesis (SPPS) methods for Boc-chemistry. The milligram quantities of protein required for conventional biophysical studies were readily accessible using the synthetic protocol described here. The folding properties of the synthetic protein L construct were also determined and found to be very similar to those of a similar wild-type protein L constructs prepared by recombinant-DNA methods. This work facilitates future unnatural amino acid mutagenesis experiments on this model protein system to further dissect the molecular basis of its folding and stability.

The YopM protein of Yersinia sp. is a type III secreted effector that is required for virulence in murine models of infection. YopM has previously been shown to contain leucine-rich repeats (LRRs) and to interact with two host kinases, RSK1 and PRK2, although the consequence of these interactions is unknown. A series of YopM proteins missing different numbers of LRRs or a C-terminal domain were produced and used for in vitro binding reactions to map domains required for interaction with RSK1 and PRK2. A C-terminal domain of YopM (from LRR12 to the C terminus) was shown to be required for interaction with RSK1, while an internal portion encompassing LRR6 to LRR15 was shown to be required for interaction with PRK2. The virulence of a Yersinia pseudotuberculosis ⌬yopM mutant in mice via an intravenous route of infection was significantly attenuated. At day 4 postinfection, there were significantly increased levels of gamma interferon and reduced levels of interleukin-18 (IL-18) and IL-10 in the serum of the ⌬yopM-infected mice compared to that of mice infected with the wild type, suggesting that YopM action alters the balance of these key cytokines to promote virulence. The PRK2 and RSK1 interaction domains of YopM were both required for IL-10 induction in vivo, irrespective of splenic colonization levels. In an orogastric model of Y. pseudotuberculosis infection, a ⌬yopM mutant was defective in dissemination from the intestine to the spleen and significantly reduced in virulence. In addition, Y. pseudotuberculosis mutants expressing YopM proteins unable to interact with either RSK1 (YopM⌬12-C) or PRK2 (YopM⌬6-15) were defective for virulence in this assay, indicating that both interaction domains are important for YopM to promote pathogenesis.

The nucleolus is a plurifunctional, nuclear organelle, which is responsible for ribosome biogenesis and many other functions in eukaryotes, including RNA processing, viral replication and tumour suppression. Our knowledge of the human nucleolar proteome has been expanded dramatically by the two recent MS studies on isolated nucleoli from HeLa cells Curr. Biol. 12, 1-11; Scherl, Coute, Deon, Calle, Kindbeiter, Sanchez, Greco, Hochstrasser and Diaz Mol. Biol. Cell 13, 4100-4109]. Nearly 400 proteins were identified within the nucleolar proteome so far in humans. Approx. 12 % of the identified proteins were previously shown to be nucleolar in human cells and, as expected, nearly all of the known housekeeping proteins required for ribo-some biogenesis were identified in these analyses. Surprisingly, approx. 30 % represented either novel or uncharacterized proteins. This review focuses on how to apply the derived knowledge of this newly recognized nucleolar proteome, such as their amino acid/peptide composition and their homologies across species, to explore the function and dynamics of the nucleolus, and suggests ways to identify, in silico, possible functions of the novel/ uncharacterized proteins and potential interaction networks within the human nucleolus, or between the nucleolus and other nuclear organelles, by drawing resources from the public domain.

The goal of training is to prepare the distance athlete to perform at his or her best during major competitions. Whatever the event, nutrition plays a substantial role in the achievement of various factors that will see a runner or walker take the starting line in the best possible form. Everyday eating patterns must supply fuel and nutrients needed to optimise their performance during training sessions and to recover quickly afterward. Carbohydrate and fluid intake before, during, and after a workout may help to reduce fatigue and enhance performance. Recovery eating should also consider issues for adaptation and the immune system which may involve intakes of protein and some micronutrients. Race preparation strategies should include preparation of adequate fuel stores, including carbohydrate loading for prolonged events such as the marathon or 50 km walk. Fluid and carbohydrate intake during races lasting an hour or longer should also be considered. Sports foods and supplements of value to distance athletes include sports drinks and liquid meal supplements to allow nutrition goals to be achieved when normal foods are not practical. While caffeine is an ergogenic aid of possible value to distance athletes, most other supplements are of minimal benefit.

Florida pompano is considered a suitable candidate for mariculture due to its ready adaptation to culture systems, acceptance of formulated feeds, and rapid growth rates. Little information on the nutritional requirements or growth rates under controlled conditions have been reported. A Ž . seven week feeding experiment was conducted with juvenile fish mean wt. 4.5 g to evaluate the effect of dietary protein level on growth, feed intake, feed utilization and survival. Four practical Ž diets were formulated to be isoenergetic and to contain increasing levels of protein 30, 35, 40 or . 45% crude protein . Although total feed intake was not significantly different among treatments, feed intake expressed as amount consumed per unit of body weight was significantly different among treatments. Fish fed the highest protein diet had lower percentage daily feed consumption values than did fish fed the lowest protein diet. Growth and feed efficiency ratios increased with dietary protein level and were highest for fish fed the diet containing 45% protein. The results obtained in the present study indicate that juvenile Florida pompano required a minimum of 45% protein for maximum growth and feed efficiency ratio when fish meal and soybean meal are the primary sources of protein. q 1998 Elsevier Science B.V. All rights reserved.

central spindle and contractile ring for cytokinesis encodes a kinesin-like protein required to organize the pavarotti References http://www.genesdev.org/cgi/content/full/12/10/1483#otherarticles Article cited in: http://www.genesdev.org/cgi/content/full/12/10/1483#References This article cites 50 articles, 28 of which can be accessed free at: service Email alerting click here top right corner of the article or Receive free email alerts when new articles cite this article -sign up in the box at the Notes http://www.genesdev.org/subscriptions/ go to: Genes and Development To subscribe to

Prolonged exposure of ␤-cells to high glucose (glucotoxicity) diminishes insulin secretion in response to glucose and has been linked to altered generation of metabolism-secretion coupling factors. We have investigated whether glucotoxicity may also alter calcium handling and late steps in secretion such as exocytosis. Clonal INS-1E ␤-cells cultured at high glucose (20 or 30 mM vs. 5.5 mM) for 72 h exhibited elevated basal intracellular calcium ([Ca 2؉ ] i ), which was K ATP -channel dependent and due to long-term activation of protein kinase A. An increased amplitude and shortened duration of depolarization-evoked rises in [Ca 2؉ ] i were apparent. These changes were probably linked to the observed increased filling of intracellular stores and to short-term activation of protein kinase A. Insulin secretion was reduced not only by acute stimulation with either glucose or KCl but more importantly by direct calcium stimulation of permeabilized cells. These findings indicate a defect in the final steps of exocytosis. To con-firm this, we measured expression levels of some 30 proteins implicated in trafficking/exocytosis of post-Golgi vesicles. Several proteins required for calcium-induced exocytosis of secretory granules were down-regulated, such as the soluble N-ethylmaleimide-sensitive factor-sensitive factor attachment receptor (SNARE) proteins VAMP-2 [vesicle (v)-SNARE, vesicle-associated membrane protein 2] and syntaxin 1 as well as complexin. VAMP-2 was also reduced in human islets. In contrast, cell immunostaining and expression levels of several fluorescent proteins suggested that other post-trans-Golgi trafficking steps and compartments are preserved and that cells were not degranulated. Thus, these studies indicate that, in addition to known metabolic changes, glucotoxicity impedes generation of signals for secretion and diminishes the efficiency of late steps in exocytosis. (Endocrinology

Meiosis pairs and segregates homologous chromosomes and thereby forms haploid germ cells to compensate the genome doubling at fertilization. Homologue pairing in many eukaryotic species depends on formation of DNA double strand breaks (DSBs) during early prophase I when telomeres begin to cluster at the nuclear periphery (bouquet stage). By fluorescence in situ hybridization criteria, we observe that mid-preleptotene and bouquet stage frequencies are altered in male mice deficient for proteins required for recombination, ubiquitin conjugation and telomere length control. The generally low frequencies of midpreleptotene spermatocytes were significantly increased in male mice lacking recombination proteins SPO11, MEI1, MLH1, KU80, ubiquitin conjugating enzyme HR6B, and in mice with only one copy of the telomere length regulator Terf1. The bouquet stage was significantly enriched in Atm −/− , Spo11 −/− , Mei1 m1Jcs/m1Jcs , Mlh1 −/− , Terf1 +/− and Hr6b −/− spermatogenesis, but not in mice lacking recombination proteins DMC1 and HOP2, the non-homologous end-joining DNA repair factor KU80 and the ATM downstream effector GADD45a. Mice defective in spermiogenesis (Tnp1 −/− , Gmcl1 −/− , Asm −/− ) showed wild-type mid-preleptotene and bouquet frequencies. A low frequency of bouquet spermatocytes in Spo11 −/− Atm −/− spermatogenesis suggests that DSBs contribute to the Atm −/− -correlated bouquet stage exit defect. Insignificant changes of bouquet frequencies in mice with defects Keywords: ATM Bouquet DSB Meiosis Mouse Recombination Telomere Spermatogenesis E X P E R I M E N T A L C E L L R E S E A R C H 3 1 2 ( 2 0 0 6 ) 3 7 6 8 -3 7 8 1 ava i l a b l e a t w w w. s c i e n c e d i r e c t . c o m w w w. e l s ev i e r. c o m / l o c a t e / yexc r in early stages of DSB repair (Dmc1 −/− , Hop2 −/− ) suggest that there is an ATM-specific influence on bouquet stage duration.

To search for virulence effector genes of the rice blast fungus, Magnaporthe oryzae, we carried out a large-scale targeted disruption of genes for 78 putative secreted proteins that are expressed during the early stages of infection of M. oryzae. Disruption of the majority of genes did not affect growth, conidiation, or pathogenicity of M. oryzae. One exception was the gene MC69. The mc69 mutant showed a severe reduction in blast symptoms on rice and barley, indicating the importance of MC69 for pathogenicity of M. oryzae. The mc69 mutant did not exhibit changes in saprophytic growth and conidiation. Microscopic analysis of infection behavior in the mc69 mutant revealed that MC69 is dispensable for appressorium formation. However, mc69 mutant failed to develop invasive hyphae after appressorium formation in rice leaf sheath, indicating a critical role of MC69 in interaction with host plants. MC69 encodes a hypothetical 54 amino acids protein with a signal peptide. Live-cell imaging suggested that fluorescently labeled MC69 was not translocated into rice cytoplasm. Sitedirected mutagenesis of two conserved cysteine residues (Cys36 and Cys46) in the mature MC69 impaired function of MC69 without affecting its secretion, suggesting the importance of the disulfide bond in MC69 pathogenicity function. Furthermore, deletion of the MC69 orthologous gene reduced pathogenicity of the cucumber anthracnose fungus Colletotrichum orbiculare on both cucumber and Nicotiana benthamiana leaves. We conclude that MC69 is a secreted pathogenicity protein commonly required for infection of two different plant pathogenic fungi, M. oryzae and C. orbiculare pathogenic on monocot and dicot plants, respectively.

Calcium-independent phospholipase A 2 (iPLA 2 ␤) has recently been suggested to regulate Ca 2+ entry by activating store-operated Ca 2+ channels. These studies have been conducted in mast cells using thapsigargin to deplete intracellular stores. In RBL 2H3 and bone marrowderived mast cells (BMMCs), Ca 2+ entry is critical for exocytosis and therefore we have examined whether the proposed mechanism would be relevant when a physiological stimulus is applied to these cells. Using an iPLA 2 ␤ antibody, we demonstrate that the 84 kDa iPLA 2 ␤ is expressed in these mast cells. As bromoenol lactone (BEL) is a suicide-based irreversible inhibitor of iPLA 2 ␤ it was used to probe this potential mechanism. We observe inhibition of exocytosis stimulated either with antigen or with thapsigargin. However, BEL also inhibits exocytosis when stimulated using a Ca 2+ ionophore A23187, which passively transports Ca 2+ down a concentration gradient and also in permeabilised mast cells where Ca 2+ entry is no longer relevant. Moreover, BEL has only a minor effect on antigen-or thapsigargin-stimulated Ca 2+ signalling, both the release from internal stores and sustained elevation due to Ca 2+ influx. These results cast doubt on the proposed mechanism involving iPLA 2 ␤ required for Ca 2+ entry. Although inhibition of exocytosis by BEL could imply a requirement for iPLA 2 ␤ activation for exocytosis, an alternative explanation is that BEL inactivates other target proteins required for exocytosis. Ca 2+ from intracellular stores. Ca 2+ is essential for exocytosis and in the non-excitable mast cells a sustained intracellular increase critically depends on influx of extracellular Ca 2+ . Ca 2+ release from stores in the absence of influx fails to elicit exocytosis [1]. Such Ca 2+ influx is thought to occur by a "store-operated" mechanism where the specific Ca 2+ conducting channel in the plasma membrane is triggered by depletion of intracellular stores . Several mechanisms that connect store depletion with Ca 2+ influx via store-operated calcium channels has been put forward (reviewed in [3]). Some possible mechanisms include: calcium influx factor (CIF) which is released in response to store-depletion and directly activates the SOCC (store-operated calcium channel), insertion of SOCCs into the plasma membrane on store depletion, regulation of the channels due to a decrease in Ca 2+ in their immediate vicinity, and finally conformational 0143-4160/$ -see front matter

The objective of this study was to evaluate the protein requirements for hand-rearing Blue-fronted Amazon parrots (Amazona aestiva). Forty hatchlings were fed semi-purified diets containing one of four (as-fed basis) protein levels: 13%, 18%, 23% and 28%. The experiment was carried out in a randomized block design with the initial weight of the nestling as the blocking factor and 10 parrots per protein level. Regression analysis was used to determine relationships between protein level and biometric measurements. The data indicated that 13% crude protein supported nestling growth with 18% being the minimum tested level required for maximum development. The optimal protein concentration for maximum weight gain was 24.4% (p = 0.08; r 2 = 0.25), tail length 23.7% (p = 0.09; r 2 = 0.19), wing length 23.0% (p = 0.07; r 2 = 0.17), tarsus length 21.3% (p = 0.06; r 2 = 0.10) and tarsus width 21.4% (p = 0.07; r 2 = 0.09). Tarsus measurements were larger in males (p < 0.05), indicating that sex must be considered when studying developing psittacines. These results were obtained using a highly digestible protein and a diet with moderate metabolizable energy levels.

The present study was designed to assess the alternate environmental friendly source of protein; here we report the suitability of soybean meal as a plant protein source for partial replacement of fishmeal in the diet of Arabian yellowfin seabream Acanthopagrus arabicus; reared in floating net cages. A total of 56 days feeding trial was conducted to evaluate the growth performance, feed utilization, survival, and carcass composition of the juveniles. Triplicate groups (30 fish/cage) of A. arabicus (initial weight 22.61 ± 0.11 g) were stocked in floating net cages (1.5 × 1.5 × 1.5 m). Five semi-purified experimental diets, each containing 42% dietary protein, were prepared where fishmeal was replaced with soybean meal: 0% (control), 20% (SBM20), 40% (SBM40), 60% (SBM60) and 80% (SBM80). Fish were fed twice daily at 7% body weight. The formulated diet was well accepted by the fish, where, 100% of survival was recorded at the end of the experiment. No significant differences (P > 0.05) were observed in final weight gain (WG) and specific growth rate (SGR) of juvenile Arabian yellowfin seabream fed on SBM20 and SBM40 diets compared to the control diet. However, a decline in growth performance was recorded in fish received SBM60 and SBM80 diets. Poor feed conversion ratio (FCR) was observed for fish fed with increasing dietary soybean meal above SBM40. No significant differences (P > 0.05) were found in the final whole-body carcass composition of proteins, moisture, and ash. However, the lipid content in the fish body increased significantly (P < 0.05) with an increasing soybean meal and fish oil in the diets. Our results showed that 40% of SBM could substitute fishmeal without jeopardizing the health and growth of Arabian yellowfin seabream.

Factorial determinations of energy and protein requirements in growing Sparus aurata were carried out at 23-24°C. The energy content in the whole fish was dependent on fish weight and ranged from 5 to 11 MJ kg -1 body mass for 1-250 g fish, whereas the protein content remained constant at 179 g kg -1

The facultative pathogenic fungus Candida albicans has to come up with dynamic metabolic adaptation programs in order to be able to survive within a variety of niches in the human host, each of which has its different nutrient availability. Using a large-scale two-dimensional (2-D) protein gel electrophoresis approach, we analyzed the adaptation mechanisms to nutrient limitation in a batch culture in complex medium with glucose as carbon source. To this end, we constructed a 2-D reference map of cytoplasmic proteins and quantitatively compared protein accumulation of growing yeast cells with those from the stationary phase. This yielded characteristic proteome signatures for each physiological state. During exponential growth, proteins required for the synthesis of RNA, DNA, and proteins, including components of purine and pyrimidine synthesis pathways and ribosomal proteins, were overrepresented. The stationary-phase signature revealed a complex reprogramming of metabolic networks: Up-regulation of glyoxylate cycle, gluconeogenesis, and glutamate degradation signaled a switch to the utilization of alternative carbon sources instead of the exhausted glucose. Induction of proteins involved in defense against oxidative and heat stress indicates a change in redox balance and reactive oxygen species concentrations.

A feeding trial was carried out where five protein concentrations [26%, 29%, 33%, 37%, and 41% crude protein (CP)] were fed to jundia fingerlings at two dietary energy concentrations [3200 and 3650 kcal metabolizable energy (ME)/kg diet] to establish the protein requirement for this species. Triplicate groups of 23 fish (average weight 1.52F0.34 g) were each stocked in 120-l aquaria and fed semipurified diets twice a day to apparent satiation. After 90 days, fish weight gain (WG), specific growth rate (SGR), feed efficiency (FE), protein efficiency ratio (PER), energy retention (ER), apparent net protein utilization (ANPU), feed consumption (% body weight) and body composition (crude protein, fat, and ash) were affected by diet composition. Weight gain, SGR, FE, ANPU and ER increased ( P<0.05) as the dietary protein concentration increased up to 33% and 37%, for fish fed diets containing 3650 and 3200 kcal, respectively. A significant interaction between dietary protein and energy was observed for WG, SGR, and PER. Feed consumption was not affected by dietary energy concentration, but decreased with increasing dietary protein concentration ( P<0.05). Body fat decreased as dietary protein increased at both energy concentrations, but was higher in fish fed the 3650 kcal diet. The opposite was observed for body protein, ER, and ANPU ( P<0.05). Our findings demonstrate that jundia presents dietary protein sparing effect when the energy concentration increases from 3200 to 3650 kcal/kg. Jundia protein requirement, determined by the broken line method, is between 32.6% and 37.3% CP, depending on dietary energy concentration. D

Every bone in the human skeleton contains a reservoir of bone-building cells (osteoblasts) of which the function is to continually deposit rigid extracellular matrix (ECM), which is subsequently converted into mineralized hard tissue. The skeleton also includes bone-resorbing cells (osteoclasts), of which the sole function is to break down the hard tissue. This delicate balance of bone building and destroying is tipped only in special circumstances, such as trauma or aging. In trauma, a bone is fractured and the healing defense stimulates osteoblast activity; bone formation outpaces bone resorption, and the tissue is repaired. The balance is tipped in the other direction -in favor of the osteoclasts -when we age; bone is destroyed faster than it is replaced, and the skeleton becomes weaker and less dense. The challenge of how to stimulate osteoblasts to respond faster, and in greater numbers to accelerate fracture healing, repair large defects the body cannot ordinarily rebuild, strengthen the spine, or even to turn back the clock on bone density lost with aging, has engaged scientists for decades. However, it appears that we are on the brink of tremendous advances in the use of bone growth factors as therapeutics, which will revolutionize how clinicians treat such problems. Bone contains a cocktail of growth factors including transforming growth factor beta (TGF-β), plateletderived growth factor (PDGF), bone morphogenetic proteins (BMPs), insulin-like growth factors I and II (IGF-I and IGF-II) and fibroblast growth factors (FGFs). BMPs differ from other growth factors in that they are OSTEOINDUCTIVE (see Glossary). Currently, local application of these potent growth factors is being investigated for several bone regeneration applications. For a clinically beneficial outcome, these growth factors require a delivery system to guide tissue regeneration and prevent rapid dispersal of the factors from the site. This article provides a perspective on how bone

Most diets for dairy cattle in the US are formulated using the mathematical model developed by the National Research Council (NRC). This model is simpler than more mechanistic models and is largely empirical. Based on the research reviewed in this paper, the simpler empirical approach is recommended for routine diet formulation at the present time. Under typical conditions using feed tables for most feed descriptions, the NRC model was more accurate than the Cornell Net Carbohydrate and Protein System, and, in its present form, the model developed by Baldwin et al. (4) is too difficult to run routinely in the field. However, the more mechanistic approaches are recommended to investigate diet and animal interactions under nonstandard environmental conditions, animals, or feeds. Because the NRC model does not address many of the potential limitations of some diets or management conditions, more complex models are needed to identify why some herds appear to underperform. Mechanistic mo...

protein folding in vivo and are essential for viability in eukaryotic cells. They share the active-site sequence C-X-X-C that forms a catalytic disulfide. The Universität Regensburg recent finding that the EUG1 protein, a PDI-related yeast protein, with UniverstätsstraPe 31

1989; Du et al., 1996) and an ion channel with an extracellular regulatory domain (Garcia-Añ overos et al., 1995). Drosophila mutations with defects in mechanosensory behavior and electrophysiology (Kernan et al., 1994) identify gene products that may be involved in mechano

This study aimed to evaluate the protein requirement of Clarias batrachus fry, were estimated at two different water temperatures, 28 and 32 °C. The influence of dietary protein level and water temperature on body composition, weight gain, food and nutrient utilization were estimated. The Asian catfish, C. batrachus fry were fed four diets containing 28% (diet 1), 32% (diet 2), 36% (diet 3) and 40% (diet 4) protein levels and reared at two water temperatures 28 and 32 °C for 60 days. Fry fed with diet 3 containing 36% protein showed the highest mean final body weight at 32 °C. Final body weight was significantly (P<0.05) affected by dietary treatments and temperatures. Clarias batrachus fry raised at 28 °C had higher feed efficiency (93.20%) than the fry reared at 32 °C (87.58%) with 28% dietary protein level. Further, feed efficiency decreased with increase in dietary protein level. Higher daily protein retention (0.089%) observed at lower (0.0217 g) daily protein intake at 28 °C than 0.0283 g at 32 °C. While, optimal (0.0282 g) daily protein intake showed higher daily weight gain at 32 °C. Productive protein value (% PPV) was maximum (1.76%) at 32 °C than at 28 °C (0.76%). Final body lipid recorded higher value than initial body lipid at both the temperatures. Hepatosomatic index (HSI) observed to have been influenced (P<0.05) by diets and temperatures, while viscerosomatic index (VSI) affected (P<0.05) by only diets and not (P>0.05) by temperatures. The study concluded that the diet 3 containing 36% protein was optimal for growth of C. batrachus fry at both the temperatures.

A screen for mutants of Saccharomyces cerevisiae secretory pathway components previously yielded sec34 , a mutant that accumulates numerous vesicles and fails to transport proteins from the ER to the Golgi complex at the restrictive temperature (Wuestehube, L.J., R. Duden, A. Eun, S. Hamamoto, P. Korn, R. Ram, and R. Schekman. 1996. Genetics . 142:393-406). We find that SEC34 encodes a novel protein of 93-kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec34-2 is suppressed by the rab GTPase Ypt1p that functions early in the secretory pathway, or by the dominant form of the ER to Golgi complex target-SNARE (soluble N -ethylmaleimide sensitive fusion protein attachment protein receptor)associated protein Sly1p, Sly1-20p. Weaker suppression is evident upon overexpression of genes encoding the vesicle tethering factor Uso1p or the vesicle-SNAREs Sec22p, Bet1p, or Ykt6p. This genetic suppression profile is similar to that of sec35-1 , a mutant allele of a gene encoding an ER to Golgi vesicle tethering factor and, like Sec35p, Sec34p is required in vitro for vesicle tethering. sec34-2 and sec35-1 display a synthetic lethal interaction, a genetic result explained by the finding that Sec34p and Sec35p can interact by two-hybrid analysis. Fractionation of yeast cytosol indicates that Sec34p and Sec35p exist in an ‫ف‬ 750-kD protein complex. Finally, we describe RUD3 , a novel gene identified through a genetic screen for multicopy suppressors of a mutation in USO1 , which suppresses the sec34-2 mutation as well.

This study assessed the protein and energy requirements of Tra catfish (Pangasianodon hypothalamus) using a bio-energetic factorial approach. To examine these parameters, a series of interrelated studies were undertaken to define the various factorial parameters. Studies included controlled laboratory experiments and field-data collection from commercial farms in the Mekong Delta region of Vietnam. Key components of the model include parameters for maintenance energy (39.7 kJ kg)0.844 day)1 at 32°C) and protein (0.467 g kg)0.833 day)1 at 32°C) demands, the effect of animal live-weight and temperature on growth and maintenance energy demands, the efficiencies of energy and protein utilization at various energy and protein intake levels and the variability in whole body composition with varying liveweight. Interestingly, both the energy [0.510AE (Energy intake)) 11.143] and protein [0.322AE(Protein intake) + 0.665] utilization efficiencies at near satiety feed intake levels were substantially lower than that observed from other fish species. Modelling using these equations was used to iteratively derive optimal diet designs and feeding rations for growing Pangasius catfish.

A ¢eld study was carried out to assess the use of the bottom-feeding grey mullet (Mugil cephalus) within benthic enclosures as a means to reduce the benthic impacts of a net cage ¢sh farm in the Gulf of Aqaba, Red Sea. Five experimental 1-m 3 net-cage enclosures were stocked with 12 grey mullets each (¢sh weights 50, 70 or 144 g). The enclosures had no bottom and the mullets had access to the enclosed organically enriched sediments and to particulate matter (PM) falling from the overlying cages. Sediment traps were used to quantify and qualify the PM falling from the ¢sh farm to the sea £oor. Simultaneously, a feeding trial was performed with mullets (50 and 70 g) in experimental tanks. In order to estimate the potential growth rate and to quantify energy and protein requirements, the ¢sh were fed a formulated diet with known composition. After approximately 70 days at sea, mullets in the enclosures had used up all the available food in the sediment and gained up to 0.78 g day À 1 ¢sh À 1. Applying the values for energy and protein requirements for maintenance and growth derived from the experimental trial, estimates indicated that the grey mullets e¡ectively removed 4.2 g organic carbon, 0.70 g nitrogen and 7.5 mg phosphorus kg À 1 mullet m À 2 day À 1 from the organically enriched sediment. Thus deployment of grey mullets may be an e⁄cient means to improve the quality of sediments below intensive net-cage ¢sh farms.

Inhibins are expressed in the adrenal cortex, but little is known of their binding or role in the adrenal. The aims of the present study were, first, to establish whether a mouse adrenocortical (AC) cell line expresses inhibins/activins and bone morphogenetic proteins (BMP), along with proteins required for inhibin to antagonise activin and BMP actions and, secondly, to characterise and compare inhibin binding sites and proteins in the rat adrenal gland and AC cells. AC cells were found to: (1) express mRNA for multiple BMPs (BMP-2, -3, -4, -6, -8a), growth/differentiation factors (GDF-1, -3, -5, -9), Lefty A and B, and the inhibin α, βA and βB subunits (2) secrete inhibin A and inhibin B and (3) express mRNA encoding the inhibin co-receptor, betaglycan, along with activin and BMP type I (ALK2–7) and type II (ActRII, ActRIIB, BMPRII) receptors, and binding proteins (follistatin, BAMBI, gremlin). When applied to sections of rat adrenal glands, [125I]inhibin A specifically bound to ce...

Structure comparisons of all representative proteins have been done. Employing the relative root mean square deviation (RMSD) from native enables the assessment of the statistical significance of structure alignments of different lengths in terms of a Z-score. Two conclusions emerge: first, proteins with their native fold can be distinguished by their Z-score. Second and somewhat surprising, all small proteins up to 100 residues in length have significant structure alignments to other proteins in a different secondary structure and fold class; i.e. 24.0% of them have 60% coverage by a template protein with a RMSD below 3.5 Å and 6.0% have 70% coverage. If the restriction that we align proteins only having different secondary structure types is removed, then in a representative benchmark set of proteins of 200 residues or smaller, 93% can be aligned to a single template structure (with average sequence identity of 9.8%), with a RMSD less than 4 Å , and 79% average coverage. In this sense, the current Protein Data Bank (PDB) is almost a covering set of small protein structures. The length of the aligned region (relative to the whole protein length) does not differ among the top hit proteins, indicating that protein structure space is highly dense. For larger proteins, non-related proteins can cover a significant portion of the structure. Moreover, these top hit proteins are aligned to different parts of the target protein, so that almost the entire molecule can be covered when combined. The number of proteins required to cover a target protein is very small, e.g. the top ten hit proteins can give 90% coverage below a RMSD of 3.5 Å for proteins up to 320 residues long. These results give a new view of the nature of protein structure space, and its implications for protein structure prediction are discussed.