Properties and regulation of glutamine transporter SN1 by protein kinases SGK and PKB

The amino acid transporter SN1 with substrate specificity identical to the amino acid transport system N is expressed mainly in astrocytes and hepatocytes where it accomplishes Na(+)-coupled glutamine uptake and efflux. To characterize properties and regulation of SN1, substrate-induced currents and/or radioactive glutamine uptake were determined in Xenopus oocytes injected with cRNA encoding SN1, the ubiquitin ligase Nedd4-2, and/or the constitutively active serum and glucocorticoid inducible kinase S422DSGK1, its isoform SGK3, and the constitutively active protein kinase B T308D,S473DPKB. The substrate-induced currents were enhanced by increasing glutamine and/or Na(+) concentrations, hyperpolarization, and alkalinization (pH 8.0). They were inhibited by acidification (pH 6.0). Coexpression of Nedd4-2 downregulated SN1-mediated transport, an effect reversed by coexpression of S422DSGK1, SGK3, and T308D,S473DPKB. It is concluded that SN1 is a target for the ubiquitin ligase Nedd4-2, which is inactivated by the serum and glucocorticoid inducible kinase SGK1, its isoform SGK3, and protein kinase B.     

Purification and properties of a predominantly female-specific protein from the hemolymph of the larva of the tobacco hornworm, Manduca sexta

A major serum protein was isolated from the hemolymph of larvae of the female tobacco hornworm, Manduca sexta, just prior to metamorphosis. After 3 or 4 days, this predominantly female-specific protein is rapidly cleared from the hemolymph and taken up and stored by the fat body. This larval serum protein was purified by density gradient ultracentrifugation, gel permeation, and ion-exchange chromatography. The purified protein exhibits a single band on native gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Chemical cross-linking with dimethylsuberimidate indicates a hexameric subunit arrangement for the native protein. The amino acid composition, relatively rich in methionine but poor in cysteine, was used to calculate a v = 0.75 cm3/g. Analytical ultracentrifugation experiments yielded S020,w = 16.9 S and D020,w = 3.23 X 10(-7) cm2/s. From these values Mr = 510,000, f/f0 = 1.22, and Stokes radius = 66.3 A were calculated. Immunoblotting experiments with anti-larval serum protein serum indicate a cross-reactivity with storage protein-1 of Bombyx mori. The amino acid composition and immunological data suggest that larval serum protein may be an example of a class of insect storage proteins distinct from the arylphorins, which are characterized by high content of aromatic amino acids.     

The Role of Dispensable Amino Acids for the Maximum Growth of Chick

Chicks were fed on the purified diets of which amino acid pattern was modeled after whole egg protein and crude protein content was 21.1%, changing the dietary ratio of indispensable amino acid nitrogen to dispensable amino acid nitrogen (I/D ratio) from 1/1.5 to 3/1 at regular intervals. The balances among amino acids in each indispensable and dispensable group of test diets were kept the same pattern as that of the whole egg, respectively. Optimum I/D ratio for normal chick growth was estimated to be in the range of between 1/1 and 1.5/1, because feed efficiency was the highest at the I/D ratio 1/1 and growth rate was the highest at the I/D ratio 1.5/1.

Chicks were killed and the serum was collected at the end of the experiment. It was shown that the I/D ratios of free amino acid in the serum of chicks were strongly influenced by that of diet.

White Leghorn chicks fed on the Scott’s reference amino acid diet grew as well as those fed on a conventional chick starter. Nitrogen retention of the former was a little less than that of the latter, but the amount of carcass fat of the former was almost twice as much as the latter.

Growth rate of chick was considerably reduced, when glutamic acid which is the only dispensable amino acid in the Scott’s diet was replaced by a mixture of glutamic acid, aspartic acid, alanine and serine, nitrogen content being kept at constant. Sufficient amount of glutamic acid in the Scott’s diet seems to be essential for the maximum growth of chick.     

Molecular characterization of the interaction between porins of Neisseria gonorrhoeae and C4b-binding protein

Neisseria gonorrhoeae, the causative agent of gonorrhea, is a natural infection only in humans. The resistance of N. gonorrhoeae to normal human serum killing correlates with porin (Por)-mediated binding to the complement inhibitor, C4b-binding protein (C4BP). The entire binding site for both porin molecules resides within complement control protein domain 1 (CCP1) of C4BP. Only human and chimpanzee C4BPs bind to Por1B-bearing gonococci, whereas only human C4BP binds to Por1A strains. We have now used these species-specific differences in C4BP binding to gonococci to map the porin binding sites on CCP1 of C4BP. A comparison between human and chimpanzee or rhesus C4BP CCP1 revealed differences at 4 and 12 amino acid positions, respectively. These amino acids were targeted in the construction of 13 recombinant human mutant C4BPs. Overall, amino acids T43, T45, and K24 individually and A12, M14, R22, and L34 together were important for binding to Por1A strains. Altering D15 (found in man) to N15 (found in rhesus) introduced a glycosylation site that blocked binding to Por1A gonococci. C4BP binding to Por1B strains required K24 and was partially shielded by additional glycosylation in the D15N mutant. Only those recombinant mutant C4BPs that bound to bacteria rescued them from 100% killing by rhesus serum, thereby providing a functional correlate for the binding studies and highlighting C4BP function in gonococcal serum resistance.     

Effects of streptozotocin-induced diabetes in pregnant rats on placental transport and tissue uptake of alpha-amino-isobutyric acid

Placental transport and tissue uptake of amino acids were studied in streptozotocin (STZ)-induced diabetic rats by using the non-metabolizable amino acid [U-14C]-alpha-amino-isobutyric acid (AIB). Fifteen minutes prior to autopsy, animals of each group, control (C), diabetic (D), diabetic-insulin treated (DI) and diabetic-T4 followed by 3-5-Dimethyl-3'-isopropyl-L-thyronine (DIMIT) treated (DTD), received an injection of the [U-14C]-AIB SC. Disintegrations per minute (DPM) were measured in serum and tissues subsequent to autopsy. There were no differences in maternal serum DPM/ml among groups. Fetal serum DPM, however, were lower in D and DTD groups than in the C group. The whole fetal tissue homogenate radioactivity was lower in the D, DTD, and DI groups than in the C group. In general, more AIB was taken up by fetal tissues of C than D animals. Maternal liver AIB uptake was reduced in D, DI, and DTD from C animals and net placental transport of AIB was less in D and DTD than C animals. Fetal liver protein concentrations were depressed in D and DTD animals from C and DI, but fetal brain protein concentrations showed no significant differences. Furthermore, the lower organ and fetal body weights of the D and DTD groups compared with the C and DI groups support the proposal that fetal anabolism is impaired. Maternal and fetal serum T4 concentrations were lower in D and DTD than in C and DI animals. Insulin therapy improved serum T4 levels in both mother and fetuses. It did not, however, correct all other measured parameters.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparison of glycopeptides from the ovotransferrin and serum transferrin of the hen

1. Glycopeptides were prepared from proteolytic digests of ovotransferrin and serum transferrin of the hen. The carbohydrate compositions and amino acid sequences of the peptides were studied. 2. The bulk of the carbohydrate of ovotransferrin is present as a single oligosaccharide composed of 4 residues of mannose and 8 residues of N-acetylglucosamine. Transferrin has most of its carbohydrate in a single unit composed of 2 residues of mannose, 2 residues of galactose, 3 residues of N-acetylglucosamine and either 1 or 2 residues of sialic acid. 3. The amino acid sequences of the glycopeptides carrying these different oligosaccharides are the same in ovotransferrin and serum transferrin, showing that the carbohydrate groups are attached to the same site on the protein molecule.     

Single amino acid (arginine) deprivation induces G1 arrest associated with inhibition of cdk4 expression in cultured human diploid fibroblasts

Withdrawal of a single amino acid (arginine) from freely cycling early passage primary human fibroblasts caused a halt to proliferation, characterized by an accumulation of cells in the G1 phase of the cell cycle. This arrest was accompanied by the suppression of cyclin D1- and cyclin E-associated kinase activities and the appearance of hypophosphorylated retinoblastoma protein. Arginine-deprived cells remained viable for in excess of 4 days and could be made to synchronously reenter the cell cycle by restoration of the amino acid, with kinetics characteristic of exit from a quiescent state. Stimulation of cells arrested by serum withdrawal did not result in S-phase entry when arginine was omitted from the culture medium. Although cyclin D1 accumulated on normal schedule, cdk4, which increased following restimulation in amino acid-replete medium, was not induced when arginine was absent. These results suggest that arginine deprivation-in common with other "suboptimal" conditions-inhibits the passage of normal human cells through the restriction point and implicate cdk4 as the key regulatory element in amino acid-sensitive cell cycle control.     

Site-specific mutations of the N-terminal threonines in the D1 protein affects photoautotrophic growth but not D1 protein stability in Synechocystis 6803

The cyanobacterium Synechocystis 6803 was engineered to produce a D1 protein where one or more of the N-terminal threonines at positions 2, 3 and 4 were replaced by other amino acid residues. No phenotypic effects were found for the T2S or T2L mutations, whereas the T2V, T2L;T4V and T2V;T3V;T4V mutations resulted in reduced photoautotrophic growth rate and oxygen evolving activity. The mutant strain T2V;T3V;T4V exhibited an oxygen evolution activity that was only half of that for the wild-type strain. Despite of that, both accumulation and stability of the D1 protein in the thylakoid membrane appeared unaffected in the mutant.     

Cytokinin effect on protein synthesis in vivo in higher plants

The influence of naphthalene-1-acetic acid and kinetin on protein synthesis in vivo was investigated by measuring the incorporation of radioactive amino acids into polypeptides of synthesizing polysomes. The second subculture of sterile pith tissue of Nicotiana tabacum L. cv. Wisconsin 38 grown on a medium containing only a minimum of growth substances was further starved in a small volume of medium lacking auxin and cytokinin for 12 h. An incubation period of 4 h with [¹⁴C]amino acids followed. The last 30 min of those incubations were carried out in the presence of actinomycin D and the last 20 min were performed under different conditions: a) without any growth substances, b) with naphthalene-l-acetic acid, c) with kinetin. By measuring the differences of the specific radioactivities of the polysomes the following results were revealed: 1) Kinetin increases the protein synthesis by an average of 35%-2) Auxin has no effect on protein synthesis.Abbreviations Act. D actinomycin D - NAA naphthalene-1-acetic acid Part of a Diplomarbeit, University of Bonn 1976     

Complement C3c and related protein biomarkers in amyotrophic lateral sclerosis and Parkinson's disease

We have used quantitative 2D gel electrophoresis to analyze serum proteins from 422 patients with neurodegenerative diseases and normal individuals in an unbiased approach to identify biomarkers. Differences in abnormal serum levels were found between amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), and related disorders for 34 protein biomarker spots, nine of which were related to the complement system. Of these nine, four spots originated from the Complement C3b-alpha-chain (C3c(1), C3c(2a), C3c(2b), and C3dg). The C3c spots (C3c(1), C3c(2a), and C3c(2b)) had the same amino acid sequence and glycosylation, though only C3c(1) was phosphorylated. In addition, Complement Factors H, Bb, and Pre-Serum amyloid protein displayed different serum concentrations in ALS, PD, and normal sera, whereas Complement C4b gamma-chain and Complement Factor I did not. The differential expression of the complement proteins provides potentially useful biomarkers as well as evidence for the involvement of inflammatory processes in the pathogenesis of ALS and PD.     

Studies on the composition of pig serum lipoproteins. Isolation and characterization of different apoproteins.

1. Different lipoprotein density fractions from pig serum were isolated by phosphotungstate precipitation followed by purification in the preparative ultra-centrifuge. 2. The protein part of very low density lipoproteins was composed of approximately 52 percent lipoprotein B apoprotein and the rest of lipoprotein C II apoprotein and other as yet unidentified peptides. 3. The protein moiety of low density lipoproteins consisted primarily of lipoprotein B apoprotein (over 95 percent); the amino acid compositions of lipoprotein B apoprotein of very low and low density lipoproteins were practically identical. 4. The predominant polypeptide of pig serum high density lipoproteins exhibited an amino acid composition and a molecular weight very similar to human liprotein A I apoprotein. In contrast to human lipoprotein A I apoprotein, the apoprotein from pigs was found to release leucine first followed by alanine, threonine, and lysine upon incubation with carboxypeptidase A. 5. In pig serum the major lipoprotein C apoprotein was found to be a polypeptide similar in amino acid composition to lipoprotein C II apoprotein from human serum. The molecular weight of this polypeptide is approximately 8000. Incubation experiments with carboxypeptidase A indicate serine to be the most likely C-terminal amino acid.     

Analysis of wild-type and mutant p21WAF-1 gene activities.

The p21WAF-1 gene is positively regulated by the wild-type p53 protein. p21WAF-1 has been shown to interact with several cyclin-dependent kinase complexes and block the activity of G1 cyclin-dependent kinases (cdks). Mutational analysis with the p21WAF-1 gene localized a site, at amino acid residues 21 and 24 in the amino terminus of the protein, for p21WAF-1 binding to cyclins D and E. This region of the protein is conserved (residues 21 to 26) in other p21WAF-1 family members, p27kip-1 and p57kip-2. The same p21WAF-121,24 mutant also fails to bind to cyclin D1-cdk 4 or cyclin E-cdk 2 complexes in vitro, suggesting that amino acid residues 21 and 24 are important for p21WAF-1-cdk-cyclin trimeric complex interactions. The p21WAF-1 wild-type protein will suppress tumor cell growth in culture while p21WAF-1 mutant proteins with defects in residues 21 and 24 fail to suppress tumor cell growth. The overexpression of cyclin D or E in these cells will partially overcome the growth suppression of wild-type p21WAF-1 protein in cells. These results provide evidence that p21WAF-1 acts through cyclin D1-cdk4 and cyclin E-cdk2 complexes in vivo to induce the growth suppression. The p21WAF-1 binding sites for cyclins (residues 21 to 26), cdk2 (residues 49 to 71), and proliferating-cell nuclear antigen (residues 124 to 164) have all been mapped to discrete sites on the protein.     

Molecular cloning and characterization of three novel cytochrome P450 2D isoforms, CYP2D20, CYP2D27, and CYP2D28 in the Syrian hamster (Mesocricetus auratus)

We cloned three novel cytochrome P450 (CYP) 2D cDNAs in the Syrian hamster (Mesocricetus auratus). Each clone contained an open reading frame of 1500 nucleotides encoding a protein of 500 amino acids. The deduced amino acid sequences of these had high identities with those of the other CYP2D members, therefore, the clones were assigned as CYP2D20, CYP2D27, and CYP2D28. Northern blot analysis showed that the CYP2D27 mRNA was expressed in liver, but not in kidney, small intestine, and brain, while the CYP2D20 and CYP2D28 mRNAs were not detected in these tissues examined. The expression of CYP2D27 mRNA in liver did not show sex difference and was not induced by either 3-methylcholanthrene or phenobarbital treatment. We characterized the enzyme activities of recombinant CYP2D27 expressed in COS-7 cells. The CYP2D27 protein had the bufuralol 1'-hydroxylase and debrisoquine 4-hydroxylase activities that are specific to the CYP2D subfamily.     

The D1 and D2 proteins of dinoflagellates: unusually accumulated mutations which influence on PSII photoreaction

Plastid encoded genes of the dinoflagellates are rapidly evolving and most divergent. The importance of unusually accumulated mutations on structure of PSII core protein and photosynthetic function was examined in the dinoflagellates, Symbiodinium sp. and Alexandrium tamarense. Full-length cDNA sequences of psbA (D1 protein) and psbD (D2 protein) were obtained and compared with the other oxygen-evolving photoautotrophs. Twenty-three amino acid positions (7%) for the D1 protein and 34 positions (10%) for the D2 were mutated in the dinoflagellates, although amino acid residues at these positions were conserved in cyanobacteria, the other algae, and plant. Many mutations were likely to distribute in the N-terminus and the D-E interhelical loop of the D1 protein and helix B of D2 protein, while the remaining regions were well conserved. The different structural properties in these mutated regions were supported by hydropathy profiles. The chlorophyll fluorescence kinetics of the dinoflagellates was compared with Synechocystis sp. PCC6803 in relation to the altered protein structure.     

Isolation and characterization of proteoglycans from porcine lungs.

Proteoglycans were extracted from porcine lungs with 4 M guanidinium chloride. The extract was subjected to associative density gradient centrifugation, and four equal fractions, labeled A1 through A4 from the bottom to the top of the gradient, were obtained. The pooled A1 fractions containing proteoglycan aggregates were further fractionated by dissociative density gradient centrifugation to yield four equal fractions labeled A1D1 through A1D4 from the bottom to the top of the gradient. These fractions were analyzed for their protein, uronic acid, glucosamine, galactosamine, hexose, and sialic acid content. The fraction A1D1 with the highest buoyant density had the highest content of uronic acid and galactosamine, and lowest content of protein, indicating the enrichment of proteoglycan monomers at the bottom of the dissociative density gradient. As the density of the gradient decreased, the protein, hexoses, and sialic acid content increased, whereas uronic acid and galactosamine content decreased. The amino acid analysis showed similar composition for all four fractions with aspartic acid, serine, glutamic acid, proline, glycine, alanine, valine, and leucine as the major constituent amino acids. No hydroxyproline was detected in any of the fractions. As the buoyant density of the fractions decreased, the aspartic acid content increased and glycine content decreased.     

Purification of the Atlantic salmon hepatic 21 kDa protein and classification as a high mobility group chromatin protein.

The 21 kDa protein of liver from Atlantic salmon (Salmo salar) has been purified. Hepatic nuclei were extracted with 0.75 M HClO4. The extracted proteins were fractionated using reversed phase high performance liquid chromatography. The purity of the protein was analysed by isoelectric focusing in the first, and SDS-polyacrylamide gel electrophoresis in the 2nd dimension. Isoelectric focusing separated the protein into 5 spots. In gel trypsin digestion after isoelectric focusing followed by SDS-polyacrylamide gel electrophoresis resulted in identical migration of the tryptic peptides. The amino acid composition of the 21 kDa protein was similar to that of high mobility group (HMG) proteins C and D from rainbow trout (Oncorhynchus mykiss). The N-terminal sequence of the amino acids 1-19 revealed a conserved region characteristic for HMG 14/17 proteins of mammals and avians, and their equivalents in rainbow trout. Considering the electrophoretic mobility, amino acid composition and N-terminal amino acid sequence it is concluded that the 21 kDa protein of Atlantic salmon is a member of the HMG protein family resembling the HMG D protein of rainbow trout.     

Effects of amino acid supplementation on the nutritive quality of fermented linseed meal protein in the diets for rohu, Labeo rohita, fingerlings

A feeding trial was conducted for 8 weeks to examine the effects of partial substitution of fish meal (FM) protein (crude protein content: 58.5%) with linseed meal protein with and without supplemental amino acids in diets for rohu Labeo rohita (Hamilton), fingerlings (mean weight: 1.50 ± 0.3 g). Prior to incorporation into the diets, linseed meal was fermented with lactic acid bacteria ( Lactobacillus acidophilus ) to reduce/eliminate the antinutritional tannin and phytic acid factors. Twelve experimental diets (diets D1–D12) were formulated to replace the FM protein from a reference diet (RD) with linseed meal protein at different levels (four sets of diets, of which each set of three diets contained 25%, 50% and 75% replacement of FM protein by linseed meal protein, respectively). Diets D1–D3 were not supplemented with any amino acid. Lysine was supplemented in diets D4–D6. Diets D7–D9 were supplemented with methionine + cystine (together), and diets D10–D12 contained lysine and methionine + cystine (together). Lysine and methionine + cystine (together) were added to the diets at 5.7% and 3.1% of dietary protein, respectively. The groups of fish fed diets without amino acid supplementation had significantly lower percentages of weight gain, specific growth rate and high feed : gain ratio than the fish groups fed other experimental diets. The addition of lysine and methionine + cystine to the diet in which 50% of the FM protein was replaced by linseed meal protein (diet D11) significantly improved fish performance. The results of the present study suggest that rohu fingerlings can effectively utilize the supplemented amino acids and that linseed meal protein can replace up to 50% of the FM protein in rohu diets if the linseed meal is properly processed (fermented) and supplemented with the lacking amino acids.     

Serotriflin, a CRISP family protein with binding affinity for small serum protein-2 in snake serum

Habu (Trimeresurus flavoviridis) serum contains 3 small serum proteins (SSP-1, SSP-2, and SSP-3) with molecular masses of 6.5 to 10 kDa. Gel filtration analysis showed that all the SSPs exist in high molecular mass forms of approximately 60 kDa in the serum. Ultrafiltration of Habu serum showed that SSPs dissociated from the complex below a pH of 4. An SSP-binding protein was purified from Habu serum by gel filtration, ion exchange, and reverse-phase HPLC. N-terminal sequencing yielded a 39-amino acid sequence, similar to the N-terminal region of triflin, which is a snake venom-derived Ca2+ channel blocker that suppresses smooth muscle contraction. The amino acid sequence of this protein, termed serotriflin, was established by peptide analysis and cDNA cloning. Serotriflin is a glycosylated protein and consists of 221 amino acids. Among the 3 SSPs, only SSP-2 formed a noncovalent complex with serotriflin. It was bound to triflin and serotriflin with high affinity, as evidenced by surface plasmon resonance. SSP-2 is considered to be a protein that prevents self injury by accidental leaking of venom into the blood.     

Scaffolding proteins altered in the ability to perform a conformational switch confer dominant lethal assembly defects

In the phiX174 procapsid crystal structure, 240 external scaffolding protein D subunits form 60 pairs of asymmetric dimers, D(1)D(2) and D(3)D(4), in a non-quasi-equivalent structure. To achieve this arrangement, alpha-helix 3 assumes two different conformations: (i) kinked 30 degrees at glycine residue 61 in subunits D(1) and D(3) and (ii) straight in subunits D(2) and D(4). Substitutions for G61 may inhibit viral assembly by preventing the protein from achieving its fully kinked conformation while still allowing it to interact with other scaffolding and structural proteins. Mutations designed to inhibit conformational switching in alpha-helix 3 were introduced into a cloned gene, and expression was demonstrated to inhibit wild-type morphogenesis. The severity of inhibition appears to be related to the size of the substituted amino acid. For infections in which only the mutant protein is present, morphogenesis does not proceed past the first step that requires the wild-type external scaffolding protein. Thus, mutant subunits alone appear to have little or no morphogenetic function. In contrast, assembly in the presence of wild-type and mutant subunits is blocked prematurely, before D protein is required in a wild-type infection, or channeled into an off-pathway reaction. These data suggest that the wild-type protein transports the inhibitory protein to the pathway. Viruses resistant to the lethal dominant proteins were isolated, and mutations were mapped to the coat and internal scaffolding proteins. The affected amino acids cluster in the atomic structure and may act to exclude mutant subunits from occupying particular positions atop pentamers of the viral coat protein.     

艳妇斑粉蝶蛹和成虫的营养成分分析

为了深层次地开发利用艳妇斑粉蝶,探寻艳妇斑粉蝶蛹和成虫的主要营养成分,评价其营养价值水平,运用国家标准检测方法测定艳妇斑粉蝶蛹和成虫的水分、脂肪、蛋白质、矿质元素和氨基酸的含量,分析氨基酸分和必需氨基酸指数,并将艳妇斑粉蝶蛹和成虫的蛋白质、氨基酸、脂肪、矿质元素含量与常见食物的含量进行比较分析.结果显示,艳妇斑粉蝶蛹和成虫具有高蛋白(含量分别为70.7％和76.8％)、低脂肪(含量分别为11.3％ ％和7.1％)、无机物质含量丰富、矿质元素含量水平高、能量值低等特点;艳妇斑粉蝶蛹和成虫的总氨基酸含量较高,分别为417.7 mg·g-1和458.0 mg·g-1,必需氨基酸总含量分别为159.7 mg·g-1和171.7 mg·g-1,必需氨基酸占总氨基酸的比例分别为38.2％和37.5％,必需氨基酸与非必需氨基酸的比值分别为0.62和0.60;艳妇斑粉蝶蛹和成虫的必需氨基酸指数分别为0.75和0.73.艳妇斑粉蝶蛹和成虫具有较高的营养价值和食用开发价值,但是其限制性氨基酸影响了氨基酸结构的平衡.