Evaluation of protein <Emphasis Type="BoldItalic">in vitro</Emphasis> digestibility of <Emphasis Type="BoldItalic">Palmaria palmata</Emphasis> and <Emphasis Type="BoldItalic">Gracilaria verrucosa</Emphasis>

Palmaria palmata and Gracilaria verrucosa are edible red seaweeds and potential protein sources for human or animal nutrition, so studies were conducted on their in vitro protein digestibility. After 30 min predigestion by pepsin followed by 6 h digestion into a cell dialysis containing porcine pancreatin, the in vitro protein digestibility of P. palmata and G. verrucosa, expressed in regard to casein digestibility, was 4.9% and 42.1%, respectively. The level of protein digestibility seems to be related to the amount of soluble fibre, which was 45.3% and 30.5%, respectively.     

Chemical analysis of phenolic glycosides: art,facts, and artifacts

Summary Phenolic glycosides have been the subject of considerable interest in recent ecological and systematic studies, especially those involving the Salicaceae. But these compounds are markedly labile in aqueous media, and the consequences of spontaneous degradation for valid interpretation of results have been largely ignored by researchers. We found that freeze-drying and oven-drying of leaf samples from several Populus and Salix species produced dramatic changes in the total and relative concentrations of specific phenolic glycosides, when compared to analyses of fresh material. Extraction in aqueous and alcoholic media for extended (24 h) periods also effected changes in glycoside concentrations. Alterations in phenolic glycoside concentrations, interconversions among glycosides, and production of artifactual glycosides result from a series of hydrolytic reactions. These deleterious effects can be best (but not entirely) avoided by the use of fresh plant material, cold, nonaqueous extraction solvents, and short extraction times. Because individual phenolic glycosides exhibit very different biological activities against herbivores, we caution ecologists to use utmost care in the performance and interpretation of phenolic glycoside assays.     

加拿大一枝黄花提取物的抗氧化活性研究

为进一步开发和利用加拿大一枝黄花(Solidago canadensis),该研究采用氯化铝显色法和福林酚法测定加拿大一枝黄花乙醇提取物及其不同极性萃取物中的总黄酮和总酚的含量;以抗坏血酸(Vitamin C,Vc)和二丁基羟基甲苯(BHT)为阳性对照,应用2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐(ABTS)、1,1-二苯基苦基苯肼(DPPH)自由基清除体系、铁离子还原能力(FRAP)法和抗氧化能力指数(ORAC)法研究其体外抗氧化活性。结果表明:乙酸乙酯萃取物中的总黄酮(202.45 mg·g~(-1))和总酚(485.94 mg·g~(-1))含量最高,且其抗氧化活性最强,并强于阳性对照Vc(P0.05)。因而,加拿大一枝黄花乙酸乙酯萃取物将有可能成为一种潜在的天然高效抗氧化剂,具有广泛的应用前景。     

The composition,nutritional status and digestibility of the diets of Sarotherodon mossambicus from nine man-made lakes in Sri Lanka

Synopsis The dietary composition and the nutritional status and the digestibility of the diets of Sarotherodon mossambicus from nine reservoirs in Sri Lanka were evaluated. The feeding habits of S. mossambicus were variable from reservoir to reservoir; they ranged from herbivory to total carnivory. The protein, total lipid, carbohydrate and total organic matter content of the ingested material were related to the dietary composition and ranged from 18.53% to 35.15% (x−24.18%), 5.94% to 9.84% (x−7.91%), 11.6% to 34.7% (x−22.34%) and 34.4% to 64.4% (x−45.71%), respectively. Irrespective of the feeding habits, the diet contained a significant proportion of organic material which cannot be accounted for by protein, total lipid and carbohydrate. As much as the ingested material was related to the feeding habit, the digestibility of the nutrient components was related to the food material devoured. For example, the mean digestibility of the total organic matter in S. mossambicus feeding on detritus, plants and animal were 36.85, 33.5 and 29.5 respectively, and compared well with observations from elsewhere. It is hypothesised that the favourable nutrient quality of the available dietary material in the reservoirs of Sri Lanka, which could be and is effectively utilized by S. mossambicus, may have been, at least partially, responsible for its almost unprecedented success in Sri Lanka.     

Lysis protein T of bacteriophage T4

Summary Lysis protein T of phage T4 is required to allow the phage's lysozyme to reach the murein layer of the cell envelope and cause lysis. Using fusions of the cloned gene t with that of the Escherichia coli alkaline phosphatase or a fragment of the gene for the outer membrane protein OmpA, it was possible to identify T as an integral protein of the plasma membrane. The protein was present in the membrane as a homooligomer and was active at very low cellular concentrations. Expression of the cloned gene t was lethal without causing gross leakiness of the membrane. The functional equivalent of T in phage is protein S. An amber mutant of gene S can be complemented by gene t, although neither protein R of (the functional equivalent of T4 lysozyme) nor S possess any sequence similarity with their T4 counterparts. The murein-degrading enzymes (including that of phage P22) have in common a relatively small size (molecular masses of ca. 18 000) and a rather basic nature not exhibited by other E. coli cystosolic proteins. The results suggest that T acts as a pore that is specific for this type of enzyme.     

优化的绿色荧光蛋白在超嗜热嗜酸古菌冰岛硫化叶菌中的表达与应用

【目的】开发可用于在极端嗜热嗜酸模式泉古菌冰岛硫化叶菌(Sulfolobus islandicus)中进行高效表达的eCGP123(enhanced consensus green protein variant 123)荧光蛋白,并用作S.islandicus的细胞内蛋白定位工具。【方法】绿色荧光蛋白突变体eCGP123具有极高的热稳定性、耐酸性和可逆的荧光特性等。本研究主要对eCGP123的基因根据S.islandicus密码子偏好性进行优化与合成,在大肠杆菌(Escherichia coli)中表达并研究其蛋白性质;通过在eCGP123的C末端分别融合具有不同细胞内定位的蛋白(包括E.coli来源的Fts Z和S.islandicus来源的Ups E、PCNA1和SiRe_1200等),构建eCGP123及其融合蛋白的表达菌株,用激光共聚焦显微镜分析eCGP123及其融合蛋白在E.coli和S.islandicus活细胞中的亚细胞定位。【结果】我们确认了在E.coli中表达并纯化密码子优化后的e CGP123具有与野生型绿色荧光蛋白相同的吸光值和较高的热稳定性。细胞学分析显示细胞分裂相关蛋白FtsZ和Si Re_1200分别主要定位于E.coli和S.islandicus分裂细胞的中间;鞭毛组分蛋白Ups E呈点状均匀分布,可能定位于细胞膜上;DNA复制滑动夹亚基PCNA1呈区域性点状分布,暗示了DNA复制区域的位置。蛋白的亚细胞定位与预期结果基本吻合。【结论】绿色荧光蛋白e CGP123可以作为报告蛋白,应用于S.islandicus细胞的蛋白定位分析中,可作为该模式菌株中功能基因研究的重要工具,但需要进一步优化条件。     

Amino acid sequence of the ribosomal protein HS23 from the halophilicHaloarcula marismortui and homology studies to other ribosomal proteins

The ribosomal protein HS23 from the 30S subunit of the extreme halophilicHaloarcula marismortui, belonging to the group of archaea, was isolated either by RP-HLPLC or two-dimensional polyacrylamide gel electrophoresis. The complete amino acid sequence was determined by automated N-terminal microsequencing. The protein consists of 123 residues with a corresponding molecular mass of 12,552 Da as determined by electrospray mass spectroscopy; the pI is 11.04. Homology studies reveal similarities to the eukaryotic ribosomal protein S8 fromHomo sapiens, Rattus norvegicus, Leishmania major, andSaccharomyces cerevisiae.Abbreviations H. marismortui Haloarcula marismortui - PVDF polyvinylidene difluoride - PTH phenylthiohydantoin - RP-HPLC reversed-phase high-performance liquid chromatography - TFA trifluoro acetic acid - TP30 total protein mixture from the 30S ribosomal subunit ofH. marismortui     

Structure,function, and biogenesis of SecY,an integral membrane protein involved in protein export

TheE. coli secY (prlA) gene, located in the operator-distal part of thespec ribosomal protein operon, codes for an integral membrane protein, SecY. The phenotypes of temperature-sensitive and cold-sensitive mutations insecY suggest that the SecY protein plays an essential rolein vivo to facilitate protein translocation, whereas theprlA mutations in this gene suggest that SecY may interact with the signal sequence of translocating polypeptides. SecY contains most probably six cytoplasmic and five periplasmic domains, as well as 10 transmembrane segments. Such membrane-embedded structure may confer the SecY protein a translocator function, in which it provides a proteinaceous pathway for passage of secreted as well as membrane proteins. Results obtained byin vitro analyses of the translocation reactions, as well as some new phenotypes of thesecY mutants, are consistent with this notion. Possible interaction of SecY with other secretion and chaperone-like factors is also discussed.     

Overexpression of bacterioferritin comigratory protein (Bcp) enhance viability and reduced glutathione level in the fission yeast under stress

The structural gene encoding bacterioferritin comigratory protein (Bcp) was amplified using PCR from the genomic DNA of Schizosaccharomyces pombe, and transferred into the shuttle vector pRS316 to generate the recombinant plasmid pBCPlO. The bcp ⁺ mRNA level in the pBCPlO-containing yeast cells was significantly higher than that in the control yeast cells, indicating that the cloned gene is functioning. The S. pombe cells harboring the plasmid pBCPIO exhibited higher survival on the solid minimal media with hydrogen peroxide, tert-BOOH or cadmium than the control yeast cells. They also exhibited enhanced cellular viability in the liquid media containing the stressful agents. The increased viabilities of the fission yeast cells harboring the plasmid pBCP10 were also obtained with 0.4% glucose or 0.4% sucrose as a sole carbon source, and nitrogen starvation, compared with those of the control yeast cells. The total glutathione (GSH) content and total GSH/GSSG ratio were significantly higher in the yeast cells harboring the plasmid pBCP10 than in the control yeast cells. In brief, the S. pombe Bcp plays a protective role in the defensive response to oxidative stress possibly via up-regulation of total and reduced glutathione levels.     

溶菌酶对瘤胃体外发酵、甲烷生成及微生物菌群结构的影响

【目的】通过体外静态模拟瘤胃发酵法研究溶菌酶对瘤胃发酵、甲烷生成及微生物菌群结构的影响。【方法】采用单因素多水平试验设计,溶菌酶添加水平分别为0(L-0,对照组)、0.1 mg/100 m L(L-0.1)、1 mg/100 m L(L-1)、10 mg/100 m L(L-10)和100 mg/100 m L(L-100),定时测定产气量和甲烷产量,培养24 h后,发酵液用于发酵参数和微生物菌群数量的q PCR测定,其中L-0、L-1和L-100三个组发酵液同时进行16S r RNA基因Illumina高通量测序。【结果】与对照组相比,低剂量溶菌酶添加(L-0.1组)不影响甲烷产量、氨氮浓度、干物质消失率、有机物消失率和总挥发性脂肪酸等瘤胃发酵参数(P0.05);随着剂量提高,L-1处理组甲烷产量、氨氮浓度显著降低(P0.05),丙酸浓度显著增加(P0.05),并且干物质消失率、有机物消失率和总挥发性脂肪酸不受影响(P0.05);而较高剂量组(L-10和L-100组)虽然甲烷产量显著降低,丙酸浓度显著增加(P0.05),但干物质消失率和有机物消失率也显著降低(P0.05)。q PCR结果显示高剂量组(L-100组)总菌、原虫、甲烷菌数量与对照组相比显著降低(P0.05),而L-0.1、L-1和L-10组总菌、真菌和原虫数量与对照组相比均无显著变化(P0.05)。高通量测序主成分分析(PCA)显示对照组与溶菌酶添加组间瘤胃细菌组成的明显区分,说明添加溶菌酶显著改变了瘤胃细菌菌群结构。溶菌酶通过增加月形单胞菌和琥珀酸弧菌等丙酸生成菌的相对丰度,使更多的氢被用于生成丙酸,导致甲烷产量降低;溶菌酶可抑制普雷沃氏菌和拟杆菌属等蛋白降解菌的生长,进而减少蛋白质过度降解,降低氨氮浓度。【结论】添加适宜浓度(1 mg/100 m L)的溶菌酶可通过调控瘤胃微生态改变瘤胃发酵模式,降低瘤胃甲烷和氨的生成,短期内并不影响饲料消化。     

Expression and purification of Listeria innocua Sv6b p60 invasion associated protein

The p60 protein of Listeria is a major extra-cellular protein which is used as indicator for the detection of these bacteria from contaminated food samples. To produce p60 in Escherichia coli, the invasion associated protein (iap) gene of L. innocua Sv6b encoding p60 was cloned and over-expressed with expression vector pMAL-C2. Recombinant pMBP-iap/innocua was induced with IPTG in E. coli. The expressed recombinant p60 protein that was fused with a maltose-binding protein (MBP) was purified by amylose resin-based affinity chromatography. The purified recombinant p60 protein was also detected as denatured and neutralized form by using a specific p60 monoclonal antibody against L. monocytogenes and it may be useful for the production of L. innocua-specific antibody.     

Characterization of Plasmodium falciparum calcium-dependent protein kinase 4

In Plasmodium berghei, the orthologous gene of P. falciparum calcium-dependent protein kinase 4 (PfCDPK4) was reported to be essential for the exflagellation of male gametocytes. To elucidate the role of PfCDPK4 in P. falciparum gametogenesis, we characterized the biological function of PfCDPK4 in vitro. PfCDPK4 was purified as a fusion protein that was labeled with [γ-³²P]ATP; this labeling was then eliminated by phosphatase. Phosphorylation activity of PfCDPK4 was eliminated when its putative catalytic lysine residue was replaced with alanine. In biochemical analyses, PfCDPK4 was found to have characteristics that were similar to those of homologous proteins from plants. PfCDPK4 phosphorylation was activated when experimental conditions were changed from those characteristic of human blood (37 °C, pH 7.4) to those of the mosquito bloodmeal (at least 5 °C below 37 °C, pH 7.6, with xanthurenic acid (XA)). PfCDPK4 was overexpressed in day 15 gametocytes exposed to XA or human serum. Thus, PfCDPK4 phosphorylation is activated by an increase in Ca²⁺ concentration or pH and by a decrease in temperature, and is associated with the Ca²⁺ signals that facilitate P. falciparum gametogenesis.     

Nutritive value and voluntary feed intake by goats of three browse fodder species in the Sahelian zone of West Africa

Browsing ruminants have access to different biomass, depending on how high they can reach. Foliage consisting of leaves and green pods from Acacia senegal, Pterocarpus lucens and Guiera senegalensis, was collected according to height above ground accessible to either sheep (0.90 m), goats (1.65 m) or cattle (1.50 m). There was a significant variation in the chemical composition of the biomass between species. The crude protein (CP) content was 114, 157 and 217 g/kg dry matter (DM) and the neutral detergent fiber (aNDF) content 604, 534 and 412 g/kg DM for G. senegalensis, P. lucens and A. senegal, respectively. There was no significant variation in chemical composition according to the height accessible by cattle, sheep or goats. The voluntary intake was studied using eight goats per diet. The six diets consisted of the three browse leaves and two pods (A. senegal and P. lucens) and a control. The leaves were fed combined with hay of Schoenefeldia gracilis (maximum 30%) and the control was pure hay. Apparent digestibilities of the same diets, with the exception of G. senegalensis, were measured using five goats per diet. All browse fodders used in the feeding and digestibility trials were high in CP (105–170 g/kg DM) and lignin (164–234 g/kg DM except A. senegal leaves) and low in fiber (322–590 g/kg DM of NDF) compared to the hay (31 g/kg DM of CP and 755 g/kg DM of NDF). The highest intake was of the P. lucens diet (864 g) and the lowest of the G. senegalensis diet (397 g). The intake of pods from A. senegal was higher (1033 g) than from P. lucens pods (691 g). The apparent digestibility of OM and CP in the browse leaves was 0.63 and 0.57 and 0.63 and 0.64 for A. senegal and P. lucens, respectively, higher than for the hay, which showed higher digestibility of NDF. A. senegal pods had higher digestibility for all nutrients than P. lucens pods. Based on the high CP content and the intake and digestibility characteristics, P. lucens leaves and A. senegal leaves and pods can be recommended as protein supplements to low quality diets.     

Studies of the gene expression in a somatic hybrid of dihaploid Solanum tuberosum and diploid S. brevidens using two-dimensional protein electrophoresis

Two-dimensional protein electrophoretic patterns of leaf, stem and microtuber were compared between a somatic hybrid (Solanum tuberosum + S. brevidens) and parental plants. Polypeptide spots observed in leaf of the somatic hybrid (BT-1) were similar to those of S. brevidens. In the stem of BT-1, the spots characteristic for each parental plants were also observed. Three specific spots (W, X, Y) found in BT-1 were identical to those of S. tuberosum, however their appearance in S. brevidens depended on the culture conditions (observed at 16h daylength regime, but not in the dark with high sucrose concentration). Potato tuber storage protein patatin was observed in small amounts in the microtubers of BT-1. The data indicated that gene expression unique to each parental plants also existed in the somatic hybrid.Abbreviations BAP 6-benzylaminopurine - 2,4-D (2,4-dichlorophenoxy) acetic acid - IAA indole-3-acetic acid - MES 2-(N-morpholino) ethanesulfonic acid - NAA 1-naphthaleneacetic acid - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecylsulfate     

Phenolic Content of Microcuttings of Adult Chestnut along Rooting Induction

From the same adult 80-year-old tree of chestnut (Castanea sativa Mill.) 2 types of material have been taken and micropropagated. This resulted in in vitro easy-to-root microshoots (basal shoot origin - BS), and hard-to-root microshoots (crown shoot origin - CR). In these shoots, the phenolic contents were analysed at 0, 2, 5 and 8 days after in vitro rooting induction by 2 minute-dipping into an indole-3-butyric acid (IBA) solution (4.9 mM) and subsequent culture in a hormone-free rooting medium. The variation of the phenolic content along the adventitious rooting process differs between CR and BS microshoots for tannin, flavonol and elagic acid concentrations, which could be related to their differential rooting capacity.     

Expression of HBsAg and preS2-S protein in different yeast based system: A comparative analysis

We have expressed both S and preS2-S genes coding for the hepatitis B small (S) and medium (M) proteins, respectively, in different yeast based expression systems and compared the production level of the recombinant proteins. In Saccharomyces cerevisiae, viral genes were expressed under the inducible Gal10/cyc1 and the constitutive PGK promoters using 2 μ replicating vectors. We showed that the yield of S protein was higher than M protein under both inducible (14.27 vs 10.9 mg/l) and constitutive (9.18 vs 6.39 mg/l) conditions, respectively. In the methylotrophic yeast Pichia pastoris, the viral genes were expressed in GS115 (Mut⁺: Methanol Utilizing) and KM71 (Mut^S: Methanol Utilizing Slow) under the control of the alcohol oxidase promoter (AOX1). In Mut^S background, both S and preS2-S genes were expressed at higher levels than in Mut⁺. In attempt to increase the yield of recombinant viral proteins in S. cerevisiae, we have co-expressed both inducible and constitutive vectors harboring the S or preS2-S genes leading to recombinant strains called UTS (containing pDP/S + pYePIT/S) and UTP (containing pDP/preS2-S + pYePIT/preS2-S). We showed that the recombinant S and preS2-S proteins were successfully detected and the production level reached 18.31 mg/l for the S and 13.22 mg/l for the M proteins.Our comparative study provides evidence that in small scale, S. cerevisiae is more suitable for HBsAg and preS2-S proteins production than P. pastoris under inducible rather than constitutive condition.     

Evolution of fraction 1 protein in the genus Lycopersicon

The large- and small-subunit polypeptide composition of fraction 1 protein contained in seven species of Lycopersicon and Solanum pennellii was determined by electrofocusing. The eight species of protein had large subunits composed of three polypeptides separated by about 0.05 pH unit, but there was no difference in the isoelectric points of the clusters of three polypeptides. By this criterion, no surviving mutations have appeared in the extranuclear DNA coding for the cluster of large-subunit polypeptides during a period of evolution which generated the eight species of plants. The genus Lycopersicon appears to be much younger than its sister genus Nicotiana in the family Solanaceae, where four types of polypeptide clusters have evolved. Three different small-subunit polypeptides whose isoelectric points are coded by nuclear DNA have arisen among the seven Lycopersicon species, and L. hirsutum and S. pennellii have proteins containing single polypeptides and are therefore considered older than L. chilense, L. chimielewskii, and L. parviflorum, whose proteins contain two polypeptides. L. cheesemanii, L. pimpinellifolium, and L. esculentum (and probably L. peruvianum) seem to be the most recently evolved species since their fraction 1 proteins have small subunits composed of three polypeptides.This research was supported by NSF Grant 75-07368 and Contract No. EY-76-S-03-0034, P. A. #8, from the Department of Energy.     

Improvement of in-rumen digestibility of alfalfa forage by genetic manipulation of lignin O-methyltransferases

Lignin inhibits forage digestibility by ruminant animals, and lignin levels and the proportion of dimethylated syringyl (S) lignin monomers increase with progressive maturity in stems of forage crops. We generated transgenic alfalfa (Medicago sativa L.) with reduced lignin content and altered lignin composition. Down-regulation of caffeic acid 3-O-methyltransferase (COMT) reduces lignin content, accompanied by near total loss of S lignin, whereas down-regulation of caffeoyl coenzyme A 3-O-methyltransferase (CCoAOMT) reduces lignin content without reduction in S lignin. These changes are not accompanied by altered ratios of cell wall polysaccharides. Analysis of rumen digestibility of alfalfa forage in fistulated steers revealed improved digestibility of forage from COMT down-regulated plants, but a greater improvement in digestibility following down-regulation of CCoAOMT. The results indicate that both lignin content and composition affect digestibility of alfalfa forage, and reveal a new strategy for forage quality improvement by genetic manipulation of CCoAOMT expression.     

Identification of a new cartilage-specific S100-like protein up-regulated during endo/perichondral mineralization in gilthead seabream

Calcium ions and calcium-binding proteins play a major role in many cellular processes, in particular skeletogenesis and bone formation. We report here the discovery of a novel S100 protein in fish and the analysis of its gene expression patterns. A 648-bp full-length cDNA encoding an 86-amino acid S100-like calcium-binding protein was identified through the subtractive hybridization of a gilthead seabream (Sparus aurata) cDNA library constructed to identify genes associated with in vitro mineralization. Deduced protein lacks an identifiable signal peptide and exhibits two EF-hand motifs characteristic of S100 proteins. Phylogenetic and bioinformatic analyses of S100 sequences suggested that gilthead seabream protein represents a novel and fish-specific member of the S100 protein family. Expression of S100-like gene was up-regulated during the in vitro mineralization of bone-derived cell lines and during seabream development, from larvae throughout adulthood, reflecting skeletogenesis. Restriction of S100-like gene expression to chondrocytes of cartilaginous tissues undergoing endo/perichondral mineralization in juvenile fish further confirmed the mineralogenic role of the protein in fish and emphasized the potential of S100-like as a marker of mineralizing cartilage in developing fish.