Comparison of protein fermentation characteristics in rumen fluid determined with the gas production technique and the nylon bag technique

In this study, a modified version of the gas production technique was used to determine protein fermentation characteristics in rumen fluid of 19 feedstuffs. Performing the incubations in a N-free environment, and with an excess of rapidly fermentable carbohydrates, made N the limiting factor to microbial growth, and so gas production profiles reflected the availability of N from the feed samples. Results showed that fermentation of protein in rumen fluid can be determined with this modified gas production technique, and that there were distinct differences in protein fermentation between the feed samples. Availability of protein for fermentation was highest in wheat, potato pieces and lupin, and lowest in Rumiraap, a formaldehyde treated rapeseed meal, palm kernel expeller and brewery grains. The protein degradation characteristics of the 19 feed ingredients were also determined with the in situ nylon bag technique. With the obtained results, the amount of rumen escape protein (REP) was calculated for each feedstuff. The results showed that the rate of degradation ranged from 0.010/h for Rumiraap to 0.151/h for wheat. The amount of REP ranged from 197 g/kg CP for lupin to 840 g/kg CP for Rumiraap. Comparing the gas production results with the results obtained with the nylon bag technique showed that there was a good relationship between the gas production after 12–25 h of incubation and the calculated amount of REP (r² = 0.83–0.85). The results show that the adapted gas production technique, being depleted of N and using an excess of rapidly fermentable carbohydrates, is suitable to recognize differences in N availability between feed samples and can be used as an alternative to the nylon bag technique and other in vitro techniques.     

Influence of drying treatment on three Sargassum species 2. Protein extractability, in vitro protein digestibility and amino acid profile of protein concentrates

The effects of different drying treatments (oven- andfreeze-drying) on protein extractability of three subtropical brownseaweeds, Sargassum hemiphyllum, S. henslowianum and S. patens, as well as in vitro protein digestibility and amino acid profileof their protein concentrates (PCs) were investigated. When comparedwith freeze-drying, oven-drying not only improved significantly (p <0.05, two-way ANOVA, Tukey-HSD) the protein extractability of thethree Sargassum species but also the protein quality (in vitroprotein digestibility) of their PCs. Therefore, oven-drying was found to bemore suitable for protein extraction of brown seaweeds. The relationshipsbetween the protein extractability and total phenolic content in seaweedsand between in vitro protein digestibility and total phenolic contentin PCs were also discussed.     

Relationship between utilization of dual translational initiation signals and protein processing in Streptomyces

Summary Two sets of the Shine-Dalgarno sequence and the initiation codon (ATG) for translation of a gene encoding the protein SSI (Streptomyces subtilisin inhibitor) were studied in vivo by site-directed mutagenesis. The result shows that each ATG can function as an initiator of translation in either Streptomyces lividans 66 or Escherichia coli. The choice of initiation codon seems dependent on the host strain and is closely related to the processing mechanism of pre-SSI protein. The upstream ATG is presumed to be utilized preferentially giving two cleavage sites in pre-SSI in S. albogriseolus S-3253, the original SSI producer strain.Abbreviations SD Shine-Dalgarno - SSI Streptomyces subtilisin inhibitor     

小麦贮藏蛋白与小麦品质性状的关系及研究进展

小麦贮藏蛋白尤其是谷蛋白与醇溶蛋白的组成及所占比例是影响小麦加工品质的主要因素。本文对近年来国内外小麦蛋白亚基和小麦品质性状的研究现状进行了综述,同时介绍了小麦品质性状的遗传规律及其与品质的关系。     

Epitope analysis of human monoclonal antibody specific for rice allergenic protein generated by in vitro immunization

We previously established an in vitro immunization protocol for generating antigen specific human monoclonal antibodies (mAbs). In vitro immunization was performed against the soluble protein of rice allergenic protein (RA), resulting in the generation of three B cell clones, AC7-1/F9, CB7-1/E2 and CB7-8/F5, all of which produce a RA-specific human monoclonal IgM antibody. We attempted to map the epitope regions recognized by thesem Abs to characterize their specificities. We performed two rounds of epitope mapping, rough mapping using 10-mer peptides covering the full-length RA with 5 amino acids overlapping, and fine mapping using 8-mer peptides covering the putative epitope regions from the rough mapping with 1amino acid overlapping. As a result of the fine mapping,we identified the epitope regions of these three mAbs as⁴⁵QVWQDCCRQ⁵⁴L, ⁵⁶AVDDGWCRCGA⁶⁷L and⁹¹FPGCRRG⁹⁸D on the RA molecule and found to be identical. Furthermore, we determined the putative core epitope regions, which are critical for mAb binding to each region, ⁴⁷WQDCC⁵²R and ⁶⁰GWC⁶³R. The information about the epitope region on the RA molecule,which might trigger the allergenic response, would be useful to establish a specific immunotherapy against rice allergy. This revised version was published online in July 2006 with corrections to the Cover Date.     

Relationships between peroxidases and in vitro bulbification in garlic (Allium sativum L.)

Summary The relationship between in vitro bulbification and peroxidase activities of garlic (Allium sativum L.) was studied. Two stages could be distinguished during in vitro bulb formation characterized by the peroxidase activity, isoenzymatic patterns especially of the soluble fractions, dry weight, and bulbification index (BI). The first stage, called the morphogenic stage, started after planting until 30d of culture with a maximum soluble peroxidase activity, BI=1–0.5 and low dry weight. At that time axillary buds preformed at the base of the leaves grew and the in vitro bulb was generated. The second stage (filling in and bulb maturation) started when the BI reached 0.5 at 30 d of the ontogenic cycle, as a result of the bulb assimilate accumulation phenomenon. During the morphogenic stage the soluble peroxidase activity was maximum and the zymograms showed higher intensity bands. The second stage presented anodic ionic peroxidases and substantial increase in staining of the anodic covalent peroxidase fraction. The putative role of the different isoforms of peroxidases in relation to the bulbification process is discussed.     

Survivin protein expression in bovine follicular oocytes and their in vitro developmental competence

This study examined the relationship between survivin expression and the stage of development of in vitro cultured bovine oocytes and embryos; and whether survivin expression is affected by the quality of cumulus–oocyte complexes (COCS) or the quality of pre-implantation embryos. A polyclonal antibody was prepared using recombinant bovine survivin protein. Expression of survivin mRNA and protein was analyzed by real-time quantitative RT-PCR and immunocytochemistry. In the first experiment, survivin mRNA expression was examined at developmental stages from germinal vesicle (GV) oocyte to blastocyst, it was significantly decreased after fertilization of matured oocytes (P < 0.05), then increased slightly to the 8-cell stage followed by rapid increases at the morula and blastocyst stages (P < 0.05). In the second experiment, the effect of oocyte quality on survivin protein, pro-apoptotic (bax, caspase-3) and anti-apoptotic (survivin, bax inhibitor) mRNA expression was examined. Survivin protein was more strongly expressed in good quality COCS than in poor quality COCS. The expression of the anti-apoptotic genes, survivin and bax inhibitor, was significantly higher (P < 0.05) and that of the pro-apoptotic genes, bax and caspase-3, was significantly lower (P < 0.05) in good compared to poor quality COCS. The developmental competence of good quality COCS (30.4% blastocysts) was significantly better than that of poor quality COCS. In the last experiment also, we confirmed that significantly higher expression of survivin and bax inhibitor genes and significantly lower expression of bax and caspase-3 genes was resulted in good quality blastocysts than in poor quality blastocysts (P < 0.05). It was concluded that the expression of survivin was related to the quality of COCS, their developmental competence and the quality of in vitro produced blastocysts. Consequently, survivin may be a good candidate marker for embryo quality.     

Functional mode of NtHSP17.6, a cytosolic small heat-shock protein fromNicotiana tabacum

Small heat-shock proteins (sHsps) are ubiquitous stress proteins with molecular chaperone activity. They share characteristic homology with the α-crystallin protein of the mammalian eye lens as well as being ATP-independent in their chaperone activity. We isolated a clone for a cytosolic class I sHsp,NtHSP17.6, fromNicotiana tabacum, and analyzed its functional mode for such activity. Following its transformation intoEscherichia coli and its over-expression, NtHSPI 7.6 was purified and examinedin vitro. This purified NtHSPI 7.6 exhibited typical chaperone activity in a light-scattering test. It was enable to protect a model substrate, firefly luciferase, from heat-induced aggregation. Non-denaturing PAGE showed that NtHSP17.6 formed a dodecamer in its native conformation, and was bound to its substrate under heat stress. A labeling test with bis-ANS indicated that this binding might be linked to newly exposed hydrophobic sites of the NtHSPI 7.6 complexes during heat shock. Based on these data, we suggest that NtHSP17.6 is a molecular chaperone that functions as a dodecamer in a heat-induced manner.     

Turnover of abnormal proteins in Bacillus megaterium and Saccharomyces cerevisiae: differences between in vivo and in vitro degradation

Degradation of abnormal proteins in Bacillus megaterium and Saccharomyces cerevisiae in vivo was compared with that in cell-free extracts. Protein degradation in vivo, when the cells were labelled with ¹⁴C-leucine during growth in the presence of ethionine, was affected by the concentration of the analogue used. Proteins synthesized in the presence of 0.2–1 mM ethionine were degraded most rapidly in both organisms. The proteolytic enzyme system of yeast degraded the analogue-containing proteins in vitro faster than the normal proteins. This holds also for proteins synthesized in the presence of 5 mM ethionine, whose degradation in vivo was impaired. The proteolytic system of B. megaterium, on the other hand, was unable in vitro to differentiate between normal and abnormal proteins. Denatured proteins underwent preferential degradation over normal and ethionine-containing proteins.Participant in the UNESCO Postgraduate Course On Modern Problems in Biology and Microbial Technology.     

硫对大蒜生理生化指标及营养品质的影响

采用盆栽试验探讨了硫对大蒜生理生化指标及营养品质的影响．结果表明,土壤硫含量8．41mg·kg^-1时,大蒜叶片硝酸还原酶、POD活性、光合色素含量、光合速率、气孔导度和蒸腾速率较高;可溶性蛋白质含量及CAT活性却是以土壤硫含量为7．92mg·kg^-1时最高;SOD活性以较低土壤硫含量6．45mg·kg^-1时较高．同时,土壤硫含量为8．41mg·kg^-1时,鳞茎中大蒜素、可溶性糖和Vc及蒜薹中大蒜素、可溶性糖和可溶性蛋白质含量分别比对照增加33．67％、23．75％、68．82％、40．95％、3．45％和7．45％;较高土壤硫含量9．48mg·kg^-1有利于蒜薹中Vc和游离氨基酸及鳞茎中游离氨基酸和可溶性蛋白质含量的提高,分别比对照高出15．17％、20．93％、6．49％和8．07％．     

Effects of protein kinase inhibitors on in vitro protein phosphorylation and cellular differentiation of Streptomyces griseus

Summary In vitro phosphorylation reactions using extracts of Streptomyces griseus cells and -[³²P]ATP revealed the presence of multiple phosphorylated proteins. Most of the phosphorylations were distinctly inhibited by staurosporine and K-252a which are known to be eukaryotic protein kinase inhibitors. The in vitro experiments also showed that phosphorylation was greatly enhanced by manganese and inhibition of phosphorylation by staurosporine and K-252a was partially circumvented by 10 mM manganese. A calcium-activated protein kinase(s) was little affected by these inhibitors. Herbimycin and radicicol, known to be tyrosine kinase inhibitors, completely inhibited the phosphorylation of one protein. Consistent with their in vitro effects the protein kinase inhibitors inhibited aerial mycelium formation and pigment production by S. griseus. All these data suggest that S. griseus possesses several protein kinases of eukaryotic type which are essential for morphogenesis and secondary metabolism. In vitro phosphorylation of some proteins in a staurosporine-producing Streptomyces sp. was also inhibited by staurosporine, K-252a and herbimycin, which suggests the presence of a mechanism for self-protection in this microorganism.     

Relationship between multiple sequence alignments and quality of protein comparative models

Comparative modeling is the method of choice, whenever applicable, for protein structure prediction, not only because of its higher accuracy compared to alternative methods, but also because it is possible to estimate a priori the quality of the models that it can produce, thereby allowing the usefulness of a model for a given application to be assessed beforehand. By and large, the quality of a comparative model depends on two factors: the extent of structural divergence between the target and the template and the quality of the sequence alignment between the two protein sequences. The latter is usually derived from a multiple sequence alignment (MSA) of as many proteins of the family as possible, and its accuracy depends on the number and similarity distribution of the sequences of the protein family. Here we describe a method to evaluate the expected difficulty, and by extension accuracy, of a comparative model on the basis of the MSA used to build it. The parameter that we derive is used to compare the results obtained in the last two editions of the Critical Assessment of Methods for Structure Prediction (CASP) experiment as a function of the difficulty of the modeling exercise. Our analysis demonstrates that the improvement in the scope and quality of comparative models between the two experiments is largely due to the increased number of available protein sequences and to the consequent increased chance that a large and appropriately spaced set of protein sequences homologous to the proteins of interest is available.     

Relation between nuclear envelope and nuclear lamina in nuclear assembly in vitro

Xenopus laevis egg extracts cell-free nuclear assembly system was used as an experimental model to study the process of nuclear lamina assembly in nuclear reconstitutionin vitro. The experimental results showed that lamin was involved in the nuclear assemblyin vitro. The assembly of nuclear lamina was preceded by the assembly of nuclear matrix, and probably, inner nuclear matrix assembly provided the basis for nuclear lamina assembly. Inhibition of normal assembly of nuclear Iknina, by preincubating egg extracts cell-free system with anti-lamin antibodies, resulted in abnormal assembly of nuclear envelope, suggesting that nuclear envelope assembly is closely associated with nuclear lamina assembly. Project supported by the National Natural Science Foundation of China.     

Application of luminescence for quality evaluation of in vitro regenerated strawberry plants

The parameters of chlorophyll a fluorescence induction were measured: F_v/F_m, S_c/F_m, Rf_d and coefficient of L_d delayed luminescence decay kinetics, related with a course of primary photosynthesis reactions on leaves of strawberry plants, cultured in vitro by means of the micropropagation methods. Strawberry plants cv. Ananasowa from in vitro cultures in optimal condition show significantly higher values of luminescence parameters indicating better condition of plants of this variety in comparison with the variety Senga Sengana. After temperature lowering, however, these values were more reduced than for plants of Senga Sengana, which can be interpreted as higher susceptibility of this variety to chill. Addition of BAP caused disturbance of primary photosynthesis reactions rate, particularly in lower temperature. Auxin 2,4-D had no effect on the luminescence parameters in comparison with control cultures. Dehydration stress strongly diminished the values of measured parameters for Ananasowa variety what indicates the inhibition of primary photosynthesis reaction in leaves. The old culture of Senga Sengana variety showed higher tolerance on linuron in comparison with the new one.     

Influence of arginine,ornithine, DFMO and polyamines on division of the generative nucleus in cultured pollen tubes of <Emphasis Type="Italic">Aechmea fasciata</Emphasis> (Bromeliaceae)

During in vitro pollen tube growth of Aechmea fasciata the second pollen mitosis (PM II) that produces two sperm cells was influenced by exogenous amino acids. Arginine (Arg) as single amino acid was the limiting factor for the second mitosis of the generative nucleus and thus the formation of sperm cells in cultured pollen tubes of A. fasciata. The involvement of Arg was probably related to protein synthesis. The need for Arg was not related to polyamine (PA) biosynthesis, since PA added to the germination medium were unfavourable for sperm cell production. Both ornithine (Orn) and difluoromethylornithine (DFMO) inhibited the second mitosis in cultured pollen tubes of A. fasciata. The addition of Arg during the first 2 h of pollen germination was necessary to establish the division of the generative nucleus 6 h later.     

Relations between auxin and cytokinin contents and in vitro rooting of tree Peony (Paeonia suffruticosa Andr.)

In this work, a combined HPLC-ELISA technique was used to associate in vitro rooting capacity of tree peony micro-cuttings with contents of cytokinin and auxin; the cytokinin mainly detected corresponded to the N⁶-benzyladenine which had been added to the multiplication medium. Rooting capacity of explants was favoured by a preliminary accumulation of endogenous IAA only when levels of the BA absorbed from the multiplication medium had decreased. Main shoots coming from a 5-weeks subculture fulfilled these hormonal conditions and were the best microcuttings for rooting (87% rooting). Main shoots coming from shorter cycles or axillary shoots coming from a 5-weeks cycle always contained high benzyladenine levels and had a low rooting capacity (25–55% rooting). Root induction was associated with an early peak of indole-3-acetic acid followed by a 10-fold lower peak of endogenous ribofuranosyl-isopentenyladenine. Only a low and transitory accumulation of isopentenyladenine occurred during root development, and this could explain the lack of shoot development. Root development was efficient, especially in a medium containing activated charcoal, which led to an almost 3-fold decrease of IAA contents in roots.Abbreviations AC activated charcoal - BA N⁶-benzyladenine - ELISA enzyme linked immunosorbent assay - HPLC high performance liquid chromatography - IAA indole-3-acetic acid - IBA indole-3-butyric acid - iP N⁶-(²-isopentenyl)adenine - RDM root development medium - RIM root induction medium - 9RIP 9--d-ribofuranosyl-iP - 9RZ 9--d ribofuranosyl-zeatin - Z zeatin     

The relative rate of synthesis and levels of single-stranded DNA binding protein during induction of SOS repair in Escherichia coli

Summary Induction of the SOS response in Escherichia coli results in an increase in the relative rate of synthesis of single-stranded DNA binding protein (SSB). In contrast to RecA protein, this increase is slow and does not lead to higher SSB levels. The significance of ssb induction to SOS repair is discussed.