Location and density labelling of acid invertase in aging discs of Daucus carota storage tissue

Cell walls prepared from aged discs by extraction in 0·1 M acetate buffer, pH 4·8, possess ionically bound acid invertase which can be removed from the wall by incubation in 1 M sodium chloride in 0·1 M acetate buffer, pH 4·8, and more firmly attached enzyme which is not removed. Cell walls prepared in 0·195 M phosphate-0·003 M citrate-buffer, pH 8·0, do not possess ionically bound enzyme. Ionically bound invertase is density labelled when discs are aged in 90% deuterium oxide suggesting that at least part of the increase in activity observed during aging is due to de novo protein synthesis.     

基因靶位操作的技术与应用

ＴｈｅＡｐｐｒｏａｃｈａｎｄＡｐｐｌｉｃａｔｉｏｎｏｆＧｅｎｅＴａｒｇｅｔｉｎｇＷＡＮＧＹａ－ＰｉｎｇＺＨＵＺｕｏ－Ｙａｎ（ＩｎｓｔｉｔｕｔｅｏｆＨｙｄｒｏｂｉｏｌｏｇｙ,ＴｈｅＣｈｉｎｅｓｅＡｃａｄｅｍｙｏｆＳｃｉｅｎｃｅｓ,Ｗｕｈａｎ４３００７２...     

Analysis of wheat mitochondrially encoded polypeptides by in organello labelling

 The in organello labeling pattern in wheat (Triticum aestivum) mitochondria isolated from imbibed embryos were compared with those from the commonly used starting material, etiolated seedlings. Mitochondria from imbibed embryos proved to be metabolically more active than those from etiolated seedlings and produced a large number of strongly in organello-labeled polypeptides. Immunoprecipitation of the labeled proteins enabled the identification of mitochondrially encoded subunits of the respiratory chain complex I, some of which could not be detected by conventional Western blotting due to their high hydrophobicity. A method for mass isolation of wheat embryos is also presented which allows easy preparation of large amounts of intact and highly active mitochondria suitable for biochemical studies. Received: 9 November 1998 / Revision received: 10 March 1999 / Accepted: 1 April 1999     

Total synthesis of zervamicin IIB and its deuterium-labelled analogues

For the first time the total synthesis of the peptaibol antibiotic zervamicin IIB is described. Synthesis of this peptaibol was achieved by the Fmoc/tert-butyl strategy in solution using a fragment condensation approach. Three fragments of zervamicin IIB were obtained by stepwise elongation with Fmoc amino acids using BOP as a coupling reagent. For the introduction of the highly sterically hindered α-aminoisobutyric acid residues BOP/DMAP activation was applied. The Fmoc group was removed by reaction with 0.1 M NaOH in dioxane/methanol/water (30/9/1, v/v/v). Peptide fragments were coupled by means of a new coupling reagent, CF₃-PyBOP. Using the strategy developed, zervamicin IIB and two analogues specifically deuterium-labelled at different positions of the glutamine-11 residue have been synthesized in 40% overall yield based on the isotopically labelled amino acid and with 98±2% of isotope enrichment. FAB mass spectroscopy, 600 MHz ¹H-NMR spectroscopy and high-performance liquid chromatography provided convincing evidence that the synthetic products, zervamicin IIB and its deuterium-labelled analogues, fully correspond to the naturally occurring zervamicin IIB. © 1997 European Peptide Society and John Wiley & Sons, Ltd.     

Growth rate correlates negatively with protein turnover in Arabidopsis accessions

Previous studies with Arabidopsis accessions revealed that biomass correlates negatively to dusk starch content and total protein, and positively to the maximum activities of enzymes in photosynthesis. We hypothesized that large accessions have lower ribosome abundance and lower rates of protein synthesis, and that this is compensated by lower rates of protein degradation. This would increase growth efficiency and allow more investment in photosynthetic machinery. We analysed ribosome abundance and polysome loading in 19 accessions, modelled the rates of protein synthesis and compared them with the observed rate of growth. Large accessions contained less ribosomes than small accessions, due mainly to cytosolic ribosome abundance falling at night in large accessions. The modelled rates of protein synthesis resembled those required for growth in large accessions, but were up to 30% in excess in small accessions. We then employed ¹³CO₂ pulse‐chase labelling to measure the rates of protein synthesis and degradation in 13 accessions. Small accessions had a slightly higher rate of protein synthesis and much higher rates of protein degradation than large accessions. Protein turnover was negligible in large accessions but equivalent to up to 30% of synthesised protein day^?1 in small accessions. We discuss to what extent the decrease in growth in small accessions can be quantitatively explained by known costs of protein turnover and what factors may lead to the altered diurnal dynamics and increase of ribosome abundance in small accessions, and propose that there is a trade‐off between protein turnover and maximisation of growth rate.     

Confirmation of cadaveric blood sample identity by DNA profiling using Short Tandem Repeat (STR) analysis

Blood samples collected from deceased tissue donors for mandatory transfusion microbiology testing may be taken either at the time of tissue donation, or residual samples may be retrieved from hospital laboratories where they were originally used for ante-mortem tests. In the latter case, sample labelling may not conform to the required standard, which stipulates that three independent identifiers be provided. If no alternative adequately labelled sample is available for testing the donated tissues may have to be discarded, which can adversely affect tissue sufficiency. An alternative method to ensure that the blood sample to be tested is from the intended deceased donor is to confirm the identity of the blood sample by Deoxyribonucleic Nucleic Acid (DNA) Short Tandem Repeats (STR) analysis, then comparing the DNA profile with the DNA from the donated tissues. If the two DNA profiles are identical, probability calculations can demonstrate the chance of the two samples of DNA being from the same or different individuals. The authors have used this approach to salvage deceased tissue donations.     

Effect of tree age on needle morphology and anatomy of Pinus uliginosa and Pinus silvestris – species-specific character separation during ontogenesis

Differences in morphological and anatomical characters of needles between seedlings, saplings and adult trees of the endangered Pinus uliginosa from the Węgliniec Nature Reserve in SW Poland were examined biometrically and statistically assessed using the Student's t-test, Tukey–Kramer test, step-wise discrimination and agglomeration on Euclidean distances according to Ward's method. Pinus sylvestris adults and seedlings were used as comparative material. The results show that needles of all three P. uliginosa generations differ significantly from each other. In seedling needles, several anatomical characters were similar to those of P. sylvestris growing in the vicinity of the reserve. However, P. uliginosa had a lower number of resin canals, lower frequency of fibre-like sclerenchyma cells and higher frequency of thin-walled sclerenchyma cells with large lumens in the spaces between vascular bundles. Needle characters of saplings and adult trees of both species were distinctly more different than it was the case in the seedling stage.     

Stability studies of hydrazide and hydroxylamine-based glycoconjugates in aqueous solution

Glycoconjugates can be readily formed by the condensation of a free-reducing terminus and a strong α-effect nucleophile, such as a hydrazide or a hydroxylamine. Further characterization of a series of glycoconjugates formed from xylose, glucose and N-acetylglucosamine, and either p-toluenesulfonyl hydrazide or an N-methylhydroxylamine, was carried out to gain insight into the optimal conditions for the formation of these useful conjugates, and their stability. Their apparent association constants (9-74 M⁻¹) at pH 4.5; as well, as rate constants for hydrolysis, at pH 4.0, 5.0 and 6.0 (37 °C), were determined. The half-lives of the conjugates varied between 3 h and 300 days. All the compounds were increasingly stable as the pH approached neutrality. Conjugate hydrolysis rates mirrored those found for O-glycoside hydrolysis where conjugates formed from electron-rich monosaccharides hydrolyzed more rapidly.     

Production of yeastolates for uniform stable isotope labelling in eukaryotic cell culture

Preparation of stable isotope-labelled yeastolates opens up ways to establish more cost-effective stable isotope labelling of biomolecules in insect and mammalian cell lines and hence to employ higher eukaryotic cell lines for stable isotope labelling of complex recombinant proteins. Therefore, we evaluated several common yeast strains of the Saccharomycetoideae family as a source of high-quality, non-toxic yeastolates with the major aim to find a primary amino acid source for insect and mammalian cell culture that would allow cost-effective uniform stable isotope labelling (¹³C, ¹⁵N). Strains of the facultative methylotrophic yeasts Pichia pastoris and Hansenula polymorpha (Pichia angusta) as well as a strain of the baker’s yeast Saccharomyces cerevisiae were compared as a source of yeastolate with respect to processing, recovery and ability to sustain growth of insect and mammalian cell lines. The best growth-supporting yeastolates were prepared via autolysis from yeast obtained from fed-batch cultures that were terminated at the end of the logarithmic growth phase. Yeastolates obtained from H. polymorpha performed well as a component of insect cell cultures, while yeastolates from S. cerevisiae and H. polymorpha both yielded good results in mammalian cell cultures. Growth of yeasts in Heine’s medium without lactic acid allows relatively low concentrations of ¹³C and ¹⁵N sources, and this medium can be reused several times with supplementation of the ¹³C source only.