Synthesis of L-Tyrosine or 3,4-Dihydroxyphenyl-L-alanine from DL-Serine and Phenol or Pyrocathechol

Reaction conditions for the synthesis of L-tyrosine or L-dopa from DL-serine and phenol or pyrocatechol were studied with intact cells of Erwinia herbicola (ATCC 21434) containing high tyrosine phenol lyase activity. The optimum pH for this reaction was around 8.0, and the optimum temperature range was between 37~40°C for the synthesis of L-tyrosine and between 15~25°C for that of L-dopa. Sodium sulfite and EDTA were added to protect the synthesized L-dopa from decomposition. As high concentrations of phenol or pyrocatechol denatured the enzyme, each substrate was fed to maintain the optimum concentration during incubation.

The reaction mixture (100 ml) containing 4.0 g of DL-serine, 1.0 g of phenol or 0.7 g of pyrocatechol, 0.5 g of ammonium acetate and the cells, was incubated. During incubation, phenol or pyrocatechol was fed at intervals to maintain the substrate at the initial concentration. 5.35 g of L-tyrosine or 5.10 g of L-dopa was synthesized in 100 ml of the reaction mixture.     

Survival of 3T3 cells expressing or co-expressing bFGF and/or IGF-I and/or IGF-II in low serum and serum free media

The aim of this study was to improve the survival ofNIH3T3 cells in low serum or serum-free media by theendogenous expression of recombinant bFGF (basicfibroblast growth factor) and/or IGF (insulin-likegrowth factor) -I and II. Expression was detected byWestern blotting, and the growth characteristics ofdifferent transfected cell lines investigated in bothserum-free and low serum conditions and also in softagar. Morphological changes and the growth rate werecompared with growth in normal serum-containingmedium. The experimental data suggested that theexpression of either bFGF alone, or the co-expressionof bFGF and either IGF-I or II could improve thesurvival of NIH3T3 cells in low serum or serum-freemedia. The use of such lines could decrease the use ofserum in cell culture and thus both reduce the costsinvolved in this technique and simplify thedown-stream purification procedure in protein harvest.Hence, such lines may be of value in both experimentaland industrial applications.     

Cd对不同种类植物生长和养分积累的影响

溶液培养法研究了不同活度Cd2 对4种植物卷心菜、黑麦草、玉米和白三叶草生长和养分积累的影响．结果表明,随Cd2 活度增加,4种植物的生长速率和干物质产量均下降．白三叶草对Cd2 最敏感;黑麦草和玉米耐性较强;卷心菜在高ACd2 时敏感．Cd2 对Fe、Mn、Cu、Zn、Ca、Mg、P、S等养分积累的影响因植物种类而异．Cd2 降低Fe、Mn、Cu、Zn、Ca、Mg等的积累．P在4种植物中和S在除卷心菜外的3种植物中的积累比对照增加．卷心菜和白三叶草对Cd毒敏感性差异与Fe、Mn、Ca、Mg的积累受Cd2 影响程度不同密切相关．     

Protection or sensitization by thiols or ascorbate in irradiated solutions of DNA or deoxyguanosine.

The initial aim of this study was to investigate how charge and other chemical properties of some radical scavengers influence the radiation-induced formation of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) in two model systems. The target molecule, deoxyguanosine (dG), was either organized in the DNA-helix form or present as a free nucleoside in an aerated aqueous phosphate buffer. Samples were irradiated with 137Cs gamma rays, alone or in the presence of different thiols, alcohols or ascorbate with net charges from -1 to +1. The formation of 8-oxo-dG was assayed with reverse-phase HPLC coupled to an electrochemical detector. In the absence of radical scavengers, the radiation-induced formation of 8-oxo-dG in DNA was extensive, and the ratio for formation of 8-oxo-dG was 20-fold higher for DNA compared to dG. The yields of 8-oxo-dG in DNA and dG were 7.7 x 10(-3) micromol J(-1) and 3.8 x 10(-4) micromol J(-1), respectively. Yield-dose plots showed that the efficiency of the positively charged thiol cysteamine to counteract the radiation-induced formation of 8-oxo-dG in DNA was significantly (P < 0.001) greater compared to the uncharged or negatively charged thiols. Uncharged thiols were significantly (0.001 < P < 0.05) more effective in protecting DNA compared to negatively charged thiols. In contrast to the protection against oxidative damage provided by thiols and ascorbate when they were present during irradiation of DNA, the formation of 8-oxo-dG was significantly increased when these compounds were present during irradiation of dG in solution. Compared to the irradiated control, the increase was 11- to 116-fold for thiols and ascorbate, respectively. The enhanced oxidative damage of dG observed in the presence of ascorbate or thiols suggests that secondarily formed radicals from thiols or ascorbate may react with dG, or that transformation of different primary sites of damage on dG to 8-oxo-dG is enhanced.     

Structural and functional roles of deamidation and/or truncation of N- or C-termini in human alpha A-crystallin

The purpose of the study was to compare the effects of deamidation alone, truncation alone, or both truncation and deamidation on structural and functional properties of human lens alphaA-crystallin. Specifically, the study investigated whether deamidation of one or two sites in alphaA-crystallin (i.e., alphaA-N101D, alphaA-N123D, alphaA-N101/123D) and/or truncation of the N-terminal domain (residues 1-63) or C-terminal extension (residues 140-173) affected the structural and functional properties relative to wild-type (WT) alphaA. Human WT-alphaA and human deamidated alphaA (alphaA-N101D, alphaA-N123D, alphaA-N101/123D) were used as templates to generate the following eight N-terminal domain (residues 1-63) deleted or C-terminal extension (residues 140-173) deleted alphaA mutants and deamidated plus N-terminal domain or C-terminal extension deleted mutants: (i) alphaA-NT (NT, N-terminal domain deleted), (ii) alphaA-N101D-NT, (iii) alphaA-N123D-NT, (iv) alphaA-N101/123D-NT, (v) alphaA-CT (CT, C-terminal extension deleted), (vi) alphaA-N101D-CT, (vii) alphaA-N123D-CT, and (viii) alphaA-N101/123D-CT. All of the proteins were purified and their structural and functional (chaperone activity) properties determined. The desired deletions in the alphaA-crystallin mutants were confirmed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometric analysis. Relative to WT-alphaA homomers, the mutant proteins exhibited major structural and functional changes. The maximum decrease in chaperone activity in homomers occurred on deamidation of N123 residue, but it was substantially restored after N- or C-terminal truncations in this mutant protein. Far-UV circular dichroism (CD) spectral analyses generally showed an increase in the beta-contents in alphaA mutants with deletions of N-terminal domain or C-terminal extension and also with deamidation plus above N- or C-terminal deletions. Intrinsic tryptophan (Trp) and total fluorescence spectral studies suggested altered microenvironments in the alphaA mutant proteins. Similarly, the ANS (8-anilino-1-naphthalenesulfate) binding showed generally increased fluorescence with blue shift on deletion of the N-terminal domain in the deamidated mutant proteins, but opposite effects were observed on deletion of the C-terminal extension. Molecular mass, polydispersity of homomers, and the rate of subunit exchange with WT-alphaB-crystallin increased on deletion of the C-terminal extension in the deamidated alphaA mutants, but on N-terminal domain deletion these values showed variable results based on the deamidation site. In summary, the data suggested that the deamidation alone showed greater effect on chaperone activity than the deletion of N-terminal domain or C-terminal extension of alphaA-crystallin. The N123 residue of alphaA-crystallin plays a crucial role in maintaining its chaperone function. However, both the N-terminal domain and C-terminal extension are also important for the chaperone activity of alphaA-crystallin because the activity was partially or fully recovered following either deletion in the alphaA-N123D mutant. The results of subunit exchange rates among alphaA mutants and WT-alphaB suggested that such exchange is an important determinant in maintenance of chaperone activity following deamidation and/or deletion of the N-terminal domain or C-terminal extension in alphaA-crystallin.     

Frequency of FMR1 premutation in individuals with ataxia and/or tremor and/or parkinsonism

A late onset neurological syndrome in carriers of premutation in FMR1 gene was recently described. The condition was named fragile-X-associated tremor/ataxia syndrome (FXTAS) and includes intentional tremor, cerebellar ataxia, parkinsonism, and cognitive deficit. We ascertained the contribution of FMR1 premutation to the phenotypes ataxia, tremor and/or parkinsonism. Sixty-six men over 45 years old presenting these symptoms, isolated or combined, were tested. Also, 74 normal men, randomly chosen in the population, formed the control group. In the patient group, no premutation carrier was found, which is in agreement with other observed frequencies reported elsewhere (0-5% variation). No significant differences were found when comparing gray zone allele frequencies among target and control groups. The FXTAS contribution in patients with phenotypic manifestations of FXTAS was 15/748 (2%). The presence of gray zone alleles is not correlated with FXTAS occurrence.     

Synthesis of l-Tyrosine or 3,4-Dihydroxyphenyl-l-alanine from Pyruvic Acid,Ammonia and Phenol or Pyrocatechol

The synthesis of l-tyrosine or 3,4-dihydroxyphenyl-l-alanine (l-dopa) from pyruvate, ammonia and phenol or pyrocatechol was studied with intact cells of Erwinia herbicola ATCC 21434 containing high tyrosine phenol lyase activity. By elemental analyses and determination of optical activity, the tyrosine or dopa synthesized was confirmed to be entirely of l-form. Maximum amount of l-tyrosine (60.5 g/liter) or l-dopa (58.5 g/liter) was formed using this enzymatic method by feeding sodium pyruvate and phenol or pyrocatechol. However, large amounts of by-products were formed in the l-dopa synthetic reaction mixture. By-products were proved to be formed from l-dopa and pyruvate by a nonenzymic reaction. pH and the temperature of reaction had intensive effects on the formation of by-products. A simple method using a boric acid-pyrocatechol complex was devised, as the feeding procedure of substrates was complicated.     

林下人参生理特性和生长与林内生态因子的关系

结合模拟试验,对林下人参的生理活动及生长过程与林内生态因子的关系进行 了观测研究．结果表明,林内光照、水分、温度等因子与林下人参生长的关系极为密切．一 般林内光照在中等条件下,即相对光照在１０～３５％左右．林分郁闭度约０．６～０．８,土壤含 水量在 ３５～４０％时人参生长最好;林下人参的物候进程及生长与温度的关系密切．林内 温度在５℃左右人参芽胞开始萌动,１５℃左右地上茎高生长进入速生期,６月上旬生长停 止．因此,人为调节各种生态因子在适宜的范围内有利于林下人参的生长．     

Safety and regulation of yeasts used for biocontrol or biopreservation in the food or feed chain

Yeasts have been important components of spontaneous fermentations in food and beverage processing for millennia. More recently, the potential of utilising antagonistic yeasts, e.g. Pichia anomala and Candida spp., for post-harvest biological control of spoilage fungi during storage of plant-derived produce (‘biopreservation’) has been clearly demonstrated. Although some yeast species are among the safest microorganisms known, several have been reported in opportunistic infections in humans, including P. anomala and bakers’ yeast, Saccharomyces cerevisiae. More research is needed about the dominant pathogenicity and virulence factors in opportunistic yeasts, and whether increased utilisation of biopreservative yeasts in general could contribute to an increased prevalence of yeast infections. The regulatory situation for yeasts used in post-harvest biocontrol is complex and the few products that have reached the market are mainly registered as biological pesticides. The qualified presumption of safety (QPS) approach to safety assessments of microorganisms intentionally added to food or feed, recently launched by the European Food Safety Authority, can lead to more efficient evaluations of new products containing microbial species with a sufficient body of knowledge or long-term experience on their safety. P. anomala is one of several yeast species that have been given QPS status, although the status is restricted to use of this yeast for enzyme and metabolite production purposes. With regard to authorisation of new biopreservative yeasts, we recommend that the possibility to regulate microorganisms for food biopreservation as food additives be considered.     

Live virus-free or die: coupling of antivirus immunity and programmed suicide or dormancy in prokaryotes

Background

mtRF1 is a vertebrate mitochondrial protein with an unknown function that arose from a duplication of the mitochondrial release factor mtRF1a. To elucidate the function of mtRF1, we determined the positions that are conserved among mtRF1 sequences but that are different in their mtRF1a paralogs. We subsequently modeled the 3D structure of mtRF1a and mtRF1 bound to the ribosome, highlighting the structural implications of these differences to derive a hypothesis for the function of mtRF1.

Results

Our model predicts, in agreement with the experimental data, that the 3D structure of mtRF1a allows it to recognize the stop codons UAA and UAG in the A-site of the ribosome. In contrast, we show that mtRF1 likely can only bind the ribosome when the A-site is devoid of mRNA. Furthermore, while mtRF1a will adopt its catalytic conformation, in which it functions as a peptidyl-tRNA hydrolase in the ribosome, only upon binding of a stop codon in the A-site, mtRF1 appears specifically adapted to assume this extended, peptidyl-tRNA hydrolyzing conformation in the absence of mRNA in the A-site.

Conclusions

We predict that mtRF1 specifically recognizes ribosomes with an empty A-site and is able to function as a peptidyl-tRNA hydrolase in those situations. Stalled ribosomes with empty A-sites that still contain a tRNA bound to a peptide chain can result from the translation of truncated, stop-codon less mRNAs. We hypothesize that mtRF1 recycles such stalled ribosomes, performing a function that is analogous to that of tmRNA in bacteria.

Reviewers

This article was reviewed by Dr. Eugene Koonin, Prof. Knud H. Nierhaus (nominated by Dr. Sarah Teichmann) and Dr. Shamil Sunyaev.     

Analysis of nonlinear physiological systems with single or multiple spike inputs and analogue or spike outputs

This paper outlines an extension of Krausz' (1975) approach to nonlinear analysis of physiological systems with random impulse train inputs. This extension concerns multiple (here two) inputs and spike train outputs in addition to analogue outputs. The respective theoretical formula are given, and the approach is illustrated by experimental data relating to the nonlinear interaction of cat motor units activated with random pulse trains. As outputs are considered muscle tension (as an example for an analogue signal) and discharge frequency of a Ia fibre (as an example for a spike output).     

棉蚜种群动态预报模型

对１９８０—１９８９年河南棉区棉蚜种群动态分析表明,棉蚜为害可分为苗蚜和伏蚜两个时期,根据环境因子（气温、相对湿度、降雨量）和不同危害期,建立了２个模糊预报模型,并取得了较满意的预测效果．