p21-activated protein kinase gamma-PAK is activated by ionizing radiation and other DNA-damaging agents. Similarities and differences to alpha-PAK.

The p21-activated protein kinase gamma-PAK is activated 2-5-fold in response to ionizing radiation (IR) in 3T3-L1 fibroblasts and U937 leukemia cells. gamma-PAK is activated in a dose- and time-dependent manner. Doses from 1 to 100 Gy result in significant stimulation of activity at 30 min, whereas maximal stimulation is observed at 120 min after irradiation. UV (80 J/m(2)) and the DNA-damaging drugs cytosine beta-D-arabinofuranoside (AraC) and cis-platinum(II)diammine dichloride (cisplatin) also induce gamma-PAK activation. The activation of gamma-PAK in response to IR or AraC is dependent on tyrosine kinase and phosphoinositide 3-kinase activity, as demonstrated by use of the inhibitors genistein and wortmannin; in contrast activation of gamma-PAK by cisplatin and UV is not affected significantly by these inhibitors, suggesting that gamma-PAK can be activated by more than one pathway in response to different types of DNA damage. In contrast to gamma-PAK, alpha-PAK and JNK are activated only by cisplatin and UV in 3T3-L1 cells, suggesting differential regulation of the protein kinases. This is the first time that members of the Ste20/PAK family of protein kinases have been shown to be involved in the cellular response to IR and other DNA-damaging agents.     

2D-NMR studies of the unnatural duplex alpha-d(TCTAAAC)-beta-d(AGATTTG).

The unnatural oligonucleotide alpha-d(TCTAAAC) was synthesized and was found more resistant towards endonucleases than its beta-analog. 2D-NMR experiments allowed the assignment of all non-exchangeable aromatic and sugar protons except for the overlapping 5' -5" resonances, as well as the exchangeable imino protons of the parallel hybrid duplex alpha-d (TCTAAAC)-beta-d(AGATTTG). NMR studies show that the strength of the association between the alpha-strand and the beta parallel strand is equivalent to that between their anti-parallel complementary beta-analogs beta-d(CAAATCT) and beta-d(AGATTTG). NOE data provide evidence that both duplexes form stable right-helical duplexes with an anti-conformation on the glycosyl linkages and a Watson-Crick pairing. NOESY and COSY spectra allowed us to determine that alpha and beta deoxyriboses adopt a 3' -exo conformation.     

Helix–coil transition in poly(γ-benzyl-L-glutamate) and poly-ε-carbobenzoxy-L-lysine

In this paper two points are considered: the methods of evaluating the helical content θ and the calculation of the parameters of the transition from experimental data and its interpretation. The parameter ΔH obtained is in good agreement with the calorimetric one and v is found to be independent of temperature and solvent and in agreement with the ordinarily accepted value for poly(γ-benzyl-L -glutamate). The different methods of estimating θ are discussed for both polypeptides.     

蚕豆叶片发育与衰老过程中超氧物歧化酶活性与丙二醛含量变化

蚕豆植株叶片随茎节自上而下表现出明显的发育与衰老顺序,可作为衰老特征的是叶绿素和蛋白质含量明显下降。蚕豆叶中SOD活性主要定位于12 000× g离心后所得的上清液和叶绿体组分。衰老叶片的SOD总活性和叶绿体组分的相对活性都有所下降,SOD同工酶谱也发生了改变。O_2~ 产生速率随叶龄增大而稍上升;而MDA含量在叶片外观表现枯黄衰老征兆前就急剧上升。可能因为衰老叶片过氧化氢酶活性大幅度下降与SOD之间的不平衡,致使O_2~ 代谢中间产物累积而引起膜的损伤.     

Examining the relationship between secondary structure and antibody recognition in immunopeptides from foot-and-mouth disease virus

Summary The conformation of a peptide that represents antigenic site A of foot-and-mouth disease virus strain C-S8c1 (residues 136–156 of VP1; YTASARGDLAHLTTTHARHLP) has been studied by circular dichroism and compared with three analogs that reproduce amino acid substitutions at position 146 (HisArg, Gln or Asp) which affect antibody recognition. Four other peptides, incorporating replacements at position 147 predicted to maintain (LeuIle, Nle and Ala) or disrupt (LeuGly) helical structure at this site, have also been studied. In aqueous solution or in 4 M urea, the spectra of all eight peptides were typical of aperiodic conformation and independent of concentration or pH. However, upon addition of solvents such as methanol or hexafluoroisopropanol, spectral patterns evidenced significant levels (ca. 50%) of helical structure. The single residue substitutions at positions 146 and 147 caused minor to significant variations in the calculated amount of -helix of the peptides. An attempt to relate these changes in helical content to the antigenic behaviour of the peptides towards five monoclonal antibodies elicited with virus and mapping at site A could not find any straightforward correspondence between the two sets of results. The parent peptide and its His¹⁴⁶Arg analog were also analyzed by circular dichroism in the presence of the Fab fragment of SD6, a monoclonal antibody mapping at site A and much less reactive with viruses carrying the referred mutation. Although a peptide-antibody interaction was evident from spectral changes, careful inspection of the difference spectra (peptide-Fab minus Fab) of both peptides failed to detect any significant distinction between them that could be attributed to their different immunoreactivity. While these findings do not necessarily conflict with previous reports that the interaction of antigenic site A with antibodies is mediated to some extent by the adoption of a helix structure, they suggest that, at least for C-serotype viruses, other structural features in addition to a helical conformation are critically involved in antigenic recognition.     

Use of olive mill wastewater compost for crop production

Vast amounts of olive mill wastewaters (OMW) are produced in Mediterranean countries, where their treatment and disposal are becoming a serious environmental problem. Increasing attention has been paid to discovering a use for OMW and a wide range of technological treatments are available nowadays for reducing their pollutant effects and for their transformation into valuable products, the most suitable procedures being found to involve recycling rather than the detoxication of these wastes. Direct application of OMW to soil has been considered as an inexpensive method of disposal and recovery of their mineral and organic components but, because of their organic acid and phenol contents, OMW are also a source of pollution. By using composting technologies, it is possible to transform either fresh OMW or sludge from pond-stored OMW mixed with appropriate plant waste waterials (carriers) into organic fertilizers (composts) with no phytotoxicity to improve soil fertility and plant production, the process involving the microbial degradation of the polluting load of the wastes. Results of field and pot experiments using OMW-composts to cultivate horticultural and other crops have shown that yields obtained with organic fertilization are similar, and sometimes higher, to those obtained with a balanced mineral fertilizer. A comparison between the macro and micronutrient contents of plants cultivated with organic or mineral fertilizers did not generally reveal important differences. However, the cases of iron and manganese are worth mentioning as their bio-availability may be linked to the soil humic complexes originated by the OMW organic fertilizers.     

Biosynthesis of lysosomal hydrolases: their synthesis in bound polysomes and the role of co- and post-translational processing in determining their subcellular distribution

By in vitro translation of mRNA’s isolated from free and membrane-bound polysomes, direct evidence was obtained for the synthesis of two lysosomal hydrolases, β-glucuronidase of the rat preputial gland and cathespin D of mouse spleen, on polysomes bound to rough endoplasmic reticulum (ER) membranes. When the mRNA’s for these two proteins were translated in the presence of microsomal membranes, the in vitro synthesized polypeptides were cotranslationally glycosylated and transferred into the microsomal lumen. Polypeptides synthesized in the absence of microsomal membranes were approximately 2,000 daltons larger than the respective unglycosylated microsomal polypeptides found after short times of labeling in cultured rat liver cells treated with tunicamycin. This strongly suggests that nascent chains of the lysosomal enzymes bear transient amino terminal signals which determine synthesis on bound polysomes and are removed during the cotranslational insertion of the polypeptides into the ER membranes. In the line of cultured rat liver cells used for this work, newly synthesized lysosomal hydrolases showed a dual destination; approximately 60 percent of the microsomal polypeptides detected after short times of labeling were subsequently processed proteolytically to lower molecular weight forms characteristic of the mature enzymes. The remainder was secreted from the cells without further proteolytic processing. As previously observed by other investigations in cultured fibroblasts (A. Gonzalez-Noriega, J.H. Grubbs, V. Talkad, and W.S. Sly, 1980, J Cell Biol. 85: 839-852; A. Hasilik and E.F. Neufeld, 1980, J. Biol. Chem., 255:4937-4945.) the lysosomotropic amine chloroquine prevented the proteolytic maturation of newly synthesized hydrolases and enhanced their section. In addition, unglycosylated hydrolases synthesized in cells treated with tunicamycin were exclusively exported from the cells without undergoing proteolytic processing. These results support the notions that modified sugar residues serve as sorting out signals which address the hydrolases to their lysosomal destination and that final proteolytic cleavage of hydrolase precursors take place within lysosome itself. Structural differences in the carbohydrate chains of intracellular and secreted precursors of cathespin D were detected from their differential sensitivity to digestion with endoglycosidases H and D. These observations suggest that the hydrolases exported into the medium follow the normal secretory route and that some of their oligosaccharides are subject to modifications known to affect many secretory glycoproteins during their passage through the Golgi apparatus.     

Depletion of polyubiquitin encoded by the UBI4 gene confers pleiotropic phenotype to Candida albicans cells

We have studied the roles of polyubiquitin in Candida albicans physiology. Heterologous expression of the C. albicans polyubiquitin (UBI4) gene in a ubi4 Saccharomyces cerevisiae strain suppressed the mutant phenotype (hypersensitivity to heat shock). A heterozygous strain UBI4/Deltaubi4::hisG, obtained following the ura-blaster procedure, was used to construct a conditional mutant using a pCaDis derivative plasmid. By serendipity we isolated the UBI4 conditional mutant as well as a UBI4 mutant containing a non-functional MET3 promoter. Depletion of polyubiquitin conferred pleiotropic effects to mutant cells: (i) a limited increased sensitivity to mild heat shock; (ii) increased formation of colony morphology variants; and (iii) induction of hyphal and pseudohypal development. These results indicate that polyubiquitin in C. albicans is involved in the negative control of switching, as well as in maintaining the yeast cell morphology, probably by silencing mechanisms triggering the hyphal and pseudohyphal development in the absence of environmental inducers.