Cytoprotective effect of arginine deiminase on taxol-induced apoptosis in DU145 human prostate cancer cells

We purified and partially sequenced a cytostatic protein from the ASC-17D Sertoli cell-conditioned media (rSCCM) showing a molecular weight of 90 kDa with homodimeric composition. N-terminal amino acid analysis revealed that the protein was homologous to the arginine deiminase (ADI) of Mycoplasma arginini. We found ADI enzyme activity in rSCCM and the abolishment of the growth inhibitory effect by the supplement of L-arginine. Thus, we confirmed that the cytostatic activity in rSCCM was due to the depletion of extracellular L-arginine by ADI. Apparent increase of cell death or DNA fragmentation was not observed in DU145 cells cultured in the presence of ADI. Incubation of DU145 cancer cells with taxol resulted in a marked DNA fragmentation, whereas pretreatment with ADI or cycloheximide protected the cells from taxol-induced apoptosis. Preincubation of the cells with ADI inhibited S35-methionine incorporation into protein synthesis in a dose dependent manner. These data suggest that ADI-induced arginine depletion may inhibit protein synthesis, and result in the protection of apoptotic cell death that requires new protein synthesis.     

High Temperature Stress Resistance of Escherichia coli Induced by a Tobacco Class I Low Molecular Weight Heat-Shock Protein

TLHS1 is a class I low molecular weight heat-shock protein (LMW HSP) of tobacco (Nicotiana tabacum). For a functional study of TLHS1, a recombinant DNA coding for TLHS1 with a hexahistidine tag at the aminoterminus was constructed and expressed in Escherichia coli. An expressed fusion protein, H₆TLHS1, was purified using a Ni²⁺ affinity column and a Sephacryl S400 HR column. A polyclonal antibody against H₆TLHS1 was produced to follow the fate of H₆TLHS1 in E. coli. The fusion protein in E. coli maintained its solubility at a temperature of up to 90°C and most of the proteins in the E. coli cell lysate with H₆TLHS1 were prevented from thermally induced aggregation at up to 90°C. We compared the viability of E. coli cells expressing H₆TLHS1 to the E. coli cells without H₆TLHS1 at a temperature of 50°C. After 8 h of high temperature treatment, E. coli cells with H₆TLHS1 survived about three thousand times more than the bacterial cells without H₆TLHS1. These results showed that a plant class I LMW HSP, TLHS1, can protect proteins of E. coli from heat denaturation, which could lead to a higher survival rate of the bacterial cells at high temperature.     

Treatment of dark-grown Arabidopsis thaliana with a brassinosteroid-biosynthesis inhibitor, brassinazole, induces some characteristics of light-grown plants

When a brassinosteroid biosynthesis inhibitor, brassinazole (Brz), was applied at concentrations ranging from 0.1 to 2 μM, Arabidopsis thaliana (L.) Heynh seedlings grown in the dark exhibited morphological features of light-grown plants, i.e. short hypocotyls, expanded cotyledons, and true leaves, in a dose-dependent manner. Control (non Brz-treated) seedlings grown in the dark for 40 d did not develop leaf primordia. However, treatment with the lowest concentration of Brz induced the development of leaf buds, although it hardly induced any short hypocotyls, and treatment with the highest concentration of Brz induced both short hypocotyls and leaves. Labeling experiments with the thymidine analogue 5-bromo-2′-deoxyuridine revealed that amplification of cell nuclei and organellar nucleoids is activated in the shoot apical meristems of dark-grown Brz-treated seedlings. These results suggest that Brz-treatment induces development of true leaves. Furthermore, condensation and scattering of plastid nucleoids, which is known to occur during the differentiation of etioplasts into chloroplasts, was observed in the plastids of dark-grown Brz-treated cotyledons. In addition, high levels of ribulose-1,5-bisphosphate carboxylase-oxygenase proteins accumulated in the plastids of the cotyledons. Electron microscopy showed that the plastids were etioplasts with a prolamellar body and few thylakoid membranes. These results suggest that Brz treatment in the dark induces the initial steps of plastid differentiation, which occur prior to the development of thylakoid membranes. This is a novel presumed function of brassinosteroids. These cytological changes seen in Brz-treated Arabidopsis were exactly the same as those seen in a brassinosteroid-biosynthesis-deficient mutant, det2, supporting the hypothesis that Brz has no side-effects except inhibiting brassinosteroid biosynthesis, and should prove a useful tool in clarifying the role of brassinosteroids. Received: 10 February 2000 / Accepted: 11 April 2000     

Retinoic acid extends the in vitro life span of normal human oral keratinocytes by decreasing p16(INK4A) expression and maintaining telomerase activity

Retinoic acid (RA) plays an important role in the regulation of cell growth and differentiation. To investigate whether RA extends in vitro the life span of human epithelial cells, we examined the effect of all-trans RA on both the cumulative population-doubling level (PDL) and the replicative senescence of cultured oral keratinocytes. When proliferating oral keratinocytes were cultured in medium containing 1 nM of all-trans RA, the in vitro life span of the cells was increased 1.5- to 1.8-fold compared to the vehicle control and the replicative senescence of the cells was significantly inhibited. Since the replicative senescence of human epithelial cells is associated with a steady increase of p16(INK4A) and a loss of telomerase activity, we expected that RA could delay the replicative senescence of oral keratinocytes by decreasing p16(INK4A) expression and/or inhibiting the loss of telomerase activity. To test this possibility, we examined the expression of replicative senescence-associated genes and the telomerase activities of different PDL numbers of oral keratinocytes exposed to 1 nM of all-trans RA. The protein level of cellular p16(INK4A) in the RA-treated oral keratinocytes was gradually but significantly enhanced by an increased PDL number; however, the level was significantly lower than that of the vehicle control at all of the same PDL numbers. In contrast, the telomerase activity was maintained in oral keratinocytes with increasing PDL numbers induced by RA treatment. Summarizing, these results indicate that RA induces the in vitro life-span extension of oral keratinocytes, which is linked to a decreased cellular level of p16(INK4A) and the maintenance of telomerase activity.     

根瘤菌4012a菌株产细胞分裂素发酵条件的研究

用酶标免疫检测法研究了根瘤菌4012a菌株细胞分裂素发酵的适宜培养基和培养条件。结果表明,其最佳培养基为（g／L）：葡萄糖10.0,（NH4）2SO41.0,K2HPO4·3H2O0．6,MgSO4·7H2O0.1,CaCl2·2H2O0.4,FeCI3·6H2O0．04,Na2MoO4·2H2O0．1mg／L,泛酸钙100μg／L,腺漂吟200mg／L。该菌株在150r／min的旋转摇床上27℃振荡培养96h,发酵液中细胞分裂素产量可达908μg／L,生物活性（萝卜子叶扩大法）为1mg／L激动素当量。     

番茄抗病基因Cf9的3′UTR区含有内含子序列

从番茄（ＬｙｃｏｐｅｒｓｉｃｏｎｅｓｃｕｌｅｎｔｕｍＭｉｌ．）抗病品种“中杂９号”基因组ＤＮＡ中扩增并克隆了番茄抗叶霉病基因Ｃｆ９。序列分析结果表明,该基因全长２７５１ｂｐ,含有一个编码８６３个氨基酸的开放阅读框架。在该基因的３′ＵＴＲ区发现了一段未曾报道的内含子序列,长１１５ｂｐ,它所处的位置与番茄另一个抗叶霉病基因Ｃｆ２内含子的位置相似,其５′和３′边界序列为一重复序列,ＴＣＣＡＧＧ（Ｔ）ＡＴＴＣ,并与Ｃｆ２基因内含子边界序列高度同源。与已报道的Ｃｆ９的ｃＤＮＡ序列相比,它的第３７１位的核苷酸Ｔ突变成了Ｃ,从而使其编码蛋白的ＬＲＲ区第１２１位的亮氨酸突变为脯氨酸。     

不同盐浓度的混合盐对羊草苗的胁迫效应

用５０～３５０ｍｍｏｌ／Ｌ含有不同比例的ＮａＣｌ、Ｎａ２ＳＯ４、ＮａＨＣＯ３和Ｎａ２ＣＯ３等４种盐成分的３０种混合盐对羊草（Ａｎｅｕｒｏｌｅｐｉｄｉｕｍｃｈｉｎｅｎｓｅ（Ｔｒｉｎ．）Ｋｉｔａｇ．）苗进行盐碱混合胁迫处理。结果表明：相对生长率、Ｋ和Ｎａ含量、脯氨酸积累量及叶片电解质外渗率等胁变反应均随盐浓度增大以及碱性盐比例增高而加剧。而且碱性盐所造成的高ｐＨ的致胁变效应与盐浓度关系极大,浓度低时作用较小,随浓度增大其作用加剧。在含有碱性盐的混合盐胁迫中,包含有盐胁迫和碱胁迫两种作用。以总盐浓度代表盐胁强,缓冲量代表碱胁强。在碱胁强较弱时胁变主要受盐浓度影响,随碱胁强增大缓冲量变为影响胁变的主导因素     

An interplay between two EGF-receptor ligands, Vein and Spitz, is required for the formation of a subset of muscle precursors in Drosophila.

Activation of the Drosophila EGF-receptor (DER) is spatially and temporally controlled by the release of its various ligands. DER and its ligand Spitz mediate the formation of specific somatic muscle precursors. We show that a second DER ligand, Vein, complements the activity of Spitz in the development of various somatic muscle precursors. In vn mutant embryos, the DER-dependent muscle precursors do not form in some of the segments. This phenotype is significantly enhanced in embryos carrying only one copy of wild type spitz. Our analysis suggests that Vein activation of DER differs qualitatively from that of Spitz in that it does not lead to the expression of the inhibitory protein Argos, possibly leading to a continuous activation of the DER signaling pathway.     

Restricted β-galactosidase expression of a hygromycin-lacZ gene targeted to the β- actin locus and embryonic lethality of β-actin mutant mice

Transgenic Research -