Endopeptidase Activity of Phage λ-Endolysin

JACOB and Fuerst^1,2 demonstrated the presence of a bacteriolytic enzyme (λ-endolysin) in the induced cultures of lysogenic Escherichia coli K12 (λ). The enzyme was later identified as the product of gene R; of phage λ³ which is involved in bacterial lysis at the end of a latent period. The enzyme is apt to form spheroplast-like structures in E. coli² and one would therefore expect its substrate to be murein.     

人粒细胞集落刺激因子（hG－CSF）cDNA3′－UTR对其表达的影响

利用人粒细胞集落刺激因子（ｈＧ－ＣＳＦ）ｃＤＮＡ３′端非翻译区（３′－ＵＴＲ）中存在的ＤｒａⅠ酶切位点,通过部分酶切与完全酶切,删除３′－ＵＴＲ不同长度,构建了四种ｈＧ－ＣＳＦｃＤＮＡ瞬时重组表达质粒。转染ＣＯＳ－７细胞后,生物活性测定结果提示,ｈＧ－ＣＳＦｃＤＮＡ３′－ＵＴＲ对其表达起负调控作用,其关键性序列位于紧接终止密码子ＴＧＡ下游的６５ｂｐ范围内,３′－ＵＴＲ对ｈＧ－ＣＳＦｃＤＮＡ表达的影响与转录水平的差别有一定关系。     

EFFECTS OF HELPER PROTEINS ENCODED BY p19 AND orf1-orf2 GENES ON Cyt1Aa PROTEIN EXPRESSION IN ACRYSTALLIFEROUS STRAIN OF BACILLUS THURINGIENSIS

A series of plasmids were constructed to examine the effects of p19 and orf1‐orf2 genes from Bacillus thuringiensis on Cyt1Aa synthesis and inclusion formation. The plasmids expressed the cyt1Aa gene along with either p19 or orf1‐orf2, or each of them coordinatively with p20 in the acrystalliferous strain of B. thuringiensis subsp. israelensis 4Q7. No effect on the expression of Cyt1Aa protein was found when P19 or Orf1‐Orf2 co‐expressed with Cyt1Aa. However, when including p20 gene, the constructs with p19 or orf1‐orf2 gene produced lower yield of Cyt1Aa proteins than without p19 or orf1‐orf2 gene. Electron microscopy observation and bioassay showed that P19 and Orf1‐Orf2 have no influence on the crystal size and toxicity of Cyt1Aa protein. It is presumed that P19 and Orf1‐Orf2 might have negative effects on Cyt1Aa synthesis in B. thuringiensis.     

稀土元素Pr~(3+)、Nd~(3+)对蚕豆(Vicia feba)叶片原生质膜上Ca~(2+)·Mg~(2+)-ATPase活性的影响

本文介绍用二相分配法制备蚕豆叶片原生质膜上的Ca~(2+)·Mg~(2+)-ATPase,用以研究镧系,稀土离子对此酶活性的影响。初步证实Pr~(3+)、Nd~(3+)对依赖于CaM的以及不依赖于CaM的蚕豆叶片原生质膜上Ca~(2+)·Mg~(2+)-ATPase活性的抑制不是CaM专一的。     

Serine utilization as a precursor of phosphatidylserine and alkenyl-(plasmenyl)-, alkyl-, and acylethanolamine phosphoglycerides in cultured glioma cells

In several tissues and cell lines, serine utilized for phosphatidylserine (PS) synthesis is an eventual precursor of the base moiety of ethanolamine phosphoglycerides (PE). We investigated the biosynthesis and decarboxylation of PS in cultured C6 glioma cells, with particular attention to 1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine (plasmenylethanolamine) biosynthesis. Incorporation of [3H]serine into PS reached a maximum within 4-8 h, and label in nonplasmenylethanolamine phosphoglyceride (NP-PE) and plasmenylethanolamine was maximal by 12-24 h and 48 h, respectively. After 8 h, label in PS decreased even though 40-60% of initial label remained in the culture medium. Serial additions of fresh [3H]serine restored PS synthesis to higher levels of labeled PS accumulation followed by a subsequent decrease in 4-8 h. High performance liquid chromatographic analyses confirmed that medium serine was depleted by 8 h, and thereafter metabolites, including acetate and formate, accounted for radioactivity in the medium. The rapid but transient appearance of labeled glycine and ATP inside the cells indicated conversion of serine by hydroxymethyltransferase. 78-85% of label from serine was in headgroup of PS or of PE formed by decarboxylation. A precursor-product relationship was suggested for label from [3H]serine appearing in the headgroup of diacyl, alkylacyl, and alkenylacyl subclasses of PE. By 48 h, a constant specific activity, ratio of approximately 1:1 was reached between plasmenylethanolamine and NP-PE, similar to the molar distribution of these lipids. In contrast, equilibrium was not achieved in cells incubated with [1,2-14C]ethanolamine; plasmenylethanolamine had 2-fold greater specific activity than labeled NP-PE by 72-96 h. These observations indicate that in cultured glioma cells 1) serine serves as a precursor of the head group of PS and of both plasmenyl and non-plasmenyl species of PE; 2) exchange of headgroup between NP-PE and plasmenylethanolamine may involve different donor pools of PE depending on whether the headgroup originates with exogenous serine or ethanolamine; 3) serine is rapidly converted to other metabolites, which limits exogenous serine as a direct phospholipid precursor.     

Identification of six phosphorylation sites in the COOH-terminal tail region of the rat neurofilament protein M.

The COOH-terminal tail domain of the neurofilament polypeptide M from rat nervous tissue contains approximately six molecules of phosphate. We report here that protein kinases in a crude cytoskeleton preparation of rat nervous tissue phosphorylated a set of tryptic peptides of M similar (but not identical) to those phosphorylated by living dorsal root ganglion cells in culture. Using these phosphopeptides as markers, we purified these same peptides from rat spinal cord and identified six specific phosphorylation sites in M by enzymatic and chemical criteria. These sites, serines 502, 506, 536, 606, 608, and 666, are all located in the COOH-terminal tail domain. Four are embedded in the repeated motif KSP whereas two are within variants of this motif, KSD and ESP. All of the sites that were preceded by lysine were resistant to alkaline phosphatase prior to modification of the lysine with citraconic anhydride. The identification of these sites should aid in investigations of the function of the phosphorylation of this protein and provides criteria for identifying the relevant kinases.     

Protection against acute paraquat toxicity by ambroxol

An expression vector for G-CSF, pASLB3-3, was constructed and introduced into Namalwa KJM-1 cells (Hosoi et al., 1988), and cells resistant to 100 nM of methotrexate (MTX) were obtained. Among them, the highest producer, clone SC57, was selected and the productivity of this clone was further characterized. The maximal production of G-CSF was at the most 1.8 g/ml/day using a 25 cm² tissue culture flask, even though the cell number was above 7×10⁵ cells/ml. The limiting factors at high density were analyzed as the deficiency of nutrients, such as glucose, cysteine and serine, and pH control. The depression of specific G-CSF productivity per cell under the batch culture conditions was overcome by using a perfusion culture system, Biofermenter^TM (Sato, 1983) with modifications of nutrients supplementation by a dialysis membrane and/or dissolved oxygen (DO) supplementation by microsilicone fibers. ITPSGF medium was modified to elevate concentrations of amino acids and glucose by 2.0- and 2.5-times, respectively. Under the control of pH at 7.4 and DO at 3 ppm, the specific G-CSF productivity was not depressed even at high cell density (above 1×10⁷ cells/ml), and the amount of G-CSF reached 41 g/ml. These results indicated the possibility of finding the optimum culture conditions for the production of recombinant proteins by Namalwa KJM-1 cells.Abbreviations ABTS 2,2-Azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid - BSA Bovine Serum Albumin - BSA-PBS Phosphate-buffered Saline without Ca²⁺ and Mg²⁺ containing Bovine Serum Albumin - dhfr Dihydrofolate Reductase - DO Dissolved Oxygen - G-CSF Granulocyte Colony-stimulating Factor - HEPES 4-(2-Hydroxyethyl)-1-piperazineethansulfonic Acid - IFN Interferon - MTX Methotrexate - PBS(-) Phosphate-buffered saline without Ca²⁺ and Mg²⁺ - Tween-PBS Phosphate-buffered saline without Ca²⁺ and Mg²⁺ containing 0.05% of Tween 20