Expression of an enhancer-binding protein in insect cells transfected with the Autographa californica nuclear polyhedrosis virus IE1 gene.

The baculovirus Autographa californica nuclear polyhedrosis virus contains an element known as homologous region 5 (hr5) which is an enhancer of delayed-early viral gene expression. To begin to identify proteins that interact with hr5, DNA-protein interactions were analyzed by using extracts from Spodoptera frugiperda cells and a fragment of DNA containing the left half of the hr5 enhancer. This 252-bp DNA fragment contains two copies of a 30-bp direct repeat (DR30) and two copies of a 24-bp imperfect palindrome contained within a 60-bp direct repeat (DR60). Extracts prepared from normal S. frugiperda cells and cells transfected with pUC8 lacked enhancer-binding proteins. However, when gel shift assays were performed with extracts from cells transfected with a plasmid containing the viral trans-activator IE1 gene, two DNA-protein complexes were formed. Both DNA-protein complexes were specifically inhibited by competition with a 60-bp oligonucleotide corresponding to DR60 but not by competition with a different oligonucleotide corresponding to DR30. Formation of the two complexes did not appear to involve cooperative interactions between binding proteins. When DR60 was used as a probe, a single complex was formed. To measure the enhancer activity of DR60, a reporter plasmid was constructed that contained DR60 cloned upstream of the reporter chloramphenicol acetyltransferase gene under the control of the delayed-early 39K promoter. Transient expression analysis indicated that the oligonucleotide increased expression of this gene 300-fold over the level obtained in the absence of any enhancer sequences.     

Changes in soil microflora associated with control of Sclerotium Rolfsii by Furfuraldehyde

The efficacy of 2‐furfuraldehyde for control of Sclerotium rolfsii was studied in laboratory and greenhouse experiments. Mycelial growth of the fungus was reduced proportionally with concentrations of 0.1–0.5 ml furfuraldehyde l^‐1 agar medium, and viability of sclerotia diminished on exposure to 2‐furfuraldehyde vapours. Detectable populations of bacteria and fungi, including Trichoderma spp., were reduced significantly (9=0.05) when furfuraldehyde was added to the agar used for soil dilution plates of untreated soil. Repeated treatments of natural soil with the fumigant significantly increased populations of Trichoderma spp. and bacteria, but diminished numbers of actinomycetes. Increasing dosages applied to soil artificially infested with S. rolfsii caused a reduction of disease on lentil, Lens culinaris. Results indicate that the compound, when applied to field soil, changes the composition of soil microflora and has potential for integrated control of S. rolfsii.     

Periodic cleavage of poly(dA) by oligothymidylates covalently linked to the 1,10-phenanthroline-copper complex

1,10-Phenanthroline (OP) was covalently attached to the 3'-terminus of two oligothymidylates via different linkers [abbreviated as T8-(OP) and T6-(OP)]. In the presence of Cu2+ and 3-mercaptopropionic acid (MPA), these reagents induce a hybridization-dependent cleavage of poly(dA) and of a 27 nucleotide long oligodeoxynucleotide containing an A8 sequence. The principal cleavage sites on the 27-mer span four residues located near the 3'-terminal phosphate group of T8-(OP). When poly(dA) was degraded by T6-(OP) and T8-(OP), a series of bands were obtained corresponding to a repeat unit of six and eight nucleotides, respectively. This periodicity reflects the cooperative binding of oligothymidylate-OP to the polynucleotide matrix and the localized nicking sites.     

Monoclonal antibodies specific for type 3 protein kinase C recognize distinct domains of protein kinase C and inhibit in vitro functional activity

Monoclonal antibodies (mAbs) which distinguish Type 3 protein kinase C (PKC) from Types 1 and 2 have been obtained from mice immunized with purified Type 3 PKC from rabbit brain cytosol. Most of these mAbs (seven out of eight) selectively recognize Type 3 versus Types 1 and 2 PKC in both enzyme-linked immunosorbent and immunoblot assays. Trypsin treatment of Type 3 PKC reduced the immunoreactivity with 82-kDa PKC and generated immunoreactive fragments of 45 and 35 kDa. The mAbs can be divided into two classes based on their ability to recognize the 45-kDa catalytic fragment (5/8) or the 35 kDa regulatory domain fragment (3/8). Each of the mAbs inhibits phosphorylation of histone or lipocortin by PKC, although the extent of the inhibition varied. Only those mAbs that recognize the 35-kDa regulatory domain inhibited phorbol ester binding. The inhibition of both kinase and binding activities by this group of mAbs was sensitive to the concentration of phospholipid used in the assay. This functional inhibition suggests that these mAbs may be useful for defining the phospholipid binding domain(s) of Type 3 PKC. The mAbs recognized 82-kDa PKC in a variety of cell types; the presence of smaller molecular weight fragments was not consistently found. Distinct immunofluorescence staining patterns were observed with mAbs directed toward different epitopes, suggesting that there may be heterogeneity in the subcellular localization of PKC. The type specificity of these mAbs will make them valuable tools for studying activation and regulation of Type 3 PKC in cell culture model systems.     

Repair of potentially lethal radiation damage in confluent and non-confluent cultures of human hybrid cells

The repair of potentially lethal damage (PLDR) in a gamma-irradiated human hybrid cell line (skin fibroblast X HeLa) and its tumourigenic segregant has been studied as a function of cell density at the time of irradiation and during the postirradiation repair period. The data show that PLDR occurs in both non-confluent and confluent cultures of both cell lines. Furthermore, there is evidence that the extent of PLDR is dependent on cell density and that cell-cell contact may be an important factor in this regard.     

An electrogenic proton pump associated with the Golgi apparatus of mouse liver driven by NADH and ATP

Golgi-apparatus membranes, isolated from mouse liver, pump protons inwards, when supplied with NADH or ATP. The acidification of Golgi-apparatus cisternae and vesicles was detected with neutral red, a permeant dye, as a difference in absorbance at 550 nm minus that at 600 nm. The maximum rates detected with NADH and ATP were between 0.0006-0.0009 and 0.0030-0.0050 delta OD units/mg of protein/min, respectively, at pH 7.5. The outside buffer used was a bovine serum albumin suspension. The acidification of Golgi apparatus was inhibited from 45 to 100% by ionophores and from 22 to 100% by uncouplers. The results implicate both ATP and a redox system coupled to NADH oxidation in the acidification of Golgi-apparatus membranes.     

cGMP、cAMP及两种cAMP衍生物对大鼠肝细胞核体外转录系统的影响

 本文介绍了以α-鹅膏蕈碱和低浓度KCl为手段建立了RNA聚合酶Ⅰ、RNA聚合酶Ⅱ活性的细胞核转录系统进而研究了cGMP、cAMP、cAMP丁酯及cAMP硫代环磷酰二乙胺对大鼠肝细胞核中RNA聚合酶Ⅰ与Ⅱ活性的影响。结果显示cGMP可以提高RNA聚合酶Ⅰ的活性;cAMP主要提高RNA聚合酶Ⅱ的活极,而cAMP分子结构变化产生的丁酯及硫代环磷酰二乙胺衍生物可增强cAMP的这种作用,为深入研究cAMP的构效关系提供了实验依据。     

Detergent Effects on the Phosphatidylinositol-Specific Phospholipase C in Rat Brain Synaptosomes

In the presence of Ca2+ (2.5 mM) and using [14C]arachidonoyl phosphatidylinositol (PI) membrane as substrate, phosphatidylinositol-specific phospholipase C (PI-PLC) (EC 3.1.4.10) in rat brain synaptosomes was activated by deoxycholate but not taurocholate. Calcium stimulated enzymic hydrolysis by both detergents, but the stimulatory effect of taurocholate was less than that of deoxycholate. Peak stimulation for deoxycholate was observed at 1 mg/ml, whereas that for taurocholate was 4 mg/ml. When 1 mM EDTA was added to the taurocholate (4 mg/ml) and Ca2+ (3.5 mM) system, synaptosomal PI-PLC activity was greatly stimulated, to almost the same level as the deoxycholate + Ca2+ system. This system required the presence of all three factors, and EGTA could not effectively replace EDTA in the stimulatory action. The detergent-induced hydrolysis of synaptosomal PI by the deoxycholate + Ca2+ and the taurocholate + Ca2+ + EDTA systems was strongly inhibited by divalent metal ions such as Zn2+, Cu2+, Pb2+, and Fe2+, whereas Mg2+ and Ca2+ were ineffective. Nevertheless, only the deoxycholate + Ca2+ system was responsive to enzyme inhibition by membrane-perturbing agents such as lysophospholipids and free fatty acids. The specific requirement for EDTA in the taurocholate system may be due to the release of a pool of inhibitory divalent metal ions from the membranes.