Electron microscopy study of viral DNA packaging with lambda head mutants

In a previous study, various intermediates in λ DNA packaging were visualized after lysis of λ-infected cells with osmotic shock and sedimentation through a sucrose formalin cushion onto electron microscope grids. Along this line, a systematic screening for intermediates accumulated in all head mutants available was performed. λA^?-infected cells accumulate only empty spherical protein shells (petit λ) bound at an intermediate point along the DNA thread. In situ digestion experiments with restriction endonuclease EcoRI show that the petit λ-DNA complexes are formed at a fixed point on the DNA concatemer. In $\text{λNu1}{}^{\mathrm{?}}\text{-}\text{infected}$ cells, however, most petit λ was not bound to DNA. In Fec^? cells, which are defective in formation of concatemers but normal in head protein synthesis, most petit λ did not sediment onto the carbon film of the grid. In $\text{D}{}^{\mathrm{?}}$ mutant, petit λ, partially full heads and empty heads with released DNA were observed. λFI^?-infected cells also accumulate petit λ and partially full heads. The present studies suggest that protein pNu1 is required for complex formation between head precursors and DNA concatemers, $\text{p}\text{A}$ for the initiation of DNA packaging, $\text{p}\text{D}$ and $\text{p}\text{F}$I for the promotion of DNA packaging, and $\text{p}\text{D}$ for stabilization of head structures. The results obtained with other head mutants involved in formation of mature proheads and head completion confirm earlier results obtained by different techniques.     

Purification and properties of a lambda operator-binding protein which is expected to be autorepressor (tof protein) from E. coli carrying lambdadv plasmid.

Summary In order to study the mode of action of the tof gene product, which is an autorepressor of the bacteriophage and plasmid dv, we have purified a DNA-binding protein which is specifically produced in bacteria carrying dv. This protein possesses characteristics expected for the product of the tof gene, since it is produced under conditions where cI-repressor is not made, and since it binds to oL and oR operators on the phage genome. The molecular weight of the native protein is 16,000–17,000 daltons, and the monomeric molecular weight as measured by gel electrophoresis in the presence of sodium dodecyl sulfate is about 10,000 daltons. Denaturation and renaturation experiments demonstrated that the native protein is a dimer of 10,000-dalton monomers. The DNA-specific binding protein is not produced in cells carrying i ²¹dv or 80dv.     

On the role of recA gene product in genetic recombination: an analysis by in vitro packaging of recombinant DNA molecules formed in the absence of protein synthesis.

Summary The role of the recA gene product of Escherichia coli in genetic recombination was examined in a system where recombination takes place in the absence of protein synthesis. recA200 bacteria were infected with two mutant strains of phage lambda in the presence of chloramphenicol and rifampin, and the resulting recombinant DNA molecules were measured by in vitro packaging. When recA200 bacteria grown at a temperature that is permissive for RecA phenotype were transferred to a temperature that is restrictive for RecA phenotype in the presence of the inhibitors, recombination of the infecting phages was severely blocked. This result shows that the recombination activity of the recA200 cells is inactivated by the change of temperature even in the absence of protein synthesis. The most likely explanation of this result is that the recA protein is directly involved in the recombination detected in the presence of chloramphenicol and rifampin.     

Mass fragmentographic determination of lofepramine and its metabolites in human plasma and urine using deuterated internal standards

A sensitive and specific method for the determination of lofepramine and its metabolites, desipramine and 2-hydroxydesipramine, in human plasma and urine is described. Lofepramine, desipramine and 2-hydroxydesipramine were derivatized to ethyl p-chlorobenzoate, the bis(heptafluorobutyryl) derivative and the N,O-bis(trifluoroacetyl) derivative, respectively, and then analysed by gas chromatography—mass fragmentography. Corresponding deuterated compounds were used as internal standards. Determination was possible at levels as low as 2 ng/ml for lofepramine and desipramine and 20 ng/ml for 2-hydroxydesipramine.     

Different responses of CR(−) and CR(+) B cells to LPS stimulation

Proliferative response of B cells with or without CR [CR(+) or CR(?) B cells] was compared in their polyclonal response when they were stimulated with lipopolysaccharide (LPS). CR(+) B cells responded better in proliferation and more poorly in polyclonal antibody formation than did CR(?) B cells. The dissociation between proliferation and antibody formation in LPS response was not due to the shift of the time kinetics nor the exhaustion of the culture medium. T cells and macrophages did not take part in the dissociation, since macrophage depletion from nu/nu mouse spleen cells could not modify the dissociation. The polyclonal antibody response of CR(?) B cells was more resistant to irradiation than that of CR(+) B cells. These results suggest that among LPS-responsive B cells there are CR(?) B-cell subset(s) more mature than CR(+) B cells.     

Ligand-exchange chromatographic resolution ofDl-amino acids in the presence of nucleic acids

Summary Nine representativeDl-amino acids were resolved using ligand-exchange chromatography whose mobile phase containe Cu (II) and ribonucleic acids. It was also found that deoxyribonucleic acids could be substituted for RNAs. In the presence of 5-GMP, 4XMP, (a mixture of 5-GMP,-AMP,-CMP, and-UMP), and oligomer of RNA,L-amino acids, which seemed to give more stable complexes, were eluted more slowly than the opposite isomers.     

Identification of Elastase as a Secretory Protease from Cultured Rat Microglia

In the course of studying the secretory products of microglia, we detected protease activity in the conditioned medium. Various proteins (casein, histone, myelin basic protein, and extracellular matrix) were digested. The protease activity was characterized by using purified myelin basic protein as a substrate. Maximal activity was observed at neutral pH levels (7-8), which was different from the optimum pH level of proteolytic activity observed in the cell homogenate. The activity was inhibited approximately 60 and 50% by 1 mM phenylmethylsulfonyl fluoride and 40 microM elastatinal, respectively. In gel filtration, the major activity, which was inhibited in the presence of N-methoxysuccinyl-Ala-Ala-Pro-Val-methyl chloride, eluted at a position corresponding to a molecular mass of approximately 25 kDa. These results suggest that the major protease present in microglial conditioned medium is elastase or an elastase-like protease. This suggestion was confirmed by the finding that the 25-kDa protein band was stained with anti-elastase antiserum by western blotting. De novo synthesis of elastase in microglia was supported by [35S]methionine incorporation. In the presence of lipopolysaccharide, the secretory elastase decreased. These results demonstrate that microglia secrete proteases, one of which was identified as elastase. The significance of this enzyme production in physiological and pathological conditions is discussed.     

Development of the Formula-Detoxification System during Adaptation to Air of Submerged Rice Seedlings

Changes in activities of enzymes and levels of antioxidant substratesinvolved in the -detoxification system in seedlings of rice (Oryza sativa L. cv. Yamabiko) inresponse to variations in the oxygen environment were studied.Activities of superoxide dismutase, ascorbate peroxidase, monodehydroascorbatereductase, dehydroascorbate reductase, glutathione reductaseand catalase, expressed either on the basis of fresh weightof shoots or relative to levels of soluble protein were muchlower in seedlings germinated under water for 6 days than inthose germinated in air for the same period of time. When submergedseedlings were exposed to air, the activities of these six enzymesincreased to or exceeded the levels in aerobically grown controlsduring 24 h of adaptation to air. Ascorbate and glutathione,which act as antioxidant substrates in the -detoxification system, were present in submergedseedlings at nearly the same levels as those found in aerobicallygrown controls. On exposure of submerged seedlings to air, thelevel of ascorbate in creased slightly, but the level of glutathioneshowed a rapid increase, reaching 7 times that in aerobicallygrown controls within 12 h of adaptation to air. Levels of allsix antioxidative enzymes and of two substrates involved inthe detoxification of the superoxide radical increased withincreases in oxygen tension in the environment. Moreover, thedevelopment of this system consisted of two steps, namely, arapid increase in the level of glutathione and a subsequentslow increase in the activities of antioxidative enzymes. ¹ Present address: Research Institute for Bioresources, OkayamaUniversity, Kurashiki, 710 Japan.     

1H NMR studies on ferricytochromec 3 fromDesulfovibrio vulgaris Miyazaki F and its interaction with ferredoxin I

Summary The¹H NMR signals of the heme methyl, propionate and related chemical groups of cytochromec ₃ fromDesulfovibrio vulgaris Miyazaki F (D.v. MF) were site-specifically assigned by means of ID NOE, 2D DQFCOSY and 2D TOCSY spectra. They were consistent with the site-specific assignments of the hemes with the highest and second-lowest redox potentials reported by Fan et al. (Biochemistry,29 (1990) 2257–2263). The site-specific heme assignments were also supported by NOE between the methyl groups of these hemes and the side chain of Val¹⁸. All the results contradicted the heme assignments forD.v. MF cytochromec ₃ made on the basis of electron spin resonance (Gayda et al. (1987)FEBS Lett.,217 57–61). Based on these assignments, the interaction of cytochromec ₃ withD.v. MF ferredoxin I was investigated by NMR. The major interaction site of cytochromec ₃ was identified as the heme with the highest redox potential, which is surrounded by the highest density of positive charges. The stoichiometry and association constant were two cytochromec ₃ molecules per monomer of ferredoxin I and 10⁸ M^–2 (at 53 mM ionic strength and 25°C), respectively.