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Biomedical subjects

H Fujisawa

Publications and source records attributed to H Fujisawa.

At least 361 records · Page 20Linked to original sources
PubMed1986

Depression of protective mechanism during the early phase of a viral infection in tumor-bearing mice and prevention by PSK.

Effector mechanisms responsible for resistance against ectromelia virus including antiviral activity of non-immune macrophages, antiviral antibody, delayed footpad reaction to viral antigen, and interferon induction after viral infection were depressed in BALB/c mice bearing syngeneic Meth A tumor. The degree of viral growth correlated well with the depression of delayed footpad reaction, antibody production, and interferon induction. Therefore, modification of macrophage functions by a tumor-bearing state and treatment with PSK may contribute to this modification of antiviral resistance, at an early phase of infection. Cytotoxic activity may not be the principal effector, since the cytotoxicity was induced in normal and tumor-bearing mice to almost the same extent yet an extensive viral growth occurred only in the latter.

Animals↗
PubMed1986

Subunit arrangement of the tail fiber of bacteriophage T3.

A tail fiber of phage T3 is a trimer of the product of gene 17 (gp17). Treatment of T3 phage particles with chymotrypsin resulted in cleavage of only the tail fiber protein, at a site near the distal end of the fiber, causing a decrease of about 10% in the size of gp17 in the treated virion. The N-terminal amino acid sequences of intact and cleaved tail fiber proteins were identical and corresponded to that deduced from the nucleotide sequence of gene 17 except for the absence of the initiation Met residue. These results indicate that cleavage of the tail fiber occurred near the C terminus and suggest that gp17 polypeptides are oriented parallel to each other in the tail fiber. Association of tail fibers with the tail involves the N-terminal region of gp17. Under mild conditions of SDS-polyacrylamide gel electrophoresis, intact tail fibers dissociated from virions but cleaved ones did not. The nucleotide sequences indicate that T3 and T7 gp17 contain many sites that are potentially sensitive to chymotrypsin. In fact, free tail fibers, purified from T3-infected cells, were cleaved to many smaller fragments by chymotrypsin. These results suggest that the attachment of the tail fibers to the tail may induce a change(s) in the configuration and/or arrangement of gp17 to mask the sensitive sites from cleavage by chymotrypsin.

Amino Acid Sequence↗
PubMed1986

Cloning and sequencing of the genetic right end of bacteriophage T3 DNA.

The genetic right end of phage T3 DNA, from the beginning of gene 17, was cloned and sequenced. Genes 17, 18, and 19 were identified by comparing the sequence with the genetic map and by comparing the calculated and observed molecular weights of gene products. N-terminal amino acid sequence of the gene 17 product (gp17) predicted from the nucleotide sequence was consistent with the data from the analysis of purified gp17. Gene 17.5 was identified as the lysis gene on the basis of the presence of a nonsense codon within an open reading frame in the sequence of DNA from an amber mutant of lysis gene. In addition, five potential genes have been identified. Sequences corresponding to a promoter for phage T7 RNA polymerase (Rosa and Andrews, 1981) and to a class-III promoter for phage T3 RNA polymerase (Sarker et al., 1985) were found. The genomic organization and the nucleotide and deduced amino acid sequences of T3 were compared with those of T7. The genomic organizations of T3 and T7 were identical in this region. The sequence comparisons of T3 and T7 DNA point out the highly conserved sequences in all genes but also heavily varied regions in some genes. From these comparisons, possible implications with regard to structural and functional domains within several genes are discussed.

Amino Acid Sequence↗
PubMed1986

Overproduction and purification of the products of bacteriophage T3 genes 18 and 19, two genes involved in DNA packaging.

The products of gene 18 (gp18) and gene 19 (gp19) of bacteriophage T3 are noncapsid proteins involved in DNA packaging. A restriction fragment containing gene 18 or 19 was cloned into the plasmid vector pNT45 under the control of the inducible leftward promoter (PL) of phage lambda. Induction of transcription of gene 18 or 19 by derepression of the PL promoter led to the synthesis of a high level of gp18 or gp19. By using complementation of T3 DNA packaging in vitro as an assay, gp18 and gp19 were purified to near homogeneity. The overall yields of gp18 and gp19 were 1.4 mg and 0.35 mg, respectively, from 1 g wet wt cells. Addition of gp18 to the in vitro DNA packaging system resulted in increased phage production with increasing amounts of gp18 until 10% of the DNA was packaged into infectious phage particles. In contrast, addition of gp19 to the packaging system initially caused an increase in phage production, but increasing amounts of gp19 inhibited DNA packaging.

Cloning, Molecular↗
PubMed1986

A defined in vitro system for packaging of bacteriophage T3 DNA.

Using purified components, we have constructed an in vitro system for packaging of mature phage T3 DNA. In addition to mature T3 DNA, the system contained T3 proheads and the products of gene 18 (gp18) and gene 19 (gp19). The reaction required Mg2+, ATP, and polyvinyl alcohol. Spermidine was stimulatory but not absolutely required for the packaging reaction. Polyvinyl alcohol could be replaced by polyethylene glycol. The packaging efficiency decreased with decreasing molecular weight of the polymer, and low molecular weight polyols such as sucrose, sorbitol, and glycerol were inactive. The packaging reaction exhibited a sigmoidal relationship with respect to the concentration of ATP with the concentration for half maximal activity about 15 microM. A nonhydrolyzable ATP analog, adenosine 5'-O-(3-thiotriphosphate), inhibited the packaging reaction.

Adenosine Triphosphate↗
PubMed1986

Plasticity and rigidity of differentiation of brain vesicles studied in quail-chick chimeras.

Transplantation of a piece of the alar plate of the prosencephalon or of the rhombencephalon of quail embryos into the roof of the mesencephalon of chick embryos was carried out at 7-10 somite stage. Results obtained were: the transplanted alar plate of the prosencephalon differentiated into tissue closely resembling the tectum when the transplants were integrated into the host mesencephalon; in all the cases, the alar plate of the rhombencephalon did not differentiate into tectum-like structure, but into rhombencephalic descendants. We conclude that the alar plate of the prosencephalon at 7-10 stage is not definitively determined and may retain an ability to differentiate into the optic tectum, whereas the prospective fate of the rhombencephalon has already been determined at 7-10 stage.

Animals↗
PubMed1986

Bacteriophage T3 gene 8 product oligomer structure.

The structure of the connector of bacteriophage T3 (built up by the product of gene 8) has been studied in two dimensions by combined use of translational and rotational image filtering procedures applied to tetragonal ordered aggregates of the former oligomers. This analysis, performed up to 1/1.6 nm-1 resolution, has revealed the existence of a 12-fold symmetry in the outermost region of the specimen (mainly between radii 5.2 and 6.7 nm), a 6-fold one in the inner region (between radii 1.7 and 3.2 nm), and a hole in its center. These features are very similar to the ones described for the connectors of other phages, such as T4, lambda, and phi 29, thus suggesting a common mechanism for the functions carried out by this viral region.

Fourier Analysis↗
PubMed1986

Analysis of expression of yeast enolase 1 gene containing a longer pyrimidine-rich region located between the TATA box and transcription start site.

Yeast ENO1 promoter was prepared by a chemical synthetic method. Two variant promoters containing a pyrimidine-rich region (CT block), located between the TATA box and the transcription start site, either 32 or 34 base pairs (bp) longer than the native ENO1 promoter were isolated during the chemical synthesis. Gene expression of variant promoters was compared with that of the native promoter by measuring the amount of mRNA and the activity of beta-galactosidase by constructing ENO1-lacZ gene fusions. No significant differences were observed between the native and variant promoters in transcription levels. The start site of transcription was mapped on CAAG, a consensus sequence of transcription start site of yeast glycolytic genes. The results suggest a longer CT block in ENO1 promoter may not affect the expression of the yeast ENO1 gene. In addition, the level of ENO1 gene expression was found to be higher in stationary phase cells than in log phase cells.

Base Sequence↗
PubMed1986

Immune protective mechanisms during pregnancy. I. Cell-mediated immunity against Listeria monocytogenes in pregnant mice.

Characteristics of protective mechanisms during pregnancy were investigated using neonatally thymectomized (NTx) and/or pregnant mice infected with sublethal doses of Listeria monocytogenes, of which the explosive growth at an early phase of 2 or 3 days after infection is prevented by non-immune macrophages, and complete elimination at a late phase from 4 to 10 days after infection is attributed to the augmented functions of macrophages in co-operation with lymphokine-producing sensitized T lymphocytes. Although in virgin control mice there was a gradual decline of bacteria from the day after infection, viable bacteria in pregnant mice showed an increase in number until Day 3. In such pregnant mice, carbon clearance was suppressed. Thus, the enhanced bacterial growth in pregnant mice within 3 days may be attributable to the suppressed functions of non-immune macrophages. Complete elimination of Listeria from Day 4 was observed in pregnant sham-operated mice as well as in non-pregnant and pregnant NTx mice. Twenty-four hour reaction of delayed-type in normal mice induced by sheep red blood cells (SRBC) in incomplete Freund's adjuvant (IFA) was not affected by pregnancy, while 48 hr reaction in mice immunized with SRBC in complete Freund's adjuvant (CFA) was suppressed by pregnancy. We have reported previously that macrophage migration inhibitory factor (MIF) was produced in the latter but not in the former, and that the tuberculin type of delayed hypersensitivity accompanied by MIF production scarcely participated in acquired resistance to Listeria. Effective elimination of Listeria in pregnant and/or NTx mice at a late phase may be attributable to the activity of cellular immunity comparable to 24 hr reaction. These results suggest that T cells showing a low degree of thymus dependency in the ontogenic development may be the major component required for acquired protective immunity against Listeria and may account for the protection in pregnant mice.

Animals↗
PubMed1985

Sequence of the T4 recombination gene, uvsX, and its comparison with that of the recA gene of Escherichia coli.

We have determined the nucleotide sequence of the uvsX gene of bacteriophage T4 which is involved in DNA recombination and damage repair, and whose product catalyzes in vitro reactions related to recombination process in analogous manners to E. coli recA gene product. The coding region consisted of 1170 nucleotides directing the synthesis of a polypeptide of 390 amino acids in length with a calculated molecular weight of 43,760. Amino acid composition, the sequence of seven NH2-terminal amino acids and molecular weight of the protein deduced from the nucleotide sequence were consistent with the data from the analysis of the purified uvsX protein. The nucleotide sequence and the deduced amino acid sequence were compared with those of the recA gene. Although a significant homology was not found in the nucleotide sequences, the amino acid sequences included 23% of identical and 15% of conservatively substituted residues.

Adenosine Triphosphatases↗
PubMed1985

Genetic analysis of subunit assembly of the tail fiber of bacteriophage T3.

Bacteriophage T3 virions have six tail fibers composed of the product of gene 17 (gp17). Each tail fiber is a trimer of gp17 polypeptide. To characterize the assembly process of the tail fiber, temperature-sensitive (ts) mutants of gene 17 (ts17) were analyzed by SDS-polyacrylamide gel electrophoresis and by extract complementation. Newly synthesized gp17 polypeptide chains matured to SDS-resistant native trimers with a half time of about 7.5 min at 30 degrees. Although all ts17 mutants had similar plating efficiencies at restrictive temperature (41.5 degrees or 42 degrees), they showed different phenotypes. tsNG75, whose mutation was located in the carboxyl-terminal region of gene 17, was defective in trimer assembly at 41.5 degrees. The ts tail fibers formed at 30 degrees lost the ability to attach to the tail upon treatment at 41.5 degrees. There was a change in temperature sensitivity of tsNG75 tail fibers upon attachment to the tail, suggesting that the tail fiber may change conformation after attachment to the tail. tsNG215 and tsNG169, whose mutation sites were located in the amino-terminal region of gene 17, were not defective in the trimer assembly and attachment to the tail at the restrictive temperature. tsNG215 tail fibers formed at 41.5 degrees appear to be aberrant because they were not active in extract complementation and their attachment to fiberless particles resulted in production of noninfectious phage. Tail fibers produced by cells infected with tsNG169 at the restrictive temperature were active in extract complementation. Phage particles were formed in tsNG169-infected cells at the restrictive temperature. These particles were infectious at the permissive temperature and the mutant was non-infectious only if infection was continued at the restrictive temperature. These phenotypic differences exhibited by different gene 17 mutants may indicate the regions within the gene 17 polypeptide that play a role(s) in the folding and assembly of gp17 and in the biological activity of the mature tail fiber.

Electrophoresis, Polyacrylamide Gel↗
PubMed1985

Purification and characterization of gene 17 product of bacteriophage T3.

Tail fiber proteins of bacteriophage T3 are encoded by gene 17. By using in vitro complementation for phage assembly as an assay, the product of gene 17 (gp17) was purified to near homogeneity from cells infected with a double mutant of T3 defective in DNA synthesis and head assembly. The purified gp17 consists of a single polypeptide having a molecular weight of 67,000. Electron microscopy of the purified gp17 showed a fiber structure similar to the tail fiber in a virion. The subunit structure of the purified, native gp17 was analyzed by using a crosslinking agent, dimethyl suberimidate. The results indicate that native gp17 is a trimer of gp17 monomer.

Macromolecular Substances↗
PubMed1985

Isolation and characterization of bacteriophage T3/T7 hybrids and their use in studies on molecular basis of DNA-packaging specificity.

In vitro DNA-packaging systems of bacteriophages T3 and T7 packaged homologous DNA more efficiently than heterologous DNA. Packaging of phage DNA proceeds by way of concatemeric intermediates (H. Fujisawa, J. Miyazaki, and T. Minagawa (1978), Virology 87, 394-400). The conversion of mature homologous and heterologous DNAs to concatemers was efficient in both the T3- and T7-packaging systems. In vitro complementation experiments indicate that the gene 19 product (gp19) specifies which DNA enters the capsid. To identify DNA regions recognized by the packaging systems, T3/T7 hybrids were constructed and physical maps of the hybrid DNAs were determined by restriction enzyme analysis. By comparing restriction maps and in vitro packaging of hybrid DNAs, it is concluded that the sequence responsible for specificity of DNA packaging is confined within 5% of the ends of the T3 and T7 genomes.

Base Sequence↗
PubMed1985

Self-regulation of calmodulin-dependent protein kinase II and glycogen synthase kinase by autophosphorylation.

Calmodulin-dependent protein kinase II from rat brain underwent autophosphorylation and the autophosphorylation caused a marked decrease in the enzyme activity. Calmodulin-dependent glycogen synthase kinase from rabbit skeletal muscle was also inactivated by incubation under autophosphorylating conditions. The inactivation of the protein kinases by the autophosphorylation may be an important self-regulatory mechanism in controlling the enzyme activities.

Adenosine Triphosphate↗