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D P Fan

Publications and source records attributed to D P Fan.

At least 37 records · Page 2Linked to original sources
PubMed1983

Isolation and characterization of a CNBr cleavage peptide of influenza viral hemagglutinin stimulatory for mouse cytolytic T lymphocytes.

A polypeptide fragment obtained by CNBr cleavage of the hemagglutinin from A/JAPAN/305/57 influenza virus has been purified by using high performance liquid chromatography. The first five N-terminal amino acids as determined by sequential Edman degradations have localized this peptide to the HA2 subunit of the hemagglutinin between residues 103 and 123. This peptide, denoted HA2(103-23), can generate both proliferative and cytolytic responses from spleen cells of BALB/c mice previously immunized with A/JAPAN/305/57. These results demonstrate that a single nonglycosylated fragment of the influenza hemagglutinin as small as 21 amino acid residues is capable of being recognized as an antigenic determinant to generate influenza CTL from primed precursors.

Amino Acid Sequence↗
PubMed1982

Purification of the peptidoglycan transglycosylase of Bacillus megaterium.

The peptidoglycan transglycosylase of Bacillus megaterium has been purified approximately 500-fold from a crude membrane fraction. This protein is likely to be the one previously called PG-II and was assayed by its ability to reconstitute with a crude phospho-N-acetyl-muramyl-pentapeptide translocase preparation and partially purified N-acetylglucosaminyl transferase to give peptidoglycan synthesis from nucleotide precursors. The protein was identified as the peptidoglycan transglycosylase by its ability to synthesize lysozyme-sensitive peptidoglycan from undecaprenylpyrophosphoryl-disaccharide-pentapeptide. The enzyme is inhibited by vancomycin but not by bacitracin, penicillin G, or tunicamycin. The enzyme has no detectable transpeptidase activity, but it does bind penicillin.

Anti-Bacterial Agents↗
PubMed1981

An adherent, radiosensitive accessory cell necessary for mouse cytolytic T lymphocyte response to viral antigen.

A cell type in addition to T lymphocytes and macrophages was found to be necessary for the in vitro stimulation of mouse anti-Sendai viral T lymphocytes in response to subviral antigens. This cell type was found to be glass and nylon wool adherent, to bear no surface immunoglobulin or Thy 1.2 antigen, and to be radiosensitive. This type of cell was not needed when the stimulatory antigen was virus-infected cells.

Animals↗
PubMed1981

Stimulation of anti-influenza cytolytic T lymphocytes by CNBr cleavage fragments of the viral hemagglutinin.

Purified hemagglutinin of influenza virus, subtype A/Jap/305/57 (H2/N2) was cleaved with CNBr, and the resulting fragments were capable of stimulating H-2 restricted cytolytic T lymphocytes in vitro from spleen cells of mice immunized previously with A/Jap/305/57. When the peptides were separated by high-performance liquid chromatography, stimulatory activity was confined primarily to a series of peptides contained within a limited chromatographic range. The majority of the lytic activity was specific for cells infected with A/Jap virus, although a small amount of cross-reactive killing of A/PR/8/34-(H0/N1) infected cells could not be excluded. These results demonstrated that H-2-restricted influenza viral-specific CTL could be generated by fragments of a complex polypeptide antigen, the influenza hemagglutinin, and offer the possibility of analyzing specific antigenic determinants recognized by cytolytic T cells.

Animals↗
PubMed1980

Dissociation and reconstitution of membranes synthesizing the peptidoglycan of Escherichia coli. Lipid dependence of the synthetic enzymes.

The peptidoglycan synthetic enzymes can be dissociated with cholate and LiCl into components with mobilities on a gel filtration column in the same ranges as bovine serum albumin. The active enzymes can be separated further from the lipids necessary for synthesis by precipitation with ammonium sulfate. The needed lipids stable to hydrolysis with base. A protein needed for peptidoglycan polymerization can be separated from the other synthetic enzymes by hydroxylapatite chromatography.

Acetylglucosamine↗
PubMed1980

Stimulation of cytolytic T cells by isolated viral peptides and HN protein coupled to agarose beads.

Sendai virus-infected mouse cells can be lysed by mouse cytolytic thymus-dependent lymphocytes (CTL) directed specifically against the infected cells. The CTL is known to recognise the H-2 antigens on the target cells together with structure(s) including at least the two viral surface glycoproteins also found on purified virus. We report here that anti-Sendai CTL can be stimulated in vitro by detergent-solubilised viral haemagglutinin-neuraminidase (HN), either as the isolated protein or coupled to agarose beads. We further show stimulation by the hydrophilic portion of a protein removed from the virus by the protease subtilisin BPN', and we demonstrate that cyanogen bromide- (CNBr-) cleaved viral peptides also produce such stimulation.

Animals↗
PubMed1980

Elicitation of anti-Sendai virus cytotoxic T lymphocytes by viral and H-2 antigens incorporated into the same lipid bilayer by membrane fusion and by reconstitution into liposomes.

We have investigated the minimal molecular requirements for elicitation of anti-Sendai virus cytotoxic T lymphocytes (CTL), and the minimal molecular requirements for the recognition and lysis processes associated with anti-Sendai virus CTL-target cell interactions. This report demonstrates a) that the hemagglutinin-neuraminidase and/or fusion glycoproteins of Sendai virus can elicit anti-Sendai virus CTL and b) that these glycoproteins and H-2 antigens must be within the same membrane lipid bilayer for effective elicitation of anti-Sendai-virus CTL and for effective recognition and lysis of target cells by anti-Sendai virus CTL.

Animals↗
PubMed1979

Purification and characterization of a carboxypeptidase-transpeptidase of Bacillus megaterium acting on the tetrapeptide moiety of the peptidoglycan.

The enzyme carboxypeptidase-IIW of Bacillus megaterium incorporates free diaminopimelate into purified bacterial walls. This enzyme can be solubilized from toluene-treated cells by LiCl extraction and has now been purified 106-fold to one major band on polyacrylamide gel electrophoresis. The enzyme has an apparent molecular weight of approximately 60,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by Sephadex G-100 gel filtration. Carboxypeptidase-IIW requires divalent cations and thiol group(s) for optimal activity. Product analysis indicates that the enzyme can hydrolyze the terminal D-alanine from the tetrapeptide of the peptidoglycan or replace it with a variety of amino acids with D-asymmetric centers for transpeptidation. Substrate specificity studies reveal that the enzymatic activity depends on the presence of N-acetyl-D-glucosamine of the GlcNAc-MurNAc-tetrapeptide. This specificity of carboxypeptidase-IIW for the N-acetyl-D-glucosamine explains in part the affinity of the enzyme for the cell wall of B. megaterium. The enzyme is compared to the carboxypeptidases-transpeptidases of other organisms with the similarities and differences discussed.

Acyltransferases↗
PubMed1979

Dissociation and reconstitution of membranes synthesizing the peptidoglycan of Bacillus megaterium. A protein factor for the polymerization step.

Cholate-solubilized Bacillus megaterium membranes can be reconstituted by dialysis in the presence of magnesium ion to regain approximately 12% of the original peptidoglycan synthetic activity. Bio-Gel A-5m filtration of the solubilized components shows that all of the compounds necessary for peptidoglycan synthesis can be dissociated into material with a molecular weight of less than approximately 68,000. Using this reconstitution system, an assay has been developed for a new protein factor, PG-II, of B. megaterium. This factor could be combined with phospho-N-acetylmuramyl pentapeptide translocase and N-acetylglucosaminyl transferase to synthesize polymerized peptidoglycan from the precursors UDP-N-acetylmuramyl pentapeptide and UDP-N-acetylglucosamine. In the absence of PG-II, the disaccharide pentapeptide substrate for the polymerase was accumulated. In the presence of this factor, the amount of the substrate was diminished and polymeric peptidoglycan was formed. Therefore, PG-II was likely to be necessary for the polymerization step and may well have been the polymerase itself. From three chromatographic steps developed for the purification of PG-II, it seemed likely that a single protein with a molecular weight of approximately 60,000 could have PG-II activity.

Bacillus megaterium↗
PubMed1979

Alloantigen bound to agarose beads and syngeneic carrier cells are capable of stimulating mouse cytolytic T lymphocytes in vitro.

Bead-bound antigen was prepared by coupling alloantigen covalently to agarose beads. Alloantigen-bearing syngeneic carrier cells were prepared by dilution of detergent solubilized alloantigen in the presence of syngeneic spleen cells. Both types of antigen were compared to spleen cells and reconstituted membrane fragments for the ability to stimulate cytolytic thymus-dependent lymphocytes in vitro. All these types of antigen could stimulate immune but not nonimmune spleen cells to form cytolytic T lymphocytes. The amount of lytic activity obtained with the bead-bound antigen was found to be only dependent upon the amount of H-2 antigen present in the culture and independent of the number of beads.

Animals↗
PubMed1978

The entry into host cells of Sindbis virus, vesicular stomatitis virus and Sendai virus.

We have compared the mechanisms of entry into host cells of three enveloped viruses: Sendai virus, vesicular stomatitis virus (VSV) and Sindbis virus. Virus entry by membrane fusion should antigenically modify the surface of a newly infected cell in such a way that it will be killed by anti-viral antibody and complement. On the other hand, virus entry by a mechanism involving uptake by the cell of the whole virion should not make cells sensitive to antibody and complement. As expected, cells newly infected with Sendai virus were readily and completely lysed by anti-Sendai antibody and complement. In marked contrast, however, cells newly infected with either Sindbis virus or VSV were killed by anti-viral antibody and complement only when infected at an extremely high multiplicity of infection, in excess of 1000 plaque-forming units per cell. We favor the following explanation for these results with Sindbis virus and VSV: a very large majority of the Sindbis and VSV virions entered the infected cells by some means other than membrane fusion, presumably engulfment of the whole particle. Efficient entry by way of membrane fusion may therefore not be a general characteristic of enveloped viruses.

Antigens, Surface↗
PubMed1978

Dissociated and reconstituted subcellular alloantigen capable of stimulating mouse cytotoxic T lymphocytes in vitro.

Reconstituted subcellular membrane fragments were prepared from sonicated murine lymphoid tissue membrane fragments by detergent solubilization and dialysis. The reconstituted subcellular antigen was compared with sonicated cell membranes and x-irradiated cells for the ability to stimulate the formation of allogeneic cytotoxic thymus-dependent lymphocytes in vitro. The results showed that the reconstituted subcellular antigen had properties similar to those reported for the sonicated antigen. Both types of antigen could stimulate immune spleen cells but not normal spleen cells. Furthermore, soluble H-2 antigens, even of membrane origin, were incapable of the stimulation unless reconstituted into membrane fragments large enough to be centrifuged at 48,000 X G for 30 min.

Animals↗
PubMed1976

Identification of an isolated protein essential for peptidoglycan synthesis as the N-acetylglucosaminyltransferase.

We have identified a protein extractable from Bacillus megaterium membranes by LiCl as the enzyme in peptidoglycan synthesis which catalyzes the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to N-acetyl-D-muramyl-L-analyl-D-gamma-glutamyl-meso-diaminopimelyl-D-alanyl-D-alanine-P-P-lipid. This identification demonstrates the isolation and characterization of a membrane-associated enzyme involved in a complex series of reactions without the ready availability of a substrate for the enzyme.

Acetylglucosamine↗
PubMed1976

Purification and properties of a protein factor stimulating peptidoglycan synthesis in toluene- and Licl-treated Bacillus megaterium cells.

A protein factor, called PG-I, can be solubilized from toluene-treated Bacillus megaterium cells by LiCl extraction. After LiCl extraction, peptidoglycan synthesis by the toluene-treated cells is decreased. Protein PG-I can be added back to the extracted cells to stimulate peptidoglycan synthesis. This factor has now been purified 124-fold. It has a molecular weight of 42,000 as estimated by Sephadex gel filtration in the presence of 0.4 M KCl and 52,000 as determined by sodium dodecyl sulfate disc gel electrophoresis. Periodate-Schiff staining of the polyacrylamide gel indicates that factor PG-I is a glycoprotein. The reconstitution of LiCl-extracted cells requires Mg2+ with an apparent Km of 1.9 X 10(-3) M. The Mg2+ ions can be replaced by Ca2+ and by Mn2+ ions to some extent; Zn2+ and Cu2+ ions had no effect. The available data suggest that factor PG-I is essential for peptidoglycan synthesis and requires at least one thiol group for stimulatory activity.

Bacillus megaterium↗
PubMed1976

Essential genes in the metB-malB region of Escherichia coli K-12.

We isolated 25 independent mutants that are deficient in essential genes located in the metB-malB region of the Escherichia coli chromosome. The mutations were mapped within this region by using several F' factors and were also classified into 11 cistrons by complementation testing. There is clustering of mutations in essential genes within the metB-malB region.

Chromosome Mapping↗
PubMed1975

Reconstitution of cell wall synthesis in toluene- and LiCl-treated Bacillus megaterium cells by addition of a soluble protein extract.

Proteins required for the synthesis of peptidoglycan and incorporation of diaminopimelic acid into cell walls have been solubilized from Bacillus megaterium toluene-treated cells. Some of these proteins might have been removed from the cytoplasmic membrane through the cell wall by extraction with LiCl. The solubilized proteins have molecular weights in the range of 40,000 to 70,000 and can be added back to B. megaterium toluene-treated cells to reconstitute the synthetic reactions.

Bacillus megaterium↗