Biomedical subjects

D D Wood

Publications and source records attributed to D D Wood.

At least 19 recordsLinked to original sources

PubMed1992

The N terminus of human myelin basic protein consists of C2, C4, C6, and C8 alkyl carboxylic acids.

Peptide 1-21, generated by cyanogen bromide cleavage of each of two highly purified components of human myelin basic protein, components 1 and 8, gave a series of peaks in the fast atom bombardment mass spectra with m/z 2299, 2327, 2355, 2383, and 2411, indicating additions of 42, 70, 98, 126, and 154 atomic mass units respectively with m/z 2327 and 2355 as the dominant species. The pentafluorobenzyl esters prepared from an acid hydrolysate analyzed by negative ion chemical ionization gas chromatography mass spectrometry confirmed that C6, C8, and C10 fatty acids were present. These data demonstrated (i) that the N terminus of a myelin basic protein is not simply acetylated but contains C2, C4, C6, C8, and C10 fatty acids with C4 and C6 as the dominant species, (ii) the two components studied (C-1 and C-8) showed different relative amounts of C2 and C8 in particular, and (iii) human myelin basic protein is the first protein to be reported with a complex N terminus consisting of several alkyl carboxylic acid species.

Amino Acid Sequence↗

PubMed1992

An immunochemical comparison of human myelin basic protein and its modified, citrullinated form, C8.

An immunochemical analysis was conducted to compare the C1 isomer of human myelin basic protein (MBP) with the newly described and less cationic, citrullinated isomer of MBP referred to as C8. Ten polyclonal antisera directed at multiple epitopes or restricted regions of MBP were used in radioimmunoassays to examine MBP-C1 and MBP-C8. Antisera reactive with MBP peptide 1-14 clearly distinguished MBP-C1 from MBP-C8. Antisera to human MBP peptides 10-19 and 90-170, but not to MBP peptide 69-89, showed modest differences between MBP-C1 and MBP-C8. The MBP-C8s from multiple sclerosis (MS) and non-MS brain reacted essentially the same. With murine monoclonal antibodies and enzyme-linked immunosorbent assay (ELISA), differences between MBP-C8 and other isomers were shown for anti-MBP 10-19 but not for anti-MBP 1-9 or anti-MBP 80-89. These findings imply differences in sequence or conformation in the structure of MBP-C7 compared to MBP-C1, most notably near the amino terminus.

Adult↗

PubMed1990

Steroidal glycolipid, L-644,257, is a potent enhancer of nonspecific host resistance.

A steroidal glycolipid that enhances the nonspecific cellular response to opportunistic infection in an immunocompromised host has been discovered. A dose dependent response with 6-(5-cholesten-3 beta-yloxy)hexyl 1-thio-beta-D-mannopyranoside, L-644,257, was observed against several infective agents including bacterial, fungal, and viral pathogens in cyclophosphamide-treated mice. A mechanism for this protective action is proposed.

Adjuvants, Immunologic↗

PubMed1990

Glycolipids as host resistance stimulators.

6-(5-Cholesten-3 beta-yloxy)hexyl 1-thio-beta-D-mannopyranoside (L-644,257) enhances natural host resistance in cyclophosphamide-treated mice against Pseudomonas aeruginosa in a dose-dependent manner. It is active sc, im, and ip but not orally. L-644,257 is substantially more protective against P. aeruginosa than its alpha anomer. The beta-L-fucose glycolipid is more effective when given im and ip than sc. The lactose and beta-D-glucose glycolipids were only marginally effective to nonprotective. The 17 beta-steroidal side chain of L-644,257 can be modified without substantial loss of protective activity.

Adjuvants, Immunologic↗

PubMed1990

A hydroxyproline-containing protein from shark brain that is related to myelin basic protein.

Myelin basic protein (MBP) from shark (Chondricthyes) consists of a simpler mixture of charge isomers than human MBP. About two-thirds of the total amount applied to a CM-52 cellulose cation-exchange column was recovered in the unbound fraction of the column; the remaining one-third bound to column and was eluted as a single OD280 peak. This bound material did not sow the usual pattern of charge microheterogeneity found with human or bovine MBP. The unbound fraction was composed of a high molecular weight protein (55-60 kDa), which constituted most of this protein fraction and a low molecular weight protein (approximately 18 kDa). The amino acid composition of our unbound fraction was similar to that reported earlier. The Glx (glutamic acid + glutamine) was increased about threefold whereas the Arg content was only about 25% of that of the 18.5 kDa variant of bovine or human origin. The presence of hydroxyproline (1.2 residues/100) in this protein was noteworthy, identification of which was achieved by amino acid analysis in two different systems and by mass spectrometry. In the precolumn derivatization method, hydroxyproline eluted at 2.7 min; in the postcolumn derivatization method it eluted at 12.2 min. Identification of hydroxyproline was completed by fast atom bombardment-mass spectral analysis. The effect of hydroxyproline on the secondary structure of this protein is being studied. Verification that this high molecular weight protein contained MBP sequences within its primary structure was confirmed by immunological methods.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

PubMed1989

Interaction of glycosylated human myelin basic protein with lipid bilayers.

Myelin basic protein (MBP), isolated from normal human myelin, was glycosylated with UDP-N-acetyl-D-galactosamine and a glycosyltransferase isolated from porcine submaxillary glands. MBP containing 0.85 mol of N-acetyl-D-galactosamine per mole of protein was oxidized at carbon 6 by galactose oxidase and complexed with a spin-label, Tempoamine, in order to study its interactions with lipids. When the spin-labeled MBP was reacted with lipid vesicles consisting of DSPG, DPPG, and DMPG, most of the spin-label was motionally restricted in the gel phase, with a correlation time greater than 10(-8)s. The motion increased with increasing temperature and was sensitive to the lipid phase transition. Interaction with the gel phase of DPPA caused much less motional restriction of the probe. However, melting of the lipid allowed increased interaction and motional restriction of the probe, which was only partially reversed on cooling back to the gel phase. The motional restriction of the probe in these lipids is attributed to its penetration partway into the lipid bilayer in both the gel and liquid-crystalline phases. The fact that the probe bound to the protein can penetrate partway into the bilayer suggests that other hydrophobic side chains and residues of the protein can similarly penetrate into the bilayer. Additional evidence for penetration was provided by digestion of the lipid-bound protein with endoproteinase Lys-C. When nonglycosylated and glycosylated MBP in solution was treated with Lys-C, extensive digestion occurred. A single radioactive peptide which eluted at 25 min was identified as residues 92-105.(ABSTRACT TRUNCATED AT 250 WORDS)

Glycosylation↗

PubMed1989

The isolation, characterization, and lipid-aggregating properties of a citrulline containing myelin basic protein.

Human myelin basic protein was fractionated into its various charge isomers by CM52 cation exchange chromatography. Approximately 25-30% of the total charge applied to the column appeared in the void volume. This material termed "C-8," was further purified by reversed phase high performance liquid chromatography. Amino acid analyses of C-8 revealed low Arg (7 residue % in C-8 compared to 11-12 residue % in C-1) and increased Glx residues. The low Arg was accounted for by a corresponding amount of citrulline. Sequence analysis after chemical fragmentation (cyanogen bromide and BNPS-skatole) and enzymatic (cathepsin D and carboxypeptidase S-1) digestion localized the citrulline at residues 25, 31, 122, 130, 159, and 170 of the amino acid sequence. The effect of this loss of positive charge on the ability of the protein to aggregate lipid vesicles was demonstrated with vesicles composed of phosphatidylcholine (92.2 mol %) and phosphatidylserine (7.8 mol %). C-1 was the most effective charge isomer, and C-8 was the least effective. The ability of these charge isomers to aggregate vesicles correlated with the net positive charge on each. Vesicles composed of phosphatidylcholine alone were not aggregated by lipophilin or any of the charge isomers. However, when lipophilin was incorporated into phosphatidylcholine vesicles (50% w/w), small, optically clear suspensions of vesicles were formed. None of C-1, C-2, or C-3 aggregated these vesicles, but C-8 produced rapid vesicle aggregation. Since the substitution of citrulline for Arg would generate several relatively long apolar sequences, these would enhance the ability of C-8 to interact with the hydrophobic lipophilin molecule, promoting vesicle aggregation by hydrophobic interactions. The mechanism by which citrulline is generated in myelin is not known, although enzymatic conversion has been described in other systems. Studies are underway to elucidate the mechanism by which this post-translational modification is generated.

Amino Acid Sequence↗

PubMed1989

Prostaglandins in inflammatory bone pathology: mechanism and therapeutic benefit of etodolac.

To investigate the role of PGE2 in the development of bone and joint pathology in rat adjuvant arthritis, hindlimb paws were evaluated by calcified tissue histologic techniques focusing on histochemical visualization of cartilage and bone lesions. Case studies of hindlimbs from normal, adjuvant arthritic, and etodolac-treated arthritic rats demonstrated the association of disease severity with inflammation, chondromalacia, replacement of adipose bone marrow with a fibroid marrow, osteoclastic bone resorption, synovial cysts, and pannus formation within the joints. Extensive periosteal intramembranous bone formation was temporally associated with joint destruction and medullary tissue pathology. In vivo data were correlated with in vitro effects of inflammatory mediators (IL-1, PGE2) on bone resorption. Etodolac blocked bone explant PGE2 accumulation at concentrations of 10(-7) M and higher, and inhibited bone resorption at concentrations of 10(-5) M and higher. The data indicate that in vitro and in vivo models of bone metabolism are well correlated regarding prostaglandin synthesis; that the inflammatory mediator PGE2 is largely responsible for the involvement of skeletal tissue in the adjuvant arthritis model; and that the effects of etodolac are specifically mediated by its ability to inhibit PGE2 accumulation in vivo.

Animals↗

PubMed1988

A monoclonal anti-type II collagen antibody with cross-reactive anti-Ig activity specific for the F(ab')2 fragment.

An IgG2a hybridoma antibody (BC-10) was obtained by a myeloma fusion with lymphocytes from B10.RIII mice immunized against native bovine type II collagen. This anti-collagen monoclonal exhibited extensive cross-reactivity with several type II collagen species. BC-10 was found to have self-associating properties, but not the specificity of a typical IgG rheumatoid factor, inasmuch as this mAb bound to F(ab')2 fragments of itself and of normal mouse IgG. Self binding was inhibited by the association of BC-10 with type II collagen, and inhibition assays indicated that antibodies with the capacity to inhibit BC-10 binding to collagen were present in the sera from B10.RIII arthritic mice, but not from DBA/1 LacJ arthritic mice. Joint inflammation and histopathologic features consistent with arthritis were observed in mice injected with the BC-10 hybridoma.

Animals↗

PubMed1988

The glycosylation of human myelin basic protein at threonines 95 and 98 occurs sequentially.

Human myelin basic protein (MBP) was glycosylated by the enzyme, UDP-GalNAc:polypeptide N-acetylgalactosaminyl transferase (EC 2.4.2.41). A maximum of 1.7 mol of GalNAc was transferred to basic protein on threonines 95 and 98 of the protein. Proton NMR studies of basic protein glycosylated with 0.48-1.7 mol of GalNAc/mol of MBP showed that the order of addition to the two threonine residues is not random but sequential. The Thr-95 resonances shifted downfield, followed by the downfield shift of the Thr-98 resonances with increasing glycosylation. Since this peptide segment of the molecule is highly structured, conformational factors are probably responsible for this directed addition.

Amino Acid Sequence↗

PubMed1988

An ELISA for PGE2 utilizing monoclonal antibody.

An ELISA for PGE2 has been developed which is sensitive to concentrations of 0.5 to 20.0 ng PGE2/ml. Mouse monoclonal anti-PGE2 ascites is utilized in a binding competition between the test sample and an adsorbed conjugate of PGE2-BSA. The antibody which remains bound to the solid phase is quantitated colorimetrically by incubation with alkaline phosphatase-conjugated goat anti-mouse IgG followed by incubation with p-nitro-phenylphosphate. PGE1, PGA1, PGA2, PGB2, 6-keto-PGF1 alpha, PGF2 alpha, 13,14-dihydro-15-keto-PGE2, thromboxane B2 and arachidonic acid showed minimal cross-reactivity with the anti-PGE2. The PGE2 ELISA permits the quantitative analysis of large numbers of samples at a fraction of the cost and time required to process a commercial RIA kit. When linked to the appropriate computer software, data collection and analysis can be performed in less than 10 minutes per 96-well plate. Furthermore, the use of an ELISA system eliminates the radioactive and toxic chemical waste generated by RIA methods.

Antibodies, Monoclonal↗

PubMed1987

Inhibition of prostaglandin biosynthesis by etodolac. I. Selective activities in arthritis.

Etodolac is the first anti-inflammatory drug belonging to the tetrahydropyranoindole class. In contrast to several other common anti-inflammatory drugs, etodolac exhibited an unusually high potency as an inhibitor of established adjuvant arthritis relative to its activity against carrageenan paw edema in the rat. This phenomenon led us to investigate whether the ability of NSAIDs to inhibit prostaglandin biosynthesis differed between cultures of macrophages, which are present in inflammatory exudates, and cultures of synoviocytes and chondrocytes, which contribute to inflammation of the articulating joint. Although other anti-inflammatory drugs were found to be equally active in all three cell types, etodolac was found to be much more effective on the cells of the joint than on the macrophage. This differential activity may be responsible for the striking efficacy of etodolac as an anti-arthritic drug.

Acetates↗

PubMed1986

The role of charge microheterogeneity of basic protein in the formation and maintenance of the multilayered structure of myelin: a possible role in multiple sclerosis.

Isolation of several of the charge isomers from both normal and multiple sclerosis (MS) myelin basic protein (MBP) was achieved on CM-52 columns at pH 10.6. In liquid x-ray diffraction and aggregation experiments, corresponding charge isomers were equally effective in the formation of multilayers, demonstrating the dominant role of overall net positive charge. These studies demonstrated that the change in overall charge of MBP of one net positive charge was sufficient to produce large changes in aggregation and in multilayer formation. The x-ray diffraction experiments showed that component 1 was twice as effective as component 2 although they differed in charge by a single positive charge. Component 3 was less effective than component 2 and component "8" was not effective at all. Vesicle aggregation also showed a dependence on net positive charge. In order of decreasing effectiveness, component 1 greater than component 2 greater than component 3 greater than component "8". Since overall charge on MBP is determined by contributions from the various charge isomers, the relative proportions of these charge isomers favoring the less cationic components could explain the observation that MBP from MS victims was less effective than MBP from normal brain in vesicle aggregation and multilayer formation. The isolation of myelin-containing white matter fractions from both normal and MS tissue in which the loss of some of the most cationic charge isomers was correlated with presence of less compact myelin supports this hypothesis.

Cell Fractionation↗

PubMed1986

Serological characterization of acidic (pI 5.5) and neutral (pI 7.1) forms of 17kD and 35kD interleukin-1.

Human interleukin-1 (IL-1) was partially purified into the acidic and neutral 17,000 dalton forms and the acidic and neutral 35,000 dalton forms. Heterologous antiserum raised against purified human IL-1 (neutral, 17,000 daltons) neutralized only the neutral 35,000 dalton and 17,000 dalton isoelectric forms. Conversely, an antiserum raised against purified porcine acidic IL-1 (21,000 daltons) neutralized only the acidic 35,000 and 17,000 dalton forms. These results confirm earlier findings that the acidic and neutral species of IL-1 are not derived from the same gene. In addition, the results suggest that each isoelectric form of the 17,000 dalton IL-1 is probably derived directly from the corresponding 35,000 dalton IL-1.

Antigen-Antibody Complex↗

PubMed1985

The role of charge microheterogeneity of human myelin basic protein in the formation of phosphatidylglycerol multilayers.

Human myelin basic protein (HBP) was fractionated into its various charge isomers by chromatography on CM-52 columns at pH 10.6. Components 1,2,3 and "8" (C-1, C-2, C-3, and C-"8") were cleanly separated. Each component was combined with phosphatidylglycerol (PG) vesicles, at neutral pH at a concentration of 30% (w/w), protein/lipid. C-1, the most cationic of the components was the most effective at inducing the formation of multilayers when studied by liquid X-ray diffraction. C-3, which differs from C-1 by 2 positive charges was less effective than C-2. C-"8" was totally ineffective since the scattering pattern with this component was no different from that of the pure lipid. Thus a seemingly small change in net charge of the protein had a dramatic effect on the ability of the protein to organize the lipid into a crystalline, multilayer arrangement characteristic of compact myelin.

Chromatography↗

PubMed1985

Release of interleukin-1 from human synovial tissue in vitro.

During the enzymatic disaggregation of human synovium, used in the process of isolating synoviocytes, a factor was liberated into the culture medium that exhibited the thymocyte mitogenic properties of interleukin-1. Like interleukin-1, this synovial-derived mitogen could be isolated using an affinity column of antihuman leukocytic pyrogen. By gel filtration and isoelectric focusing, the mitogen cofractionated with human monocyte-derived interleukin-1. Finally, the isolated mitogen was shown to exhibit other properties of interleukin-1: stimulation of the secretion of interleukin-2, enhancement of the titer of acute-phase proteins in vivo, and stimulation of the release of prostaglandin E2 from human synoviocyte cultures. These observations suggest that interleukin-1 can be derived from the human synovium.

Chromatography, Affinity↗

PubMed1985

Reduction of serum Interleukin-1-like activity after treatment with dexamethasone.

The ability of steroids to modulate the appearance of Interleukin-1(IL-1) in vivo was evaluated in a model of endotoxin shock. High levels of IL-1 were found in serum from A/J mice which were sensitized with P. acnes and challenged with bacterial lipopolysaccharide (LPS). The factor appeared in the serum 2-4 hours after LPS challenge and was dependent on the period of P. acnes sensitization and the dose of LPS. Treating the mice with dexamethasone prior to LPS challenge resulted in significantly lower thymocyte proliferative activity in the serum. Three experiments demonstrated that this reduced activity reflects a decrease in IL-1. 1) The reduced activity was not due to the presence of proliferation inhibitors since mixing the serum from dexamethasone-treated mice with purified IL-1 or adding the equivalent amount of steroid directly to thymocyte cultures did not reduce the degree of proliferation. 2) When the serum was fractionated by gel filtration, the proliferative activity for both control and steroid treated sera eluted at 10-16 kilodaltons; however, the activity was nearly 50% less in the sample from steroid-treated mice. 3) In addition to thymocyte proliferative activity, IL-1 induces an increase in the serum titer of the acute phase protein known as serum amyloid A. Both serum- and gel-purified samples were able to induce the SAA, but again the samples from steroid-treated mice were much less active. We conclude that the factor produced in vivo has the properties of IL-1 and that the serum titre of the factor is reduced by dexamethasone treatment.

Animals↗

PubMed1985

Stimulation of the proliferation of human bone cells in vitro by human monocyte products with interleukin-1 activity.

The process of induction of bone formation, which follows bone resorption during normal and pathological bone turnover, is well documented. However, the mechanisms responsible for this process are unclear. Mononuclear phagocytes present at the sites of bone remodeling could play a role in this "coupling" of bone formation to bone resorption. This study was designed to investigate such a possibility. By measuring both the increase in [3H]thymidine incorporation and in cell number, we found that human monocytes in culture released factors capable of stimulating the proliferation of osteoblast-like cells derived from human bone. Rapidly dividing cells exhibited a greater response to interleukin 1 (IL-1) than confluent cells. The factors are similar to IL-1 in that they exhibited the same molecular weight and isoelectric point, were present in fractions that contained IL-1 activity after gel filtration chromatography and isoelectric focusing, and showed similar dose-response characteristics. Proliferation was more marked when prostaglandin production by the cells, which was also stimulated by these factors, was inhibited by indomethacin. A factor produced by monocytes that affects osteoblast activity may be important in the coupling of osteoclast and osteoblast actions.

Bone and Bones↗