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Biomedical subjects

M A Moscarello

Publications and source records attributed to M A Moscarello.

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

Reactivity of two anti-galactosyl ceramide antibodies towards myelin basic protein.

Two anti-galactosyl ceramide antibodies (polyclonal Ab142 and a monoclonal antibody) were characterized in terms of their reactivity towards purified lipids and myelin basic protein. Polyclonal Ab142 is a rabbit anti-mouse galactosyl ceramide (Gal C) IgG. Antigenic recognition is dependent on both galactose and ceramide since neither could inhibit galactosyl ceramide binding by more than 10%. MAb-Gal C is a monoclonal antibody raised in mice against galactosyl ceramide. Binding of MAb-Gal C to Gal-C was equally inhibited by ceramide and galactose to approximately 50%, indicating that both groups are important for antibody recognition. MAb-Gal C was also shown to be reactive with the structurally related lipids, sphingomyelin and sulfatide. Polyclonal Ab142, although raised against Gal C, was shown to be 3-fold more reactive with component 8 (C-8) of myelin basic protein than Gal C. On the other hand, the MAb-Gal C which also reacted with C-8 was 2-fold less reactive with C-8 than with Gal C. Neither of these antibodies were reactive with component 1 (C-1) of myelin basic protein. An anti-MBP IgG was shown to be reactive with C-1 and C-8 but unreactive with Gal C. In competitive inhibition ELISA, C-8 was able to compete out 44% and 41% of Gal C binding to polyclonal Ab142 and MAb, respectively. The reverse competition demonstrated that Gal C could inhibit 75% of C-8 binding to both antibodies. D-galactose was unable to inhibit C-8 binding to either antibody, whereas ceramide was as efficient as Gal C.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
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↗
PubMed1992

An antibody specific for component 8 of myelin basic protein from normal brain reacts strongly with component 8 from multiple sclerosis brain.

Myelin basic protein (MBP) consists of several components or charge isomers (C-1 through C-8) generated by one or a combination of posttranslational modifications. One of these, C-8, has been shown to contain citrulline (Cit) at defined sites formed by deimination of six arginyl residues. This unusual modification has allowed us to raise antibodies specific for this charge isomer only. To do this, a synthetic peptide, Gly-Cit-Cit-Cit-Cit, was coupled to keyhole limpet hemocyanin and injected into rabbits. The antibodies so generated reacted only with C-8 and not with any of the other charge isomers. A second antibody fraction was raised against the synthetic peptide ACitHGFLPCitHR naturally occurring between residues 24 and 33 of C-8 (all other charge isomers contain R instead of Cit at positions 25 and 31). These antibodies preferred C-8 but reacted with the other charge isomers, to the extent of approximately 25-30% of the reactivity shown with C-8. In studies with C-8 from multiple sclerosis (MS) MBP, much greater reactivity was obtained with these antibodies when compared with their reactivity with C-8 from normal MBP. Because the total number of Cit residues in C-8 from MS and normal MBP is the same, the difference in reactivity may be related to structural factors. The antibodies raised with the tetra-Cit peptide were reacted with three pairs of synthetic peptides: 24ARHGFLPRHR33 and ACitHGFLPCitHR; 120GQRPGFGYGGRAS132 and GQCitPGFGYGGCitAS; and 157GGRDSRSGSPMARR170 and GGCitDSRSGSPMACitR. They reacted only with the Cit-containing peptides in the order 157-170 greater than 120-130 greater than 24-33.(ABSTRACT TRUNCATED AT 250 WORDS)

Alzheimer Disease↗
PubMed1991

A 57-kDa phosphatidylinositol-specific phospholipase C from bovine brain.

A phosphatidylinositol-specific phospholipase C (PI-PLC) has been isolated from bovine brain (purification factor of 5.6 x 10(4)). By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it had a Mr of 57,000. Neither amino nor neutral sugars were detected in the purified enzyme. The pH optimum was 7.0-7.5, and the activity decreased only slightly at pH 8.0. When phosphatidylinositol was used as a substrate, the optimum Ca2+ requirement was 4 mM, and Km was 260 microM. When phosphatidylinositol 4,5-bisphosphate was used, the optimum Ca2+ requirement was 10(-7) M, and the Km was reduced to 90 microM. Lipid specificity studies showed that equal amounts of inositol phosphate and diacylglycerol were released from phosphatidylinositol but 4 times as much inositol 1,4,5-trisphosphate was released from phosphatidylinositol 4,5-bisphosphate. Other lipids, phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin, were not substrates. Failure to detect phosphatidic acid confirmed the absence of a phospholipase D activity in the purified enzyme. Myelin basic protein (MBP) stimulated the PI-PLC activity between 2- and 3-fold. Histone had a small effect only, whereas bovine serum albumin and cytochrome C had no effect. Phosphorylation of MBP reduced the stimulatory effect. Protein-protein interactions between MBP and PI-PLC have been demonstrated both immunologically and by sucrose density gradients. A stoichiometry of 1:1 has been suggested by the latter method. A number of peptides have been prepared by chemical, enzymatic, and synthetic methods. Peptides containing the MBP sequences consisting of residues 24-33 and 114-122 stimulated the PI-PLC but were less effective than the intact protein.

Amino Acid Sequence↗
PubMed1991

Characterization of myelin basic protein charge isomers from adult mouse brain.

ADULT mouse MBP charge isomers (C1 or component 1, C2 or component 2, etc.) were purified from an acid soluble brain protein fraction by cation exchange chromatography. They were characterized by their elution profiles, their migration rates in alkaline-urea gels and by SDS-PAGE. Mouse C1 and C2 were both 14 kD in size, while C3, C4 and C5 consisted of the 18.5, 17 and 14 kD isoforms. Comparison of mouse and human MBP charge isomers showed that they were similar in that they both showed extensive charge heterogeneity, but different in that mouse MBP charge isomers were more cationic than their human counterparts. Finally, a possible explanation for the presence of the 14 kD MBP isoform in mouse myelin was suggested.

Animals↗
PubMed1991

Localization of CD44 (P80) on the external surface of a human astrocytoma cell.

A brain antigen, originally identified by a MAb 44D10, has been shown to be a glycoprotein with an Mr of 80 kDa. Cellular localization studies of sections of brain showed that the antigen was associated with the membrane of astrocytes. In the present study we demonstrate its localization to the membrane of an astrocytoma cell line by fluorescence and electron microscopic immunogold methods. Heavy labelling with immunogold was found along cellular processes. Labelling of the smooth surfaces of cells and the microvilli was also observed.

Antibodies, Monoclonal↗
PubMed1991

Localization of the CD44 glycoprotein to fibrous astrocytes in normal white matter and to reactive astrocytes in active lesions in multiple sclerosis.

The CD44 antigen is a proteoglycan recently implicated in several adhesion events including that of lymphocytes to endothelium. The CD44 antigen, reactive with monoclonal antibody (MAb) 44D10, has been shown previously to be expressed in normal human white matter homogenates and to be found at higher concentrations in brain homogenates of victims of multiple sclerosis (MS). The cellular localization of CD44 in human brain of normal individuals and in those afflicted with MS has now been determined. Monoclonal antibody 44D10 reacted with astrocyte-like cells in 40 microns thick paraformaldehyde-fixed sections but not in thin (6 microns) fixed sections. A double labeling experiment performed on a frozen brain section with MAb 44D10 and rabbit anti-glial fibrillary acidic protein (GFAP), a cytoplasmic marker of astrocytes, confirmed the co-localization of these two antigens. The reactivity with brain tissue sections of a rabbit antiserum produced against lymphocyte-CD44 could be absorbed by a preparation of the CD44 glycoprotein, purified 2,100-fold from a white matter homogenate. The antiserum was shown by Western blot analysis to be specific for p80 glycoprotein in brain extracts derived from a normal and MS patients. This antibody reacted with fibrous astrocytes predominantly in white matter; staining was also noted in subependymal and subpial regions. Inhibition studies using a cellular radioimmunoassay indicated that the highest concentrations of CD44 in three MS victims were found in plaques, followed by periplaques and non-involved areas of white matter which were higher than normal white matter. Reactive astrocytes, identified in active lesions, expressed high levels of CD44 on their surfaces. Thus, CD44 is associated with astrocytes in human brain and the increased expression observed in MS brain may reflect activation and/or proliferation of astrocytes implicated in the pathogenesis of this disease.

Astrocytes↗
PubMed1991

Aminoaciduria as a marker of acute renal transplant rejection--a patient study.

Over 12 months, urine samples were systematically collected from 40 children who underwent renal transplantation for the treatment of end-stage renal disease. Sequential determinations of the excretion of individual amino acids relative to that of creatinine were carried out on 15 subjects. Nine of these (including three who sustained episodes of acute rejection) retained a native kidney in-situ, while in six patients (including three who underwent an episode of acute rejection) both native kidneys had been removed. In both subgroups, the amino acid/creatinine ratios of early morning urine samples were higher shortly before clinical manifestations of acute rejection became evident than in patients who, following renal transplantation, had stable kidney function, chronic graft rejection, or acute tubular necrosis, with one exception: a patient with one native kidney in-situ in whom acute tubular necrosis developed immediately after transplantation. The amino acids showing the greatest increase included Thr, Ser, Gly, and Ala. These values fell dramatically immediately prior to the clinical episode of acute rejection, with Thr, Ala, and Phe showing the most consistent changes. These alterations in urinary amino acid excretion occurred several days before changes in urinary protein excretion or the serum concentrations of urea and creatinine, and may have a role to play in the monitoring of renal transplant recipients.

Adolescent↗
PubMed1991

Aminoaciduria is an earlier index of renal tubular damage than conventional renal disease markers in the gentamicin-rat model of acute renal failure.

There are currently no reliable early markers of renal tubular damage. Since aminoaciduria is an accompanying feature of this condition, the usefulness of increased urinary amino acid excretion as a marker was investigated by inducing renal tubular necrosis in male Wistar rats by the administration of gentamicin (40 mg/kg/d) for 14 d. Plasma amino acids, urea, creatinine, protein and electrolytes, and urine amino acids, protein and N-acetylglucosaminidase (a lysosomal enzyme) were measured over a 20 d period. Amino acid excretion increased dramatically within 24 h of the initial dose for 14 of the 16 amino acids measured. The conventional renal disease markers listed above did not increase until after day 7. Glomerular damage caused by puromycin aminonucleoside did not induce aminoaciduria until marked proteinuria occurred (day 9), and even then amino acid excretion was much less than that caused by gentamicin. To distinguish whether the gentamicin-induced aminoaciduria was a consequence of tubular damage or inhibition of amino acid transport, isolated rat kidneys were perfused with a Krebs-Henseleit albumin buffer with and without gentamicin for 20 min, during which time urinary amino acids were quantitated. Gentamicin did not inhibit amino acid reabsorption. Thus, it appears that in the rat-gentamicin model of acute renal failure, urinary amino acids are early markers of renal tubular damage.

Acetylglucosaminidase↗
PubMed1990

Myelin basic protein is affected by reduced synthesis of myelin proteolipid protein in the jimpy mouse.

Myelin basic proteins (MBPs) from 6-day-old, 10-day-old, 20-day-old and adult normal mouse brain were compared with those from 20-day-old jimpy (dysmyelinating mutant) mouse brain to determine the effect of reduced levels of proteolipid protein (PLP) on MBPs. Alkaline-urea-gel electrophoresis showed that 6-day-old and 10-day-old normal and jimpy MBPs lacked charge microheterogeneity, since C8 (the least cationic of the components; not be confused with complement component C8) was the only charge isomer present. In contrast, MBPs from 20-day-old and adult normal mouse brain displayed extensive charge microheterogeneity, having at least eight components. A 32 kDa MBP was the major isoform observed on immunoblots of acid-soluble protein from 6-day-old and 10-day-old normal and 20-day-old jimpy mouse brain. There were eight bands present in 20-day-old and adult normal mouse brain. Purified human MBP charge heteromers C1, C2, C3 and C4 reacted strongly with rat 14 kDa MBP antiserum, whereas the reaction with human C8 was weak. This suggested that MBPs from early-myelinating and jimpy mice did not react to MBP antisera because C8 was the major charge isomer in these animals. Purification of MBPs from normal and jimpy brain by alkaline-gel electrophoresis showed that both normal and jimpy MBPs have size heterogeneity when subjected to SDS/PAGE. However, the size isoforms in normal mouse brain (32, 21, 18.5, 17 and 14 kDa) differed from those in jimpy brain (32, 21, 20, 17, 15 and 14 kDa) in both size and relative amounts. Amino acid analyses of MBPs from jimpy brain showed an increase in glutamic acid, alanine and ornithine, and a decrease in histidine, arginine and proline. The changes in glutamic acid, ornithine and arginine are characteristic of the differences observed in human C8 when compared with C1.

Aging↗
PubMed1990

The preparation of antibodies reactive against citrulline-containing charge isomers of myelin basic protein but not against the arginine-containing charge isomers.

Human myelin basic protein (MBP) is composed of several charge isomers, the result of various post-translational modifications. One of the charge isomers C-8, has been shown in our laboratory to contain six citrullinyl residues which replace arginyl residues at selected sites in the MBP. In order to determine the disposition of the citrulline-containing charge isomers in the myelin stack, we prepared specific antisera against the citrullinyl group. Since 9-fluorenylmethoxycarbonyl (Fmoc)-citrulline, required for the preparation of the synthetic peptides to be used for antibody production, was not commercially available, synthesis of the Fmoc-citrulline was a necessary prerequisite. The synthesis and purification of the N-9-fluorenylmethyloxycarbonyl derivative of citrulline is described. It was characterized by thin layer chromatography, 1H and 13C NMR spectroscopy, fast-atom bombardment mass spectroscopy, and thermal analyses. It was used in the automated peptide synthesis of a peptide Ala-Cit-His-Gly-Phe-Leu-Pro-Cit-His-Arg corresponding to residues 24-33 and Gly-Cit-Asp-Ser-Arg-Ser-Gly-Ser-Pro-Met-Ala-Cit-Arg, corresponding to residues 158-170 of the C-8 sequence, a naturally occurring charge isomer of human myelin basic protein, and a tetracitrulline peptide, Cit-Cit-Cit-Cit-Gly. The tetracitrulline peptide was used for the production of an antibody shown to react only with synthetic peptides and proteins containing citrulline. This antibody was used to distinguish between a citrulline-containing protein, C-8, a naturally occurring charge isomer of MBP, and a non-citrulline-containing charge isomer of MBP, C-1.

Amino Acid Sequence↗
PubMed1990

Conformational correlates of the epitopes of human myelin basic protein peptide 80-89.

Different epitopes residing within the decapeptide of residues 80-89 of human myelin basic protein (MBP) exist in the MBP-like material detected in human CSF and urine. In the present study, the structure of human MBP peptide 80-89 was examined by a combination of physical measurements and correlated with its varying immunochemical reaction with three polyclonal antisera. At least two epitopes are present in the decapeptide. Progressive shortening and reduction in net negative charge of MBP peptide 80-89 to form peptides 81-89, 82-89, 83-89, and 84-89 revealed an epitope not present in intact MBP. Circular dichroism and Fourier-transform infrared of these MBP peptides in water demonstrated random structure that was partially changed to beta-structure in the shorter peptides. In methanol, used as a model for a lipid environment, the random structure was diminished and was replaced by alpha-helix and beta-structure, especially in the shorter peptides. The findings indicate that the range of epitopes present in this decapeptide is influenced by conformation, which, unexpectedly, becomes progressively less random as the peptide becomes smaller, especially in a hydrophobic environment. This behavior has implications for the immunochemical detection of small antigens or antibodies to them in tissue extracts or body fluids.

Circular Dichroism↗
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↗
PubMed1990

Phosphorylation of charge isomers (components) of human myelin basic protein: identification of phosphorylated sites.

Myelin basic protein isolated from normal human brain was resolved into its various components (charge isomers) by CM-52 column chromatography. Two of the components C-1 and C-4, were phosphorylated in vitro with a soluble preparation of brain protein kinase C. For each component, the peptides phosphorylated were identified. In both components a major site of phosphorylation was found at Ser7 in the N-terminal portion of the protein. Both the specific activity and the rate of phosphorylation were greatest at this site in both components when compared with the other sites. The rate of phosphorylation of peptide 5-13 was approximately 10 times greater than that of any of the other peptides derived from C-1, while the rate of phosphorylation of peptide 5-13 derived from C-4 was 10-20 times greater than that of any of the other peptides derived from C-4. In addition, peptide 5-13, which contained a major phosphorylation site in both C-1 and C-4, was phosphorylated at a faster rate in C-4 (460 cpm/nM/min) compared with C-1 (285 cpm/nM/min). Both the specific activity and the rate data presented in the present communication were correlated with the proportion of beta-structure in a previous study. In that study, C-1, which contained about 13% beta-structure before phosphorylation, increased to approximately 40% after phosphorylation. Construction of a model peptide of this N-terminal region, which included the phosphorylation site at Ser7, demonstrated that the beta-structure was stabilized by electrostatic interactions between the phosphate on Ser7 and the guanidyl groups of Arg5 and Arg9.(ABSTRACT TRUNCATED AT 250 WORDS)

Humans↗
PubMed1989

Secondary structure of charge isomers of myelin basic protein before and after phosphorylation.

Human myelin basic protein (MBP) was fractionated into several of its charge isomers (components). Of these, the secondary structures of four isomers before and after phosphorylation have been studied by circular dichroism (CD). None of the four showed any alpha-helical structure. All of the components showed varying amounts of beta-structure, random structure, and turns. Component 1 (C-1), the most cationic of the components, showed 13%; component 2 (C-2) had 19%; C-3, 17%; and C-4, 24% of beta-structure. Each of the four components was phosphorylated with protein kinase C, from human brain. The extent of phosphorylation varied considerably from 2.8 +/- 0.6 mol of PO4/mol of protein in C-1 to 5.2 +/- 0.8 mol of PO4/mol of protein in C-4. The effect of phosphorylation on the secondary structure was to induce beta-structure in all the components. The largest change in beta-structure was in C-1 and the least in C-4. The surprising result is that although the components were phosphorylated to different extents, the amount of beta-structure in all four components increased to a final proportion of 35-40%. Treatment of phosphorylated C-1 with acid phosphatase removed 50% of the total radioactivity. Although the remainder represented approximately 1 mol of PO4/mol of protein, the proportion of beta-structure was unaltered. We concluded that a single phosphorylation site identified as residues 5-13 represented a critical size for stabilization of beta-structure of MBP in solution and that phosphorylation at the other sites had little influence on secondary structure.

Binding Sites↗