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J Zaia

Publications and source records attributed to J Zaia.

At least 19 recordsLinked to original sources
PubMed2003

Should HIV-positive patients with lymphoma be offered stem cell transplants?

Advances in effective antiretroviral therapy for HIV infection have made high-dose therapy and autologous stem cell transplantation possible in patients with HIV-associated lymphomas. Regimen-related toxicity is not significantly increased when antiretroviral therapy is combined with high-dose chemoradiotherapy. Durable engraftment can be seen with autologous stem cell rescue. Infectious complications can be managed with a combination of surveillance and prophylaxis. Long-term remissions of these high-risk lymphomas can be achieved with this approach. This suggests that patients with HIV-associated lymphomas should be considered for autologous transplantation in a manner similar to HIV-negative lymphoma patients.

Antiretroviral Therapy, Highly Active↗
PubMed2001

Tandem mass spectrometric determination of the 4S/6S sulfation sequence in chondroitin sulfate oligosaccharides.

Chondroitin sulfate (CS) is a glycosaminoglycan consisting of repeating uronic acid, N-acetylgalactosamine sulfate disaccharide units [-UroA(beta1,3)-GalNAcS(beta1,4)]n. Chondroitin sulfate type A (CSA) contains glucuronic acid, and 90% of the GalNAc residues are sulfated at the 4-position with 10% at the 6-position. Chondroitin sulfate type C (CSC) contains glucuronic acid, and 90% of the GalNAc residues are sulfated at the 6-position with 10% sulfated at the 4-position. These molecules are fragile due to their high degree of sulfation and are challenging to analyze as a result. This work presents the first evidence that tandem mass spectrometry can be used for the determination of a CS oligosaccharide sequence with respect to the positions of GalNAc sulfation. Using this technique, it is possible to analyze individual components from mixtures, saving much purification effort. Oligosaccharides produced from CSA and CSC are used in this work to demonstrate that CID MS/MS can be used to distinguish positional sulfation isomers. For charge states where charge equals the number of sulfates, abundant odd-numbered Bn and Yn ions are observed. The percent total ion abundances of these ions indicate the position of sulfation.

Carbohydrate Sequence↗
PubMed2001

Autologous stem cell transplantation for HIV-associated lymphoma.

Is peripheral stem cell mobilization followed by autologous stem cell transplantation (ASCT) feasible in patients with human immunodeficiency virus (HIV)- associated lymphoma (HIV-L)? Studies have demonstrated that, in the HIV- negative (HIV(-)) setting, ASCT may improve lymphoma-free survival in high-risk non-Hodgkin lymphoma (NHL) or relapsed Hodgkin disease (HD) and NHL. Given the poor prognosis of HIV-L with conventional chemotherapy, this dose-intensive approach was explored. Nine patients with HIV-HD or NHL mobilized a median of 10.6 x 10(6) CD34(+) cells/kg and engrafted after ASCT. CD4 counts recovered to pretransplantation levels and HIV viral loads were controlled in patients compliant with antiretroviral therapy. Seven of 9 patients remain in remission from their lymphoma at a median of 19 months after transplantation. Thus, patients with HIV-L on antiretroviral therapy can engraft following ASCT. Prolonged lymphoma remissions, without significant compromise of immune function, can be seen, suggesting that ASCT can be used in selected patients with HIV-L.

Adolescent↗
PubMed2001

The SH-3 and SH-4 antibodies recognize distinct epitopes on CD73 from human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are pluripotent cells in the bone marrow that have the capacity to differentiate along a number of connective tissue lineages, including cartilage, bone, adipose tissue, and stroma. The SH-3 and SH-4 monoclonal antibodies recognize epitopes present on the surface of human MSCs. This study describes the isolation and characterization of the antigen that is recognized by these antibodies. A protein of molecular weight approximately 67 kDa was immunoprecipitated from a solubilized membrane preparation of human MSCs using the SH-3 antibody. Analysis of peptides derived from this protein by mass spectrometry and sequencing identified it as CD73 (ecto-5'-nucleotidase). The SH-4 antibody was also shown to react with purified bovine CD73 by immunoblotting, but the SH-3 antibody failed to react with the bovine protein. These results indicate that both SH-3 and SH-4 epitopes are present on CD73, but they are distinct. CD73, present in lymphoid tissue, plays a role in the activation of B-lymphocytes and in signal transduction in the hematopoietic compartment of bone marrow. The role that CD73 may play in bone marrow stromal interactions and in the differentiation of MSCs is discussed.

5'-Nucleotidase↗
PubMed2001

Posttranslational modifications to human bone sialoprotein determined by mass spectrometry.

Bone sialoprotein (BSP) is an acidic 301 amino acid protein expressed by osteoblasts and at a low level by hypertrophic chondrocytes. Its expression is highest during early stages of bone formation, and it is particularly abundant in the cells lining the surface of newly formed trabeculae. BSP contains numerous substituents which are anionic in nature and apparently essential for the function of the protein. Thus, the proposed role of BSP in hydroxyapatite nucleation and growth may depend on such modifying groups. The posttranslational modifications include several acidic oligosaccharides as well as phosphate and sulfate groups. This work combines matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry with selective enzyme treatment of BSP to provide new information on the precise distribution and structure of oligosaccharides, sulfate, and phosphate groups in BSP isolated from human bone. The results provide a high level of detail in the location of these modifying groups toward the end of providing a basis for further understanding the function of BSP in bone nucleation.

Adult↗
PubMed2001

Compositional analysis of glycosaminoglycans by electrospray mass spectrometry.

The purpose of this work is to analyze glycosaminoglycans (GAGs) directly from complex mixtures without the need to purify individual components. Novel conditions for negative ion electrospray MS of chondroitin sulfate (CS) oligosaccharides are described in which sodium adduction and fragmentation are avoided. Differentiation between positional sulfation isomers is demonstrated for CS disaccharides, and a selected reaction monitoring scheme is used to quantify sulfation isomers in disaccharides liberated from decorin and biglycan. A size exclusion chromatography LC/MS method is shown to be effective for compositional analysis of longer CS oligosaccharides. The SEC step serves to simplify the composition of GAGs entering the mass spectrometer at any time, thus allowing the masses of the constituent molecules to be extracted. Mass spectrometric detection produces far more information than conventional UV or fluorescent detectors and allows the monosaccharide composition of individual components to be determined.

Chromatography, Gel↗
PubMed2000

Alternative O-glycosylation/O-phosphorylation of the murine estrogen receptor beta.

Estrogen receptor beta, a homologue to estrogen receptor alpha, is a new member of the steroid hormone receptor family. Recently, we documented that estrogen receptor alpha, like other transcription factors, is modified by O-linked N-acetylglucosamine (O-GlcNAc), a ubiquitous transitory posttranslational modification on nuclear and cytoplasmic proteins. Here, we report that estrogen receptor beta is alternatively modified by either O-GlcNAc or O-phosphate. Lectin chromatography of in vitro translated protein first suggested that murine estrogen receptor beta (mER-beta) is O-GlcNAcylated. Structural characterization of the carbohydrate moieties on mER-beta, overexpressed in insect Sf9 cells, confirmed the presence of O-GlcNAc. mER-beta, overexpressed in mammalian cells, is also O-GlcNAcylated. The major site of O-GlcNAc on mER-beta from Sf9 cells is Ser(16) near the N-terminus. Concomitant analyses also documented the O-phosphorylation of mER-beta at Ser(16). MALDI-TOF mass spectrometry showed alternative occupancy of this locus by these two abundant and dynamic posttranslational modifications. The localization of a major O-GlcNAc/O-phosphate site in proximity of the transactivation domain and as part of a PEST region (target sequences for rapid protein degradation) on mER-beta suggests that these modifications may play a role in regulating estrogen receptor beta transactivation and turnover.

Acetylglucosamine↗
PubMed2000

High dose therapy and autologous stem cell transplantation for human immunodeficiency virus-associated non-Hodgkin lymphoma in the era of highly active antiretroviral therapy.

BACKGROUND: The advent of highly active antiretroviral therapy (HAART) has allowed the exploration of more dose-intensive therapy such as autologous stem cell transplantation (ASCT) in selected patients with human immunodeficiency virus (HIV)-associated non-Hodgkin lymphoma (NHL). METHODS: The authors report on the use of myeloablative chemotherapy with ASCT in two HIV positive patients with NHL. The first patient underwent ASCT at the time of first disease remission for poor risk, diffuse, large cell NHL and the second patient had multiply recurrent, chemosensitive Burkitt lymphoma. ASCT was performed in both patients using a transplant conditioning regimen of high dose cyclophosphamide, carmustine, and etoposide (CBV). RESULTS: The target dose of >/= 5 x 10(6)/kg CD34 positive peripheral blood stem cells (PBSC) utilized for ASCT was collected using granulocyte-colony stimulating factor (G-CSF) after chemotherapy for mobilization while both patients were receiving concomitant HAART for HIV infection. HAART was continued during CBV conditioning. Prompt hematopoietic recovery was observed after ASCT. Both patients remained in clinical disease remission from their lymphoma at 28 months and 20 months after transplant, respectively. CONCLUSIONS: ASCT is feasible in patients with HIV-associated NHL. Adequate numbers of CD34 positive PBSC can be procured from patients receiving HAART and chemotherapy for NHL. Selected patients with HIV-related lymphoma can tolerate the high dose CBV myeloablative chemotherapy regimen without increased acute regimen-related toxicity. Reinfusion of G-CSF-mobilized PBSC can lead to rapid recovery of hematologic function and sustained engraftment after ASCT. Given the poor prognosis of patients with HIV-associated NHL treated with conventional chemotherapy, further investigation of this approach should be considered.

Adult↗
PubMed2000

Structural analysis of cartilage proteoglycans and glycoproteins using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Cartilage extracellular matrix molecules synthesized and maintained by chondrocytes form a strong, elastic tissue functioning to cushion and protect the subchondral bone. Osteoarthritis is characterized by degradation of cartilage extracellular matrix molecules resulting in fibrillation, irreversible erosion, and eventual failure of the tissue. With recent interest in the degradation of cartilage extracellular matrix molecules, a need for more detailed structural information exists. Posttranslational modifications are believed to play a role in determining the susceptibility of these molecules to proteolytic degradation during the development of osteoarthritis. The purpose of this paper is to show how the application of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to extracellular matrix protein and proteoglycan structure will help elucidate problems in extracellular matrix biochemistry. Methodological issues relating to the high molecular weight, polydispersity, and high degree of posttranslational modification of these molecules are discussed. MALDI-TOF mass spectrometry provides an improved level of detail for extracellular matrix protein and proteoglycan structure and is useful in addressing issues surrounding the causes of degradation during osteoarthritis.

Aggrecans↗
PubMed1999

The monoclonal antibody SH-2, raised against human mesenchymal stem cells, recognizes an epitope on endoglin (CD105).

Mesenchymal stem cells are multipotent cells resident in the bone marrow throughout adulthood which have the capacity to differentiate into cartilage, bone, fat, muscle, and tendon. A number of monoclonal antibodies raised against human MSCs have been shown to react with surface antigens on these cells in vitro. A protein of molecular mass 92 kDa was immunoprecipitated using the SH-2 monoclonal antibody. This was purified and identified by peptide sequencing analysis and mass spectrometry as endoglin (CD105), the TGF-beta receptor III present on endothelial cells, syncytiotrophoblasts, macrophages, and connective tissue stromal cells. Endoglin on MSCs potentially plays a role in TGF-beta signalling in the control of chondrogenic differentiation of MSCs and also in mediating interactions between MSCs and haematopoietic cells in the bone marrow microenvironment.

Adult↗
PubMed1998

Monitoring metal ion flux in reactions of metallothionein and drug-modified metallothionein by electrospray mass spectrometry.

The capabilities of electrospray ionization mass spectrometry are demonstrated for monitoring the flux of metal ions out of and into the metalloprotein rabbit liver metallothionein and, in one example, chlorambucil-alkylated metallothionein. Metal ion transfers may be followed as the reactions proceed in situ to provide kinetic information. More uniquely to this technique, metal ion stoichiometries may be determined for reaction intermediates and products. Partners used in these studies include EDTA, carbonic anhydrase, a zinc-bound hexamer of insulin, and the core domain of bacteriophage T4 gene 32 protein, a binding protein for single-stranded DNA.

Alkylation↗
PubMed1998

Copurification of P6, MRP8, and MRP14 from human granulocytes and separation of individual proteins.

A method is described for purification of P6, MRP8, and MRP14, three calcium-binding proteins assigned to the S100 protein family. The purification procedure included preparation of human granulocytes, ammonium sulfate precipitation, and anion-exchange chromatography and resulted in the copurification of P6, MRP8, and MRP14. Individual proteins were separated by either preparative isoelectric focusing or preparative SDS-PAGE. The procedure was carried out in the course of 4 days and yielded several milligrams of essentially pure P6, MRP8, and MRP14 in either native or denatured form.

Amino Acid Sequence↗
PubMed1998

Mesenchymal stem cell surface antigen SB-10 corresponds to activated leukocyte cell adhesion molecule and is involved in osteogenic differentiation.

Bone marrow contains a rare population of mesenchymal stem cells (MSCs) capable of giving rise to multiple mesodermal tissues including bone, cartilage, tendon, muscle, and fat. The cell surface antigen recognized by monoclonal antibody SB-10 is expressed on human MSCs but is lost during their developmental progression into differentiated phenotypes. Here we report on the immunopurification of the SB-10 antigen and its identification as activated leukocyte-cell adhesion molecule (ALCAM). Mass spectrometry establishes that the molecular mass of ALCAM is 80,303 +/- 193 Da and that it possesses 17,763 +/- 237 Da of N-linked oligosaccharide substituents. Molecular cloning of a full-length cDNA from a MSC expression library demonstrates nucleotide sequence identity with ALCAM. We also identified ALCAM homologs in rat, rabbit, and canine MSCs, each of which is over 90% identical to human ALCAM in their peptide sequence. The addition of antibody SB-10 Fab fragments to human MSCs undergoing osteogenic differentiation in vitro accelerated the process, thereby implicating a role for ALCAM during bone morphogenesis and adding ALCAM to the group of cell adhesion molecules involved in osteogenesis. Together, these results provide evidence that ALCAM plays a critical role in the differentiation of mesenchymal tissues in multiple species across the phylogenetic tree.

Activated-Leukocyte Cell Adhesion Molecule↗
PubMed1997

Post-translational modifications in cartilage oligomeric matrix protein. Characterization of the N-linked oligosaccharides by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Analysis of the carboxymethylated subunit of human cartilage oligomeric matrix protein (COMP) by matrix-assisted laser desorption time-of-flight mass spectrometry indicated a protonated molecular mass of 86949 +/- 149 Da, compared with 83547.0 Da calculated from the sequence. Treatment with N-glycanase caused a reduction in mass of 3571 +/- 219 Da, but there was no loss of mass after treatment with O-glycanase or neuraminidase. Peptides containing two putative sites of N-glycosylation were purified and characterized. Analysis of the masses of these after N-glycanase treatment indicated that one was substituted at Asn-101 with an oligosaccharide of mass 1847. 2 +/- 6.6 Da, and the other was unsubstituted at Asn-124. The remaining site of attachment, at Asn-721, was, therefore, also substituted with an oligosaccharide of mass 1724 +/- 226 Da. Analysis of the total monosaccharide content by chemical methods indicated that there were no additional oligosaccharide substituents. The MALDI-TOF mass spectra of COMP from bovine fetal and adult cartilage were compared, indicating a more heterogeneous pattern of substitution at Asn-101 in the fetal form. Since COMP is distributed throughout the pericellular and territorial environments in developing cartilage but occupies the interterritorial zone in mature cartilage, these changes in glycosylation may allow for different intermolecular interactions.

Amino Acid Sequence↗
PubMed1996

A binding site for chlorambucil on metallothionein.

It is of interest to test the hypothesis that induced metallothionein (MT) acts in acquired drug resistance by covalent sequestration. In this study MT was incubated in vitro with chlorambucil (CHB) under conditions where only 1:1 covalent adducts were formed. The proteolytic products of these adducts were analyzed by HPLC and mass spectrometry to reveal two major sites of modification. These were the sulfur atoms of cysteines 33 and 48, which cochelate the same metal atom in native MT. The time course of the reaction was followed using on-line electrospray ionization with a double-focusing mass spectrometer. These experiments showed that drug-modified MT binds seven metal ions, as does the unmodified protein. Molecular docking experiments showed that the selectively of drug binding is influenced by the presence of the aziridinium ion in the drug structure and complementary charge densities in the protein structure.

Amino Acid Sequence↗
PubMed1992

The correct molecular weight of myoglobin, a common calibrant for mass spectrometry.

Myoglobins from horse heart muscle, horse skeletal muscle and sperm whale are widely used as calibration standards or test compounds for various mass spectrometric methodologies. In all such cases reported in the literature, a molecular weight value is used (16,950.5 and 17,199, respectively) which is based on the assumption that amino acid 122 in this 153 amino-acid-long protein is asparagine, overlooking a published suggestion that it is aspartic acid instead. Since the mass assignment accuracy for matrix-assisted laser desorption mass spectrometry is reported to be +/- 0.01% and for electrospray ionization +/- 0.0025%, and error of one mass unit in approximately 17,000 would be significant. The mass-to-charge ratio of ions of the tryptic peptide encompassing amino acid 122 derived from commercially available horse heart and horse skeletal myoglobins, the apomyoglobin of the latter, and the tryptic and chymotryptic peptide of sperm whale myoglobin proved that in both proteins amino acid 122 is indeed aspartic acid, rather than asparagine. This finding was further confirmed by the collision-induced dissociation spectra of the [M + H]+ ions of the tryptic peptides from the horse myoglobins and the chymotriptic peptide from sperm whale myoglobin. Thus, the correct molecular weight of horse myoglobin is 16,951.49 and that of the sperm whale protein is 17,199.91.

Amino Acid Sequence↗
PubMed1991

Exploring the use of antisense, enzymatic RNA molecules (ribozymes) as therapeutic agents.

Antisense catalytic RNAs that specifically base-pair with and cleave target RNA sequences have potential for use as therapeutic agents against viral as well as endogenous gene expression. With the ultimate goal of developing anti-human immunodeficiency virus type 1 (HIV-1) ribozymes for therapeutic use, we have been exploring ways to improve upon the functional activity of ribozymes in living cells. This is being done by the systematic exploration of parameters that affect antisense, and hence ribozyme, function. These include target accessibility, stability of the catalyst, methods for delivery, and intracellular localization of the ribozyme. In addition, we have been examining the kinetic consequences of having extra, nontargeted sequences appended to the ribozyme flanking sequences. Perhaps the single most important consideration for ribozyme effectiveness in an intracellular environment is the accessibility of the target RNA for cleavage. By exploiting the mechanisms by which naturally occurring antisense RNAs interact with their target sequences, we hope to be able to address this problem of targeting and fully capitalize upon the potential of ribozymes as therapeutic agents.

Antiviral Agents↗