Biomedical subjects

J A Leary

Publications and source records attributed to J A Leary.

At least 37 records · Page 2Linked to original sources

PubMed1999

Determining anomericity of the glycosidic bond in Zn(II)-diethylenetriamine-disaccharide complexes using MSn in a quadrupole ion trap.

Zinc-diethylenetriamine (Zn-dien) N-glycoside complexes of four 1,4 and four 1,6 linked disaccharides are prepared. Each reaction mixture is ionized by electrospray and the resulting species [Zn(dien)(disaccharide)-H]+ is allowed to undergo collision-induced dissociation in a quadrupole ion trap. An MS3 analysis is used to differentiate alpha versus beta anomericity of the glycosidic bond in the disaccharide moiety. In addition, the MS2 and MS3 spectra can be used together to determine the linkage position of this glycosidic bond.

Disaccharides↗

PubMed1998

Stereochemical differentiation of mannose, glucose, galactose, and talose using zinc(II) diethylenetriamine and ESI-ion trap mass spectrometry.

Diastereomeric diethylenetriamine N-glycoside zinc(II) complexes are investigated using electrospray ionization followed by tandem mass spectrometry in a quadrupole ion trap. Dissociation ions specific to stereochemical differences at C2 and C4 in hexose complexes are observed in the MS2 and MS3 spectra, thus allowing unambiguous differentiation of glucose, galactose, mannose, and talose. Labeling studies incorporating 2H and 13C are used to probe the mechanisms of dissociation involved with these diastereomers, and MS2 studies on deoxyglucose complexes are implemented to support proposed sites of deprotonation within the complexes.

Galactose↗

PubMed1998

Evidence for linkage position determination in cobalt coordinated pentasaccharides using ion trap mass spectrometry.

A methodology to determine the linkage position of oligosaccharides is presented. In order to illustrate this technique, several oligosaccharides and disaccharides were ionized by electrospray and analyzed in a Paul trap mass spectrometer. Multiple stage tandem mass spectrometry experiments were used to determine linkage and structural information for the following four cobalt coordinated and singly charged ([M + Co-H]+) pentasaccharides: Lacto-N-fucopentaose I, II, III, and V. In order to differentiate between linkage positions, multiple low energy collision induced experiments with mass selected C type ions have been carried out in an ion trap mass spectrometer. Because of the coordination with cobalt, which directs the dissociation pathways, these C type ions undergo specific fragmentation reactions upon low energy collision induced dissociation. These dissociation pathways are unambiguously dependent on their linkage position, thus allowing differentiation between 1-->2, 1-->3, 1-->4, and 1-->6 linkage positions throughout the oligomers. Studies on various linked disaccharides and N-acetyl-disaccharides, which are smaller constituents of the pentasaccharides, were used to verify and confirm the results obtained from the pentasaccharides.

Carbohydrate Sequence↗

PubMed1998

Immunohistochemical detection of progesterone receptors in archival breast cancer.

The progesterone receptor (PR) is an important marker of response to endocrine agents in breast cancer. Immunohistochemical demonstration of PR in formalin fixed tissue has previously proved difficult, and heat pretreatment is considered necessary to retrieve the antigen. There are few data on the effectiveness of autoclaving in unmasking PR, however, and it is not known whether all PR epitopes are equally unmasked. The objectives of this study were to compare the efficacy of autoclaving and microwaving to retrieve PR antigen in archival breast tumors, to determine whether there is an epitope-dependent variability in the pretreatment required, and to examine different slide types and adhesives to reduce the problem of section loss frequently associated with these procedures. Paraffin embedded sections were cut at 2 or 4 microm, mounted onto various slide types with or without the addition of adhesive, and heat pretreated prior to immunoperoxidase staining. Whereas PR immunoreactivity was clearly demonstrated in tissue after both autoclaving and microwaving, autoclaving produced a significantly stronger staining intensity under the conditions used in this study. The duration of autoclaving required to reveal PR fully differed for different epitopes examined. In the absence of heat pretreatment, PR was not detected. Section retention was improved by the use of adhesives and by cutting tissue at 2 microm. Maximum retention was obtained using positively charged slides coated with Mayer albumen adhesive. We conclude that for maximal tissue preservation autoclave pretreatment is the preferred method of PR antigen retrieval from archival breast tumors, that there is epitope-dependent variability in pretreatment required, and that section loss during this procedure can be minimized by choice of slide type, the use of adhesive, and by cutting sections at 2 microm.

Adhesives↗

PubMed1997

Analysis of loss of heterozygosity and KRAS2 mutations in ovarian neoplasms: clinicopathological correlations.

The molecular events that give rise to ovarian epithelial neoplasms are not well understood. In particular, it is not known whether adenocarcinomas arise from benign or low malignant potential (LMP) precursors. We have examined a large series of benign (25) and LMP (31) ovarian tumors for loss of heterozygosity (LOH) at multiple loci on 17 chromosomes. LOH was observed in benign tumors on chromosomes 6 (14%) and 9 (5%) and on the X chromosome (33%) only. LOH on these chromosomes was also detected in a small number of LMP neoplasms, suggesting that these may derive sometimes from benign precursors. In addition, we examined LOH in 93 adenocarcinomas. Analysis of associations between LOH events showed that LOH on chromosomes 5 and 17 (P = 0.0002) and on chromosomes 17 and 18 (P = 0.00007) were associated significantly with each other, which suggests that these may represent cooperative, progressive events. No novel significant associations were identified between LOH events and stage, grade, or histology, which would indicate the existence of genetic heterogeneity in ovarian neoplasms. KRAS2 mutations were detected more often in LMP neoplasms than in malignant tumors (P = 0.004) and were detected more often in Stage I/II malignant tumors than in Stage III/IV malignant tumors (P = 0.033), suggesting that LMP tumors with KRAS2 mutations are unlikely to progress to frank malignancy. Univariate (but not multivariate) survival analysis showed that LOH of chromosomes 11 (P = 0.039) and 17 (P = 0.04) was associated with a significantly worse prognosis. Replication of these novel findings is necessary, and the identification, isolation, and characterization of the critical genes affected by LOH will determine their importance in the pathogenesis of ovarian malignancies.

Chromosome Deletion↗

PubMed1996

Allelic loss on chromosome 7q in ovarian adenocarcinomas: two critical regions and a rearrangement of the PLANH1 locus.

The presence of a tumour suppressor gene on chromosome 7q is indicated by cytogenetic, loss of heterozygosity (LOH) and chromosome transfer studies. One candidate gene in this region is Plasminogen Activator Inhibitor-1 (PAI-1). The PAI-1 gene product is involved in proteolysis and may therefore influence tumour spread and invasion. We have analysed a series of 139 ovarian epithelial tumours at four loci in the region 7q21-q31 which includes the PAI-1 gene. The highest rates of loss were found in malignant tumours (FIGO stages I-IV) at markers D7S471 (38%, 20/52 informative cases) and D7S522 (34%, 15/44). No loss was seen in benign tumours and only one out of 27 (4%) informative LMP tumours demonstrated LOH. The smallest region of overlap (SRO) lies between D7S471 and PAI-1. We also identified a rearrangement in one tumour in the PAI-1 gene, suggesting that this may be the inactivated gene in this region. In addition LOH at the more distal marker, D7S522, which lies outside the SRO, shows significant association with stage (P=0.0343) and with LOH on chromosome 13 (P=0.0024). This is in contrast to all other markers examined. These data suggest the presence of two critical regions on 7q which may be important in subsets of epithelial ovarian tumours.

Adenocarcinoma↗

PubMed1996

MDR1 and multidrug resistance-associated protein (MRP) gene expression in epithelial ovarian tumors.

Clinical drug resistance poses a major problem in the successful treatment of ovarian cancer. Two genes, MDR1 and MRP, have been found to play a role in the multidrug resistance phenotype of a number of human tumors. Using a sensitive semiquantitative PCR assay we examined 53 primary untreated ovarian tumors for expression of both MDR1 and MRP genes. Approximately one-third of the tumors expressed MDR1 while all the tumors expressed varying levels of the MRP gene.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

LC/MS/MS of carbohydrates with postcolumn addition of metal chlorides using a triaxial electrospray probe.

A new technique for on-line postcolumn addition (PCA) formation and tandem electrospray mass spectrometry of carbohydrate-metal complexes is presented. A metal chloride solution is added to a carbohydrate sample directly within the ion source of the mass spectrometer. Using a triaxial electrospray probe, this technique can be applied to form carbohydrate-metal complexes on-line, without the need of previously mixing the carbohydrate and metal chloride. Two basic tasks may be accomplished: structural analysis and sensitivity enhancement. The performance of this approach is demonstrated through PCA of LiCl, NaCl, KCl, RbCl, CsCl, and CoCl2, introduced via the triaxial probe after chromatographic separation of two four-component carbohydrate mixtures. Each metal-carbohydrate complex is subsequently analyzed by on-line MS and MS/MS. This technique is used to enhance sensitivity and also, in the case of cobalt coordination, to assist in carbohydrate structural elucidation. On-line LC/MS with PCA of LiCl was achieved with as little as 1.7 pmol of oligosaccharide (average consumed amount, 1.7 pmol with 1 microL of a 10 pmol/microL carbohydrate test mixture injection).

Carbohydrates↗

PubMed1995

Increased expression of the NME1 gene is associated with metastasis in epithelial ovarian cancer.

The genetic events involved in the development of metastases of epithelial ovarian cancer are largely unknown. One gene postulated to play a role in tumour metastasis suppression is NME1 (nm23-H1), and an inverse relationship between NME1 expression and metastatic potential has been observed for some solid tumours. In this study we have investigated the levels of mRNA expression of the 2 isoforms of the NME gene, NME1 and NME2. A maximum of 45 tumours samples from 33 patients were available for Northern blot analysis. We observed variable levels expression of NME1 and NME2 mRNA. The average level of NME1, but not NME2, mRNA expression was statistically higher in metastatic biopsies when compared with primary tumour biopsies. To examine the possible tumour suppressor gene role of NME1 in ovarian tumours, 76 patients were investigated by Southern blot analysis to determine the rate of allelic deletion. Allele loss at 5 other chromosome 17 loci (D17S5, TP53, NF1, D17S74, D17S4) was also evaluated for many of these 76 patients. Allele loss was observed in 22/30 (73%) informative patients at the NME1 locus. We also observed high rates of allele loss at the other loci evaluated. No correlations with clinical stage, histological subtype or patient survival were observed in either mRNA or DNA analyses. We have established that tumour progression in ovarian cancer is accompanied by over-expression of the NME1 gene; however, despite high rates of allele loss at the NME1 locus, the concept that NME1 may be a candidate tumour suppressor gene in ovarian cancer cannot be confirmed by this study.

Base Sequence↗

PubMed1993

Differentiation of Ca(2+)- and Mg(2+)-coordinated branched trisaccharide isomers: an electrospray ionization and tandem mass spectrometry study.

Three isomeric branched trisaccharides coordinated to calcium and magnesium were analyzed by electrospray ionization followed by tandem mass spectrometry. Studies were undertaken to optimize the singly charged metal coordinated precursor prior to MS/MS and MS/MS/MS experiments. The positioning of the glycosidic linkages between reducing and nonreducing rings could be determined in all three isomers, and the linkage of the fucose ring was obvious in two of the three isomers when calcium was used as the coordinating metal. The ionic radius and charge of the metal appear to be very important in obtaining structural information in these isomers.

Calcium↗

PubMed1992

Characterization of phycocyanin produced by cpcE and cpcF mutants and identification of an intergenic suppressor of the defect in bilin attachment.

Mutants of the cyanobacterium Synechococcus sp. PCC 7002 constructed by the insertional inactivation of either the cpcE or cpcF gene produce low levels of spectroscopically detectable phycocyanin. The majority of the phycocyanin produced in these strains appears to lack the alpha subunit phycocyanobilin (PCB) chromophore (Zhou, J., Gasparich, G. E., Stirewalt, V. L., de Lorimier, R., and Bryant, D. A. (1992) J. Biol. Chem. 267, 16138-16145). Purification of the phycocyanin produced in the mutants revealed two fractions each with an aberrant absorption spectrum. Tryptic peptide maps of the major fraction showed that the alpha-84 PCB peptide was absent. The two PCB peptides derived from the beta subunit were normal. Tryptic digests of the less abundant phycocyanin fraction contained a family of bilin peptides derived from the alpha subunit. Several distinct bilin adducts were present. A major component was a mesobiliverdin adduct, a previously described product of the in vitro reaction of PCB and apophycocyanin. The same results were obtained with both the cpcE mutant and the cpcF mutant. In vitro reactions with PCB and the fractions containing apo alpha subunit showed that the alpha-84 bilin attachment site was unmodified and competent for adduct formation. Pseudo-revertants of both strains were observed to arise at high frequency. Analysis of the phycocyanin from a cpcE pseudo-revertant, which produced a near wild-type level of phycocyanin with alpha subunit carrying PCB, revealed a single amino acid substitution, alpha-Tyr129----Cys. This residue, which is conserved in all phycocyanins sequenced to date, forms part of the alpha-84 bilin binding site and lies within 5 A of alpha-Cys84. A mutated cpcA gene containing this substitution was constructed by site-directed mutagenesis and transformed, along with cpcB, into a cpcBAC deletion strain containing an insertionally inactivated cpcE. This strain produces high levels of phycocyanin and the majority of the alpha subunit carries PCB at alpha-Cys84.

Amino Acid Sequence↗

PubMed1992

Amplification of HER-2/neu oncogene in human ovarian cancer.

Amplification and/or increased expression of the HER-2/neu oncogene has been reported to occur in ovarian tumors and possibly to correlate with biologic behavior and prognosis. The frequency with which amplification is reported to occur is quite variable ranging from 0-30% in different series and this variability is probably accounted for by technical and methodologic factors. The variability and lack of reproducibility has raised questions about the usefulness of assessing amplification of the HER-2/neu oncogene and in particular its clinical relevance. In this study by using strict criteria for amplification and using multiple controls we could demonstrate unequivocal amplification of the HER-2/neu oncogene by Southern blot analysis in only 11% of malignant ovarian tumors. The potential pitfalls with the techniques used to detect HER-2/neu oncogene amplification and overexpression are reviewed and possible ways to overcome some of the problems are suggested.

Journal Article↗

PubMed1989

Amplification of c-ras-Ki oncogene in human ovarian tumours.

Amplification of the c-ras-Ki oncogene has been the most consistent finding reported in studies on oncogene activation in ovarian cancer, but for the most part the studies have been small and the results conflicting. In order to determine whether amplification occurred de novo in primary tumours or was associated with tumour progression and metastasis, 81 tumour samples from different sites in 26 patients with ovarian tumours and 7 xenografted ovarian tumour cell lines were assayed. Amplification of c-ras-Ki occurred infrequently and was apparent in a single metastatic site in a patient with poorly differentiated serous cystadenocarcinoma of the ovary. Ten-fold amplification of the c-ras-Ki oncogene was also evident in a benign ovarian fibroma. Amplification of the c-ras-Ki oncogene is rare in ovarian tumours and does not appear to play a fundamental role in tumor development or progression.

Blotting, Southern↗

PubMed1987

Immunohistochemical detection of proliferating cells in vivo.

Incorporation of the thymidine analogue 5-bromo-2'-deoxyuridine (BrUdR) into newly synthesized DNA provides the basis of a simple technique for identifying proliferating cells. BrUdR was administered to C57BL/6 mice by continuous infusion for 1-7 days, or by intraperitoneal injection for shorter intervals. Various tissue types, including gut, kidney, and liver, were excised, fixed in neutral buffered formalin, and paraffin-embedded for sectioning. De-paraffinized 4-micron tissue sections and bone marrow samples were incubated with an anti-BrUdR antibody and cells that had traversed S-phase during the BrUdR exposure period were identified immunohistochemically. Proliferation and migration of intestinal epithelial cells were identified by antibody staining after continuous in vivo exposure to BrUdR for 1-4 days, and BrUdR incorporation into proliferating marrow cells was detected within 30 min. Tissues such as normal liver, known to have low levels of proliferation, remained unstained after 3 days' exposure to BrUdR. After we established that normal proliferating cells could be identified using this technique, BrUdR was administered to mice bearing B16 melanomas. Again, proliferating tumor cells were clearly identified in histological sections. The nuclei from these paraffin-embedded tumors were also collected for flow cytometric analysis after de-waxing, rehydration, and pepsin treatment. This combination of techniques made possible the comparison in adjacent tissue sections of labeling index, obtained from stained sections, with percentage S-phase, measured using DNA flow cytometry. The % S-phase was consistently higher than the labeling index obtained with immunocytochemistry, and two-parameter DNA vs BrUdR flow cytometry showed that this difference could be accounted for by a population of unlabeled cells with an S-phase DNA content.

Animals↗

PubMed1987

Chemical and toxicological characterization of residential oil burner emissions: I. Yields and chemical characterization of extractables from combustion of No. 2 fuel oil at different Bacharach Smoke Numbers and firing cycles.

Particulates and complex organic mixtures were sampled from the exhaust of a flame retention head residential oil burner combusting No. 2 fuel oil at three firing conditions: continuous at Bacharach Smoke No. 1, and cyclic (5 min on, 10 min off) at Smoke Nos. 1 and 5. The complex mixtures were recovered by successive Soxhlet extraction of filtered particulates and XAD-2 sorbent resin with methylene chloride (DCM) and then methanol (MeOH). Bacterial mutagenicity [see Paper II (8)] was found in the DCM extractables. Samples of DCM extracts from the two cyclic firing conditions and of the raw fuel were separated by gravity column chromatography on alumina. The resulting fractions were further characterized by a range of instrumental methods. Average yields of both unextracted particulates and of DCM extractables, normalized to a basis of per unit weight of fuel fired, were lower for continuous firing than for cyclic firing. For cyclic firing, decreasing the smoke number lowered the particulates emissions but only slightly reduced the average yield of DCM extractables. These and similar observations, here reported for two other oil burners, show that adjusting the burner to a lower smoke number has little effect on, or may actually increase, emissions of organic extractables of potential public health interest. Modifications of the burner firing cycle aimed at approaching continuous operation offer promise for reducing the amount of complex organic emissions. Unburned fuel accounted for roughly half of the DCM extractables from cyclic firing of the flame retention head burner at high and low smoke number. Large (i.e., greater than 3 ring) polycyclic aromatic hydrocarbons (PAH) were not observed in the DCM extractables from cyclic firing. However, nitroaromatics, typified by alkylated nitronaphthalenes, alkyl-nitrobiphenyls, and alkyl-nitrophenanthrenes were found in a minor subfraction containing a significant portion of the total mutagenic activity of the cyclic low smoke samples (8). Oxygen-containing PAH, typified by phenalene-1-one and its alkyl derivatives, are important mutagens from cyclic firing at high smoke conditions. Thus, oil burner effluents differ markedly from those of several other combustors, including the automotive diesel engine, where multiring PAH, typified by fluoranthene and alkylated phenanthrenes, account for a significant portion of the effluent mutagenicity. Implications for combustion and emissions source identification are discussed.

Air Pollutants↗

PubMed1987

Metastatic adeno or undifferentiated carcinoma from an unknown primary site--natural history and guidelines for identification of treatable subsets.

Two hundred and eighty-six patients presenting with metastatic adenocarcinoma or undifferentiated carcinoma whose primary site was not identified by clinical history, physical examination and chest radiograph have been studied. Median survival from presentation was 22 weeks. Factors independently predicting improved survival were lymph node presentations, good performance status and body weight loss of less than 10 per cent. In 88 (31 per cent) patients the primary tumour site was subsequently identified, in 58 (20 per cent) during life. Lung cancer was the most frequently identified primary tumour, and in only 32 (11 per cent) of the patients was a 'treatable' primary tumour (i.e. germ cell, breast, ovarian, prostate, thyroid cancer or lymphoma) identified. Among the treatable primary tumours were those in eight out of 16 female patients presenting with axillary metastases who were subsequently shown to have primary breast cancer and four of 13 females presenting with ascites who were found to have primary ovarian cancer. Prostatic cancer was confirmed in five out of 13 men with raised serum acid phosphatase. Of 22 patients with elevated serum alphafoetoprotein (AFP) or beta-human chorionic gonadotrophin levels (beta HCG) 18 had some features of the 'atypical teratoma syndrome'. Of the total of 32 patients with treatable tumour types, 29 (90 per cent) were identified during life. Median survival for patients with treatable tumour types identified during life was 104 weeks, compared with 22 weeks for the group as a whole. Retrospective immunocytochemical staining of the original biopsy showed that prostatic specific antigen and antibodies to beta HCG and AFP were diagnostically useful, but a series of organ site non-specific markers of histogenesis or cellular differentiation (carcinoembryonic antigen, secretory component for IgA, peanut lectin binding, epithelial membrane antigen and keratin) showed no significant correlations with identified primary sites, responsiveness to empirical chemotherapy or survival. Metastatic undifferentiated carcinoma or adenocarcinoma from an unknown primary site represents 6.5 per cent of all referrals to the medical oncology unit, Royal Prince Alfred Hospital, Sydney. We offer guidelines for the rapid identification of the limited number of primary sites for which effective and specific forms of systemic treatment are available.

Adenocarcinoma↗

PubMed1986

Post-translational methylation of asparaginyl residues. Identification of beta-71 gamma-N-methylasparagine in allophycocyanin.

A novel post-translationally modified residue, gamma-N-methylasparagine, was detected in the beta subunit of Anabaena variabilis allophycocyanin. Structure determination was accomplished by isolating a decapeptide, AP-beta (63-72) shown to have the following structure: Ser-Asp-Ile-Thr-Arg-Pro-Gly-Gly- Asn[N-CH3]-homoserine lactone Fast atom bombardment-mass spectrometry established that the residue corresponding to position 71 in the protein (DeLange, R. J., Williams, L. C., and Glazer, A. N. (1981) J. Biol. Chem. 256, 9558-9566) contained 13 mass units more than expected for aspartic acid though aspartic acid was recovered after acid hydrolysis. The 1H NMR spectrum of AP-beta (63-72) revealed a strong methyl single at 2.71 ppm characteristic of the methyl derivative of an amide nitrogen. Confirmation of this bond arrangement was obtained by detection of a stoichiometric amount of methylamine in acid hydrolysates of the peptide. This is the first report of gamma-N-methylasparagine in a protein. Amino acid analysis of A. variabilis allophycocyanin subunits showed that the derivative at position 71 can account for the total methylamine released from the beta subunit, while hydrolysis of the alpha subunit released no methylamine. The beta subunits of the allophycocyanins from the cyanobacterium Synechococcus PCC 6301 and the red alga Porphyridium cruentum each released 1 eq of methylamine upon acid hydrolysis. No methylamine was released from the alpha subunits.

Amino Acid Sequence↗

PubMed1986

Isolation and identification of 1(3),2-diacylglyceryl-(3)-O-4'-(N,N,N-trimethyl)homoserine from the soil amoeba, Acanthamoeba castellanii.

A polar lipid accounting for 12.5% of the total lipid nitrogen has been isolated from the protozoan Acanthamoeba castellanii. On the basis of thin-layer chromatography and mass spectral analysis, the lipid has been identified as diacylglyceryltrimethylhomoserine (DGTS). Fast atom bombardment (FAB) mass spectra of DGTS are reported for the first time and are compared to the FAB mass spectra of phosphatidylcholines and the electron ionization (EI) and field desorption (FD) mass spectra of DGTS. Gas-liquid chromatographic-mass spectrometric (GLC-MS) analysis of the acyl chain composition of this lipid has shown that 87.5% consists of cis-9-octadecenoic acid. Plasma membrane isolated from this organism has shown that labeled DGTS appears in the plasma membrane but is not enriched in this fraction. DGTS has been isolated previously only from a limited number of green plants and one species of fungus. Identification of this lipid in Acanthamoeba indicates that this lipid is distributed among a diverse group of lower eucaryotes.

Amoeba↗