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Biomedical subjects

J Roboz

Publications and source records attributed to J Roboz.

At least 19 recordsLinked to original sources
PubMed2003

Putative protein markers in the sera of men with prostatic neoplasms.

OBJECTIVE: To describe the preliminary identification of serum proteins that may be diagnostic markers in prostate cancer. PATIENTS AND METHODS: The study included 11 men referred for treatment of localized prostate cancer, 12 with benign prostatic hyperplasia (BPH) and 12 disease-free controls. For serum protein analysis, the protein-chip array surface-enhanced laser desorption/ionization (SELDI) technique was used (Ciphergen Biosystems, Fremont, CA). SELDI combines protein-chip technology with time-of-flight mass spectrometry, and offers the advantages of speed, simplicity and sensitivity. RESULTS: Three protein peaks were identified in the serum of men with prostate cancer and BPH, but not in controls, with relative molecular masses of 15.2, 15.9 and 17.5 kDa. These three proteins were significantly associated with BPH and prostate cancer when compared with controls (P = 0.001, 0.004, and 0.011, respectively, Kruskal-Wallis test). Interestingly, the 17.5 kDa protein was more abundant in five men with stage T1 prostate cancer than in eight with stage T2 (P = 0.016, two tailed Mann-Whitney U-test corrected for ties). CONCLUSIONS: These proteins, particularly the 15.9 kDa one, may be used for the diagnosis or monitoring of prostate cancer and differentiation from BPH, and have the potential for antibody-based chip SELDI-TOF technology. Identified proteins may be targets for immunotherapy.

Biomarkers, Tumor↗
PubMed1998

Inhibition of microtubule assembly in tumor cells by 3-bromoacetylamino benzoylurea, a new cancericidal compound.

We have synthesized a new compound, 3-bromoacetylamino benzoylurea (3-BAABU), which showed strong cancericidal activity by inducing irreversible mitotic arrest and subsequently apoptosis in human T cell leukemic cells (CEM), human biphenotypic leukemic cells (SP), a human prostate cancer cell line (PC-3), murine melanoma cells (B-16), and murine lymphoma/leukemia cells (EL4) in vitro with an ID50 in the range of 0.013-0.07 microg/ml (0.04-0.22 microM). Treatment of tumor cells for 12-24 h with 3-BAABU resulted in mitotic arrest at prometaphase/metaphase/anaphase, with separation and dispersion of chromosomes and with the absence of mitotic spindle apparatus in cytoplasm. Treatment with 3-BAABU had no cytotoxic and mitotic blocking effect in normal human lymphocytes, proliferating fibroblast cells (3T3), or proliferating myocardial cells (MOT). Cell cycle analyses showed that most treated leukemic cells accumulated at M phase 12 h after treatment. By the end of 48 h of treatment, the cells underwent apoptosis with DNA fragmentation. 3-BAABU inhibited the assembly of microtubules from tubulin but did not interfere with the disassembly of microtubules. The presence and the position of bromine and urea groups on the benzoic ring are the determining factors for its inhibition of microtubule assembly. Replacing bromine with chlorine yielded much less mitotic blocking activity and increased the ID50 40-fold. Substitution of the urea group with ethyl ester abrogated the activity of blocking mitosis but induced apoptosis. Moving the bromoacetylamino group from the 3-position to the 4-position removed blocking activity for mitosis but induced necrosis. These results suggest that 3-BAABU possesses a unique and functional structure and is a potential agent for cancer chemotherapy.

Antineoplastic Agents↗
PubMed1998

Electrospray ionization mass spectrometric and liquid chromatographic-mass spectrometric studies on the metabolism of synthetic dynorphin A peptides in brain tissue in vitro and in vivo.

Metabolic stability of synthetic dynorphins [N-terminal fragments of dynorphin A (Dyn A)] were evaluated in vitro and in vivo. These peptides were applied at concentrations 100-1000 times higher than those of the endogenous dynorphins. Degradation kinetics of these peptides were studied in rat brain homogenate by using microbore gradient RP-LC assay, and limited information on their metabolism was obtained by electrospray ionization mass spectrometry (ESI-MS) of the isolated metabolites. In vivo cerebral microdialysis, in which the peptides were introduced via the probe placed in striatum region of the brain of the experimental animals, was used to circumvent contamination arising from autoproteolysis of brain during incubation of the samples in vitro. Metabolites of Dyn A (1-13) and Dyn A (1-11) were identified from electrospray ionization mass spectra of the microdialysates without chromatographic separation; the identification of peptides in the mixtures were supported by medium resolution ESI Fourier-transform ion cyclotron resonance MS. LC-MS was used to fully characterize the complex peptide mixture obtained after the striatal perfusion of Dyn A (1-12).

Amino Acid Sequence↗
PubMed1998

Investigation of suramin-albumin binding by electrospray mass spectrometry.

Suramin, an organic polyanion with six sulphonic groups, is under clinical trials as an agent against hormone-refractory prostate cancer. The drug binds strongly to serum albumin. The objectives were to use electrospray to measure the molecular masses of the intact complexes of albumin and suramin to determine the number of suramin molecules bound under different molar ratios, and to investigate the binding of suramin in human serum. With albumin in excess (2:1 to 25:1 ratio), only 1 and 2 bound suramins were found; with suramin excess (2:1 to 1000:1) up to 20 bound suramin molecules/albumin were found. Up to 5 bound suramins were found in human serum with 4:1 suramin:albumin ratio, which corresponds to recommended therapeutic doses (200-300 micrograms/mL). At 8:1 ratio, which would be toxic, complexes with up to ten bound suramin molecules were found, and unreacted albumin diminished.

Humans↗
PubMed1998

Synthesis, cancericidal and antimicrotubule activities of 3-(haloacetamido)-benzoylureas.

The four title compounds (not hitherto reported) were synthesized from 3-aminobenzoic acid through its trifluoroacetic acid-acid chloride derivative, reaction with urea and aminolytic deprotection to yield 3-aminobenzoylurea, followed by unconventional haloacetylation. Three key factors were found essential for antitumor activity: (i) the cytotoxic nature of the halogen: I > Br > Cl > F (ID90 0.014->10 microM); (ii) the position of the halogen: only the 3-position (meta) expressed relevant activity; and (iii) the presence of the urea group (1-position). The selectivity of the bromo and iodo compounds were higher than those of vinblastine and paclitaxel in terms of cytotoxicity (ID50 ratios in nonmalignant myocardial fibroblasts and CEM leukemia cells) and therapeutic indices (P338 leukemia bearing mice). Relevant mechanisms of bioactivity were mitotic arrest and apoptosis. Complete inhibition of microtubule assembly occurred in cell-free systems (at 2.8 versus 2.1 microM for vinblastine); in contrast to paclitaxel, the target compounds did not interfere with microtubule disassembly. The strong cancericidal and antimicrotubular activities of the bromine and iodine compounds justify further exploration of their potential in antineoplastic chemotherapy.

Animals↗
PubMed1997

Selective tumor apoptosis by MF13, L-prolyl-L-m-[bis(chloroethyl)amino]-phenylalanyl-L-norvaline ethyl ester, a new sarcolysin containing tripeptide.

ID50 and ID90 values for L-prolyl-L-m-[bis(chloroethyl)amino]-phenylalanyl-L-norvaline ethyl ester HCl (MF13), were determined in four murine (leukemia, lymphoma, melanoma, and lung) and eight human cancer cell lines (two leukemia, prostate, kidney, colon, two melanoma, and breast). Cytotoxic activity was 2-5 times higher than that of sarcolysin [(L-3-[bis(2-chloroethyl)amino]-L-phenylalanine] against all leukemias and lymphomas, ID50 0.5-0.9 microM, and against human solid tumors, ID50 0.4-2.1 microM. Sensitivities of L-phenylalanine mustard-resistant and methotrexate-resistant L1210 cells were the same as the naive lines, ID50 0.5 microM. Apoptosis was confirmed by: (a) morphology, revealing chromatin condensation and nuclear fragmentation; (b) flow cytometry, showing changes in cell size and DNA integrity; and (c) DNA electrophoresis, demonstrating multiples of 180-200-bp DNA units. MF13 had no cytotoxicity against human peripheral blood lymphocytes at concentrations lethal to tumor cells (ID50, 13.3 microM without and 11 microM with phytohemagglutinin stimulation) and failed to induce apoptosis. s.c. MF13 treatment of mice with advanced EL4 leukemic ascites yielded extensive apoptosis, with DNA degradation identical to that seen in vitro, and resulted in complete tumor regression in all treated mice. These results suggest MF13 as a potential chemotherapeutic agent.

Animals↗
PubMed1996

Characterization of digoxigenin derivatives of human coagulation factors VIIa and X by electrospray mass spectrometry and enzyme kinetics.

Digoxigenin ester (Dig) derivatives of coagulation factors VIIa and X facilitate staining studies to localize tissue factor activity in human atherosclerotic plaques. The larger the number of attached Dig units the easier it is to form highly visual stains. Electrospray ionization was used to characterize the Dig derivatives, as a function of excess derivatization reagent, to determine the optimal derivatives, i.e. the highest number of Dig units attached with the product still retaining enzymatic activity. The enzymatic activity of factor VIIa derivatized at 50-fold Dig excess (with 28, 34, 48 or 52 Dig units attached, masses to 79 kDa) remained the same as that of native factor VIIa. In contrast, the enzymatic activity of factor X derivatives diminished above 15-fold Dig excess (15 Dig units attached, mass 65.6 kDa). The distribution of derivatized lysine, histidine, arginine, tryptophan and tyrosine residues was estimated for several possible configurations.

Amino Acids↗
PubMed1996

Determination of hyaluronidase activity in venoms using capillary electrophoresis.

The technique used in this study was based on the addition of a known quantity of hyaluronic acid (HA) to an aliquot of crude venom sample, followed by obtaining capillary electrophoresis profiles both immediately after the mixing and after a known period of incubation. The presence of hyaluronidase (HYASE) and the degree of its activity were determined from the change in the abundance (peak height) of intact HA at its retention time. Quantitative and/or comparative enzyme activity could also be obtained from determining the incubation time needed either to achieve a selected percentage decrease in the size of the intact HA peak or to complete the digestion process as determined by the attainment of a constant profile of the oligosaccharide end products. The detection limit was 3 x 10(-6) U/microliter HYASE, defined as the decrease of the peak height of the added standard quantity of HA (0.008 mg/ml) from the initial signal-to-noise ratio of 6 down to 2; with respect to sample size, 1.5 x 10(-8) U of HYASE could be detected in 5 nl of incubated sample. The utility of the technique was illustrated by the rapid detection of HYASE activity in HYASE standards, crude bee venom and several snake venoms, the HYASE content of which has not yet been reported, and in collected high-performance liquid chromatography-separated venom fractions.

Animals↗
PubMed1996

In situ localization of tissue factor in human atherosclerotic plaques by binding of digoxigenin-labeled factors VIIa and X.

The mechanism responsible for the thrombotic complications of atherosclerotic plaques is not well understood. Although a role for tissue factor (TF) has been hypothesized, there are scant data on the presence, location, quantity, and activity of TF in atherosclerotic plaques. The purpose of this study was to show the localization of TF in human atherosclerotic plaques. Digoxigenin-labeled factors VIIa and X were used to demonstrate their specific binding sites in formalin-fixed, paraffin-embedded human arteries by incubation of sections with the labeled factor and localization of TF:factor(s) complexes by immunohistochemical staining for digoxigenin. In sections of atherosclerotic plaques, diffuse staining was most intense in the relatively acellular, lipid-rich core but was also present intracellularly in macrophages and smooth muscle cells and, to a lesser extent, in the relatively acellular fibrous tissue of the plaque. Endothelial cells overlying plaques and occasional medial smooth muscle cells stained positively as well. The adventitia routinely stained for TF in both normal and diseased artery segments. Staining for labeled factor VIIa was blocked when sections were preincubated with a 10-fold excess of unlabeled factor VIIa or with a polyclonal antihuman TF antibody. Binding of labeled factors VIIa and X was Ca(2+)-dependent. In conclusion, binding of digoxigenin-labeled factors VIIa and X shows that the lipid rich core of atherosclerotic plaques contains high levels of extracellular TF. This location may be responsible for the rapid initiation of thrombosis when lipid rich atherosclerotic plaques rupture and the core contents are exposed to flowing blood.

Animals↗
PubMed1994

Purification and characterization of three isolectins of soybean agglutinin. Evidence for C-terminal truncation by electrospray ionization mass spectrometry.

Soybean agglutinin (SBA) is a tetrameric D-Gal/D-GalNAc-specific lectin possessing one Man9 oligomannose-type chain/monomer. SBA exists as multiple isolectins having similar binding and immunochemical properties. The present study shows that native SBA consists of at least five isolectins. Three of these isoforms have been purified by chromatofocusing and designated as SBA-I, SBA-II and SBA-III in order of their elution from a chromatofocusing column. The pI of the isolectins are 7.0, 6.85 and 6.7, respectively, as determined by isoelectric focusing. Each isolectin was denatured in 6 M guanidine hydrochloride into their individual subunits which were separated by reverse-phase high performance liquid chromatography (RP-HPLC). The HPLC profiles were similar for all three isoforms which showed two major peaks (peak 1 and peak 3) along with a minor peak (peak 2). The first peak of SBA-II existed as a double labeled as 1 a and 1 b. Each peak was analyzed by electrospray ionization mass spectrometry to characterize each isoform and determine their structural differences. The calculated mass of an intact lectin monomer from the amino acid sequence (253 residues) derived from cDNA of the lectin including a Man9 oligomannose chain is 29438 Da. The present results show that peak 3 of each isoform corresponds to an intact subunit (alpha) while peak 1 of each isoform shows lower masses which are assigned to C-terminal fragmentation of the protein. Peak 1 of SBA-I has a molecular mass of 28000Da corresponding to a fragmented subunit (beta) consisting of 240 residues (calculated molecular mass 28001Da). Peak 1a of SBA-II shows a molecular mass of 28000Da corresponding to a fragmented beta subunit, while peak 1b showed two major species: a 28000-Da (beta subunit) and a 28327-Da subunit which corresponds to 243 residues (calculated mass 28326Da) designated as a gamma subunit. In addition, peak 1b showed the presence of a molecular species of 28627Da corresponding to a 246-residue subunit (gamma'). Peak 1 of SBA-III showed a major molecular species corresponding to a fragmented gamma subunit. The minor peak in the HPLC profile (peak 2) represented a subunit of 252 residues for all three isoforms. The results suggest that the subunit compositions of SBA-I, SBA-II and SBA-III are approximately alpha 2 beta 2, alpha 2 beta gamma and alpha 2 gamma 2, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1994

Diagnosis and monitoring of disseminated candidiasis based on serum/urine D/L-arabinitol ratios.

Disseminated candidiasis, a devastating disease with high morbidity and mortality in immunosuppressed patients, is difficult to diagnose because of the protean nature of symptoms and the lack of rapid and reliable laboratory diagnostic procedures. The subject of this review is the status of gas chromatographic-mass spectrometric techniques for the determination of D-arabinitol, a unique metabolite of pathogenic Candida species, in serum and urine. The enantiomers are separated by chiral chromatography followed by specific and sensitive detection using chemical ionization and selected ion monitoring. Using D/L-arabinitol ratios, instead of individual concentrations, eliminates the need for knowing the volume of samples and for calibration curves. A new filter paper technique requires only an unmeasured drop of whole blood (venous or finger/heel puncture) or urine; paper spots are mailable. Parallel determinations of D/L-arabinitol ratios in serum and urine in normal subjects and cancer patients with both normal and increased D/L-arabinitol ratios revealed constant (1.2-1.3 range) ratios of serum D/L-arabinitol/urine D/L-arabinitol for all populations studied. Analyzing two body fluids taken at the same time increases reliability by reducing false positives.

Biomarkers↗
PubMed1994

On-line buffer removal and fraction selection in gradient capillary high-performance liquid chromatography prior to electrospray mass spectrometry of peptides and proteins.

The technique for on-line removal of buffers and for fraction selection to eliminate source clogging and suppression of electrospray ionization by unwanted constituents consisted of: (i) selecting the initial mobile phase composition to retain analytes on top of a capillary high-performance liquid chromatography (HPLC) column; (ii) washing the buffer isocratically to waste, using a two-way micro dump valve; (iii) starting the gradient and switching the dump valve to introduce analytes into the source. Peptide and protein mixtures were prepared in 0.05-0.5 M phosphate and 0.55 mM tris-based buffers, and water. After buffer removal, chromatograms and electrospray spectra were indistinguishable from those of aqueous controls, down to 1 pmol (acidic fibroblast growth factor) consumed, and up to 78 kDa (bovine transferrin) molecular weight. Aided by the dump valve, 100 fmol of angiotensin could be fractionated and identified on the slope of a 10 x 10(6) fmol of leucine enkephalin HPLC peak. On-line buffer removal and fraction selection eliminate the need for additional preparation steps than can lead to excessive sample losses.

Buffers↗
PubMed1992

Diagnosis of disseminated candidiasis based on serum D/L-arabinitol ratios using negative chemical ionization mass spectrometry.

The main objective was to appraise the diagnostic specificity of the serum D/L-arabinitol ratio technique in a patient population biased for renal dysfunction. The D/L ratio (mean +/- S.D.) in normal serum (n = 29) was 1.76 +/- 0.47 (range 0.77-2.75). D/L-Arabinitol greater than 3.18 (mean + 3 S.D.) are considered indicative of disseminated candidiasis. Of 49 patients without candidiasis, but 46% with serum creatinine greater than 1.5 mg/dl, diagnostic specificity was 88%. In confirmed candidiasis (n = 16) sensitivity was 94% (D/L range 3.2-50.1). Switching from positive to negative chemical ionization permits the use of as little as 5 microliters sample (20 microliters used routinely; D/L ratios constant in the 5-200 microliters range) permitting the extension of the technique to pediatric applications. Results can obtained in 2 h. Suggested areas of clinical application include aiding diagnosis, monitoring patients as risk so that treatment could be initiated while fungus load is still small, and following the course of antifungal chemotherapy.

Candidiasis↗
PubMed1992

Zidovudine pharmacokinetics during pregnancy.

As the indications for zidovudine (ZDV) treatment in human immunodeficiency virus-infected individuals expand, we anticipate an increased use of this drug during pregnancy. We report pharmacokinetics data from a patient studied both in the third trimester and intrapartum. ZDV peak-plasma levels and serum half-lives were comparable to nonpregnant adults. High concentrations of ZDV and its glucuronide metabolite were found in umbilical cord blood and in amniotic fluid.

Adult↗
PubMed1991

Arabinitol enantiomers in cerebrospinal fluid.

D-Arabinitol is a metabolite of Candida species, and its presence in serum above endogenous concentration may indicate disseminated candidiasis. The o-trifluoroacetylated derivatives of arabinitol enantiomers in cerebrospinal fluid (CSF) were separated using perpentylated cyclodextrin capillary columns and measured by selected ion monitoring using negative chemical ionization mass spectrometry. The presence of D-arabinitol was confirmed using highly specific D-arabinitol dehydrogenase. The mean D/L-arabinitol ratio, 16.7 +/- 4.8 (range: 8.6-22.8), in CSF of the "controls" is approximately 10-fold higher than the ratio previously found in normal serum and urine. At the same time, the mean L-arabinitol concentration, 0.13 +/- 0.05 (range: 0.09-0.2), is virtually identical to that in serum. Therefore, the high D/L-arabinitol ratio in CSF is attributed to D-arabinitol. Persistently high D/L ratios were found in a variety of diseases (without Candida infection). The finding of D-arabinitol in CSF suggests that serum D-arabinitol may originate from the brain or the spinal cord, rather than from resident Candida species in the gut, and that the accumulation of D-arabinitol in CSF may be caused by non-consumption or, conversely, the high concentration may be maintained in order to have it readily available for metabolism in the brain.

Biomarkers↗
PubMed1990

Separation and quantification by gas chromatography-mass spectrometry of arabinitol enantiomers to aid the differential diagnosis of disseminated candidiasis.

To differentiate increased arabinitol due to fungal (only D-arabinitol) and non-fungal origin, O-trifluoroacetyl derivatives of the enantiomers were separated using alpha-perpentylated cyclodextrin columns and measured by selected ion monitoring. Mean +/- S.D. D/L in normal serum: 1.40 +/- 0.42. D/L ratios greater than 2.24, defined as normal mean + 2S.D., were considered outside normal range. D/L was greater than 2.2 in 10 of 12 confirmed candidiasis cases with one false negative and one borderline. Renal dysfunction without candidiasis yielded normal D/L despite high arabinitol concentrations. D/L in normal urine was nearly identical to that in serum despite 60 times larger concentration. D/L ratios, determined by peak heights or areas, could be used without the need to determine concentrations.

Candidiasis↗
PubMed1990

Polyol concentrations in serum during hemodialysis.

Polyol concentrations were determined by selected-ion monitoring in 10 sample sets taken before dialysis and hourly for 4 h during dialysis. Predialysis concentrations (mumol/L) exceeding the upper limit of normal (mean + 2 SD; n = 33) were found for erythritol (25.4-59.0), threitol (11.5-46.7), and arabinitol (21.0-49.3) in all 10 cases; mannitol (2.7-62.0) in nine cases; and xylitol (0.7-1.3) in eight cases. Concentrations of adonitol, galactitol, and sorbitol were all within normal limits. During dialysis, the concentration of six polyols decreased by approximately 50%, but there were no significant changes for xylitol and adonitol. Erythritol, threitol, and arabinitol concentrations remained more than double the upper limit of normal in all patients after 4 h of dialysis. High residual concentrations were found for mannitol (seven cases) and xylitol (six cases). The results suggest fundamental differences in the homeostasis as well as the dialyzability of different polyols. The potential toxic significance of the high residual concentrations after a 4-h dialysis needs to be investigated.

Homeostasis↗