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.

High anticancer efficacy of L-proline-m-bis (2-chloroethyl) amino-L-phenylalanyl-L-norvaline ethyl ester hydrochloride (MF13) in vivo.

The anticancer efficacy of the new anticancer tripeptide, L-proline-m-bis (2-chloroethyl) amino-L-phenylalanyl-L-norvaline ethyl ester hydrochloride (MF13), was investigated in mice. MF13 showed a therapeutic effect in liquid tumors and induced complete remission even in late stage malignancies. MF13 also inhibited human colon cancer growth in nude mice by more than 85% (volume, p<0.001). It acted in a dose-dependent manner and induced a complete regression of tumor in 20% of the mice when the initial dose was high (15 mg/kg, i.p.). Human melanoma exhibited a response to MF13 similar to colon cancer. Activity of MF13 in murine hepatoma in vivo was stronger than its precursor m-sarcolysin (p<0.001). Tumor cells in peritoneal cavities of the MF13 treated (s.c.) mice underwent an irreversible apoptosis. Side effects of MF13 were the transient depression of hemopoiesis and loss of body weight, which vanished within 9-10 days. LD50 of MF13 of a single i.p. injection was 27 mg/kg (94 mg/m2), 11 times higher than the therapeutic dose of a single injection.

Mode of action of methotrexate-albumin in a human T-cell leukemia line and activity against an MTX-resistant clone.

Limitations of low mol. wt anticancer drugs are short tumor exposure times and toxicity to normal tissue. Methotrexate (MTX) covalently linked to human serum albumin (HSA) as a macromolecular carrier caused tumor regressions concomitant with a favorable toxicity profile in a clinical phase I trial (Hartung et aL, Clin. Cancer Res., 1999, 5, 753). We examined the uptake, intracellular degradation, metabolism and thymidylate synthase (TS) inhibition of MTX-HSA in the T-cell leukemia line CCRF-CEM and the MTX transport resistant clone CCRF-CEM/MTX. The number of MTX molecules per albumin molecule was determined by electrospray mass spectrometry. A loading ratio (LR) of approximately 1.4 mol MTX/albumin revealed intact complexes with one and two MTX molecules/albumin. In the complex with an LR of 5.7, albumin with up to 16 MTX molecules was seen. MTX-HSA was taken up by CCRF-CEM cells via endocytosis and cleaved by lysosomal enzymes. Liberated MTX was a poor substrate of folylpolyglutamate synthetase and was exported into the medium. TS was inhibited and cell survival was impaired by MTX-HSA in a time- and concentration-dependent manner. CCRF-CEM/MTX cells exhibited a growth inhibition of 30-40% after MTX-HSA treatment, but no TS inhibition. The alternative uptake route via endocytosis enables MTX-HSA to overcome transport resistance to MTX.

3-m-bromoacetylamino benzoic acid ethyl ester: a new cancericidal agent that activates the apoptotic pathway through caspase-9.

The mechanism underlying the cancericidal activity of 3-m-bromoacetylamino benzoic acid ethyl ester (3-BAABE) was investigated. 3-BAABE exerted a strong cancericidal effect on human leukemia and lymphoma cells (IC(50) < 0.2 microgram/mL) and on cell lines of prostate, colon, ductal, and kidney cancer (IC(50) 0.8 to 0.88 microgram/mL). Multiple drug resistance (MDR) had no effect on the susceptibility of human lymphoma cells to 3-BAABE, since Daudi/MDR(20) and wild-type Daudi cells had a similar susceptibility to the cytotoxic effect of 3-BAABE. The cancericidal effect of 3-BAABE, which was not associated with changes in the cell cycle, was mediated by apoptosis. Thus, cells exposed to 3-BAABE displayed the DNA fragmentation ladder characteristic for apoptosis, associated with a marked increase of the activity of apoptosis effector caspases-3 and -6, which was followed by proteolytic cleavage of DNA fragmentation factor (DFF) and poly(ADP-ribose) polymerase (PARP). Exposure of tumor cells to 3-BAABE increased the activity of apical caspase-9, but had no effect on caspase-8. Complete inhibition of 3-BAABE-induced apoptosis was exerted by LEHD-FMK, a caspase-9 inhibitor. DEVD-FMK, a caspase-3 inhibitor, and VEID-FMK, a caspase-6 inhibitor, partially inhibited 3-BAABE-induced apoptosis, whereas exposure to IETD-FMK, a caspase-8 inhibitor, had no effect. The fragmentation and elevated activity of caspase-9 in 3-BAABE-treated cells and the fact that only an inhibitor of caspase-9 abrogated 3-BAABE-induced apoptosis indicate that 3-BAABE is a distinctive compound that elicits apoptosis through a pathway that is limited specifically to activation of apical caspase-9.

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.

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.

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.

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).

Synthesis of diacylamines and the preparation of alpha-amino-acylureas, a new type of alpha-amino acid derivatives.

Sixteen new and one known unsymmetrical open-chain diacylamines were synthesized by sodium methoxide catalyzed acylation of amides with carboxylic esters and acylamino-carboxylic esters, or acylureas with acylamino-carboxylic esters and alpha-amino acid esters.

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.

Concentrations of soluble CD95 and CD8 antigens in the plasma and levels of CD8+CD95+, CD8+CD38+, and CD4+CD95+ T cells are markers for HIV-1 infection and clinical status.

Apoptosis mediated via the CD95 (FAS/APO-1) receptor is thought to play a role in the depletion of CD4+ T cells in HIV infection. In the present study expression of the CD95 antigen on lymphocyte subsets and the plasma level of soluble CD95 (sCD95) were determined in HIV-1-infected adults. The expression of CD95 was increased on CD8 cells in all groups of HIV+ individuals, while increased expression of CD95+ cells on CD4 cells was limited to individuals with CD4 counts of < 200 mm3. The proportion of CD4+ that expressed CD95 was inversely correlated with the percentage of CD4+ PBL. The concentration of sCD95 was significantly higher in the plasma of HIV-infected individuals than in normal controls. The level of sCD95 in HIV-infected subjects showed no correlation with the percentage of PBL expressing CD95, indicating that the increased level of sCD95 did not reflect release from CD95+ PBL. The plasma sCD95 concentration was significantly correlated with the percentage of CD8+ cells and, particularly, with CD8+ CD38- cells. A striking inverse correlation was found between the sCD95 plasma concentration and the proportion of CD4+ CD95+ cells out of the total CD4+ population. There was no correlation between the serum level of sCD95 and that of soluble CD8 (sCD8), both of which were increased in the plasma of HIV+ individuals. Unlike the level of sCD95, the level of sCD8 in the plasma of HIV+ individuals. Unlike the level of sCD95, the level of sCD8 in the plasma of HIV+ individuals was correlated with the percentage of CD95+ and CD8+ CD38+ cells. The present study indicates that plasma sCD95 may be one of the factors that regulate apoptotic death of lymphocytes in HIV infection.

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.

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.

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.

Increased expression of activation markers on CD8 lymphocytes in children with human immunodeficiency virus-1 infection.

The aims of the present study were to analyze the impact of perinatal human immunodeficiency virus (HIV)-1 infection on lymphocyte maturation in children, to determine the expression of activation markers on CD8+ cells, and to define predictors of survival in HIV-infected children. Seventy-one children presenting HIV-related symptoms were included in the study; 29 were less than 2 y old and 42 were 2 to 12 y of age. Results were compared with those obtained in normal children of a similar age. In HIV-infected children the proportion of CD4+ and CD8+CD45RA+ cells was significantly decreased, whereas that of CD8+, CD8+CD38+, and CD8+CD45RO+ cells was strikingly increased compared with controls. In children less than 2 y old the absolute number of CD4+ and CD8+CD45RA+ cells decreased, and the number of CD8+CD45RO+ cells increased significantly, whereas the number of CD8+ and CD8+CD38+ cells did not change. The absolute number of CD4+ T cells declined with age both among controls and among HIV-infected children. In contrast, the absolute number of CD8+ cells and CD8 subsets decreased with age only in controls but not in infected children. In HIV-1-infected children the expression of the CD38 and CD45RO markers on CD8+ cells was significantly correlated, indicating that these were activated cells. The survival of less than 2-y-old children with AIDS symptoms was positively correlated with the total number of CD8 cells and CD8+CD38+ cells at time of entry into the study.(ABSTRACT TRUNCATED AT 250 WORDS)

A distinctive form of soluble CD8 is secreted by stimulated CD8+ cells in HIV-1-infected and high-risk individuals.

The aim of the present study was to investigate the biochemical structure and pathogenic significance of the soluble CD8 (sCD8) present in the serum of HIV-1-infected individuals. In a longitudinal study of a cohort of HIV-infected homosexuals and the amount of sCD8 detected in the plasma was correlated with changes in lymphocyte subsets and with the clinical course of HIV infection. The level of sCD8 in the plasma, the percentage, and the absolute number of CD8+CD38+ cells were increased in HIV-seronegative, high-risk homosexuals and in seropositive HIV+ individuals. The plasma concentration of serum sCD8 showed a significant correlation with the absolute number of CD8+ and CD8+CD38+ cells in HIV+ homosexuals. In addition to a molecule with a molecular weight (m.w.) of 30 kDa, sCD8 isolated from the plasma of HIV-1-infected individuals and of healthy controls was found to consist of two molecules, one with a m.w. of 57 to 62 kDa and another with a m.w. of 66 to 70 kDa. The former was the predominant molecule in normal individuals, while the latter was the predominant molecule in HIV-negative high-risk homosexuals and in HIV-infected individuals. The latter molecule, secreted by chronically stimulated CD8+ cells, seems to be present in the circulation as a dimer. While it was previously shown that CD8 can be shed from the cell membrane in vitro, the present study indicates that in vivo-stimulated CD8+ cells release a distinctive form of soluble CD8.

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.

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)

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.

Characterization of a unique mucin-like glycoprotein secreted by a human endometrial adenocarcinoma cell line (Ishikawa).

A human endometrial adenocarcinoma cell line (Ishikawa) has been shown to incorporate [3H]glucosamine and to secrete a radiolabeled high molecular weight compound which is excluded from a Sepharose CL-2B column. The excluded material was resistant to hyaluronidase, chondroitinase ABC, and heparinase. These findings rule out the possibility of this material being a proteoglycan. The susceptibility of this material to digestion with pronase, neuraminidase, and alkaline borohydride treatment strongly suggests that the excluded material is an O-glycosidic glycoprotein. The glycoprotein secreted by Ishikawa cells (ICGP) did not react immunologically with antibodies against either lactoferrin or fibronectin, but did react with an antibody made against tracheal mucin. Conversely, immunoblot analysis revealed that an antibody made against ICGP did not recognize hyaluronic acid, chondroitin, heparin, nasal turbinate mucin, bovine submaxillary gland mucin, lactoferrin, or fibronectin, but did recognize tracheal mucin. Analysis of ICGP amino acid and carbohydrate composition showed that it is rich in serine, threonine, glutamic acid, aspartic acid, and N-acetylneuraminic acid. In this respect, ICGP differs from other mucins, even though it is immunologically similar to respiratory mucin; hence we may consider ICGP to be a mucin-like glycoprotein. Secretion of ICGP can be modulated by Ca(2+)-ionophore and other mucus secretagogues, such as platelet activating factor, carbachol, and monocyte/macrophage mucus secretagogue, all mediators of lung inflammation. Ishikawa cells and anti-ICGP antibody may be used in studies on in vitro regulation of mucin-like glycoprotein synthesis and secretion in the respiratory tract as well as in the endometrium.