The prognostic value of serum MMP-7 levels in prostate cancer patients who received docetaxel, abiraterone, or enzalutamide therapy.

OBJECTIVES: The rapidly changing treatment landscape in metastatic castration-resistant prostate cancer (mCRPC) calls for biomarkers to guide treatment decisions. We recently identified MMP-7 as a potential serum marker for the prediction of response and survival in mCRPC patients who received docetaxel (DOC) chemotherapy. Here, we aimed to test this finding in an independent patient cohort and in addition to explore the prognostic potential of serum MMP-7 in abiraterone (ABI) or enzalutamide (ENZA) treated patients. METHODS AND MATERIALS: MMP-7 levels were measured in 836 serum samples from 320 mCRPC patients collected before and during DOC (n = 95), ABI (n = 140), or ENZA (n = 85) treatment by using the ELISA method. Results were correlated with clinical and follow-up data. RESULTS: MMP-7 baseline levels were similar between the 3 treatment groups. In the ABI and ENZA cohorts, baseline MMP-7 levels were lower in patients with prior radical prostatectomy (P = 0.058 and P = 0.041, respectively). Baseline MMP-7 levels above the median were associated with shorter overall survival for the DOC (P = 0.001) and ENZA (P = 0.006) cohorts. Multivariable analyses in the DOC and ENZA cohorts revealed that high pretreatment MMP-7 level is an independent risk factor for patients' survival. In addition, in DOC-treated patients with high baseline MMP-7 level, marker decrease at the third DOC cycle was associated with improved survival. Patients with high baseline MMP-7 levels had better survival when treated with ABI compared to DOC or ENZA. CONCLUSIONS: We confirmed the prognostic value of pretreatment MMP-7 serum level and its changes as independent predictors of survival in DOC-treated mCRPC patients. In addition, high MMP-7 was a negative predictor in ENZA-treated but not in ABI-treated patients. These results warrant further research to confirm the predictive value of serum MMP-7 and to explore the potential mechanistic involvement of MMP-7 in DOC and ENZA resistance of mCRPC patients.

Development of immunosensor based on OWLS technique for determining Aflatoxin B1 and Ochratoxin A.

Mycotoxins are toxic secondary metabolites produced by a number of different fungi, and can be present in a wide range of food and feed commodities including cereal grains, oil seeds, dried fruits, apple juice, wine and meat products from animals fed contaminated meal. Many mycotoxins are highly resistant, and survive food processing, and therefore enter the food chain and provide a threat to human health. The optical waveguide lightmode spectroscopy (OWLS) technique has been applied to the detection of Aflatoxin and Ochratoxin in both competitive and in direct immunoassays. After immobilizing the antibody or antigen conjugate for the direct or indirect measurement, respectively, the sensor chip was used in flow-injection analyser (FIA) system. When using non-competitive method, sensor responses were obtained first only at analyte concentrations of 5-10 ng ml(-1). In both cases, the responses were very unstable. For competitive sensor investigation with the sensitized chip first the optimal dilution rate of monoclonal antibodies was determined, for the measurement of Ochratoxin A and Aflatoxin B1 the monoclonal antibody stock solution was diluted to 1 microg ml(-1) and to a 1:400 dilution, respectively. During the competitive measurement standard solutions were mixed with monoclonal antibodies at the appropriate concentration, the mixture was incubated for 1 min and injected into the OWLS system. The sensitive detection range of the competitive detection method was between 0.5 and 10 ng ml(-1) in both cases. After the establishment of the indirect method, barley and wheat flour samples were measured, and the results were in good correlation by those measured by enzyme linked immuno-sorbent assay (ELISA). Regression coefficient between the two methods for Ochratoxin and Aflatoxin was determined as 0.96 and 0.89, respectively.

Space charge effect on the electrostatic collection of thoron decay products.

Investigations on electrostatic collection of thoron decay products as a function of field strength in a special source-electrode arrangement have been performed. Yields of the decay products vs. field strength were measured by solid state silicon and CR-39 track-etched detectors. Results show a significant space charge effect on the yield of collected products if an insulator is placed between the electrodes. The intensities of the collected thoron daughters are influenced strongly by the air stream present in the cell.

Low or absent SPARC expression in acute myeloid leukemia with MLL rearrangements is associated with sensitivity to growth inhibition by exogenous SPARC protein.

Secreted protein, acidic and rich in cysteine (SPARC), is a matricellular glycoprotein with growth-inhibitory and antiangiogenic functions. Although SPARC has been implicated as a tumor suppressor in humans, its function in normal or malignant hematopoiesis has not previously been studied. We found that the leukemic cells of AML patients with MLL gene rearrangements express low to undetectable amounts of SPARC whereas normal hematopoietic progenitors and most AML patients express this gene. SPARC RNA and protein levels were also low or undetectable in AML cell lines with MLL translocations. Consistent with its tumor suppressive effects in various solid tumor models, exogenous SPARC protein selectively reduced the growth of cell lines with MLL rearrangements by inhibiting cell cycle progression from G1 to S phase. The lack of SPARC expression in MLL-rearranged cell lines was associated with dense promoter methylation. However, we found no evidence of methylation-based silencing of SPARC in primary patient samples. Our results suggest that low or absent SPARC expression is a consistent feature of AML cells with MLL rearrangements and that SPARC may function as a tumor suppressor in this subset of patients. A potential role of exogenous SPARC in the therapy of MLL-rearranged AML warrants further investigation.

Modification of the surface of integrated optical wave-guide sensors for immunosensor applications.

Methods have been developed which enable attachment amino and epoxy groups to the surface of integrated optical wave-guide sensors for immunosensor applications. The SiO2-TiO2 surfaces were modified by use of the trifunctional silane reagents gamma-aminopropyltriethoxysilane (APTS) and gamma-glycidoxypropyltrimethoxysilane (GOPS) in organic and/or in inorganic phases. Silanization methods were optimized taking into consideration the concentration of silane reagent used and the temperature and time of reaction. To evaluate the layers formed, immobilization experiments were undertaken on the modified surfaces using the bovine serum albumin (BSA)-anti-BSA IgG antibody model molecule pair. The regenerability of the sensitized surfaces was also studied.

Determination of the ratio of D- and L-amino acids in brewing by an immobilised amino acid oxidase enzyme reactor coupled to amperometric detection.

To determine the quantity of free amino acids, the D- and L-forms separately, is an important task in modern nutritional studies. The aim of our present work was to develop rapid, routine methods for fast determination of the different forms of free amino acids. We utilized two enzymes (L-amino acid oxidase, D-amino acid oxidase) with broad specificity. In our home-made reactors, the enzymes were immobilized in a thin-layer Plexi-cell on natural protein membrane. The enzyme-cell was built into a FIA system and the hydrogen peroxide generated during the enzymatic reaction was determined by an amperometric detector. The electrode potential was fixed at +100 mV. The parameters for the biochemical and electrochemical reactions were optimized in each case. The optimal pH value for measuring L- and D-amino acids was found ca. 8.8 and 9.5, respectively. The LAO reactor could be used for more than 900 measurements, while the DAO reactor for about 1000 measurements. The working concentration range was between 0.1-3 and 0.2-3 mM, respectively. The same standard solution (L- and D-Methionine, 1 mM) was injected 25 times sequentially and the standard deviations were 2 and 2.7%, respectively. After determining the optimal parameters, the specificity of the immobilized enzyme preparations towards different amino acids and in samples from different stages of brewing was investigated.

Critical role of conserved proline residues in the transmembrane segment 4 voltage sensor function and in the gating of L-type calcium channels.

The fourth transmembrane segment (S4) has been shown to function as a voltage sensor in voltage-gated channels. On membrane depolarization, a stretch of S4 moves outward and initiates a number of conformational changes that ultimately lead to channel opening. Conserved proline residues are in the middle of the S4 of motifs I and III in voltage-dependent Ca2+ channels. Because proline often introduces a "kink" into a helical structure of proteins, these residues might have an intrinsic function in the voltage sensor. Here, we report that the removal of S4 prolines results in a dramatic shortening of channel open time whereas the introduction of extra prolines to the corresponding positions in motif IIS4 and IVS4 lengthens channel open time. The number of S4s with a proline residue showed a clear positive correlation with the mean open time of the channel. The mean open time was >11-fold longer for a channel mutagenized to have prolines in all four S4s compared with a channel that had no prolines in the S4 region. Additionally, prolines in the S4s slowed activation kinetics and shifted the voltage dependence of activation and inactivation in a hyperpolarized direction. Our results strongly suggest that proline residues in the S4s are critical for stabilizing the open state of the channel. Moreover, it is suggested that motif IS4 and IIIS4 contribute to the channel opening more efficiently than motif IIS4 and IVS4.

cAMP-dependent phosphorylation sites and macroscopic activity of recombinant cardiac L-type calcium channels.

The involvement of cAMP-dependent phosphorylation sites in establishing the basal activity of cardiac L-type Ca2+ channels was studied in HEK 293 cells transiently cotransfected with mutants of the human cardiac alpha1 and accessory subunits. Systematic individual or combined elimination of high consensus protein kinase A (PKA) sites, by serine to alanine substitutions at the amino and carboxyl termini of the alpha1 subunit, resulted in Ca2+ channel currents indistinguishable from those of wild type channels. Dihydropyridine (DHP)-binding characteristics were also unaltered. To explore the possible involvement of nonconsensus sites, deletion mutants were used. Carboxyl-terminal truncations of the alpha1 subunit distal to residue 1597 resulted in increased channel expression and current amplitudes. Modulation of PKA activity in cells transfected with the wild type channel or any of the mutants did not alter Ca2+ channel functions suggesting that cardiac Ca2+ channels expressed in these cells behave, in terms of lack of PKA control, like Ca2+ channels of smooth muscle cells.

Application of biosensor for monitoring galactose content.

The quality and quantity of different sugars play a very important role in studying the carbohydrate metabolism of yeast. During the bioprocesses there is a need to follow the concentrations of these sugars. Authors have reported on the development of biosensors for determination of glucose and maltose previously. The aim of this research was to construct a sensor for determining galactose in fermentation broths to prepare the basis for an online monitoring system. Using a modified thin-layer enzyme cell connected to an electrochemical detector cell, a biosensor has been developed for this purpose. Galactose was oxidized with immobilized galactose oxidase enzyme (EC 1.1.3.9) and the hydrogen peroxide generated during the enzyme reaction was determined with an amperometric detector. The parameters for the biochemical and electrochemical reactions were optimized. The pH optimum of 6.6 was found when using phosphate buffer. The buffer solution completed by micro elements (Mg2+, Se2+) gave more stable signs. The activities for raffinose, lactose, glycerol and dihydroxyacetone were 68, 16, 6 and 430%, respectively. With the thin-layer cell more than 900 samples were measured in 6 weeks. Samples obtained from different fermentations were measured with the newly developed galactose sensor and the results were compared with the standard UV method. The correlation coefficient was 0.991. The results showed that the application of the new biosensor was successful.

Endogenous cardiac Ca2+ channels do not overcome the E-C coupling defect in immortalized dysgenic muscle cells: evidence for a missing link.

The expression of subunit genes of the Ca2+ channel complex was studied in differentiating, immortalized mouse mdg cells. These cells expressed alpha 1 and alpha 2/delta transcripts of the skeletal muscle Ca2+ channel genes, a cardiac Ca2+ channel alpha 1 subunit gene and several known transcript variants of skeletal, cardiac and brain beta genes. The mdg mutation is retained in the 129DA3 cell line and occurs exclusively at nucleotide position 4010 in the skeletal alpha 1 transcript in which a cytosine residue is deleted. In early stages of differentiation and fusion, Ba2+ currents were detected in dysgenic myotubes the same as the cardiac L-type Ca2+ channel. These data provide specific structural evidence [Chaudhari, N. (1992) J. Biol. Chem. 267, 25636-25639] for the major genetic defect in mouse muscular dysgenesis and show a change in the expression levels of alpha 1S and alpha 1C. The upregulation of the expression of alpha 1C results in functional Ca2+ channel activity, however, presumably not sufficient for excitation-contraction coupling.

Involvement of the carboxyl-terminal region of the alpha 1 subunit in voltage-dependent inactivation of cardiac calcium channels.

Intracellular application of proteases increases cardiac calcium current to a level similar to beta-adrenergic stimulation. Using transiently transfected HEK 293 cells, we studied the molecular mechanism underlying calcium channel stimulation by proteolytic treatment. Perfusion of HEK cells, coexpressing the human cardiac (hHT) alpha 1, alpha 2, and beta 3 subunits, with 1 mg/ml of trypsin or carboxypeptidase A, increased the peak amplitude of the calcium channel current 3-4-fold without affecting the voltage dependence. Similar results were obtained in HEK cells cotransfected with hHT alpha 1 and alpha 2 or with alpha 1 alone, suggesting that modification of the alpha 1 subunit itself is responsible for the current enhancement by proteolysis. To further characterize the modification of the alpha 1 subunit by trypsin, we expressed a deletion mutant in which part of the carboxyl-terminal tail up to amino acid 1673 was removed. The expressed calcium channel currents no longer responded to intracellular application of the proteases; however, a 3-fold higher current density as well as faster inactivation compared with the wild type was observed. The results provide evidence that a specific region of the carboxyl-terminal tail of the cardiac alpha 1 subunit is an important regulatory segment that may serve as a critical component of the gating machinery that influences both inactivation properties as well as channel availability.

Coexpression of skeletal muscle voltage-dependent calcium channel alpha 1 and beta cDNAs in mouse Ltk- cells increases the amount of alpha 1 protein in the cell membrane.

Coexpression of alpha 1 and beta subunits for all tissue-specific isotypes of calcium channels results in an increase in Ca2+ channel current density and drug receptor function. The quantification of the photoaffinity labeled skeletal muscle alpha 1 subunit polypeptide by two alternative immunoadsorption techniques showed that 3 times higher amount of calcium channel alpha 1 protein appears in the cell membrane when alpha 1 and beta subunits were coexpressed compared to alpha 1 alone. Using Western blotting and immunodetection techniques, we identified two polypeptide bands of alpha 1 with 210 kDa and 170 kD molecular mass, respectively. The combined intensity of these bands was 7-9 times higher when alpha 1 and beta were coexpressed compared to alpha 1 alone. The data provide evidence that coexpression of alpha 1 and beta results in an increased amount of alpha 1 protein in the cell membrane.

Studying the bienzyme reaction with amperometric detection for measuring maltose.

If a fermentation process is followed by a change in the concentration of maltose, information on the level of the maltose is important. A new type of sensor was developed for measuring the content of maltose in fermentation broth. The base of the sensor is a thin-layer reactor, in which a protein membrane was chosen for the immobilization of enzymes. To measure maltose, we investigated the influence of enzymes such as alpha-glucosidase (EC 3.2.1.20) and amyloglucosidase (EC 3.2.1.3) on the effectiveness of conversion to glucose. Because amyloglucosidase proved to be more effective, a mixture of amyloglucosidase and glucose oxidase was applied for the determination of maltose. To develop the best measuring technique, the consequences of changes in different parameters, such as the optimal ratio of enzymes, role of pH value and that of the flow rate, were studied. An amperometric measuring cell with Pt-Ag/AgCl-Pt electrodes was used at +600 mV operating potential. The results indicate that the maltose content in different types of fermentation broth can be determined by the new measuring cell in the range of 0.2-4 mM maltose. The cell was successfully tested in the fermentation of brewer's yeast.

Characterization of beta subunit modulation of a rabbit cardiac L-type Ca2+ channel alpha 1 subunit as expressed in mouse L cells.

Functional properties of a rabbit cardiac alpha 1 Ca2+ channel subunit (CARD alpha 1) were investigated using the patch-clamp technique in mouse L cells, a recipient cell line which is devoid of any Ca2+ channel subunits. Cell lines resulting from stable transfection of the CARD alpha 1 subunit as well as in coexpression with a beta subunit (CARD alpha 1 beta) derived from skeletal muscle (SKM beta) were characterized. The results show that while the CARD alpha 1-Ca2+ channel activity is negligible, the Ba2+ current density is dramatically increased in the presence of beta subunit (approximately 20-fold). CARD alpha 1- and CARD alpha 1 beta-Ba2+ currents were both sensitive to the 1,4-dihydropyridine (DHP) agonist, Bay K 8644 (5- to 8-fold increase). Activation kinetics of CARD alpha 1- and CARD alpha 1 beta-Ba2+ currents were comparable. The inactivation time-course was faster (3- to 4-fold) for CARD alpha 1 beta-Ba2+ currents. We conclude that the main role of the beta subunit in heart is to modulate the L-type current density and present several lines of evidence that SKM alpha 1 and CARD alpha 1 are differentially regulated by the beta subunit.

Acceleration of activation and inactivation by the beta subunit of the skeletal muscle calcium channel.

The L-type voltage-dependent calcium channel is an important link in excitation-contraction coupling of muscle cells (reviewed in refs 2 and 3). The channel has two functional characteristics: calcium permeation and receptor sites for calcium antagonists. In skeletal muscle the channel is a complex of five subunits, alpha 1, alpha 2, beta, gamma and delta. Complementary DNAs to these subunits have been cloned and their amino-acid sequences deduced. The skeletal muscle alpha 1 subunit cDNA expressed in L cells manifests as specific calcium-ion permeation, as well as sensitivity to the three classes of organic calcium-channel blockers. We report here that coexpression of the alpha 1 subunit with other subunits results in significant changes in dihydropyridine binding and gating properties. The available number of drug receptor sites increases 10-fold with an alpha 1 beta combination, whereas the affinity of the dihydropyridine binding site remains unchanged. Also, the presence of the beta subunit accelerates activation and inactivation kinetics of the calcium-channel current.