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PubMed2005

EGFR gene copy number heterogeneity in fine-needle aspiration cytology from breast carcinomas determined by chromogenic in situ hybridization.

Most studies have shown epidermal growth factor receptor (EGFR) overexpression to be associated with poor prognostic factors in breast carcinomas. The relationship to EGFR gene copy number is unclear. The aim of our study was to investigate the heterogeneity of the EGFR gene copy number in breast carcinomas. The material consisted of air-dried smears from 29 breast carcinomas and 3 breast cancer cell lines (MCF-7, SKBR3, and T47D). Chromogenic in situ hybridization (CISH) was done using chromogenic detection. The mean signal numbers for EGFR gene and chromosome 7 as well as the EGFR gene/chromosome 7 centromere probe (CEP7) ratio were recorded. Immunohistochemical (IHC) staining was done on the corresponding paraffin sections. The copy number of the EGFR gene in each tumor/cell line ranged from 1.2 to 5.6. The EGFR gene/CEP7 ratio showed a biological continuum ranging from 0.59 to 1.94 with a mean of 1.04. EGFR gene copy loss was found in 16.6% of cases whereas copy gain was demonstrated in 19.4%. There was no relationship between IHC protein expression of EGFR and EGFR gene copy number or EGFR gene/CEP7 ratio.In conclusion, most breast carcinomas had a balanced EGFR gene/CEP7 copy number with a mean ratio of 1.04. Almost equal subpopulations revealed limited copy gain and copy loss. EGFR high dosage amplification, like in HER-2, was not demonstrated. Demonstration of EGFR gene copy loss might have a potential as a surrogate marker for EGFR gene mutation and/or deletion.

Adenocarcinoma↗
PubMed2006

Enhanced sensitivity and stability in two-color in situ hybridization by means of a novel chromagenic substrate combination.

Double in situ hybridization analysis is a fundamental technique for studying the expression of two genes with high temporal and spatial resolution. However, due to the lack of sensitivity in current detection methods, this approach is powerful only when at least one transcript is abundantly expressed. Here, we report a new enzyme/chromagenic substrate combination that provides sufficient sensitivity for detecting two less abundant transcripts and stability for subsequent paraffin sectioning.

Animals↗
PubMed1991

Luminography--a new, highly sensitive visualization method for electrophoresis.

A highly sensitive method for protein visualization following electrophoresis and protein blotting was developed. The method is based on the light-emitting reaction of luminol and hydrogen peroxide catalyzed by horseradish peroxidase. The luminescent assay can be applied either to the native gel or after protein blotting, and it has a sensitivity two orders of magnitude higher than that achieved with chromogenic detection systems. Analogous to autoradiography the luminescent signal is recorded on an X-ray film with similar sensitivity. We present several examples of application emphasizing the general versatility of this innovative method.

Animals↗
PubMed1993

Mutagenicity of methyl methanesulfonate (MMS) in vivo at the Dlb-1 native locus and a lacI transgene.

Methyl methanesulfonate (MMS) is an extraordinarily poor mutagen compared to ethylnitrosourea (ENU) or even X-rays. In lung fibroblasts in vivo, MMS has been shown to induce many micronuclei but few, if any, mutations at the hpt locus. We wondered if the lack of mutations might be due to the lack of division and DNA synthesis in fibroblasts in vivo, which would permit substantial time for differential repair of DNA lesions. This idea was tested in the small intestine, a tissue in which the cells are actively dividing. Two loci were examined: a native locus (Dlb-1) which determines the presence or absence of a lectin binding site on the surface of the epithelial cells, and a lacl transgene which controls beta-galactosidase synthesis. Locl mutations were detected after in vitro packaging of DNA isolated from the intestinal epithelium into lambda phage and expression in suitable bacteria. Although the epithelial cells are proliferating, acute treatments produced no significant increase in mutations at either locus. Subacute treatments produced low but significant increases in mutation frequency at both loci. The results confirm that MMS is a far more potent clastogen than it is a mutagen and should be regarded as a super-clastogen in the same manner as ENU is a super-mutagen. The carcinogenicity of MMS is probably the result of its potent clastogenicity rather than its weak activity as a point mutagen.

Animals↗
PubMed2001

Retroviral labeling of Schwann cells: in vitro characterization and in vivo transplantation to improve peripheral nerve regeneration.

Transplantation of Schwann cells (SCs) is a promising treatment modality to improve neuronal regeneration. Identification of the transplanted cells is an important step when studying the development of this method. Genetic labeling is the most stable and reliable method of cell identification, but it is still unclear whether it has deleterious effect on SC characteristics. Our aim was to achieve a stable population of SCs transduced with the lacZ gene at a high frequency using a retroviral vector in vitro, and to follow the labeled SC in vitro to assess their viability and phenotypic marker expression. Furthermore, we transplanted lacZ-labeled SCs in a conduit to repair peripheral nerve to investigate their effect on nerve regeneration in vivo. Rat and human SCs were cultured and transduced with an MFG lacZ nls marker gene, achieving a transduction rate of 80% and 70%, respectively. Rat SCs were kept in culture for 27 weeks and examined every 4 weeks for expression of lacZ, viability, and phenotypic marker expression of GFAP, p75, MHC I and II. Throughout this period, transduced rat SCs remained viable and continued to proliferate. The proportion of cells expressing lacZ dropped only by 10% and the expression of phenotypic markers remained stable. Transduced human SCs were followed up for 4 weeks in culture. They proliferated and continued to express the lacZ gene and phenotypic marker expression of GFAP and p75 was preserved. Primary culture of transduced rat SCs were transplanted, syngeneically, in a conduit to bridge a 10 mm gap in sciatic nerve and the grafts were examined after 3 weeks for the presence and participation of labeled SCs and for axonal regeneration distance. Transplanted transduced rat SCs were clearly identified, taking part in the regeneration process and enhancing the axonal regeneration rate by 100% (at the optimal concentration) compared to conduits without SCs. Thus, retroviral introduction of lacZ gene has no deleterious effect on SCs in vitro and these SCs take part and enhance nerve regeneration in vivo.

Animals↗
PubMedDate not supplied

Endotoxin levels measured by a chromogenic assay in portal, hepatic and peripheral venous blood in patients with cirrhosis.

Endotoxin concentrations were measured in the portal, hepatic and peripheral venous blood of two groups of patients with cirrhosis using a limulus-based chromogenic assay. The high sensitivity of chromogenic detection allowed measurement of endotoxin as low as 10 to 15 pg per ml, an order of magnitude greater than previously possible by gelation studies. Group 1 consisted of 56 patients with cirrhosis undergoing angiographic evaluation. In this group, there was wide variability in hepatic venous concentration [73 +/- 110 pg per ml (mean +/- S.D.)] and peripheral venous concentration [31 +/- 58 pg per ml]. However, paired t test showed peripheral venous concentration was significantly (p less than 0.001) lower than hepatic venous concentration. Neither hepatic or peripheral venous endotoxin levels correlated significantly with a variety of clinical, biochemical or radiological parameters. Group 2 consisted of 21 patients with cirrhosis undergoing shunt surgery. Endotoxin levels again showed a wide range, with portal venous concentration (142 +/- 167 pg per ml) and simultaneous peripheral venous concentration (82 +/- 150 pg per ml). Paired t test in this group showed a significant (p less than 0.001) portal to peripheral venous gradient. This study showed the feasibility of measuring endotoxin in plasma to low concentrations by a chromogenic assay technique. It supports the concept of relatively high levels of endotoxin in the portal circulation. In the presence of liver disease, systemic endotoxemia occurs, which is augmented by stressful situations.

Chromogenic Compounds↗
PubMed2004

Splice-site genetic polymorphism of the human kallikrein 12 (KLK12) gene correlates with no substantial expression of KLK12 protein having serine protease activity.

The human kallikrein 12 (KLK12) gene is a new member of the KLK gene family, some members of which are implicated in the initiation and progression of cancer. In this study, we examined 50 non-cancerous tissues from Japanese patients with primary gastric cancer to determine the presence of genetic polymorphisms in the KLK12 gene using polymerase chain reaction (PCR)-single-strand conformation polymorphism and sequencing. Four different types of genetic polymorphisms were identified: one at a splice-donor site of intron 4 (c.457+2T>C), two in exon 6 (c.618_619delTG:p.Cys206fsX72 and c.735G>A:p.Met245Ile), and one in intron 3. The c.457+2T>C polymorphism was observed at a high frequency (allele frequency:0.63), compared to the frequencies of the two polymorphisms in exon 6 (allele frequency:0.01). Reverse transcriptase (RT)-PCR and Western blot analyses revealed that the c.457+2T>C polymorphism was associated with a splicing abnormality and that the expression of the human KLK12 protein (hK12), corresponding to the putative serine protease, was absent in individuals with a c.457+2C/C genotype but not in individuals with the T/T or T/C genotypes. We also found that recombinant His6-tagged hK12 has activity that cleaves chromogenic substrate (H-D-Pro-L-Phe-L-Arg-p-nitroaniline dihydrochloride), that is, serine protease activity. These results indicate that individuals with the c.457+2C/C genotype have no substantial expression of hK12 serine protease.

Carcinoma↗
PubMed1994

Factor XII fragment and kallikrein generation in plasma during incubation with biomaterials.

Blood biocompatibility of medical devices is in many ways dependent on surface characteristics and biochemical blood material interactions. In this study, the contact system, in which the activation of factor XII and plasma kallikrein is included, is highlighted. This article describes a simple chromogenic assay to determine the Hageman Factor fragment (HFf, or factor XIIf) and kallikrein activity in vitro. The assay is based on conversion of Z-Lys-Phe-Arg-pNA.2HCl to which human factor XIIf and kallikrein appeared to have a high affinity. To discriminate between the serine proteases factor XIIf and kallikrein to cleave this substrate, aprotinin was added to one of two complementary samples. In this in vitro study, standardized disks from glass, high-density polyethylene (HDPE), polytetrafluoro ethylene (PTFE), and polydimethyl siloxane (PDMS) were studied for their capacity to generate factor XIIf and kallikrein in plasma. Kaolin was used as positive control. On glass disks the highest and on HDPE the lowest generation of factor XIIf and kallikrein were found, both with a ratio of 1:1. On PDMS and on PTFE disks protease activities were intermediate, but with a factor XIIf and kallikrein activity ratio of 1:2 and 1:4, respectively. Apparently because of the hydrophobic surface character of PDMS and PTFE, these surfaces absorb or fail to produce the factor XIIf. This assay appeared to be discriminative even for materials that are considered mild activators of the contact system and can therefore be used as a standard method to qualify biomaterials.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1987

Chromogenic reactions of tertiary amines with polycarboxylic acids and acetic anhydride: carbon suboxide as the reactive species in the malonic acid reagent.

Analytical methods based on the title reactions are reviewed, and the malonic acid-acetic anhydride system was selected for detailed study. It is postulated that carbon suboxide, O = C = C = C = O, formed by the action of acetic anhydride on malonic acid, is the effective reactive species in this system. Carbon suboxide was prepared and identified, and spectrophotometric observations of its reactions with tertiary amines are described. Aliphatic and aromatic tertiary amines generate colored products upon reaction with carbon suboxide in the presence of acetic anhydride. It was found that aliphatic tertiary amines form colors upon reaction with carbon suboxide in the absence of acetic anhydride, whereas aromatic tertiary amines require the presence of acetic anhydride.

Acetates↗
PubMed2004

Serine proteases in the spiny lobster olfactory organ: their functional expression along a developmental axis, and the contribution of a CUB-serine protease.

Several serine proteases and protease inhibitors have been identified in the crustacean olfactory organ, which is comprised of the lateral flagellum of the antennule and its aesthetascs sensilla that house olfactory receptor neurons and their supporting cells. The function of these proteases in the olfactory organ is unknown, but may include a role in perireception (e.g., odor activation or inactivation) or in the development or survival of olfactory receptor neurons. To examine directly the function of proteases in the olfactory organ of the Caribbean spiny lobster Panulirus argus, we used different tissue fractions from the lateral flagellum in an enzyme activity assay with a variety of protease substrates and inhibitors. Trypsin-like serine protease activity occurs throughout the lateral flagellum but is enriched in the cell membranes from aesthetascs. Cysteine- and metalloprotease activities also occur in olfactory tissue, but are more abundant in tissue fractions other than aesthetascs. To assess the contribution of one of the olfactory serine proteases--CUB-serine protease (Csp)--Csp was immunoprecipitated using an antibody; results with the remaining fraction suggest that Csp accounts for at least 40% of the total serine protease activity in the olfactory organ. The amount of total serine protease activity follows a developmental axis in the lateral flagellum. Total protease activity is lowest in the proximal zone, which lacks aesthetascs, and the proliferation zone, where olfactory receptor neurons and associated cells are born, and highest in aesthetascs of the distally-located senescence zone, which has the oldest olfactory tissue.

Animals↗
PubMed2006

Use of a fluorescent internal protein standard to achieve quantitative two-dimensional gel electrophoresis.

2-DE is a powerful separation method for complex protein mixtures. However, large intergel variations in spot intensity limit its use for quantitative proteomics studies. To address this issue, we developed a fluorescent internal protein standard for use in 2-DE analysis. Protein samples are spiked with an Alexa-labeled internal standard (ALIS) prior to separation with 2-DE. Due to the high extinction coefficient of the Alexa-fluor, incorporation of 0.1% of total protein is sufficient to allow visualization of the internal standard yet low enough to avoid interference in subsequent quantification and identification steps. Following 2-DE, total proteins are visualized with fluorescent postelectrophoretic stains spectrally separated from ALIS. Four protein stains, Deep Purple, Sulforhodamine G, ruthenium II-tris(bathophenanthroline disulfonate) (RuTBS), and SYPRO Ruby, including improved purification and staining protocols for RuTBS and ten-fold dilutions of SYPRO Ruby were evaluated. All staining protocols were compatible with the ALIS method and had similar LODs (1-4 ng) and dynamic ranges (10(3)). ALIS is a powerful normalization method for quantitative 2-DE which avoids potential problems associated with dual spot migration patterns observed in the DIGE method. Furthermore, ALIS provides significantly improved normality in the distribution of spot abundance-variance compared to normalization through division by the total spot volume.

Animals↗
PubMed1993

Exploration of subsite binding specificity of human cathepsin D through kinetics and rule-based molecular modeling.

The family of aspartic proteinases includes several human enzymes that may play roles in both physiological and pathophysiological processes. The human lysosomal aspartic proteinase cathepsin D is thought to function in the normal degradation of intracellular and endocytosed proteins but has also emerged as a prognostic indicator of breast tumor invasiveness. Presented here are results from a continuing effort to elucidate the factors that contribute to specificity of ligand binding at individual subsites within the cathepsin D active site. The synthetic peptide Lys-Pro-Ile-Glu-Phe*Nph-Arg-Leu has proven to be an excellent chromogenic substrate for cathepsin D yielding a value of kcat/Km = 0.92 x 10(-6) s-1 M-1 for enzyme isolated from human placenta. In contrast, the peptide Lys-Pro-Ala-Lys-Phe*Nph-Arg-Leu and all derivatives with Ala-Lys in the P3-P2 positions are either not cleaved at all or cleaved with extremely poor efficiency. To explore the binding requirements of the S3 and S2 subsites of cathepsin D, a series of synthetic peptides was prepared with systematic replacements at the P2 position fixing either Ile or Ala in P3. Kinetic parameters were determined using both human placenta cathepsin D and recombinant human fibroblast cathepsin D expressed in Escherichia coli. A rule-based structural model of human cathepsin D, constructed on the basis of known three-dimensional structures of other aspartic proteinases, was utilized in an effort to rationalize the observed substrate selectivity.

Amino Acid Sequence↗
PubMed2004

Characterization of Lys-698-to-Met substitution in human plasminogen catalytic domain.

Streptokinase (SK) is a human plasminogen (Pg) activator secreted by streptococci. The activation mechanism of SK differs from that of physiological Pg activators in that SK is not a protease and cannot proteolytically activate Pg. Instead, it forms a tight complex with Pg that proteolytically activates other Pg molecules. The residue Lys-698 of human Pg was hypothesized to participate in triggering activation in the SK-Pg complex. Here, we report a study of the Lys-698 to Met substitution in the catalytic domain of Pg (microPg) containing the proteolytic activation-resistant background (R561A). While it remains competent in forming a complex with SK, maintaining a comparable equilibration dissociation constant (K(D)), the recombinant protein shows a nearly 60-fold reduction in amidolytic activity relative to its R561A background when mixed with native SK. A 2.3 A crystal structure of this mutant microPg confirmed the correct folding of this recombinant protein. Combined with other biochemical data, these results support the premise that Lys-698 of human Pg plays a functional role in the so-called N-terminal insertion activation mechanism by SK.

Amino Acid Substitution↗
PubMed1995

Biochemical characterization of basilase, a fibrinolytic enzyme from Crotalus basiliscus basiliscus.

Snake venoms, especially from the Crotalidae family, contain a variety of enzymes that prevent blood coagulation by virtue of their fibrinolytic enzymes. Nineteen snake venoms were screened for fibrinolytic activity and the highest activity was found in the venom of Crotalus basiliscus basiliscus venom. The active principle, basilase, was isolated, purified, and found to have fibrinolytic and fibrinogenolytic activity. It had a molecular weight of 22,000 and 1 mol of zinc per mole of protein associated with it. The proteolytic activity of the enzyme against dimethyl casein was inhibited by ethylenediaminetetraacetic acid and alpha 2-macroglobulin. It did not inactivate alpha 2-macroglobulin. Basilase did not have any of the following activities: thrombin-like, factor X-like, protein C activating, or urokinase-like. It caused neither hemorrhage nor platelet aggregation. In spite of its proteolytic activity, basilase did not hydrolyze the membranes of platelets. Basilase had 24% alpha-helix, 31% beta-sheet, 25% turns, and 20% unordered structure, as determined by Fourier Transform Infrared spectroscopy. Basilase is an enzyme that hydrolyzes fibrin directly without activation of plasminogen.

Amino Acid Sequence↗
PubMed2002

Kinetic peculiarities of human tissue kallikrein: 1--substrate activation in the catalyzed hydrolysis of H-D-valyl-L-leucyl-L-arginine 4-nitroanilide and H-D-valyl-L-leucyl-L-lysine 4-nitroanilide; 2--substrate inhibition in the catalyzed hydrolysis of N alpha-p-tosyl-L-arginine methyl ester.

Hydrolysis of D-valyl-L-leucyl-L-lysine 4-nitroanilide (1), D-valyl-L-leucyl-L-arginine 4-nitroanilide (2), and N alpha-p-tosyl-L-arginine methyl ester (3) by human tissue kallikrein was studied throughout a wide range of substrate concentrations. At low substrate concentrations, the hydrolysis followed Michaelis-Menten kinetics but, at higher substrate concentrations, a deviation from Michaelis-Menten behavior was observed. With the nitroanilides, a significant increase in hydrolysis rates was observed, while with the ester, a significant decrease in hydrolysis rates was observed. The results for substrates (1) and (3) can be accounted for by a model based on the hypothesis that a second substrate molecule binds to the ES complex to produce a more active or an inactive SES complex. The deviation observed for substrate (2) can be explained as a bimolecular reaction between the enzyme-substrate complex and a free substrate molecule.

Catalysis↗
PubMed1993

Detection of c-type cytochromes using enhanced chemiluminescence.

A rapid, nontoxic method was devised for staining c-type cytochromes on filters. Bacterial and mitochondrial c-type cytochromes from sodium dodecyl sulfate-polyacrylamide gels were transferred to nitrocellulose filters and visualized by a staining system that relies on their heme peroxidase activity. This involved the use of enhanced chemiluminescence (ECL). This system was compared with the 3,3',5,5'-tetramethylbenzidine (TMBZ) staining of cytochromes transferred to nitrocellulose filters and found to be equally sensitive. The ECL assay has the following advantages: (i) the signals can be quantitated with a densitometer; (ii) it is much quicker than the TMBZ-staining protocol (20 min versus 2 h); (iii) the generated signals are detected on a film providing a convenient record; (iv) after removal of the reagents, filters can be reused for immunoassays; and (v) it uses nonhazardous components.

Animals↗
PubMed1993

Continuous spectrophotometric assay for ascorbate oxidase based on a novel chromophoric substrate, 2-aminoascorbic acid.

Recently, we reported the development of a sensitive continuous spectrophotometric assay for the ascorbate-dependent mammalian enzyme dopamine beta-monooxygenase based on the novel chromophoric electron donor 2-aminoascorbic acid [K. Wimalasena and D.S. Wimalasena (1991) Anal. Biochem. 197, 353-361]. We now report that ascorbate oxidase (EC 1.10.3.3, L-ascorbate:O2 oxidoreductase) also catalyzes the oxidation of 2-aminoascorbic acid to chromophoric 2,2'-nitrilodi-2(2')-deoxy-L-ascorbic acid (red pigment). The reaction is kinetically well behaved, displaying the expected stoichiometry for an oxidase-catalyzed reaction with respect to oxygen and the oxidation product (red pigment), demonstrating that 2-aminoascorbic acid is a well-behaved alternative substrate for the enzyme. Ascorbate oxidase is a very efficient enzyme toward its natural substrate, ascorbic acid. Although 2-amino-ascorbic acid is a significantly weak substrate for the enzyme in comparison to ascorbic acid, as indicated by the apparent initial rate kinetic parameters, the high extinction coefficient of the red pigment under our assay conditions suggests that this novel reactivity of the enzyme could be used to design a sensitive, convenient, and continuous spectrophotometric assay for ascorbate oxidase. While this assay is more convenient than the existing oxygen monitor assay, its adaptability to measure the activity of the enzyme in the immobilized form may be helpful in the development of technologies for the automated detection of ascorbic acid in biological fluids for industrial or clinical applications. In addition, this novel reactivity of the enzyme may be used to examine the substrate specificity and the mechanism of action of the enzyme.

Ascorbate Oxidase↗