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PubMed2004

Isolation of Citrobacter sp. mutants defective in decolorizing malachite green.

To identify genes involved in the decolorization of malachite green, random mutants generated by transposon insertion in the malachite green-decolorizing bacterium, Citrobacter sp. were isolated. The resulting mutant bank yielded 24 mutants with complete defects in their abilities to decolorize malachite green. Southern hybridization with a Tn5 fragment as a probe showed a single hybridized band in 7 mutants, which appeared to have insertions at different sites of the chromosome. The Tn5-inserted genes were isolated and the DNA sequence flanking Tn5 was determined. Based on a sequence database, the putative protein products encoded by the mg genes were identified as follows. mg3, an ABC transporter homolog; mg6, a LysR-type regulatory protein; m11, an oxidoreductase; mg17, a MalG protein in the maltose transport system; and mg21, a sugar kinase. The deduced sequences from two mg genes (mg7 and mg18) showed no significant similarity to any protein with a known function, suggesting that these two mg genes encode unidentified proteins that are responsible for the decolorization of malachite green.

Bacterial Proteins↗
PubMed2002

Phase I study on sentinel lymph node mapping in colon cancer: a preliminary report.

BACKGROUND AND OBJECTIVES: Lymph node (LN) metastasis is one of the most significant prognostic factor in colorectal cancer. In fact, therapeutic decisions are based on LN status. However, multiple studies have reported on the limitations of the conventional pathological LN examination techniques, and therefore, the actual number of patients with LN positive colorectal cancer is probably underestimated. We assume that lymphatic tumor dissemination follows an orderly sequential route. We report here a simple and harmless coloration technique that was recently elaborated, and that allows us to identify the sentinel LN(s) (SLN) or first relay LNs in colorectal cancer patients. The main endpoint of this clinical trial is the feasibility of the technique. METHODS: Twenty patients treated by surgery for a colic cancer were admitted in this protocol. A subserosal peritumoral injection of lymphazurin 1% was performed 10 min before completing the colic resection. A pathologist immediately examined the specimens, harvested the colored SLN, and examined them by serial cuts (200 microm) with H&E staining, followed by immunohistochemical staining (AE1-AE3 cytokeratin markers), when serial sections were classified as cancer free. RESULTS: The preoperative identification of the SLN was impossible in at least 50 of the cases, however, SLNs were identified by the pathologist in 90% of cases. In two patients (10%) SLN was never identified. The average number of SLN was 3.9. Immunohistochemical analysis of the SLN has potentially changed the initial staging (from Dukes B to Dukes C) for 5 of the 20 patients (25%). On the other hand, there was one patient (5%) with hepatic metastasis from adenocarcinoma for whom SLN pathology was negative for metastasis (skip metastasis). CONCLUSIONS: SLN biopsy is readily feasible with identification of SLN in at least 90% of patients with colorectal cancers. Our results indicate that 45% of patients initially staged as Dukes B had tumor cells identified in their SLN when these were subjected to our protocol. This represented a 25% upgrading rate when our complete study population is considered. However, controversy persist about the clinical significance and metastatic potential of these often very small clusters of tumor cells.

Biomarkers, Tumor↗
PubMed2001

Interstitial MR lymphangiography for the detection of sentinel lymph nodes.

BACKGROUND AND OBJECTIVES: The challenge for implementation of sentinel lymph node biopsy is to develop a reliable minimally invasive technique that identifies all possible sentinel nodes with high temporal and spatial resolution. This study evaluated the use of a magnetic resonance imaging (MRI) contrast agent (USPIO) for preoperative sentinel node detection. METHODS: Anesthetized pigs received interstitial or intradermal injections of ultra small superparamagnetic of iron oxide (USPIO) (0.2 or 5 mg Fe) in the L/R posterior tongue and stifles (knee) respectively. MRI was done before, during injection and at 0.25, 0.5, 1, 2, 4, 6, 24, and 48 hr after which isosulfan blue sentinel node mapping was done. RESULTS: In the tongue, both doses of USPIO identified the sentinel node in the early images. No additional nodes were detected by MR at 24 or 48 hr. In the hind limb, sentinel nodes identified on the early MR images were also identified by the isosulfan blue. In both locations, the higher dose also identified secondary nodes some of which were also identified by the isosulfan blue. All sentinel nodes that were identified by USPIO on MRI were noted to be stained brown at the time of dissection. CONCLUSIONS: Interstitial MR lymphangiography is a useful technique for the detection of sentinel lymph nodes. This method provides excellent simultaneous temporal and spatial resolution, is minimally invasive, and can be performed preoperatively.

Animals↗
PubMed1983

The distribution of acidic coomassie blue-stained proteins from uninvolved human liver, hepatoma, normal colon, primary colon cancer, and colon metastases to the liver, determined by two-dimensional protein electrophoresis.

The distribution of Coomassie blue-stained proteins from uninvolved regions of 4 human livers, from 1 hepatocellular carcinoma, and from 4 samples each of uninvolved colon, primary adenocarcinoma of the colon, and colon cancer metastatic to the liver was analyzed by two-dimensional protein electrophoresis. From a comparison of acidic proteins between pI 3.5 and 6.5, we conclude (1) that the majority (66 of 82) of denoted acidic proteins from 4 normal liver samples were represented in the hepatocellular carcinoma. Fifty-one of 58 proteins denoted in the 4 colon samples were detected in each of the 4 primary colon cancers; (2) that the "normograms" of proteins from normal colon and normal liver differed in many details, and their dissimilar patterns identified the source of the sample; (3) that hepatoma and primary adenocarcinoma of the colon were easily distinguished by their distribution of proteins; (4) that colon cancer metastatic to the liver contained a majority (50/58) of acidic proteins enumerated in primary colon cancer. These results indicate that uninvolved liver and colon and their primary or secondary cancers can be identified by their distribution of electrophoresed acidic proteins.

Adenocarcinoma↗
PubMed1995

Absorption cytometric DNA analysis of sections of gastric carcinomas and a comparison with cytofluorometry of single-cell suspensions and flow cytometry.

Measurements were made of cellular deoxyribonucleic acid (DNA) content in paraffin-embedded materials from 44 gastric carcinomas, using three different methods; static cytometric light absorbance technique using tissue sections (SCM-abs), static cytometric fluorescence technique using single-cell suspensions (SCM-flu), and flow cytometry (FCM). The DNA ploidy determined by SCM-abs was grouped into low or high ploidy according to dispersion on the DNA histogram, and the DNA ploidy determined by SCM-flu and FCM was grouped into diploidy or aneuploidy, according to the peak DNA value. Accord between SCM-abs and SCM-flu or between SCM-abs and FCM was confirmed when low ploidy by SCM-abs showed diploidy by SCM-flu or FCM and high ploidy by SCM-abs showed aneuploidy by SCM-flu or FCM. An incomplete but good accord of DNA ploidy was obtained with these methods.

Absorption↗
PubMed2002

Effects of Er:YAG laser irradiation on human dentin: polarizing microscopic, light microscopic and microradiographic observations, and FT-IR analysis.

BACKGROUND AND OBJECTIVES: The effects of Er:YAG laser irradiation on dentin have not been sufficiently investigated. The purpose of this study was to investigate the effects of Er:YAG laser irradiation on dentin. STUDY DESIGN/MATERIALS AND METHODS: After cavities were prepared using Er:YAG laser irradiation or rotary cutting instruments, histological observations of cavity-floor dentin utilizing polarizing microscopy, microradiography and light microscopy, and analysis of composition of cavity-floor dentin using Fourier-transformed (FT-IR) spectrometry were conducted. RESULTS: In the laser-treated side, a deeply stained basophilic layer was observed. The number of odontoblastic processes present was obviously less in the laser-treated side than in the bur-treated side. FT-IR analysis revealed that compared to the bur-treated side, a broad background peak at around 1,600 cm(-1) was present. CONCLUSIONS: Er:YAG laser irradiation might have denatured the organic materials of dentin.

Basophils↗
PubMed1991

Acrosome reaction induced by immunoaggregation of a proteinase inhibitor bound to the murine sperm head.

ZP3, a glycoprotein of the murine zona pellucida, functions both to bind acrosome intact sperm and to induce the acrosome reaction. Solubilized whole zonae as well as purified ZP3 are able to induce acrosome reactions in capacitated sperm. Pronase digests of whole zonae yield glycopeptides that bind to sperm but are unable to induce acrosome reactions. However, immunoaggregation of these glycopeptides results in the exocytosis of the acrosome in the majority of treated sperm. The data suggest that ZP3 triggers the acrosome reaction by the aggregation of ZP3 binding sites on the sperm head. If aggregation of ZP3 binding sites is important in the induction of the acrosome reaction, then it may be possible to induce the acrosome reaction in the absence of zona by immunoaggregation of the sites. This presentation deals with the immunoaggregation of a proteinase inhibitor of seminal vesicle origin (SVI) that binds to a site on the sperm head known to participate in zona binding. We show that capacitated murine sperm, pretreated with the SVI, will acrosome react, as determined by Coomassie brilliant blue staining, when incubated with rabbit antiinhibitor antiserum (anti-SVI). The percentage of SVI-treated sperm displaying an acrosome reaction is dependent on the concentration of the immune serum. Sperm stain positive for intact acrosomes when anti-SVI Fab fragments or normal rabbit serum is substituted for the immune serum. However, when capacitated sperm, treated with both SVI and anti-SVI Fab fragments, are incubated with goat antirabbit IgG, the majority of sperm acrosome react. The data suggest that the aggregation of SVI bound to the sperm surface, in the absence of zona glycoproteins, is sufficient to induce the acrosome reaction.

Acrosome↗
PubMed1994

Computer-assisted chromatin texture characterization of Feulgen-stained nuclei in a series of 331 transitional bladder cell carcinomas.

The chromatin patterns of Feulgen-stained nuclei in a series of six specimens of normal mucosa and 331 transitional bladder carcinomas, including 293 superficial (Ta and T1) and 38 invasive (T2-T4) cases, were quantitatively described by means of eight parameters relating to densitometric, run-length distribution, and co-occurrence matrix features. The results show that the chromatin texture of the superficial lesions was markedly different from that of the invasive tumours, which exhibited a distinctly more dense and heterogeneous chromatin pattern. The data also show that the increasing level of malignancy, as revealed by the increasing clinical stage, was accompanied by an increase in the overall chromatin condensation level. Only some areas of the nucleus actually increased in density; other pale areas appeared concomitantly with these increasingly denser chromatin areas. This chromatin density increase corresponded to a marked increase in the frequency of small dense chromatin clumps; these joined together into very large dense chromatin clumps, which were distributed more and more heterogeneously in the nucleus as the clinical stage of the tumour increased.

Adult↗
PubMed2004

Differential protein composition of bovine whey: a comparison of whey from healthy animals and from those with clinical mastitis.

During clinical mastitis in dairy cows, the quantity of milk produced decreases and the composition of the milk is altered. As the severity of inflammation associated with the disease increases, the chemical composition of milk approaches that of blood as a consequence of increased permeability of the blood mammary barrier, or de novo intramammary synthesis, as has been suggested for mammary associated serum amyloid A3. A better understanding of these events may provide new approaches for the diagnosis and treatment of mastitis. The objective of this study was to document the changes in the protein composition of milk during clinical mastitis using a proteomic approach, with the objective of identifying new diagnostic markers of mastitis. Whey from dairy cows with clinical mastitis was compared to whey from healthy animals by two-dimensional gel electrophoresis (2-DE) with colloidal Coomassie staining and matrix-assisted desorption/ionization mass spectrometry. Increases in the concentrations of proteins of blood serum origin, including serotransferrin and albumin, were identified in mastitic whey compared to normal whey, while concentrations of the major whey proteins alpha-lactalbumin and beta-lactoglobulin were reduced in mastitic whey. Mass spectrometry subsequently confirmed the location of albumin, alpha-lactalbumin and beta-lactoglobulin on the 2-DE gels at M(r)/pI of 69 294/5.8, 14 200/4.5 and 19 883/4.9 respectively.

Animals↗
PubMed2004

Assessing factors for reliable quantitative proteomics based on two-dimensional gel electrophoresis.

We statistically analysed various factors to get accurate estimates of protein quantities from two-dimensional gels. Yeast proteins were labelled with (35)S or stained with Coomassie Brilliant Blue G-250, and spots were automatically quantified with software packages Kepler, ImageQuaNT, Melanie 3.0 and Progenesis. The different software packages proved to have very similar performances. With (35)S-labelled actin spot as a reference, we studied the staining efficiency of colloidal Coomassie blue as a function of amino acid composition of the protein, and derived an equation to estimate the number of molecules per cell from blue-stained proteins. Absolute quantification of most glycolytic enzymes was carried out in two yeast strains.

Actins↗
PubMed2004

Proteome reference maps of Medicago truncatula embryogenic cell cultures generated from single protoplasts.

Using a combination of two-dimensional gel electrophoresis (2-DE) protein mapping and mass spectrometry (MS) analysis, we have established proteome reference maps of Medicago truncatula embryogenic tissue culture cells. The cultures were generated from single protoplasts, which provided a relatively homogeneous cell population. We used these to analyze protein expression at the globular stages of somatic embryogenesis, which is the earliest morphogenetic embryonic stage. Over 3000 proteins could reproducibly be resolved over a pI range of 4-11. Three hundred and twelve protein spots were extracted from colloidal Coomassie Blue-stained 2-DE gels and analyzed by matrix-assisted laser desorption/ionization-time of flight MS analysis and tandem MS sequencing. This enabled the identification of 169 protein spots representing 128 unique gene products using a publicly available expressed sequence tag database and the MASCOT search engine. These reference maps will be valuable for the investigation of the molecular events which occur during somatic embryogenesis in M. truncatula. The proteome reference maps and supplementary materials will be available and updated for public access at http://semele.anu.edu.au/.

Amino Acid Sequence↗
PubMed2003

Wheat proteomics: relationship between fine chromosome deletion and protein expression.

To explore the relationship between fine chromosome deletion and protein expression in common wheat, the changes in protein composition of wheat seed proteome were investigated by using chromosome 1B. A momosomic alien chromosome addition line of common wheat was used to produce the fine deletion lines. Endosperm and embryo proteins were separated by two-dimensional gel electrophoresis (2-DE) and visualized by staining with Commassie Brilliant Blue, and gel images were analyzed with a computer assisted image analyzer. For the first time, fine gene locations of a few endosperm and embryo proteins were identified on the chromosome 1B. These proteins with their specific gene location on the chromosome can be used as protein markers in breeding programs for quality of wheat proteins. To identify wheat seed proteins and to understand their expression in relation to chromosome deletion, the feasibility of a new analytical approach based on isotope coded affinity tag labeling (ICAT) of peptides in tryptic digests followed by electrospray ionization mass spectrometry has been described. Simplification of the complex tryptic digest prior to mass spectral analysis was performed by treating the samples with light and heavy ICAT labeling reagents. A clear separation of peptide fragment containing the light and heavy reagents was achieved in mass spectral analysis. Out of the 14 peptides detected by mass fragment analysis of the euploid, four were down-regulated, nine up-regulated and one did not show any change due to the terminal deletion of chromosome 1B. Selected peptide fragments were subjected to tandem mass spectrometry analysis for sequence information and the resulting sequence information was submitted to databases for protein identification. Of the five proteins submitted, four were identified as alpha-amylase inhibitor, alpha-amylase/subtilisin inhibitor precursor, proteasome subunit alpha-type 7 and 1,4 alpha-glucan-D-maltohydrolase. With this approach it is possible to identify wheat seed proteins and to understand their expression, which have been reported to be difficult by 2-DE due to cosynthesis of proteins by genes from three genomes, A, B and D.

Chromosome Deletion↗
PubMed2006

Mass spectrometric profiling of O-linked glycans released directly from glycoproteins in gels using in-gel reductive beta-elimination.

Glycosylation is a widespread PTM of proteins; the carbohydrate moieties provide various functional, immunological and structural aspects of both eukaryotic and prokaryotic glycoproteins. Traditional strategies used to analyse glycoprotein O-glycans involve glycoprotein isolation, followed by glycan release using solution-phase base-catalysed beta-elimination. However, in a proteomics context, mixtures of proteins and glycoproteins are routinely separated using SDS-PAGE. We have therefore developed a method to enable the profiling of O-linked glycans directly from glycoproteins on gels. This is achieved using in-gel reductive beta-elimination followed by mass spectrometric analysis of the released glycans. Here we describe our demonstration of the feasibility of this approach, our development and optimisation of the procedure using bovine submaxillary gland glycoproteins as a standard, and then show its usefulness by applying the developed procedure to the analysis of the O-glycans from a glycoprotein band from a Coomassie-stained SDS-PAGE separation of a mixture of Mycobacterium avium capsular proteins and glycoproteins. The procedure has been shown to be applicable to both CBB- and silver-stained gels. The method offers a quick and easy way to identify the O-glycans from gel-separated glycoproteins within gel-based proteomics workflows.

Animals↗
PubMed2006

Analysis of membrane protein complexes by blue native PAGE.

Blue native polyacryamide gel electrophoresis is a special case of native electrophoresis for high resolution separation of enzymatically active protein complexes from tissue homogenates and cell fractions. The method is powerful between 10 and 10,000 kDa. Also membrane protein complexes are separated well after solubilization of complexes with mild neutral detergents. The separation principle relies on binding of Coomassie blue G250 which provides negative charges to the surface of the protein. During migration to the anode, protein complexes are separated according to molecular mass and/or size and high resolution is obtained by the decreasing pore size of a polyacrylamide gradient gel. The principles of 2-dimensional blue native sodium dodecyl sulfate polyacrylamide gel electrophoresis are presented here together with a practical step-by-step guide to performing the method in the laboratory.

Detergents↗
PubMed2006

Sensitive, quantitative, and fast modifications for Coomassie Blue staining of polyacrylamide gels.

In spite of the high sensitivity of silver staining and the wide dynamic range of various fluorescent detection methods, Coomassie Brilliant Blue staining is still the most widely used protein detection technique for proteins separated by polyacrylamide gel electrophoresis. There are several reasons: Low price, Visible with the eye, Desk top scanners can be employed for image acquisition, Better for quantitative analysis than silver staining, Possible modifications for fast or highly sensitive staining, Mass spectrometry compatible.

Colloids↗
PubMed1994

Nuclear shape and prognosis following orchiectomy in stage D2 prostate cancer.

In this study, we have examined whether tumor grade and morphometric nuclear features can predict the outcome of treatment by orchiectomy in patients with stage D2 prostate cancer. Two outcome groups based on duration of survival postorchiectomy were examined, a bad outcome group of 63 patients who died from prostate cancer within 12 months and a good outcome group of 34 patients who survived beyond 5 years. Tumors were histologically classified as well (17%), moderate (17%), or poorly differentiated (66%). Tumor grade and patient outcome were significantly associated (Mann-Whitney test; P < 0.005), with 76% of poorly differentiated tumors in the bad outcome group, and 65% of well-differentiated tumors in the good outcome group. Using discriminant function analysis, tumor grade correctly predicted outcome in 70% of cases. A statistically significant difference was also detected in nuclear shape values between the two outcome groups (P < 0.05) and histological grades (P < 0.05). Using discriminant function analysis, 51% of cases were correctly classified into outcome groups using nuclear shape factors, a figure which rose to 65% when all nuclear morphometric features were used. This demonstrates that nuclear morphometric features are of no clinical value in predicting the outcome of treatment in stage D2 disease. Furthermore, these evaluations cannot select patients who might be spared orchiectomy on the basis of a predicted poor response. However, nuclear shape and variance measurements of benign glandular epithelial cells within cancerous prostates were significantly different from those of malignant cells (P < 0.005). We conclude that, while video image analysis of prostatic nuclear shape can reliably discriminate between benign and malignant cells, nuclear morphometric features are of minimal prognostic value in men with stage D2 prostate cancer treated by androgen ablation.

Aged↗
PubMed2006

No-discharge atmospheric pressure chemical ionization: evaluation and application to the analysis of animal drug residues in complex matrices.

Alternative ionization methods are increasingly being utilized to increase the versatility and selectivity of liquid chromatography/mass spectrometry (LC/MS). One such technique is the practice of using commercially available atmospheric pressure chemical ionization (APCI) sources with the corona discharge turned off, a process termed no-discharge APCI (ND-APCI). The relative LC/MS responses for several different classes of veterinary drugs were obtained by using ND-APCI, electrospray ionization (ESI), and APCI. While the ND-APCI-MS and -MSn spectra for these compounds were comparable with ESI, ND-APCI provided advantages in sensitivity and selectivity for some compounds. Drugs that were charged in solution as cations or sodium adducts responded particularly well with this technique. Instrumental parameters such as temperatures, gas and liquid flow rates, and source design were investigated to determine their effect on the process of ND-APCI. This paper explores advantages of using ND-APCI for the determination and confirmation of drug residues that might be found in food matrices, including malachite green residues in fish tissue and avermectin residues in milk.

Animals↗