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PubMed2003

Combined phage typing and amperometric detection of released enzymatic activity for the specific identification and quantification of bacteria.

Here, we describe a novel electrochemical method for the rapid identification and quantification of pathogenic and polluting bacteria. The design incorporates a bacteriophage, a virus that recognizes, infects, and lyses only one bacterial species among mixed populations, thereby releasing intracellular enzymes that can be monitored by the amperometic measurement of enzymatic activity. As a model system, we used virulent phage typing and cell-marker enzyme activity (beta-D-galactosidase), a combination that is specific for the bacterial strain Escherichia coli (K-12, MG1655). Filtration and preincubation before infecting the bacteria with the phage enabled amperometric detection at a wide range of concentrations, reaching as low as 1 colony-forming unit/100 mL within 6-8 h. In principle, this electrochemical method can be applied to any type of bacterium-phage combination by measuring the enzymatic marker released by the lytic cycle of a specific phage.

Bacteria↗
PubMed1985

Subcellular compartmentation of prolactin in rat lactotrophs.

The intracellular localization of different molecular forms of prolactin was studied in various experimental models covering a wide range of secretory states. By correlating electron microscopy, morphometry and quantification of monomeric (small) and polymeric (big) prolactin after differential extraction procedures, big prolactin was found stored in secretory granules while small prolactin was loosely associated with all organelles involved in hormone synthesis and processing. No correlation with levels of lactotrophic secretory activity was detected by either the number of secretory granules or prolactin content in lactotrophs.

Animals↗
PubMed2004

Locking of 3' ends of single-stranded DNA templates for improved Pyrosequencing performance.

In Pyrosequencing, a DNA strand complementary to a single-stranded DNA (ssDNA) template is synthesized, whereby each incorporated nucleotide yields detectable light, and the light intensity is proportional to the incorporated nucleotides. Correct data interpretation (i.e., signal-to-noise ratio of light intensities) is hampered by artifacts due to the formation of secondary structures of single-stranded templates. Critical among these is the looping back of the template's nonbiotinylated 3' end to itself In the resulting structure, the 3' end functions as a primer, the extension of which results in background signals. We present two ways of preventing the self-priming of a template's 3' end: (i) the use of a modified oligonucleotide, called blOligo, which is complementary to the template's 3' end and (ii) the extension of the template's 3' end with a ddNMP. In contrast to unprotected 3' ends of ssDNA templates, causing inconsistent results, we show that protecting the 3' end of an ssDNA template using either blOligos or ddNMP enables the correct interpretation of signals and results in reliable quantification.

Alleles↗
PubMed1982

Deoxynucleotide-interconverting enzymes and the quantification of deoxynucleoside triphosphates in mammalian cells.

We have demonstrated that methanol extracts of human cells are heterogeneous with regard to content of dNDP (deoxynucleoside diphosphate) and dNMP (deoxynucleoside monophosphate) kinases. The presence of these enzymes can affect the reliability of techniques used to measure intracellular pools of deoxynucleotides. An optimized extraction procedure and enzymic assay for dNTP species in haematopoietic cells are described which provide sensitivity to measure 0.1-40pmol of dATP, dTTP and dGTP, and 1.0-40pmol of dCTP. The extraction and assay give linear results with (2.5-15)x10(6) nucleated cells and (0.1-1.5)x10(9) red blood cells. Under these conditions, extracts equivalent to ~0.5x10(6) nucleated haematopoietic cells catalyse the phosphorylation of 0-8% of dNDP and dNMP standards to dNTP and incorporate them into deoxynucleotide polymer under circumstances where 100% of an equimolar dNTP standard would be incorporated. By contrast, extracts of 0.4x10(6) HeLa cells totally converted dADP, dTDP and dGDP into dNTP with subsequent polymerization. Conversion of dCDP was somewhat less efficient. The results demonstrate conclusively that the activities of deoxynucleotide interconverting enzymes differ in different types of human cells. They can interfere with assay of nucleotides, but may not do so in many types of cell extracts. In particular, dNTP concentrations can be measured in human haematopoietic cells after extraction with 60% (v/v) methanol and are not artificially elevated by deoxynucleotide interconversions. It is apparent that extraction and assay procedures for measurement of dNTP species should be analysed for each cell type in order to minimize contaminating enzyme activities and ensure accuracy of dNTP quantification.

Cell Line↗
PubMed2005

Highly sensitive simultaneous detection of cultured cellular thiols by laser induced fluorescence-capillary electrophoresis.

We have recently described a new method to determine physiological thiols, in which the quantification of plasma homocysteine, cysteine, cysteinylglycine, glutathione, and glutamylcysteine was achieved after derivatization with 5-iodoacetamidofluorescein. Samples were separated and measured by capillary electrophoresis with laser-induced fluorescence in an uncoated fused-silica capillary, using a phosphate/borate run buffer and the organic base N-Methyl-D-glucamine as effective electrolyte addictive to obtain a baseline peak separation. In this paper, we propose an improvement of our method useful for the analysis of the intracellular thiols in different cultured cells. In particular, we studied run buffer and injection conditions in order to increase the sensitivity of the assay and we found that, by incrementing two times the injected volume and using the water plug before the sample injection, the sensitivity of our previous method was increased by about ten times. To maintain a good resolution between peaks, particularly between homocysteine and the internal standard d-penicillamine, we lengthened the run time by incrementing the concentration of the electrolyte buffer and the organic base d-glucamine and by decreasing the cartridge temperature from 40 to 25 degrees C. After these changes in electrophoretical parameters, cellular thiols were baseline-resolved in less than 14 min instead of 9 min as in our previous method, but the limit of quantification is increased from 50 to 1 nmol/L. This new procedure allows also to measure the intracellular thiols commonly found at low concentration, such as cysteinylglycine, glutamylcysteine, and homocysteine. The new analytical method performance was assessed by measuring the intracellular thiols in three different cell lines, i.e., HUVEC, ECV304, and R1 stem cells.

Animals↗
PubMed2005

Quantitative comparison of intracellular trafficking and nuclear transcription between adenoviral and lipoplex systems.

To develop nonviral gene vectors that are sufficient for clinical application, it is necessary to understand why and to what extent nonviral vectors are inferior to viral vectors, which in general show a more efficient transfection activity. This study describes a systematic and quantitative comparison of the cellular uptake and subsequent intracellular distribution (e.g., endosome/lysosome, cytosol, and nucleus) of exogenous DNA transfected by viral and nonviral vectors in living cells, using a combination of TaqMan PCR and a recently developed confocal image-assisted three-dimensionally integrated quantification method. As a model, adenovirus (Ad) and Lipofectamine Plus (LFN) were used for comparison since they are highly potent and widely used viral and nonviral vectors, respectively. The findings indicate that the efficiency of cellular uptake for LFN is significantly higher than that for Ad. Once taken up by a cell, Ad exhibited comparable endosomal escape and slightly higher nuclear transfer efficiency compared with LFN. In contrast, LFN requires 3 orders of magnitude more intranuclear gene copies to exhibit a transgene expression comparable to that of the Ad, suggesting that the difference in transfection efficiency principally arises from differences in nuclear transcription efficiency and not from a difference in intracellular trafficking between Ad and LFN.

Adenoviridae↗
PubMed2002

Metabolic physiology of aroma-producing Kluyveromyces marxianus.

Kluyveromyces marxianus has a high potential for industrial production of aroma compounds, such as 2-phenylethanol, which is derived in a bioconversion from L-phenylalanine. In the present work the product yield of K. marxianus in batch cultivation was estimated as 0.65 mol 2-phenylethanol/mol L-phenylalanine and thus significantly below the theoretical optimum of 1 mol/mol. By a comprehensive approach of stoichiometric balancing and GC-MS analysis of various substrates and products of K. marxianus a detailed insight into its metabolism was gained. For this purpose ring-labelled ((13)C(6)) L-phenylalanine and naturally labelled glucose were applied as substrates in tracer studies in batch culture. The produced aroma compounds 2-phenylethanol and 2-phenylethylacetate stem exclusively from the supplied L-phenylalanine, whereas glucose was not converted into these products because of efficient feed-back inhibition of prephenate dehydratase in the L-phenylalanine biosynthetic pathway. It could be further shown that the supplied L-phenylalanine completely covers the anabolic cellular demand for this amino acid. Quantification of (13)CO(2) in the exhaust gas provided clear evidence for catabolic breakdown of L-phenylalanine during cultivation. Metabolic balancing around the pool of free intracellular L-phenylalanine revealed a significant loss of L-phenylalanine into catabolic and anabolic pathways. While 73.3% of L-phenylalanine was converted into 2-phenylethanol or 2-phenylethylacetate, 22.4% was catabolized through the cinnamate pathway and 4.3% was directed towards protein biosynthesis. Catabolic breakdown of L-phenylalanine via hydroxylation to L-tyrosine could be excluded. In addition to an insight into metabolic functioning and regulation of 2-phenylethanol-producing K. marxianus, the approach presented here provides important information on potential targets for genetic optimization of 2-phenylethanol-producing yeasts.

Biotechnology↗
PubMed1998

Quantification of human dopamine D2s receptor interactions with G alpha(i,1,2)- and G alpha(o)-proteins.

A simple and rapid in vitro method for qualitative and quantitative estimation of the G alpha-subunits interaction with the third intracellular loop of human D2s dopamine receptor has been developed. For this purpose, D2s-CL3 was cloned in pGEX-2T vector and expressed in E. coli BL21 DE3 as a fusion protein with glutathione-S-transferase (D2s-CL3-GST). The resulting soluble construct was purified by affinity chromatography on glutathione-Sepharose. G alpha-subunits were expressed and purified as His-tagged proteins. For the assay of G alpha/D2s-CL3-GST interactions, varying concentrations of pure His-tagged G alpha-proteins were immobilized on His-Bind Resin and titrated with D2s-CL3-GST fusion protein. G alpha/D2s-CL3-GST interactions were quantified by GST activity determination assay. It was shown that the fusion protein interacts specifically with different G alpha proteins, especially with G alpha(i) proteins. Based on saturation binding analyses, Kd values were determined revealing the highest affinity of His-G alpha(i,2) binding to the fusion protein. The affinities for G alpha(i)/D2s-CL3-GST protein interactions estimated in this way were in nanomolar range of concentrations.

Escherichia coli↗
PubMed2000

[Ca(2+)](i) oscillations regulate type II cell exocytosis in the pulmonary alveolus.

Pulmonary surfactant, a critical determinant of alveolar stability, is secreted by alveolar type II cells by exocytosis of lamellar bodies (LBs). To determine exocytosis mechanisms in situ, we imaged single alveolar cells from the isolated blood-perfused rat lung. We quantified cytosolic Ca(2+) concentration ([Ca(2+)](i)) by the fura 2 method and LB exocytosis as the loss of cell fluorescence of LysoTracker Green. We identified alveolar cell type by immunofluorescence in situ. A 15-s lung expansion induced synchronous [Ca(2+)](i) oscillations in all alveolar cells and LB exocytosis in type II cells. The exocytosis rate correlated with the frequency of [Ca(2+)](i) oscillations. Fluorescence of the lipidophilic dye FM1-43 indicated multiple exocytosis sites per cell. Intracellular Ca(2+) chelation and gap junctional inhibition each blocked [Ca(2+)](i) oscillations and exocytosis in type II cells. We demonstrated the feasibility of real-time quantifications in alveolar cells in situ. We conclude that in lung expansion, type II cell exocytosis is modulated by the frequency of intercellularly communicated [Ca(2+)](i) oscillations that are likely to be initiated in type I cells. Thus during lung inflation, type I cells may act as alveolar mechanotransducers that regulate type II cell secretion.

Animals↗
PubMed1995

Plasminogen activator inhibitor type 2: an intracellular keratinocyte differentiation product that is incorporated into the cornified envelope.

Human epidermal keratinocytes synthesize a complex plasminogen activator proteolytic cascade, consisting of two plasminogen activating enzymes and two inhibitors, that is thought to play a role in epidermal migration and differentiation as well as in several cutaneous diseases. Quantification of the plasminogen activator cascade proteins in keratinocytes reveals that plasminogen activator inhibitor type 2 (PAI-2) is distinct from the other components (i.e., urokinase and tissue-type plasminogen activators and inhibitor type 1) in several respects: (i) PAI-2 remains mostly cell-associated, rather than secreted; (ii) The level of cell-associated PAI-2 is at least 50-fold greater than that of the other components; (iii) PAI-2 is the only component whose level is enhanced upon elevation of the Ca2+ concentration, which is well known to induce a more differentiated phenotype in keratinocyte culture. Immunocytochemical localization experiments reveal that most keratinocytes contain PAI-2, which in a subpopulation of more differentiated cells is resistant to detergent extraction. Additional immunocytochemical localization and immunoblot experiments demonstrate that some of the PAI-2 becomes incorporated into the cornified envelope during terminal differentiation of the keratinocyte. These studies raise the possibility that PAI-2 may have an intracellular role associated with the terminal stage of keratinocyte differentiation.

Cell Differentiation↗
PubMed2002

Quantification of G6PD in small and large intestine of rat during aging.

Numerous studies have demonstrated a decrease in glucose-6-phosphate dehydrogenase (G6PD) activity during aging in many cell types, including red blood cells, fibroblasts and lens cells. Moreover, the intracellular activity of G6PD has been shown to be regulated by binding to cell organelles. To investigate whether binding of G6PD to cell organelles is related with the decrease in its activity during aging, distribution patterns of G6PD activity and protein were assessed in small (SI) and large (LI) intestine of 3-month-old and 28-month-old rats. Enzyme activity, as measured spectrophotometrically, did not show any significant change with aging in SI or LI. Enzyme histochemistry, performed by subtracting activity staining of 6-phosphogluconate dehydrogenase (6PGD) from that of G6PD, showed a lower net G6PD activity in SI and LI epithelium of old rats in comparison with young rats. G6PD activity did not change significantly with aging in the muscularis externa of SI and LI. Immunoelectron microscopic analysis of G6PD protein allowed us to measure the density of G6PD molecules in cellular compartments, and the fraction of enzyme bound to cell organelles. In SI and LI epithelia, density of G6PD molecules was higher in old rats than in young rats; however, the fraction of enzyme bound to cell organelles also increased with aging. These data suggest that G6PD activity in epithelium of SI and LI decreases with aging due to the accumulation of significant amounts of enzyme bound to cell organelles, a condition which makes it less active than the soluble enzyme.

Aging↗
PubMed1999

Intracellular expression of a cloned antibody fragment interferes with hepatitis B virus surface antigen secretion.

We evaluated the potential of an intracellularly expressed antibody fragment to interfere with hepatitis B virus (HBV). Sequences coding for the immunoglobulin variable regions of the HBV surface antigen (HBsAg) specific monoclonal antibody 5C3 were isolated and characterized. A secretory pathway-targeted, 5C3 derived single chain Fv (sFv) fragment was expressed in HuH-7 hepatocellular carcinoma cells together with HBsAg. Quantification of extracellular HBsAg levels in the cell culture supernatant demonstrated that the presence of the 5C3 sFv equipped with a secretory pathway retention signal SEKDEL reduced extracellular HBsAg levels by a mean of 85%. Co-immunoprecipitation studies revealed that the 5C3 sFv targeted to the secretory pathway physically interacted with its target antigen, HBsAg. Confocal microscopy studies confirmed the intracellular expression and colocalization of the 5C3 sFv and HBsAg. We conclude that certain intracellularly expressed antibody fragments will substantially interfere with HBV antigen secretion from the cell.

Amino Acid Sequence↗
PubMed2004

Calpain-mediated proteolysis of talin regulates adhesion dynamics.

Dynamic regulation of adhesion complexes is required for cell migration and has therefore emerged as a key issue in the study of cell motility. Recent progress has been made in defining some of the molecular mechanisms by which adhesion disassembly is regulated, including the contributions of adhesion adaptor proteins and tyrosine kinases. However, little is known about the potential contribution of proteolytic mechanisms to the regulation of adhesion complex dynamics. Here, we show that proteolysis of talin by the intracellular calcium-dependent protease calpain is critical for focal adhesion disassembly. We have generated a single point mutation in talin that renders it resistant to proteolysis by calpain. Quantification of adhesion assembly and disassembly rates demonstrates that calpain-mediated talin proteolysis is a rate-limiting step during adhesion turnover. Furthermore, we demonstrate that disassembly of other adhesion components, including paxillin, vinculin and zyxin, is also dependent on the ability of calpain to cleave talin, suggesting a general role for talin proteolysis in regulating adhesion turnover. Together, these findings identify calpain-mediated proteolysis of talin as a mechanism by which adhesion dynamics are regulated.

Animals↗
PubMed2002

[Structural and functional analysis of 2.2 kb spliced variant of hepatitis B virus genomes isolated from liver tissues from hepatocellular carcinoma patients].

BACKGROUND: To study the structure and function of 2.2 kb spliced variant of HBV genome from liver tissues of hepatocellular carcinoma patients. METHODS: HBV genomes were amplified by using PCR from paired hepatocellular carcinoma tissues and peritumor tissues. The 3.2 kb full-length HBV genome and 2.2 kb spliced variant were separately cloned and sequenced. Hep G2 cells were co-transfected with full-length HBV DNA and 2.2 kb spliced variant, and after transfection, HBV DNAs from intracellular core particles were harvested and specific primers were used in PCR to evaluate the interactions between spliced variant and full-length counterpart in replication. RESULTS: Semi-quantification by scanning density showed that 2.2 kb spliced variant was present in all tumor and peri-tumor samples studied. Sequence analyes revealed that the 5 terminus packaging signal for pregenomic and X and PreC/C genes were retained. When full-length HBV DNA was co-transfected with 2.2 kb, the replication signal of 3.2 kb HBV genome was increased 3-7 times. CONCLUSIONS: The 2.2 kb HBV spliced variant was present in liver tissues, and relative content was higher in tumor tissues than that in the peri-tumor tissues. This spliced variant could enhance the replication of full-length HBV genome, which suggested the possible role of the variant in the pathogenesis of development of hepatocellular carcinoma.

Adult↗
PubMed1987

[Contrast and quantitative characteristics of biological tissues in experimental neoplastic processes based on biexponential relaxation analysis of NMR tomograms].

A method of contrast enhancement and quantification of experimental tumors in vivo by estimation of tissue proton transverse magnetic relaxation nonexponentiality is proposed. Relaxation time and percentage of a rapidly descending magnetization component due to intracellular water protons are followed for tumor and uninvolved tissues in a course of subcutaneously transplanted solid mouse melanoma B16 development. The exponential approximation of a relaxation curve within the 6-90 ms time range is shown to fit melanoma B16 tissue characterization. Contrast-enhanced calculated MR-images are presented.

Animals↗
PubMed2003

Quantification and cytokine production of circulating lymphoid and myeloid cells in acute myelogenous leukaemia.

A simple assay was developed to assess the potential of patients with acute myelogenous leukaemia (AML) to respond to immunotherapy. Lymphocytes, monocytes and leukaemic blasts with their corresponding intracellular cytokine profiles were evaluated by four-colour flow cytometry. In 50 microl samples of whole blood, surface labelling for CD45, CD8 and CD3 was used for cell identification prior to intracellular staining for interleukin (IL)-4, IL-10, IL-12 and interferon (IFN)-gamma. Absolute numbers of CD8(+) and CD8(-) (putative CD4(+)) T-cells, NK cells (CD8(+)/CD3(-)) and monocytes were determined by reference to a fixed number of added fluorescent beads. The absolute numbers of CD8(-) and CD8(+) T-cells in the blood of patients with AML were similar to those of normal controls. More of the lymphocytes in the blood of leukaemic patients spontaneously produced cytokines compared with those of controls. Furthermore, primary AML blasts secreted predominantly IFN-gamma. After recovery from chemotherapy, lymphocyte counts tended to be lower than in normals and reduction of NK cells reached significance after the second chemotherapy (P=0.01). A prominent CD8(lo)/CD3(lo-int) lymphocyte subset appeared after recovery in some patients. This laboratory application of the study of cell subsets and intracellular cytokines in patients undergoing treatment may be helpful in monitoring immunological responses in AML.

Acute Disease↗
PubMed2004

Incorporation of OSI-7836 into DNA of Calu-6 and H460 xenograft tumors.

OSI-7836 (4'-thio-beta-D-arabinofuranosylcytosine) is a novel nucleoside analog in phase I clinical development for the treatment of cancer. As with other nucleoside analogs, the proposed mechanism of action involves phosphorylation to the triphosphate form followed by incorporation into cellular DNA, leading to cell death. This hypothesis has been examined by measuring and comparing the incorporation of ara-C, OSI-7836, and gemcitabine (dFdC) into DNA of cultured cells and by investigating the role of deoxycytidine kinase in OSI-7836 toxicity. We report here additional studies in which the role of cell cycling on OSI-7836 toxicity was investigated and incorporation of OSI-7836 into DNA of xenograft tumors measured. The role of the cell cycle was examined by comparing the toxicity of OSI-7836 in A549 NSCLC cells that were either in log phase growth or had reached confluence. A novel validated LC-MS/MS assay was developed to quantify the concentrations of OSI-7836 in DNA from Calu-6 and H460 human tumor xenografts in mice. Results showed that apoptosis induced by OSI-7836 was markedly greater in cycling cells than in confluent non-cycling cells despite only a modest increase in intracellular OSI-7836 triphosphate concentration. The LC-MS/MS assay developed to measure OSI-7836 incorporation into DNA had an on-column detection limit of 0.25 fmol, a quantification limit of 0.5 fmol, and a sensitivity of approximately 0.1 pmol OSI-7836/micromol dThy. Concentrations of OSI-7836 in splenic DNA (0.4 pmol OSI-7836/micromol dThy) averaged fivefold less than the average concentration in Calu-6 and H460 xenograft DNA (3.0 pmol OSI-7836/micromol dThy) following a 400 mg/kg dose of OSI-7836. Concentrations of OSI-7836 in Calu-6 tumor DNA isolated 24 h following a dose of 400, 1000, or 1600 mg OSI-7836/kg were approximately 1.3, 1 and 1.3 pmol OSI-7836/micromol dThy, respectively. Concentrations of OSI-7836 in DNA from H460 and Calu-6 xenografts did not appear to increase during repeated administration of 400 mg OSI-7836/kg on days 1, 4, 7, and 10. The majority of OSI-7836 in DNA from Calu-6 and H460 tumors of mice dosed with 1600 mg/kg was located at internal nucleotide linkages, similar to dFdC and ara-C. In conclusion, cell cycling studies supported the hypothesis that OSI-7836 cytotoxicity is dependent upon DNA synthesis. A validated LC-MS/MS assay was developed that could quantify OSI-7836 in DNA from tissues. The assay was used to show that OSI-7836 was incorporated in internal linkages in tumor DNA in a manner that was dose-independent at the doses tested and did not appear to accumulate during repeated dosing. The results suggest that if DNA incorporation is a toxic event, the relationships between administered dose, DNA incorporation, and toxicity are complex.

Animals↗