Human Tissue Extracts Research Articles

Transmission of Synucleinopathies in the Enteric Nervous System of A53T Alpha-Synuclein Transgenic Mice

Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are characterized by abnormal deposition of α-synuclein aggregates in many regions of the central and peripheral nervous systems. Accumulating evidence suggests that the α-synuclein pathology initiates in a few discrete regions and spreads to larger areas in the nervous system. Recent pathological studies of PD patients have raised the possibility that the enteric nervous system is one of the initial sites of α-synuclein aggregation and propagation. Here, we evaluated the induction and propagation of α-synuclein aggregates in the enteric nervous system of the A53T α-synuclein transgenic mice after injection of human brain tissue extracts into the gastric walls of the mice. Western analysis of the brain extracts showed that the DLB extract contained detergent-stable α-synuclein aggregates, but the normal brain extract did not. Injection of the DLB extract resulted in an increased deposition of α-synuclein in the myenteric neurons, in which α-synuclein formed punctate aggregates over time up to 4 months. In these mice, inflammatory responses were increased transiently at early time points. None of these changes were observed in the A53T mice injected with saline or the normal brain extract, nor were these found in the wild type mice injected with the DLB extract. These results demonstrate that pathological α-synuclein aggregates present in the brain of DLB patient can induce the aggregation of endogenous α-synuclein in the myenteric neurons in A53T mice, suggesting the transmission of synucleinopathy lesions in the enteric nervous system.

MMSAT: Automated Quantification of Metabolites in Selected Reaction Monitoring Experiments

Selected reaction monitoring (SRM) is a mass spectrometry-based approach commonly used to increase analytical sensitivity and selectively for specific compounds in complex metabolomic samples. While the goal of well-designed SRM methods is to monitor for unique precursor-product ion pairs, in practice this is not always possible due to the diversity of the metabome and the resolution limits of mass spectrometers that are capable of SRM. Isobaric or near-isobaric precursor ions with different chromatographic properties but identical product ions often arise in complex samples. Without analytical standards, such metabolites will go undetected by conventional data analysis methods. Furthermore, a single SRM method may include simultaneous monitoring of tens to hundreds of different metabolites across multiple samples making quantification of all detected ions a challenging task. To facilitate the analysis of SRM data from complex metabolomic samples, we have developed the Metabolite Mass Spectrometry Analysis Tool (MMSAT). MMSAT is a web-based tool that objectively quantifies every metabolite peak detected in a set of samples and aligns peaks across multiple samples to enable quantitative comparison of each metabolite between samples. The analysis incorporates quantification of multiple peaks/ions that have different chromatographic retention times but are detected within a single SRM transition. We compare the performance of MMSAT against existing tools using a human glioblastoma tissue extract and illustrate its ability to automatically quantify multiple precursors within each of three different transitions. The Web-interface and source code is avaliable at http://www.cancerresearch.unsw.edu.au/crcweb.nsf/page/MMSAT .

Adrenomedullin in Heart Failure: Molecular Mechanism and Therapeutic Implication

Many neurohumoral factors play an important role in regulation of the cardiovascular system and in the pathophysiology of heart failure. Adrenomedullin (AM) is a potent long-lasting vasodilatory peptide that was discovered in acid extracts of human pheochromocytoma tissues. Both AM and its gene expression are widely distributed in the cardiovascular system, including the heart, vessels, and kidneys. AM co-localizes with its receptor components such as calcitonin receptor-like receptor (CRLR), receptor activity modifying protein (RAMP)2 and RAMP3 in the heart, vessels and kidneys, suggesting that it plays an important role in the regulation of cardiovascular function through an autocrine and/or paracrine mechanism. AM has inotropic action in vitro and in vivo and inhibits cardiac hypertrophy in myocytes as well as proliferation and collagen production in cardiac fibroblasts. These results suggest that AM functions as an antifibrotic, antihypertrophic and positive inotropic factor in the failing heart. In addition, AM has anti-apoptotic, angiogenic, anti-inflammatory and anti-oxidant effects. Several mechanisms such as cAMP/PKA, NO/cGMP, PI-3K/Akt and/or ERK are thought to mediate cellular AM signaling, suggesting that increased AM levels play a protective role in heart failure. Indeed, acute AM administration exerts beneficial vasodilatory, diuretic, natriuretic and inotropic effects on experimental and human heart failure. A recent pilot study has shown that AM has potential as a therapeutic drug in the clinical setting of acute decompensated heart failure. Keywords: Adrenomedullin, heart failure, antifibrotic effects, cardiovascular diseases, neurohumoral factors, cAMP response

Abstract C14: Tissue expression profile of novel tumor biomarkers identified by monoclonal antibodies.

Abstract Background: Monoclonal antibodies (mAbs) were raised against human colon tumor tissue extracts. Antibodies that specifically recognized tumor tissues, but did not cross-react with normal tissues, were selected for further characterization and development as diagnostic reagents and therapeutic candidates. Three mAbs that react with variant forms of MUC5AC, CEACAM molecules, and A33 were identified. The immunohistochemical staining profiles of these antibodies (NEO-101, NEO-201, and NEO-301) will be discussed. Methods: The three antibodies (NEO-101, NEO-201, and NEO-301) were each biotin-labeled, and used to stain a variety of tumor and normal human tissue types, typically at 10 ug/mL. Binding of the primary antibody was detected using streptavidin-HRP conjugate. Slides were reviewed and scored by light microscopy. Results: The NEO-101 antibody recognizes a variant form of MUC5AC expressed by colorectal (79%) and pancreatic (48%) cancer tissues. NEO-101 did not significantly cross-react with normal colon or pancreas tissues. Staining appeared to be cytoplasmic, membrane-associated, and elaborated into the lumenal spaces of positively stained tissues. The NEO-201 antibody recognizes variant forms of both CEACAM-5 and CEACAM-6. NEO-201 reacts with a wider variety of tumor tissue types including colorectal (98%), pancreatic (80%), lung (71%), stomach (100%), esophagus (61%), breast (31%), and ovarian (31%). Cross-reactivity to normal tissues was weak and sporadic. The NEO-301 antibody recognizes a variant form of A33, that is expressed by colorectal (46%), pancreatic (52%), lung (65%), and breast (45%) tumor tissues. NEO-301 also does not cross-react significantly with normal tissues. Antibodies NEO-201 and NEO-301 stained both cytoplasm and membranes of tissues examined. Conclusions: NEO-101, NEO-201, and NEO-301 antibodies react specifically with tumor tissues without significant cross-reactivity to normal tissues, an attractive characteristic for developing effective therapeutic and diagnostic products for oncology indications. We suggest that the antigens they react with are novel variants of commonly known wild type proteins that can be used as biomarker since they do not cross-react with healthy tissues. Interestingly, although these 3 antibodies were generated from extracts of pooled human colon tumor tissues, the variant antigens they cross-react with are not restricted to expression by colon tumor tissues, supporting the idea that variant protein expression may be a general property of tumor cells compared to normal cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C14.

Protein-Binding Microarray Analysis of Tumor Suppressor AP2α Target Gene Specificity

Cheap and massively parallel methods to assess the DNA-binding specificity of transcription factors are actively sought, given their prominent regulatory role in cellular processes and diseases. Here we evaluated the use of protein-binding microarrays (PBM) to probe the association of the tumor suppressor AP2α with 6000 human genomic DNA regulatory sequences. We show that the PBM provides accurate relative binding affinities when compared to quantitative surface plasmon resonance assays. A PBM-based study of human healthy and breast tumor tissue extracts allowed the identification of previously unknown AP2α target genes and it revealed genes whose direct or indirect interactions with AP2α are affected in the diseased tissues. AP2α binding and regulation was confirmed experimentally in human carcinoma cells for novel target genes involved in tumor progression and resistance to chemotherapeutics, providing a molecular interpretation of AP2α role in cancer chemoresistance. Overall, we conclude that this approach provides quantitative and accurate assays of the specificity and activity of tumor suppressor and oncogenic proteins in clinical samples, interfacing genomic and proteomic assays.

A novel ELISA for eosinophil peroxidase provides a sensitive high throughput assay for eosinophil degranulation in either mouse or human biological samples (65.29)

Abstract Eosinophils comprise only 1-3% of circulating leukocytes but are often a diagnostic feature associated with a variety of inflammatory and disease states, including parasitic and fungal infections, allergic diseases (e.g., asthma, rhinitis and sensitivities to specific foods), cancer, and transplant rejection. The accurate evaluations of eosinophilia as well as the release of stored eosinophil granule proteins are important for the monitoring disease progression and/or assessing the effectiveness of a given treatment strategy(ies). Despite the availability of detection methods for each of the eosinophil granule proteins logistical difficulties limiting either the assays specificity and/or sensitivity have prevented their extensive use. We have generated unique eosinophil peroxidase specific monoclonal antibodies (EPX-mAb) and a high throughput sandwich ELISA assay as a means of overcoming these difficulties. Our EPX-mAb based ELISA is ~10 times more sensitive than traditional OPD based activity assays and allows detection of EPX in mouse BAL fluid after an acute OVA protocol as well as EPX release from mouse eosinophils stimulated ex vivo. The assay is highly specific and gives no signal in samples from EPX deficient mice even with massive eosinophilia (e.g., EPX deficient IL-5 transgenic mice). More significantly, this assay detects EPX in human tissue extracts and biological fluids and thus represents novel diagnostic assay previously unavailable in clinical settings.

B-Type Natriuretic Peptide Signal Peptide Circulates in Human Blood

The diagnosis of cardiac necrosis such as myocardial infarction can be difficult and relies on the use of circulating protein markers like troponin. However, there is a clear need to identify circulating, specific biomarkers that can detect cardiac ischemia without necrosis. Using specific immunoassay and tandem mass spectrometry, we show that a fragment derived from the signal peptide of B-type natriuretic peptide (BNPsp) not only is detectable in cytosolic extracts of explant human heart tissue but also is secreted from the heart into the circulation of healthy individuals. Furthermore, plasma levels of BNPsp in patients with documented acute ST-elevation myocardial infarction (n=25) rise to peak values ( approximately 3 times higher than the 99th percentile of the normal range) significantly earlier than the currently used biomarkers myoglobin, creatine kinase-MB, and troponin. Preliminary receiver-operating characteristic curve analysis comparing BNPsp concentrations in ST-elevation myocardial infarction patients and other patient groups was positive (area under the curve=0.97; P<0.001), suggesting that further, more rigorous studies in heterogeneous chest pain patient cohorts are warranted. Our results demonstrate for the first time that BNPsp exists as a distinct entity in the human circulation and could serve as a new class of circulating biomarker with the potential to accelerate the clinical diagnosis of cardiac ischemia and myocardial infarction. Clinical Trial Registration- URL: http://www.anzctr.org.au. Unique identifier: ACTRN12609000040268.

Development of an ELISA for quantification of tumstatin in serum samples and tissue extracts of patients with lung carcinoma

Background Tumstatin, an angiogenesis inhibitor with anti-tumor activity in mice, is the bioactive NC1 domain of Col IVa3, the potential significance of tumstatin as an endogenous angiogenesis inhibitor in human is not yet completely understood. This study aimed to develop an enzyme-linked immunoassay (ELISA) for tumstatin to accurately measure its concentrations in biological samples. Methods Recombinant tumstatin as an immunogen was expressed by E. coli. The purified tumstatin was injected into mice for antibody generation. An ELISA was developed with the monoclonal antibodies. Results The detection limit of the ELISA was 1.4 ng/ml, and the intra- and inter- assay CVs were within 10%. Recovery of tumstatin added to sera was 92.7–115%. Patients with metastases had serum tumstatin levels 50% lower than patients without metastases ( P = 0.039). Tumstatin levels in poorly differentiated tumor tissues were significantly lower than in nontumorous tissues and well-differentiated tumor tissues ( P < 0.001). Conclusions The development of a highly sensitive and reliable ELISA method capable of quantifying tumstatin in human serum samples and tissue extracts is reported. This assay for tumstatin in biological samples may be helpful in evaluating the therapeutic and/or prognostic value of tumstatin in cancer patients.

Circulating and tissue-derived isoforms of bone alkaline phosphatase in Paget's disease of bone

Background. Alkaline phosphatase (ALP) is routinely used in the assessment of Paget's disease of bone (PDB); however, the individual bone ALP isoforms (B/I, B1, and B2) have not been investigated in this disorder. Methods. Subjects comprised 37 patients (mean age 74 years) with symptomatic PDB confirmed by radiograph and stratified into high and low total ALP activity groups and 66 healthy individuals (mean age 64 years). Extracts of human cancellous and cortical bone tissues were also investigated. The bone ALP isoforms, measured by HPLC, were compared with two bone ALP immunoassays (Metra® and Ostase®), and the bone formation marker intact amino-terminal procollagen type I propeptide (iPINP). Results. All bone ALP isoforms were increased in high ALP activity PDB compared with the low ALP activity and control groups (p < 0.0001). The B2 isoform had the greater relative activity representing 36%, 50%, and 71%, of the total ALP activity in the control, low and high ALP activity groups, respectively. Compared with controls, B2 was increased in the low ALP activity PDB group (p < 0.05). ROC analysis showed a validity of approximately 80% for B2 to discriminate patients with PDB. Conclusion. All bone ALP isoforms were increased in patients with high ALP activity PDB and the B2 isoform was even elevated in the low ALP activity PDB group. The bone ALP isoform B2 may be of use in the management of PDB but that has to be further elucidated in subsequent studies.

The microenvironment in chronic pancreatitis and pancreatic cancer induces neuronal plasticity

Pancreatic neuropathy in chronic pancreatitis (CP) and pancreatic cancer (PCa) is characterized by pancreatic neuropathy, i.e. increased neural density and hypertrophy, which are associated with neuropathic pain. To better understand the mechanism of these neuropathic alterations, we aimed at achieving an in-vitro simulation of the intrapancreatic neuroplasticity. Dissociated myenteric plexus (MP) and dorsal root ganglia (DRG) neurons of newborn rats were treated with normal human pancreas (NP), CP or PCa tissue extracts. Furthermore, MP and DRG neurons were cultured in supernatants from different pancreatic cancer cell lines (PCC) and human pancreatic stellate cells (hPSC) obtained from either CP or PCa tissues. For analysis, the neurite density, outgrowth, neuronal branching capacity and perikaryonal size were quantified. Myenteric plexus and DRG neurons grown in CP and PCa tissue extracts built denser networks than in NP extracts. Both neuronal types showed a strong neurite outgrowth, more complex branching pattern and a somatic hypertrophy in CP and PCa extracts. Pancreatic cancer cell supernatants induced a prominent neurite outgrowth, increased neurite density and perikaryonal hypertrophy in MP and DRG neurons. Supernatants of CP-derived hPSC strongly stimulated neurite outgrowth. Glial density in MP cultures was strikingly increased by PCa tissue extracts. Intrapancreatic microenvironment in CP and PCa induces neuroplastic alterations under in-vitro conditions, leading to increased neural density and hypertrophy. Thus, due to its neurotrophic attributes, the intrapancreatic microenviroment in CP and PCa seems to be a key player in the generation of pancreatic neuropathy and neuroplasticity.

Receptor affinity CE for measuring bioactive inflammatory cytokines in human skin biopsies

A chip-based receptor affinity CE system has been employed to measure the concentrations of bioactive pro-inflammatory cytokines in biopsy materials obtained from human atopic skin lesions. The device employs a replaceable affinity disk to which recombinant cytokine receptors have been chemically immobilized. Homogenates obtained from micro-dissected human skin samples were injected into the system where the bioactive cytokines were captured in the receptor affinity port and labeled in situ with a laser dye. The captured cytokines were released and separated by CE, the resolved peaks being detected and measured by laser-induced fluorescence. When compared with conventional cell-based bioassays, the affinity receptor chip showed reasonable correlation with r(2) values of 0.998 for interferon gamma, 0.994 for IL-6 and 0.991 for tumor necrosis factor alpha. The complete process including cytokine capture, labeling, and analysis took approximately 12.5 min with intra- and inter-assay CVs below 5.3% and recoveries of 84.9-98.4% at the 100 pg/mL concentration in buffer solutions and 84.5-95% in normal human tissue extract. The system could indicate clear differences between the various clinical stages of atopic dermatitis in human patients and could run 4-6 samples per hour. This system, like previous chip-based systems designed in our laboratory, holds the potential for being modified to be a portable unit that could be used in clinics and other biomedical screening studies.

A new easy method for specific measurement of active myeloperoxidase in human biological fluids and tissue extracts

The SIEFED (“Specific Immunological Extraction Followed by Enzymatic Detection”) method already developed for the specific detection of the activity of equine myeloperoxidase (MPO) was adapted for the specific measurement of active human MPO in biological fluids or tissue extracts. The method consists of the extraction of MPO from aqueous solutions by immobilized anti-MPO antibodies followed by a washing (to eliminate the extraction medium and the biological fluid with their possible interfering molecules) and the measurement of the activity of MPO with a detection system containing a fluorogenic substrate, H 2O 2 and nitrite ions as reaction enhancer. The SIEFED was applied to study active MPO in human biological fluids (plasma, bronchoalveolar lavage fluid and supernatant from carotids extracts). The SIEFED for human MPO has a sensitivity limit of 0.080 mU/mL and showed good precision with intra- and inter-assay coefficients of variation below 10 and 20% respectively within a broad range of MPO activities establish from 0.156 to 473 mU/mL. The SIEFED for human MPO will be useful for the specific detection of active MPO in complex fluids and can be complementary to an ELISA to determine an active/total MPO ratio in healthy volunteers and patients especially in case of chronic or acute inflammatory diseases.

Thyroglobulin may affect telomerase activity in thyroid follicular cells

Telomerase (TA) activity is known to be present in malignant tumor cells, but not in most somatic differentiated cells. TA shows relatively high activity in thyroid cancer cells, but reports vary. This fact prompted us to elucidate whether cell component inhibitors of TA in the thyroid follicles can modulate its activity. The activity of TA extracted from Hela cells was inhibited by mixing with the supernatant fraction of human thyroid tissue extract. To examine the effect of iodine, thyroid hormones (ℓ-T3 and ℓ-T4) and human thyroglobulin (hTg) contained in the thyroid follicles, ℓ-T3, ℓ-T4 and hTg were added to the TRAP assay system in vitro, using TA from Hela cells. Iodine, ℓ-T3 and ℓ-T4 did not affect TA activity, but hTg inhibited the TA activity in a dose-dependent manner (IC50 of hTg: ca 0.45 μM: inhibiting concentration of hTg was from 0.15 μM to 3.0 μM). The hTg inhibition was not evident in the RT-PCR system, suggesting no effect of hTg on Taq DNA polymerase activity. The hTg inhibition of TA activity was attenuated by dNTP but not significantly by TS primer. These data suggest that hTg contained in thyroid follicular cells of various thyroid diseases may affect the TA activity measured in biopsied thyroid specimens, and that the reduction of the TA activity by hTg may induce slow progression and growth, and low grade malignancy of thyroid cancer, particularly differentiated carcinoma.

Analysis of Cardiac Myofibrillar Troponin I Phosphorylation in Normal and Failing Human Hearts Using Phos-Tags

Recently, we have used phosphate-affinity SDS-PAGE gels containing Phos-tag-acrylamide (a phosphate-chelating molecule), to determine the level of cardiac troponin I (cTnI) phosphorylation in human myofibrillar extracts. The Phos-tag moiety binds to, and retards, the mobility of phosphoproteins through the gel and results in the separation of the phosphoprotein bands according to their phosphorylation level.Samples from time-courses of in-vitro PKA catalytic subunit-treated recombinant human cTnI and myofibrillar extracts from non-failing donor, hypertrophic obstructive cardiomyopathy (HOCM) and end-stage failing human heart tissue were analysed by phosphate-affinity SDS-PAGE. Separate gel bands corresponding to 1P, 2P, 3P, 4P and 5P cTnI were observed for the PKA-treated recombinant cTnI. Western blotting probed with the anti-cTnI antibody 14G5 and several different site specific phospho-cTnI antibodies demonstrated that all five of these phospho-species bound to a Ser24P-specific antibody, while a Thr144P-specific antibody only reacted with the 3P, 4P and 5P phospho-species of cTnI.We observed 3 phospho-species of cTnI in the human heart tissue extracts, which correspond to 0P, 1P and 2P cTnI. Ratios of 0P cTnI were significantly higher in failing and HOCM (both 63±4%) compared to donor (8±2%) while ratios of 2P were significantly lower (failing = 6±2%, HOCM = 8±2%, donor 73±6%). Western blots demonstrated that in human heart cTnI phosphorylation of Ser23/24 was mainly present in the 2P species (with a very small proportion in the 1P species) and that there was no phosphorylation at Thr144. Calculated levels of total cTnI phosphorylation in both HOCM (0.37±0.03, n=50, p<0.0001) and failing heart (0.38±0.03, n=24, p<0.0001) were significantly reduced from levels in donor heart (1.65±0.04, n=38) and were comparable to previously determined measurements obtained from Pro-Q Diamond phosphoprotein gel staining.

CGI-58 Is an α/β-Hydrolase within Lipid Transporting Lamellar Granules of Differentiated Keratinocytes

CGI-58 is the causative molecule underlying Dorfman-Chanarin syndrome, a neutral lipid storage disease exhibiting apparent clinical features of ichthyosis. CGI-58, associated with triacylglycerol hydrolysis, has an alpha/beta-hydrolase fold and is also known as the alpha/beta-hydrolase domain-containing protein 5. The purpose of this study was to elucidate the function of CGI-58 and the pathogenic mechanisms of ichthyosis in Dorfman-Chanarin syndrome. Using an anti-CGI-58 antibody, we found CGI-58 to be expressed in the upper epidermis, predominantly in the granular layer cells, as well as in neurons and hepatocytes. Immunoelectron microscopy revealed that CGI-58 was also localized to the lamellar granules (LGs), which are lipid transport and secretion granules found in keratinocytes. CGI-58 expression was markedly reduced in the epidermis of patients with harlequin ichthyosis, demonstrating defective LG formation. In cultured keratinocytes, CGI-58 expression was mildly up-regulated under high Ca(2+) conditions and markedly up-regulated in three-dimensional, organotypic cultures. In the developing human epidermis, CGI-58 immunostaining was observed at an estimated gestational age of 49 days, and CGI-58 mRNA expression was up-regulated concomitantly with both epidermal stratification and keratinocyte differentiation. CGI-58 knockdown reduced expression of keratinocyte differentiation/keratinization markers in cultured human keratinocytes. Our results indicate that CGI-58 is expressed and packaged into LGs during keratinization and likely plays crucial role(s) in keratinocyte differentiation and LG lipid metabolism, contributing to skin lipid barrier formation.

A novel enzyme immunoassay specific for ABCA1 protein quantification in human tissues and cells

ATP-binding cassette transporter A1 (ABCA1) mediates the transport of cholesterol and phospholipids from cells to lipid-poor HDL and maintains cellular lipid homeostasis. Impaired ABCA1 function plays a role in lipid disorders, cardiovascular disease, atherosclerosis, and metabolic disorders. Despite the clinical importance of ABCA1, no method is available for quantifying ABCA1 protein. We developed a sensitive indirect competitive ELISA for measuring ABCA1 protein in human tissues using a commercial ABCA1 peptide and a polyclonal anti-ABCA1 antibody. The ELISA has a detection limit of 8 ng/well (0.08 mg/l) with a working range of 9-1000 ng/well (0.09-10 mg/l). Intra- and interassay coefficient of variations (CVs) were 6.4% and 9.6%, respectively. Good linearity (r = 0.97-0.99) was recorded in serial dilutions of human arterial and placental crude membrane preparations, and fibroblast lysates. The ELISA measurements for ABCA1 quantification in reference arterial tissues corresponded well with immunoblot analysis. The assay performance and clinical utility was evaluated with arterial tissues obtained from 15 controls and 44 patients with atherosclerotic plaques. ABCA1 protein concentrations in tissue lysates were significantly lower in patients (n = 24) as compared with controls (n = 5; 9.37 +/- 0.82 vs. 17.03 +/- 4.25 microg/g tissue; P < 0.01). The novel ELISA enables the quantification of ABCA1 protein in human tissues and confirms previous semiquantitative immunoblot results.

Rapid and sensitive HPLC assay for simultaneous determination of procaine and para-aminobenzoic acid from human and rat liver tissue extracts

A sensitive and rapid high-performance liquid chromatography method has been developed for simultaneous determination of procaine and its metabolite p-aminobenzoic acid (PABA) from human and rat liver tissue extracts. The method has been validated according to ICH guidelines in terms of selectivity, linearity, lower limit of detection, lower limit of quantitation, accuracy, precision and recovery from human and rat liver tissue extracts. Chromatography was carried out on a Discovery ® C 18 column using 10 mM ammonium acetate at pH 4.0 and acetonitrile as mobile phase. Retention times for procaine and PABA were 6.6 and 5.3 min, respectively. Linearity for each calibration curve in both tissue extracts was observed across a range from 10 μM to 750 μM for procaine and PABA. The lower limit of detection for both procaine and PABA was 5 μM and the lower limit of quantitation was 10 μM in both tissue extracts. The intra- and inter-day relative standard deviations (R.S.D.) for both procaine and PABA were <6%. Recoveries of procaine and PABA from human and rat liver tissue extracts were determined by two different methods with a single-step protein precipitation technique being employed in both methods. Recoveries for both procaine and PABA were greater than 80% from both human and rat liver tissue extracts.

Microscale solution IEF combined with 2‐D DIGE substantially enhances analysis depth of complex proteomes such as mammalian cell and tissue extracts

Current gel-based protein profiling methods such as 2-DE and fluorescent 2-D difference in gel electrophoresis (DIGE) evaluate small portions of complex proteomes. Hence, sample prefractionation is essential for more comprehensive proteome coverage and detection of low-abundant proteins. In this study, we describe the combination of DIGE labeling with microscale solution IEF (MicroSol-IEF) fractionation and subsequent analysis on slightly overlapping narrow pH range 2-D gels. By fluorescently tagging and mixing samples and controls prior to prefractionation, complications resulting from minor run-to-run variations during MicroSol-IEF separations of multiple samples are avoided. This greatly improves the reliability of quantitative comparisons. To illustrate its utility, this 3-D DIGE strategy was applied to analysis of human melanoma cells and mouse lung tissue extracts. Approximately 1000 reproducible spots can be obtained from narrow range 2-D gels of individual MicroSol-IEF fractions, and approximately 6000 spots can be obtained from entire proteomes. Quantitative changes in closely related samples could be more reliably detected and the method has a greatly increased capacity to distinguish between closely related protein isoforms. Thus the 3-D DIGE strategy produces a powerful method for more comprehensive and more reliable quantitative comparisons of protein profiles of very complex proteomes.

Seminal Plasma Lipocalin-Type Prostaglandin D Synthase: A Potential New Marker for the Diagnosis of Obstructive Azoospermia

We examined the relationship between L-PGDS (lipocalin-type prostaglandin D synthase) levels in seminal plasma and the presence or absence of obstruction in the male seminal tract. Semen samples were collected and analyzed from 1) 10 patients with normal semen parameters, 2) 9 with obstructive azoospermia, 3) 20 after vasectomy and 4) 14 with nonobstructive azoospermia. Seminal L-PGDS was measured using an enzyme-linked immunosorbent assay technique. We found that seminal plasma L-PGDS in the groups with obstruction was significantly lower than in any of the other groups (p <0.001). Using a cutoff of 100 microg/l all men with obstructive azoospermia had L-PGDS less than 100 microg/l, while none with normal sperm parameters did. Men with nonobstructive azoospermia had less homogeneity of L-PGDS levels, including 29.6% with L-PGDS more than 100 microg/l. Our results suggest that seminal L-PGDS level can potentially be a biomarker for assessing patency in the seminal tract in men with azoospermia. In men with azoospermia and high seminal L-PGDS (more than 100 microg/l) the diagnosis of nonobstructive azoospermia can be potentially made without biopsy. Our study shows that using semen L-PGDS levels provides a diagnosis of nonobstructive azoospermia in almost 30% of these men.

First molecular characterization and immunolocalization of keratoepithelin in adult human skeletal muscle

Keratoepithelin (KE) is an extracellular matrix protein that binds collagens, fibronectin, decorin, biglycan and integrins, interconnecting extracellular matrix components with resident cells in several tissues. KE has a molecular mass of 68 kDa and harbours four FAS1 domains named after those identified in the insect cell adhesion molecule fasciclin I. In humans, KE is preferentially expressed by the corneal epithelial layer and liberated towards the corneal stroma but it was also detected in the lung and in the bladder smooth muscle. No detailed information is available on the distribution of this protein in other human tissues. In this work, we have raised a polyclonal antibody against the recombinantly expressed human fourth FAS1 domain which is able to specifically detect KE in human skeletal muscle tissue extracts. Immunofluorescence experiments indicate that KE is localized around the perimysium and endomysium of each skeletal muscle fiber. The same kind of analysis shows that in muscle sections from patients affected by different forms of muscular dystrophy KE is upregulated and widely distributed in fibrotic tissues. The muscle specific expression of KE was also demonstrated by RT-PCR. In human skeletal muscle, KE may help to build up a bridge between collagen VI and yet unidentified muscle receptor(s), adding to the complexity of the adhesive molecular network established between muscle fibers and the surrounding basement membrane.