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PubMedDate not supplied

Determination of nitroimidazole residues in poultry tissues, serum and eggs by high-performance liquid chromatography.

A simple high-performance liquid chromatographic (HPLC) method was developed for determination of dimetridazole and metronidazole residues in poultry muscle, liver, serum and eggs. The drug residues were extracted from tissues and egg samples with acetonitrile followed by solid-phase extraction clean-up and HPLC analysis with photodiode array detector. Serum samples were treated with trichloroacetic acid, centrifuged and analysed without further clean-up. The detection limits were 2 ng/g and 5 ng/mL of analysed tissue (egg) and serum samples, respectively. Average recoveries for spike levels of 10 and 20 ng/g ranged from 82 to 94%, and coefficients of variation were below 10%.

Animals↗

PubMed2005

MyoD targets chromatin remodeling complexes to the myogenin locus prior to forming a stable DNA-bound complex.

The activation of muscle-specific gene expression requires the coordinated action of muscle regulatory proteins and chromatin-remodeling enzymes. Microarray analysis performed in the presence or absence of a dominant-negative BRG1 ATPase demonstrated that approximately one-third of MyoD-induced genes were highly dependent on SWI/SNF enzymes. To understand the mechanism of activation, we performed chromatin immunoprecipitations analyzing the myogenin promoter. We found that H4 hyperacetylation preceded Brg1 binding in a MyoD-dependent manner but that MyoD binding occurred subsequent to H4 modification and Brg1 interaction. In the absence of functional SWI/SNF enzymes, muscle regulatory proteins did not bind to the myogenin promoter, thereby providing evidence for SWI/SNF-dependent activator binding. We observed that the homeodomain factor Pbx1, which cooperates with MyoD to stimulate myogenin expression, is constitutively bound to the myogenin promoter in a SWI/SNF-independent manner, suggesting a two-step mechanism in which MyoD initially interacts indirectly with the myogenin promoter and attracts chromatin-remodeling enzymes, which then facilitate direct binding by MyoD and other regulatory proteins.

Acetylation↗

PubMed2004

Probing microRNAs with microarrays: tissue specificity and functional inference.

MicroRNAs (miRNAs) are short, stable, noncoding RNAs involved in post-transcriptional gene silencing via hybridization to mRNA. Few have been thoroughly characterized in any species. Here, we describe a method to detect miRNAs using micro-arrays, in which the miRNAs are directly hybridized to the array. We used this method to analyze miRNA expression across 17 mouse organs and tissues. More than half of the 78 miRNAs detected were expressed in specific adult tissues, suggesting that miRNAs have widespread regulatory roles in adults. By comparing miRNA levels to mRNA levels determined in a parallel microarray analysis of the same tissues, we found that the expression of target mRNAs predicted on the basis of sequence complementarity is unrelated to the tissues in which the corresponding miRNA is expressed.

Animals↗

PubMed2006

Identification of primary tumors of brain metastases by infrared spectroscopic imaging and linear discriminant analysis.

This study applies infrared (IR) spectroscopy to distinguish normal brain tissue from brain metastases and to determine the primary tumor of four frequent brain metastases such as lung cancer, colorectal cancer, breast cancer, and renal cell carcinoma. Standard methods sometimes fail to identify the origin of brain metastases. As metastatic cells contain the molecular information of the primary tissue cells and IR spectroscopy probes the molecular fingerprint of cells, IR spectroscopy based methods constitute a new approach to determine the primary tumor of a brain metastasis. IR spectroscopic images were recorded by a FTIR spectrometer equipped with a macro sample chamber and coupled to a focal plane array detector. Unsupervised cluster analysis of IR images revealed variances within each sample and between samples of the same tissue type. Cluster averaged IR spectra of tissue classes with known diagnoses were selected to develop a metric with eight variables. These data trained a supervised classification model based on linear discriminant analysis that was used to identify the origin of 20 cryosections including one brain metastasis with an unknown primary tumor.

Brain Neoplasms↗

PubMed2003

The impact of parietal cells on Helicobacter pylori tropism and host pathology: an analysis using gnotobiotic normal and transgenic mice.

Helicobacter pylori infection of the human stomach is common and typically benign, although a subset of hosts develops severe pathology. Infection occurs in an organ with distinct microenvironments characterized by pronounced differences in the composition of acid-producing parietal cells. In this study, we examine determinants of bacterial tropism to various gastric niches by using germ-free normal and transgenic mice with an engineered parietal cell ablation. Mice were colonized for 8 weeks with a clinical isolate (Hp1) that expresses adhesins recognized by epithelial NeuAcalpha2,3Galbeta1,4 glycan receptors. In normal mice, Hp1 has tropism for a parietal cell-deficient niche where sialylated glycans are expressed by a narrow band of pit cells positioned at the boundary between the squamous epithelium (forestomach) and the proximal glandular epithelium. Lymphoid aggregates that develop in this niche, but not elsewhere in the stomach, were analyzed by GeneChip and quantitative RT-PCR studies of laser capture microdissected mucosa and yielded a series of biomarkers indicative of immune cell activation and maturation. Genetic ablation of parietal cells produced a new source of NeuAcalpha2,3Galbeta1,4 glycans in amplified gastric epithelial lineage progenitors, with accompanying expansion of Hp1 within the glandular epithelium. Lymphoid aggregates that develop in this formerly acid-protected epithelium have molecular features similar to those observed at the forestomach/glandular junction. These findings demonstrate the important roles played by parietal cells and glycan receptors in determining the positioning of H. pylori within the gastric ecosystem, and emphasize the need to consider the evolution of pathology within a given host in a niche-specific context.

Animals↗

PubMed2006

Loss of intercellular adhesion activates a transition from low- to high-grade human squamous cell carcinoma.

The relationship between loss of intercellular adhesion and the biologic properties of human squamous cell carcinoma is not well understood. We investigated how abrogation of E-cadherin-mediated adhesion influenced the behavior and phenotype of squamous cell carcinoma in 3D human tissues. Cell-cell adhesion was disrupted in early-stage epithelial tumor cells (HaCaT-II-4) through expression of a dominant-negative form of E-cadherin (H-2Kd-Ecad). Three-dimensional human tissue constructs harboring either H-2Kd-Ecad-expressing or control II-4 cells (pBabe, H-2Kd-EcadDeltaC25) were cultured at an air-liquid interface for 8 days and transplanted to nude mice; tumor phenotype was analyzed 2 days and 2 and 4 weeks later. H-2Kd-Ecad-expressing tumors demonstrated a switch to a high-grade aggressive tumor phenotype characterized by poorly differentiated tumor cells that infiltrated throughout the stroma. This high-grade carcinoma revealed elevated cell proliferation in a random pattern, loss of keratin 1 and diffuse deposition of laminin 5 gamma2 chain. When II-4 cell variants were seeded into type I collagen gels as an in vitro assay for cell migration, we found that only E-cadherin-deficient cells detached, migrated as single cells and expressed N-cadherin. Function-blocking studies demonstrated that this migration was matrix metalloproteinase-dependent, as GM-6001 and TIMP-2, but not TIMP-1, could block migration. Gene expression profiles revealed that E-cadherin-deficient II-4 cells demonstrated increased expression of proteases and cell-cell and cell-matrix proteins. These findings showed that loss of E-cadherin-mediated adhesion plays a causal role in the transition from low- to high-grade squamous cell carcinomas and that the absence of E-cadherin is an important prognostic marker in the progression of this disease.

Animals↗

PubMed2001

Preprocessing implementation for microarray (PRIM): an efficient method for processing cDNA microarray data.

cDNA microarray technology is useful for systematically analyzing the expression profiles of thousands of genes at once. Although many useful results inferred by using this technology and a hierarchical clustering method for statistical analysis have been confirmed using other methods, there are still questions about the reproducibility of the data. We have therefore developed a data processing method that very efficiently extracts reproducible data from the result of duplicate experiments. It is designed to automatically filter the raw results obtained from cDNA microarray image-analysis software. We optimize the threshold value for filtering the data by using the product of N and R, where N is the ratio of the number of spots that passed the filtering vs. the total number of spots, and R is the correlation coefficient for results obtained in the duplicate experiments. Using this method to process mouse tissue expression profile data that contain 1,881,600 points of analysis, we obtained clustered results more reasonable than those obtained using previously reported filtering methods.

Animals↗

PubMed1991

Ultrasonic tissue characterization of chronic liver disease using cepstral analysis.

To evaluate the morphological changes in chronic liver disease by means of ultrasonography, cepstral analysis was applied to the ultrasonic waveforms obtained with a 10-MHz transducer during peritoneoscopy. Assuming that liver tissue was a collection of semiregularly arrayed small scatters of ultrasound, the spaces between scatterers of the liver could be measured by means of cepstral analysis. From the distribution of the spaces between scatterers, two statistical parameters, mode and kurtosis, were determined for each patient. The mode of spaces between scatterers for liver cirrhosis was significantly larger than that for chronic hepatitis and nonspecific change. The mode of spaces between scatterers was correlated with the size of hepatic lobules or nodules. The kurtosis of spaces between scatterers for chronic hepatitis and liver cirrhosis was significantly smaller than that for nonspecific change. As a preliminary study for noninvasive application of this technique, cepstral analysis was also applied to the ultrasonogram obtained through the abdominal wall with a 3.5-MHz transducer. The results obtained with a 3.5-MHz transducer were correlated well with those obtained with a 10-MHz transducer. These results suggest the possibility of noninvasive evaluation of the structural changes in chronic liver disease.

Chronic Disease↗

PubMed2000

Fatty acid synthesis: from CO2 to functional genomics.

For over 25 years there has been uncertainty over the pathway from CO(2) to acetyl-CoA in chloroplasts. On the one hand, free acetate is the most effective substrate for fatty acid synthesis by isolated chloroplasts, and free acetate concentrations reported in leaf tissue (0.1-1 mM) appear adequate to saturate fatty acid synthase. On the other hand, a clear mechanism to generate sufficient free acetate for fatty acid synthesis is not established and direct production of acetyl-CoA from pyruvate by a plastid pyruvate dehydrogenase seems a more simple and direct path. We have re-examined this question and attempted to distinguish between the alternatives. The kinetics of (13)CO(2) and (14)CO(2) movement into fatty acids and the absolute rate of fatty acid synthesis in leaves was determined in light and dark. Because administered (14)C appears in fatty acids within < 2-3 min our results are inconsistent with a large pool of free acetate as an intermediate in leaf fatty acid synthesis. In addition, these studies provide an estimate of the turnover rate of fatty acid in leaves. Studies similar to the above are more complex in seeds, and some questions about the regulation of plant lipid metabolism seem difficult to solve using conventional biochemical or molecular approaches. For example, we have little understanding of why or how some seeds produce >50% oil whereas other seeds store largely carbohydrate or protein. Major control over complex plant biochemical pathways may only become possible by understanding regulatory networks which provide 'global' control over these pathways. To begin to discover such networks and provide a broad analysis of gene expression in developing oilseeds, we have produced microarrays that display approx. 5000 seed-expressed Arabidopsis genes. Sensitivity of the arrays was 1-2 copies of mRNA/cell. The arrays have been hybridized with probes derived from seeds, leaves and roots, and analysis of expression ratios between the different tissues has allowed the tissue-specific expression patterns of many hundreds of genes to be described for the first time. Approx. 10% of the genes were expressed at ratios > or = 10-fold higher in seeds than in leaves or roots. Included in this list are a large number of proteins of unknown function, and potential regulatory factors such as protein kinases, phosphatases and transcription factors. The arrays were also found to be useful for analysis of Brassica seeds.

Acetyl Coenzyme A↗

PubMed2004

Genomic portraits of the nervous system in health and disease.

As the human genome project moves toward its goal of sequencing the entire human genome, gene expression profiling by DNA microarray technology is being employed to rapidly screen genes for biological information. In this review, we will introduce DNA microarray technology, outline the basic experimental paradigms and data analysis methods, and then show with some examples how gene expression profiling can be applied to the study of the central nervous system in health and disease.

Algorithms↗

PubMed2000

Identification of age-dependent changes in expression of senescence-accelerated mouse (SAMP8) hippocampal proteins by expression array analysis.

Aging is associated with extensive cognitive impairments, although the biochemical and physiological basis of these deficits are unknown. As the hippocampus plays a vital role in cognitive functions, we have selected this tissue to analyze changes in gene expression at two different ages. Array technology is utilized to explore how gene expression in hippocampus is affected by accelerated cognitive impairment in Senescence-Accelerated Mouse (SAM P8) strain. We show that the expression of genes associated with stress response and xenobiotic metabolism are strongly affected at a time when cognitive impairment occurs. Affected genes include those involved both in signaling and chaperone function. The effector and regulator family of chaperones, which play an important role in protein folding, and also the xenobiotic metabolizing enzymes that play crucial role in antioxidant systems, show significant changes in gene expression between 4 and 12 months.

Aging↗

PubMed2002

High-density microarray analysis of hippocampal gene expression following experimental brain injury.

Behavioral, biophysical, and pharmacological studies have implicated the hippocampus in the formation and storage of spatial memory. Traumatic brain injury (TBI) often causes spatial memory deficits, which are thought to arise from the death as well as the dysfunction of hippocampal neurons. Cell death and dysfunction are commonly associated with and often caused by altered expression of specific genes. The identification of the genes involved in these processes, as well as those participating in postinjury cellular repair and plasticity, is important for the development of mechanism-based therapies. To monitor the expression levels of a large number of genes and to identify genes not previously implicated in TBI pathophysiology, a high-density oligonucleotide array containing 8,800 genes was interrogated. RNA samples were prepared from ipsilateral hippocampi 3 hr and 24 hr following lateral cortical impact injury and compared to samples from sham-operated controls. Cluster analysis was employed using statistical algorithms to arrange the genes according to similarity in patterns of expression. The study indicates that the genomic response to TBI is complex, affecting approximately 6% (at the time points examined) of the total number of genes examined. The identity of the genes revealed that TBI affects many aspects of cell physiology, including oxidative stress, metabolism, inflammation, structural changes, and cellular signaling. The analysis revealed genes whose expression levels have been reported to be altered in response to injury as well as several genes not previously implicated in TBI pathophysiology.

Animals↗

PubMed2002

Expression profiling of microdissected pancreatic adenocarcinomas.

Pancreatic ductal adenocarcinoma is characterized by a paucity of neoplastic cells embedded in a densely desmoplastic stroma. Therefore, laser capture microdissection was performed to obtain homogeneous populations of normal and neoplastic ductal cells. These were subjected to a comparative study of gene expression utilizing human cDNA arrays. A variety of dysregulated genes were identified, comprising cell cycle and growth regulators, invasion regulators, signalling and developmental molecules. In addition to genes already found to be overexpressed in pancreatic cancer, such as TIMP1, MMP7, CD59, rhoC and NDKA, we present evidence to implicate genes which have not previously been reported in this tumour type. These include the overexpressed genes ABL2, Notch4 and SOD1, as well as XRCC1, a DNA repair gene whose transcript was found downregulated. Quantitative real-time RT-PCR (QRT-PCR) was employed to confirm differential expression of ABL2, Notch4 and SOD1 and immunohistochemical analysis was used to verify decreased protein expression of XRCC1 using a custom-built pancreatic tissue array. Combining microarray-derived gene expression profiles of pure pancreatic cell populations, QRT-PCR and pancreas-specific tissue arrays therefore proved to be highly informative in elucidating the molecular pathology of this highly malignant tumour type.

Adenocarcinoma↗

PubMed2005

Comparative gene and protein expression in primary cultures of epithelial cells from benign prostatic hyperplasia and prostate cancer.

Primary cultures are widely used to investigate the disease-specific biology of prostate cancer and benign prostatic hyperplasia (BPH). To identify genes differentially expressed between epithelial cells cultured from adenocarcinomas versus BPH tissues, we used probe array technology. Gene expression profiles were evaluated on Affymetrix Human Cancer G110 Array Chips containing approximately 1900 cancer-related genes. After defined statistical analysis, genes that were over-expressed in cancer cultures were identified. Protein expression of four of the differentially expressed genes was measured in immunoblots, and the expression of two other genes was measured by real-time reverse transcription-polymerase chain reaction (RT-PCR). While no gene or protein was consistently over-expressed in all cancer versus BPH cell cultures, cytokeratin 16 protein was highly elevated in several of the cancer cultures, suggesting that a hyperproliferative phenotype may be characteristic of prostate cancer cells.

Adenocarcinoma↗

PubMedDate not supplied

A novel cell array technique for high-throughput, cell-based analysis.

Microarray technology has burgeoned over the past few years from nucleic acid-based arrays to tissue microarrays (TMAs). This study aimed to develop a technique to incorporate cell lines into an array and to demonstrate the usefulness of this technique by performing immunohistochemistry for beta-catenin. Cell suspensions were prepared from 23 tumor cell lines. These were fixed in formalin, suspended in agar, and embedded in paraffin to produce a cell block. A "tissue microarrayer" was used to remove triplicate, 0.6 mm-cores from each cell block and to transfer these into a recipient paraffin block at precise coordinates. Immunohistochemistry was used to identify cell lines positive for beta-catenin. Cultured cells were successfully incorporated into the microarray, with preservation of cell architecture and even distribution of cells within each core. A total of 18 of 69 cores (26%) were lost in processing. A total of 16 of 23 cell lines were identified as positive for membrane and cytoplasmic beta-catenin, and 6 of 23 were negative. Only one cell line was unscorable because of complete core loss. We have developed a "cell microarray" technique for analyzing antigen expression by immunohistochemistry in multiple cell lines in a single experiment. This novel application of microarrays permits high-throughput, cost-efficient analysis, with the potential to rapidly identify markers with potential diagnostic and therapeutic implications in human disease.

Biomarkers, Tumor↗

PubMed2003

Gene expression in donor corneal endothelium.

OBJECTIVE: To report gene expression profiles of normal human corneal endothelium with microarray analysis and serial analysis of gene expression (SAGE). METHODS: Corneal endothelium was removed from normal human corneas obtained from eye banks. Total RNA was isolated and SAGE analysis was performed. The same RNA source was used to construct a complementary DNA library that was hybridized to microarrays containing 12 558 transcripts. RESULTS: A total of 9530 SAGE tags were sequenced, representing 4724 unique tags. Microarray analysis identified 542 distinct transcripts. A database of human corneal endothelial gene expression was compiled. Of the SAGE tags, 1720 matched known genes, 478 corresponded to expressed sequence tags, and 2526 had no known match to public databases. The 5 most abundantly expressed SAGE tags were cytochrome c oxidase subunit II, adenosine triphosphate synthase F(0) subunit 6, carbonic anhydrase XII, 12S ribosomal RNA, and ferritin, heavy polypeptide 1. Thirty-four percent of the transcripts (n = 1616) were specific to the corneal endothelium, when compared with other publicly available SAGE libraries. The 5 most abundant unique tags were keratin 12, angiopoietinlike factor, annexin A8, and 2 tags with no match to the database. Many endothelial pump function enzymes were confirmed, including several plasma membrane Na( +)/K(+) adenosine triphosphatases and a recently reported bicarbonate transporter. CONCLUSIONS: Corneal endothelial gene expression profiles by the current analysis provide an understanding of endothelial metabolism, structure, and function; enable comparisons to diseased endothelium; and provide baseline data that may lead to the discovery of novel endothelial genes.

Aged↗

PubMed2001

Anabolic and catabolic gene expression pattern analysis in normal versus osteoarthritic cartilage using complementary DNA-array technology.

OBJECTIVE: To understand changes in gene expression levels that occur during osteoarthritic (OA) cartilage degeneration, using complementary DNA (cDNA)-array technology. METHODS: Nine normal, 6 early degenerated, and 6 late-stage OA cartilage samples of human knee joints were analyzed using the Human Cancer 1.2 cDNA array and TaqMan analysis. RESULTS: In addition to a large variability of expression levels between different patients, significant expression patterns were detectable for many genes. Cartilage types II and VI collagen were strongly expressed in late-stage specimens, reflecting the high matrix-remodeling activity of advanced OA cartilage. The increase in fibronectin expression in early degeneration suggests that fibronectin is a crucial regulator of matrix turnover activity of chondrocytes during early disease development. Of the matrix metalloproteinases (MMPs), MMP-3 appeared to be strongly expressed in normal and early degenerative cartilage and down-regulated in the late stages of disease. This indicates that other degradation pathways might be more important in late stages of cartilage degeneration, involving other enzymes, such as MMP-2 and MMP-11, both of which were up-regulated in late-stage disease. MMP-11 was up-regulated in OA chondrocytes and, interestingly, also in the early-stage samples. Neither MMP-1 nor MMP-8 was detectable, and MMP-13 and MMP-2 were significantly detectable only in late-stage specimens, suggesting that early stages are characterized more by degradation of other matrix components, such as aggrecan and other noncollagenous molecules, than by degradation of type II collagen fibers. CONCLUSION: This investigation allowed us to identify gene expression profiles of the disease process and to get new insights into disease mechanisms, for example, to develop a picture of matrix proteinases that are differentially involved in different phases of the disease process.

Adult↗

PubMed2005

Interferon-alpha/beta-mediated innate immune mechanisms in dermatomyositis.

Dermatomyositis has been modeled as an autoimmune disease largely mediated by the adaptive immune system, including a local humorally mediated response with B and T helper cell muscle infiltration, antibody and complement-mediated injury of capillaries, and perifascicular atrophy of muscle fibers caused by ischemia. To further understand the pathophysiology of dermatomyositis, we used microarrays, computational methods, immunohistochemistry and electron microscopy to study muscle specimens from 67 patients, 54 with inflammatory myopathies, 14 with dermatomyositis. In dermatomyositis, genes induced by interferon-alpha/beta were highly overexpressed, and immunohistochemistry for the interferon-alpha/beta inducible protein MxA showed dense staining of perifascicular, and, sometimes all myofibers in 8/14 patients and on capillaries in 13/14 patients. Of 36 patients with other inflammatory myopathies, 1 patient had faint MxA staining of myofibers and 3 of capillaries. Plasmacytoid dendritic cells, potent CD4+ cellular sources of interferon-alpha, are present in substantial numbers in dermatomyositis and may account for most of the cells previously identified as T helper cells. In addition to an adaptive immune response, an innate immune response characterized by plasmacytoid dendritic cell infiltration and interferon-alpha/beta inducible gene and protein expression may be an important part of the pathogenesis of dermatomyositis, as it appears to be in systemic lupus erythematosus.

Adult↗