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PubMed2007

CYCLONET--an integrated database on cell cycle regulation and carcinogenesis.

Computational modelling of mammalian cell cycle regulation is a challenging task, which requires comprehensive knowledge on many interrelated processes in the cell. We have developed a web-based integrated database on cell cycle regulation in mammals in normal and pathological states (Cyclonet database). It integrates data obtained by 'omics' sciences and chemoinformatics on the basis of systems biology approach. Cyclonet is a specialized resource, which enables researchers working in the field of anticancer drug discovery to analyze the wealth of currently available information in a systematic way. Cyclonet contains information on relevant genes and molecules; diagrams and models of cell cycle regulation and results of their simulation; microarray data on cell cycle and on various types of cancer, information on drug targets and their ligands, as well as extensive bibliography on modelling of cell cycle and cancer-related gene expression data. The Cyclonet database is also accessible through the BioUML workbench, which allows flexible querying, analyzing and editing the data by means of visual modelling. Cyclonet aims to predict promising anticancer targets and their agents by application of Prediction of Activity Spectra for Substances. The Cyclonet database is available at http://cyclonet.biouml.org.

Animals↗
PubMed2006

Transcriptome of the subcutaneous adipose tissue in response to oral supplementation of type 2 Leprdb obese diabetic mice with niacin-bound chromium.

The effects of oral niacin-bound chromium (NBC) supplementation on the subcutaneous fat tissue of type 2 Lepr(db) obese diabetic mice were examined using high-density comprehensive mouse genome (45,101 probe sets) expression arrays. The influence of such supplementation on the plasma cardiovascular risk factors of these mice was also investigated. Supplementation of NBC had no significant effect on age-dependent weight gain in the Lepr(db) obese diabetic mice. However, NBC lowered total cholesterol (TC), TC-to-HDL ratio, LDL cholesterol, and triglyceride levels while increasing HDL cholesterol in the blood plasma. No effect of NBC supplementation was observed on fasting blood glucose levels. Oral glucose tolerance test revealed a significantly improved clearance of blood glucose between 1 and 2 h of glucose challenge in NBC-supplemented mice. Unbiased genome-wide interrogation demonstrated that NBC resulted in the upregulation of muscle-specific gene expression in the fat tissue. Genes encoding proteins involved in glycolysis, muscle contraction, muscle metabolism, and muscle development were specifically upregulated in response to NBC supplementation. Genes in the adipose tissue that were downregulated in response to NBC supplementation included cell death-inducing DNA fragmentation factor (CIDEA) and uncoupling protein-1, which represent key components involved in the thermogenic role of brown adipose tissue and tocopherol transfer protein, the primary carrier of alpha-tocopherol to adipose tissue. The observation that CIDEA-null mice are resistant to obesity and diabetes suggests that the inhibitory role of NBC on CIDEA expression was favorable. Further studies testing the molecular basis of NBC function and long-term outcomes are warranted.

Adipose Tissue↗
PubMed2026

Dynamic effects of short-term storage temperature and duration on the transcriptome and functional pathways of umbilical cord blood-derived NK cells.

OBJECTIVE: This study aims to elucidate the dynamic impact of different storage conditions (temperature and time) on the function of natural killer (NK) cells derived from umbilical cord blood (UCB) at the transcriptome level, providing a theoretical basis for optimizing the standardized post-collection processing protocol of UCB in clinical settings. METHODS: Four healthy full-term UCB samples were collected and assigned to a fresh control group (0H) and experimental groups stored at 4 °C (4C) or 25 °C (RT) for 24 h (24H) and 72 h (72H). Umbilical cord blood mononuclear cells (CBMCs) were isolated and expanded in vitro to derive NK cells. Using RNA sequencing (RNA-seq) technology, combined with principal component analysis (PCA), screening of differentially expressed genes (DEGs), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses, the effects of storage conditions on the transcriptome of NK cells were comprehensively analyzed. RESULTS: The results indicated that storage time was the primary factor causing transcriptomic differences. Compared to the 0H group, storage for 24 h induced a limited number of Differentially Expressed Genes (DEGs) (only 11 in the 24H-RT group), indicating overall transcriptome stability. In contrast, storage for 72 h triggered profound transcriptomic reprogramming, with DEGs significantly enriched in immune activation-related pathways. Specifically, at the 72-h time point, storage at 4 °C compared to 25 °C prevented the downregulation of key metabolic pathways such as 'response to decreased oxygen levels' and 'regulation of leukocyte cell-cell adhesion'. CONCLUSION: CBMC-derived NK cells can tolerate up to 24 h of ex vivo storage while maintaining transcriptome and core functional stability. However, extending the ex vivo storage period to 72 h leads to significant reprogramming in the expression of immune and metabolism-related genes. Notably, the degree of metabolic suppression under refrigerated conditions at 4 °C was less pronounced than that at 25 °C room temperature storage. This study recommends that the ex vivo storage time of UCB should ideally be limited to within 24 h, with room temperature storage being a viable option. If prolonged storage is necessary, refrigeration at 4 °C is advised.

Humans↗
PubMed2006

Transcriptome analyses of human genes and applications for proteome analyses.

By utilizing recently developed full-length cDNA technologies, large-scale cDNA sequencing was carried out by several cDNA projects. Now full-length cDNA resources cover the major part of the protein-coding human genes. Comprehensive analyses of the collected full-length cDNA data revealed not only the complete sequences of thousands of novel gene transcripts but also novel alternatively spliced isoforms of hitherto identified genes. However, it was not as easy as expected to deduce their encoded amino acid sequences based solely on the full-length cDNA sequences. It was neither always the case that the longest open reading frame corresponded to the real protein coding region nor that the first ATG was the translation initiator codon. Also, proteome-wide mass-spectrometry analysis has shown that there is an unexpectedly large population of small proteins, encoded by so-called upstream open reading frames, within the cell. Since sound manual annotations by experts were still indispensable to address these problems, an international meeting to make transcriptome-wide functional annotations of cDNAs was held, namely the H-invitational. In this meeting, functional annotations were made both manually and computationally for most of the pre-existing full-length cDNAs collected from world-wide cDNA projects. The achieved integrated information for each of the cDNAs was published as a database. It was also shown that the full-length cDNA data were useful for identifying alternative splicing variants, exact transcriptional start sites of the mRNAs and the adjacent promoter regions. Rapidly accumulating genome data as well as versatile use of the transcriptome information will shortly lay a firm foundation for proteome-level understanding of human gene networks.

Alternative Splicing↗
PubMed2006

Comprehensive assessment of DNA copy number alterations in human prostate cancers using Affymetrix 100K SNP mapping array.

Although multiple recurrent chromosomal alterations have been identified in prostate cancer cells, the specific genes driving the apparent selection of these changes remain largely unknown. In part, this uncertainty is due to the limited resolution of the techniques used to detect these alterations. In this study, we applied a high-resolution genome-wide method, Affymetrix 100K SNP mapping array, to screen for somatic DNA copy number (CN) alterations among 22 pairs of samples from primary prostate cancers and matched nonmalignant tissues. We detected 355 recurrent deletions and 223 recurrent gains, many of which were novel. As expected, the sizes of novel alterations tend to be smaller. Importantly, among tumors with increasing grade, Gleason sum 6, 7, and 8, we found a significant trend of larger number of alterations in the tumors with higher grade. Overall, gains are significantly more likely to occur within genes (74%) than are deletions (49%). However, when we looked at the most frequent CN alterations, defined as those in > or =4 subjects, we observed that both gains (85%) and deletions (57%) occur preferentially within genes. An example of a novel, recurrent alteration observed in this study was a deletion between the ERG and TMPRSS2 genes on chromosome 21, presumably related to the recently identified fusion transcripts from these two genes. Results from this study provide a basis for a systematic and comprehensive cataloging of CN alterations associated with grades of prostate cancer, and the subsequent identification of specific genes that associated with initiation and progression of the disease. This article contains supplementary material available via the Internet at http://www.interscience.wiley.com/jpages/1045-2257/suppmat

Aged↗
PubMed2026

transfactor: transcription factor activity estimation via probabilistic gene expression deconvolution.

Gene expression is a primary modality being studied to differentiate between biological cells. Contemporary single-cell studies simultaneously measure genome-wide transcription levels for thousands of individual cells in a single experiment. While the characterization of cell population differences has often occurred through differential gene expression analysis, tiny effect sizes become statistically significant when thousands of cells are available for each population, compromising biological interpretation. Moreover, these large studies have spurred the development of methods to infer gene regulatory networks (GRNs) directly from the data, and GRN databases are becoming more comprehensive. In this work, we propose a statistical model for gene expression measures and an inference method that leverage GRNs to deconvolve transcription factor (TF) activity from gene expression, by probabilistically assigning mRNA molecules to TFs. This shifts the paradigm from investigating gene expression differences to regulatory differences at the level of TF activity, aiding interpretation and allowing prioritization of a limited number of TFs responsible for significant contributions to the observed gene expression differences. The inferred TF activities result in intuitive prioritization of TFs in terms of the (difference in) estimated number of molecules they produce, in contrast to other widely used methods relying on arbitrary enrichment scores. Our model allows the incorporation of prior information on the regulatory potential between each TF and target gene and is able to deal with both repressing and activating interactions. We compare our approach to other TF activity estimation methods using two simulation experiments and two case studies. Single-cell RNA-sequencing; TF activity; bioinformatics; GRN.

Transcription Factors↗
PubMed2003

Expression changes in mRNAs and mitochondrial damage in lens epithelial cells with selenite.

An overdose of sodium selenite induces cataracts in young rats. The mid-stage events producing the cataract include calpain-induced hydrolysis and precipitation of lens proteins. Apoptosis in lens epithelial cells has been suggested as an initial event in selenite cataracts. Expression levels of two genes associated with apoptosis were altered in lens epithelial cells from selenite-injected rats. The purpose of the present experiment was to perform a more comprehensive search for changes in expression of mRNAs in lens epithelial cells in order to more fully delineate the early events in selenite-induced cataracts. Lens epithelial cells were harvested at 1 and 2 days after a single subcutaneous injection of sodium selenite (30 mumol/kg body weight) into 12-day-old rats. Gene expression was analyzed using a commercial DNA array (Rat Genome U34A GeneChip array, Affymetrix). Of approximately 8000 genes assayed by hybridization, 13 genes were decreased and 27 genes were increased in the rat lens epithelial cells after injection of selenite. Some of the up-regulated genes included apoptosis-related genes, and a majority of the down-regulated genes were mitochondrial genes. Previously observed changes in expression of EGR-1 mRNA were also confirmed. Changes in the expression patterns of mRNAs were also confirmed by RT-PCR. To determine the mechanism for damage of lens epithelial cells (alpha TN4 cell) by culture in selenite, leakage of cytochrome c from mitochondria was measured. Selenite caused significant leakage of cytochrome c into the cytosol of alpha TN4 cells. Our data suggested that the loss of integrity of lens epithelial cells by selenite might be caused by preferential down-regulation of mitochondrial RNAs, release of cytochrome c, and impaired mitochondrial function. Up-regulation of mRNAs involved in maintenance of DNA, regulation of metabolism, and induction of apoptosis may also play roles.

Animals↗
PubMed2024

Analysis of different expression RNA binding protein genes in mouse microglia cell from the brains of mice 72 h after subarachnoid hemorrhage or sham operation.

BACKGROUND: The prognosis of brain injury caused by subarachnoid hemorrhage (SAH) is poor. Previous studies showed that abnormal function of RBPs might be involved in brain injury, neuroinflammation and further affect microglia homeostasis. However, no studies have systematically analyzed the genome-wide abnormal expression of RBPs genes in microglia during SAH. METHODS: RNA-seq data of microglia from the SAH mouse group (SAH) and control sham-operated mouse group (sham) were downloaded from the GEO database in GSE167957, including four samples from the sham group and four samples from the SAH group for subsequent analysis.Utilizing GO and KEGG functional enrichment analyses, we conducted a comprehensive study of differentially expressed genes (DEGs), alternative splicing patterns, and co-expression networks to gain deeper insights into the differential expression of RNA-binding proteins (RBPs) and differential alternative splicing events (ASEs) between the SAH (subarachnoid hemorrhage) and sham groups. This analysis aimed to elucidate the potential mechanisms underlying the aberrant expression of RBPs in microglia during brain injury caused by SAH. RESULTS: ASEs and co-expression analyses of differentially expressed RBPs and differential ASEs were carried out in microglia in terms of gene expression. GO and KEGG functional enrichment analysis showed that aberrantly expressed RBPs such as Mcm7, Mtdh, SRSF3, and Hnrnpa2b1 may affect and regulate downstream Csnk1d, Uckl1 and other protein phosphorylation-related genes by alterative splicing. CONCLUSION: RBPs were aberrantly expressed in microglia during the development of brain injury secondary to SAH, regulating alterative splicing of downstream genes and influencing the progression of SAH brain injury in this study. This implies that RBPs are important for the identification of new therapeutic targets for brain injury after SAH.

Animals↗
PubMed2001

Toward a catalog of human genes and proteins: sequencing and analysis of 500 novel complete protein coding human cDNAs.

With the complete human genomic sequence being unraveled, the focus will shift to gene identification and to the functional analysis of gene products. The generation of a set of cDNAs, both sequences and physical clones, which contains the complete and noninterrupted protein coding regions of all human genes will provide the indispensable tools for the systematic and comprehensive analysis of protein function to eventually understand the molecular basis of man. Here we report the sequencing and analysis of 500 novel human cDNAs containing the complete protein coding frame. Assignment to functional categories was possible for 52% (259) of the encoded proteins, the remaining fraction having no similarities with known proteins. By aligning the cDNA sequences with the sequences of the finished chromosomes 21 and 22 we identified a number of genes that either had been completely missed in the analysis of the genomic sequences or had been wrongly predicted. Three of these genes appear to be present in several copies. We conclude that full-length cDNA sequencing continues to be crucial also for the accurate identification of genes. The set of 500 novel cDNAs, and another 1000 full-coding cDNAs of known transcripts we have identified, adds up to cDNA representations covering 2%--5 % of all human genes. We thus substantially contribute to the generation of a gene catalog, consisting of both full-coding cDNA sequences and clones, which should be made freely available and will become an invaluable tool for detailed functional studies.

3' Untranslated Regions↗
PubMed2006

Growth phase- and nutrient limitation-associated transcript abundance regulation in Bordetella pertussis.

To survive in a host environment, microbial pathogens must sense local conditions, including nutrient availability, and adjust their growth state and virulence functions accordingly. No comprehensive investigation of growth phase-related gene regulation in Bordetella pertussis has been reported previously. We characterized changes in genome-wide transcript abundance of B. pertussis as a function of growth phase and availability of glutamate, a key nutrient for this organism. Using a Bordetella DNA microarray, we discovered significant changes in transcript abundance for 861 array elements during the transition from log phase to stationary phase, including declining transcript levels of many virulence factor genes. The responses to glutamate depletion exhibited similarities to the responses induced by exit from log phase, including decreased virulence factor transcript levels. However, only 23% of array elements that showed at least a fourfold growth phase-associated difference in transcript abundance also exhibited glutamate depletion-associated changes, suggesting that nutrient limitation may be one of several interacting factors affecting gene regulation during stationary phase. Transcript abundance patterns of a Bvg+ phase-locked mutant revealed that the BvgAS two-component regulatory system is a key determinant of growth phase- and nutrient limitation-related transcriptional control. Several adhesin genes exhibited lower transcript abundance during stationary phase and under glutamate restriction conditions. The predicted bacterial phenotype was confirmed: adherence to bronchoepithelial cells decreased 3.3- and 4.4-fold at stationary phase and with glutamate deprivation, respectively. Growth phase and nutrient availability may serve as cues by which B. pertussis regulates virulence according to the stage of infection or the location within the human airway.

Bacterial Proteins↗
PubMed2003

A comprehensive analysis of the expression of crystallins in mouse retina.

PURPOSE: Crystallins are expressed at high levels in lens fiber cells. Recent studies have revealed that several members of the alpha, beta, and gamma-crystallin family are also distributed in many non-lens tissues, though at lower levels. We observed that the use of retinal RNA as target for both custom I-Gene microarrays and Affymetrix GeneChips revealed significant expression of many crystallin genes. This prompted us to undertake a comprehensive investigation to delineate the baseline expression of crystallin genes in the adult mouse retina. METHODS: Quantitative RT-PCR was carried out using gene specific primers (derived from the mouse genomic sequence) for each crystallin gene. Immunofluorescence studies using frozen sections of the mouse retinas were performed with crystallin-specific antibodies. Retinal lysates were analyzed by immunoblotting using antibodies specific to alphaA and alphaB crystallins and those produced against total beta-crystallin and gamma-crystallin fractions of bovine lenses. RESULTS: Microarray analysis followed by quantitative RT-PCR revealed that mouse retinal cells express transcripts for 20 different members of the crystallin gene family; these are alphaA, alphaA-INS, alphaA-nov1, alphaB, betaA1, betaA2, betaA3, betaA4, betaB1, betaB2, betaB3, gammaA, gammaC, gammaD, gammaE, gammaF, gammaS, mu, zeta, and lambda-crystallin. The gene products of alphaA, alphaB, beta-, and gamma-crystallins are detected in the outer and inner nuclear layers of the retina by immunofluorescence analysis. In addition, alpha and beta-crystallins are detected in the photoreceptor inner segments. Retinal expression of these proteins was further confirmed by immunoblot analysis. Interestingly, our studies also showed a significant animal-to-animal variation in the expression level of some of the crystallins. CONCLUSIONS: Our results establish the expression of many crystallins in the adult mouse retina. Detection of crystallins in the retinal nuclear layers, though surprising, is consistent with their proposed role in cell survival and genomic stability. We suggest that crystallins play vital functions in protecting retinal neurons from damage by environmental and/or metabolic stress.

Animals↗
PubMed2026

Effect of Tertiary Lymphoid Structures on Immune Cell Infiltration in the Tumor Microenvironment and Prognosis in Lung Adenocarcinoma.

Tertiary lymphoid structures (TLSs) modulate immune responses in various solid tumors, but their comprehensive role in lung adenocarcinoma (LUAD) remains unclear. In this study, we analyzed RNA-seq data from 539 LUAD patients in The Cancer Genome Atlas (TCGA) and microarray data from 223 samples from the Gene Expression Omnibus (GEO, GSE13213, and GSE37745). TLS signatures were evaluated via unsupervised consensus clustering based on 12 chemokine transcriptome signatures. The relationships between TLS and clinical characteristics, tumor microenvironment (TME) cell infiltration, and prognosis were assessed using ESTIMATE and CIBERSORT. A prognostic model was established using LASSO regression and validated with external datasets. Additionally, H&E and IHC analyses were performed to explore associations between intratumoral TLS density, immune-related molecular expression, and patient prognosis in LUAD. Consensus clustering of the TCGA cohort revealed two distinct LUAD patient clusters according to TLS abundance. Cluster 1 exhibited greater immune cell infiltration, more favorable prognosis, and increased expression of immune checkpoint molecules. We developed a prognostic model comprising eight survival-associated genes that act as independent prognostic factors for patient survival. H&E/IHC analyses revealed that TLS density-regardless of pathological stage-was associated with better prognosis; higher intratumoral TLS density/proportion was also related to more favorable outcomes. IHC confirmed that survival-associated genes (CD5, HLA-DMB, and P2RY13) are independent prognostic indicators in LUAD. Our study demonstrated the close relationship between TLS signatures and an active immune microenvironment, highlighting their potential as independent prognostic indicators in LUAD.

Humans↗
PubMed2005

Overview of molecular, cellular, and genetic neurotoxicology.

It has become increasingly evident that the field of neurotoxicology is not only rapidly growing but also rapidly evolving, especially over the last 20 years. As the number of drugs and environmental and bacterial/viral agents with potential neurotoxic properties has grown, the need for additional testing has increased. Only recently has the technology advanced to a level that neurotoxicologic studies can be performed without operating in a "black box." Examination of the effects of agents that are suspected of being toxic can occur on the molecular (protein-protein), cellular (biomarkers, neuronal function), and genetic (polymorphisms) level. Together, these areas help to elucidate the potential toxic profiles of unknown (and in some cases, known) agents. The area of proteomics is one of the fastest growing areas in science and particularly applicable to neurotoxicology. Lubec et al, provide a review of the potential and limitations of proteomics. Proteomics focuses on a more comprehensive view of cellular proteins and provides considerably more information about the effects of toxins on the CNS. Proteomics can be classified into three different focuses: post-translational modification, protein-expression profiling, and protein-network mapping. Together, these methods represent a more complete and powerful image of protein modifications following potential toxin exposure. Cellular neurotoxicology involves many cellular processes including alterations in cellular energy homeostasis, ion homeostasis, intracellular signaling function, and neurotransmitter release, uptake, and storage. The greatest hurdle in cellular neurotoxicology has been the discovery of appropriate biomarkers that are reliable, reproducible, and easy to obtain. There are biomarkers of exposure effect, and susceptibility. Finding the appropriate biomarker for a particular toxin is a daunting task. The appropriate biomarker for a particular toxin is a daunting task. The advantage to biomarker/toxin combinations is they can be detected and measured shortly following exposure and before overt neuroanatomic damage or lesions. Intervention at this point, shortly following exposure, may prevent or at least attenuate further damage to the individual. The use of peripheral biomarkers to assess toxin damage in the CNS has numerous advantages: time-course analysis may be performed, ethical concerns with the use of human subjects can partially be avoided, procedures to acquire samples are less invasive, and in general, peripheral studies are easier to perform. Genetic neurotoxicology comprises two focuses--toxin-induced alterations in genetic expression and genetic alterations that affect toxin metabolism, distribution, and clearance. These differences can be beneficial or toxic. Polymorphisms have been shown to result in altered metabolism of certain toxins (paraoxonase and paraoxon). Conversely, it is possible that some polymorphisms may be beneficial and help prevent the formation of a toxic by-product of an exogenous agent (resistance to ozone-induced lung inflammation). It has also become clear that interactions of potential toxins are not straightforward as interactions with DNA, causing mutations. There are numerous agents that cause epigenetic responses (cellular alterations that are not mutagenic or cytotoxic). This finding suggests that many agents that may originally have been thought of as nontoxic should be re-examined for potential "indirect" toxicity. With the advancement of the human genome project and the development of a human genome map, the effects of potential toxins on single or multiple genes can be identified. Although collectively, the field of neurotoxicology has recently come a long way, it still has a long way to go reach its full potential. As technology and methodology advances continue and cooperation with other disciplines such as neuroscience, biochemistry, neurophysiology, and molecular biology is improved, the mechanisms of toxin action will be further elucidated. With this increased understanding will come improved clinical interventions to prevent neuronal damage following exposure to a toxin.

Animals↗
PubMed2004

Function-informed transcriptome analysis of Drosophila renal tubule.

BACKGROUND: Comprehensive, tissue-specific, microarray analysis is a potent tool for the identification of tightly defined expression patterns that might be missed in whole-organism scans. We applied such an analysis to Drosophila melanogaster Malpighian (renal) tubule, a defined differentiated tissue. RESULTS: The transcriptome of the D. melanogaster Malpighian tubule is highly reproducible and significantly different from that obtained from whole-organism arrays. More than 200 genes are more than 10-fold enriched and over 1,000 are significantly enriched. Of the top 200 genes, only 18 have previously been named, and only 45% have even estimates of function. In addition, 30 transcription factors, not previously implicated in tubule development, are shown to be enriched in adult tubule, and their expression patterns respect precisely the domains and cell types previously identified by enhancer trapping. Of Drosophila genes with close human disease homologs, 50 are enriched threefold or more, and eight enriched 10-fold or more, in tubule. Intriguingly, several of these diseases have human renal phenotypes, implying close conservation of renal function across 400 million years of divergent evolution. CONCLUSIONS: From those genes that are identifiable, a radically new view of the function of the tubule, emphasizing solute transport rather than fluid secretion, can be obtained. The results illustrate the phenotype gap: historically, the effort expended on a model organism has tended to concentrate on a relatively small set of processes, rather than on the spread of genes in the genome.

Animals↗
PubMed2002

Analysis of B cell memory formation using DNA microarrays.

DNA microarray analysis of B cell subsets has identified comprehensive programs of gene expression that distinguish B cells at discrete stages of differentiation. The next task is to identify key genetic signals within these complex programs that regulate the dynamic cellular events during B cell activation in vivo. After stimulation with antigen, naïve B cells proliferate and differentiate, and then produce antibodies. Crucial qualitative differences in antibody responses are observed depending on whether or not B cells receive T cell help during activation. Proteins, lipopolysaccharides, and polysaccharides stimulate T-dependent (TD), T-independent type 1 (TI-1), and type 2 (TI-2) antibody responses, respectively. Only TD responses generate somatically mutated antibody-forming (plasma) cells and memory B cells, which produce high affinity anamnestic responses to subsequent antigen challenge. Somatic mutation of immunoglobulin genes occurs during B cell proliferation in germinal centres (GC), which are typical in TD responses but rare in TI responses. However, we have described a model, which is exceptional because numerous large GC form in response to a model TI-2 antigen, (4-hydoxy-3-nitrophenyl) acetyl (NP)-Ficoll. Significantly, these GC undergo involution before memory B cells are generated. This model provides an opportunity to investigate the genetic signals that drive memory cell formation, and we have compared global gene expression in TI and TD GC to identify a relatively small number of genes that are differentially expressed between the two prototypic B cell responses. This model demonstrates how genome-scale technology can be adapted to investigate specific aspects of B cell biology.

Animals↗
PubMed2026

Genome-wide identification and expression analysis of the UGT gene family in honeysuckle.

BACKGROUND: The UGT gene family plays critical roles in regulating plant growth, development, stress responses, and secondary metabolite synthesis. Although UGT proteins have been studied in numerous plant species, research on the UGT family in honeysuckle (Lonicera japonica Thunb.) remains limited. RESULTS: In this study, a comprehensive genome-wide analysis of the UGT gene family was performed in honeysuckle. A total of 224 unique LjUGT genes were identified and classified into 21 distinct subfamilies (T71-T92 without T77) based on the phylogenetic analysis. These genes were unevenly distributed on the 9 chromosomes. Eighteen segmental duplication events and 61 tandem duplications were identified, of which only 3 were positive selection. Integrated analysis of promoter cis-acting elements, transcription factors, targeted miRNAs, and interacting proteins suggested that the expression and function of the LjUGT genes may be regulated by transcription factors and proteins through binding to the various binding sites and cis-acting elements, thereby putatively participating in diverse biological processes, including hormone signaling, stress response, and metabolism. The expression pattern analysis of LjUGTs in different tissues and under stress conditions indicated that Lj2A1135G32, Lj5A236T61, Lj6A350T83, and Lj7A737T47 emerged as candidate genes potentially associated with development, 46 genes showed expression changes under all 6 abiotic stresses, suggesting broad stress responsiveness. Additionally, there 7 genes were identified as candidate hub genes that may correlate with the low temperature stress tolerance in honeysuckle according to the WGCNA results, and further verification by qRT-PCR confirmed that Lj4A99G61 and Lj9A591T82 can be regarded as key candidate genes for in-depth research. CONCLUSIONS: This study systematically identified 224 LjUGT genes in honeysuckle for the first time and characterized their physicochemical properties, phylogenetic relationship, and expression patterns. These findings provide a foundational resource for hypothesis-driven investigations into the functions and action mechanisms of LjUGTs.

Lonicera↗
PubMed2002

Molecular techniques for studying gene expression in carcinogenesis.

Many genes and signaling pathways controlling cell proliferation, death, differentiation, and genomic integrity are involved in cancer development. Various methods are available for detection and quantification of messenger RNA. Older methods such as Northern blots, nuclease protection, plaque hybridization, and slot blots suffer from being inherently serial, measure a single mRNA at a time, or being difficult to automate. New techniques for analysis of gene expression include: (a) comprehensive open systems such as serial analysis of gene expression (SAGE), differential display (DD) analysis, RNA arbitrarily primer (RAP)-PCR, restriction endonucleolytic analysis of differentially expressed sequences (READS), amplified restriction fragment-length polymorphism (AFLP), total gene expression analysis (TOGA), and use of internal standard competitive template primers (CTs) in a quantitative multiplex RT-PCR method [StaRT-(PCR)], and (b) focused closed systems such as: high density cDNA filter hybridization (HDFCA) analysis, suppression subtractive hybridization (SSH), differential screening (DS), several forms of high-density cDNA arrays, or oligonucleotide chips, and tissue microarrays. Sometimes, a combination of these systems is used to enhance the sensitivity and specificity of the assays. While closed systems are excellent for the initial screening of large number of sequences, the value of the information generated is generally limited to an often arbitrarily chosen known sequence. On the other hand, only the open system platform has the potential to evaluate the expression patterns of tens of thousands of genes that have not yet been cloned or partially sequenced in a quantitative manner. A cost analysis of the most commonly used expression technologies is provided. A method for purifying tumors from surrounding stroma and normal tissue employing laser microdissection, and subsequent RNA isolation/amplification from few cells employing sensitive kits are also discussed.

Cell Transformation, Neoplastic↗
PubMed2004

A comprehensive assessment of p53-responsive genes following adenoviral-p53 gene transfer in Bcl-2-expressing prostate cancer cells.

The p53 protein can induce cell cycle arrest or apoptosis following activation in response to DNA damage. The function of p53 is largely mediated by regulating the expression of downstream target genes. Adenoviral-p53 gene transfer (Ad-p53) is currently being evaluated in clinical trials as a therapeutic intervention. Tumor response is likely to be influenced by context-dependent variables, such as expression of bcl-2. Bcl-2 is upregulated in a variety of neoplasms, and can inhibit p53-dependent apoptosis. It was therefore of interest to use a global genomic strategy to assess gene expression following Ad-p53 gene transfer and to determine if the expression of specific Ad-p53-responsive genes could be modulated in the context of bcl-2 gene deregulation. cDNA arrays were used to identify p53-responsive genes following Ad-p53 gene transfer in control and bcl-2-overexpressing PC3 prostate cancer cells. A total of 40 transcripts were significantly upregulated by Ad-p53 in both control and bcl-2-transfectant PC3 cells. Conversely, 19 transcripts were significantly repressed in both cell lines. These Ad-p53-responsive transcripts included previously identified p53 targets, known genes representing candidate p53 targets, and transcripts identified as expressed sequence tags. A subset of 15 transcripts was differentially modulated by Ad-p53 in the context of bcl-2. Some of these genes were also differentially modulated in LNCaP (wt p53) cells following DNA damage. These results document a number of potential p53 targets and mediators of therapeutically relevant genotoxic stress. The findings further suggest that bcl-2 may inhibit cell death at multiple points downstream of p53 activation.

Adenoviridae↗