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PubMedDate not supplied

Survivin gene expression positively correlates with proliferative activity of cancer cells in esophageal cancer.

OBJECTIVE: The correlation between survivin gene expression and the occurrence of spontaneous apoptosis, proliferative activity of cancer cells, tumor angiogenesis, and abnormal p53 nuclear accumulation was evaluated in esophageal cancer. METHODS: A total of 57 patients, on whom surgical esophageal resection had been performed between 1993 and 2000, were enrolled in this study. Total RNA was extracted from tumors and noncancerous epithelia. Expression levels of survivin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNA (mRNA) were analyzed quantitatively by real-time reverse transcriptase polymerase chain reaction (quantitative PCR). The proliferative activity of cancer cells, apoptotic cancer cells, microvessel density, and abnormal p53 nuclear accumulation were analyzed in these tumors. RESULTS: The expression level of tumor survivin mRNA described as survivin/GAPDH (s/G) ratio was higher than that of noncancerous tissue (p = 0.0003), but did not correlate with lymph node metastasis, with the depth of tumor invasion, with the occurrence of apoptosis or with tumor angiogenesis. However, the tumor s/G ratio positively correlated with the proliferative activity and p53 nuclear accumulation in cancer cells. The 5-year survival rate of 53 patients was 37% (we excluded 4 patients who died from operative complications from this survival study). The 5-year survival rate of 27 patients with a high tumor s/G ratio (28.6%) was lower than that of 26 patients with a low tumor s/G ratio (46.2%, p = 0.041). High tumor s/G ratio was detected as an important prognostic factor independent of tumor stage. CONCLUSION: Detection of survivin mRNA by quantitative PCR provided us with important prognostic and biological information regarding esophageal cancer patients.

Aged↗
PubMed2002

Retinoid receptors in the developing human lung.

Nuclear receptors and their ligands are known to play very important roles in lung development. Among these receptors, retinoid receptors, members of the steroid/thyroid hormone receptor superfamily, are classified into retinoic acid receptor (RAR) isoforms alpha, beta, and gamma and retinoid X receptor (RXR) isoforms alpha, beta, and gamma. In addition, isoforms I and II of the orphan receptor chicken ovalbumin upstream promoter-transcription factor (COUP-TF) have been shown to negatively regulate the activation of retinoid receptors. Both of these receptors have been shown to regulate lung development in the mouse. In the present study we utilized immunohistochemistry and real-time quantitative PCR to examine the expression of RAR-alpha, -beta and -gamma, RXR-alpha, -beta and -gamma and COUP-TFII in the human fetal lung at 13-16 gestational weeks, a very critical stage of human pulmonary development, in order to study possible roles in pulmonary morphogenesis by comparing these findings with those of the adult lung. RXR-gamma immunoreactivity was detected at both proximal (epithelia and mesenchyme of the trachea and bronchi associated with cartilage) and distal (epithelia and mesenchyme of smaller distal bronchi) sites in the fetal lung, but was markedly weaker in the adult lung. RAR-beta immunoreactivity was detected in distal mesenchymal cells of the fetal lung, but was not discernible in distal mesenchymal cells in the adult lung (bronchioles, alveolar ducts and alveolus). Relatively intense RAR-gamma immunoreactivity was detected in the chondrocytes of bronchial cells. COUP-TFII immunoreactivity was detected with a similar pattern to that of RAR-beta. Real-time quantitative PCR analyses revealed that mRNA levels of RXR-gamma at proximal and distal sites (ratio of fetal lung/adult lung: 3.4+/-0.05-fold and 3.1+/-0.03-fold respectively; P <0.01), RAR-beta at distal sites (2.4+/-0.01-fold; P <0.05) and RAR-gamma at proximal sites (2.2+/-0.11-fold; P <0.05) were significantly higher in the fetus than in the adult.

Adult↗
PubMed2004

Disruption of estrogen-regulated gene expression by dioxin: downregulation of a gene associated with the onset of non-insulin-dependent diabetes mellitus (type 2 diabetes).

Expression of an estrogen-regulated reporter gene, growth of MCF-7 cells in the presence of 17beta-estradiol (E2) or E2 plus TCDD, and DNA microarray plus real time quantitative PCR analyses of gene expression in MCF-7 cells were used to evaluate the effects of TCDD, a known E2 antagonist, on E2-regulated gene expression in human cells. TCDD added simultaneously with E2 exhibited significantly decreased E2-associated upregulation of reporter gene expression compared with cells treated with E2 alone, and decreased E2 enhancement of mitosis in MCF-7 cells. MCF-7 cells treated with E2 or E2 plus TCDD and DNA microarray-evaluated to determine patterns of gene expression, showed substantial differences in gene expression in TCDD-treated cells compared with E2-treated cells. Of the 2400 genes on the Perkin Elmer global array microchip utilized for this analysis, a minimum of 317 were significantly upregulated and 488 were significantly downregulated. Of these, the gene encoding insulin receptor substrate-1 (IRS-1), the protein product of which has been previously reported to be decreased, missing, altered, or defective in persons with type 2 diabetes mellitus, was evaluated by real time quantitative PCR to corroborate the array data. An evaluation of the potential consequences of TCDD-altered IRS-1 downregulation is presented.

Animals↗
PubMed2025

Survival and safety evaluation of Bifidobacterium longum subsp. longum ZS-8 in healthy adults, determined using PMAxx-qPCR and amplicon sequencing.

UNLABELLED: Species-level quantitative PCR (qPCR) provides in-depth knowledge of oral probiotics in the human gastrointestinal tract (GIT). However, it lacks the capability to differentiate exogenous strains from native microbiota, nor can it distinguish between live and dead bacteria. In this study, we employed improved propidium monoazide (PMAxx)-qPCR to evaluate the survival and colonization of Bifidobacterium longum subsp. longum ZS-8 (designated ZS-8) on the strain level in the GIT and its impact on human gut microbiota. By spiking in live and dead ZS-8, we demonstrated that strain-level PMAxx-qPCR could identify and quantify the viable ZS-8 in fecal samples accurately. Using this method, we found that, in healthy humans, oral administration of ZS-8 can transiently survive in the GIT, and multi-layer seamless capsules (MLSC) significantly improve the gastrointestinal tolerance and survivability of ZS-8 compared to its powder form. Furthermore, through selective cultivation and PMAxx-microbiome sequencing, we investigated the response of gut viable microbiome to ZS-8. Results showed that, while the microbiota diversity and total viable counts of Bifidobacterium and Lactobacillus remained stable, certain indigenous species of Bifidobacterium and Lactobacillus increased in abundance, confirming ZS-8's probiotic potential in healthy individuals. Overall, our study demonstrates the effectiveness of combining strain-specific comparative genomics with PMAxx-qPCR for evaluating probiotic survival and colonization in the human gut and highlights the safety of ZS-8 oral administration in healthy individuals. IMPORTANCE: The survival and colonization of probiotics in the gut are critical for their functional efficacy, yet conventional species-level quantitative PCR (qPCR) fails to distinguish exogenous strains from native microbiota or differentiate live from dead bacteria. By integrating strain-specific comparative genomics with propidium monoazide (PMAxx)-qPCR, we precisely quantified the viability of Bifidobacterium longum ZS-8 at the strain level in the human gut after its oral administration. Our study demonstrated that 1.53-6.90% of cells surviving transit and multi-layer seamless capsules (MLSC) significantly enhanced the gastrointestinal tolerance of ZS-8. While ZS-8 administration did not alter gut microbiota diversity or total viable counts of Bifidobacterium and Lactobacillus, it selectively increased the abundance of specific indigenous beneficial species. This method overcomes the dual limitations of traditional techniques (strain-level specificity and viability discrimination), providing a robust tool for probiotic research. Furthermore, our findings confirm the safety of ZS-8 in healthy individuals and its potential to modulate gut ecology, offering a scientific foundation for personalized probiotic development and clinical translation.

Humans↗
PubMed2004

Downregulation of prostaglandin E receptor subtype EP3 during colon cancer development.

BACKGROUND AND AIMS: Involvement of prostaglandin E(2) (PGE(2)) receptors EP(1), EP(2), and EP(4) in the formation of aberrant crypt foci (ACF) and/or intestinal polyps has been suggested. In contrast, EP(3) appears to have no influence on the early stages of colon carcinogenesis. In the present study, we examined expression of PGE(2) receptor subtypes EP(1), EP(2), EP(3), and EP(4) in normal colon mucosa and colon cancers, and assessed the contribution of EP(3) to colon cancer development. METHODS: mRNA expression of PGE(2) receptor subtypes EP(1), EP(2), EP(3), and EP(4) in normal colon mucosa and colon cancers in azoxymethane (AOM) treated mice and rats, and in humans, were examined by reverse transcription-polymerase chain reaction (RT-PCR), quantitative real time RT-PCR, and immunohistochemical analyses. Evaluation of the role of EP(3) was performed by intraperitoneal injection of AOM, using EP(3) receptor knockout mice. Effects of EP(3) receptor activation on cell growth of human colon cancer cell lines were examined using ONO-AE-248, an EP(3) selective agonist. Moreover, EP(3) expression in colon cancer cell lines was analysed with or without 5-aza-2'-deoxycytidine (5-aza-dC) treatment. RESULTS: Expression levels of EP(1) and EP(2) mRNA were increased in cancer tissues. EP(4) mRNA was constantly expressed in normal mucosa and cancers. In contrast, expression of EP(3) mRNA was markedly decreased in colon cancer tissues, being 5% in mice, 9% in rats, and 28% in humans compared with normal colon mucosa, analysed by quantitative real time RT-PCR. Immunohistochemical staining demonstrated the rat EP(3) receptor protein to be expressed in epithelial cells of normal mucosa and some parts of small carcinomas but hardly detectable in large carcinomas of the colon. Colon cancer development induced by AOM in EP(3) receptor knockout mice was enhanced compared with wild-type mice, with a higher incidence of colon tumours (78% v 57%) and mean number of tumours per mouse (2.17 (0.51) v 0.75 (0.15); p<0.05). Expression of EP(3) mRNA was detected in only one of 11 human colon cancer cell lines tested. Treatment with 5 microM of an EP(3) selective agonist, ONO-AE-248, resulted in a 30% decrease in viable cell numbers in the HCA-7 human colon cancer cell line in which EP(3) was expressed. Treatment with 5-aza-dC restored EP(3) expression in CACO-2, CW-2, and DLD-1 cells but not in WiDr cells, suggesting involvement of hypermethylation in the downregulation of EP(3) to some extent. CONCLUSION: The PGE(2) receptor subtype EP(3) plays an important role in suppression of cell growth and its downregulation enhances colon carcinogenesis at a later stage. Hypermethylation of the EP(3) receptor gene could occur and may contribute towards downregulating EP(3) expression to some extent in colon cancers.

Animals↗
PubMed1997

Quantitative enriched PCR (QEPCR), a highly sensitive method for detection of K-ras oncogene mutation.

We have developed a rapid and highly sensitive method for the detection of mutant K-ras codon 12 allele in the presence of 10(5) copies of the wild-type alleles. This sensitivity is achieved by selective amplification of mutant K-ras sequences, using a two-stage procedure with modified primers. In the first stage, primers consist of K-ras sequences in the 3' portion and polyomavirus sequence (to minimize homology with human genome) on the 5' portion. The 3' portion also consists of mismatch sequence that generates an MvaI site in normal, but not mutant, K-ras codon 12 alleles. Thus, following the first round of 20 cycles, restriction enzyme cleavage is carried out to selectively digest normal K-ras codon 12 alleles. To enrich mutant alleles, a second amplification is performed using tail primers that recognize the polyoma, but not human sequences. This design ensures that in the second amplification only mutant alleles that were pre-amplified in the first round would serve as template for this reaction. Ethidium bromide-stained polyacrylamide gel electrophoresis (PAGE) of second-stage PCR product that has been digested with MvaI is used to monitor the presence of mutant alleles, detected at sensitivity of 1/10(5). This technique offers high sensitive detection of mutant K-ras alleles using a new concept of tail-primer design and is likely to assist in identifying patients at risk to develop pancreatic, colon, or lung cancer, which harbor high incidence of mutant ras alleles.

Base Sequence↗
PubMed2003

55 kDa nuclear matrix protein (nmt55) mRNA is expressed in human prostate cancer tissue and is associated with the androgen receptor.

Most prostate cancer grows in a hormone-dependent manner. Most patients, however, show hormone-independent growth after several years of hormone therapy. The mechanism of hormone-refractory prostate cancer remains unknown. It is important, therefore, to identify gene(s) related to prostate cancer that are up- or downregulated. We studied differences in gene expression in pairs of prostate cancer and normal prostate tissue utilizing the differential display method. Expression of the identified gene was examined by RT-PCR and real time quantitative PCR (TaqMan-PCR) using 26 pairs of human prostate cancer and normal tissues. We identified a specific upregulated gene encoding a 55 kDa nuclear matrix protein (nmt55) in human prostate cancer. nmt55 gene expression in human prostate cancer tissue was higher (20/26 cases) than that in normal prostate tissue. Moreover, the relationship between nmt55 and androgen receptor (AR) expression showed a positive correlation. In another experiment, transcriptional activity of the prostate specific antigen (PSA) promoter was upregulated by nmt55 in 293 cells. nmt55 showed high expression in prostate cancer compared to normal tissue and its expression showed a positive correlation with AR expression. The PSA promoter was activated by nmt55 expression. These results suggest the possibility that nmt55 expression is related to hormone-dependency or -independence associated with the AR.

Cell Nucleus↗
PubMed2007

Expression of a novel alternatively spliced variant of NADP(H)-dependent retinol dehydrogenase/reductase with deletion of exon 3 in cervical squamous carcinoma.

NADP(H)-dependent retinol dehydrogenase/reductase (NRDR) plays an important role in maintaining the homeostasis of retinoid. Aberrations in retinoid metabolism are considered as early events in carcinogenesis. We identified a novel alternatively spliced variant, NRDRB1, in HeLa cell and human cervical squamous carcinoma tissues, which is characterized by a complete deletion of exon 3. The latter resulted in changes in subcellular localization of NRDRB1 when compared with the peroxisomal localization of NRDR. To clarify the clinical significance of NRDRB1, we investigated its mRNA and protein expressions in normal cervical and cervical squamous carcinoma tissues, using RT-PCR, quantitative real-time PCR, Gateway expressing system, immunoprecipitation, immunoblotting, MALDI-TOF mass spectrometry and immunohistochemistry. We detected NRDRB1 mRNA in 14 of 26 (53.9%) cervical cancer tissues, but in none of the 12 normal cervical tissues. NRDRB1 protein was expressed in NRDRB1 mRNA-positive cases. While the full-length NRDR mRNA was observed in both normal and neoplastic cervical tissues, its protein was only expressed in normal cervical epithelium. The results presented here provide evidence that metabolic disturbances of retinal and retinoic acid, due to abnormal splicing and functional disorder of NRDR, may be involved in cervical tumorigenesis.

Alcohol Oxidoreductases↗
PubMed2006

Differential expression of HOX genes in neoplastic and non-neoplastic human astrocytes.

HOX genes are a large family of regulatory genes implicated in the control of developmental processes. HOX genes are involved in malignant transformation and progression of different types of tumour. Despite intensive efforts to delineate the expression profiles of HOX genes in other cell types, nothing is known regarding the global expression profile of these genes in normal human astrocytes and astrocytomas. The present study has analysed the expression profile of the 39 class I HOX genes in normal human astrocytes (NHA and E6/E7), two well-established glioblastoma cell lines (U-87 MG and U-1242-MG), as well as neoplastic (WHO grades II/III and IV) and non-neoplastic temporal lobe specimens with hippocampal sclerosis and medically intractable epilepsy. RT-PCR, quantitative real-time PCR, immunocytochemistry, and western blot analyses revealed differential expression of nine HOX genes (A6, A7, A9, A13, B13, D4, D9, D10, and D13) in normal human astrocytic cell lines and non-neoplastic temporal lobe specimens. The data show that HOX genes are differentially expressed in neoplastic and non-neoplastic astrocytes and that multiple HOX genes are overexpressed in glioblastoma cell lines, astrocytomas (II/III), and glioblastoma multiforme. The differential expression of HOX genes in normal and neoplastic astrocytes suggests a role for these genes in brain tumourigenesis.

Astrocytes↗
PubMed1996

Evidence that Th1 lymphocytes predominate in islet inflammation and thyroiditis in the BioBreeding (BB) rat.

Th1 cytokines are thought to play a key role in islet inflammation and destruction in insulin-dependent diabetes mellitus (IDDM). We studied this hypothesis in the diabetes-prone (DP)-BB and the diabetes-resistant (DR)-BB rats that are used as a model of human IDDM. The DP-BB rat develops spontaneous autoimmune diabetes at the age of 11-14 weeks. In the DR-BB rat, diabetes is inducible by depletion of RT6+ lymphocytes and coadministration of polyinosinic:polycytidylic acid (Poly I:C). We used reverse transcriptase-polymerase chain reaction (RT-PCR) and semi-quantitative PCR techniques to examine mRNA expression of Th1 and Th2 cytokines in inflamed islets and thyroids from DP-BB and DR-BB rats. We observed that in DP-BB and in treated DR-BB rats, the levels of TCR beta, IFN-gamma and IL-12p40 mRNA increase with disease progression. In contrast, expression of message for IL-2 and IL-4 is minimal to undetectable in DP-BB and RT6-depleted DR-BB animals at any age. Message for IL-10 is detectable in DP and DR islets; however, its level of expression does not change with disease progression. A similar cytokine mRNA profile is observed in inflamed thyroids from acutely diabetic RT6-depleted DR-BB rats. Incubation of 10 wk old DP islets for 48 h in the presence of anti-CD3 antibody, followed by an incubation with rIL-2 for an additional 5-7 days, results in an expansion of T lymphocytes, and these cells express high levels of IFN-gamma and IL-10 mRNA. Our results suggest that autoimmunity in DP-BB and DR-BB rats is mediated by Th1 lymphocytes and that IFN-gamma and IL-12 are likely to play a key role in islet and thyroid inflammation and destruction in IDDM.

Animals↗
PubMed2006

[Research into the human genome driven by improved methods].

The enormous progress made by research of the human genome is mainly driven by newly established or improved methods for the analysis of nucleic acids and proteins. Among the methods that have gained a wide-spread use within a comparably short time are fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR) including methods for quantitative PCR, and the use of short interfering RNA (siRNA) molecules aimed at gene silencing. The increasing significance of the analysis of secondary modifications of nucleic acids and proteins (genomic imprinting by DNA methylation, posttranslational protein modification) is reflected by an increasing use of mass spectrometry for the analysis and characterization of these biomolecules. Overall, in the future the research into the human genome and the interpretation of data will further benefit from these and other refined tools.

Animals↗
PubMed2004

Identification and isolation of a pectin methylesterase isoform that could be involved in flax cell wall stiffening.

Pectin methylesterases (PMEs) are ubiquitous enzymes present in the plant cell wall. They catalyse the demethylesterification of homogalacturonic acid units of pectins, which, in turn, can be associated with different physiological phenomena. In this study, different flax (Linum usitatissimum L.) PME isoforms were observed: neutral (pI 7.0 and 7.5, MW: 110 kDa), basic (pI 8.3 and 8.5, MW: 110 kDa) and very basic (pI>9.5, MW: 38 kDa). In an attempt to identify most of the expressed cell wall LuPME isoforms, polyclonal antibodies were raised against a conserved region of PME. These antibodies allowed the purification of the very basic PME isoform (pI 9.5, MW: 36 kDa) from flax cells, designated LuPME5. This isoform corresponds to the Lupme5 cDNA isolated, at the same time, from flax hypocotyls, by using the RACE-PCR technique. Semi-quantitative PCR experiments showed that the Lupme5 transcript was highly expressed in the hypocotyl zones where elongation is being achieved. Thus, this enzyme may be involved in cell wall stiffening.

Amino Acid Sequence↗
PubMed1992

PCR-based quantitation of low levels of HIV-1 DNA by using an external standard.

Beginning with 10(3)-10(5) molecules of a purified HIV-1 target sequence as a starting template, we have examined the effects of starting template concentration and cycle number on the amplification efficiency of the polymerase chain reaction. An external standard DNA sequence has been designed that when added to a DNA sample enables a determination of the starting concentration of HIV-1 target sequence in that sample of DNA. Varying ratios of external standard and target DNA sequences were amplified for 22 cycles. When the starting concentration of the external standard was within 50-fold of the starting concentration of the target, the amplifications of both sequences were proportional. These same results were obtained when the two templates were amplified in the presence of an excess of heterogeneous genomic DNA. Using this quantitative method, the number of starting target molecules in a DNA sample can be calculated to within a two-fold range of accuracy.

Base Sequence↗
PubMed2006

A rapid quantitative PCR-based assay for testing antiviral agents against human adenoviruses demonstrates type specific differences in ribavirin activity.

Human adenovirus (HAdV) infections are increasingly frequent and potentially fatal as a disseminated disease in highly immunocompromised patients. Determining the in vitro sensitivity of HAdV to antiviral agents is not an easy task because HAdV CPE reduction assays are difficult to interpret and may take more than 1 week. We developed a phenotypic assay for testing the antiviral activity during the first round of replication using HAdV DNA concentration as an objective readout within 30 h. After evaluating the assay with cidofovir, we focused on determining the antiviral of ribavirin against different HAdV serotypes because clinical response of HAdV infections towards ribavirin treatment varied considerably. Several HAdV prototypes (1, 2, 5, 11, 31, 34, 48) associated with disseminated infections and clinical isolates were tested. Predominating HAdV of species C were more sensitive to ribavirin (HAdV-2 and -5: EC(50)<10 microM, EC(99) 111 and 104 microM, respectively) than HAdV of other species, for example HAdV-31 (EC(50) 56 microM, EC(99)>500 microM). Differential ribavirin sensitivity of HAdV types may contribute to the variable outcome of ribavirin therapy. Rapid screening of antiviral agents with the rapid qPCR-based assay against a multitude of HAdV serotypes may also facilitate development of future antiviral agents.

Adenovirus Infections, Human↗
PubMed2004

Aberrant expression of interleukin-7 (IL-7) and its signalling complex in human breast cancer.

Interleukin-7 (IL-7), a haematopoietic growth factor, is known to induce the differentiation and proliferation of some haematological malignancies including certain types of leukaemias and lymphomas. However, little is known about its role in solid tumours, including breast cancer. In this study, the expression level of IL-7, IL-7 receptor (IL-7R) and their downstream signalling molecules, including the Janus kinases (Jak-1 and Jak-3), phosphoinositide 3-kinase (PI3-K) and signal transducers and activators of transcription (Stat-5) were analysed using the reverse transcriptase-polymerase chain reaction (RT-PCR), real-time quantitative PCR and immunohistochemistry in a cohort of patients with breast cancer. The results were analysed in relation to tumour grade, TNM stage, patients' prognosis (using the Nottingham Prognostic Index (NPI)) and survival. The levels of expression of IL-7, IL-7R, Jak-1, Jak-3, PI3-K and Stat-5 were significantly higher in the most aggressive tumours. With the exception of Stat-5 expression, the transcript copies of IL-7 and all other signalling molecules were higher in patients with the worst prognoses (NPI3) and in patients who died from breast cancer after 72 months of follow-up. This aberrant expression of IL-7 and its signalling intermediates in invasive breast cancers could have significant diagnostic and prognostic implications. Measuring these molecules in breast cancer tissues may provide, for the first time, important molecular indicators of tumour differentiation, aggressiveness, nodal status, prognosis and patient survival.

Breast Neoplasms↗
PubMed2004

Comparative analysis of gene expression mechanisms between group IA and IB phospholipase A2 genes from sea snake Laticauda semifasciata.

Phospholipase A(2) (PLA(2)) genes expressed in the venom glands of the sea snake, Laticauda semifasciata, were investigated. Both mRNAs, encoding group IA (without a pancreatic loop) and group IB (with pancreatic loop), were detected from venom glands by Northern blot hybridization analysis and RT-PCR. The results of quantitative PCR analysis indicated that the expression amount of group IA genes was around 100-300 times greater than that of group IB genes. Sequence analysis of 5'-upstream regions and a reporter gene assay of the genes (groups IA and IB) previously cloned showed that the functional sequence (411 bp) was inserted in the 5'-flanking region of the group IA PLA(2) genes. It seemed that the contribution of the inserted sequence to the amount of transcribed mRNAs was greater than that of number of genes present in the genome. Comparative analysis of the 5'-flanking sequences from several snake genes encoding toxic PLA(2)s revealed that this sequence was probably inserted into an ancestral gene of PLA(2) with a pancreatic loop. After the duplication of the gene, which contained the inserted sequence, the PLA(2) gene without a pancreatic loop evolved from one of the duplicate genes. This inserted sequence might determine the future of the genes expressed in the venom glands.

5' Flanking Region↗
PubMed2026

Identification and validation of an alternatively spliced novel isoform of maspin that modulate genes involved in inflammatory and apoptotic pathways.

Maspin regulates cellular adhesion, migration, apoptosis, angiogenesis, and tumor suppression in a tissue and context-dependent manner. Its functional diversity is governed largely thorough extracellular matrix interactions, subcellular localization, and the reactive center loop (RCL), although the structural details are not well understood. To examine whether alternative splicing contributes to this heterogeneity, we analysed the SERPINB5 gene using a computational genomics approach and identified a novel 80&#x202f;bp coding exon upstream of the first coding exon (E1). The alternatively spliced transcript was validated in human skin and esophagus by semi-nested touchdown PCR, quantitative real-time PCR, and Sanger sequencing. Recombinant B5N displayed a red-shifted fluorescence emission spectrum, indicating a more solvent-exposed conformation, which was supported by molecular dynamics simulations showing greater exposure of the nuclear localization signal (NLS) and the reactive center loop. Enzyme kinetic assays demonstrated concentration-dependent enhancement of tissue plasminogen activator (tPA) activity by both isoforms. In HaCaT cells, wildtype maspin produced stronger antiproliferative and anti-migratory effects, whereas B5N was only mildly antiproliferative. Annexin V/7-AAD staining revealed that wildtype maspin induced higher early apoptosis and cell death, while B5N produced lower overall cell death but a greater proportion of late apoptotic cells. RNA-seq of transfected HaCaT cells identified differentially expressed genes enriched in inflammatory, antiviral, and apoptotic pathways, which was validated by qPCR, and several of these were markedly upregulated in SARS-CoV-2 infected A549 cells. Thus, a novel N-terminally extended maspin isoform with differentially regulated gene profile is identified and validated in this study.

Apoptosis↗
PubMed2004

Immune activation status of CD8+ T cells infiltrating non-small cell lung cancer.

In a variety of human cancers, the presence of tumor-infiltrating T lymphocytes (TILs) is associated with tumor regression and favorable prognosis. Local interferon (IFN)-gamma secretion from activated T cells is supposed to induce a specific immune response leading to tumor-specific cytotoxicity. Nonetheless, significance and properties of TILs still remains controversial in lung cancer patients. We determined CD8+ T cell counts in 31 patients with non-small cell lung cancer (NSCLC) by immunohistochemistry, and assessed T-cell immune activation status in a subset of patients by measuring IFN-gamma mRNA expression by quantitative PCR (TaqMan). Semi-quantitative immunohistochemical analysis revealed significantly higher CD8+ T cell counts within the tumor as when compared to the invasive margin. CD8+ T cells immune activation status, represented in the IFN-gamma/CD8 mRNA ratio, correlated with the median number of CD8+ T cells presented at the tumor-host interface. Neither tumor histology and grade, nor CD8+ T cell counts and IFN-gamma/CD8 ratio could demonstrate an influence on overall survival in these patients. Our results indicate that CD8+ T cells infiltrating the tumor cell nests may be inadequately activated and thus incapable of mounting an effective anti-tumor immune response.

CD8-Positive T-Lymphocytes↗