Expression Levels Of A1 Research Articles

Extraction, phytochemical characterization and anti-cancer mechanism of Haritaki churna: An ayurvedic formulation.

Haritaki churna (HC), a single herb ayurvedic formulations is known to be prescribed for various gastro-intestinal disorders in Ayurveda. Haritaki churna aqueous extract (HCAE) has anti-cancer activity against different types of cancer cells with an IC50 in the range of 50-97 μg/ml. Bioavailability of Haritaki Churna is very high in digestive track and treatment of colorectal cancer cells HCT-116, DLD1, HT-29 with HCAE reduces its cellular viability with anti-cancer IC50 70μg/ml. HCAE consumption is safe for human as it didn't affect the cellular viability of primary human PBMCs or non-cancerogenic HEK-293 cells. Haritaki churna was found to be stable in biological gastric fluids and bioactive agents are not losing their anti-cancer activity under such harsh conditions. The HPLC Chromatogram of HCAE is giving 13 major peaks and 11 minor peaks. Exploiting LC-MS, IR and NMR spectroscopic techniques, a total of 13 compounds were identified from HCAE namely Shikimic acid, Chebulic acid, gallic acid, 5-hydroxymethylfurfural, Protocatechuic acid, 4-O-galloyl-shikimic Acid, 5-O-galloyl-shikimic Acid, Methylgallate, corilagin, 1, 2, 6, Tri-O-galloyl β-D-glucose, chebulagic acid, chebulinic acid, and Ellagic acid. Reconstitution and subtraction of phytochemicals from the mixture indicate that Ellagic acid significantly contribute into anti-cancer effect of HCAE. Cancer cells treated with ellagic acid from HCAE were incapable of completing their cell-cycle and halted the cell-cycle at DNA synthesis S-Phase, as demonstrated by decreased cyclin A2 expression levels with increasing ellagic acid concentration. Halting of cells at S-phase causes induction of apoptosis in cancer cells. Cancer cells exhibiting DNA fragmentation, changes in expression of several apoptotic proteins such as Bcl2, cytochrome-c and formation of cleaved products of caspase 3 and PARP-1 suggests ellagic acid induces cell death via mitochondrial pathway of apoptosis.

Characteristics and Significance of Gene Expression Changes in Peripheral Blood of Lumbar Disc Extrusion Patients before and after Nonoperative Treatment

To define the gene expression characteristics in the peripheral blood of patients with lumbar disc extrusion (LDE) and the effect of nonoperative treatment on the gene expression. DNA microarray was used to identify semi-quantitatively the differentially expressed genes (DEGs) in the peripheral blood of patients with LDE and that of the healthy controls and the variation trend of these DEGs after nonoperative treatment. Enrichment analysis was done to reveal the functional characteristics of these DEGs, and network analysis was done to identify key genes that contribute to gene dysregulation. The levels of these key genes were measured by qRT-PCR to examine their expression in LDE patients and the controls, and the effect of nonoperative treatment on the expression level. We identified 153 DEGs in the peripheral blood of LDE patients and healthy controls, including 131 upregulated genes and 22 downregulated genes. Enrichment analysis revealed that most of the DEGs were related to immunity and the inflammatory response. Network analysis revealed that toll-like receptor 4 ( TLR4 ), matrix metallopeptidase 9 ( MMP9) and myeloperoxidase ( MPO), cathelicidin antimicrobial peptide ( CAMP), resistin ( RETN), toll-like receptor 5 ( TLR5) were the key genes in the protein-protein interaction network. These key genes were all enriched into the terms releated to immunity and the inflammatory response. The patients experienced pain relief after nonoperative treatment. Among the 153 DEGs, TLR5 , interleukin 1 receptor antagonist ( IL1 RN) and solute carrier family 8 member A1 ( SLC8 A1) were downregulated after nonoperative treatment. qRT-PCR revealed that the levels of TLR4, MMP9 , MPO, CAMP, RETN, TLR5, IL1 RN and SLC8 A1 in the peripheral blood of the LDE patients were higher than those of the healthy control group ( P<0.05). In addition, TLR5 , IL1 RN and SLC8 A1 expression levels decreased after treatmentin in comparison with the levels before treatment ( P<0.05). Gene expression in the peripheral blood of LDE patients was characterized by the dysregulation of immune and inflammatory response-related genes, among which, TLR4, MMP9, MPO, CAMP, RETN and TLR5, the genes relevant to immune and inflammatory response, played a key role in the dysregulation of gene expression in the peripheral blood of LDE patients. The outcome of non-operative treatment may be related to the downregulation of the overexpressed TLR5, IL1 RN and SLC8 A1 in the peripheral blood of patients.

Prognostic value of using glucosylceramide synthase and cytochrome P450 family 1 subfamily A1 expression levels for patients with triple-negative breast cancer following neoadjuvant chemotherapy.

Neoadjuvant chemotherapy (NACT) has been considered to be the preferred treatment option for early operable triple-negative breast cancer (TNBC). However, resistance to drugs remains to be the barrier to the efficacy of NACT. Glucosylceramide synthase (GCS) and cytochrome P450 family 1 subfamily A1 (CYP1A1) have been previously associated with drug resistance in breast cancer. The present study aimed to explore whether the expression levels of GCS and/or CYP1A1 are associated with the prognosis of TNBC after NACT. Immunohistochemistry was used to detect and measure GCS and CYP1A1 expression. Associations between GCS or CYP1A1 expression and the clinicopathological characteristics, pathological complete response (pCR), clinical complete response (cCR) and disease-free survival (DFS) were analyzed. GCS expression was found to be associated with tumor size (P=0.021) and TNM staging (P=0.042), whilst CYP1A1 expression was associated with lymph node metastasis (P = 0.026) and TNM staging (P=0.034). The expression levels of GCS (P=0.024) and CYP1A1 (P=0.027) were upregulated after NACT. GCS and CYP1A1 expression were positively correlated (P=0.003; r=0.327). No difference was observed between the GCS+ (P=0.188) or CYP1A1+ group (P=0.073) and the GCS- or CYP1A1- group in terms of pCR. However, compared with that in the GCS+CYP1A1+ group, the pCR was markedly increased in the GCS-CYP1A1- group (P=0.031). The cCR was lower in the GCS+ (P=0.021) and CYP1A1+ groups (P=0.016) compared with in the GCS- or CYP1A1- group. The DFS rate (57.9 vs. 65.4%; P=0.049) was lower in the GCS+CYP1A1+ group compared with that in the GCS-CYP1A1- group. However, there was no statistical significance after P-value was adjusted for multiple comparisons using Bonferroni correction. In conclusion, co-expression of GCS and CYP1A1 was associated with pCR and DFS in TNBC, which may serve a role in the prediction of the prognosis of patients with TNBC following treatment with NACT.

Functional complementation of V-ATPase a subunit isoforms in osteoclasts.

In osteoclasts, the a3 isoform of the proton-pumping V-ATPase plays essential roles in anterograde trafficking of secretory lysosomes and extracellular acidification required for bone resorption. This study examined functional complementation of the a isoforms by exogenously expressing the a1, a2 and a3 isoforms in a3-knockout (KO) osteoclasts. The expression levels of a1 and a2 in a3KO osteoclasts were similar, but lower than that of a3. a1 significantly localized to lysosomes, whereas a2 slightly did. On the other hand, a2 interacted with Rab7, a regulator of secretory lysosome trafficking in osteoclasts, more efficiently than a1. a1 partly complemented the functions of a3 in secretory lysosome trafficking and calcium phosphate resorption, while a2 partly complemented the former but not the latter function.

Expression of A1 and A3 adenosine receptors in human breast tumors.

Adenosine receptors (A1, A2A, A2B, A3) play an important role in the regulation of growth, proliferation and death of cancer and normal cells. We recently showed the expression profile of A2A and A2B receptors in normal and tumor breast tissues. In the present study, we used semiquantitative RT-PCR to measure the A1 and A3 gene expression levels in normal and tumor breast tissues. Breast tumors (n = 18) and non-neoplastic mammary tissues (n = 10) were collected and histologically confirmed to be neoplastic or non-neoplastic, respectively. Total RNA was extracted and reverse transcribed into cDNA, and PCR was performed under optimized condition for each receptor subtype. Amplification of beta-actin mRNA served as control for RT-PCR. The PCR products were separated on 1.7% agarose gels. The intensity of the bands was quantitated with ImageJ software after normalization against beta-actin expression. All breast tumor and normal tissue specimens expressed A1 and A3 adenosine receptor transcripts. However, we observed that the expression level of the A3 receptor in tumor tissues was 1.27-fold that of normal tissues, whereas there was no significant difference between the expression levels of A1 in normal and tumor tissues. Interestingly, the results of the present study indicate that breast tumors exhibit a higher level of A3 transcripts (than normal tissues) and support the possible key role of A3 adenosine receptor in tumor development. However, further studies based on real-time quantitative RT-PCR are needed to identify the exact gene expression levels.

Anti-tumorigenic and anti-angiogenic effects of natural conifer Abies sibirica terpenoids in vivo and in vitro.

The natural terpenoid compound was explored in vitro and in vivo to investigate the anti-HCC properties. For our study we used Abisilin®- a novel natural pharmacological terpenoid compound extracted and purified from coniferous Pinaceae trees. Anti-tumorigenic properties of different concentrations of Abisilin® were tested on murine hepatoma Hepa 1-6 cell lines. The analysis of proliferation and apoptosis was performed using immunofluorescence microscopy, FACS and qPCR. As an in vivo approach, we tested Abisilin® (400mg/kg/day, 14 days, orally) in xenograft mouse models of liver cancer and investigated tumor growth, proliferation, apoptosis and angiogenesis by means of Western blotting, immunofluorescence microscopy and qPCR. Application of Abisilin® for 24h at a dosage ranging from 0.03 to 0.045mg significantly reduced the number of viable Hepa 1-6 cells and induced apoptotic cell death with microscopic evidence of changes in cell morphology, and positive TUNEL, cleaved caspase 3 and Annexin V/Propidium Iodide (PI) stainings. Furthermore, treatment with Abisilin® strongly inhibited proliferation, impaired mitosis and prompted cell cycle arrest by down-regulation of the Cyclin D1, E1 and A2 expression levels. In Hepa 1-6 xenograft in vivo model, Abisilin® considerably decreased the xenograft tumor size and tumor volume. Consistently with in vitro Abisilin® administration elicited apoptosis and inhibit proliferation in the xenograft tumor. We also found that Abisilin® remarkably decrease microvessel density, diminished tumor angiogenesis and reduced expression of ICAM-1. Moreover, the expression of pAMPK, a cellular energy sensor, was up-regulated after Abisilin® application. Anti-proliferative, pro-apoptotic activity and anti-angiogenic potential of natural conifer terpenoids might turn these compounds into an attractive drug candidate for combination therapy against liver cancer.

Metabolomics profiling reveals novel markers for leukocyte telomere length.

Leukocyte telomere length (LTL) is considered one of the most predictive markers of biological aging. The aim of this study was to identify novel pathways regulating LTL using a metabolomics approach. To this end, we tested associations between 280 blood metabolites and LTL in 3511 females from TwinsUK and replicated our results in the KORA cohort. We furthermore tested significant metabolites for associations with several aging-related phenotypes, gene expression markers and epigenetic markers to investigate potential underlying pathways. Five metabolites were associated with LTL: Two lysolipids, 1-stearoylglycerophosphoinositol (P=1.6×10(-5)) and 1-palmitoylglycerophosphoinositol (P=1.6×10(-5)), were found to be negatively associated with LTL and positively associated with phospholipase A2 expression levels suggesting an involvement of fatty acid metabolism and particularly membrane composition in biological aging. Moreover, two gamma-glutamyl amino acids, gamma-glutamyltyrosine (P=2.5×10(-6)) and gamma-glutamylphenylalanine (P=1.7×10(-5)), were negatively correlated with LTL. Both are products of the glutathione cycle and markers for increased oxidative stress. Metabolites were also correlated with functional measures of aging, i.e. higher blood pressure and HDL cholesterol levels and poorer lung, liver and kidney function. Our results suggest an involvement of altered fatty acid metabolism and increased oxidative stress in human biological aging, reflected by LTL and age-related phenotypes of vital organ systems.

Transcriptional regulation of Annexin A2 promotes starvation-induced autophagy.

Autophagy is an important degradation pathway, which is induced after starvation, where it buffers nutrient deprivation by recycling macromolecules in organisms from yeast to man. While the classical pathway mediating this response is via mTOR inhibition, there are likely to be additional pathways that support the process. Here, we identify Annexin A2 as an autophagy modulator that regulates autophagosome formation by enabling appropriate ATG9A trafficking from endosomes to autophagosomes via actin. This process is dependent on the Annexin A2 effectors ARP2 and Spire1. Annexin A2 expression increases after starvation in cells in an mTOR-independent fashion. This is mediated via Jun N-terminal kinase activation of c-Jun, which, in turn, enhances the trans-activation of the Annexin A2 promoter. Annexin A2 knockdown abrogates starvation-induced autophagy, while its overexpression induces autophagy. Hence, c-Jun-mediated transcriptional responses support starvation-induced autophagy by regulating Annexin A2 expression levels.

Dynamic expression of tgf-β2, tgf-β3 and inhibin βA during muscle growth resumption and satellite cell differentiation in rainbow trout (Oncorhynchus mykiss)

Members of the TGF-β superfamily are involved in numerous cell functions; however, except for myostatin, their roles in the regulation of muscle growth in fish are completely unknown. We measured tgf-β1, tgf-β2, tgf-β3, inhibin βA (inh) and follistatin (fst) gene expression during muscle growth recovery following a fasting period. We observed that tgf-β1a and tgf-β2 expression were quickly down-regulated after refeeding and that tgf-β3 reached its highest level of expression 7days post-refeeding, mirroring myogenin expression. Inh βA1 mRNA levels decreased sharply after refeeding, in contrast to fst b2 expression, which peaked at day 2. No significant modification of expression was observed for tgf-β1a, tgf-β1b, tgf-β1c and tgf-β6 during refeeding. In vitro, tgf-β2 and inh βA1 expression decreased during the differentiation of satellite cells, whereas tgf-β3 expression increased following the same pattern as myogenin. Surprisingly, fst b1 and fst b2 expression decreased during differentiation, whereas no variation was observed in fst a1 and fst a2 expression levels. In vitro analyses also indicated that IGF1 treatment up-regulated tgf-β3, inh βA1 and myogenin expression, and that MSTN treatment increased fst b1 and fst b2 expression. In conclusion, we showed that the expression of tgf-β2, tgf-β3 and inh βA1 is dynamically regulated during muscle growth resumption and satellite cell differentiation, strongly suggesting that these genes have a role in the regulation of muscle growth.

Expression of the Annexin A1 gene is associated with suppression of growth, invasion and metastasis of nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) has a highly increased incidence rate (20/100,000) in Southern regions of China, while being rare in the rest of the world. NPC is a malignant type of cancer due to its high occurrence rate of metastasis; however, biomarkers for effective diagnosis and treatment are yet to be identified. Annexin A1 is a glucocorticoid‑regulated member of a large superfamily of calcium and phospholipid‑binding proteins and has been shown to have important roles in tumor development and progression, and was demonstrated to be a prognostic biomarker for head and neck cancer types. A previous study by our group showed that Annexin A1 was decreased in NPC tissue as compared with normal adjacent tissue. To investigate whether Annexin A1 is a potential biomarker for NPC, the present study assessed the effect of the Annexin A1 on the biological behavior (i.e., invasion and metastasis) of the highly metastatic NPC cell line 5‑8F and the non‑metastatic NPC cell line 6‑10B. The expression levels of Annexin A1 in the above two cell lines were determined by western blot analysis. Next, the recombinant plasmid pEGFP‑C1‑Annexin A1 and the small interfering (si)RNA plasmid pRNAT‑U6.1‑Annexin A1 were used and stably transfected into 5‑8F and 6‑10B cells, respectively. These established recombinant cell lines were then used to study the up- and downregulation of Annexin A1, respectively. The correlation of Annexin A1 expression levels with the biological behavior of NPC cell lines was analyzed using a cell proliferation assay, flow cytometry, soft agar colony formation assay, as well as Transwell invasion and migration assays. The results demonstrated that upregulation of Annexin A1 suppressed the proliferation, invasion and migration of NPC cells, while downregulation of Annexin A1 promoted the proliferation, invasion and migration of NPC cells. These findings suggested that Annexin A1 may be a potential biomarker for the development and prognosis of NPC, and its dysregulation may have an important role in its underlying pathogenesis.

Antiproliferative effects of selective adenosine receptor agonists and antagonists on human lymphocytes: evidence for receptor-independent mechanisms.

The effects of standard adenosine receptor (AR) agonists and antagonists on the proliferation of human T lymphocytes, unstimulated and phytohemagglutinin-stimulated human peripheral blood lymphocytes (PBL), and Jurkat T cells were investigated. Real-time PCR measurements confirmed the presence of all four AR subtypes on the investigated cells, although at different expression levels. A2A ARs were predominantly expressed in PBL and further upregulated upon stimulation, while malignant Jurkat T cells showed high expression levels of A1, A2A, and A2B ARs. Cell proliferation was measured by [(3)H]-thymidine incorporation assays. Several ligands, including the subtype-selective agonists CPA (A1), BAY60-6583 (A2B), and IB-MECA (A3), and the antagonists PSB-36 (A1), MSX-2 (A2A), and PSB-10 (A3) significantly inhibited cell proliferation at micromolar concentrations, which were about three orders of magnitude higher than their AR affinities. In contrast, further investigated AR ligands, including the agonists NECA (nonselective) and CGS21680 (A2A), and the antagonists preladenant (SCH-420814, A2A), PSB-1115 (A2B), and PSB-603 (A2B) showed no or only minor effects on lymphocyte proliferation. The anti-proliferative effects of the AR agonists could not be blocked by the corresponding antagonists. The non-selective AR antagonist caffeine stimulated phytohemagglutinin-activated PBL with an EC50 value of 104 μM. This is the first study to compare a complete set of commonly used AR ligands for all subtypes on lymphocyte proliferation. Our results strongly suggest that these compounds induce an inhibition of lymphocyte proliferation and cell death through AR-independent mechanisms.

Expression of A1 and A3 Adenosine Receptors in Human Breast Tumors

Adenosine receptors (A1, A2A, A2B, A3) play an important role in the regulation of growth, proliferation and death of cancer and normal cells. We recently showed the expression profile of A2A and A2B receptors in normal and tumor breast tissues. In the present study, we used semiquantitative RT-PCR to measure the A1 and A3 gene expression levels in normal and tumor breast tissues. Breast tumors (n = 18) and non-neoplastic mammary tissues (n = 10) were collected and histologically confirmed to be neoplastic or non-neoplastic, respectively. Total RNA was extracted and reverse transcribed into cDNA, and PCR was performed under optimized condition for each receptor subtype. Amplification of beta-actin mRNA served as control for RT-PCR. The PCR products were separated on 1.7% agarose gels. The intensity of the bands was quantitated with ImageJ software after normalization against beta-actin expression. All breast tumor and normal tissue specimens expressed A1 and A3 adenosine receptor transcripts. However, we observed that the expression level of the A3 receptor in tumor tissues was 1.27-fold that of normal tissues, whereas there was no significant difference between the expression levels of A1 in normal and tumor tissues. Interestingly, the results of the present study indicate that breast tumors exhibit a higher level of A3 transcripts (than normal tissues) and support the possible key role of A3 adenosine receptor in tumor development. However, further studies based on real-time quantitative RT-PCR are needed to identify the exact gene expression levels.

Correlation of overexpression of HMGA1 and HMGA2 with poor tumor differentiation, invasion, and proliferation associated with let-7 down-regulation in retinoblastomas

In addition to RB1, the causative genes involved in the tumorigenesis and progression of retinoblastomas remain to be elucidated. High-mobility group A1 and high-mobility group A2 proteins are expressed at high levels in various benign and malignant tumors and are associated with expressions of malignant phenotypes and poor prognoses. Reduction in let-7 expression levels was detected in cancers; it may be related to high-mobility group A1 and high-mobility group A2 overexpressions. Little is known about the correlations among high-mobility group A1, high-mobility group A2, and let-7 expression and clinicopathologic features of retinoblastoma. In our study, the expressions of high-mobility group A1 and high-mobility group A2 were studied in 44 retinoblastomas by immunohistochemical analysis. Semiquantitative reverse transcription-polymerase chain reaction was used to assay the let-7 expression levels in 28 nontumor retina and 44 tumor samples. Nuclear immunostaining of high-mobility group A1 and high-mobility group A2 was frequently observed in retinoblastomas (68% and 75%, respectively). Expression levels of both high-mobility group A1 and high-mobility group A2 were significantly higher in poorly differentiated retinoblastomas than in well-differentiated retinoblastomas (P < .05 and P < .0001, respectively). In addition, overexpressions of high-mobility group A1 and high-mobility group A2 were more frequently detected in poorly differentiated tumors than in well-differentiated tumors (P < .01 and P = .0001, respectively). High-mobility group A2 expression levels were significantly higher in invasive tumors than in noninvasive tumors (P < .05). In addition, the MIB-1 labeling index was higher in poorly differentiated tumors than in well-differentiated tumors (P < .0001). Our study revealed that high-mobility group A1 and high-mobility group A2 expressions correlated with the MIB-1 labeling index (R = 0.327, P = .029; R = 0.602, P < .0001; respectively). The let-7 was expressed in high levels in all 28 nontumor retina samples. However, reduced expression levels of let-7 were observed in 17 (39%) tumors. A potentially inverse correlation exists between the expression levels of let-7 and high-mobility group A1 (r = -0.247, P = .105). In addition, a significantly inverse association was detected between let-7 and high-mobility group A2 and MIB-1 labeling index (r = -0.31, P = .04; r = -0.392, P = .007, respectively). Our findings imply that the overexpressions of high-mobility group A1, high-mobility group A2, and down-regulation of let-7 may be associated with tumorigenesis and progression of retinoblastomas.

Comparative proteomic analysis of differentially expressed proteins in an in vitro cellular carcinogenesis model of oral squamous cell carcinoma.

In vitro cellular model is an important tool to be used to investigate the cellular events related to pathophysiological conditions in humans. We have developed an in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC). In this study, we performed comparative proteomic analysis using 2-DE and LC-tandem mass chromatography to separate and identify differentially expressed proteins. Forty-five proteins were identified, including 24 proteins with decreased expression and 19 proteins with increased expression during carcinogenesis from immortalized oral epithelial cells to squamous cancerous cells. The identified known proteins were classified into three ontologies of cellular component, molecular function, and biological process. Further validation of five identified proteins (ANXA1, ANXA2, CTSB, KRT17, and S100A6) in the cellular carcinogenesis model and cancerous tissues from OSCC patients confirmed the comparative proteomic results. Moreover, Annexin A1 and A2 expression levels correlated with the pathological differentiation grade of cancerous tissues. Thus, this work provides a dynamic protein file of differentially expressed proteins in oral squamous carcinoma cells, which could provide clues to study the mechanisms of OSCC carcinogenesis and possibly be developed as potential biomarkers for clinical diagnosis or prognostic monitoring.

Serine/Arginine Protein–Specific Kinase 2 Promotes Leukemia Cell Proliferation by Phosphorylating Acinus and Regulating Cyclin A1

Serine/arginine (SR) protein-specific kinase (SRPK), a family of cell cycle-regulated protein kinases, phosphorylate SR domain-containing proteins in nuclear speckles and mediate the pre-mRNA splicing. However, the physiologic roles of this event in cell cycle are incompletely understood. Here, we show that SRPK2 binds and phosphorylates acinus, an SR protein essential for RNA splicing, and redistributes it from the nuclear speckles to the nucleoplasm, resulting in cyclin A1 but not A2 up-regulation. Acinus S422D, an SRPK2 phosphorylation mimetic, enhances cyclin A1 transcription, whereas acinus S422A, an unphosphorylatable mutant, blocks the stimulatory effect of SRPK2. Ablation of acinus or SRPK2 abrogates cyclin A1 expression in leukemia cells and arrest cells at G(1) phase. Overexpression of acinus or SRPK2 increases leukemia cell proliferation. Furthermore, both SRPK2 and acinus are overexpressed in some human acute myelogenous leukemia patients and correlate with elevated cyclin A1 expression levels, fitting with the oncogenic activity of cyclin A1 in leukemia. Thus, our findings establish a molecular mechanism by which SR splicing machinery regulates cell cycle and contributes to leukemia tumorigenesis.

Specific interaction of heterogeneous nuclear ribonucleoprotein A1 with the -219T allelic form modulates APOE promoter activity.

The polymorphic -219T/G variant in the APOE promoter has been associated with variations in basal transcriptional activity as well as with the risk of developing Alzheimer's disease, myocardial infarction and early-onset coronary heart disease. The molecular mechanisms underlying these effects are presently unknown. In this report, we show that nuclear extracts from Jurkat cells form a T-specific complex with a motif including the -219 site within the APOE promoter. By DNA-affinity chromatography and mass spectrometry, the human heterogeneous nuclear ribonucleoprotein hnRNPA1(A1) was identified as one component of the complex. In vitro binding analysis indicated that a fragment of A1 had a marked binding specificity for the T form. Interaction of A1 with this region is driven by an adjacent telomeric-like sequence; however, the presence of G, but not T, at -219 position inhibited this interaction. The differences in transcriptional activity between the -219T and -219G promoter allelic forms correlated with the expression levels of A1 in several cell lines; also, over-expression of A1 increased the activity of the T form relative to that of the G form. These results indicate that A1 transactivates APOE promoter activity by direct and specific interaction with the -219T site.

Quantification of cyclin A1 and glyceraldehyde-3-phosphate dehydrogenase expression in testicular biopsies of infertile patients by fluorescence real-time RT-PCR.

The objective of this study was to establish a classification of meiotic disorders by detection of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and cyclin A1 mRNA in patients presenting with non-obstructive azoospermia. GAPDH and cyclin A1 expression levels were detected in 37 histologically classified testicular tissue specimens by one-line RT-PCR. Levels of cyclin A1 expression (NCyclinA1) were high in tissue specimens with full spermatogenesis [3.519 +/- 3.141 (mean +/- SD)] but only minimal in those without haploid germ cells. A lack of cyclin A1 expression was seen in most sertoli-cell-only syndrome (SCOS) specimens. The criteria for an ideal internal standard were only partially met by GAPDH, as we detected decreased expression in tissue samples with maturation arrest [(268.2 +/- 106.2 relative gene expression (mean +/- SD)] and SCOS (201.7 +/- 95.2) compared to those with normal histological findings (325.1 +/- 129.3). It was concluded that the level of cyclin A1 mRNA expression predicts meiotic disorders during spermatogenesis. GAPDH is not an ideal internal standard for specific gene expression in testicular tissue specimens.