Reverse Transcriptase-polymerase Chain Reaction Data Research Articles

Acetylcholine and Molecular Components of its Synthesis and Release Machinery in the Urothelium

ObjectivesPrevious studies provided indirect evidence for urothelial synthesis and release of acetylcholine (ACh). We aimed to determine directly the ACh content in the urothelium and to characterize the molecular components of its synthesis and release machinery. MethodsThe study was performed on mouse bladder and abraded urothelium, and human mucosal bladder biopsies. ACh content was measured by high-performance liquid chromatography-electrochemical. Reverse transcriptase–polymerase chain reaction (RT-PCR) and immunohistochemistry served to investigate expression of ACh-synthesizing enzymes—choline acetyltransferase (ChAT) and carnitine acetyltransferase (CarAT)—vesicular ACh transporter (VAChT), and polyspecific organic cation transporters (OCTs; isoforms 1–3). Transfected cells served to investigate whether the anticholinergic drug trospium chloride interferes with ACh-transporting OCTs. ResultsACh is present in the urothelium in a nanomolar range per gram of wet weight. RT-PCR data support the presence of CarAT but not ChAT. VAChT, used by neurons to shuffle ACh into synaptic vesicles, is detected in subepithelial cholinergic nerve fibres, but not by RT-PCR or immunohistochemistry in the urothelium. OCT1 and OCT3 are expressed by the urothelium. The quarternary ammonium base trospium chloride inhibits cation transport by OCTs with a potency rank order of OCT2 (IC50=0.67±0.42μmol/l)>OCT1 (IC50=6.2±2.1μmol/l)>OCT3 (IC50=871±177μmol/l). ConclusionsThis study demonstrates a urothelial non-neuronal cholinergic system that differs widely from that of neurons with respect to molecular components of the ACh synthesis and release machinery. Consequently, these two systems might be differentially targeted by pharmacologic approaches.

Analysis of in vivo expressed genes in Mannheimia haemolytica A1

The expression of Mannheimia haemolytica A1 genes during in vivo growth was examined by reverse transcriptase-polymerase chain reaction (RT-PCR) using total RNA extracted directly from M. haemolytica A1 recovered from pneumonic lungs of cattle. Primers specific for three groups of genes were used. Group 1 includes virulence-related genes: lktC, tbpB, ahs, nmaA, gs60 and gcp. Group 2 includes genes that code for putative two-component regulatory systems: narP, narQ, ttrR, ttrS, phoB and phoR. Group 3 includes genes involved in regular cellular functions such as plp4, thiL and rrf. The RT-PCR data were examined in conjunction with the percent pneumonic lesion in each lung scored during necropsy. The analysis showed that lungs with a higher percent pneumonic score exhibit expression of more M. haemolytica A1 genes. For group 1 genes, lktC was expressed in the majority of samples, whereas the other genes were only expressed in some samples. This was not unexpected as the leukotoxin is a major virulence factor of the bacterium. The genes encoding the response regulators for the putative two-component regulatory systems were found to be expressed in more samples than the genes encoding the sensor proteins. The regulator proteins may be required in higher levels to regulate expression of target genes.

Overexpression of stathmin in oral squamous-cell carcinoma: correlation with tumour progression and poor prognosis

Stathmin is an intracellular phosphoprotein that is overexpressed in a number of human malignancies. Our previous study using proteomic profiling showed that significant upregulation of stathmin occurs in oral squamous-cell carcinoma (OSCC)-derived cell lines. In the current study, to determine the potential involvement of stathmin in OSCC, we evaluated the state of stathmin protein and mRNA expression in OSCC-derived cell lines and human primary OSCCs. A significant increase in stathmin expression was observed in all OSCC-derived cell lines examined compared to human normal oral keratinocytes. In immunohistochemistry, 65% of the OSCCs were positive for stathmin, and no immunoreaction was observed in corresponding normal tissues. Real-time quantitative reverse transcriptase–polymerase chain reaction data were consistent with the protein expression status. Moreover, stathmin expression status was correlated with the TNM stage grading. Furthermore, we found a statistical correlation between the protein expression status and disease-free survival (P=0.029). These results suggest that expression of stathmin could contribute to cancer progression/prognosis, and that stathmin may have potential as a biomarker and a therapeutic target for OSCC.

Plasma membrane Ca2+ ATPase isoform 1 down-regulated in human oral cancer

The plasma membrane Ca(2+) ATPase (PMCA) is an essential regulator of free intracellular calcium. Recent studies have reported aberrant expression of the PMCA1 gene, a member of the PMCA family, in several cancer cell types. To elucidate the contribution of PMCA1 to oral carcinogenesis, we analyzed genetic and epigenetic changes and mRNA and protein expression in primary oral squamous cell carcinomas (OSCCs), oral premalignant lesions (OPLs), and OSCC-derived cell lines. The PMCA1 gene was epigenetically inactivated, but not mutated in the eight OSCC-derived cell lines tested. In clinical samples, frequent down-regulation of PMCA1 protein expression was found not only in primary OSCCs (43%), but also in OPLs (40%). Real-time quantitative reverse transcriptase-polymerase chain reaction data were consistent with the protein expression status. These results suggest that inactivation of the PMCA1 gene is a frequent and early event during oral carcinogenesis, and gene expression may be regulated by an epigenetic mechanism.

Nitric oxide production is regulated by fever-range thermal stimulation of murine macrophages

As macrophages are often called to function at times of elevated ambient temperature (e.g., during local inflammation or systemic fever), it is possible that their production of critical effector molecules, such as nitric oxide (NO) or inducible NO synthase (iNOS), is sensitive to physiological changes in temperature. To test this possibility, the threshold requirements for production of NO and iNOS in murine peritoneal macrophages maintained under normothermic conditions (37 degrees C) or following mild (fever-range) hyperthermia (39.5 degrees C) were compared. We found that hyperthermia alone had no observable effect on basal NO production or iNOS protein or message. However, although interferon (IFN)-gamma and lipopolysaccharide (LPS) were needed to induce NO at 37 degrees C, we observed that addition of only LPS was sufficient for production of NO if there were a pretreatment at 39.5 degrees C. Further, if IFN-gamma and LPS were given after thermal exposure, a substantial increase in NO and iNOS was observed over that seen using cells kept at normothermic conditions. Macrophages isolated from mice lacking heat shock factor-1 did not attenuate the ability of mild thermal stress to modulate NO production. Reverse transcriptase-polymerase chain reaction data revealed that thermal regulation of iNOS expression is not entirely at the transcriptional level, suggesting possible points of post-transcriptional thermal sensitivity. These data support the concept that altering the thermal microenvironment is an important means by which the host can manipulate macrophage responses. Increases in temperature (e.g., during fever) may function to lower the activation threshold needed for production of effector molecules in times of infection.

Shh signaling in limb bud ectoderm: Potential role in teratogen‐induced postaxial ectrodactyly

A variety of teratogens induce the loss of postaxial forelimb structures when administered during mid-gestation to the mouse. Previous studies demonstrated that teratogen exposure is associated with a reduction in zone of polarizing activity (ZPA) -related polarizing activity without a noticeable loss of Shh expression. Herein, we quantitatively confirm that expression of Shh, Ptch1, and Gli3 are unaltered by teratogen exposure and demonstrate that sonic hedgehog (Shh) translation is unaffected. Examination of the polarizing response of host chick wings to teratogen-exposed ZPA tissue revealed an induced growth response and ectopic induction of Fgf4, Bmp2, Ptch1, and Gli1 expression similar to control ZPA tissue. Control ZPA tissue altered the fate of cells destined to die in the anterior necrotic zone, whereas cell death ensued in hosts receiving teratogen-exposed grafts. Immunohistochemical studies localized Shh protein in the mouse limb to the posterior mesoderm and overlying ectoderm. We postulate that teratogen exposure alters the ability of Shh to signal to the ectoderm and present microarray and reverse transcriptase-polymerase chain reaction data, indicating that Shh signaling could occur in the limb bud ectoderm.

Clinical Significance of Tissue Levels of Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases in Gastric Cancer

Matrix metalloproteinases (MMPs) are one of the major classes of proteolytic enzymes involved in tumor invasion and metastasis, being inhibited by naturally occurring tissue inhibitors of metalloproteinases (TIMPs). In this study, we examined the expression of MMP-2, MMP-9, membrane-type 1 (MT1)-MMP, TIMP-1, and TIMP-2 in biopsy tissues of gastric cancer, and the correlation between their expression and clinicopathological parameters. Biopsy specimens from 66 patients with gastric carcinoma were available for this study. To determine the expression of MMP-2, MMP-9, MT1-MMP, TIMP-1, and TIMP-2, semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was carried out on tumor and normal tissues, respectively, sampled during diagnostic gastroscopic examination. Immunohistochemical staining of representative samples using monoclonal antibody directed against MT1-MMP was done, and the clinicopathological variables were reviewed retrospectively. The expression level of MMPs and TIMPs was evaluated using the tumor : normal (T/N) ratios of MMPs and TIMPs. The T/N ratio of MT1-MMP mRNA showed a significant correlation with lymph node metastasis and tumor stage (P < 0.05). The other RT-PCR data of MMP-2, MMP-9, TIMP-1, and TIMP-2 did not show any significant correlation with clinicopathological parameters. Immunohistochemistry for MT1-MMP showed a positive immunoreaction in gastric adenocarcinoma and negative staining in normal mucosa. The correlation between the increased expression of MT1-MMP and clinicopathological variables reflects a role in predicting the aggressive behavior of gastric cancer. Because an RT-PCR assay can be performed on biopsy specimens obtained before surgery, an evaluation of MT1-MMP expression in biopsy specimens by RT-PCR may provide useful preoperative information on tumor aggressiveness.

Expression of Matrix Metalloproteinases in Supraglottic Carcinoma and Its Clinical Implication for Estimating Lymph Node Metastases

Twenty to 30% of clinically node-negative patients with squamous cell carcinoma (SCC) of supraglottic larynx were pathologically confirmed with occult lymph node metastasis after operation. Therefore, preoperative judgment on the neck node metastasis should be very important for making an appropriate surgical plan. The purpose of this study is to explore the role of matrix metalloprotineases (MMPs) in the metastatic potential of supraglottic carcinoma in this regard. Examination of MMPs expression in supraglottic carcinoma and establishment of the mathematic model for diagnosis of occult lymph node metastases in a prospective manner. Thirty-two patients with supraglottic SCC underwent supraglottic horizontal partial laryngectomy and neck dissection, and the samples of primary cancer and the corresponding adjacent non-neoplastic tissues were evaluated for both mRNA and protein expression of MMP-1, MMP-2, MMP-7, MMP-8, MMP-9, and MMP-10 using reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. In addition, cervical lymph nodes were examined by routine histopathology to determine whether there was any evidences of node metastasis. RT-PCR and immunohistochemical data were analyzed by multivariate logistic regression, and then two mathematical models were established according to the multivariate logistic regression analysis. Among the six kinds of MMPs, both mRNA and protein expression of MMP-2, MMP-7, and MMP-9 were found up-regulated in more than 63% of the tumor tissues as compared with the adjacent non-neoplastic tissues, and this was significantly correlated with lymph node metastasis (all P < .05). Twenty patients, including seven cases with N0, were pathologically confirmed as having cervical lymph node metastasis. Using RT-PCR and immunohistochemical data of MMP-2, MMP-7, and MMP-9, two mathematic models were tested, and their diagnostic efficiencies for lymph node metastasis were 84% and 88%, respectively. Expressions of MMP-2, MMP-7, and MMP-9 are up-regulated in supraglottic carcinoma tissues, suggesting that MMPs may be useful in the detection of occult lymph node metastasis but that more research is needed.

High Sequence Variability Among Little cherry virus Isolates Occurring in British Columbia.

The LC5 isolate of Little cherry virus (LChV-LC5) is one of at least two distinct viruses contributing to a severe disease of cherry (Little cherry disease [LChD]) in British Columbia. A near-complete nucleotide sequence of LChV-LC5 is available as well as polyclonal antibodies against LChV-LC5 coat protein produced in bacterial cells. A survey for LChV-LC5-infected trees in the Okanagan Valley and Kootenay region of British Columbia was carried out using enzyme-linked immunosorbent assays (ELISA) and LChV-LC5 antibodies. Reverse transcriptase polymerase chain reaction (RT-PCR) and sequence analysis of four different regions of the genomes of 31 of these isolates have been conducted. A high level of sequence variability was found: nucleotide sequence divergence between LChV-LC5 and the other sequenced isolates ranged from 0 to 19.7%, and amino acid sequence divergence ranged from 0 to 9.1%. Further examination of RT-PCR and sequence data identified six discrete groups of isolates, including a group identical to LChV-LC5. The high level of divergence in LChV-LC5 isolates occurring in British Columbia suggests that caution should be used in the selection of methods used for diagnosis during surveys for this virus.

Significance of mRNA levels of connexin37, connexin43, and connexin45 in luteinized granulosa cells of controlled hyperstimulated follicles

Objective To evaluate the mRNA levels of connexins in different sizes of luteinized follicles. Design Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to determine the connexin mRNA levels in the granulosa cells of 91 luteinized follicles. Setting Academic tertiary care medical center and research unit of university. Patient(s) Ninety-one female patients on controlled ovarian hyperstimulation (COH). Intervention(s) Sonoguided aspiration to collect the oocytes and the granulosa cells simultaneously. Main outcome measure(s) The RT-PCR data are normalized by comparing the intensity of the connexins to the intensity of internal controls (β-actin). The follicles are grouped according to the size and the mRNA levels of the connexins. The correlations among the size of the follicles, the outcome of oocytes, and the mRNA levels of the connexins are compared by Fisher's exact test and Mann-Whitney test. Result(s) The mRNA levels of the connexins are low in the follicles equal or larger than 5.5 mL. High cx43 levels are linked to good prognosis of oocytes. Conclusion(s) The luteinized granulosa cells from the large follicles are relatively quiescent in the connexin mRNA expression. In addition to the volume, the mRNA levels of cx43 may serve as a marker to predict the outcome of oocytes.

Gene expression and functional characterization of melatonin receptors in the spinal cord of the rat: implications for pain modulation.

Recently, a species-dependent distribution of melatonin binding sites have been found in lamina I-V and lamina X of the spinal cord. In order to learn more about the function of spinal melatonin receptors, we investigated (i) the gene expression for melatonin receptor subtypes in lumbar and thoracal spinal cord tissue by means of the reverse-transcriptase polymerase chain reaction (RT-PCR) technique, and (ii) the electrophysiological and pharmacological properties of melatonin receptors heterologously expressed in Xenopus oocytes after injection of spinal cord mRNA by means of the voltage clamp technique. Because ample evidence indicates an antinociceptive effect of melatonin, (iii) the role of spinal melatonin receptors for maintaining mechanical and thermal hyperalgesia was studied in a rat model for postoperative pain. The RT-PCR data revealed that transcripts for MT1 and MT2 melatonin receptors are present in the dorsal and ventral horn of lumbar and thoracal spinal cord tissue. Injection of mRNA from lumbar spinal cord tissue into Xenopus oocytes led to the functional reconstitution of melatonin receptors which activate calcium-dependent chloride inward currents. Melatonin responses were abolished by simultaneous administration of the antagonists, 2-phenylmelatonin and luzindole and were unaffected by the MT2 antagonist 4-phenyl-2-propionamidotetralin. Intrathecal administration of different melatonin doses (10-100 nmol) did not inhibit mechanical or thermal hyperalgesia. However, intrathecal application of a low dose of morphine together with melatonin caused a brief antinociceptive effect suggesting an enhanced morphine analgesia by melatonin. In conclusion, the present study demonstrated for the first time the presence of transcripts of MT1 and MT2 receptors located in the dorsal and ventral horn of the spinal cord. Furthermore, spinal melatonin enhanced the antinociceptive effect of morphine indicating that melatonin acts as a neuromodulator in the spinal cord.

Exercise-induced HSP27, HSP70 and MAPK responses in human skeletal muscle.

The present work examined protein and messenger RNA (mRNA) expression of intramuscular heat shock protein 27 (HSP27), heat shock cognate (HSC70) and HSP70 in human biceps brachii (BB) and vastus lateralis (VL) subsequent to two different exercises. Untrained subjects performed 50 high-force eccentric contractions with their non-dominant BB and ran downhill (-10 degrees) for 30 min. The 48-h PX stress response was evaluated with immunoblotting and reverse transcriptase-polymerase chain reaction (RT-PCR). Muscle damage was indicated indirectly at 48 h post-exercise (PX) [loss of mobility, muscle soreness and serum creatine kinase (CK) activity]. On the protein level, HSP27 and HSP70 increased significantly PX in the BB (384 and 227%, respectively; P < 0.01), but there were no significant HSP changes in the VL or in HSC70 in either muscle. The RT-PCR data complemented these findings: BB HSP27 and HSP70C mRNA levels increased (135 and 128%, respectively; P < 0.05); in the VL only HSP70B increased (206%; P < 0.05). Phosphorylation of e-jun NH2-terminal kinase (JNK) and extracellular regulated kinase (ERK) increased significantly in the BB (226 and 200%, respectively; P < 0.05) but not in the VL, indicating activation of these pathways only after the resistance exercise. These data indicate that the PX HSP and mitogen-activated protein kinase responses are exercise-specific and local, not systemic. Further, only the resistance exercise induced HSP expression (protein and mRNA) and JNK/ERK activation at 48 h PX, suggesting that these molecules may be important to long-term skeletal muscle adaptations such as hypertrophy.

Role of death receptor pathway in estradiol-induced T-cell apoptosis in vivo.

In the current study we investigated the mechanism by which beta-estradiol-17-valerate (E2) induces apoptosis in T cells. To this end, C57BL/6 wild-type (+/+), Fas-deficient (C57BL/6-lpr/lpr), and FasL-deficient (C57BL/6-gld/gld) mice were treated with various concentrations of E2, including 75, 25, 5, 1, or 0.1 mg/kg body weight or the vehicle. The thymi from these mice were harvested on days 1, 4, or 7 following treatment, and cellularity and apoptosis were determined. Treatment with E2 caused a decrease in thymic cellularity at all doses except 0.1 mg/kg in all three groups of mice, particularly on days 4 and 7. Interestingly, however, the degree of thymic atrophy in C57BL/6-lpr/lpr and C57BL/6-gld/gld mice was significantly less than that seen in C57BL/6 wild-type mice. When thymocytes were analyzed for apoptosis, cells from C57BL/6-lpr/lpr and C57BL/6-gld/gld mice showed decreased levels of apoptosis. Moreover, cDNA array analysis of gene expression revealed that treatment with E2 upregulated several genes involved in apoptosis, including FasL, caspases, TRAIL, and iNOS, but not bcl-2 gene family. Reverse transcriptase-polymerase chain reaction data also demonstrated the increased expression of Fas and FasL genes following E2 treatment. Caspase 8 inhibitor blocked the E2-induced apoptosis of thymocytes in vitro. These data suggested that E2 may induce apoptosis by activating the death-receptor rather than the mitochondrial pathway. E2 treatment decreased the expansion of peripheral Vbeta3+ T cells to the bacterial superantigen SEA in vivo and their subsequent in vitro proliferative response to SEA, thereby suggesting increased induction of apoptosis in Vbeta3+ T cells. The current study suggests that E2 may trigger the death receptor pathway in vivo in T cells, thereby inducing apoptosis.

Expression of somatostatin receptor types 1-5 in 81 cases of gastrointestinal and pancreatic endocrine tumors. A correlative immunohistochemical and reverse-transcriptase polymerase chain reaction analysis.

Somatostatin receptors (SSTRs) have been extensively mapped in human tumors by means of autoradiography, reverse-transcriptase polymerase chain reaction (RT-PCR), in situ hybridization (ISH) and immunohistochemistry (IHC). We analyzed the SSTR type 1-5 expression by means of RT-PCR and/or IHC in a series of 81 functioning and non-functioning gastroenteropancreatic (GEP) endocrine tumors and related normal tissues. Moreover, we compared the results with clinical, pathological and hormonal features. Forty-six cases (13 intestinal and 33 pancreatic) were studied for SSTR 1-5 expression using RT-PCR, IHC with antibodies to SSTR types 2, 3, 5 and ISH for SSTR2 mRNA. The vast majority of tumors expressed SSTR types 1, 2, 3 and 5, while SSTR4 was detected in a small minority. Due to the good correlation between RT-PCR and IHC data on SSTR types 2, 3, and 5, thirty-five additional GEP endocrine tumors were studied with IHC alone. Pancreatic insulinomas had an heterogeneous SSTR expression, while 100% of somatostatinomas expressed SSTR5 and 100% gastrinomas and glucagonomas expressed SSTR2. Pre-operative biopsy material showed an overlapping immunoreactivity with that of surgical specimens, suggesting that the SSTR status can be detected in the diagnostic work-up. It is concluded that SSTRs 1-5 are heterogeneously expressed in GEP endocrine tumors and that IHC is a reliable tool to detect SSTR types 2, 3 and 5 in surgical and biopsy specimens.

Expression of somatostatin receptor types 2, 3 and 5 in biopsies and surgical specimens of human lung tumours. Correlation with preoperative octreotide scintigraphy.

The increasingly popular use of somatostatin analogs in clinical practice for both diagnostic and therapeutic purposes prompted extensive investigations on somatostatin receptor (sst) expression in human tumors by autoradiography, nucleic acid analysis and, recently, immunohistochemistry (IHC). The currently employed radiotracer for scintigraphy (Octreoscan) is octreotide, a somatostatin analog having a high affinity for sst types 2, 3, and 5. In this study on 25 patients, we compared sst 2, 3, and 5 expression in surgical and biopsy specimens of lung tumors, as revealed by immunohistochemical and reverse transcriptase polymerase chain reaction (RT-PCR), with the octreoscan outcome (which was positive in 20/25 cases). By IHC, the tumors mainly expressed sst2 (17/25, 68%) at the cell membrane level, while sst 3 and 5 were detected in a fraction of cases (24% and 20%, respectively). Comparing RT-PCR and IHC data, a correlation was found in 83.3% of cases, while octreoscan findings and sst expression were correlated in 22/25 cases (88%). In addition, cytological and biopsy specimens expressed the same sst type found in the corresponding surgical sample, thus indicating that a cell membrane sst immunoreactivity in a biopsy reliably predicts the tumor-receptor profile before its resection. Finally, sst expression was not restricted to neuroendocrine lung tumors, but was also a feature of some non-neuroendocrine carcinomas, although to a lesser extent. The occasional expression of sst subtypes in intratumoral lymphocytes, endothelia and necrotic areas is an additional feature to be considered in the interpretation of Octreoscan findings, since the in vivo procedure does not allow to define the sst cellular distribution. IHC can therefore be usefully coupled to radionuclear investigations to better characterize the sst cellular location and subtype in lung tumors.

In vivo and in vitro expression of somatostatin receptors in two human thymomas with similar clinical presentation and different histological features.

[(111)In-DTPA0]octreotide scintigraphy allows the in vivo visualization of several types of SS receptor (SSR)-expressing tumors. Among these, thymomas have been recently detected. Here we report on 2 patients admitted for myasthenia gravis and radiological evidence of thymic mass. Although these patients had similar clinical presentation, in vivo SSR scintigraphy displayed a difference in the degree of the [(111)In-DTPA0]octreotide uptake. Considering that both thymic masses had comparable volume, [(111)In-DTPA0]octreotide level was significantly higher in one of the 2 tumors (tumor/background ratio of 5.7 vs 2.6). The SSR subtype expression pattern was studied in vitro on the surgically resected specimens by ligand binding techniques, quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. According to the recent World Health Organization classification, the 2 tumors were classified A and B2 thymomas respectively. In membrane homogenates, we found a higher number of high affinity [125I-Tyr11]-SS-14 binding sites in the B2 thymomas (23.5+/-2.5 vs 12.0+/-0.4 fmol/mg membrane protein; p<0.05). RT-PCR analysis showed sst1, sst2A and sst3 mRNA in the 2 thymoma tissues, whereas SS mRNA was detectable only in the A thymoma. Quantitative evaluation of RT-PCR data showed a comparable expression of the relative amount of sst2A mRNA in both tumors, whereas a significant higher expression of sst3 mRNA in the B2 thymoma. Sst2A immunoreactivity was localized mainly on the endothelium of intratumoral vessels, whereas sst3 was present on either tumoral epithelial cells or normal reactive thymocytes. The expression of sst2A receptors in these tumors is in line with the in vivo visualization by [(111)In-DTPA0]octreotide, which is considered a sst2-preferring ligand. However, since radioligand uptake was significantly higher in the B2 thymoma, which expressed the largest sst3 mRNA levels, it might be possible that this subtype is involved in determining the tumor visualization during SSR scintigraphy. Apart from the affinity of the radioligand for the receptor, also the efficacy of the internalization of the radioligand-receptor complex might play a role in radioactivity accumulation during in vivo SSR scintigraphy. In fact, although octreotide binds with lower affinity to sst3 receptors, this subtype displayed the highest amount of agonist-dependent receptor internalization compared to the other SSR subtypes. Moreover, sst3 was localized on both tumor cells and reactive thymocytes, and these latter cells are characterized by a very active turnover of membrane molecules. Finally, although more cases need to be evaluated, the lack of detection of SS mRNA in the tumor presenting a more aggressive phenotype (B2 thymoma) might have physiopathological or prognostic significance.

Soluble biodegradable polymer-based cytokine gene delivery for cancer treatment.

Transgene expression and tumor regression after direct injection of plasmid DNA encoding cytokine genes, such as mIL-12 and mIFN-gamma, remain very low. The objective of this study is to develop nontoxic biodegradable polymer-based cytokine gene delivery systems, which should enhance mIL-12 expression, increasing the likelihood of complete tumor elimination. We synthesized poly[alpha-(4-aminobutyl)-l-glycolic acid] (PAGA), a biodegradable nontoxic polymer, by melting condensation. Plasmids used in this study encoded luciferase (pLuc) and murine interleukin-12 (pmIL-12) genes. PAGA/plasmid complexes were prepared at different (+/-) charge ratios and characterized in terms of particle size, zeta potential, osmolality, surface morphology, and cytotoxicity. Polyplexes prepared by complexing PAGA with pmIL-12 as well as pLuc were used for transfection into cultured CT-26 colon adenocarcinoma cells as well as into CT-26 tumor-bearing BALB/c mice. The in vitro and in vivo transfection efficiency was determined by luciferase assay (for pLuc), enzyme-linked immunosorbent assay (for mIL-12, p70, and p40), and reverse transcriptase-polymerase chain reaction (RT-PCR) (for Luc and mIL-12 p35). PAGA condensed and protected plasmids from nuclease degradation. The mean particle size and zeta potential of the polyplexes prepared in 5% (w/v) glucose at 3:1 (+/-) charge ratio were approximately 100 nm and 20 mV, respectively. The surface characterization of polyplexes as determined by atomic force microscopy showed complete condensation of DNA with an ellipsoidal structure in Z direction. The levels of mIL-12 p40, mIL-12 p70, and mIFN-gamma were significantly higher for PAGA/pmIL-12 complexes compared to that of naked pmIL-12. This is in good agreement with RT-PCR data, which showed significant levels of mIL-12 p35 expression. The PAGA/pmIL-12 complexes did not induce any cytotoxicity in CT-26 cells as evidenced by 3-¿4, 5-dimethylthiazol-2-yl¿-2,5-diphenyltetrazolium bromide assay and showed enhanced antitumor activity in vivo compared to naked pmIL-12. PAGA/pmIL-12 complexes are nontoxic and significantly enhance mIL-12 expression at mRNA and protein levels both in vitro and in vivo.

Vascular development in the mouse embryonic pancreas and lung

Purpose: The purpose of this study was to analyze the formation of blood vessels in the developing mouse pancreas and lung by studying two ligands, angiopoietin-1 (ang1) and angiopoietin-2 (ang2), which are thought to play a role as angiogenesis-activating factors in development. Understanding the role of vasculogenic peptides in normal embryonic development also may have important implications for common clinical problems regarding neonatal pulmonary vasculature.Methods: Reverse transcriptase-polymerase chain reaction (RT-PCR) as well as Southern blotting was used to determine the ontogeny of angiopoietin-1 and angiopoietin-2 gene expression in the embryonic mouse pancreas and lung. Immunohistochemistry was performed for von Willebrand factor, a known marker of endothelial cells, to chronicle the development of the vasculature in these organs.Results: The authors determined the temporal expression of angiopoietin-1 and angiopoietin-2 as a function of gestational age. RT-PCR data demonstrated expression of ang1 and ang2 in the developing mouse lung between gestational day E9.5 and postnatal day 1, and in the developing pancreas between gestational days E12.5 and E18.5. Southern blot analysis confirmed PCR data for ang2 expression in both the lung and pancreas. The authors also traced the spatial development of the vascular system by von Willebrand factor staining. For both lung and pancreas specimens, no blood vessels were identifiable by immunohistochemistry until embryonic day 12.5. With increased gestational age, the blood vessel networks grew larger.Conclusion: The authors have demonstrated that ang1 and ang2 may be involved in the mechanisms of vascular development in the embryonic mouse lung and pancreas.

DNA topoisomerase IIalpha and -beta expression in human ovarian cancer.

To study DNA topoisomerase IIα (Topo-IIα) and -β expression and regulation in human ovarian cancer, 15 ovarian tumour samples were investigated. To compare different levels of expression, the samples were screened for topo IIα and -β mRNA with Northern blotting and a quantitative reverse transcriptase polymerase chain reaction (RT-PCR) assay for Topo-IIα mRNA. Additionally, protein levels were determined with Western blotting and topoisomerase II activity levels with the decatenation assay. The results obtained were compared with each other and with the tumour volume index of the samples. In tumours with a tumour volume index ≥ 50%, the mRNA levels (as determined by Northern blotting) and protein levels for each isozyme were in accordance. Additionally, correlations were found between Topo-IIα RT-PCR data and Topo-IIα Northern blot results, and between Topo-IIα RT-PCR data and Topo-IIα protein levels. Interestingly, Topo-IIβ protein levels correlated better with Topo-II activity than Topo-IIα protein levels. In eight ovarian cystadenoma samples, no Topo-IIα protein could be found. In only three out of eight of these cystadenomas, Topo-IIβ protein could be detected. These findings suggest that Topo-IIα and Topo-IIβ protein levels are up-regulated in ovarian cancer and may indicate that Topo-IIβ is an interesting target for chemotherapy in ovarian tumours. © 1999 Cancer Research Campaign

Presence of four tissue inhibitors of matrix metalloproteinases (TIMP-1, -2, -3 and -4) in human fetal membranes.

Matrix metalloproteinases play a critical role in fetal membrane extracellular matrix (ECM) homeostasis. Remodeling of the ECM during normal placental development is a balanced activity between various matrix metalloproteinases and their tissue-specific counter-regulatory proteins (tissue inhibitors of matrix metalloproteinases [TIMPs]). We have reported the presence of TIMP-1 and TIMP-2 in placental membranes in culture. In this study we have investigated the membrane expression of TIMP-1 and TIMP-2 during labor and nonlabor conditions and also the presence of two novel TIMP family members (TIMP-3 and TIMP-4). Amniochorionic membranes collected from women undergoing Cesarean section and were cultured in an organ explant system. Membranes were also collected from laboring women after vaginal delivery. Samples were subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) using primers specific for TIMP-1, TIMP-2, TIMP-3, and TIMP-4. Localization of TIMP mRNAs was accomplished by in situ hybridization, and peptides were localized by immunocytochemistry. RT-PCR data demonstrated the expression of all the TIMPs in tissues from laboring and nonlaboring women as well as in cultured membranes. TIMP-4 expression was seen in RT-PCR, however, only a faint band was visible in all the tissues tested. In situ hybridization localized the TIMP mRNAs to the amnion, chorion, and to scattered cells in the connective tissue. Human fetal membrane cells (amniochorion and decidua) express mRNA for all the TIMPs studied so far.