Effect of synbiotics on thyroid hormones, intestinal histomorphology, and heat shock protein 70 expression in broiler chickens reared under cyclic heat stress.

This study examined effect of a dietary synbiotic supplement on the concentrations of plasma thyroid hormones, expressions of heat shock protein 70 (HSP70), and intestinal histomorphology in broiler chickens exposed to cyclic heat stress (HS). Three hundred and sixty day old male Ross 708 broiler chicks were randomly distributed among 3 dietary treatments containing a synbiotic (PoultryStar meUS) at 0 (control), 0.5 (0.5×), and 1.0 (1.0×) g/kg. Each treatment contained 8 replicates of 15 birds each housed in floor pens in a temperature and lighting controlled room. Heat stimulation was established from days 15 to 42 at 32°C for 9 h daily. The results indicated that under the HS condition, both synbiotic fed groups had lower liver and hypothalamus HSP70 levels (P < 0.001) compared to control group; however, HSP70 mRNA expression was not different among treatments (P > 0.05). There were no treatment effects on the levels of triiodothyronine (T3) and thyroxine (T4) as well as T3/T4 ratio (P > 0.05). Compared to controls, 1.0× HS broilers had greater villus height in the duodenum (P < 0.01), and greater villus height and villus height:crypt depth ratios in the ileum (P < 0.01). There were no differences among treatments on the measured intestinal parameters in the jejunum (P > 0.05). The results suggest that the synbiotic may ameliorate the negative effects of HS on chicken health as indicated by the changes in the intestinal architecture and the levels of HSP70. Dietary synbiotic supplement could be a feasible nutritive strategy for the poultry industry to improve the health and welfare of chickens when exposed to hot environmental temperature.

Effects of probiotic (Bacillus subtilis) supplementation on meat quality characteristics of breast muscle from broilers exposed to chronic heat stress.

The aim of this study was to determine the impact of probiotic feeding and chronic heat stress on meat quality, total lipid and phospholipid contents, lipid oxidation, antioxidant capacity, and heat shock protein abundance of broiler breast muscle. A total of 240 male broilers (5 birds per pen) were subjected to 4 treatments consisting of a 2 × 2 factorial design. Broilers were kept at 21-32-21°C for 10 h daily (heat stress, HS) or 21°C (thermoneutral condition) and fed a regular diet or the diet mixed with probiotic (250 ppm of Sporulin containing 3 strains of Bacillus subtilis). A total of 48 broilers (12 birds/treatment) were harvested at 46 d. Neither HS nor probiotic had substantial impacts on water-holding capacity, shear force, and color characteristics. HS induced lipid oxidation as increased 2-thiobarbituric acid reactive substances (TBARS), in which probiotic feeding decreased TBARS value (P = 0.002) and phospholipid contents (P = 0.0033) in breast muscle of HS broilers. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity was increased with HS (P < 0.0001), but no significant impact of probiotic supplementation was found. Neither probiotic nor HS affected catalase activity, but superoxide dismutase and glutathione peroxidase activities were lower in HS broilers compared to thermoneutral controls (P < 0.0001) and in probiotics-fed broilers (P < 0.0001) compared to their counterparts. In addition, a significant interaction between probiotic and HS was found at glutathione peroxidase activities, in which breast muscle of broilers fed probiotic at thermoneutral condition showed the highest activity (P < 0.05). Regarding heat shock protein (HSP) determination, HS slightly increased the levels of both HSP70 (P = 0.08) and HSP27 (P = 0.05), but no significant impacts of probiotic supplementation were found. Our results indicate that probiotic feeding could improve breast muscle weight without adverse impacts on meat quality attributes, as well as alleviate oxidative deterioration of breast muscle of broilers undergoing heat stress.

Does Labral Size Correlate With Degree of Acetabular Dysplasia?

BACKGROUND: Hip dysplasia has been shown to be a cause of early arthritis. The decrease in bony coverage has shown increased stress on the acetabular labrum as it shares an increased load. PURPOSE/HYPOTHESIS: The purpose of this study was to divide a cohort of patients by radiographic measures of dysplastic and nondysplastic hips for comparison with regard to labral size at 4 anatomic locations. The hypothesis was that dysplastic hips will have significantly larger labral size compared with nondysplastic hips. STUDY DESIGN: Cross-sectional study; Level of evidence, 3. METHODS: A prospective study was conducted at a single institution. A total of 130 patients underwent hip arthroscopy during the study period from September 2011 to February 2012. Intraoperatively, arthroscopic measurements were taken at 4 quadrants on the acetabular clockface: anterosuperior (12-3 o'clock), anteroinferior (3-6 o'clock), posterosuperior (9-12 o'clock), and posteroinferior (6-9 o'clock). Three radiographic parameters for dysplasia were used to substratify the study population base: lateral center-edge angle (LCEA) ≤25° and LCEA >25°, acetabular inclination (AI) ≤10° and AI >10°, and anterior center-edge angle (ACEA) ≤20° and ACEA >20°. RESULTS: For the LCEA ≤25° group, there were 28 hips with mean LCEA of 20.96° ± 3.40°. Patients with LCEA ≤25° had larger labral width in all 4 quadrants (P < .05). For AI >10°, there were 12 hips with the mean AI 12.92° ± 2.50°. Patients with AI >10° had larger labral size in the posteroinferior quadrant only (P < .05). For ACEA ≤20°, there were 4 hips with a mean ACEA of 11.25° ± 5.19°. The anteroinferior and posteroinferior quadrants had a significant increase in labral size when substratified by ACEA ≤20° (P < .05). CONCLUSION: Labral size was significantly larger in dysplastic hips compared with nondysplastic hips. The posteroinferior quadrant labrum was larger in size in dysplastic hips, as measured by any of the 3 radiographic measurements of dysplasia. Hips with LCEA ≤25° had larger labra in all 4 quadrants.

Return to sport after hip arthroscopy: aggregate recommendations from high-volume hip arthroscopy centers.

Hip arthroscopy is a minimally invasive surgical technique often performed in athletes who want an expeditious return to sport. To the authors' knowledge, no studies in the literature provide a time frame or criteria for return to sport after hip arthroscopy. The purpose of this study was to develop an aggregate recommendation for return to sport after hip arthroscopy based on data assimilated from high-volume hip arthroscopy centers. Twenty-seven orthopedic surgeons from high-volume hip arthroscopy centers completed a survey regarding return to sport after hip arthroscopy. The questionnaire asked surgeons to give a time frame for return to sport and to choose meaningful criteria that an athlete must meet prior to return to sport. Surgeons were asked to categorize various common sports as high, medium, or low risk with regard to the hip. The aggregate results were used to create standardized recommendations for time, criteria, and risk for return to competitive sports. Regarding time frame for return to sport, 70% of surgeons recommended 12 to 20 weeks. In addressing criteria for return to sport, 85% of surgeons recommended that patients need to be able to reproduce all motions involved in their sport without pain. A majority of surgeons recommended criteria of pain-free running, jumping, lateral agility drills, and single-leg squats. Finally, surgeons categorized sports requiring the most movement and impact of the hip joint (football, basketball, wrestling, and martial arts) as high-risk sports. Sports with less impact on the hip, such as golf, were ranked as low risk.

Heat stress attenuates new cell generation in the hypothalamus: a role for miR-138.

The anterior hypothalamus (Ant Hyp) of the brain serves as the main regulator of numerous homeostatic functions, among them body temperature. Fine-tuning of the thermal-response set point during the critical postnatal sensory-developmental period involves neuronal network remodeling which might also be accompanied by alterations in hypothalamic cell populations. Here we demonstrate that heat stress during the critical period of thermal-control establishment interferes with generation of new cells in the chick hypothalamus. Whereas conditioning of the 3-day-old chicks under high ambient temperatures for 24h diminished the number of newborn cells in anterior hypothalamic structures 1 week after the treatment, mild heat stress did not influence the amount of new cells. Phenotypic analysis of these newborn cells indicated a predominant decrease in non-neuronal cell precursors, i.e. cells that do not express doublecortin (DCX). Furthermore, heat challenge of 10-day-old previously high-temperature-conditioned chicks abolished hypothalamic neurogenesis and significantly decreased the number of cells of non-neural origin. As a potential regulatory mechanism for the underlying generation of new cells in the hypothalamus, we investigated the role of the microRNA (miRNA) miR-138, previously reported by us to promote hypothalamic cell migration in vitro and whose levels are reduced during heat stress. Intracranial injection into the third ventricle of miR-138 led to an increase in the number of newborn cells in the Ant Hyp, an effect which might be partially mediated by inhibition of its direct target reelin. These data demonstrate the role of ambient temperature on the generation of new cells in the hypothalamus during the critical period of thermal-control establishment and highlight the long-term effect of severe heat stress on hypothalamic cell population. Moreover, miRNAs, miR-138 in particular, can regulate new cell generation in the hypothalamus.

Improved hemostasis in hemophilia mice by means of an engineered factor Va mutant.

BACKGROUND: Factor (F)VIIa-based bypassing not always provides sufficient hemostasis in hemophilia. OBJECTIVES: To investigate the potential of engineered activated factor V (FVa) variants as bypassing agents in hemophilia A. METHODS: Activity of FVa variants was studied in vitro using prothrombinase assays with purified components and in FV- and FVIII-deficient plasma using clotting and thrombin generation assays. In vivo bleed reduction after the tail clip was studied in hemophilia A mice. RESULTS AND CONCLUSIONS: FVa mutations included a disulfide bond connecting the A2 and A3 domains and ones that rendered FVa resistant to inactivation by activated protein C (APC). '(super) FVa,' a combination of the A2-A3 disulfide (A2-SS-A3) to stabilize FVa and of APC-cleavage site mutations (Arg506/306/679Gln), had enhanced specific activity and complete APC resistance compared with wild-type FVa, FVL eiden (Arg506Gln), or FVaL eiden (A2-SS-A3). Furthermore, (super) FVa potently increased thrombin generation in vitro in FVIII-deficient plasma. In vivo, (super) FVa reduced bleeding in FVIII-deficient mice more effectively than wild-type FVa. Low-dose (super) FVa, but not wild-type FVa, decreased early blood loss during the first 10 min by more than two-fold compared with saline and provided bleed protection for the majority of mice, similar to treatments with FVIII. During the second 10 min after tail cut, (super) FVa at high dose, but not wild-type FVa, effectively reduced bleeding. These findings suggest that (super) FVa enhances not only clot formation but also clot stabilization. Thus, (super) FVa efficiently improved hemostasis in hemophilia in vitro and in vivo and may have potential therapeutic benefits as a novel bypassing agent in hemophilia.

The growing complexity of HIF-1α's role in tumorigenesis: DNA repair and beyond.

Lack of oxygen (hypoxia) is a central hallmark of cancer and a pivotal driving force of malignant progression. Transcriptional activators of the hypoxia-inducible factor α (HIFα) family represent the principal molecular mediators of hypoxia under both physiological and pathophysiological conditions. While HIF-2α is expressed in a tissue- and cell-type-restricted manner, stabilization of HIF-1α was reported in tumours of widely different origin, and functional analyses led to the perception of HIF-1α as an oncoprotein. In this review, we aim to acknowledge HIFα's growing complexity by outlining its functional relevance for genomic integrity and tumour heterogeneity, two features of paramount importance for basic and clinical oncology. Pharmaceutical companies around the globe are ambitiously hunting for HIF-1α-inhibiting compounds, some of which are currently being evaluated in phase 1 trials. To avoid the rather disappointing clinical efficacy emblematic of most targeted therapeutics, potential resistance mechanisms of, as well as potential combination partners for, HIF-1α-inhibiting drugs should be evaluated. In this regard, the interrelation of HIF-1α with genomic integrity and tumour heterogeneity offers ample possibilities, potentially resulting in more efficient clinical translation of HIF-1α's pathobiology.

Water-gated organic field effect transistors - opportunities for biochemical sensing and extracellular signal transduction.

There is a quest for electronic biosensors operated in water for biomedical applications and environmental monitoring. Water is an aggressive medium for standard electronics materials and devices due to its strong polarizability and electrochemical activity. Thick dielectric encapsulation provides necessary stability while it damps the sensitivity of the device to sensing events occurring in the aqueous environment. Organic electronics provides materials that exhibit stable electronic conduction in direct contact with water combined with other desirable properties like mechanical softness, biocompatibility and processability onto flexible substrates. In this review, we introduce an emerging class of organic transistors, in which the current across the organic film is gated by the electric field of the Debye-Helmholtz layer. We discuss the device physics, the sensing mechanism and the relevant electrochemical processes. Applications of water-gated transistors range from the sensing of biologically relevant molecules like DNA, proteins or hormones to non-invasive recording and stimulation of electrical activity of neurons. Materials chemistry is crucial to control properties of electrically active films and to allow the introduction of specific chemical functionalities and receptors at sensing interfaces of the device.

Two-dimensional charge transport in disordered organic semiconductors.

We analyze the effect of carrier confinement on the charge-transport properties of organic field-effect transistors. Confinement is achieved experimentally by the use of semiconductors of which the active layer is only one molecule thick. The two-dimensional confinement of charge carriers provides access to a previously unexplored charge-transport regime and is reflected by a reduced temperature dependence of the transfer curves of organic monolayer transistors.

Membrane gas transfer of methane and carbon dioxide in submerged coal deposits.

Membrane degassing technology may prove to be a viable alternative to current coal bed methane recovery. The proposed approach involves supplying a CO2 sweep gas to membrane fibres placed directly within a saturated coal seam to provide simultaneous CO2 sequestration and CH4 recovery. A system of ordinary differential equations derived from a mass balance on an infinitesimal fibre element enabled the calculation of lumen gas composition as a function of fibre length. The results were verified through the use of a bench-scale vessel. The model agreement appears reasonable for CH4 recovery; however, agreement for CO2 recovery declines as liquid flow decreases and lumen flow increases. To further evaluate the feasibility of the membrane degassing technology, model predictions were normalized to an average conventional CH4 recovery rate of 1.56 x 10(4) m3 d(-1). Assuming a hypothetical coal seam with a groundwater velocity of 100 cm d(-1), thickness of 36.6 m and an average depth of 107 m, 290,000 m2 or 7.73 km of fibre fabric is required, resulting in 4.11 x 10(5) m3 of CO2 transfer daily and an outlet gas composition of 95% CH4, 4.4% CO2 and 0.6% H2O vapour. Increasing groundwater velocities reduce the required membrane surface area with diminishing effect, stabilizing at 100 cm d(-1). Greater pore pressures also reduce required membrane areas, and predictions indicate that a deeper coal seam under 4.3 times greater pressure would require 98% fewer fibres as compared with the hypothetical coal seam and only 0.206 km of membrane fabric.

HIF-1alpha determines the metastatic potential of gastric cancer cells.

Gastric adenocarcinoma is characterised by rapid emergence of systemic metastases, resulting in poor prognosis due to vanished curative treatment options. Better understanding of the molecular basis of gastric cancer spread is needed to design innovative treatments. The transcription factor HIF-1alpha (hypoxia-inducible factor 1alpha) is frequently overexpressed in human gastric cancer, and inhibition of HIF-1alpha has proven antitumour efficacy in rodent models, whereas the relevance of HIF-1alpha for the metastatic phenotype of gastric adenocarcinoma remains elusive. Therefore, we have conducted a comprehensive analysis of the role of HIF-1alpha for pivotal metastasis-associated processes of human gastric cancer. Immunhistochemistry for HIF-1alpha showed specific staining at the invading tumour edge in 90% of human gastric cancer samples, whereas normal gastric tissue was negative and only a minority of early gastric cancers (T1 tumours) showed specific staining. Hypoxia-inducible factor 1alpha-deficient cells showed a significant reduction of migratory, invasive and adhesive properties in vitro. Furthermore, the HIF-1alpha-inhibitor 2-methoxy-estradiol significantly reduced metastatic properties of gastric cancer cells. The accentuated expression at the invading edge together with the in vitro requirement of HIF-1alpha for migration, invasion and adherence argues for a pivotal role of HIF-1alpha in local invasion and, ultimately, systemic tumour spread. These results warrant the exploration of HIF-1alpha-inhibiting substances in clinical treatment studies of advanced gastric cancer.

IgM colocalises with complement and C reactive protein in infarcted human myocardium.

AIMS: Reperfusion of ischaemic myocardium after acute myocardial infarction (AMI) can induce ischaemia/reperfusion (I/R) injury, as a result of local activation of the complement system. C reactive protein (CRP) is involved in this activation. This study analysed the potential role of IgM in complement activation in the infarcted human myocardium. METHODS: Immunochemical analysis was carried out on heart specimens from 59 patients who died from AMI. Serial slides of frozen tissue from the infarction site were stained for IgM, complement factors C3d and C5b-9 (membrane attack complex), and CRP. RESULTS: IgM deposits were found on the plasma membrane, cross striations, and in the cytoplasm of jeopardised cardiomyocytes in infarcts of one to five days duration. IgM depositions were remarkably similar to those of CRP and both complement factors. The relative staining intensities of IgM and CRP varied greatly among patients. CONCLUSIONS: Similar to CRP, IgM targets complement locally to jeopardised cardiomyocytes in the human heart after AMI. Localisation patterns and relative staining intensities suggest that IgM and CRP recognise similar epitopes in the ischaemic heart, but that the relative contribution of each protein to complement activation in the ischaemic myocardium differs among patients.

Expression of VEGF isoforms by epiphyseal chondrocytes during low-oxygen tension is HIF-1 alpha dependent.

OBJECTIVE: To establish the role of hypoxia and HIF-1 alpha for VEGF expression of murine epiphyseal chondrocytes. To analyze the effect of hypoxia on VEGF isoform expression. MATERIALS AND METHODS: VEGF mRNA and VEGF isoform expression was investigated in epiphyses of murine newborns by in situ hybridization and real-time PCR. Further, epiphyseal chondrocytes were isolated from newborn mice with homozygous flanking of the HIF-1 alpha gene with lox-P sites. HIF-1 alpha was deleted by infection with adenovirus containing cre-recombinase. After chondrocytes reached confluency they were exposed to 0.5% or 20% oxygen, respectively. Total VEGF and VEGF isoform mRNA expression levels were measured by real-time PCR. Secreted VEGF protein was determined by ELISA. RESULTS: VEGF mRNA signals were detected in the hypertrophic zone and in the center of the proliferative zone of the murine epiphysis, which is considered to be hypoxic. Real-time PCR revealed that VEGF(120)is the dominant isoform in vivo. In cultured epiphyseal chondrocytes strongly increased VEGF gene expression levels were detected after exposure to hypoxia. Furthermore, secretion of VEGF protein was significantly enhanced under 0.5% oxygen. Remarkably, functional inactivation of HIF-1 alpha abolished the hypoxic increase of VEGF expression in chondrocytes completely. Furthermore, the soluble isoforms VEGF(120)and VEGF(164)are the most abundantly expressed splice variants in chondrocytes exposed to low oxygen levels. CONCLUSIONS: The data presented here clearly indicate that hypoxia is able to induce the synthesis of soluble VEGF isoforms by epiphyseal chondrocytes, most likely through stabilization of HIF-1 alpha. Thus it can be speculated that HIF-1 alpha is an essential prerequisite for hypoxic VEGF synthesis in the epiphysis, thereby contributing to the formation and invasion of blood vessels in long bone development.

Vascular endothelial growth factor in articular cartilage of healthy and osteoarthritic human knee joints.

OBJECTIVE: To determine the levels of vascular endothelial growth factor (VEGF) mRNA and protein expression in normal and osteoarthritic (OA) human articular cartilage, and whether VEGF expression alters during the progression of OA. METHODS: Sections from normal and OA human knee cartilage were immunotained with a polyclonal antibody recognising VEGF. In addition, total RNA was isolated from normal and osteoarthritic human knee cartilage and analysed by reverse transcriptase-polymerase chain reaction (RT-PCR) for VEGF mRNA expression. RESULTS: VEGF was found to be present in normal and OA human knee cartilage in all cartilage layers. A significant increase of VEGF immunopositive chondrocytes to up to approximately 82% was detected in severe OA cartilage compared with normal articular cartilage (approximately 56% of immunopositive chondrocytes). RT-PCR analysis showed the expression of VEGF also on the mRNA level. CONCLUSIONS: VEGF is expressed by articular chondrocytes in normal and OA human knee cartilage. The percentage of VEGF immunopositive chondrocytes significantly increases in late stages of the disease. The VEGF transcript levels encoding all four isoforms shows a big variability in samples from different donors, suggesting a distinct regulation of the expression of the four VEGF isoforms in normal and OA cartilage.

Neuroendocrine-specific and gastrin-dependent expression of a chromogranin A-luciferase fusion gene in transgenic mice.

BACKGROUND AND AIMS: Chromogranin A (CgA) is a multifunctional acidic protein specifically expressed in neuroendocrine cells. In the stomach, CgA is found predominantly in enterochromaffin-like (ECL) cells, where it is regulated by gastrin. We investigated the ability of a promoter fragment comprising 4.8 kb of 5'-flanking DNA of the mouse CgA (mCgA) gene to direct cell-specific expression as well as gastrin responsiveness in the gastroenteropancreatic neuroendocrine system. METHODS: Two independent lines of mCgA 4.8 kb-luc transgenic mice were created. Transgene expression was assessed by determination of luciferase activity and reverse-transcription polymerase chain reaction analysis of luciferase messenger RNA. Cell specificity of transgene expression was investigated by immunohistochemical analysis. The influence of hypergastrinemia on transgene expression was determined after repeated omeprazole injections. RESULTS: In both transgenic lines, mCgA 4.8 kb-luc expression paralleled the expression pattern of the endogenous CgA gene. ECL cells were identified as the major gastric cell population expressing the transgene. Omeprazole treatment stimulated expression of the transgene and the endogenous CgA gene selectivity in the gastric corpus (3-4-fold). CONCLUSIONS: mCgA 5'-flanking DNA (4.8 kb) contain the major cis-regulatory element(s) required for cell-specific CgA expression in the neuroendocrine system and gastrin-responsiveness in the gastric corpus. Further analysis of the CgA promoter in transgenic studies may elucidate the general molecular mechanisms underlying cell-specific gene expression in the gastroenteropancreatic neuroendocrine system.

Gastrin induces expression and promoter activity of the vesicular monoamine transporter subtype 2.

Gastric enterochromaffin-like cells produce histamine in response to the antral hormone gastrin and accumulate the biogenic amine in secretory organelles via vesicular monoamine transporter subtype 2. The putative effects of gastrin on vesicular monoamine transporter subtype 2 expression and promoter activity are poorly understood. In the present study we used highly enriched rat enterochromaffin-like cells (purity, >90%) and rat pheochromocytoma cells stably transfected with a gastrin/cholecystokinin B receptor to investigate the expression and transcriptional regulation of vesicular monoamine transporter subtype 2. Stimulation of vesicular monoamine transporter subtype 2 mRNA and protein expression was observed in isolated enterochromaffin-like cells after 3- to 7-h incubation with gastrin (10(-7) M), forskolin (10(-5) M), or ionomycin (10(-5) M). Deletion analysis of the rat vesicular monoamine transporter subtype 2 promoter defined the minimal promoter sequence necessary for full basal activity as a -121 bp segment upstream of exon 1 containing two Sp1 sites (-97 to -88 bp and -68 to -59 bp) and a cAMP-responsive element (-44 to -35 bp). Gastrin (10(-7) M) stimulated extracellular signal related kinase1/2 phosphorylation, activated Sp1 and cAMP-responsive element-binding protein, and further induced activity of the complete rat vesicular monoamine transporter subtype 2 promoter (-800 bp) in gastrin/cholecystokinin B receptor cells. The -121-bp fragment was able to confer full gastrin responsiveness, and site-directed mutagenesis of the Sp1 and cAMP-responsive element motifs demonstrated their crucial importance for basal and inducible activities. Comparison of promoter activity of histidine decarboxylase, chromogranin A, or vesicular monoamine transporter subtype 2 in transfected cell lines revealed significant differences in basal and gastrin-stimulated activities. Our current study provides the first evidence that gastrin directly stimulates the expression and promoter activity of vesicular monoamine transporter subtype 2. Sp1 and cAMP-responsive element-binding protein recognition motifs located within 121 bp upstream of exon 1 appear to be indispensable for full basal and inducible promoter activities. Diverging effects of gastrin on histidine decarboxylase, chromogranin A, and vesicular monoamine transporter subtype 2 promoter may account for the coordinated synthesis and storage of histamine in this neuroendocrine cell type.

Dual mechanism of vascular endothelial growth factor upregulation by hypoxia in human hepatocellular carcinoma.

BACKGROUND/AIMS: Vascular endothelial growth factor (VEGF) plays a key role in regulation of tumour associated angiogenesis. In the current study we analysed expression of VEGF and its receptors in human hepatocellular carcinoma (HCC) and investigated the molecular mechanisms of VEGF regulation by hypoxia. METHODS: VEGF, kinase domain region (KDR)/fetal liver kinase 1 (flk-1), and flt-1 expression were examined by immunohistochemistry and in situ hybridisation in 15 human HCC tissues. Expression of VEGF and regulation by hypoxia were assessed in three human HCC cell lines using a quantitative competitive reverse transcription-polymerase chain reaction, ELISA, and a series of 5' deletion reporter gene constructs of the human VEGF promoter in transient transfection assays. RESULTS: We observed over expression of VEGF mRNA and protein in HCC compared with cirrhosis or normal liver. Expression of VEGF in tumour cells was strongly increased in areas directly adjacent to necrotic/hypoxic regions. Both VEGF receptors were detected in vascular endothelia of HCC while only KDR/flk-1 receptors were detected in endothelial cells of cirrhotic livers. Expression of VEGF was observed in all human HCC cell lines examined. Hypoxia (1% oxygen) resulted in profound upregulation of VEGF mRNA and protein levels. Furthermore, hypoxia treatment resulted in a doubling of VEGF mRNA stability. Deletion analysis of the human VEGF 5' flanking region -2018 and +50 demonstrated induction of VEGF promoter activity under hypoxic conditions which was significantly decreased following deletion of the region -1286 and -789 suggesting a substantial contribution of the -975 putative hypoxia inducible factor 1 binding site to hypoxia mediated transcriptional activation of the VEGF gene. CONCLUSION: These data suggest hypoxia as a central stimulus of angiogenesis in human HCC through upregulation of VEGF gene expression by at least two distinct molecular mechanisms: activation of VEGF gene transcription and an increase in VEGF mRNA stability.

Cloning of a neonatal calcium atpase isoform (SERCA 1B) from extraocular muscle of adult blue marlin (Makaira nigricans).

Complete cDNAs for the fast-twitch Ca2+ -ATPase isoform (SERCA 1) were cloned and sequenced from blue marlin (Makaira nigricans) extraocular muscle (EOM). Complete cDNAs for SERCA 1 were also cloned from fast-twitch skeletal muscle of the same species. The two sequences are identical over the coding region except for the last five codons on the carboxyl end; EOM SERCA 1 cDNA codes for 996 amino acids and the fast-twitch cDNAs code for 991 aa. Phylogenetic analysis revealed that EOM SERCA 1 clusters with an isoform of Ca2+ -ATPase normally expressed in early development of mammals (SERCA 1B). This is the first report of SERCA 1B in an adult vertebrate. RNA hybridization assays indicate that 1B expression is limited to extraocular muscles. Because EOM gives rise to the thermogenic heater organ in marlin, we investigated whether SERCA 1B may play a role in heat generation, or if 1B expression is common in EOM among vertebrates. Chicken also expresses SERCA 1B in EOM, but rat expresses SERCA 1A; because SERCA 1B is not specific to heater tissue we conclude it is unlikely that it plays a specific role in intracellular heat production. Comparative sequence analysis does reveal, however, several sites that may be the source of functional differences between fish and mammalian SERCAs.

De novo expression of vascular endothelial growth factor in human pancreatic cancer: evidence for an autocrine mitogenic loop.

BACKGROUND & AIMS: The role of vascular endothelial growth factor (VEGF) and its receptors in tumor angiogenesis has been well established. We analyzed the expression pattern and biologic significance of VEGF and its receptors in human pancreatic cancer. METHODS: VEGF, KDR/flk-1, and flt-1 expression were examined by immunohistochemistry, in situ hybridization, reverse-transcription polymerase chain reaction, enzyme-linked immunosorbent assay, and receptor phosphorylation. VEGF-stimulated mitogenesis was investigated by mitogen-activated protein kinase (MAPK) phosphorylation, transactivation of a c-fos promoter reporter construct, DNA synthesis assays, and stable transfection of a dominant-negative flk-1 complementary DNA (cDNA) construct. RESULTS: Compared with normal pancreas and chronic pancreatitis, VEGF and its receptors were overexpressed in pancreatic cancer. KDR and flt-1 were detected not only in endothelial cells but also in tumor cells. VEGF expression was observed in all human pancreatic tumor cell lines examined, and the KDR/flk-1 and flt-1 receptor was detected in 2 cell lines. VEGF treatment results in phosphorylation of MAPKs, transactivation of a c-fos promoter construct, and growth stimulation in KDR/flk-1-expressing cell lines, which could be blocked by VEGF antagonists. Furthermore, stable transfection of a dominant-negative flk-1 cDNA significantly inhibited tumor cell growth. CONCLUSIONS: These results not only support the important role of the VEGF/VEGF receptor system in pancreatic tumor biology but also suggest the existence of an autocrine/paracrine mitogenic loop for pancreatic cancer cells.