[COMBINED LAPAROSCOPICALLY AND RETROPERITONEOSCOPICALLY ASSISTED PANCREATONECRSEQUESTRECTOMY].

Combined method of laparoscopically and retroperitoneoscopically assisted necrsequestrectomy, consisting of staged application of miniinvasive methods with simultaneous laparoscopic and retroperitoneoscopic control of necrsequestrectomy, was elaborated with the objective to improve surgical treatment of an acute pancreatitits. The procedure has significant advantages over open operative intervention in purulent complications of necrotic purulent pancreatitis: reduction of the local and systemic operative treatment severity, minimization of microbial metabolites coming into the blood, total visual control of intervention, reduction of the vascular injuries risk, аdequate surgical sanation with saving of viable pancreatic parenchyma, absence of conditions for the purulent complications occurrence while the operative wound healing is going on, preservation of possibility for an adequate draining, using drains of a large diameter.

PHD3 regulates differentiation, tumour growth and angiogenesis in pancreatic cancer.

PURPOSE: Tumour hypoxia activates hypoxia-inducible factor-1 (HIF-1) and indluences angiogenesis, cell survival and invasion. Prolyl hydroxylase-3 (PHD3) regulates degradation of HIF-1α. The effects of PHD3 in tumour growth are largely unknown. EXPERIMENTAL DESIGN: PHD3 expression was analysed in human pancreatic cancer tissues and cancer cell lines by real-time quantitative PCR and immunohistochemistry. PHD3 overexpression was established by stable transfection and downregulation by short interfering RNA technology. VEGF was quantified by enzyme-linked immunosorbent assay. Matrigel invasion assays were performed to examine tumour cell invasion. Apoptosis was measured by annexin-V staining and caspase-3 assays. The effect of PHD3 on tumour growth in vivo was evaluated in an established orthotopic murine model. RESULTS: PHD3 was upregulated in well-differentiated human tumours and cell lines, and regulated hypoxic VEGF secretion. PHD3 overexpression mediated tumour cell growth and invasion by induction of apoptosis in a nerve growth factor-dependent manner by the activation of caspase-3 and phosphorylation of focal adhesion kinase HIF-1 independently. In vivo, PHD3 inhibited tumour growth by abrogation of tumour angiogenesis. CONCLUSION: Our results indicate essential functions of PHD3 in tumour growth, apoptosis and angiogenesis and through HIF-1-dependent and HIF-1-independent pathways.

A p47-phox pseudogene carries the most common mutation causing p47-phox- deficient chronic granulomatous disease.

The predominant genetic defect causing p47-phox-deficient chronic granulomatous disease (A47 degrees CGD) is a GT deletion (DeltaGT) at the beginning of exon 2. No explanation exists to account for the high incidence of this single mutation causing a rare disease in an unrelated, racially diverse population. In each of 34 consecutive unrelated normal individuals, both the normal and mutant DeltaGT sequences were present in genomic DNA, suggesting that a p47-phox related sequence carrying DeltaGT exists in the normal population. Screening of genomic bacteriophage and YAC libraries identified 13 p47-phox bacteriophage and 19 YAC clones. The GT deletion was found in 11 bacteriophage and 15 YAC clones. Only 5 exonic and 33 intronic differences distinguished all DeltaGT clones from all wild-type clones. The most striking differences were a 30-bp deletion in intron 1 and a 20-bp duplication in intron 2. These results provide good evidence for the existence of at least one highly homologous p47-phox pseudogene containing the DeltaGT mutation. The p47-phox gene and pseudogene(s) colocalize to chromosome 7q11.23. This close linkage, together with the presence within each gene of multiple recombination hot spots, suggests that the predominance of the DeltaGT mutation in A47 degrees CGD is caused by recombination events between the wild-type gene and the pseudogene(s).

Thrombin activates the hypoxia-inducible factor-1 signaling pathway in vascular smooth muscle cells: Role of the p22(phox)-containing NADPH oxidase.

The heterodimeric transcription factor hypoxia-inducible factor-1 (HIF-1) is activated under hypoxic conditions, resulting in the upregulation of its target genes plasminogen activator inhibitor-1 (PAI-1) and vascular endothelial growth factor (VEGF). PAI-1 and VEGF are also induced in response to vascular injury, which is characterized by the activation of platelets and the coagulation cascade as well as the generation of reactive oxygen species (ROS). However, it is not known whether HIF-1 is also stimulated by thrombotic factors. We investigated the role of thrombin, platelet-associated growth factors, and ROS derived from the p22(phox)-containing NADPH oxidase in the activation of HIF-1 and the induction of its target genes PAI-1 and VEGF in human vascular smooth muscle cells (VSMCs). Thrombin, platelet-derived growth factor-AB (PDGF-AB), and transforming growth factor-beta(1) (TGF-beta(1)) upregulated HIF-1alpha protein in cultured and native VSMCs. This response was accompanied by nuclear accumulation of HIF-1alpha as well as by increased HIF-1 DNA-binding and reporter gene activity. The thrombin-induced expression of HIF-1alpha, PAI-1, and VEGF was attenuated by antioxidant treatment as well as by transfection of p22(phox) antisense oligonucleotides. Inhibition of p38 mitogen-activated protein kinase and phosphatidylinositol-3-kinase significantly decreased thrombin-induced HIF-1alpha, PAI-1, and VEGF expression. These findings demonstrate that the HIF-1 signaling pathway can be stimulated by thrombin and platelet-associated growth factors and that a redox-sensitive cascade activated by ROS derived from the p22(phox)-containing NADPH oxidase is crucially involved in this response.

Efficient translation of mouse hypoxia-inducible factor-1alpha under normoxic and hypoxic conditions.

The heterodimeric hypoxia-inducible factor-1 (HIF-1), consisting of the subunits HIF-1alpha and HIF-1beta/ARNT, is a master transcriptional regulator of oxygen homeostasis. Under hypoxic conditions, HIF-1alpha levels very rapidly increase, mostly due to protein stabilization. However, translational regulation of HIF-1alpha has not been directly analyzed so far. Mouse HIF-1alpha exists as two mRNA isoforms (termed mHIF-1alphaI.1 and mHIF-1alphaI. 2) containing structurally different 5'-termini which might modulate translation initiation. Whereas the in vitro translation efficiency of these two mRNA isoforms was about equal, the mHIF-1alphaI.2 5'-untranslated region (5'-UTR) conferred significantly higher in vivo luciferase reporter gene activity than the mHIF-1alphaI.1 5'-UTR. Similar corresponding luciferase mRNA levels indicate translational rather than transcriptional alterations. Reporter gene expression was not affected upon exposure of transiently transfected cells to hypoxia (1% oxygen). Direct assessment of translational regulation by polysomal profile analysis of HeLaS3 cells showed that HIF-1alpha (and to a lower extent ARNT) mRNA was found mainly in the translationally active polyribosomal fractions under both normoxic and hypoxic conditions. In contrast, the association of mRNAs for beta-actin and ribosomal protein L28 with the polyribosomal fractions was substantially reduced under hypoxic conditions, suggesting decreased overall protein synthesis. Thus, efficient translation of mouse HIF-1alpha in a situation where the general translation efficiency is reduced represents a prerequisite for the very rapid accumulation of HIF-1alpha protein upon exposure to hypoxia.

Thrombin causes vascular endothelial growth factor expression in vascular smooth muscle cells: role of reactive oxygen species.

Vascular endothelial growth factor (VEGF) has been implicated in the reendothelialization of the vascular wall after balloon injury. This study investigated whether thrombin, which is formed during activation of the coagulation cascade at sites of vascular injury, upregulates VEGF expression in vascular smooth muscle cells (VSMCs). VEGF expression was assessed in native and cultured VSMCs by Northern blot analysis and reverse transcription-polymerase chain reaction and the release of VEGF protein by immunoassay. alpha-Thrombin time- and concentration-dependently increased VEGF mRNA levels, mainly that mRNA coding for the soluble splice variant VEGF(164/165), and stimulated the release of VEGF protein. These effects required the proteolytic activity of thrombin and were mimicked by a thrombin receptor activating-peptide. Upregulation of VEGF expression was also induced by conditioned medium from alpha-thrombin-stimulated VSMCs. Both the early and the delayed alpha-thrombin-induced VEGF expressions were attenuated by antioxidants and by diphenyleneiodonium. alpha-Thrombin-induced VEGF release was significantly reduced by a platelet-derived growth factor (PDGF)-, a transforming growth factor (TGF)-beta-, and a basic fibroblast growth factor (bFGF)-neutralizing antibody. Thrombin caused a redox-sensitive upregulation of expression of VEGF in VSMCs through a direct and an indirect effect, which was dependent on the endogenous formation of PDGF, TGF-beta, and bFGF. Upregulation of VEGF expression may represent an important mechanism by which the coagulation cascade contributes to the regeneration of the endothelial lining at sites of balloon injury.

Effects of cobalt on haem proteins of erythropoietin-producing HepG2 cells in multicellular spheroid culture.

The hypoxia-induced increase of spectrophotometrically measured light absorption at 560 nm, considered as reduced cytochrome b, in HepG2 cells is diminished after exposure to cobalt chloride (50 or 100 microM) for 18-36 h. The redox state of cytochrome c and cytochrome aa3, however, remains stable, indicating a particular affinity of cytochrome b for cobalt. Erythropoietin production of HepG2 cells increases after application of cobalt chloride, whereas H2O2 production, as measured by the dihydrorhodamine technique, decreases. It is concluded that cobalt stimulates a signal cascade with cytochrome b as receptor and H2O2 as second messenger for regulating erythropoietin production.

Thrombin-induced MCP-1 expression involves activation of the p22phox-containing NADPH oxidase in human vascular smooth muscle cells.

UNLABELLED: Activation of vascular smooth muscle cells (VMSC) by thrombin induces the expression of the chemokine, monocyte chemoattractant protein-1 (MCP-1). We investigated in cultured human and rat VSMC whether reactive oxygen species (ROS) derived from the vascular NADPH oxidase contribute to this effect. Exposure of cultured VSMC to thrombin rapidly increased ROS formation, phosphorylation of p38 MAP kinase as well as the expression of MCP-1. Specific inhibition of the p22phox subunit of the vascular NADPH oxidase using either p22phox neutralizing antibody or p22phox antisense oligonucleotides attenuated thrombin-induced ROS generation. Furthermore, thrombin-induced p38 MAP kinase activation as well as MCP-1 expression were impaired by antioxidants as well as by p22phox antisense oligonucleotides. Inhibition of p38 MAP kinase diminished the thrombin-induced expression of MCP-1. CONCLUSION: Thrombin, by activating a p22phox-containing NADPH oxidase, elicits ROS generation and activation of p38 MAP kinase in VSMC. The subsequent induction of MCP-1 expression highligts the crucial role of the p22phox-containing NADPH oxidase in thrombin-induced signal transduction in VSMC.

Changes in growth, po(2) and ph after exposure to oxamate - studies of 2 human tumor-cell lines growing as multicellular spheroids.

The basic metabolism and pO(2) and pH gradients in spheroids were characterized and, in some cases, changed by the addition of the pyruvate analogue oxamate. Two human tumour spheroid types, colon adenocarcinoma HT29 and malignant glioma U118MG, were applied as models. Microelectrode measurements in HT29 spheroids showed steep pO(2) gradients with large differences between surface and center, Delta pO(2), and low central pO(2) values. The HT29 spheroids had rather flat pH gradients. The U118MG spheroids had less steep pO(2) gradients but steeper pH gradients. Determinations of 1-C-14 and 2-C-14 pyruvate oxidation rates, for characterization of the oxidative glucose breakdown as well as of lactate dehydrogenase kinetics, showed consistent results with the microelectrode measurements in that there was high oxidative metabolism in the HT29 spheroids whereas the U118MG spheroids relied more on glycolysis. Western blot investigations of the LDH isoenzyme composition showed different isoenzyme patterns in the two spheroid types with a lack of LDH1 in U118MG spheroids. Addition of 40 mM of oxamate gave decreased 1-C-14- and 2-C-14-pyruvate oxidation rates in the HT29 cells and inhibition of LDH activity in the U118MG cells. Oxamate increased the central pO(2) values in HT29 spheroids and the central pH values in U118MG spheroids. One example of experimental therapy was applied and oxamate acted as a radiation sensitizer in the U118MG system and as a radioprotective substance in the HT29 system. This has to be analysed in more detail.

Visual perception and stimulus orientation in cattle.

The pupil in the eye of adult cattle is oval under contraction with the long axis nearly horizontal. Based on simple optophysical facts it is hypothesised that visual perception in such eyes is different for stimuli with vertically-separated details rather than stimuli with horizontally-separated details. This hypothesis was tested with three adult dairy bulls using an operant conditioning technique. The bulls had to discriminate a solid white line from broken white lines with decreasing interspaces. They solved this task better when the stimuli were presented vertically rather than horizontally. This result is discussed in terms of visual acuity and related to the topographical anatomy of the eye, particularly the pupil.

[Experiences in the changes of biotechnology in veterinary practice]. / Erfahrungen beim Umsetzen von Biotechniken in die tierzüchterische Praxis.

The transmission of old methods and the transformation of new knowledge in to animal breeding practice has an cultural tradition lasting millenniums. Each epoch had its own strategy of solving problems because progress is independent of religions and ideologies, and therefore, it can not be hindered. In the past different biotechnologies have increased progress in animal breeding exponentially, and it can be supposed that the course of the exponential curve will not change in the foreseeable future. Anyone who intends to compete internationally is compelled to use the new technologies, wherever old methods can not longer compete. The transition to the new biotechnology especially in Germany, hits limitations an two levels: on the one hand, an exaggerated desire for safety that has created the German gene law and, on the other hand the animal breeding industry that can hardly afford the expensive biotechnology because of its economic condition. It seems advisable to revise both the gene law and the expense of the technology. Animal breeding research should foster simplification (make less expensive) of biotechnology, and politicians should ask weather it is necessary for the gene sector to migrate further abroad. In animal breeding, the gene transfer is practical (e.g., liposome mediated gene transfer via sperm cells) because of its low costs, and so it is absurd not to allow its use. In Germany, transgenic individuals have to bee discarded at the knackery, whereas in the Netherlands heifers derived from the bull HERMAN, that is transgenic for lactoferrin are allowed to calf (and produce milk). Currently, gene transfer is not a viable alternative because of the negative emotional impact of biotechnology. Only someone who uses this technique may be responsible for handling and creating it without having, therefore, to overstep ethical borders.

The unfolded protein response controls induction and activation of ADAM17/TACE by severe hypoxia and ER stress.

The family of ADAM (a disintegrin and metalloproteinase) proteins has been implicated in tumor initiation and progression. ADAM17/tumor necrosis factor-α (TNFα)-converting enzyme (TACE) has been initially recognized to release TNFα as well as its receptors (TNFRs) from the membrane. ADAM17, TNFα and TNFR have been found upregulated in cancer patients, although the underlying mechanisms remain largely unknown. As hypoxia is a hallmark of cancer that can lead to severe stress conditions accumulating in endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), we investigated the role of these stress conditions in the regulation of ADAM17 and release of TNFR1.We found that severe hypoxia induced ADAM17 expression and activity. Although hypoxia-inducible factor 1α (HIF1α) was important to maintain basal ADAM17 mRNA levels during moderate hypoxia, it was not sufficient to induce ADAM17 levels under severe hypoxia. Instead, we found that ADAM17 induction by severe hypoxia can be mimicked by ER stressors such as Thapsigargin and occurs as a consequence of the activation of the PERK/eIF2α/ATF4 and activating transcription factor 6 (ATF6) arms of UPR in several tumor cell lines. ADAM17 expression was also increased in xenografts displaying ER stress because of treatment with the vascular endothelial growth factor (VEGF) inhibitory antibody Bevacizumab. Additionally, severe hypoxia and ER stress activated ADAM17 and ectodomain shedding of TNFR1 involving mitogen-activated protein (MAP) kinases and reactive oxygen species (ROS). Collectively, these results show that ADAM17 is a novel UPR-regulated gene in response to severe hypoxia and ER stress, which is actively involved in the release of TNFR1 under these conditions. These data provide a novel link between severe hypoxic stress conditions and inflammation in the tumor environment.

NADPH oxidases as a source of oxidative stress and molecular target in ischemia/reperfusion injury.

Ischemia/reperfusion injury (IRI) is crucial in the pathology of major cardiovascular diseases, such as stroke and myocardial infarction. Paradoxically, both the lack of oxygen during ischemia and the replenishment of oxygen during reperfusion can cause tissue injury. Clinical outcome is also determined by a third, post-reperfusion phase characterized by tissue remodeling and adaptation. Increased levels of reactive oxygen species (ROS) have been suggested to be key players in all three phases. As a second paradox, ROS seem to play a double-edged role in IRI, with both detrimental and beneficial effects. These Janus-faced effects of ROS may be linked to the different sources of ROS or to the different types of ROS that exist and may also depend on the phase of IRI. With respect to therapeutic implications, an untargeted application of antioxidants may not differentiate between detrimental and beneficial ROS, which might explain why this approach is clinically ineffective in lowering cardiovascular mortality. Under some conditions, antioxidants even appear to be harmful. In this review, we discuss recent breakthroughs regarding a more targeted and promising approach to therapeutically modulate ROS in IRI. We will focus on NADPH oxidases and their catalytic subunits, NOX, as they represent the only known enzyme family with the sole function to produce ROS. Similar to ROS, NADPH oxidases may play a dual role as different NOX isoforms may mediate detrimental or protective processes. Unraveling the precise sequence of events, i.e., determining which role the individual NOX isoforms play in the various phases of IRI, may provide the crucial molecular and mechanistic understanding to finally effectively target oxidative stress.