ABSTRACT
During metastasis, malignant cells escape the primary tumor, intravasate lymphatic vessels, and reach draining sentinel lymph nodes before they colonize distant organs via the blood circulation. Although lymph node metastasis in cancer patients correlates with poor prognosis, evidence is lacking as to whether and how tumor cells enter the bloodstream via lymph nodes. To investigate this question, we delivered carcinoma cells into the lymph nodes of mice by microinfusing the cells into afferent lymphatic vessels. We found that tumor cells rapidly infiltrated the lymph node parenchyma, invaded blood vessels, and seeded lung metastases without involvement of the thoracic duct. These results suggest that the lymph node blood vessels can serve as an exit route for systemic dissemination of cancer cells in experimental mouse models. Whether this form of tumor cell spreading occurs in cancer patients remains to be determined.
Subject(s)
Blood Vessels/pathology , Lymph Nodes/blood supply , Lymph Nodes/pathology , Lymphatic Metastasis/pathology , Neoplasm Seeding , Neoplastic Cells, Circulating/pathology , Animals , Cell Line, Tumor , Female , Humans , Lung/pathology , Mammary Neoplasms, Experimental , Mice , Neoplasms, Experimental , Thoracic Duct/pathologyABSTRACT
Sepsis is a life threatening condition that arises when the body's response to an infection injures its own tissues and organs. Sepsis can lead to shock, multiple organ failure and death especially if not recognized early and treated promptly. Molecular mechanisms underlying the systemic inflammatory response syndrome associated with sepsis are still not completely defined and most therapies developed to target the acute inflammatory component of the disease are insufficient. In this study we investigated a possibility of combating sepsis in a mouse model by intravenous treatment with recombinant human tissue non-specific alkaline phosphatase (rhTNAP) derived from transgenic rabbit milk. We induced sepsis in mice by intraperitoneal injection of LPS and three hours later treated experimental group of mice by intravenous injection with rhTNAP derived from transgenic rabbits. Such treatment was proved to be physiologically effective in this model, as administration of recombinant rhTNAP successfully combated the decrease in body temperature and resulted in increased survival of mice (80 % vs. 30 % in a control group). In a control experiment, also the administration of bovine intestinal alkaline phosphatase by intravenous injection proved to be effective in increasing survival of mice treated with LPS. Altogether, present work demonstrates the redeeming effect of the recombinant tissue non-specific AP derived from milk of genetically modified rabbits in combating sepsis induced by LPS.
Subject(s)
Alkaline Phosphatase/therapeutic use , Lipopolysaccharides/toxicity , Sepsis/chemically induced , Sepsis/drug therapy , Animals , Animals, Genetically Modified , Cattle , Female , Humans , Male , Mice , Mice, Inbred BALB C , Pilot Projects , Rabbits , Recombinant Proteins/therapeutic use , Sepsis/mortality , Survival Rate/trendsABSTRACT
BACKGROUND: Organs intended for transplantation are generally stored in the cold for better preservation of their function. However, following transplantation and reperfusion, the microvasculature of transplanted organs often proves to be activated. Extensive leukocyte adhesion and microthrombus formation contribute to failure of the transplanted organ. OBJECTIVES: In this study we analyzed cold-induced changes to the activation status of cultured endothelial cells, possibly contributing to organ failure. METHODS: We exposed human umbilical vein endothelial cells (HUVECs) to temperatures below 37 °C (mostly to 8 °C) for 30 min and upon rewarming to 37 °C kept incubating them for up to 24 h. We also in vivo locally exposed mice to cold. RESULTS: The exposure to low temperatures induced, in HUVECs, expression of the prothrombotic factors plasminogen activator inhibitor-1 (PAI-1) and tissue factor (TF) and of the inflammatory adhesion molecules, E-selectin, intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Furthermore, upon rewarming for 30 min, we detected activation of the inflammatory NF-κB pathway, as measured by transient NF-κB translocation to the nucleus and IκBα degradation. Using butylated hydroxytoluene (BHT), a scavenger of reactive oxygen species (ROS), we further demonstrated that cold-induced NF-κB activation depends on ROS production. Local exposure to cold also, in vivo, induced ROS production and ICAM-1 expression and resulted in leukocyte infiltration. CONCLUSIONS: Our results point to a causative link between ROS production and NF-κB activation, suppression of which had been shown to be beneficial during hypothermic storage and subsequent rewarming of organs for transplantation.
Subject(s)
Cold Temperature , Endothelium, Vascular/metabolism , Inflammation/metabolism , NF-kappa B/metabolism , Reactive Oxygen Species/metabolism , Animals , Base Sequence , Cell Adhesion Molecules/metabolism , Cells, Cultured , DNA Primers , Endothelium, Vascular/cytology , Flow Cytometry , Humans , Male , Mice , Mice, Inbred C57BL , Real-Time Polymerase Chain ReactionABSTRACT
Sepsis is still a major burden for our society with high incidence of morbidity and mortality each year. Molecular mechanisms underlying the systemic inflammatory response syndrome (SIRS) associated with sepsis are still ill defined and most therapies developed to target the acute inflammatory component of the disease are insufficient. Recently the role of nuclear receptors (NRs) became a major topic of interest in transcriptional regulation of inflammatory processes. Nuclear receptors, such as the peroxisome proliferators-activated receptors (PPARs), have been demonstrated to exert anti-inflammatory properties by interfering with the NFκB pathway. We identified the nuclear envelope protein, interferon stimulated gene 12 (ISG12), which directly interacts with NRs. ISG12 is a co-factor stimulating nuclear export of NRs, thereby reducing the anti-inflammatory potential of NRs such as NR4A1. To examine the role of ISG12 in acute inflammatory processes we used recently generated ISG12 deficient mice. We can clearly demonstrate that lack of ISG12 prolongs survival in experimental sepsis and endotoxemia. Furthermore we can show that several acute inflammatory parameters, such as systemic IL6 cytokine levels, are downregulated in septic ISG12-/- animals. Consistently, similar results were obtained in in vitro experiments in peritoneal macrophages derived from ISG12 deficient mice. In contrast, mice deficient for the nuclear receptor NR4A1 exhibited an exacerbated innate immune response, and showed a significantly higher mortality after lethal endotoxemic challenge. This dramatic phenotype could be restored in ISG12/NR4A1 double deficient mice. We conclude from our data in vitro and in vivo that ISG12 is a novel modulator of innate immune responses regulating anti-inflammatory nuclear receptors such as NR4A1.
Subject(s)
Macrophages, Peritoneal/immunology , Nuclear Receptor Subfamily 4, Group A, Member 1/metabolism , Proteins/metabolism , Sepsis/immunology , Animals , Cell Line , Disease Models, Animal , Gene Expression Regulation/genetics , Humans , Immunity, Innate , Immunomodulation , Interleukin-6/genetics , Interleukin-6/metabolism , Lipopolysaccharides/immunology , Mice , Mice, Knockout , Nuclear Receptor Subfamily 4, Group A, Member 1/genetics , Nuclear Receptor Subfamily 4, Group A, Member 1/immunology , Proteins/genetics , Proteins/immunologyABSTRACT
OBJECTIVES: Members of the glycoprotein 130 (gp130) receptor-gp130 ligand family play a role in angiogenesis in different tissues. We tested the effect of this cytokine family on the angiopoietin (Ang)-Tie system, which is involved in blood vessel maturation, stabilization, and regression. RESULTS: Oncostatin M (OSM) increased Ang2 expression in human umbilical vein endothelial cells via Janus kinase/signal transducer and activator of transcription (JAK/STAT) and mitogen-activated protein (MAP) kinase activation. Furthermore, OSM induced Ang2 expression in macrovascular endothelial cells isolated from the human aorta and in microvascular endothelial cells isolated from human heart. Our in vivo experiments revealed that mRNA expression of Ang2 in hearts of mice injected with OSM increased significantly, and levels of OSM mRNA significantly correlated with mRNA levels of Ang2 in human hearts. In addition, OSM increased the expression of its own receptors, gp130 and OSM receptor, in endothelial cells in vitro and in mice in vivo, and levels of OSM mRNA significantly correlated with mRNA levels of gp130 and OSM receptor in human hearts. CONCLUSION: Our data, showing the effects of OSM on the Ang-Tie system in endothelial cells, in hearts of mice, and in human heart tissue, provide yet another link between inflammation and angiogenesis.
Subject(s)
Angiopoietin-2/metabolism , Endothelial Cells/metabolism , Inflammation Mediators/metabolism , Oncostatin M/metabolism , Angiopoietin-2/genetics , Animals , Cells, Cultured , Coronary Vessels/immunology , Coronary Vessels/metabolism , Cytokine Receptor gp130/metabolism , Endothelial Cells/immunology , Humans , Inflammation Mediators/administration & dosage , Injections, Intraperitoneal , Janus Kinases/metabolism , Ligands , Male , Mice , Mice, Inbred C57BL , Mitogen-Activated Protein Kinases/metabolism , Oncostatin M/administration & dosage , Oncostatin M Receptor beta Subunit/metabolism , RNA, Messenger/metabolism , Recombinant Proteins/metabolism , STAT Transcription Factors/metabolism , Signal Transduction , Time Factors , Tissue Culture Techniques , Umbilical Veins/immunology , Umbilical Veins/metabolism , Up-RegulationABSTRACT
The mouse has become the preferred animal for genetic manipulations. Because of the diverse genetic backgrounds of various mouse strains, these can manifest strikingly different characteristics. Here, we studied the functional properties of currents through voltage-gated sodium channels in primary cultures of skeletal myocytes and cardiomyocytes derived from the three commonly used mouse strains BL6, 129/Sv, and FVB, by using the whole-cell patch-clamp technique. We found strain-specific sodium current function in skeletal myocytes, which could partly be explained by differences in sodium channel isoform expression. In addition, we found significant effects of cell source (neonatal or adult animal-derived) and variation of the differentiation time period. In contrast to skeletal myocytes, sodium current function in cardiomyocytes was similar in all strains. Our findings are relevant for the design and proper interpretation of electrophysiological studies, which use excitable cells in primary culture as a model system.
Subject(s)
Muscle Fibers, Skeletal/physiology , Myocytes, Cardiac/physiology , Sodium Channels/physiology , Aging , Animals , Animals, Newborn , Cells, Cultured , Female , Ion Channel Gating/physiology , Mice , Patch-Clamp Techniques , Species SpecificityABSTRACT
Stromal derived factor 1 (SDF-1) is a CXC chemokine important in the homing process of stem cells to injured tissue. It has been implicated in healing and tissue repair. Growing evidence suggests that the glycoprotein-130 (gp130) ligand family is involved in repair processes in the heart. The aim of our study was to determine whether gp130 ligands could affect SDF-1 expression in cardiac cells. Human adult cardiac myocytes (HACMs) and fibroblasts (HACFs) were treated with gp130 ligands. Protein and mRNA levels of SDF-1 were determined using ELISA and RT-PCR, respectively. mRNA levels of SDF-1 were determined in human and mouse heart samples by RT-PCR. HACMs and HACFs constitutively express SDF-1, which was significantly up-regulated by the gp130 ligand oncostatin M (OSM). This effect was counteracted by a p38 inhibitor and to a lesser extent by a PI3K inhibitor. mRNA expression of SDF-1 in hearts of mice injected with OSM increased significantly. Levels of OSM and SDF-1 mRNA correlated significantly in human failing hearts. Our data, showing that OSM induces SDF-1 protein secretion in human cardiac cells in vitro and murine hearts in vivo, suggest that OSM via the induction of SDF-1 might play a key role in repair and tissue regeneration.
Subject(s)
Chemokine CXCL12/metabolism , Inflammation/metabolism , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Oncostatin M/metabolism , Adult , Animals , Cells, Cultured , Chemokine CCL1/genetics , Chemokine CCL1/metabolism , Chemokine CCL5/genetics , Chemokine CCL5/metabolism , Fibroblasts/drug effects , Fibroblasts/metabolism , Flavonoids/pharmacology , Humans , Ligands , Male , Mice , Mice, Inbred C57BL , Oncostatin M/administration & dosage , Oncostatin M/genetics , Time Factors , Up-RegulationABSTRACT
OBJECTIVES: It is believed that adipose tissue acts as an endocrine organ by producing inflammatory mediators and thereby contributes to the increased cardiovascular risk seen in obesity. A link between adipose tissue mass and angiogenesis has been suggested. Vascular endothelial growth factor (VEGF) seems to be implicated in this process. Members of the glycoprotein (gp)130 ligand family regulate VEGF expression in other cells. METHODS AND RESULTS: We used tissue explants as well as primary cultures of preadipocytes and adipocytes from human subcutaneous and visceral adipose tissue to investigate whether the gp130 ligands oncostatin M (OSM), interleukin-6 (IL-6), leukemia inhibitory factor (LIF), and cardiotrophin-1 (CT-1) regulate VEGF expression in human adipose tissue. Human subcutaneous and visceral adipose tissue responded to treatment with IL-6 and OSM with a significant increase in VEGF production. Human preadipocytes were isolated from subcutaneous and visceral adipose tissue. Adipocyte-differentiation was induced by hormone-supplementation. All cell types responded to IL-6 and OSM with a robust increase in VEGF protein production and a similar increase in VEGF-specific mRNA. Furthermore, IL-1beta synergistically enhanced the effect of OSM on VEGF production. AG-490, a JAK/STAT inhibitor, abolished the OSM-dependent VEGF induction almost completely. In mice, IL-6 and OSM increased serum levels of VEGF and VEGF mRNA and vessel density in adipose tissue. CONCLUSION: We speculate that the inflammatory cytokines IL-6 and OSM might support angiogenesis during adipose tissue growth by upregulating VEGF.
Subject(s)
Adipocytes/metabolism , Cytokine Receptor gp130/metabolism , Interleukin-6/pharmacology , Oncostatin M/pharmacology , Vascular Endothelial Growth Factors/drug effects , Adipocytes/drug effects , Animals , Antigens, CD34/metabolism , Cells, Cultured , Humans , In Vitro Techniques , Inflammation Mediators/metabolism , Mice , Models, Animal , RNA, Messenger/analysis , Sensitivity and Specificity , Up-Regulation , Vascular Endothelial Growth Factors/metabolismABSTRACT
We described a procedure for multiple genotype analysis (determination of sex and of three genetic markers) from a single cell derived from bovine preimplantation embryo. It consists of primer extension preamplification-polymerase chain reaction (PEP-PCR) and subsequent single assay or multiplex PCR. A single blastomere that was isolated by microaspiration from bovine embryos at the 16- to 32-cell stage then was lysed and was subjected to the PEP-PCR. When testing 75 embryos, efficiency of genotyping by standard PCR for kappa-casein, growth hormone (GH) and prolactin (PRL) polymorphic alleles was 91, 88 and 89%, respectively. Sexing efficiency in the multiplex PCR was 91%, based on the amplification of Y-specific locus using kappa-casein internal standard. The microaspiration of a single blastomere was shown not to be invasive for the embryos. It did not alter their development potential in vitro (P > 0.05), as was seen by obtaining a similar percentage of embryos developing further into the blastocyst stage in the group subjected to biopsy (44/75, 59%) and in the control group of embryos (30/50, 60%).
Subject(s)
Blastomeres/physiology , Cattle/genetics , Polymerase Chain Reaction/veterinary , Sex Determination Analysis/veterinary , Animals , Caseins/analysis , Caseins/genetics , Cattle/physiology , Female , Fertilization in Vitro/veterinary , Genetic Markers , Genotype , Growth Hormone/analysis , Growth Hormone/genetics , Male , Polymorphism, Restriction Fragment Length , Prolactin/analysis , Prolactin/genetics , Sex Determination Analysis/methods , Y Chromosome/geneticsABSTRACT
Protein C inhibitor (PCI) is a nonspecific, heparin-binding serpin (serine protease inhibitor) that inactivates many plasmatic and extravascular serine proteases by forming stable 1:1 complexes. Proteases inhibited by PCI include the anticoagulant activated protein C, the plasminogen activator urokinase, and the sperm protease acrosin. In humans PCI circulates as a plasma protein but is also present at high concentrations in organs of the male reproductive tract. The biological role of PCI has not been defined so far. However, the colocalization of high concentrations of PCI together with several of its target proteases in the male reproductive tract suggests a role of PCI in reproduction. We generated mice lacking PCI by homologous recombination. Here we show that PCI(-/-) mice are apparently healthy but that males of this genotype are infertile. Infertility was apparently caused by abnormal spermatogenesis due to destruction of the Sertoli cell barrier, perhaps due to unopposed proteolytic activity. The resulting sperm are malformed and are morphologically similar to abnormal sperm seen in some cases of human male infertility. This animal model might therefore be useful for analyzing the molecular bases of these human conditions.
Subject(s)
Gene Deletion , Infertility, Male , Protein C Inhibitor/genetics , Protein C Inhibitor/metabolism , Spermatogenesis , Animals , Disease Models, Animal , Female , Fertilization , Fertilization in Vitro , Gene Targeting , Histocytochemistry , Male , Mice , Mice, Knockout , Microscopy, Electron , Oligopeptides/metabolism , Ovary , Protein C Inhibitor/deficiency , RNA, Messenger/analysis , RNA, Messenger/genetics , Serine Endopeptidases/metabolism , Spermatozoa/metabolism , Spermatozoa/pathology , Testis/enzymology , Testis/metabolism , Testis/pathologyABSTRACT
Protein C inhibitor is a member of the serpin family that inhibits a variety of serine proteases. Protein C inhibitor is present in numerous body fluids and is produced in the liver and by various epithelial cells. To determine if this epithelial serpin is present in skin, immunohistochemical studies were performed that showed strong staining for protein C inhibitor antigen in the epidermis. Protein C inhibitor mRNA was detected in the keratinocyte cell line HaCaT and the epidermoid carcinoma cell line A431 using reverse transcription-polymerase chain reaction suggesting that also in normal skin protein C inhibitor is derived from keratinocytes. Conditioned media from these cell lines were analyzed on immunoblots, which revealed a protein C inhibitor-antigen band that comigrated with protein C inhibitor derived from the hepatoma cell line HepG2. Using an enzyme-linked immunosorbent assay specific for total protein C inhibitor antigen the accumulation of protein C inhibitor in the cell culture supernatants of HaCaT keratinocytes was found to be 0.3 ng per h per 1 million cells. This is similar to the amount of plasminogen activator inhibitor-1 produced by these cells, which also produce tissue plasminogen activator and urokinase. Fluorescence-activated cell sorter analysis revealed similar expression of intracellular protein C inhibitor antigen in proliferating and confluent HaCaT cells. These findings demonstrate that protein C inhibitor antigen is present in the normal epidermis and that protein C inhibitor is constitutively expressed by keratinocytes in culture. Therefore, protein C inhibitor may provide protease inhibitory activity not only to internal, but also to the external surface of the body. Additionally, protein C inhibitor could contribute to the regulation of retinoid supply in the epidermis, as we have shown recently that retinoic acid binds specifically to protein C inhibitor.
Subject(s)
Keratinocytes/chemistry , Protein C Inhibitor/analysis , Skin/chemistry , Cell Line , Culture Media, Conditioned/chemistry , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Gene Expression , Humans , Immunohistochemistry , Keratinocytes/cytology , Keratinocytes/metabolism , Plasminogen Activator Inhibitor 1/analysis , Protein C Inhibitor/genetics , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Skin/cytology , Skin/metabolism , Tissue Plasminogen Activator/analysis , Tumor Cells, Cultured , Urokinase-Type Plasminogen Activator/analysisABSTRACT
Impaired fibrinolysis due to increased plasminogen activator inhibitor-1 (PAI-1) is observed in up to 40% of patients with venous thromboembolism and might be causally related to the disease. There is evidence that genetic variations in the promoter of the PAI-1 gene and metabolic factors contribute to increased plasma PAI-1 levels. A single nucleotide insertion/deletion (4G/5G) polymorphism in the promoter region of the PAI-1 gene and metabolic factors were studied in 158 unrelated patients below the age of 61 years (43 +/- 11 years, mean +/- standard deviation) with history of objectively confirmed venous thromboembolism and in 145 apparently healthy controls. Patients had on average two times higher PAI activity (11.9 vs. 6.1 IU/ml) and by 40% higher PAI-1 antigen (14.8 vs. 10.7 ng/ml) than healthy controls, and also higher body mass index, lipid levels, fasting glucose and insulin. Patients differed significantly from healthy controls neither in the frequency of the 4G and 5G alleles (0.57/0.43 in patients and 0.52/0.48 in controls) nor in the distribution of the 4G/5G genotypes. Possession of the 4G/4G or the 4G/5G genotype did not increase relative risk for venous thromboembolic disease and the distribution of the 4G/5G genotypes was neither associated with recurrent nor with spontaneous disease. In patients association between the 4G/5G genotypes and PAI activity (adjusted for body mass index, triglyceride and glucose level) was observed, with the highest PAI activity values in the 4G/4G genotype (14.6 IU/ml), intermediate in the 4G/5G genotype (13.3 IU/ml) and the lowest in the 5G/5G genotype (5.2 IU/ml, all values means). Association between PAI activity and triglyceride level was the strongest in the 4G/4G genotype (correlation coefficient r = 0.47, p < 0.01) and the weakest in the 5G/5G genotype (r = -0.04, not significant). In conclusion, the present case-control study shows an association between the 4G/5G polymorphism in the promoter of the PAI-1 gene and plasma PAI-1 levels in patients with venous thromboembolism. Similar distribution of the 4G/5G genotypes in patients and healthy controls suggests that this genetic variation by itself is not a major risk factor for venous thromboembolism.
Subject(s)
Plasminogen Activator Inhibitor 1/genetics , Polymorphism, Genetic , Thrombophlebitis/genetics , Adult , Humans , Middle Aged , Plasminogen Activator Inhibitor 1/blood , Promoter Regions, Genetic , Risk Factors , Thrombophlebitis/bloodABSTRACT
The gene encoding mouse protein C inhibitor (mPCI) was isolated and its nucleotide sequence determined. Alignment of the genomic sequence with that of a cDNA obtained from mouse testis revealed that the mPCI gene (like the human counterpart) is composed of five exons and four introns with highly conserved exon/intron boundaries. It encodes a pre-polypeptide of 405 amino acids, which shows 63% identity with human PCI (hPCI). The putative reactive site is identical to that of hPCI from P5 to P3', suggesting a similar protease specificity. Also the putative heparin binding sites and 'hinge' regions are highly homologous in mouse and hPCI.
Subject(s)
Protein C Inhibitor/genetics , Protein C Inhibitor/metabolism , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Cloning, Molecular , Humans , Mice , Molecular Sequence Data , Restriction Mapping , Sequence Analysis, DNA , Sequence Homology, Amino AcidSubject(s)
Fertilization in Vitro , Protein C Inhibitor/pharmacology , Serine Proteinase Inhibitors/pharmacology , Animals , Female , Male , Mice , Mice, Inbred BALB C , Protein C Inhibitor/biosynthesis , Protein C Inhibitor/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Serine Proteinase Inhibitors/biosynthesis , Serine Proteinase Inhibitors/genetics , Sperm-Ovum Interactions/drug effects , Sperm-Ovum Interactions/genetics , Sperm-Ovum Interactions/physiologyABSTRACT
PCI is a non-specific serpin that inhibits several proteases of the coagulation and fibrinolytic systems as well as plasma- and tissue kallikreins and the sperm protease acrosin. The precise physiological role of PCI has not been defined yet. Heparin stimulates most PCI/protease reactions, but interferes with the tissue kallikrein/PCI-interaction. Thereby heparin not only regulates PCI-activity but also its specificity in systems containing two or more of its target proteases. This effect is not restricted to heparin, but is also seen with other glycosaminoglycans (GAGs) and large, negatively charged molecules. PCI also binds to GAGs present on the surface of epithelial kidney cells, and GAGs isolated from these cells have a similar effect on PCI activity as heparin. Studies analyzing the role of PCI as an acrosin inhibitor revealed that endogenous PCI is immunocytochemically localized to disrupted acrosomal membranes of morphologically abnormal sperms, while intact sperms are negative for PCI-antigen. In a mouse in vitro fertilization model human PCI inhibited sperm/egg binding and decreased the fertilization rate. Northern blotting of human and mouse mRNA using human and mouse PCI-cDNA probes revealed that in the mouse PCI is exclusively synthesized in the genital tract (testis, seminal vesicle, ovary), while in humans PCI is additionally synthesized in many other organs (e.g., liver, pancreas, heart). Therefore PCI might regulate enzymes involved in fertilization (e.g. acrosin) in both species. Other proteases (e.g., tissue kallikrein) are possibly regulated in a species specific manner by different inhibitors.
Subject(s)
Protein C Inhibitor/metabolism , Protein C Inhibitor/physiology , Serine Proteinase Inhibitors/metabolism , Serine Proteinase Inhibitors/physiology , Animals , HumansABSTRACT
Purified annexin VI migrates as a closely spaced doublet when separated by SDS-PAGE. Immunolocalization of annexin VI in heart demonstrates staining at different defined subcellular compartments. Moss et al. identified two cDNAs, one having an insert of 18 bases encoding VAAEIL at the beginning of repeat domain seven. We have identified the splicing site of the murine annexin VI gene. It contains a single small exon of 18 bases. PCR amplification of reverse transcribed (RT) mRNA demonstrates that, in all tissues tested, the mRNA isoform containing the insert is predominant. Site-directed antibody was produced and affinity purified against peptides reflecting the insert and deletion sequences. The steady-state isoform ratio of the annexin VI protein is consistent with the RT-PCR data. Chromatographic experiments demonstrate that the annexin VI protein isoforms have biochemical differences. These differences may target the individual isoforms to unique cellular compartments or alter functional properties.
Subject(s)
Annexin A6/metabolism , Myocardium/metabolism , Amino Acid Sequence , Animals , Annexin A6/chemistry , Annexin A6/genetics , Base Sequence , DNA, Complementary/analysis , Fluorescent Antibody Technique , Gene Expression , Mice , Molecular Sequence Data , Polymerase Chain Reaction , RNA, Messenger/analysisABSTRACT
We have tested the ability of the 5' flanking region of the lactogen-regulated annexin Icp35 gene to drive the expression of the bacterial chloramphenicol acetyltransferase (CAT) gene in T-47D, HBL-100 and HC11 cells. A construct containing 1,400 bp of the 5' flanking region including the putative promoter, and the first two exons and intervening sequence showed lower activity compared with a similar construct containing only 250 bp of the 5' flanking region. This indicated the presence of negative regulatory elements in the distal 5' flanking region. Experiments were done to determine the effect of lactogens (either ovine prolactin, or human growth hormone) on the cp35 promoter using various hormone treatments, growth conditions and serum supplements. We did not observe any effect of hormones on the expression of the cp35/CAT constructs under the conditions tested.
Subject(s)
Annexin A1/biosynthesis , Promoter Regions, Genetic , RNA, Messenger/metabolism , TATA Box , Transfection , Animals , Blotting, Northern , Breast , Cell Line , Chloramphenicol O-Acetyltransferase/biosynthesis , Columbidae , Exons , Female , Gene Expression Regulation/drug effects , Growth Hormone/pharmacology , Humans , Introns , Prolactin/pharmacology , Recombinant Proteins/biosynthesis , Restriction MappingABSTRACT
1. Fibre type composition and phosphate metabolites were studied in m. longissimus thoracis (MLT), m. rectus femoris (MRF) and m. triceps brachii (MTB) in trained (N = 6) and sedentary (N = 6) pigs. 2. Samples were analyzed histochemically and by means of in vitro 31P NMR spectroscopy. 3. Training (duration 11 weeks) consisted of treadmill running at a speed of 1.1 m/sec. The daily exercise time of trained animals gradually increased from 10 min during the very first days to 60 min at the end of the 4th week. 4. During the final 7 weeks exercise time remained unchanged. Sedentary animals were not subjected to training. 5. A higher proportion of type beta R fibres in MRF, MTB and MLT and a lower proportion of type alpha W fibres were found in the trained group of animals compared to the control group. 6. In MLT no significant differences in the proportion of type alpha W were observed between both groups. 7. No significant differences in average fibre diameter of muscle fibres were found between groups. 8. No differences in concentration of phosphate compounds were observed between trained and sedentary groups. 9. Muscles with a higher proportion of type IIb fibres in both groups of pigs contained higher amounts of phosphocreatine (PCr) and were also characterized by a higher ratio of PCr to inorganic phosphate (Pi).
Subject(s)
Muscles/metabolism , Phosphates/metabolism , Physical Conditioning, Animal , Swine/metabolism , Analysis of Variance , Animals , Magnetic Resonance Spectroscopy , Muscles/cytology , RestABSTRACT
31P NMR spectroscopy was used to evaluate interspecies differences in muscle fibre types and related postmortem metabolism. M. longissimus thoracis (MLT) and m. pectoralis superficialis (MPS) of bulls and MLT of pigs were investigated. In perchloric acid extracts NMR resonances for sugar phosphates (SP), inorganic phosphate (Pi), glycerophosphorylcholine (GPC), phosphocreatine (PCr), adenosine triposphate (ATP), adenosine diphosphate (ADP) as well as for NAD+/NADH could be distinguished. Also, glycogen and lactate contents and pH were determined. The relative contents of phosphorus compounds in bovine muscles of similar participation of muscle fibre are similar. Bovine muscles contain a relatively large proportion of PCr (48% of all phosphates 15 minutes post-mortem in MPS) whereas porcine MLT show lower PCr content (11% 15 minutes post-mortem). On the other hand, the ATP content is relatively higher in porcine MLT when compared with bovine muscles in the early phases of the postmortem processes. No NMR-detectable levels of GPC were measured in porcine MLT in contrast to bovine muscles. This suggests that the GPC content does not depend solely on the fibre participation but is also animal species determined. The 24 hour postmortem metabolism patterns of bovine and porcine muscles have many common traits. CP disappeared first followed by ATP. Simultaneously, the Pi concentrations increased. However, the content of SP remained relatively constant in porcine, but not in bovine muscles where it increased only gradually. The significantly higher concentrations of SP and lactate as well as the lower values of glycogen and pH measured for porcine as compared with bovine muscles suggest an enhanced glycolysis during the early phases of postmortem processes in porcine muscles.