Identification and complete genome analysis of a virus variant or putative new foveavirus associated with apple green crinkle disease.

A virus identified as "apple green crinkle associated virus" (AGCaV) was isolated from Aurora Golden Gala apple showing severe symptoms of green crinkle disease. Evidence was obtained of a potential causal relationship to the disease. The viral genome consists of 9266 nucleotides, excluding the poly(A) tail at the 3'-terminus. It has a genome organization similar to that of members of the species Apple stem pitting virus (ASPV), the type species of the genus Foveavirus, family Betaflexiviridae. ORF1 of AGCaV encodes a replicase-complex polyprotein with a molecular mass of 247 kDa; the proteins of ORFs 2, 3, and 4 (TGB proteins) are estimated to be 25.1 kDa, 12.8 kDa, and 7.4 kDa, respectively; and ORF5 encodes the CP, with an estimated molecular mass of 43.3 kDa. Interestingly, AGCaV utilizes different stop codons for ORF1, ORF3, and ORF5 compared to the ASPV type isolate PA66, and between the two viruses, six distinct indel events were observed within ORF5. AGCaV has four non-coding regions (NCRs), including a 5'-NCR (60 nt), a 3'-NCR (134 nt), and two intergenic (IG) NCRs: IG-NCR1 (69 nt) and IG-NCR2 (91 nt). A conserved stable hairpin structure was identified in the variable 5'-NCR of members of the genus Foveavirus. AGCaV may be a variant or strain of ASPV with unique biological properties, but there is evidence that it may be a distinct putative foveavirus.

Erythropoietin and the vascular wall: the controversy continues.

BACKGROUND AND AIMS: Erythropoietin (EPO) stimulates erythropoiesis through its specific receptor (EPO-R). Preclinical work has assigned a role for the EPO/EPO-R system in the heart and blood vessels. The potential use of erythropoiesis-stimulating agents (ESAs) for nonhematopoietic indications is a focus of current research. This article considers proven actions of EPO in the cardiovascular system, with emphasis on the human responses. DATA SYNTHESIS: By use of specific anti-EPO-R antibody no EPO-R protein was detected by Western blotting in normal non-erythroid tissues. Clinical trials failed to demonstrate clear beneficial effects of high-dosed ESAs in patients with coronary syndrome or myocardial infarct. While ESA therapy may lead to an elevation in arterial blood pressure in previously anemic patients, several studies have reported no effects on vessels/blood pressure with ESAs. EPO has been reported to stimulate angiogenesis. EPO-R mRNA is detectable in human vascular endothelium. However, in most vitro studies very high concentrations of EPO were applied and well-designed studies have failed to show direct effects of ESAs on endothelial cells. Whether EPO promotes the mobilization of myeloid progenitor cells into the blood stream still needs to be studied in more detail, as this effect may prove useful for augmenting the neovascularization of ischemic tissues. With respect to the administration of ESAs to tumor patients, a deeper insight into the role of EPO for tumor angiogenesis is desirable. CONCLUSIONS: The enthusiastic reports of the nonhematopoietic cytoprotective potential of EPO and its derivatives in the cardiovascular system have not yet been confirmed in placebo-controlled clinical trials.

Effects of erythropoietin receptors and erythropoiesis-stimulating agents on disease progression in cancer.

Erythropoiesis-stimulating agents (ESAs) increase red blood cell (RBC) production in bone marrow by activating the erythropoietin receptor (EpoR) on erythrocytic-progenitor cells. Erythropoiesis-stimulating agents are approved in the United States and Europe for treating anaemia in cancer patients receiving chemotherapy based on randomised, placebo-controlled trials showing that ESAs reduce RBC transfusions. Erythropoiesis-stimulating agent-safety issues include thromboembolic events and concerns regarding whether ESAs increase disease progression and/or mortality in cancer patients. Several trials have reported an association between ESA use and increased disease progression and/or mortality, whereas other trials in the same tumour types have not provided similar findings. This review thoroughly examines available evidence regarding whether ESAs affect disease progression. Both clinical-trial data on ESAs and disease progression, and preclinical data on how ESAs could affect tumour growth are summarised. Preclinical topics include (i) whether tumour cells express EpoR and could be directly stimulated to grow by ESA exposure and (ii) whether endothelial cells express EpoR and could be stimulated by ESA exposure to undergo angiogenesis and indirectly promote tumour growth. Although assessment and definition of disease progression vary across studies, the current clinical data suggest that ESAs may have little effect on disease progression in chemotherapy patients, and preclinical data indicate a direct or indirect effect of ESAs on tumour growth is not strongly supported.

Complete genome sequences of three Erwinia amylovora phages isolated in north america and a bacteriophage induced from an Erwinia tasmaniensis strain.

Fire blight, a plant disease of economic importance caused by Erwinia amylovora, may be controlled by the application of bacteriophages. Here, we provide the complete genome sequences and the annotation of three E. amylovora-specific phages isolated in North America and genomic information about a bacteriophage induced by mitomycin C treatment of an Erwinia tasmaniensis strain that is antagonistic for E. amylovora. The American phages resemble two already-described viral genomes, whereas the E. tasmaniensis phage displays a singular genomic sequence in BLAST searches.

Erythropoiesis stimulating agents and techniques: a challenge for doping analysts.

Recombinant human erythropoietin (rHuEPO) engineered in Chinese hamster ovary (CHO) cell cultures (Epoetin alfa and Epoetin beta) and its hyperglycosylated analogue Darbepoetin alfa are known to be misused by athletes. The drugs can be detected by isoelectric focusing (IEF) and immunoblotting of urine samples, because "EPO" is in reality a mixture of isoforms and the N-glycans of the recombinant products differ from those of the endogenous hormone. However, there is a plethora of novel erythropoiesis stimulating agents (ESAs). Since the originator Epoetins alfa and beta are no longer protected by patent in the European Union, rHuEPO biosimilars have entered the market. In addition, several companies in Asia, Africa and Latin America produce copied rHuEPOs for clinical purposes. While the amino acid sequence of all Epoetins is identical, the structure of their glycans differs depending on the mode of production. Some products contain more acidic and others more basic EPO isoforms. Epoetin delta is special, as it was engineered by homologous recombination in human fibrosarcoma cells (HT-1080), thus lacking N-glycolylneuraminic acid like native human EPO. ESAs under development include EPO fusion proteins, synthetic erythropoiesis stimulating protein (SEP) and peptidic (Hematide(), CNTO 528) as well as non-peptidic EPO mimetics. Furthermore, preclinical respectively clinical trials have been performed with small orally active drugs that stimulate endogenous EPO production by activating the EPO promoter ("GATA-inhibitors": diazepane derivatives) or enhancer ("HIF-stabilizers": 2-oxoglutarate analogues). The prohibited direct EPO gene transfer may become a problem in sports only in the future.

Dexamethasone impairs hypoxia-inducible factor-1 function.

Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription-factor composed of alpha- and beta-subunits. HIF-1 is not only necessary for the cellular adaptation to hypoxia, but it is also involved in inflammatory processes and wound healing. Glucocorticoids (GC) are therapeutically used to suppress inflammatory responses. Herein, we investigated whether GC modulate HIF-1 function using GC receptor (GR) possessing (HepG2) and GR deficient (Hep3B) human hepatoma cell cultures as model systems. Dexamethasone (DEX) treatment increased HIF-1alpha levels in the cytosol of HepG2 cells, while nuclear HIF-1alpha levels and HIF-1 DNA-binding was reduced. In addition, DEX dose-dependently lowered the hypoxia-induced luciferase activity in a reporter gene system. DEX suppressed the hypoxic stimulation of the expression of the HIF-1 target gene VEGF (vascular endothelial growth factor) in HepG2 cultures. DEX did not reduce hypoxically induced luciferase activity in HRB5 cells, a Hep3B derivative lacking GR. Transient expression of the GR in HRB5 cells restored the susceptibility to DEX. Our study discloses the inhibitory action of GC on HIF-1 dependent gene expression, which may be important with respect to the impaired wound healing in DEX-treated patients.

Activity in fetal bovine serum that stimulates erythroid colony formation in fetal mouse livers is insulinlike growth factor I.

In the present study, the erythropoietic activity of fetal serum was characterized. Using fetal bovine serum (FBS) as a source of the erythropoietic activity and serum-free cultures of fetal mouse livers (FMLC assay) as a detection system, we found that FBS stimulated colony formation from late erythroid progenitor cells (CFU-E) in a dose-dependent fashion. The slope of the dose-response curve, however, was significantly different from that for erythropoietin (Ep), the best-characterized erythropoietic activity so far. The erythropoietic activity of FBS was found in the 120-160- and 40-70-kD range at neutral pH. In the presence of 1 M acetic acid, however, the erythropoietic activity had an apparent molecular mass between 3 and 13 kD. From ion exchange experiments with DEAE-cellulose, the isoionic point of the activity was estimated to about pH 5. Furthermore, the erythropoietic activity of FBS was found to be co-eluted on Sephadex G-150 with the binding proteins of insulinlike growth factors (IGF). The IGF I concentration determined by radioimmunoassay was 70 ng IGF I/ml. The Ep activity of FBS was less than 5 mU/ml when determined with the posthypoxic polycythemic mouse assay for Ep. These results suggest that the erythropoietic activity of FBS is related to IGF and not to Ep. The erythropoietic activity of FBS was abolished by an antiserum against IGF I. Furthermore, IGF I was a factor of approximately 40 more potent than IGF II in stimulating erythroid colony formation. All of these findings suggest that the erythropoietic activity of FBS is IGF I.

Hydroxylation of hypoxia-inducible transcription factors and chemical compounds targeting the HIF-alpha hydroxylases.

The hypoxia-inducible transcription factors (HIFs) are central components in the cellular responses to a lack of O(2), i.e. hypoxia. Homologs of the HIF system (HIF-1, -2 and -3) are detectable in all nucleated cells of multicellular organisms. Active HIFs are heterodimers (HIF-alpha/ beta). In hypoxia the O(2)-labile alpha-subunit is translocated to the nucleus where it binds HIF-beta. Over 100 HIF target genes have already been identified. The translational products of these genes increase O(2) delivery to hypoxic tissues, such as erythropoietin which stimulates the production of red blood cells, and they adapt cellular metabolism to hypoxia, such as glycolytic enzymes. HIFs are inactive in normoxia because of O(2)-dependent enzymatic hydroxylation and subsequent degradation of their alpha-subunit. Three HIF-alpha prolyl hydroxylases (PHD1, 2 and 3) initiate proteasomal degradation while an asparaginyl hydroxylase (factor inhibiting HIF-1, FIH-1) inhibits the function of the C-terminal transactivation domain of HIF-alpha. In addition to O(2) and 2-oxoglutarate, the HIF-alpha hydroxylases require Fe(2+) and ascorbate as co-factors. Products of glycolysis can act as endogenous inhibitors of HIF hydroxylases which may lead to sustained activation of HIFs in cancer cells. The cofactor requirements define the routes to inhibition of the enzymes when HIF activation is desirable. In particular, 2-oxoglutarate analogues have emerged as promising tools for stimulation of erythropoiesis and angiogenesis ("HIF-stabilizers"). However, as the HIF system promotes the transcription of many genes, and other 2-oxoglutarate dependent dioxygenases are likely to be inhibited by the same analogues, careful evaluation of the inhibitors seems mandatory prior to their clinical use.

Net charge and oxygen affinity of human hemoglobin are independent of hemoglobin concentration.

The dependence of net charge and oxygen affinity of human hemoglobin upon hemoglobin concentration was reinvestigated. In contrast to earlier reports from various laboratories, both functional properties of hemoglobin were found to be independent of hemoglobin concentration. Two findings indicate a concentration-independent net charge of carbonmonoxy hemoglobin at pH 6.6: (A) The pH value of a given carbonmonoty hemoglobin solution remains constant at 6.6 when the hemoglobin concentration is raised from 10 to 40 g/dl, indicating that there is no change in protonation of titratable groups of hemoglobin: (b) the net charge of carbonmonoxy hemoglobin as estimated from the Donnan distribution of 22Na+ shows no dependence on hemoglobin concentration in this concentration range. The oxygen affinity of human hemoglobin was determined from measurements of oxygen concentrations in equilibrated samples using a Lex-O2-Con apparatus (Lexington Instruments, Waltham, Mass.). P50 averaged 11.4 mm Hg at 37 degrees C, pH = 7.2, and ionic strength approximately 0.15. Neither P50 nor Hill's n showed any variation with hemoglobin concentrations increasing from 10 to 40 g/dl.

Effects of erythropoietin on brain function.

This article is a selective extension of a review on recombinant human erythropoietin (rHu-EPO) as an anti-anaemic drug, published in this journal in 2000. It summarises the recent advances in understanding the molecular mechanisms by which the hypoxia-inducible transcription factor 1 (HIF-1) regulates O(2)-dependent genes, including the EPO gene in brain. With respect to brain integrity, EPO exerts positive effects in two different ways. First, rHu-EPO raises the blood haemoglobin concentration and, hence, the O(2) capacity of the blood in anaemic patients. The restored O(2) supply ameliorates attention difficulties and psychomotor slowing, improves memory capacities and normalises neuroendocrine functions. Second, EPO can act as a neurotrophic and neuroprotective factor directly in brain. EPO and its receptor are expressed in the cerebral cortex, cerebellum, hippocampus, pituitary gland and spinal cord. In vitro EPO protects against glutamate-induced cell death in a dose-dependent way. In animal models it reduces volumes of brain ischaemia, protects the cortex from hypoxic damage and leads to survival of neurons and synapses. One can expect that in the near future rHu-EPO will be used therapeutically in cerebral ischaemia, brain trauma, inflammatory diseases, and neural degenerative disorders. A first clinical trial has shown the neuroprotective effectiveness of the drug in cerebral ischaemia.

beta 2-Adrenergic stimulation of erythropoiesis in busulfan treated mice.

The capability of chronic beta 2-adrenoceptor activation to effectively stimulate erythrocyte production in vivo was investigated in mice which had been treated with the hematopoiesis inhibiting agent busulfan. A relatively low dose (5 mg/kg i.p.) of busulfan produced moderate depression of erythropoiesis 10 days after a single injection, as determined by 59Fe-incorporation into erythrocytes. Administration of albuterol (1 mg/kg s.c. twice daily), a selective beta 2-adrenergic agonist, significantly enhanced erythropoiesis for 5--10 days after the injection of busulfan. In a long-term study with albuterol at the same dose a significant increase in hematocrit values as well as in the circulating erythrocyte mass was found in busulfan (5 mg/kg i.p. weekly) treated mice when compared to saline-busulfan treated control mice. Simultaneous injections of the beta-adrenergic blocking agent propanolol (4 mg/kg i.p.) diminished the effect of albuterol on erythropoiesis. Albuterol at a lower dose (0.1 mg/kg) had no significant effect on erythrocyte mass. In view of recent findings, which have shown that the proliferation of the pluripotent hemopoietic stem cell pool is blocked by busulfan, it is concluded that the main site of beta 2-adrenergic action on erythropoiesis is on the erythroid committed stem cell pool. In addition, enhanced release of erythropoietin from the kidney following the application of albuterol may contribute to beta 2-adrenergic stimulation of erythropoiesis.

Tumor necrosis factor p55 receptor (TNF-RI) mediates the in vitro inhibition of hepatic erythropoietin production.

Tumor necrosis factor alpha (TNFalpha) is thought to contribute to the blunted erythropoietin (Epo) production in inflammatory diseases. The present study was carried out to find out as to whether the 55 kD (TNF-RI) or the 75 kD (TNF-RII) receptor is responsible for the TNFalpha-induced inhibition of hepatic Epo synthesis. When the effects of two receptor-specific mutants were compared, only the TNF-RI-specific isoform proved to suppress the formation of immunoreactive Epo in the human hepatoma cell lines HepG2 and Hep3B, similar to the effect of wild-type TNFalpha. Anti-TNFalpha antibody restored Epo production in TNFalpha- or TNF-RI mutant-treated cultures. By gel shift assay NF-kappaB binding to DNA was demonstrated following the addition of TNFalpha or TNF-RI-specific mutant to HepG2 cells, while the TNF-RII-specific mutant was ineffective. Finally, immunoreactive TNF-RI, but not TNF-RII, fragments were measurable in cell culture supernatants. Taken together, these results suggest that the inhibition of hepatic Epo production by TNFalpha is mediated by TNF-RI signaling.

Extraction of erythropoietin from isolated renal glomeruli of hypoxic rats.

We investigated whether erythropoietin (Ep) can be extracted from renal glomeruli of hypoxic rats. Hypoxia was induced in a hypobaric chamber at 0.42 atmospheres for usually 6 h. Glomeruli were isolated with a sieving technique from kidneys that were flushed free of blood. Ep activities in glomeruli homogenates were determined in the fetal mouse liver cell assay and in the hypoxia exposed mouse assay. We found in vitro erythropoietic activity of glomerular extracts, which increased about 25-fold during hypoxia. Incubation of the extracts with anti-Ep serum abolished the activity. Its binding characteristics on Wheat germ lectin. Ricinus communis lectin and DEAE-cellulose suggested that glomeruli contained Ep precursor(s) that lacked terminal sugar residues. Such an incomplete carbohydrate portion seems also compatible with the finding that glomerular extracts did not stimulate erythropoiesis in hypoxia exposed mice.

Thyroid hormones enhance hypoxia-induced erythropoietin production in vitro.

Effects of thyroid hormones on the production of erythropoietin (Epo) were investigated in isolated perfused rat kidneys and in the human hepatoma cell line, HepG2. Epo protein was measured by radioimmunoassay. L-triiodothyronine and L-thyroxine stimulated hypoxia-induced Epo formation both in the kidney and in HepG2 cells in a dose-dependent fashion. Quantitation of Epo mRNA by competitive polymerase chain reaction (PCR) showed that hypoxic HepG2 cells had three-fold higher Epo messenger RNA levels when treated with thyroid hormones for 3 hours. Measurements of oxygen consumption revealed that this effect was not due to an increase in the degree of hypoxia. Thus, apart from the known direct effect on erythroid precursors, thyroid hormones appear to stimulate erythropoiesis by a noncalorigenic increase in Epo production.

Erythropoietin induces Ca2+ mobilization and contraction in rat mesangial and aortic smooth muscle cultures.

Reportedly, recombinant human erythropoietin (rhEpo) can induce constriction of isolated resistance vessels. We have studied whether rhEpo affects cytosolic calcium concentration, [Ca2+]i, and contraction of cultured smooth-muscle cells grown from rat renal corpuscles and aortae. rhEpo at high dose (> or = 20 U/mL) induced a transient increase in [Ca2+]i as detected by fura-2 fluorescence analysis. The number of cells responding with an increase in [Ca2+]i was dose-dependent. No significant changes of [Ca2+]i occurred when lower doses of rhEpo (< 20 U/mL) were applied. The effect of Epo on contraction was studied by phase-contrast microscopy. The number of cells responding with contraction was dose-dependent, too (76% mesangial cells contracting at 200 U rhEpo per mL). The receptor mechanism of this unusual action of Epo still needs to be clarified.

The influence of acute sterile inflammation on erythropoiesis in rats.

Many different cell types, coordinated by proinflammatory mediators, take part in the acute inflammatory reaction, but there is a lack of evidence regarding the role of erythroid cells in such conditions. In this study, changes in bone marrow, splenic, and peripheral blood erythroid cells and in erythropoietin (Epo) blood levels were investigated up to 72 hours after polyvinylpyrrolidone (PVP)-induced sterile inflammation in male Wistar rats (two intraperitoneal injections of 15 mL 3.5% PVP at 18-hour intervals). Transient changes within progenitor erythroid cells were observed in the bone marrow. Significant increases in the number of splenic immature erythroid progenitors (BFU-E) 6 hours and mature erythroid progenitors (CFU-E), erythroblasts, and orthochromatic erythroblasts 48 and 72 hours after the induction of inflammation pointed to stimulated splenic erythropoiesis. This was confirmed by semiquantitative assessment of splenic smears, which demonstrated expansion of erythroid cells at hours 48 and 72. The changes observed in the bone marrow and spleen indicated that during acute inflammation erythropoiesis was stimulated and that the spleens of PVP-treated rats were favorable to erythroid development. The significant increase in the percentage of peripheral blood reticulocytes 48 and 72 hours after PVP-induced inflammation provided evidence that effective erythropoiesis occurred. In spite of the stimulated erythropoiesis, serum levels of Epo remained unchanged, implying that other non-Epo regulatory molecules may be responsible for erythroid cellular changes.

Isolated serum-free perfused rat kidneys release immunoreactive erythropoietin in response to hypoxia.

The renal glycoprotein hormone erythropoietin (Epo) interacts with erythrocytic progenitors to stimulate their proliferation and differentiation in the bone marrow. The renal O2-sensing mechanism in the control of the synthesis of Epo is still poorly understood. Therefore, the capacity of isolated rat kidneys to produce Epo during hypoxic and anemic perfusion was studied. The kidneys were perfused at a constant perfusion pressure of 100 mm Hg with a substrate-enriched Krebs-Henseleit solution containing 60 g/liter BSA and freshly drawn human erythrocytes. Epo was measured by RIA. When the kidneys were perfused at an arterial pO2 of 720 or 150 mm Hg (hematocrit, 5%), Epo production was very low (0.1-0.2 U/g kidney within 3 h of perfusion). When the arterial pO2 was lowered to 35 or 20 mm Hg, Epo production increased to 0.4 and 0.9 U/g kidney, respectively. The release of Epo during hypoxic perfusion (pO2 35 and 20 mm Hg) was little affected by changes in the hematocrit, i.e. the O2-carrying capacity of the perfusion medium over a wide range (0-40%). These results indicate that the production of Epo in the isolated perfused kidney depends on the availability of O2 and can be modulated by changes in the arterial pO2.

Erythropoietin production by fetal mouse liver cells in response to hypoxia and adenylate cyclase stimulation.

This study was done to investigate aspects of control of extrarenal erythropoietin (Ep) production. To this end we studied the effects of three stimuli of renal Ep production in the adult, i.e. hypoxia, cobalt, and activation of adenylate cyclase on Ep generation by cultured fetal mouse liver cells. The fetal liver was taken as a model for extrarenal Ep production because this organ is considered the predominant site of extrarenal Ep production. We found that Ep production by the cells increased as the oxygen concentration was decreased in the incubation atmosphere from 20% to 1%. Cobalt (10(-4)-10(-5) M) had no effect on Ep production. Activation of adenylate cyclase by forskolin (10(-5) M) or isoproterenol (10(-5) M) greatly enhanced Ep production. These findings indicate that the Ep-stimulating effect of cobalt is specific for the kidney. However, oxygen depletion and activation of adenylate cyclase seem to be more general stimuli in Ep-producing cells. Furthermore we found that Ep production in hypoxia correlated with lactate formation in the cultured liver cells. This finding suggests that Ep production in fetal livers under hypoxic conditions parallels the shift from aerobic to anaerobic cellular energy metabolism.

A new candidate for the regulation of erythropoiesis. Insulin-like growth factor I.

The effect of pure human insulin-like growth factor I (IGF I) on the colony formation of late stage erythroid precursor cells (CFU-e) from fetal mouse liver and adult bone marrow was studied in a serum-free culture system. We found that IGF I in physiological concentrations stimulated erythroid colony formation. The combined effect of IGF I and erythropoietin was smaller than the sum of their single effects. The number of colonies induced by IGF I was linearly dependent on the number of plated cells. Our results indicate that IGF I is the first clearly defined mitogen that stimulates the late stages of erythroid differentiation independently of erythropoietin.

Proinflammatory cytokines lowering erythropoietin production.

The plasma level of erythropoietin (Epo) in anemic patients suffering from inflammation is often low in relation to the blood hemoglobin concentration. Various proinflammatory cytokines have been tested for their action on the synthesis of Epo. Interleukin 1 (IL-1) and tumor necrosis factor-alpha(TNF-alpha) suppress Epo gene expression in isolated perfused rat kidneys and in human hepatoma cell cultures. IL-6 inhibits in the kidney, and conflicting results have been reported for its effect on Epo synthesis in hepatic cells. Several other cytokines tested were without effect. Thus, mainly IL-1 and TNF-alpha seem to be responsible for the defect in Epo production in severe systemic and renal inflammatory diseases.