EFFECT OF ERYTHROPOIETIN ON PROLIFERATION OF ERYTHROPOIETIN-RESPONSIVE CELLS

A single dose of Myleran suppressed CFU in polycythemic mice to around 1% of normal for a period of 2 weeks and permitted the study of effects of erythropoietin on unipotential, erythroid stem cells (erythropoietin-responsive cells, ERC) in the absence of cell inflow from the CFU compartment. Without erythropoietin no ERC were detectable for 12 days after Myleran. Injections of erythropoietin had no effect on CFU but restored ERC populations in proportion to the dose of erythropoietin. Hydroxyurea given after erythropoietin markedly inhibited ERC repopulation and the latter is attributed to a stimulation of ERC proliferation by erythropoietin. Evidence in support of an age structure in the ERC population is presented. Daily erythropoietin injection resulted in stable ERC populations, indicating that ERC loss through differentiation and ERC self-replication were in balance.     

AUTORADIOGRAPHIC DETERMINATION OF S35 IN TISSUES AFTER INJECTION OF METHIONINE-S35 AND SODIUM SULFATE-S35

The loss of "bound" S³⁵ that occurs during various mounting procedures used in autoradiography was studied in healing surface wounds of rats treated with either methionine-S³⁵ or Na₂S³⁵O₄. Valid autoradiography of bound S³⁵ in this tissue is not possible until 48 hours after radiosulfate and 24 hours after radiomethionine injection, when the S³⁵ is almost entirely bound in large protein and polysaccharide molecules. Autoradiograms of S³⁵ given in both the organic and inorganic form reveal substantial over-all loss of the bound isotope from sections subjected to contact with solvents prior to autoradiography. A comparison of autoradiograms prepared by dry-mounting sections of frozen-dried tissue with autoradiograms of wet-mounted sections of the same tissue suggest that the loss is proportional to the extent of the contact with solvents. Evidence suggests that loss of the isotope occurs during contact of the ribbon or section itself with solutions after fixation and cutting and prior to radiation exposure. No appreciable loss of the bound isotope seems to occur during contact of the intact tissue specimen with a variety of fluid fixatives except for a marginal zone at the excision edges of the tissue. The potential hazard of displacement of the isotope during fixation, however, remains. Technics which prevent loss of the isotope and fogging of the nuclear emulsion permit the use of thinner sections and emulsion films and the fine resolution of image rendered possible by the physical properties of S³⁵.     

Interferon-γ exerts its negative regulatory effect primarily on the earliest stages of murine erythroid progenitor cell development

Interferon-γ (INFγ) has been shown to suppress erythropoiesis and perhaps to contribute to the anemia of chronic disease. In this study we demonstrated that the concentration of INFγ required to suppress murine burst forming unit-erythroid (BFU-E) growth was significantly less than that required to suppress colony forming unit-erythroid (CFU-E) growth. INFγ acted at the most primitive step in erythroid progenitor cell differentiation and proliferation, as inhibition was maximal when added at the time of BFU-E culture initiation. Inhibition was progressively less if INF-γ addition was delayed after culture initiation. The effects of INFγ on BFU-E did not require the presence of interleukin-1α (IL-1α), tumor necrosis factor-α (TNFα), or granulocyte macrophage colony stimulating factor (GM-CSF), as its effects were not neutralized by monoclonal antibodies against IL-1α, TNFα, or GM-CSF. This applied whether INFγ was added to culture with individual antibodies or with a combination of all three antibodies. INFγ was not required for IL-1α- or TNFα-induced suppression of BFU-E, as their effects were not neutralized by a monoclonal anti-INFγ antibody. In contrast, GM-CSF—induced suppression of BFU-E was negated by the simultaneous addition of anti-INFγ. We have previously shown that the addition of TNFα does not suppress BFU-E growth in cultures from marrow depleted of macrophages. Suppression did occur, however, if a small concentration of INFγ that does not inhibit and increasing concentrations of TNFα were added to culture, suggesting a synergistic effect between INFγ and TNFα. These observations suggest that INFγ is a potent direct inhibitor of erythroid colony growth in vitro. It exerts its negative regulatory effect primarily on the earliest stages of erythroid progenitor cell differentiation and proliferation, as much higher doses are required to suppress late erythroid cell development. INFγ is also involved in GM-CSF—induced inhibition of BFU-E colony growth. © 1995 Wiley-Liss, Inc.

1 This artilce is a US Government work and, as such, is in the public domain in the United States of America.

     

Pharmacokinetic study of niosome encapsulated insulin

Pharmacokinetic profile and hypoglycemic effect, after intraperitoneal injection of insulin and insulin encapsulated in niosomes were determined in diabetic rats. Niosomes (non-ionic surfactant vesicles) of different doses and different lipid compositions were prepared by lipid layer hydration method. Plasma samples were collected at specified time intervals and plasma concentration of insulin was determined by HPLC. Blood glucose level was estimated spectrophotometrically using commercial glucose assay kit. In vitro release and pharmacokinetic profile of niosomal formulation and free insulin were evaluated. Though there was a slight delay in the in vitro drug release due to cholesterol content in the niosomes, there was no difference between the two preparations when plasma levels were compared in vivo. Niosomes significantly reduced the blood glucose level in diabetic rats. Fall in blood glucose level was almost 92% of initial value. In case of the niosomal form the half-life of insulin was prolonged by 4 -5 hr in contrast to 2 hr for free drug. Niosomes maintained the plasma insulin level up to 12 hr, but free drug was cleared quickly. The area under the plasma concentration-time curve for niosomal forms was, 26.07 degrees +/- 0.99 mIU. hr/ml and for free insulin was 11.722 +/- 1.00 mIU. hr/ml. More than 80% of the drug was successfully encapsulated to give a formulation with sustained release characteristics. Entrapment efficiency increased with increasing lipid concentration and decreased with increasing drug concentration. The results showed that insulin entrapped in niosomes prolongs the existence of drug in the body therefore increasing its therapeutic value.     

Chiral synthesis of alpha-phenylpyridylmethanols with Rhizopus arrhizus

The bioreduction of 2-benzoylpyridine (1a) with Rhizopus arrhizus afforded (S)-(+)-alpha-phenyl-2-pyridylmethanol (2a) in 82% enantiomeric excess (e.e.) while the asymmetric hydrolysis of its racemic acetate resulted in the antipode (R)-(-)-2a with 24% optical purity.     

Phenotypic and genetic diversity in <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> and <Emphasis Type="Italic">S. medicae</Emphasis> from drought and salt affected regions of Morocco

Background  

Sinorhizobium meliloti and S. medicae are symbiotic nitrogen fixing bacteria in root nodules of forage legume alfalfa (Medicago sativa L.). In Morocco, alfalfa is usually grown in marginal soils of arid and semi-arid regions frequently affected by drought, extremes of temperature and soil pH, soil salinity and heavy metals, which affect biological nitrogen fixing ability of rhizobia and productivity of the host. This study examines phenotypic diversity for tolerance to the above stresses and genotypic diversity at Repetitive Extragenic Pallindromic DNA regions of Sinorhizobium nodulating alfalfa, sampled from marginal soils of arid and semi-arid regions of Morocco.     

A genetic linkage map of Lens sp. based on microsatellite and AFLP markers and the localization of fusarium vascular wilt resistance

Microsatellites have currently become the markers of choice for molecular mapping and marker-assisted selection for key traits such as disease resistance in many crop species. We report here on the mapping of microsatellites which had been identified from a genomic library of lentil (Lens culinaris Medik.). The majority of microsatellite-bearing clones contained imperfect di-nucleotide repeats. A total of 41 microsatellite and 45 amplified fragment length polymorphism (AFLP) markers were mapped on 86 recombinant inbred lines derived from the cross ILL 5588 × L 692-16-1(s), which had been previously used for the construction of a random amplified polymorphic DNA and AFLP linkage map. Since ILL 5588 was resistant to fusarium vascular wilt caused by the fungus Fusarium oxysporum Shlecht. Emend. Snyder & Hansen f.sp. lentis Vasud. & Srini., the recombinant inbreds were segregating for this character. The resulting map contained 283 markers covering about 751 cM, with an average marker distance of 2.6 cM. The fusarium vascular wilt resistance was localized on linkage group 6, and this resistance gene was flanked by microsatellite marker SSR59-2B and AFLP marker p17m30710 at distances of 8.0 cM and 3.5 cM, respectively. These markers are the most closely linked ones known to date for this agronomically important Fw gene. Using the information obtained in this investigation, the development and mapping of microsatellite markers in the existing map of lentil could be substantially increased, thereby providing the possibility for the future localization of various loci of agronomic interest.     

Effect of Ocimum sanctum Linn. on normal and dexamethasone suppressed wound healing

Ethanolic extract of leaves of O. sanctum was investigated for normal wound healing and dexamethasone depressed healing using incision, excision and dead space wound models in albino rats. The extract of O. sanctum significantly increased the wound breaking strength in incision wound model. The extract treated wounds were found to epithelialize faster and the rate of wound contraction was significantly increased as compared to control wounds. Significant increase in wet and dry granulation tissue weight, granulation tissue breaking strength and hydroxyproline content in dead space wound model was observed. The extract significantly decreased the antihealing activities of dexamethasone in all the wound models. The results indicated that the leaf extract promotes wound healing significantly and able to overcome the wound healing suppressing action of dexamethasone. Histological examination of granulation tissue to determine the pattern of lay-down for collagen confirmed the results.     

Erythropoietin enhancement of rat pancreatic tumor cell proliferation requires the activation of ERK and JNK signals

Erythropoietin (EPO) regulates the proliferation and differentiation of erythroid cells by binding to its specific transmembrane receptor EPOR. Recent studies, however, have shown that the EPOR is additionally present in various cancer cells and EPO induces the proliferation of these cells, suggesting a different function for EPO other than erythropoiesis. Therefore, the purpose of the present study was to examine EPOR expression and the role of EPO in the proliferation and signaling cascades involved in this process, using the rat pancreatic tumor cell line AR42J. Our results showed that AR42J cells expressed EPOR, and EPO significantly enhanced their proliferation. Cell cycle analysis of EPO-treated cells indicated an increased percentage of cells in the S phase, whereas cell numbers in G0/G1 phase were significantly reduced. Phosphorylation of extracellular regulatory kinase 1/2 (ERK1/2) and c-Jun NH₂ terminal kinase 1/2 (JNK1/2) was rapidly stimulated and sustained after EPO addition. Treatment of cells with mitogen-activated protein/ERK kinase (MEK) inhibitor PD98059 or JNK inhibitor SP600125 significantly inhibited EPO-enhanced proliferation and also increased the fraction of cells in G0/G1 phase. Furthermore, the inhibition of JNK using small interference RNA (siRNA) suppressed EPO-enhanced proliferation of AR42J cells. Taken together, our results indicate that AR42J cells express EPOR and that the activation of both ERK1/2 and JNK1/2 by EPO is essential in regulating proliferation and the cell cycle. Thus both appear to play a key role in EPO-enhanced proliferation and suggest that the presence of both is required for EPO-mediated proliferation of AR42J cells. erythropoietin receptor; cell signaling; mitogen-activated protein kinase induction