Residual erythroid progenitors in W/W mice respond to erythropoietin in the absence of steel factor signals.

Erythropoiesis is severely impaired in mice with inactivating mutations in the Steel factor (SF) gene (Sl/Sl mice) or in c-kit, in the SF receptor gene (W/W mice), and in mice with null mutations in the genes for either erythropoietin (EPO) or the erythropoietin receptor (EPO-R). Previous studies indicated that EPO is sufficient for colony development from colony-forming units-erythroid (CFU-E). However, recent studies have shown that there is a physical association between these 2 receptors and that c-kit can phosphorylate EPO-R. To examine the role SF and EPO play in regulating erythropoiesis, we examined the effect of SF and EPO on colony development from cells of the embryonic aorta-gonad-mesonephros (AGM) region, yolk sac, and liver of fetal wild-type and W/W mice. The maturation of CFU-E from these sites did not require the addition of SF to clonal cultures, whereas the efficient development of erythroid bursts required both EPO and SE The number of erythroid colony-forming cells was reduced in both the AGM region and liver of fetal W/W mice. The residual CFU-E present in W/W mice were dependent on EPO and independent of SF. These results indicate that EPO/EPO-R can function to support colony formation in the absence of an SF signal.

Cell-cell contact mechanisms.

Exciting new findings link characteristic properties of the inflammatory process previously not linked functionally. For example, it is now clear that oxygen radicals and leukocyte adhesion are intimately related in a carefully transduced and orchestrated series of events that culminates in release of granule contents, but not before the leukocyte has safely transversed the vessel wall. In addition to defining separate heterocellular contacts and homocellular aggregation we must now consider equilibrium events that allow associations among cell-cell partnerships involving different cell types.

Molecular cloning of the CD9 antigen. A new family of cell surface proteins.

The CD9 antigen was described originally as a 24-kDa surface protein of non-T acute lymphoblastic leukemia cells and developing B-lymphocytes. It is also strongly expressed on platelets, among other cells, where it shows the property of mediating platelet activation and aggregation upon binding with mAbs. The primary structure has been elucidated by cloning the cDNA from a lambda gt11 expression vector library constructed with megakaryocytic mRNA. Monoclonal antibodies were used as probes with an APAAP amplification of the signal. The 5' region was further cloned in a lambda gt10 randomly primed cDNA library. The initiation codon was immediately followed by a sequence coding for the tetrapeptide corresponding to the NH2-terminal sequence identified in a microsequencing procedure. Only one species of mRNA was found with an estimated size of 1.4 kilobase. CD9 antigen appears to be a 227-amino acid molecule with four hydrophobic domains and one N-glycosylation site. Sequence and structural comparisons showed extensive similarity of the CD9 antigen with a 237-amino acid molecule described previously as the human melanoma-associated antigen ME491 and a Schistosoma mansoni membrane protein of 218 amino acids. These three proteins identify a new family of cell-surface proteins.

Extensive C1q-complement initiated lysis of human platelets by IgG subclass murine monoclonal antibodies to the CD9 antigen.

Several monoclonal antibodies (MAbs) to CD9, a surface membrane glycoprotein of 24 kDa found on platelets as well as several other hematopoietic and nonhematopoietic tissues, have the property of activating platelets. We have recently shown that with two of these MAbs (ALB-6 and SYB-1) this activation is mediated by interaction of the Fc portion of these IgG1 subclass MAbs with the Fc gamma II receptor (FcRII) and is blocked by a MAb to this receptor (IV-3). In this report we show that several MAbs to the CD9 antigen (BA-2, BU-16, MM2/57) also cause extensive and rapid platelet lysis in plasma. This lysis is mediated by the classical complement pathway dependent on C1q fixation. Lysis was not blocked by inhibiting platelet activation with prostaglandin E1 or by the MAb to FcRII (IV-3). The CD9 MAb BU-16 can also activate platelets through the FcRII when complement lysis is prevented by removal of C1q using specific antisera or by isolation of the platelets from plasma.

Stimulus-response coupling in human platelets activated by monoclonal antibodies to the CD9 antigen, a 24 kDa surface-membrane glycoprotein.

The CD9 molecule is a 24 kDa surface-membrane glycoprotein present on platelets and a variety of haematopoetic and non-haematopoetic tissues. In the present study we utilized specific inhibitors of thromboxane A2 (TxA2) formation (aspirin), protein kinase C [H-7 [1-(5-isoquinolinesulphonyl)-2-methylpiperazine]] and autocrine stimulation by secreted ADP (apyrase) to modify platelet activation by a monoclonal antibody ALB-6 to the CD9 antigen. This activation is only partially inhibited by aspirin alone but, in combination with either H-7 or apyrase, more than 50% inhibition of platelet aggregation and secretion was observed. This combination of inhibitors was also required to inhibit effectively the phosphorylation of myosin light chain and the 47 kDa substrate of protein kinase C. Intracellular Ca2+ flux monitored by the fluorescent dye fura-2 showed that this was almost completely mediated by the aspirin-sensitive TxA2 pathway. We suggest that the aspirin-insensitive pathway is primarily mediated by phospholipase C formation of diacylglycerol to activate protein kinase C. The inhibition by apyrase suggests a strong dependency on autocrine stimulation by secreted ADP to fully activate both phospholipase C and express fibrinogen-binding sites mediating platelet aggregation. This alternate pathway of phospholipase C activation by ALB-6 may be mediated by cytoplasmic alkalinization [monitored by SNARF-1 (5'(6')-carboxy-10-bismethylamino-3-hydroxy-spiro-[7H- benzo[c]xanthine-1',7(3H)-isobenzofuran]-3'-one) fluorescence of the dye]. Both activation pathways are dependent on intact antibodies, since F(ab')2 fragments of SYB-1, a monoclonal antibody against the CD9 antigen with activation characteristics identical with those of ALB-6, do not elicit activation. Besides thrombin, collagen is another physiological agonist shown to induce aspirin-insensitive activation. Similarities to ALB-6 in collagen sensitivity to apyrase in combination with aspirin inhibitors were noted with respect to aggregation and secretion, as well as a complete block of Ca2+ flux by aspirin. However, it is unlikely that collagen activation is mediated by the CD9 antigen, since SYB-1 F(ab')2 fragments had no effect on collagen activation and aspirin also completely blocked the alkalinization response to collagen, in contrast with ALB-6.

Platelet activation by CD9 monoclonal antibodies is mediated by the Fc gamma II receptor.

The function of the human cell surface CD9 antigen is not known, yet monoclonal antibodies (mAbs) of the IgG1 subclass in the CD9 cluster induce activation of platelets. Previously it had been shown that this activation pathway is comparable both in kinetics and extent to physiological agonists such as thrombin. Here it is demonstrated that activation with CD9 mAbs depends on interaction of the Fc part of the CD9 antibody molecule with Fc receptors on the platelet surface, since: (i) mAb directed against the Fc receptor totally blocked the platelet response to CD9 mAb; and (ii) F(ab')2 fragments of the CD9 mAb SYB-1 which bound to platelets, as demonstrated by flow cytometry, failed to activate them. Furthermore, platelet activation by CD9 mAb closely paralleled the activation caused by cross-linking Fc receptors when comparing: (i) kinetics and extent of aggregation; (ii) thromboxane synthesis; (iii) calcium flux; and (iv) the cytoplasmic alkalinization response. Thus it is concluded that CD9 antigen itself does not necessarily participate in stimulus-response coupling leading to platelet activation by CD9 mAbs, and that this activation can be entirely accounted for by the Fc receptor pathway mechanism. The results suggest a possible novel mechanism for platelet consumption in cases of immune thrombocytopenia.

Calcium response to erythropoietin in erythroleukemia cells.

Erythroleukemic mouse spleen cells were analyzed by flow cytometry at three fluorescence wavelengths, two emissions for the calcium indicator indo 1 and immunofluorescence with an antiserum directed against murine leukemia retrovirus antigen. The antigen-positive subpopulation of cells responded to EPO by transiently changing indo 1 fluorescence emissions, indicating a change in cytoplasmic calcium concentration. EGTA blocked the EPO response, suggesting that Ca2+ influx may have followed EPO exposure in these cells. Antigen-negative cells showed no Ca2+ response to EPO. Infected mice all contained antigen-positive spleen cells, but, as measured by changes in indo 1 fluorescence, these subpopulations varied in their ability to respond to EPO.

Effects of dietary peanut oil on serum lipoprotein patterns of rats.

Oils prepared from two varieties of peanuts and from a hybrid corn having linoleic acid concentrations substantially different from the respective commercial oils were compared with commercial oils for their effects on serum lipids of weanling female rats. In the first experiment, serum lipid patterns appeared to reflect linoleic acid content of the dietary oil. However, with a longer feeding period in the second experiment, serum lipid patterns were determined by the plant source of the dietary oil rather than its linoleic acid content; all peanut oils differed from both corn oils in their physiological effects. Diets containing triglyceride, hydrocarbon and sterol fractions obtained by liquid chromatography of peanut and corn oils were fed to female rats. The data provide no evidence that the hydrocarbon or sterol fractions of peanut oil are responsible for its unusual atherogenicity when fed as the sole fat source or that similar fractions from corn oil are protective against the effects of peanut oil.

Quantitation of erythroid differentiation in vitro using a sensitive colorimetric assay for hemoglobin.

We have developed a new colorimetric method to detect early erythroid clonogenic progenitor cells by virtue of their hemoglobin-containing progeny in vitro. The method relies upon the specific oxidation of 2,7-diaminofluorene (DAF) by the pseudoperoxidase activity of hemoglobin (Hb). Generation of fluorene blue (FB), the chromophore generated upon oxidation of DAF, was a linear function of Hb and erythrocyte concentration. Measurement of FB absorbance at 610 nm was 80 times more sensitive than measurement of Hb absorbance at 530 nm. The limit of resolution was approximately 8 pmol of Hb corresponding to the Hb content of approximately 5000 murine peripheral blood erythrocytes. FB concentration closely paralleled eight-day BFU-E colony formation as a function of erythropoietin dose. However, the method was unable to detect a response for CFU-E to erythropoietin because of persistently high background levels of Hb in two-day cultures. Generation of FB by cells likely to contain myeloperoxidase was negligible, and FB production was not correlated with the number of CFU-GM colonies. DAF oxidation by hemoglobin showed a higher hydrogen peroxide optimum than oxidation by microperoxidase, so that, under the conditions used, FB generation by peroxidase was not favored. As a further confirmation of specificity, peritoneal exudate cells failed to show significant generation of FB. The DAF reagent was also used to stain both CFU-E and BFU-E colonies in situ, and was more sensitive than benzidine dihydrochloride in the histochemical detection of Hb. DAF can replace hazardous benzidine-related compounds for the staining erythroid colonies in culture dishes, and the method offers a new quantitative approach to the study of erythropoietin responsiveness in vitro.

Resistance to erythroleukemia in immunodeficient xid- CBA/N mice with susceptibility after immune stimulation.

CBA/N (xid-) mice failed to develop erythroleukemia when inoculated with an NB-tropic, anemia-causing Friend virus stock (FVA), while Fv-2ss mouse strains succumbed rapidly to the characteristic Friend disease, even after a virus dose 30-fold lower than that given to CBA/N mice. Immunization with bacterial antigens or with spleen cell allografts prior to FVA inoculation rendered CBA/N mice highly susceptible to FVA. Transplantation studies confirmed that non-immunized CBA/N mice were able to both support erythroleukemic cells and permit erythroleukemic transformation, thus arguing against host defense mechanisms as a cause of resistance. On the basis of early erythroid progenitor cell sensitivity to hydroxyurea in vivo, the CBA/N strain appeared to have the FVA sensitive genotype (Fv-2ss). These results imply that CBA/N mice are not intrinsically resistant to FVA and that an as yet unknown type of immunological activity, evoked both by various immunizations and allogeneic transplantation, is required for Friend leukemogenesis in this immunodeficient inbred strain. These findings further suggest that the erythroid target cells transformed by Friend viruses are influenced by immunological activity.

Flow cytometric analysis of megakaryocyte differentiation.

Megakaryocytes were isolated quantitatively from rat bone marrow by centrifugal elutriation (CE). CE-enriched megakaryocytes were stained supravitally for either DNA content with Hoechst 33342, surface membrane immunofluorescence with fluorescein isothiocyanate (FITC)-conjugated antiplatelet antibody, or both. The cells were then measured using a Becton Dickinson FACS IV flow cytometer. The following correlations were analyzed: DNA content and light scatter, light scatter and antiplatelet immunofluorescence, and DNA content and antiplatelet immunofluorescence. Although the range of light scatter increased as a function of DNA content, discrete subpopulations of megakaryocytes with different light scatter properties were detected within each of the three principal ploidy classes (8C, 16C, and 32C). Other discrete megakaryocyte subpopulations were revealed in the analysis of antiplatelet surface immunofluorescence as a function of degree of light scatter. The nonlinear relationship between the latter suggested that the degree of membrane immunofluorescence did not bear a simple relationship to cell size as reflected in light scatter. Megakaryocyte DNA content, on the other hand, varied in a linear fashion with membrane immunofluorescence, supporting the conclusion that there may be a proportional increase in the expression of platelet antigens with DNA content. The use of multiple markers, correlated multiparameter flow cytometry and multivectorial analysis to define differentiation on a single cell basis have revealed new complexities in this process. Flow cytometric analysis holds promise as a useful method for further characterization of megakaryocyte differentiation.

Dibutyryl cyclic guanosine monophosphate elevates bovine endothelial cell thromboxane production without affecting prostacyclin metabolism.

Cloned bovine aortic endothelial cells (BAEC) were grown to confluence then treated for 24 hours with dibutyryl cyclic AMP (dbcAMP) or dibutyryl cyclic GMP (dbcGMP) in serum free medium. Submillimolar concentrations of dbcGMP caused a significant enhancement of thromboxane (TXB2) synthesis in washed cells exposed to arachidonate. DbcAMP had no effect on the production of either metabolite. TXB2 synthesis was inhibited by 3 micrograms/ml cycloheximide, whether or not the cells were pretreated with dbcGMP. Prostacyclin production was inhibited to a much lesser extent by cycloheximide. We conclude that dbcGMP elevates TXB2 production by increasing the amount of thromboxane synthetase available, and that this effect is inhibited by cycloheximide. Data are described which suggest that dbcGMP increases the degradation rate of TXB2 by BAEC, so that the observed increase in TXB2 may be an underestimate of the true effects of dbcGMP on TXB2 production.

Effects of Sorbate, Benzoate, Sulfur Dioxide and Temperature on Growth and Patulin Production by Byssochlamys nivea in Grape Juice.

The influence of potassium sorbate, sodium benzoate, sulfur dioxide (SO2) and temperature on biomass and patulin production by Byssochlamys nivea in grape juice was investigated. Growth of B. nivea was monitored over a 25-d incubation period at 21, 30 and 37°C. Approximately 2,500 mg (dry weight) of biomass per 100 ml of juice was obtained in controls at 30 and 37°C; significantly lower amounts were observed at 21°C. Based on concentration, SO2 had the most significant effect on reducing biomass production followed by potassium sorbate and sodium benzoate, respectively. Patulin was produced in the highest concentrations (10 mg/100 ml) at 21°C after 20 d of incubation. Production was less at 30 and 37°C, with a fairly rapid decrease after reaching a maximum concentration. As in the biomass study, SO2 had the most significant influence on inhibiting patulin production followed by potassium sorbate and sodium benzoate.

Aspirin inhibits rat megakaryocyte thromboxane synthesis.

This study examines the question of whether the aspirin-induced delay in the recovery of platelet cyclooxygenase pathway activity, as measured by RIA of thromboxane B2, results from a direct effect on megakaryocyte cycloxygenase. From our measurement of recovery of TXB2 and information on megakaryocyte transit time in rats, we propose that thromboxane synthesis may represent a relatively late step in the differentiation of megakaryocytes. Megakaryocyte thromboxane production was depressed by 70% and that of platelets by 85% at two hr after 20 mg/kg oral aspirin dissolved in DMSO. Full megakaryocyte thromboxane recovery occurred by 72 hr and preceded complete platelet thromboxane recovery by 24 hr. Whereas megakaryocyte thromboxane synthesis showed substantial recovery by 36 hr after aspirin, platelet recovery did not begin for 24 hr and achieved a maximal recovery rate over the following 12 hr. This finding is consistent with predictions based upon human data for both megakaryocyte labeling studies and pot-aspirin platelet recovery. We conclude from our data and from estimates of megakaryocyte maturation times in marrow, that thromboxane synthesis develops in rat megakaryocytes after approximately 48 hr of cytoplasmic differentiation toward platelet shedding. This metabolic capacity therefore serves as a marker of megakaryocyte differentiation.

Thromboxane synthesis in megakaryocytes isolated by centrifugal elutriation.

A systematic approach to the optimization of centrifugal elutriation is described for the purification of rat femoral marrow megakaryocytes, which has permitted the first direct demonstration of thromboxane synthesis in these cells. The rapidity, simplicity, and capacity of this technique has made it possible to process 2 x 10(9) marrow cells in the absence of platelet aggregation inhibitors in 20 min to obtain 10(6) megakaryocytes at a recovery of about 80% with a concentration of up to 282-fold. Incubation of these isolated megakaryocytes with 14C-arachidonic acid and subsequent thin-layer radiochromatography has revealed thromboxane B2 as the major cyclooxygenase pathway metabolite of arachidonic acid in megakaryocytes.

Arachidonic acid: Occurrence in the reproductive tract of the male house cricket (Acheta domesticus) and field cricket (Gryllus spp.).

Reproductive tissues from male house and field crickets were investigated for the presence of arachidonic acid. Arachidonic acid was identified by retention behavior on 3 gas liquid chromatographic column systems-SE-30, OV 225, and Silar 10C-and by gas liquid/positive ion chemical ionization mass spectrometry. Arachidonic acid constituted 0.3 and 1.2% of total fatty acids in the house and field cricket, respectively. Freeze-dried male reproductive tissues from house and field cricket contained ca. 0.02 and 0.16% arachidonic acid and 8.2 and 8.6% total fatty acids. Other fatty acids found in reproductive tracts of both species of crickets were 16∶0, 18∶0, 18∶1, 18∶2, 20∶0, 20∶1 and 20∶3.

Patulin production by Byssochlamys spp. in fruit juices.

Ten strains of Byssochlamys fulva and three strains of B. nivea were cultured in a laboratory medium and tested for their ability to produce patulin. Two strains of B. fulva and all three strains of B. nivea produced the mycotoxin. One strain of B. fulva produced patulin in 11 of 13 processed fruit juices, with greatest amounts being produced in blueberry, red raspberry, and boysenberry juices, whereas no patulin was detected in prune or tomato juices. Grown in Concord grape juice at 18, 25, 30, and 38 degrees C, this strain produced the highest patulin concentration at 18 degrees C after 25 days, whereas biomass production was greatest at 25 and 30 degrees C after 20 and 25 days.

Selective inhibition of osmotic water flow by general anesthetics to toad urinary bladder.

Vasopressin increases the permeability of the total urinary bladder, an analogue of the mammalian renal collecting duct, to water and small solutes, especially the amide urea. We have observed that three general anesthetic agents of clinical importance, the gases methoxyflurane and halothane and the ultrashortacting barbiturate methohexital, reversibly inhibit vasopressin-stimulated water flow, but do not depress permeability to urea, or the the lipophilic solute diphenylhydantoin. In contrast to their effects in vasopressin-treated bladders, the anesthetics do not inhibit cyclic AMP-stimulated water flow, consistent with an effect on vasopressin-responsive adenylate cyclase. The selectivity of the anesthetic-induced depression of water flow suggests that separate adenylate cyclases and cyclic AMP pools may exist for control of water and urea permeabilities in to toad bladder. Furthermore, theophylline's usual stimulatory effect on water flow, but not its effect on urea permeability, was entirely abolished in methoxyflurane-treated bladders, suggesting that separate phosphodiesterases that control water and urea permeabilities are present as well. We conclude that the majority of water and urea transport takes place via separate pathways across the rate-limiting luminal membrane of the bladder cell, and that separate vasopressin-responsive cellular pools of cyclic AMP appear to control permeability to water and to urea.