Calcium/calmodulin transduces thrombin-stimulated secretion: studies in intact and minimally permeabilized human umbilical vein endothelial cells. Thrombin stimulates cultured endothelial cells (EC) to secrete stored von Willebrand factor (vWF), but the signal transduction pathways are poorly defined. Thrombin is known to elevate the concentration of intracellular calcium ([Ca2+]i) and to activate protein kinase C (PKC) in EC. Since both calcium ionophores and phorbol esters release vWF, both second messenger pathways have been postulated to participate in vWF secretion in response to naturally occurring agonists. We find that in intact human EC, vWF secretion stimulated by either thrombin or by a thrombin receptor activating peptide, TR(42-55), can be correlated with agonist-induced elevations of [Ca2+]i. Further evidence implicating calcium in the signal transduction pathway is suggested by the finding that MAPTAM, a cell-permeant calcium chelator, in combination with the extracellular calcium chelator EGTA, can inhibit thrombin-stimulated secretion. In contrast, the observation that staurosporine (a pharmacological inhibitor of PKC) blocks phorbol ester- but not thrombin-stimulated secretion provides evidence against PKC-mediated signal transduction. To examine further the signal transduction pathway initiated by thrombin, we developed novel conditions for minimal permeabilization of EC with saponin (4-8 micrograms/ml for 5-15 min at 37 degrees C) which allow the introduction of small extracellular molecules without the loss of large intracellular proteins and which retain thrombin-stimulated secretion. These minimally permeabilized cells secrete vWF in response to exogenous calcium, and EGTA blocks thrombin-induced secretion. Moreover, in these cells, thrombin-stimulated secretion is blocked by a calmodulin-binding inhibitory peptide but not by a PKC inhibitory peptide. Taken together, these findings demonstrate that thrombin-stimulated vWF secretion is transduced by a rise in [Ca2+]i and provide the first evidence for the role of calmodulin in this process.


A novel endothelial-specific membrane protein is a marker of cell-cell contacts. mAbs were raised in mice against cultured human endothelial cells (EC) and screened by indirect immunofluorescence for their ability to stain intercellular contacts. One mAb denoted 7B4 was identified which, out of many cultured cell types, specifically decorated cultured human EC. The antigen recognized by mAb 7B4 is bound at the appositional surfaces of cultured EC only as they become confluent and is stably expressed at intercellular boundaries of confluent monolayers. EC recognition specificity was maintained when the antibody was assayed by immuno-histochemistry in tissue sections of many normal and malignant tissues and in blood vessels of different size and type. The antigen recognized by 7B4 was enriched at EC intercellular boundaries similarly in vitro and in situ. In vitro, addition of mAb 7B4 to confluent EC increased permeation of macromolecules across monolayers even without any obvious changes of cell morphology. In addition, when EC permeability was increased by agents such as thrombin, elastase, and TNF/gamma IFN, its distribution pattern at intercellular contact rims was severely altered. mAb 7B4 immunoprecipitated a major protein of 140 kD from metabolically and surface-labeled cultured EC extracts which appeared to be an integral membrane glycoprotein. On the basis of its distribution in cultured cells and in tissues in situ, 7B4 antigen is distinct from other described EC proteins enriched at intercellular contacts. NH2-terminal sequencing of the antigen, immunopurified from human placenta, and sequencing of peptides from tryptic peptide maps revealed identity to the cDNA deduced sequence of a recently identified new member of the cadherin family (Suzuki, S., K. Sano, and H. Tanihara. 1991. Cell Regul. 2:261-270.) These data indicate that 7B4 antigen is an endothelial-specific cadherin that plays a role in the organization of lateral endothelial junctions and in the control of permeability properties of vascular endothelium.


Regulation of cell substrate adhesion: effects of small galactosaminoglycan-containing proteoglycans. Cell adhesion is a process which is initiated by the attachment of cells to specific sites in adhesive matrix proteins via cell surface receptors of the integrin family. This is followed by a reorganization of cytoskeletal elements which results in cell spreading and the formation of focal adhesion plaques. We have examined the effects of a class of small galactosaminoglycan-containing proteoglycans on the various stages of cell adhesion to fibronectin-coated substrates. Our results indicate that dermatan sulfate proteoglycans (DSPGs) derived from cartilage, as well as other related small proteoglycans, inhibit the initial attachment of CHO cells and rat embryo fibroblasts to substrates composed of the 105-kD cell-binding fibronectin fragment, but do not affect cell attachment to intact fibronectin. Although this effect involves binding of DSPGs to the substrate via the protein core, the intact proteoglycan is necessary for the observed activity. Isolated core proteins are inactive. The structural composition of the galactosaminoglycan chain does not appear to be functionally significant since both chondroitin sulfate and various dermatan sulfate proteoglycans of this family inhibit cell attachment to the fibronectin fragment. Neither the percentage of cells spread nor the mean area of spread cells adhering to substrates of intact fibronectin was significantly affected by the DSPGs. However, significantly fewer cells formed focal adhesions in the presence of DSPGs as compared with untreated control cells. These results suggest that the binding of small galactosaminoglycan-containing proteoglycans to a fibronectin substrate may affect several stages in the cell adhesion process.


Interleukin 6 promotes epithelial migration by a fibronectin-dependent mechanism. We investigated the effect of interleukin 6 (IL-6) on the migration of rabbit corneal epithelium in vitro and on the attachment of dissociated corneal epithelial cells to a fibronectin matrix. When corneal blocks were cultured with IL-6 for 24 hours, the length of the path of epithelial migration over exposed corneal stroma increased significantly (p less than 0.005 at the concentration of 10 ng/ml) in proportion to the concentrations of IL-6 (0.1-10.0 ng/ml). The addition of antiserum against fibronectin or of GRGDSP abolished the stimulatory effect of IL-6 on epithelial migration. When corneal epithelial cells were cultured with various concentrations of IL-6, suspended, and plated on wells coated with fibronectin (10 micrograms/ml), the number of cells attached to the wells increased in a dose-dependent manner. The presence of antibody against fibronectin or of GRGDSP during the attachment assay decreased the number of cells attached to the fibronectin matrix, regardless of the fact that the cells had been cultured with IL-6 or not. IL-6 stimulated the attachment of corneal epithelial cells to collagen type IV and to laminin matrices. However, the presence of GRGDSP did not affect the cell attachment to collagen type IV and to laminin. These findings strongly indicate that IL-6 stimulates epithelial migration in the cornea by a fibronectin-dependent mechanism, presumably the increased expression of fibronectin receptors.


Heparin inhibition of autonomous growth implicates amphiregulin as an autocrine growth factor for normal human mammary epithelial cells. Normal human mammary epithelial cells (HMECs) proliferate in a serum-free defined growth medium in the absence of epidermal growth factor (Li and Shipley, 1991). Amphiregulin (AR) is a heparin-regulated, EGF-like growth factor. Our observation that one strain of HMECs produce AR mRNA (Cook et al., 1991 a) stimulated us to determine whether AR expression was a common phenomenon in HMECs and whether AR could act as an autocrine growth factor to support the EGF-independent growth of these cells. In this study, we detected high levels of AR expression in four separate HMEC strains while one immortal mammary cell line (HBL-100) and six mammary tumor-derived cell lines had low to undetectable levels of AR. The EGF-independent growth of HMECs was blocked by the addition of heparin or a monoclonal anti-EGF receptor antibody to the culture medium, implicating AR as an autocrine growth mediator. This hypothesis is further supported by the fact that medium conditioned by HMECs contains secreted AR protein. A mammary tumor-derived cell line, Hs578T, which proliferates in an EGF-independent manner, does not express detectable levels of AR and is not growth inhibited by heparin. Examination of the same cell types for expression of transforming growth factor type-alpha (TGF-alpha) mRNA revealed coordinate expression of AR and TGF-alpha in these cells. These data suggest that both AR and TGF-alpha mRNA are produced in much greater abundance by normal HMECs than in tumor-derived cells in culture, and that AR is an important autostimulatory factor for the growth of normal HMECs.


Apoptosis of murine BW 5147 thymoma cells induced by cold shock. Exposure of thymoma BW 5147 cells to cold (0-2 degrees C) followed by rewarming at 37 degrees C (cold shock) resulted in internucleosomal DNA cleavage. Sensitivity to cold shock-induced cell death was critically dependent on the serum concentration in the medium and limited to serum-deficient medium (2% serum concentration), whereas cells in the complete growth medium (10%) were completely resistant. RNA/protein-synthesis inhibitors (cycloheximide and actinomycin D) had no effect on cold shock-induced DNA cleavage in BW 5147 cells. The DNA fragmentation seems to be independent of increase in the cytosolic Ca2+ level. Moreover, reduction in the calcium content of the external medium by EGTA induced DNA cleavage. Incubation of BW 5147 cells in the presence of colchicine and cytochalasin B led to the apoptosis. The latter suggests that the internucleosomal DNA cleavage induced by cold shock may be concerned with the disruption of some cytoskeletal network caused by cooling. The results are discussed in relation to cell proliferation.


Differential modulation of vascular cell integrin and extracellular matrix expression in vitro by TGF-beta 1 correlates with reciprocal effects on cell migration. In vitro, BAEC and BASMC migratory phenotypes are known to be reciprocally modulated by both soluble factors and extracellular matrix proteins. In addition, integrin matrix receptors mediate endothelial and smooth muscle cell attachment and migration. To further elucidate these phenomena, we studied the effects of TGF-beta 1 on integrin expression by vascular BASMC and BAEC in tissue culture. TGF-beta 1 upregulated mRNA levels and surface pools of BASMC beta 3 integrin classes without modulating beta 1 integrin mRNA levels or expression of beta 1 integrin organization. In contrast to its effects on BASMC, TGF-beta 1 increased BAEC mRNA levels and surface expression of beta 1 and beta 3 integrins without altering their organization. Conversely, extracellular matrix components (fibronectin, laminin, and fibrinogen) organized cell surface integrins in both BASMC and BAEC without affecting the size of their cell surface pools. These data are consistent with the hypothesis that SMC and EC behavior in neointimal lesions may be modulated, in part, through a coordination of soluble factor and extracellular matrix protein regulation of integrin surface expression and organization.


Plasminogen activator inhibitor-1 is induced in migrating endothelial cells. It has been proposed that a finely tuned protease-anti-protease equilibrium must be maintained during processes of cell migration in order to limit extracellular proteolysis to the close proximity of the cell surface, and thereby to prevent excessive extracellular matrix degradation. We have previously shown that urokinase-type plasminogen activator (u-PA) activity is induced in microvascular endothelial cells migrating from the edges of a wounded monolayer in vitro (Pepper et al., J. Cell Biol., 105:2535-2541, 1987). By Northern analysis, we now demonstrate that plasminogen activator inhibitor 1 (PAI-1) mRNA is increased in multiple-wounded monolayers of bovine microvascular (BME) or aortic (BAE) endothelial cells, with a maximal 7- and 9-fold increase 4 h after wounding, respectively. By in situ hybridization, we demonstrate that the increase in PAI-1 mRNA is localized to cells at the edge of a wounded BME or BAE cell monolayer. The increase in PAI-1 mRNA observed in BME cells is independent of cell division and is inhibited by antibodies to basic fibroblast growth factor (bFGF), suggesting that PAI-1 induction in migrating cells is mediated by the autocrine activity of bFGF. Taken together with our previous observations on the induction of u-PA, these results support the hypothesis that the proteolytic balance in the pericellular environment of migrating cells is regulated through the concomitant production of proteases and protease inhibitors.


Regulation of cell proliferation: late down-regulation of c-myb preceding myelo-monocytic cell differentiation. Expression of the c-myb nuclear oncogene during the cell proliferation and differentiation of HL-60 human promyelocytic leukemia cells was characterized and compared to the expression of c-fos, another nuclear oncogene with transcriptional regulatory activity. During progression through the cell cycle, the amount of c-myb protein increased. The increase was commensurate with total cell size, thus preserving the relative abundance of c-myb protein present at the onset of the cell cycle. In HL-60 cells, the induced metabolic cascade leading to terminal myeloid or monocytic differentiation segregates into two steps occurring over two division cycles. Expression of c-myb did not diverge from the control until late in this metabolic cascade when it declined prior to onset of terminal differentiation. This course of expression was similar for both the retinoic acid induced myeloid or the 1,25-dihydroxy vitamin D2 induced monocytic terminal differentiation of the cells. Bromodeoxyuridine, which induces proliferative arrest but not phenotypic differentiation of these cells, induced the same course of c-myb expression as the inducers of terminal differentiation. The same course of c-myb expression with growth arrest induced by these three different means is consistent with a potential proliferation regulatory role for c-myb in late but not early events leading to terminal differentiation. The dynamics of c-myb expression during this process were qualitatively, but not quantitatively, similar to the course of c-fos expression. Thus, taken with previous results, then amongst the nuclear oncogenes or tumor suppressor genes, c-myc, RB, c-fos, and c-myb, only c-myc and RB expression exhibit early regulation during induced HL-60 cell differentiation.


Use of peanut lectin and rat mammary stem cell lines to identify a cellular differentiation pathway for the alveolar cell in the rat mammary gland. The presence of the carbohydrate receptor for PNL has been used to identify the previously described morphological types of epithelial cell produced as the stem cell line rat mammary 25 (Rama 25) differentiates to casein secretory alveolar-like cells in vitro. Thus when cultures of the epithelial stem cell line Rama 25 are treated with neuraminidase, fluorescently-conjugated PNL fails to stain cuboidal cells, stains weakly grey cells, and stains strongly the surface of dark cells. When superconfluent cultures of Rama 25 are treated with dimethyl sulfoxide or retinoic acid and prolactin, estradiol, hydrocortisone, and insulin to induce differentiation to alveolar cells, PNL stains strongly the untreated surfaces of droplet cells and casein-secreting vacuolated cells. PNL-staining of the derivative cell lines with truncated cellular pathways, and quantitative binding of [125I]-labeled PNL to the cultured cells are consistent with this cellular staining pattern. The presence of the carbohydrate receptor for peanut lectin (PNL) has also been used to identify specific epithelial cell types in different mammary structures of the developing rat mammary gland, as they differentiate to casein secretory alveolar cells in vivo. Thus when different structures of the developing rat mammary gland are treated with neuraminidase, peroxidase-conjugated PNL fails to stain histochemically the majority of epithelial cells in ducts, stains the cytoplasm of the majority of epithelial cells in terminal end-buds (TEBs), and stains strongly the luminal surfaces of the majority of epithelial cells in alveolar buds (ABs). PNL also stains the untreated luminal surfaces of alveolar cells, whether or not the cells can be stained with a monoclonal antibody to rat beta-casein. Stimulation of mammary differentiation by an analogue of ethyl retinoate or by perphenazine causes cells in end-buds to bind PNL without the necessity for their desialylation similar to that seen in casein secretory alveoli of lactating rats. In conclusion the different interconverting cell types of Rama 25 which form a pathway to casein-secretory cells in vitro are thus equated with recognisable epithelial cell types in vivo. These results suggest that casein-secretory cells in vivo are generated by similar successive interconversions between the major epithelial cell types present in the different mammary structures in the order: ducts, TEBs, ABs, alveoli, and secretory alveoli.


Calcium transport by sarcoplasmic reticulum of vascular smooth muscle: II. Effects of calmodulin and calmodulin inhibitors. The role of calmodulin (CaM) in modulating calcium (Ca) uptake by sarcoplasmic reticulum (SR) of vascular smooth muscle was studied in saponin skinned strips of rat caudal artery. Exogenous CaM concentrations ranging from 0.3-1.8 microM did not statistically change the steady state MgATP-dependent Ca content, the MgATP-independent Ca content, or the oxalate-stimulated Ca influx. Calmidazolium (CDZ), W-7, and trifluoperazine (TFP) were used to examine the potential effect of an endogenous CaM pool on inward Ca transport. The IC50 of these antagonists for inhibition of Ca-CaM-stimulated phosphodiesterase activity and Ca-activated superprecipitation of canine aortic actomyosin was measured and found to be in the low micromolar range with a rank order of potency for inhibition of CDZ greater than TFP greater than W-7. In skinned tissues, micromolar concentrations of antagonists that inhibited CaM-mediated reactions in isolated enzyme systems did not reduce Ca content or oxalate-stimulated Ca influx. At higher concentrations of 100-200 microM, the MgATP-dependent Ca content was significantly reduced by TFP and W-7 but not by CDZ. The order of potency for inhibition of Ca uptake was TFP greater than W-7 greater than CDZ. The MgATP-independent Ca content was significantly decreased only by 200 microM TFP. Although none of these inhibitors significantly altered Ca efflux at concentrations up to 100 microM, Ca release was significantly stimulated by all three at 200 microM. The TFP-stimulated Ca release was partially inhibited by ruthenium red. The results indicate that neither exogenous CaM nor an endogenous CaM pool directly modulates inward Ca transport by the SR of saponin skinned caudal artery. The inhibition of Ca uptake produced by hundred micromolar concentrations of CaM antagonists fails to correlate with the order of and with the potency of inhibition measured in isolated enzyme systems. This suggests that the inhibition of Ca uptake produced by high concentrations of these antagonists may be independent of a specific interaction with CaM. The activation of Ca release by high concentrations of CaM antagonists may involve a nonspecific increase in membrane permeability as well as modulation of a membrane Ca channel.


Potassium transport in nonpigmented epithelial cells of ocular ciliary body: inhibition of a Na+, K+, Cl- cotransporter by protein kinase C. The mechanisms by which 86Rb+ (used as a tracer for K+) enters human nonpigmented ciliary epithelial cells were investigated. Ouabain-inhibitable bumetanide-insensitive 86Rb+ transport accounted for approximately 70-80% of total, whereas bumetanide-inhibitable ouabain-insensitive uptake accounted for 15-25% of total. K+ channel blockers such as BaCl2 reduced uptake by approximately 5%. Bumetanide inhibited 86Rb+ uptake with an IC50 of 0.5 microM, while furosemide inhibited with an IC50 of about 20 microM. Bumetanide-inhibitable 86Rb+ uptake was reduced in Na(+)-free or Cl(-)-free media, suggesting that Na+ and Cl- were required for optimal uptake via this mechanism. These characteristics are consistent with a Na+, K+, Cl- cotransporter in NPE cells. Treatment of NPE cells for 15 min with phorbol 12-myristate, 13-acetate (PMA), an activator of protein kinase C, caused a 50-70% decrease in 86Rb+ uptake via the Na+, K+, Cl- cotransporter. Other 86Rb+ uptake mechanisms were not affected. 86Rb+ uptake via the Na+, K+, Cl- cotransporter could be inhibited by other phorbol esters and by dioctanoylglycerol, an analog of diacylglycerol, but not by 4 alpha phorbol didecanoate, an ineffective activator of protein kinase C. Staurosporine, a protein kinase C inhibitor, blocked phorbol ester inhibition of 86Rb+ uptake. These data suggest that a Na+, K+, Cl- cotransporter in NPE cells is inhibited by activation of protein kinase C.


Electrogenic Na-independent HCO3 transport in OK cells. We have shown previously that OK cells recover from an acid load in a medium nominally CO2-free by extruding H via a Na/H exchanger and a passive H-conductive pathway. In this work, the regulation of cell pH (pHi) was studied after addition or withdrawal of CO2/HCO3 (5% CO2, 95 mM HCO3, pH = 8) using the fluoroprobe BCECF. In the presence of Na and amiloride to inhibit Na/H exchange, the recovery of pHi after CO2 entry and CO2 exit were found to depend in part on HCO3 entry and exit, respectively. Efflux of H per se also contributed to restoring pHi after CO2 addition, whereas H influx may have played a smaller role to normalize pHi after CO2 removal. DIDS, 0.5 mM, significantly inhibited both recovery phases of pHi. Removal of Na failed to inhibit the recovery of pHi after CO2 addition and removal. Cl removal also failed to inhibit pHi recovery after CO2 removal. Cell depolarization in the presence of Na moderately stimulated the pHi recovery rate after CO2 addition whereas it markedly inhibited the normalization of pHi after CO2 removal. Cell depolarization in the absence of sodium had only a slight effect to increase pHi recovery after CO2 addition but markedly prevented the pHi recovery after CO2 removal. These results indicate that OK cells lack Na or Cl-dependent HCO3 transport systems. The OK cell possesses a novel stilbene-sensitive electrogenic HCO3 transport system that is involved in the regulation of cell pH.


TGF-beta inhibits proliferation of and promotes differentiation of human promonocytic leukemia cells. Transforming growth factor-beta 1 (TGF-beta 1) has been implicated in a variety of responses associated with wound healing and inflammation. Thus, TGF-beta 1 enhances production of several extracellular matrix proteins both in vitro and in vivo, is chemotactic for monocytes, and alters the functioning of lymphocytes. We have examined the ability of TGF-beta 1 to affect the behavior of human THP-1 promonocytic leukemia cells, a cell line with the capacity to differentiate into macrophage-like cells. TGF-beta 1 reduces the growth rate of these cells, induces morphologic changes, and promotes adherence to culture surfaces. In addition, the adherent cell population expresses high levels of esterase activity, acquires the ability to ingest latex beads, and releases elevated levels of interleukin 1. TGF-beta 1-treated cells also express elevated levels of the beta 2 family of integrins. Taken together, these results suggest that TGF-beta 1 is capable of promoting the maturation of promonocytic cells into macrophages. This outcome has implications at wound sites where TGF-beta 1 and a myriad of other factors interact with many cell types to facilitate healing.


Amnion cell biosynthesis of interleukin-8: regulation by inflammatory cytokines. The cellular constituents of the placenta are important participants in the recruitment and trafficking of inflammatory cells within the placenta. In infection-induced labor, gestational tissues synthesize and release a variety of inflammatory cytokines whose effects include increased prostaglandin biosynthesis and the initiation of uterine contractions. Interleukin-8 (IL-8), a potent neutrophil chemoattractant, has been recently described as being elevated in the amniotic fluid of mothers with chorioamnionitis. We investigated the biosynthesis of IL-8 by human amnion cells and its regulation by other inflammatory cytokines. Cultured amnion cells obtained from normal term placentae were found to produce IL-8 in response to pathophysiologic concentrations of interleukin 1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha). Treatment of amnion cells stimulated by IL-1 beta with cycloheximide resulted in increased IL-8 production, while incubation of IL-1 beta treated amnion cells with actinomycin D resulted in a concentration-dependent decrease in detectable amounts of IL-8. Northern blot analysis of cultured amnion cells stimulated with IL-1 beta demonstrated a rapid increase in IL-8 mRNA which peaked at 2-4 hr. These in vitro results suggest inflammation of gestational tissues in vivo may result in locally produced IL-8 and, in association with other inflammatory mediators, may be important in the pathophysiology of infection-induced labor.


Proliferation-associated oxygen consumption and morphology of tumor cells in monolayer and spheroid culture. The oxygen consumption rate, proliferative activity, and morphology of EMT6/Ro mouse mammary sarcoma cells in monolayer and multicellular spheroid culture have been investigated in a comparative study. During the transition of monolayer cells from the exponential into the plateau growth phase, there is a distinct decrease in the cellular volume that is associated with a corresponding decrease in the proliferative and respiratory activity of the cells. The decline in cell volume is mainly due to a decrease in the content of cytoplasm, whereas the size of the nucleus is only slightly reduced. A concomitant decrease in the number of mitochondria per cell obviously accounts for the reduction in cellular oxygen uptake. Despite a continuous decrease of cell proliferation from the surface to interior regions of EMT6 spheroids reflected by a gradient in tritiated thymidine labeling, volume-related oxygen consumption is rather uniform in viable regions of these aggregates. The finding can be explained by the results of the morphometric evaluation showing a uniform volume density of mitochondria, i.e., of oxygen-consuming sites within these spheroids.


Protein kinase C phosphorylates caldesmon77 and vimentin and enhances albumin permeability across cultured bovine pulmonary artery endothelial cell monolayers. Cytoskeletal protein (CSP) interactions are critical to the contractile response in muscle and non-muscle cells. Current concepts suggest that activation of the contractile apparatus occurs through selective phosphorylation by specific cellular kinase systems. Because the Ca(2+)-phospholipid-dependent protein kinase C (PKC) is involved in the regulation of a number of key endothelial cell responses, the hypothesis that PKC modulates endothelial cell contraction and monolayer permeability was tested. Phorbol myristate acetate (PMA), a direct PKC activator, and alpha-thrombin, a receptor-mediated agonist known to increase endothelial cell permeability, both induced rapid, dose-dependent activation and translocation of PKC in bovine pulmonary artery endothelial cells (BPAEC), as assessed by gamma-[32P]ATP phosphorylation of H1 histone in cellular fractions. This activation was temporally associated with evidence of agonist-mediated endothelial cell contraction as demonstrated by characteristic changes in cellular morphology. Agonist-induced activation of the contractile apparatus was associated with increases in BPAEC monolayer permeability to albumin (approximately 200% increase with 10(-6) MPMA, approximately 400% increase with 10(-8) M alpha-thrombin). To more closely examine the role of PKC in activation of the contractile apparatus, PKC-mediated phosphorylation of two specific CSPs, the actin- and calmodulin-binding protein, caldesmon77, and the intermediate filament protein, vimentin, was assessed. In vitro phosphorylation of both caldesmon and vimentin was demonstrated by addition of exogenous, purified BPAEC PKC to unstimulated BPAEC homogenates, to purified bovine platelet caldesmon77, or to purified smooth muscle caldesmon150. Caldesmon77 and vimentin phosphorylation were observed in intact [32P]-labeled BPAEC monolayers stimulated with either PMA or alpha-thrombin, as detected by immunoprecipitation. In addition, BPAEC pretreatment with the PKC inhibitor, staurosporine, prevented alpha-thrombin- and PMA-induced phosphorylation of both cytoskeletal proteins, attenuated morphologic evidence of contraction, and abolished agonist-induced barrier dysfunction. These results demonstrate that agonist-stimulated PKC activity results in cytoskeletal protein phosphorylation in BPAEC monolayer, an event which occurs in concert with agonist-mediated endothelial cell contraction and resultant barrier dysfunction.


Quantitative fatty acid analyses in cultured porcine pulmonary artery endothelial cells: the combined effects of fatty acid supplementation and oxidant exposure. Supplemental fatty acids can modify the oxidant susceptibility of pulmonary artery endothelial cells (PAEC) in monolayer culture. In addition, in vivo dietary modifications have altered tissue and animal susceptibility to a variety of forms of oxidant stress. These modifications of oxidant injury have been attributed to changes in the numbers of fatty acid double bonds in cell lipids. We tested this hypothesis by incubating porcine PAEC in culture medium supplemented with either 0.1 mM oleic acid (18:1 omega 9) or with an equivalent volume of ethanol vehicle alone (ETOH-0.1%) for 3 h. After supplementation, PAEC were exposed to either oxidant stress, 100 microM hydrogen peroxide (H2O2) in Hanks' balanced salt solution (HBSS), or to control condition, HBSS alone, for 30 min. Supplemental PAEC were exposed to HBSS or H2O2 either immediately or 24, 48, or 72 h after supplementation. Supplementation with 18:1 protected PAEC from H2O2-induced injury at all time points. The fatty acid composition of PAEC phospholipid (PL), triglyceride (TG), and free fatty acid (FFA) subclasses was determined using thin layer and gas chromatography. The PL fraction contained the majority of PAEC fatty acids, and H2O2 reduced the polyunsaturates in this fraction regardless of supplementation. Supplementation with 18:1 increased the 18:1 content of PAEC PL, TG, and FFA at all time points, modified other fatty acids to a lesser extent, but failed to alter the overall number of fatty acid double bonds at all time points. These results indicate that modification of double bond number does not fully explain the mechanisms by which changes in lipid composition can modulate oxidant injury.


Morphological and immuno-cytochemical characterization of a hetero-spheroid composed of fibroblasts and hepatocytes. A novel method for the preparation of spheroids containing two types of cells (hetero-spheroid) has been successfully developed by utilizing a collagen-conjugated thermo-responsive polymer, poly-N-isopropyl acrylamide (PNIPAAm), as a cell substratum. PNIPAAm solidifies above its lower critical solution temperature (LCST, about 30 degrees C), and instantly dissolves into the culture medium below its LCST. We firstly seeded and cultured human dermal fibroblasts on the substratum up to a confluent state and then seeded rat primary hepatocytes onto the fibroblast monolayer. The heterospheroid was prepared by detaching the hepatocyte-attached fibroblast monolayer at a temperature below LCST and culturing it on the non-adhesive substratum. The surface area of the substratum and the seeding population ratio of each cell precisely and reproducibly regulated the size and the cell composition of the resulting hetero-spheroid, respectively. Histological and immuno-cytochemical observations of spheroids revealed characteristic organizations of fibroblasts and hepatocytes within a spheroid because the latter cells expressed albumin for up to at least 3 weeks. TEM study of the hetero-spheroid showed the presence of structures morphologically similar to the Disse's space and the bile canaliculus, which are features characteristic of liver. These findings suggest that the method described above is useful for making a hetero-spheroid that morphologically and functionally resembles tissues or organs in vivo, i.e. an organoid.


A method for determining the periodicity of a troponin component in isolated insect flight muscle thin filaments by gold/Fab labelling. Insect flight muscle has a large component (Tn-H) in the tropomyosin-troponin complex that is not present in vertebrate striated muscle thin filaments. Tn-H is shown by gold/Fab labelling to be present at regular intervals in insect flight muscle thin filaments. The Fab fragment of a monoclonal antibody to Tn-H was conjugated directly with colloidal gold and this probe used to label isolated thin filaments from the flight muscle of Lethocerus indicus (water bug). The distribution of gold particles seen in electron microscope images of negatively stained thin filaments was analysed to show that the probe bound to sites having a periodicity of approximately 40 nm, which is the expected value for the tropomyosin-troponin repeat. Conjugates of Fab with colloidal gold particles of 3 nm diameter labelled almost all sites. Conjugates with gold particles of 5 nm and 10 nm diameter labelled less efficiently (70% and 30%, respectively) but analysis of the distribution of inter-particle intervals among a number of filaments again gave the same fundamental spacing of 40 nm. The error in the measurements (standard deviation approximately +/- 4.2 for 5 nm gold/Fab) is less than earlier estimates for the size of the gold/Fab complex. Measurements on gold/Fab in negative stain suggest that the bound Fab contributes a shell about 2 nm in thickness around the gold particle. The radius of the probe (about 4.5 nm for 5 nm gold/Fab) would then be consistent with the value of error found. The size of the probe suggests that the gold particle binds to the side of the Fab molecule, rather close to the antibody combining site. The potential resolution of the technique may thus be better than originally expected.


Patterns of DNA replication in Drosophila polytene nuclei replicating in Xenopus egg and oocyte extracts. We have used Xenopus laevis cell-free extracts to study patterns of DNA replication in polytene nuclei isolated from salivary glands of Drosophila melanogaster 3rd instar larvae. Replication was visualized by supplementation with biotin-dUTP so that nascent DNA became labelled, thus allowing detection with fluorescein or Texas-Red-conjugated streptavidin. Biotin incorporation was dependent on incubation in extracts. Transverse bands were labelled in high-speed supernatants of eggs or oocytes in which replication does not initiate de novo. These patterns corresponded to the patterns of endogenous replication forks in polytene nuclei, monitored by bromodeoxyuridine incorporation in intact salivary glands. By contrast, when nuclei were incubated in low-speed supernatants of eggs, they underwent more extensive chromatin decondensation and initiated replication. The spatial patterns of replication are strikingly different from the endogenous patterns. Instead they closely resemble patterns of clustered replication forks seen in Xenopus sperm nuclei replicating in the extract. This indicates that the egg extract can impose its pattern of replication foci even when the template is presented in the highly organized form of a polytene nucleus.


Cytoskeletal and DNA structure abnormalities result from bypass of requirement for the cdc10 start gene in the fission yeast Schizosaccharomyces pombe. The cdc10 gene of the fission yeast S. pombe is required for traverse of the start control in late G1 and commitment to the mitotic cell cycle. To increase our understanding of the events which occur at start, a pseudoreversion analysis was undertaken to identify genes whose products may interact with cdc10 or bypass the requirement for it. A single gene, sct1+ (suppressor of cdc ten), has been identified, mutation of which suppresses all conditional alleles and a null allele of cdc10. Bypass of the requirement for cdc10+ function by sct1-1 mutations leads to pleiotropic defects, including microtubule, microfilament and nuclear structural abnormalities. Our data suggest that sct1 encodes a protein that is dependent upon cdc10+ either for its normal function or expression, or is a component of a checkpoint that monitors execution of p85cdc10 function.


Mutants of the microtubule motor protein, nonclaret disjunctional, affect spindle structure and chromosome movement in meiosis and mitosis. The Drosophila microtubule motor protein, nonclaret disjunctional (ncd), is required for proper chromosome distribution in meiosis and mitosis. We have examined the meiotic and mitotic divisions in wild-type Drosophila oocytes and early embryos, and the effects of three ncd mutants (cand, ncd and ncdD) on spindle structure and chromosome movement. The ncd mutants cause abnormalities in spindle structure early in meiosis I, and abnormal chromosome configurations throughout meiosis I and II. Defective divisions continue in early embryos of the motor null mutant, cand, with abnormal early mitotic spindles. The effects of mutants on spindle structure suggest that ncd is required for proper meiotic spindle assembly, and may play a role in forming or maintaining spindle poles in meiosis. The disruption of normal meiotic and mitotic chromosome distribution by ncd mutants can be attributed to its role as a spindle motor, although a role for ncd as a chromosome-associated motor protein is not excluded. The ncd motor protein functions not only in meiosis, but also performs an active role in the early mitotic divisions of the embryo.


The upright position of brush border-type microvilli depends on myosin filaments. We have studied the correlation between the actomyosin organization and microvillar position in an epithelial cell line derived from the proximal pig kidney tubule (LLC-PK1). When grown on glass, these cells are approximately 5-6 microns in height and develop numerous microvilli that project from the dorsal membrane. A fairly homogeneous distribution of microvilli was achieved by synchronization of the cell cycle. These microvilli are of the brush border type, as defined by their content of villin and their anchorage in a myosin-rich terminal web-like structure. When LLC-PK1 cells were injected with two monoclonal antibodies against pig brain nonmuscle myosin, in concentrations yielding a 1:1 ratio of antibody to myosin, neither microvillar number nor length was affected. However, when we examined the cells by scanning electron microscopy 1-3 h after microinjection, we found that one of the antibodies (a-PBM 4) had a profound effect on microvillar position: more than 50% were seen tilted or lying prone on the plasma membrane. The microvilli of cells injected with the other antibody (a-PBM 9) were not significantly different from those of cells injected with control antibodies. This difference correlates with in vitro properties of the antibodies: a-PBM 4 decreases the actin-activated Mg(2+)-ATPase of pig brain nonmuscle myosin quite substantially, while a-PBM 9 affects it only moderately. These differential effects are probably a consequence of the different epitope location as determined for both antibodies, not of differences in antibody affinity. Our data are compatible with the hypothesis that a-PBM 4 also interferes with the actomyosin interaction in situ, thus decreasing the effective cross-linking of microvillar rootlets by myosin filaments in the terminal web. On the basis of this model, we suggest that myosin filaments are essential for the upright position of brush-border type microvilli.


The contribution of the extracellular matrix to gravisensing in characean cells. The cell-extracellular matrix junction, which includes the cell wall and the outer surface of the plasma membrane, may be an essential region for the perception of gravity by the internodal cells of Chara corallina. Typically, when an internodal cell is oriented vertically, the downwardly directed cytoplasmic stream travels at a velocity that is 10% faster than that of the upwardly directed stream. However when the cells are treated with impermeant hydrolytic enzymes that partially digest cellulose or hemicellulose, the cells lose their ability to respond to gravity even though streaming continues. By contrast, enzymes that digest pectins have no effect on the gravity-induced polarity of cytoplasmic streaming. Furthermore, gravisensing is sensitive to protease treatment; Proteinase K, thermolysin and collagenase but not trypsin, alpha-chymotrypsin or carboxypeptidase B, inhibit gravisensing. These findings indicate that proteins in the cell-extracellular matrix junction may be required for gravisensing. Moreover, the tetrapeptide Arg-Gly-Asp-Ser (RGDS) inhibits gravisensing in a concentration-dependent manner, indicating that the gravireceptor may be an integrin-like protein. The macromolecules necessary for gravisensing have been localized to the cell ends. As a consequence of the exoplasmic site of action of the enzymes and the tetrapeptides, we interpret the results to mean that they are acting on the gravireceptor, although we cannot eliminate the possibility that they are acting on the signal transduction chain. On the whole, our observations indicate that the cell-extracellular matrix junction is a sine qua non for graviperception in statolith-free Chara internodal cells and we suggest that the gravireceptor is located in this region.


The 14 kDa beta-galactoside binding lectin in myoblast and myotube cultures: localization by confocal microscopy. The endogenous 14 kDa beta-galactoside-specific lectin has been localised in myoblasts and myotubes by indirect immunofluorescence using confocal microscopy. The antibodies used in these experiments suggest that in myoblast cultures the lectin is abundant on the cell surface and concentrated at ruffled edges of migrating cells. The quantity and distribution of cell surface lectin is independent of the nature of the substratum and undiminished by brief trypsinisation or lactose-washing of the cell suspension. The lectin is also abundant intracellularly, apparently relatively free in the cytoplasm until the cells approach confluency, when the lectin is concentrated into particular areas of cytoplasm resulting in a 'patchy' appearance of stained cells. After fusion to form multinucleate myotubes, intracellular lectin is less abundant and concentrated at the periphery of myotubes, from where lectin appears to be released in vesicles packed with lectin which 'bud off' from the myotube surface. In recolonising cultures, lectin is apparently more abundant and lectin-packed vesicles can also be seen 'budding off' from myoblasts. In differentiated cultures, extracellular lectin is detected both on the myotube surface and in fibrillar arrays of extracellular material.


The interendothelial junction in myocardial capillaries: evidence for the existence of regularly spaced, cleft-spanning structures. Water and hydrophilic solutes cross the endothelium of continuous capillaries via the paracellular cleft and possibly other routes. This pathway shows a selectivity to molecule size and charge. However, it is not yet known which systems confer this selectivity. Isolated rat hearts were perfusion-fixed through the coronary circulation, stained with lanthanum or tannic acid, and further processed for transmission electron microscopy. Thin sections viewed at x 160,000 magnification revealed regularly spaced, cleft-spanning structures in the wider zone of a small percentage of clefts in addition to at least one zonula occludens. Goniometric tilting of the specimen in steps of 5 degrees perpendicular to the plane of the wide zone showed that such "linkers" can be revealed in at least 40% of all clefts. They become visible at some tilt angles, although the same area of the cleft is featureless at other angles. Single linker spacing measurements were obtained using a computerized image analysis system, and compiled in a frequency distribution chart. On the basis of these data, two models of a regular linker distribution within the cleft are illustrated. Our results provide evidence for the presence of regularly spaced, cleft-spanning structures within the interendothelial cleft which may have implications for endothelial cell-cell adhesion and permeability.


Redistribution of nuclear lamin A is an early event associated with differentiation of human promyelocytic leukemia HL-60 cells. The nuclear lamina of mammalian somatic cells is characterized by the constitutive presence of lamin B polypeptides while the appearance of lamins A and C generally occur during establishment of a differentiated phenotype. We have used antibodies specific to the unique carboxy-terminal domain of lamin A, i.e. distinct from the shared domains of lamins A and C, to study the individual behaviour of lamin A during establishment of a macrophage phenotype in human HL-60 cells. Lamin A was present as a nuclear cap in the majority of undifferentiated cells and it was redistributed to a full peripheral nuclear location very early after induction of differentiation by phorbol esters, even in the presence of a protein synthesis inhibitor. Induction of the cells into a reversible precommitment state by bromodeoxyuridine was accompanied by a similar redistribution of lamin A that however reverted to a cap after removal of inducer. No changes were observed in the uniform peripheral nuclear location of lamin C under all of these conditions. These results strongly suggest that lamin A plays a role in the early events of cell differentiation. Taken together with previous results on the interaction of A-type lamins with chromatin, these findings offer experimental evidence consistent with the proposed role of A-type lamins, and particularly lamin A, in the process of chromatin reorganization that accompanies the expression of a differentiated phenotype.


Protein kinase C isotypes in human erythroleukemia cell proliferation and differentiation. The human erythroleukemia (K562) cell line is induced to differentiate into megakaryocytic cells by treatment with the tumor promoter phorbol myristate acetate (PMA). PMA-induced differentiation is characterized by (1) almost complete cessation of cellular proliferation, (2) expression of the megakaryocytic cell surface marker glycoprotein IIb/IIIa (gpIIIa), (3) increased secretion of granulocyte/macrophage-colony stimulating factor (GM-CSF) and (4) increased secretion of interleukin-6 (IL-6). PMA-induced differentiation is dose-dependent with maximal activity seen at 10 nM PMA. In contrast, bryostatin (bryo), a structurally distinct protein kinase C (PKC) activator, fails to induce megakaryocytic differentiation or growth arrest at the concentrations tested (0.01-100 nM). Rather, bryo inhibits PMA-induced growth arrest and megakaryocytic differentiation in a dose-dependent fashion (full inhibition at 100 nM). The divergent biological effects of PMA and bryo correspond to the differential activation and translocation of PKC isotypes in K562 cells. PKC isotype analysis demonstrates that undifferentiated cells express both alpha and beta II PKC but no detectable beta I, gamma or epsilon PKC. Treatment of cells with either PMA or bryo leads to rapid translocation of both alpha and beta II PKC from the cytosol to the non-nuclear particulate fraction. However, bryo also induces selective translocation of beta II PKC to the nuclear membrane.(ABSTRACT TRUNCATED AT 250 WORDS)


Tumour infiltrating macrophages are capable of bone resorption. The cell types responsible for osteolysis associated with skeletal metastasis of solid malignancies are unclear. Tumour infiltrating macrophages (TIMs) isolated from primary mammary carcinomas of C3H/Avy mice were cultured on bone slices to assess their ability to resorb bone. After 14 days in co-culture with murine marrow stromal cell line ST2 and added 1,25-dihydroxyvitamin D3 and dexamethasone, TIMs showed increased tartrate-resistant acid phosphatase activity and formed numerous lacunar resorption pits. In the absence of ST2 cells, TIMs did not form lacunar resorption pits but produced roughening of the bone surface with exposure of mineralized collagen fibres. Normal alveolar macrophages, in both the presence and absence of ST2 cells similarly produced only surface resorption. TIMs are thus capable of both low-grade (surface) and high-grade (lacunar) pathological bone resorption, a specific interaction with stromal cells being necessary for the latter to occur. TIM-mediated bone resorption could account for different clinical and pathological patterns of tumour osteolysis.


Interference in vimentin assembly in vitro by synthetic peptides derived from the vimentin head domain. The importance of the amino-terminal domain ("head") of type III intermediate filament (IF) proteins in IF assembly has been examined by testing the influence of synthetic peptides representing a highly conserved decameric motif, KSSSYRRIMFGG, located near the amino terminus of vimentin. When added to soluble vimentin subunits this peptide induces, at fourfold molar excess or slightly above, the appearance of short, regular rod-like structures as determined by electron microscopy of negatively stained and rotary-shadowed preparations as well as by viscometry. At higher peptide concentrations large, irregularly shaped aggregates of mostly non-IF structures formed, but this aggregation was reversible by prolonged dialysis against low ionic strength buffer. The aggregating effect of this peptide was highly sequence-specific and was not seen with point-mutated sequences such as RR----TR or with unrelated peptides containing a central diarginine, indicating that it is not simply ionic. When different hexapeptides representing different "head" positions were compared, only the central sequence, SYRRXF, was as effective as the decamer. The addition of peptide during IF assembly did not prevent filament formation, although 50-fold molar excess of peptide resulted in a drastic increase (up to 40 nm) in the width of the filaments, which also appeared less regular, thus reflecting some interference with assembly. In contrast to the effects on soluble vimentin, the decameric peptide did not disturb IFs, indicating that the binding domain is "masked" or stabilized in the filaments. To identify the domain to which the peptide binds, three different binding assays using vimentin fragments and genetically engineered vimentin deletion mutants were employed. The results indicate that the binding domain of the near-amino-terminal peptide is located at the start of the alpha-helical "rod" domain of the protein. Possible mechanisms of interaction of these two portions of vimentin during IF assembly are discussed.


Screening and identification of a gene, PSE-1, that affects protein secretion in Saccharomyces cerevisiae. Utilizing a screening method designed for the identification of genes involved with enhanced protein secretion in Saccharomyces cerevisiae we identified a gene, which we named PSE-1 (Protein Secretion Enhancer). Overexpression of PSE-1 in a multi-copy plasmid, as shown by Northern hybridization, gave a fourfold enhancement in total protein secretion. The repertoire of proteins that are found to be secreted in greater quantities include three known biologically active proteins: k1 killer toxin, alpha-factor, and acid phosphatase. The PSE-1 gene is located on chromosome XII of the yeast genome and codes for a hydrophobic protein containing 1089 amino acids. Haploid yeast cells that contained a LEU2 insertion mutation in PSE-1 grow very poorly, a phenotype similar to other conditional SEC mutants at restrictive temperature.


The effects of glucocorticoid therapy.

The effects of glucocorticoid therapy. The therapeutic benefit of glucocorticoids appears to derive chiefly from their suppression of immunologic and inflammatory responses, but beyond these generalized phenomena, there are a number of tissue-specific effects. Increasing knowledge of these actions is shedding light on such issues as steroid resistance and steroid-induced adrenocortical insufficiency.


Current status of HIV therapy: II. Opportunistic diseases. Infections and malignancies account for most deaths in patients with AIDS and will continue to do so as long as HIV-induced immunosuppression is progressive and irreversible. Co-trimoxazole has emerged as the preferred agent for prevention of Pneumocystis carinii pneumonia. As appropriate broad-spectrum agents are developed, multiple opportunistic pathogen prophylaxis could become effective.


Effective management of penetrating head injury. Although head injuries are usually easily recognized, other, less obvious lesions should also be promptly investigated. In either case, outcome is often determined in the first few minutes and hours of management.


Determination of sulphonamides in pork meat extracts by capillary zone electrophoresis. For sixteen sulphonamides the effective mobility was measured as a function of pH and from the effective mobilities determined in two different electrolyte systems the pK value and mobility at infinite dilution were calculated. A pH of 7.0 was found to be the optimum pH for the separation for both standard mixtures and mixtures of sulphonamides dissolved in pork meat extracts. For the determination of the sulphonamides in pork meat only a very simple pretreatment consisting of extraction with acetonitrile and centrifugation is suitable, as the matrix effects at pH 7.0 do not affect the separation. Calibration graphs for five sulphonamides were constructed, and regression coefficients of at least 0.999 were obtained. The limit of detection for the method varies from 2 to 9 ppm for a pressure injection time of 10 s (injection volume ca. 18 nl) using a Polymicro Technology capillary of length 116.45 cm, distance between injection and detection 109.75 cm and I.D. 50 microns.


Apparent inter-channel interference in dual-electrode electrochemical detection. During the course of routine high-performance liquid chromatographic analyses of brain catecholamines using dual-electrode electrochemical detection, we encountered an unusual negative peak in the lower-voltage channel. Subsequent investigations suggested that this peak was caused by tyrosine which produced a positive peak in the higher-voltage channel. Our investigations indicate that compounds that generate a peak in one channel appear to be responsible for complex peaks in a second channel set at a lower voltage, close to or below that necessary for oxidation. The complex peaks are biphasic; a sharp negative peak coinciding with the positive peak on the higher-voltage channel, followed by a positive peak. This effect was not specific for tyrosine, but was observed on the lower-voltage channel with all compounds tested that produced signals on the high-voltage channel. The cause of the problem is unknown, but it appears to be an artifact of the electrical coupling of the two electrode channels in a dual-channel system.


Counter-current chromatography of lipoproteins with a polymer phase system using the cross-axis synchronous coil planet centrifuge. Lipoproteins were separated by counter-current chromatography using the type-XLL coil planet centrifuge. The separation was performed with a polymer phase system composed of 16% (w/w) polyethylene glycol 1000 and 12.5% (w/w) dibasic potassium phosphate by eluting the lower phase at a flow-rate of 0.5 ml/min. About 5 ml of the sample solution containing approximately 150 mg of a lipoprotein mixture were loaded. High- and low-density lipoproteins were resolved within 12 h. Each component was detected by gel electrophoresis with oil red staining.


Direct high-performance liquid chromatographic separation of enantiomeric peptidoleukotriene antagonists. Enantiomeric peptidoleukotriene antagonists, SK&F R-106203 and SK&F S-106203 can be effectively separated on a cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phase. The utility of this chiral high-performance liquid chromatographic method in assigning absolute stereochemistry to SK&F S-106203-Z2, a non-crystalline amorphous compound which is not amenable to single crystal X-ray analysis, is demonstrated by correlation with the absolute configuration determined crystallographically for a second salt form.


DNA electrophoresis in uncross-linked polyacrylamide solution, studied by epifluorescence microscopy. Electrophoresis of human DNA fragments (approximately 1 x 10(5) to 1 x 10(7) bases in size) was conducted in a solution of uncross-linked polyacrylamide contained in a horizontally mounted 1 mm diameter glass tube and monitored by epifluorescence microscopy. In presence of the polymer, molecular conformations described as a "trailing network" of DNA and a globular "head" were observed. The migration velocity varies between species differing in the size of the "head", and in the ratio between the size of the "head" and that of the trailing "network". By contrast, in pure buffer, lambda phage DNA migrates in a globular form at a mobility consistent with known macroscopic data. When electrophoresis in the polymer solution of an agarose plug preparation of Schizosaccharomyces pombe DNA was carried out after melting at 70 degrees C, a migrating DNA-agarose complex was observed. The complex was not fully dissociated by an agarose-hydrolyzing enzyme (Gelase).


Ion- and ligand-exchange chromatography of proteins using porous zirconium oxide supports in organic and inorganic Lewis base eluents. The applicability of an eluotropic scale pertaining to the desorption of low molecular weight Lewis base solutes from zirconium oxide is examined for its ability to rationalize the retention of proteins on this substrate. The strongest Lewis base eluents (phosphate and fluoride) are able to bring about elution of nearly all proteins provided that their initial mobile phase concentration almost saturates the eluent's adsorption isotherm. In contrast, weaker Lewis bases such as borate, sulfate and bromide are able to elute only those proteins which are retained primarily by ionic interactions. In weak eluents, proteins that contain a large number of accessible Lewis base sites are not eluted from the support. The effect of ionic strength and a variety of Lewis base eluents were also examined.


Simultaneous purification of the neuroproteins synapsin I and synaptophysin. A procedure for the simultaneous purification of synapsin I and synaptophysin from calf brain was developed. Demyelinated membranes were extracted with 2% Triton X-100 and 2 M KCl. The extracted proteins were separated by weak cation-exchange chromatography on carboxymethyl-Sepharose Fast Flow. Synaptophysin was finally purified by preparative sodium dodecyl sulphate-polyacrylamine gel electrophoresis and synapsin I by affinity chromatography using a calmodulin-Sepharose column. The recovery obtained was 40 micrograms/g in brain for synaptophysin and 25 micrograms/g in brain for synapsin I.


Determination of nitroxynil in cow milk by reversed-phase high-performance liquid chromatography with dual-electrode coulometric detection. A sensitive and specific method is described for the determination of nitroxynil (fasciolicide) residues in cow milk. The milk samples were extracted with acetone and acetonitrile, following clean-up using a simple liquid-liquid extraction step. Nitroxynil was separated from the matrix peaks by reversed-phase high-performance liquid chromatography and detected using dual-electrode coulometric detection. The mobile phase was a mixture (20:80, v/v) of acetonitrile and 0.05 M potassium dihydrogenphosphate with the pH adjusted to 4.0. The flow-rate was 1 ml/min at 40 degrees C. The applied potentials of detectors 1 and 2 were maintained at -0.7 and +0.2 V, respectively. Average recoveries (n = 5) of nitroxynil from milk samples spiked at concentrations of 0.01 and 0.1 micrograms/ml were found to be 92.0% and 97.0% with coefficients of variation of 6.2% and 2.2%, respectively. The detection limit of nitroxynil in milk was 0.7 ng/ml. During the analysis of 30 raw cow milk and 140 market milk samples, nitroxynil was detected at a level of 4 ng/ml in one raw cow milk sample.


Reversed-phase high-performance liquid chromatography of several metal-8-quinolinethiol complexes. The reversed-phase high-performance liquid chromatography of 8-quinolinethiol (Hqt) complexes of Fe, Co, Ni, Cu, Zn, Hg and Pb on an octadecyl-bonded silica gel stationary phase was examined. The Pb complex dissociated in the column. The retention and separation of other complexes depended on the composition of the mobile phase. EDTA as an additive displaced the Zn complex and eliminated its peak. All the other metal complexes and also Hqt and its disulphide were separated in 23 min by using methanol-water (82:18, v/v) as the mobile phase. The complexes formed by the reaction of Co(II) and Hqt gave three peaks, which were assigned as fac(S)-CoIII(qt)3, mer(S)-CoIII(qt)3 and CoII(qt)2, respectively. This method is applicable to the simultaneous determination of Fe, Co, Ni, Cu and Hg.


Structure-retention index relationships for derivatized monosaccharides on non-polar gas chromatography columns. A gas chromatographic method for predicting the retention index of a derivatized monosaccharide is presented. The procedures are especially useful to detect and predict minute quantities of sugars in biological or chemical samples. Monosaccharides are first converted to the alditols and then derivatized by acetylation, permethylation or silylation. The derivatized monosaccharide structure-retention index relationship that has been developed is useful in the identification of unknown monosaccharides that can be readily confirmed by gas chromatography-mass spectrometry.


Micellar electrokinetic chromatographic study of hydroquinone and some of its ethers. Determination of hydroquinone in skin-toning cream. The separation of hydroquinone and some of its ether derivatives was studied by micellar electrokinetic chromatography with sodium dodecyl sulphate as an anionic surfactant in the background electrolyte. The optimized method was used for the determination of hydroquinone in a sample of skin-toning cream. On-column detection at 254 nm with caffeine as an internal standard gave good quantitative results.


Embryological observations on the female genital tract. The embryology of the genital tract and urinary system is described, and the hypothesis is advanced that the vagina is an organ embryologically derived from the mesonephric or Wolffian ducts in addition to the Müllerian tubercle. This is based on experimental studies and case reports in the literature and our own cases of genital malformations, especially in patients with unilateral renal agenesis and an ipsilateral blind vagina. The importance of the mesonephric ducts as guides or 'inductor' elements for adequate Müllerian development is emphasized. A new embryological classification of female genital malformations is proposed, based on these embryological concepts.


A stereological study of the human ovarian surface epithelium. The surface of the ovary has been found to be composed of two types of epithelial cells called A and B cells which are found in their own respective zones, the A and B zones. A quantitative study was undertaken to determine the mean cell volumes and cell ultrastructure. Ovarian biopsies were taken from six women and A and B zones, having been identified by scanning electron microscopy, were re-embedded for transmission electron microscopy. Stereological measurements using point sampled intercepts were made on vertical sections and showed that B cells are significantly larger than A cells. The volume weighted mean cell volumes of the A and B cells were 237.3 microns 3 and 676.8 microns 3 respectively. The volume fractions of the nucleus and mitochondria were similar in the two cell types. Although the vesicle content of each type was similar, a large variation between cases made the interpretation difficult. The stereological tools used in this study proved to be easy and efficient estimators of surface cell ultrastructure and give an important direction for ultrastructural research.


Implications of ultrasonically diagnosed polycystic ovaries. I. Correlations with basal hormonal profiles. The incidence of ultrasonically diagnosed polycystic ovaries (PCO) was studied in 389 Arab patients with different types of menstrual dysfunction and 100 normal women with regular menstruation. Two-hundred-and-forty-six patients (63.2%) were found to have PCO but only 206 (53.0%) were confirmed as cases of polycystic ovarian disease (PCOD) on endocrine grounds. Polycystic ovaries were diagnosed in 50% of patients with hyperprolactinaemia, 36.4% with hypothyroidism, 23.7% with hypothalamic dysfunction, 100% with adrenal 21-hydroxylase deficiency and in 16.0% of normal women. More women with PCOD presented with oligomenorrhoea or dysfunctional uterine bleeding (77.7%) and hirsutism (72.3%) but obesity had no discriminating value between the groups with different diagnoses. Ultrasonic diagnosis of PCO should be supplemented with an endocrine biochemical assessment to prevent overdiagnosis of PCOD and to exclude other endocrine dysfunctions.


The correlation of interleukin 1 and tumour necrosis factor to oestradiol, progesterone and testosterone levels in periovulatory follicular fluid of in-vitro fertilization patients. Data has accumulated suggesting reciprocity between cytokines and the reproductive system. The present study was performed in order to evaluate the correlation between interleukin 1 (IL-1) and tumour necrosis factor (TNF) concentrations in follicular fluid and its oestradiol, progesterone and testosterone levels. A total of 39 follicular fluid samples, from eight patients undergoing in-vitro fertilization and embryo transfer were evaluated. All of the patients were treated by a midluteal (long) protocol involving a gonadotrophin releasing hormone agonist (GnRHa) coupled with follicular phase human menopausal gonadotrophin. Mean levels in follicular fluid of IL-1, TNF, oestradiol, progesterone and testosterone were 1.58 +/- 0.42 fmol/0.1 ml, 4.69 +/- 4.18 pg/ml, 28.5 +/- 58.1 ng/ml, 2360.5 +/- 2846.3 ng/ml and 7.22 +/- 7.08 ng/ml respectively. There was a significant (P less than 0.01) positive correlation between IL-1 and progesterone levels. There was no significant correlation between the different lymphokines and oestradiol secretion, oocyte fertilization, embryo quality and pregnancy rates. It is concluded that IL-1 and TNF exist in follicular fluid. It may be hypothesized that IL-1 has a local regulatory action, possibly promoting luteinization.


Positive feedback effect of oestradiol in superovulated women. To investigate the mechanism of blockage of the luteinizing hormone (LH) surge in superovulated women, six normally ovulating women were studied in three cycles: a spontaneous cycle treated with exogenous oestrogen (oestradiol benzoate cycle), a cycle treated with follicle stimulating hormone (FSH; 225 IU/day; FSH cycle) and a cycle treated with FSH plus exogenous oestrogen (FSH + oestradiol benzoate cycle). Oestradiol benzoate was injected i.m. on cycle days 4 (0800 and 2000 h), 5 (0800 h) and 6 (0800 h) at doses of 0.5, 1.0, 2.0 and 2.5 mg respectively to achieve supraphysiological levels of serum oestradiol. Exogenous oestrogen (supraphysiological oestradiol levels) induced an LH surge in all six women in the oestradiol benzoate cycles, but failed to stimulate an LH surge in three of the six patients during treatment with FSH. In three patients treated with FSH, an LH surge was stimulated both by supraphysiological (FSH + oestradiol benzoate cycles) and 'high normal' oestradiol levels (FSH cycles), while in three patients treated with FSH only, the LH surge was blocked, although the threshold level for the positive feedback effect had been exceeded by cycle day 9. We conclude that in women, supraphysiological concentrations of oestradiol exert a positive feedback effect on LH secretion. It is suggested that the occurrence of an LH surge in cycles superovulated with FSH is not dependent on serum oestradiol concentrations, but mainly on the strength of ovarian inhibitory substances.


The effect of follicle maturing drugs on side of ovulation in successive cycles. Our previous study has demonstrated that ovulation in unstimulated cycles is random with an equal likelihood of ipsilateral or contralateral ovulation occurring in the following cycle. This study evaluated women taking ovulation-inducing drugs to see if the side of ovulation in the preceding cycle has an influence on the side of ovulation in the succeeding cycle. Ovulatory patterns in consecutive pairs of cycles in anovulatory women treated with ovulation-inducing drugs were evaluated through sonographic studies of follicular maturation. The results demonstrated that when unilateral ovulation occurred, there was an equal likelihood of ipsilateral or contralateral ovulation in the succeeding cycle. However, ovulation-inducing drugs increase the incidence of bilateral or multiple ovulation.


Prerequisite work-up of the couple before in-vitro fertilization. Prerequisite examinations, including immune status for rubella and hepatitis B antigens, cultures for Chlamydia trachomatis and mycoplasmas, Pap smear and hystersalpingogram were performed in 227 couples before their enrollment into an in-vitro fertilization (IVF) programme. The examinations were completed in 187 couples. Immune status for rubella had already been documented in 45% of the women: of the remainder, 11 patients were not immune (6%). A screening test for hepatitis B antigens had already been documented in only 10% of the patients; eight of the remaining women (5%) were carriers of hepatitis B antigen. C. trachomatis and Mycoplasma hominis were each isolated from 14% of the patients and Ureaplasma urealiticum from 16%. Pap smears had been previously performed in only 35% of the enrolled women. Six (5%) of the 122 newly referred cases had cervical intra-epithelial dysplasia. In 21 (11%) cases some pathology in the uterine cavity was demonstrated. One patient conceived after diagnostic hysteroscopy; another two patients conceived following lysis of adhesions and before IVF treatment. The results of this study show the importance of the preparatory examinations before the IVF and embryo transfer procedure, and raise the medical and medico-legal aspects of this prerequisite work-up.


Luteal phase support in in-vitro fertilization using gonadotrophin releasing hormone analogue before ovarian stimulation: a prospective randomized study of human chorionic gonadotrophin versus intramuscular progesterone. This study was conducted to compare the endocrine milieu and pregnancy rates in an in-vitro fertilization and embryo transfer (IVF-ET) programme employing a gonadotrophin-releasing hormone agonist (GnRHa) and human menopausal gonadotrophin (HMG) when either human chorionic gonadotrophin (HCG) or progesterone were used for luteal phase support. A total of 121 IVF-ET treatment cycles were prospectively studied. All patients started leuprolide acetate in the midluteal phase and it was continued for at least 10 days. When oestradiol levels were less than 150 pmol/l, HMG was started. When at least three follicles were greater than or equal to 17 mm in diameter, HCG 5000 IU i.m. was given. Oocytes were retrieved using transvaginal ultrasound and embryos were transferred 48 h later. The patients' cycles were prospectively randomized to receive HCG (72 cycles) or progesterone (49 cycles) luteal support. The HCG group received 1500 IU i.m. on days 3, 6 and 9 after the initial trigger. The progesterone group received 12.5 mg i.m. q.d. starting from the day after the HCG trigger. The dose of progesterone was increased to 25 mg i.m. q.d. starting on the day of embryo transfer and continued for 17-21 days. If the patient became pregnant, this dose of progesterone was continued until fetal heart activity was visualized by ultrasound. Mean ages, number of eggs retrieved, embryos transferred, oestradiol levels on the day of the HCG trigger, oestradiol and progesterone at the time of embryo transfer were the same in both groups.(ABSTRACT TRUNCATED AT 250 WORDS)


A prospective controlled study of in-vitro fertilization, gamete intra-fallopian transfer and intrauterine insemination combined with superovulation. The relative effectiveness of in-vitro fertilization (IVF), gamete intra-Fallopian transfer (GIFT) and intrauterine insemination (IUI) combined with superovulation in the treatment of infertility were compared in 151 couples undergoing a single cycle of treatment. Treatment was selected as appropriate (IVF for tubal disease, GIFT or IUI/superovulation for nontubal infertility) but possible bias due to non-randomization was overcome by all couples having had favourable fertilization in a previous cycle of IVF. Furthermore, in a preliminary study of initial IVF treatment in 265 couples from whom the study patients were drawn, implantation and pregnancy rates in the diagnostic groups were similar. In the definitive study comparing IVF, GIFT and IUI/superovulation, the pregnancy rate observed with GIFT was highest (40%) but this was not significantly higher than with IVF (28%) or IUI/superovulation (20%). However, the implantation rate per egg transferred by GIFT (21%) was significantly higher than the implantation rate per embryo transferred by IVF (11%). Although the pregnancy rates with GIFT were not statistically greater than with IVF, a significant advantage is likely to be observed in larger groups in view of the better implantation rate. The lower pregnancy rates with IUI superovulation are to be expected because of limited ovarian stimulation, they are nevertheless of comparative interest.


Different phenotypes in two cases of an apparently identical familial (Yq;13p) translocation. A chromosome 13 with extra material on the short arm was observed in a 17-year-old boy showing defects in skeletal growth, an altered hormone profile and asthenoteratozoospermia, and in a 46 XX fetus subjected to prenatal diagnosis. The abnormal chromosome 13 had been transmitted from phenotypically normal parents who were the mother (case 1) and the father (case 2). The extra material on the abnormal chromosome 13 was brightly fluorescent after Q-banding, and positive in C-banding (CBG) and distamycin A-Dapi (DA-Dapi) banding. Staining of the nucleolus organizer region indicated its retention. In-situ hybridization of a Yq-specific repetitive DNA probe to chromosomal spreads from both cases demonstrated that the der(13) chromosome contains sequences of the Yq heterochromatic region. However, the apparently identical unbalanced (Y;13) translocation may either interfere (case 1) or not (father of case 2) with meiotic or postmeiotic sperm cell development.


Purification and localization of a calcium-binding protein in human spermatozoa. The purpose of this study was to purify a calcium-binding protein (CalBP) from human spermatozoa of 226 men and to determine the localization of this protein in spermatozoa. The sperm cells were extracted with 0.6 M KCl, and the KCl extract was then subjected to gel filtration and high performance liquid chromatography. Two CalBPs of Mr = 38 kDa and 52 kDa were found by 45Ca(2+)-binding on blotted proteins. Only a 52 kDa CalBP (CalBP-52) was purified to a final yield of 0.007%. The isoelectric point of this protein was 5.1. The human CalBP-52 reacted with rabbit antiserum directed against rat 52 kDa CalBP. An immunocytochemical study showed that this protein was localized on the sperm head. It is postulated that this protein may have important functions related to Ca(2+)-transport into sperm cells.


Recovery of artificially inseminated spermatozoa from the fallopian tubes of a woman undergoing total abdominal hysterectomy. Investigation of the process of sperm transport within the human female reproductive tract must involve the recovery of spermatozoa from the oviducts of women post-coitum or insemination. This case report describes the insemination of a 40-year-old woman with her partner's semen and the subsequent recovery of spermatozoa from her Fallopian tubes, removed at hysterectomy, 2 days post-ovulation. The number of spermatozoa present within the tubes and their location was ascertained using a flushing technique and scanning electron microscopy (SEM). The total number of spermatozoa present within the flushed tube was calculated to be 142, whilst the total number of spermatozoa calculated to be present within the scanned tube was 673. The proportional distribution of spermatozoa within the tubes was virtually identical by the two techniques used. The largest population of spermatozoa resided within the ampulla (66.1% SEM, 68.3% flush), and the smallest population resided within the intramural region (14.9% SEM, 13.4% flush).


Quantitative ultrastructural analysis of sperm tails reveals flagellar defects associated with persistent asthenozoospermia. Sperm tail morphology was examined in 10 infertile asthenozoospermic men to determine whether poor sperm motility was caused by ultrastructural defects of the flagellum. In this quantitative analysis, the numbers of outer doublet and central pair microtubules, outer and inner dynein arms and radial spokes were counted in transverse sections of 75 axonemes from each patient and compared with similar data previously collected from 10 men with normal semen characteristics. Four patients had axonemal defects: two had severe microtubule abnormalities and two had more subtle but statistically significant deficiencies of dynein arms. These abnormalities would not have been detected by more commonly used qualitative examination. Three patients had no detectable ultrastructural abnormalities of the sperm tail, possibly indicating a metabolic deficiency. A further three patients had mid-piece abnormalities. Two had few, if any, flagellar mitochondria and the third patient had irregular and disorganized mitochondria. Quantitative ultrastructural analysis has revealed axonemal abnormalities in seven of 10 patients with previously unexplained asthenozoospermia.


The removal of morphologically abnormal sperm forms by phagocytes: a positive role for seminal leukocytes? A preliminary investigation was undertaken further to determine the function of the leukocytic cells found in semen. We performed semen analysis and quantified leukocyte subsets using immunocytochemical staining techniques in ejaculates of 351 patients. Leukocyte profiles were examined in relation to sperm morphological data for evidence of a sperm removal/selection process. Three types of seminal phagocytic cell were found to contain spermatozoa: small polymorphonuclear leukocytes (approximately 10-12 microns), monocytes of similar size and much larger (30-40 microns) macrophages capable of engulfing multiple sperm heads. The total leukocyte count (P less than 0.01), the numbers of phagocytic cells i.e. polymorphonuclear leukocytes (P less than 0.05), monocyte/macrophages (P less than 0.01) and HLA-DR positive cells (P less than 0.01), were significantly higher in those samples with greater than 50% ideal sperm forms. Significantly fewer of these same cell types were observed in samples with greater than 50% head defects. There was no difference in the number of tail or midpiece defects between leukocytospermic (greater than 10(6)/ml) and non-leukocytospermic semen samples. Oligozoospermic samples contained significantly fewer leukocytes (P less than 0.005), although above a concentration of 5 x 10(6)/ml, the sperm number was not correlated with leukocyte number. These data, along with repeated observation of spermatozoa or sperm fragments within phagocytic cells, support the hypothesis that leukocytes have a role in the removal of abnormal spermatozoa from the ejaculate.


Origin, development and ultrastructure of boar spermatozoa with folded tails and with two tails. Spermatozoa from the three epididymal regions (head, body and tail) of healthy and sexually mature boars have been examined by light microscopy, and scanning and transmission electron microscopy. The origin, development and external and internal morphologies of aberrant spermatozoa with folded tails and spermatozoa with one or two heads and two fused tails have been established. A count carried out in each region of the epididymis indicated that significant differences (P less than 0.01) exist in the frequencies of each type of malformation and the epididymal region from which the spermatozoa come. Spermatozoa with folded tails at Jensen's ring originate in the cauda of the epididymis from immature spermatozoa that have not ejected the distal cytoplasmic droplet. The plasma membrane which covers the main piece is fused with the membranes of the midpiece, the connecting piece and the head. The fibrous sheath deforms the mitochondrial sheath and is placed between the plasma membrane and the postacrosomal dense lamina. Spermatozoa with one head and two fused tails originate in the epididymal body from spermatozoa with one head and two unfused tails coming from the cephalic region of the epididymis. Spermatozoa with two heads and two fused tails originate in the cephalic region of the epididymis by head-to-head agglutination of two spermatozoa and later fusion of their tails. The frequency of spermatozoa with two fused tails increases as they progress through the epididymal duct. Their tails, parallel in monocephalic spermatozoa and helicoid in bicephalic spermatozoa, have two complete axonemal axes. In their midpiece, the mitochondrial sheaths of the two axes are fused, producing an 8-shaped sheath.


Cruciate thinning of the zona pellucida for more successful enhancement of blastocyst hatching in the mouse. Implantation rates remain low following human in-vitro fertilization (IVF). Suboptimal culture conditions may limit the ability of embryos to hatch as blastocysts, and artificial opening of the zona pellucida has been proposed as a means to promote subsequent hatching (assisted hatching). Such techniques must have minimal adverse effects on the embryos, while maximizing the potential for an embryo to hatch fully as a blastocyst. In a mouse model, we compared embryonic development after zona drilling, and cruciate thinning of the zona (CTOZ) intended to simulate the natural thinning of the zona pellucida. Using acidic Tyrode's solution both zona drilling and cruciate-thinning were performed on day 3 morulae. On day 4 the rates of complete hatching of blastocysts were 0/165, 24/172 and 72/175 in control, zona drilled and thinned groups respectively (P less than 0.0001). On day 5 the rates of complete hatching in the same groups were 20/165, 54/172 and 120/175 respectively (P less than 0.00001) and by day 6, 66/165, 74/172 and 130/175 respectively (P less than 0.00001). The rate of arrest at the morula stage was 24/172 versus 8/175 in the zona drilled and thinned groups respectively (P less than 0.005, whilst the rate of arrest at the blastocyst stage was 21/172 versus 14/175 respectively (NS). Hence cruciate thinning of the zona appears less detrimental at the morula stage than zona drilling, but eventual rates of arrest at the blastocyst stage were comparable. Both techniques significantly increased the rate of hatching, but zona drilling did not guarantee complete hatching.(ABSTRACT TRUNCATED AT 250 WORDS)


Usefulness of partial dissection of the zona pellucida in a human in-vitro fertilization programme. The influence of partial zona dissection (PZD) on the fertilization rate was studied in 34 couples with a history of fertilization failure and/or severe sperm deficiency. Overall, PZD improved the rate of monospermic fertilization compared to controls (41/254 versus 6/111: P less than 0.001) and fertilization was achieved in 50% of cases. However, the results differed according to the seminal characteristics. In 10 couples with at least two in-vitro fertilization (IVF) trials entailing total fertilization failure and with semen defined as normal, PZD did not significantly improve the monospermic fertilization rate (6/44 in the PZD group versus 2/39 among controls). A benefit related to PZD was evident in 33 attempts with severe sperm deficiency. In this group, only four of 72 unmanipulated control oocytes were fertilized but the monospermic fertilization rate was 14.6% for PZD oocytes. The rates of polyspermy were 40% and 14.6% in the groups with normal and abnormal semen parameters respectively. Of 33 trials with defective spermatozoa, 20 reached the stage of embryo transfer and three pregnancies were obtained, of which one aborted at 9 weeks.


Secretion of alpha-immunoreactive inhibin by human pre-embryos cultured in vitro. alpha-Immunoreactive inhibin was measured using an enzyme immunoassay kit in the culture medium (Ham's F-10 medium supplemented with 14% heat-inactivated human serum) from day 3 or 4 to day 14 post-fertilization of 31 surplus pre-embryos from eight women participating in an in-vitro fertilization programme. Inhibition secretion was demonstrated in all of them from the fourth day after fertilization (mean +/- SEM: 3.0 +/- 0.7 U) and was independent of the morphological development of pre-embryos (2-4 cells, n = 4; 6-8 cells, n = 4; 8-10 cells, n = 9; 10-12 cells, n = 4; morulae, n = 5 and blastocysts, n = 4). On days 7, 10, 13 and 14 post-fertilization, mean inhibin values +/- SEM for non-disintegrated pre-embryos were respectively: 6.5 +/- 0.9 U, 12.3 +/- 2.0 U, 16.8 +/- 3.2 U and 20.2 +/- 3.7 U; however, when disintegration was noted on days 10 and 13 after fertilization, inhibin mean values were 9.0 +/- 1.4 U and 8.4 +/- 1.7 U respectively. Inhibin levels were significantly correlated with human chorionic gonadotrophin levels in the same culture media only on day 13, while correlation with pregnancy specific beta 1-glycoprotein occurred on day 7 post-fertilization. In conclusion, early human pre-embryos secrete alpha-immunoreactive inhibin before the cytotrophoblast is formed. This secretion increases significantly with time when development is continued, while disintegration is followed by a net decline in the rate of inhibin release.


Inositol stimulates DNA and protein synthesis, and expansion by rabbit blastocysts in vitro. The effect of different concentrations (0, 0.6, 3, 15, 75 and 375 microM) of myo-inositol on the development of rabbit morulae to expanded blastocysts was investigated in terms of blastocyst expansion and synthesis of DNA and protein, as measured by incorporation of [3H]thymidine and [14C]amino acids into acid-precipitable material. A concentration of 15 microM inositol caused a 2.8-fold increase in blastocyst expansion (P less than 0.01), a 9.9-fold increase in thymidine incorporation into DNA (P less than 0.01) and a 3.6-fold increase in amino acid incorporation into protein (P less than 0.01). There were no significant differences in the range from 15 to 375 microM inositol.


In-vitro co-culture of early stage caprine embryos with oviduct and uterine epithelial cells. Early stage caprine embryos were incubated with goat oviduct and uterine cells to evaluate whether these cells could be used as a somatic cell culture system to enhance development through the developmental block at the 8- to 16-cell stage during in-vitro culture. Following gonadotrophin treatment and natural mating, 2- to 4-cell embryos were surgically recovered from donor females for in-vitro culture studies. In Experiment 1, embryos were equally and randomly allotted to culture treatments of either culture medium plus caprine oviduct cells or culture medium alone. In both treatment groups, embryos were incubated in Medium-199 with 10% fetal bovine serum, 0.25% lactalbumin and 1% antibiotic-antimycotic at 37 degrees C in a humidified atmosphere of 5% CO2 in air. In Experiment 2, similar embryos were cultured in the same medium with either caprine oviduct cells, caprine uterine cells or sequentially incubated with oviduct cells and then uterine cells during a corresponding incubation interval. The culture conditions in Experiment 2 were the same as in Experiment 1. Following 72 h in culture, (Experiment 1), significantly more embryos developed through the in-vitro developmental block into blastocysts and hatched blastocysts when cultured with oviduct cells compared with no embryos developing through the in-vitro block when incubated with medium alone. In Experiment 2, caprine embryos co-cultured with oviduct cells alone resulted in more embryos developing into blastocysts and hatched blastocysts compared with those co-cultured with uterine cells alone.(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of incubation volume and embryo density on the development and viability of mouse embryos in vitro. The morphology, cleavage rate and viability of preimplantation embryos from random bred Swiss mice were assessed after culture in different incubation volumes and embryo densities. Decreasing the incubation volume, from 320 to 20 microliters, significantly increased blastocyst cell number (P less than 0.01) and embryo development after transfer (P less than 0.01). Increasing the number of embryos incubated per drop from 1 to 16 significantly increased the number of two-cell embryos reaching the blastocyst stage in 5 or 320 microliters. Culturing embryos in groups significantly increased blastocyst cell numbers in all volumes employed and elevated embryo viability. Such observations are consistent with the hypothesis that the preimplantation mammalian embryo produces a factor(s) which can stimulate its own development. The results of this study have implications for clinical in-vitro fertilization, where embryos are routinely cultured individually in relatively large volumes.


The expression and localization of mRNA for colony-stimulating factor (CSF)-1 in human term placenta. A 4-kb mRNA for colony-stimulating factor 1 (CSF-1) was detected in normal human placenta at term by Northern blot analysis. In-situ hybridization revealed that the mRNA for CSF-1 was localized in the mesenchymal cells of the chorionic villous stroma, but not in the trophoblasts or capillary epithelial cells. Because there are significant numbers of tissue macrophages (Hofbauer cells) in the placental stroma and because the receptor for CSF-1 (the c-fms proto-oncogene product) is known to be expressed by trophoblasts, our results suggest that CSF-1 produced by placental stromal cells may act as a growth and survival factor for human placental macrophages and trophoblasts.


Hydatidiform mole with coexisting twin pregnancy after gamete intra-fallopian transfer. The pregnancy of a 31-year-old infertility patient is described. After gamete intra-Fallopian transfer, her pregnancy evolved uneventfully until the 18th week of gestation, when vaginal bleeding occurred. Ultrasonographic findings suggested a molar pregnancy with two live fetuses. At 24 weeks gestation, two male infants were spontaneously delivered. Fetal (46 XY) and molar (46 XX) karyotypes and post-mortem findings were consistent with a bizygotic twin pregnancy associated with a complete hydatidiform mole. The pathogenesis and obstetrical management are discussed.


The personality and motivation of semen donors: a comparison with oocyte donors. Seventeen consecutively recruited candidates for semen donation were evaluated by a psychologist with testing and a structured interview. Most men (71%) were motivated by financial compensation. Only 29% would donate semen if records were open to potential offspring. Fifty-nine per cent of the men were rated as excellent candidates from a psychological perspective and 35% were rated as acceptable with slight reservations. One was excluded as a donor. Psychological testing revealed mildly abnormal subscale scores for 35% of donors. Forty-seven per cent had histories of minor depressive or anxiety episodes and 35% had had periods of heavy alcohol use. Compared to oocyte donors at the same institution, the men were less altruistic, more affluent, and more likely to have abused alcohol. Women had more traumatic family and reproductive histories. Psychological evaluation can be a valuable tool in gamete donor selection.


Regulation of catalase in Neisseria gonorrhoeae. Effects of oxidant stress and exposure to human neutrophils. We studied the effects of oxidant stress on the catalase activity and hydrogen peroxide sensitivity of Neisseria gonorrhoeae. N. gonorrhoeae is an obligate pathogen of man that evokes a remarkable but ineffective neutrophil response. Gonococci make no superoxide dismutase but express high catalase activity. Gonococcal catalase activity increased threefold when organisms were subjected to 1.0 mM hydrogen peroxide. This increase in catalase activity was marked by a parallel increase in protein concentration recognized by a rabbit polyclonal antibody raised against the purified gonococcal enzyme. Catalase was primarily localized to the gonococcal cytoplasm in the presence or absence of stress; only a single isoenzyme of catalase could be identified. Exposure of gonococci to neutrophil-derived oxidants was accomplished by stimulating neutrophils with phorbol myristate acetate or by using gonococcal Opa variants that interacted with neutrophils with different degrees of efficiency. Gonococci exposed to neutrophils demonstrated a twofold increase in catalase activity in spite of some reduction in viability. Exposure of gonococci to 1.0 mM hydrogen peroxide made the organisms significantly more resistant to higher concentrations of hydrogen peroxide and to neutrophils than control organisms. These results suggest that catalase is an important defense for N. gonorrhoeae during attack by human neutrophils. The rapid response of this enzyme to hydrogen peroxide should be taken into consideration in studies designed to evaluate the interaction between neutrophils and gonococci.


Organ distribution of the three rat endothelin messenger RNAs and the effects of ischemia on renal gene expression. To determine the organ distribution of production of the three endothelin (ET) isopeptides, we have developed three ribonuclease protection assays specific for the messenger RNAs (mRNAs) of rat ETs 1, 2, and 3.12 organs from adult Sprague-Dawley rats were examined: heart, lung, liver, spleen, kidney, stomach, small intestine, large intestine, testis, muscle, salivary gland, and brain. The mRNA for ET1 was five times more abundant in the lung than in any other organ studied, moderate expression was seen in the large intestine, and lower levels of mRNA were detected in each of the other organs examined. ET2 was expressed at high level in both large and small intestine and at low level in stomach, muscle, and heart, but ET2 mRNA could not be detected elsewhere. ET3 mRNA was found in all organs, particularly in small intestine, lung, kidney, and large intestine. Because of reports suggesting that ETs might be involved in the hypoperfusion and hypofiltration observed in postischemic kidneys, we have also studied levels of mRNA in kidneys that had previously been subjected to 25 or 45 min of clamping of the renal pedicle. At 6 h after 45 min of ischemia, ET1 mRNA increased to a peak of 421 +/- 69% (mean +/- SEM, n = 3) of that in a standard renal RNA preparation. By contrast, ET3 mRNA decreased in the postischemic organ, falling to a value of 19 +/- 2% of standard at the same time point. The effects of ischemia on ET1 and ET3 mRNAs were long-lasting, with elevation of ET1 and depression of ET3 persisting for days. ET2 mRNA remained undetectable throughout. These findings (a) support a role for ET1 in postischemic renal vascular phenomena and (b) demonstrate a situation in which the expression of ET isoforms is clearly subject to differential regulation.


Antibodies to T- and L-isoforms of the cytoskeletal protein, fimbrin, in patients with systemic lupus erythematosus. The cytoskeleton is a complex network of proteins that maintain cell shape, mobility, and organelle function. Its components can be divided into three distinct classes: microfilaments, microtubules, and intermediate filaments. Fimbrins are microfilament proteins, a family of cytoplasmic phosphoproteins. Expression of the L-fimbrin isoform is restricted to replicating blood cells and expression of the T-fimbrin isoform to replicating cells of solid tissues. Sera from normals and from patients with systemic lupus erythematosus (SLE), juvenile arthritis, rheumatoid arthritis, Sjögren's syndrome, osteoarthritis, vasculitis, scleroderma, and mixed connective tissue disease were tested for the presence of antibodies to T- and L-fimbrin by ELISA, using purified recombinant fimbrin. The mean OD of sera from SLE patients was significantly higher than in normals (T-fimbrin, P less than 0.0001; L-fimbrin, P less than 0.001). 48 of 98 SLE sera had antibodies to T-fimbrin; 32 had antibodies to L-fimbrin; 20 had antibodies to both; 28 had only anti-T, and 12 had only anti-L-fimbrin. The mean OD for sera of the other rheumatic diseases was not significantly different from normals. The presence of either L- or T-fimbrin antibody was associated with pleuropericarditis (P = 0.015), photosensitivity (P = 0.011), and anti-Sm antibody (P = 0.010). Central nervous system SLE was associated with the presence of the L-fimbrin antibody alone (P = 0.016). There was a strong association between DR7 (but not other MHC alleles) and anti-L-fimbrin antibodies in SLE patients (chi square = 18; P less than 0.00002). No MHC association was observed with anti-T-fimbrin antibodies.