Isolation and interrelationships of the multiple molecular tissue-type and urokinase-type plasminogen activator forms produced by cultured human umbilical vein endothelial cells

Primary and early subcultures (1st- to 3rd passage) of human umbilical vein endothelial cells produce tissue-type plasminogen activator (t-PA) antigen, consisting only of a major Mr 110,000 t-PA form. Later subcultures (greater than 4th passage) produce increasing amounts of t-PA antigen, consisting of a major Mr 110,000 and a minor Mr 68,000 form as well as increasing amounts of urokinase-type plasminogen activator (u-PA) antigen, consisting of a minor Mr 95,000 and major Mr 54,000 form. All of the major plasminogen activator forms were purified to homogeneity from 72 h serum-free conditioned media (3 liters, 1-1.8 x 10(9) cells) by a combination of immunoaffinity and gel filtration chromatography. Typically, 4th to 6th passage cultures produced/secreted t-PA-type proteins consisting of an inactive Mr 110,000 (220 IU/mg) and active Mr 68,000 (76,500 IU/mg) form representing about 39 and 8%, respectively, of the total starting sodium dodecyl sulfate stable t-PA activity, and u-PA-type proteins consisting of an inactive Mr 95,000 (700 IU/mg) and active Mr 54,000 (81,000 IU/mg) form representing about 9 and 38%, respectively, of the total starting sodium dodecyl sulfate stable u-PA activity. The isolated Mr 68,000 t-PA and Mr 54,000 u-PA proteins, exist only as two-chain forms in the absence of aprotinin and as mixtures of single- and two-chain proteins in the presence of aprotinin. Treatment with nucleophilic agents completely dissociated the Mr 110,000 t-PA and Mr 95,000 u-PA proteins into their respective Mr 68,000 t-PA and Mr 54,000 u-PA activity forms and a common Mr 46,000 protein, confirming the enzyme-inhibitor complex nature of these inactive plasminogen activator forms.     

Platelet-activating factor induces glycogen degradation in fetal rabbit lung in utero

The role of platelet-activating factor (PAF, 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in initiating glycogen breakdown in the fetal rabbit lung was assessed by intraperitoneal administration of this potent ether-linked glycerophospholipid. Forty-five min after in utero injection of PAF (2.5 X 10(-7) mol), fetal pulmonary and hepatic glycogen concentrations were reduced from 326 to 256 and from 9.8 to 6.6 micrograms of glycogen/mg protein, respectively. Glycolytic activity was similarly increased as judged by an elevation of lactate (2-fold) in lung, liver, and plasma upon PAF injection. These actions of PAF were dose- and time-dependent. The glycogenolytic response did not occur when an equimolar dose of the inactive enantiomer, D-PAF was injected. Pretreatment of the fetus with a specific PAF receptor antagonist, SRI-63-441, prevented the PAF response. We have previously demonstrated (Hoffman, D. R., Truong, C. T., and Johnston, J. M. (1986) Biochim. Biophys. Acta 879, 88-96) that PAF biosynthesis and PAF concentrations increase significantly on day 24 of fetal rabbit lung development. A concurrent decrease in pulmonary glycogen concentration at this point of gestation is potentially reflective of the PAF-induced action. Thus, these observations would suggest a role for PAF in the normal physiology of fetal lung maturation.     

57Fe and 1H electron-nuclear double resonance of three doubly reduced states Escherichia coli sulfite reductase

We have employed electron-nuclear double resonance (ENDOR) spectroscopy to study the 57Fe hyperfine interactions in the bridged-siroheme [4Fe-4S] cluster that forms the catalytically active center of the two-electron-reduced hemoprotein subunit of Escherichia coli NADPH-sulfite reductase (SiR2-). Previous electron paramagnetic resonance (EPR) and M?ssbauer studies have shown that this enzyme oxidation state can exist in three distinct spectroscopic forms: (1) a "g = 2.29" EPR species that predominates in unligated SiR2-, in which the siroheme Fe2+ is believed to be in an S = 1 state; (2) a "g = 4.88" type of EPR species that predominates in SiR2- in the presence of small amounts of guanidinium sulfate, in which the siroheme Fe2+ is in an S = 2 state; and (3) a classical "g = 1.94" type of EPR species that is seen in SiR2- ligated with CO, in which the siroheme Fe2+ is in an S = 0 state. In all three species, the cluster is in the [4Fe-4S]1+ state, and two distinct types of Fe site are seen in M?ssbauer spectroscopy. ENDOR studies confirm the M?ssbauer assignments for the cluster 57Fe in the g = 1.94 state, with A values of 37, 37, and 32 MHz for site I and ca. 19 MHz for site II. The hyperfine interactions are not too different on the g = 2.29 state, with site I Fe showing more anisotropic A values of 32, 24, and 20 MHz (site II was not detected).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of temperature and photoperiod upon adult eclosion of the sweetpotato whitefly, Bemisia tabaci

A series of experiments were conducted to characterize patterns of eclosion by Bemisia tabaci (Genn.) (Homoptera: Aleyrodidae) to their adult stage and to determine how these patterns are influenced by certain environmental parameters. Under a constant temperature of 29.5±0.6°C and a photoperiod of 14:10LD, 90% of the adults emerged from their pupal cases between 0600 and 0930 h (with lights on occurring at 0600 h). Few emerged during hours of darkness. The peak time of adult emergence was delayed when temperatures were fluctuated. Under a series of constant temperatures, a significant inverse correlation was found between the time of median emergence (i.e., eclosion of 50% of the total number of adults) and temperature (P<0.001). No emergence was observed at temperatures below 17±0.3°C. Emergence patterns persisted under conditions of continuous light and continuous darkness, suggesting the presence of a circadian system.

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Zusammenfassung Um das Verständnis über den Lebenslauf von Bemisia tabaci zu ergänzen, wurde eine Serie von Experimenten durchgeführt, deren Zweck die Charakterisierung des Ausschlüpfvorgangs in das Endstadium war und die Feststellung, wie dieser Vorgang von gewissen Umweltparametern beeinflusst wird. Bei einer konstanten Temperatur von 29.5±0.6°C und einem Beleuchtungszyklus von 14: 10 LD (Licht/Dunkelheit) schlüpften 90% der Ausgewachsenen zwischen 0600 Uhr and 0930 Uhr (ab 0600 Uhr mit Licht) aus ihren Puppenhüllen aus. Wenig Ausschlüpfen geschah während der unbeleuchteten Stunden. Der Höhepunkt des Ausschlüpfens wurde bei wechselnden Temperaturen verschoben. Bei einer Serie von gleichbleibenden Temperaturen wurde eine bedeutende inverse Korrelation zwischen der medianen Ausschlüpfzeit (d.h. 50% der gesamten Ausgewachsenen schlüpften aus) und der Temperatur festgestellt (P<0.001). Kein Ausschlüpfen wurde beobachtet bei Temperaturen unter 17°C. Das Ausschlüpfschema war gleichbleibend bei dauerndem Licht oder dauernder Dunkelheit, was auf das Vorhandensein eines circadianen Systems hinweist.

     

Reversal of Con A-induced suppression by a platelet-derived factor which binds to activated suppressor T cells

A platelet-derived factor found in serum as well as in platelet releasate prepared either with calcium ionophore or with thrombin was shown to reverse Con A-induced suppression of the plaque forming cell (PFC) response to sheep erythrocytes (SRBC) in vivo in (CB6)F1 mice. In addition, as shown previously, lymphoma cell-induced suppression in SJL mice was similarly reversed. The factor could be injected prior to Con A on the day before SRBC injection, or on the same day as antigen with comparable results. It also enhanced PFC responses in the absence of Con A. Suppressor cell induction by Con A in vivo, as demonstrated by assay on PFC responses of normal spleen cells in vitro, was abrogated by simultaneous injection of the platelet factor. Cells from mouse spleen and lymph node, but not from thymus could absorb the factor from human serum at 4 degrees C. The phenotype of the relevant spleen cells was L3T4-, Ly1-, Ly2+, Thy1+, Ly22+, Qa1+, Qa4+, Qa5+, and Ly6.IE+. These results suggest that this factor binds to activated peripheral T cells of the suppressor cell phenotype.     

Effects of altering the ATP/ADP ratio on pump-mediated Na/K and Na/Na exchanges in resealed human red blood cell ghosts

Resealed human red blood cell ghosts were prepared to contain a range of ADP concentrations at fixed ATP concentrations and vice versa. ATP/ADP ratios ranging from approximately 0.2 to 50 were set and maintained (for up to 45 min) in this system. ATP and ADP concentrations were controlled by the addition of either a phosphoarginine- or phosphocreatine-based regenerating system. Ouabain-sensitive unidirectional Na efflux was determined in the presence and absence of 15 mM external K as a function of the nucleotide composition. Na/K exchange was found to increase to saturation with ATP (K 1/2 approximately equal to 250 microM), whereas Na/Na exchange (measured in K-free solutions) was a saturating function of ADP (K 1/2 approximately equal to 350 microM). The elevation of ATP from approximately 100 to 1,800 microM did not appreciably affect Na/Na exchange. In the presence of external Na and a saturating concentration of external K, increasing the ADP concentration at constant ATP was found to decrease ouabain-sensitive Na/K exchange. The decreased Na/K exchange that still remained when the ADP/ATP ratio was high was stimulated by removal of external Na. Assuming that under normal substrate conditions the reaction cycle of the Na/K pump is rate-limited by the conformational change associated with the release of occluded K [E2 X (K) X ATP----E1 X ATP + K], increasing ADP inhibits the rate of these transformations by competition with ATP for the E2(K) form. A less likely alternative is that inhibition is due to competition with ATP at the high-affinity site (E1). The acceleration of the Na/K pump that occurs upon removing external Na at high levels of ADP evidently results from a shift in the forward direction of the transformation of the intermediates involved with the release of occluded Na from E1P X (Na). Thus, the nucleotide composition and the Na gradient can modulate the rate at which the Na/K pump operates.     

Antibody-induced antigenic modulation is antigen dependent: characterization of 22 proteins on a malignant human B cell line

Expression of several of the surface antigens on normal and malignant hematopoietic cells is reduced or is modulated by incubation with specific antibodies. Although antigenic modulation provides a means by which cells can escape antibody-mediated immune destruction, the physiologic significance and frequency of this phenomenon are both poorly understood. To begin to address these issues, we identified and characterized surface antigens on the malignant B cell line Laz 221 established from a patient with acute lymphoblastic leukemia (ALL). Indirect immunofluorescence analysis with the use of 26 hematopoietic cell populations and immune precipitation studies with the use of iodinated ALL cells indicate that 163 monoclonal antibodies (MoAb) identify 22 different proteins on this cell line, including at least six previously described surface molecules. Seven of these antigens are expressed by all nucleated cells examined, whereas only the mu chain of immunoglobulin is B cell specific. Incubation of specific MoAb with cultures of Laz 221 cells at 37 degrees C reduces or modulates surface expression of five of these 22 antigens (p45, immunoglobulin mu chain, transferrin receptor, common ALL antigen (CD10), and p105). Studies that made use of multiple MoAb specific for the same antigen suggest that the capacity for antigenic modulation is an intrinsic property of individual antigens. These studies also suggest that the murine immune response to shared human antigens varies from one immunizing cell population to another. For example, three of the antigens present on Laz 221 cells were only identified by MoAb raised to the Burkitt's cell line Ramos and vice-versa. Only one of these six shared antigens is present in greater amounts on the immunogenic cell population. Immunogenicity of individual human antigens in the mouse may be a function of their cell surface environment.     

Dopamine Metabolism in Hypoxanthine-Guanine Phosphoribosyltransferase-Deficient Variants of PC12 Cells

Lesch-Nyhan syndrome results from a deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT). It is manifest by behavioral abnormalities, including self-mutilation, and evidence of abnormal 3,4-dihydroxyphenylethylamine (dopamine) metabolism. To assess whether an HPRT deficiency in a dopaminergic cell can adversely affect dopamine metabolism in that cell, dopamine metabolism was examined in HPRT-deficient variants of PC12 pheochromocytoma cells and in cells that had regained HPRT activity by virtue of transformation with a recombinant retrovirus containing the human gene for HPRT. There was no correlation between HPRT activity and endogenous dopamine levels, dopamine uptake, dopamine release, or monoamine oxidase activity. Transformation with the HPRT retrovirus did not adversely affect dopamine metabolism.     

Characterization of a progressive neurodegenerative disease induced by a temperature-sensitive Moloney murine leukemia virus infection.

A progressive neurodegenerative disease occurred following infection of mice with a temperature-sensitive (ts) isolate of Moloney (Mo) murine leukemia virus (MuLV), ts Mo BA-1 MuLV. This NB-tropic ecotropic MuLV, which was ts for a late function, induced a syndrome of tremor, weakness of the hind limbs, and spasticity following infection of several strains of laboratory neonatal mice, including NFS, C3H/He, CBA, SJL, and BALB/c. The latent period of 8 to 16 weeks was considerably longer than that observed for the acute paralytic diseases observed following neonatal infection with other ts Mo-MuLV, rat-passaged Friend MuLV, and some wild mouse ecotropic MuLVs. Spongiform pathology without inflammation and degeneration of neurons devoid of budding virions occurred in the cerebellar grey matter, brain stem, and upper spinal cord; but lower spinal cord anterior horn cells were less obviously affected than in other MuLV-associated neuroparalytic syndromes. ts Mo BA-1 MuLV differed from other ts Mo-MuLV mutants that are capable of inducing a neuroparalytic syndrome in that while infected nervous system tissue contained high levels of MuLV p30 and gp70, no evidence of precursor accumulation or abnormal processing of MuLV p30 or gp70 could be demonstrated. The localization of virus within the nervous system suggests that direct neuronal infection may not be the etiologic mechanism in this MuLV-induced neurodegenerative disease.