A novel potent Fas agonist for selective depletion of tumor cells in hematopoietic transplants.

There remains a clear need for effective tumor cell purging in autologous stem cell transplantation (ASCT) where residual malignant cells within the autograft contribute to disease relapse. Here we propose the use of a novel Fas agonist with potent pro-apoptotic activity, termed MegaFasL, as an effective ex-vivo purging agent. MegaFasL selectively kills hematological cancer cells from lymphomas and leukemias and prevents tumor development at concentrations that do not reduce the functional capacity of human hematopoietic stem/progenitor cells both in in vitro and in in vivo transplantation models. These findings highlight the potential use of MegaFasL as an ex-vivo purging agent in ASCT.

Pharmacokinetics and pharmacodynamics of BB-10153, a thrombin-activatable plasminogen, in healthy volunteers.

BACKGROUND: BB-10153 is an engineered variant of human plasminogen that is activated to plasmin by thrombin. Thrombus-selective induction of reperfusion and prevention of reocclusion have been demonstrated following bolus administration in animal models of thrombosis. OBJECTIVE AND METHODS: The objective of the study was to examine the pharmacokinetics and pharmacodynamics of BB-10153 administered as an intravenous bolus to healthy male human volunteers. Cohorts of four were dosed with BB-10153 (n = 3) or placebo (n = 1). In total, placebo was received by eight volunteers and 0.08, 0.2, 0.6, 1.2, 1.8, 2.4, 3.6 and 4.8 mg kg(-1) BB-10153 by three volunteers each. RESULTS: There was a linear relationship between AUC/Cmax and dose. The half-life of BB-10153 was approximately 3-4 h and all the BB-10153 in the circulation retained the ability to be activated by thrombin. There was a dose-related increase in plasma fibrin D-dimers. Ex vivo plasma clot lysis was observed at doses of 3.6 and 4.8 mg kg(-1), whereas lysis of clots formed from euglobulin-fractionated plasma was first evident at 0.6 mg kg(-1) and activity increased with dose. This activity decreased with time in line with the half-life. BB-10153 had no effect on plasma alpha2-antiplasmin or fibrinogen levels, coagulation assays or bleeding time. An increase in plasminogen was observed as BB-10153 was detected by the enzyme-linked immunosorbent assay (ELISA) for human plasminogen. CONCLUSIONS: BB-10153 was well tolerated and had a 3-4-h plasma half-life. Fibrinolytic activity was demonstrated by dose-related ex vivo clot lysis and in vivo production of fibrin D-dimers. These effects were not accompanied by consumption of alpha2-antiplasmin or fibrinogen.

Thrombolytic activity of BB-10153, a thrombin-activatable plasminogen.

BACKGROUND: BB-10153 is an engineered variant of human plasminogen, modified to be activated to plasmin by thrombin. Thrombin-activatable plasminogen was designed as a novel thrombolytic agent which would persist in the blood as a prodrug and be activated to plasmin only at fresh or forming thrombi by the thrombin that is tightly localized there. We previously described the construction of several thrombin-activatable plasminogens and their in vitro clot lysis activity. OBJECTIVES AND METHODS: The current study was an examination of the thrombolytic properties of BB-10153 in vivo; comparison was made with tissue-type plasminogen activator (t-PA) in a femoral artery copper coil thrombosis model in the anesthetized dog and rabbit. Heparin was not coadministered so that the fundamental activity of the agents could be compared. RESULTS: BB-10153, administered as an intravenous bolus of 5 mg kg(-1) in the dog and 10 mg kg(-1) in the rabbit, produced a comparable incidence of reperfusion to 3 mg kg(-1) t-PA. Reocclusion at these doses occurred in 4/4 dogs and 5/7 rabbits treated with t-PA and in 2/6 dogs and 0/10 rabbits treated with BB-10153. There was no reocclusion in three dogs dosed with 10 mg kg(-1) BB-10153. BB-10153 did not affect plasma alpha2-antiplasmin levels or the bleeding time, whereas 3 mg kg(-1) t-PA caused marked depletion of alpha2-antiplasmin and fibrinogen and increased the bleeding time. The plasma half-life of BB-10153 was 3-4 h. CONCLUSIONS: The long half-life and thrombus-selective thrombolytic activity of BB-10153 might allow it to overcome the bleeding and reocclusion shortfalls in the performance of current thrombolytics.

Osmolarity-dependent glycine accumulation indicates a role for glycine as an organic osmolyte in early preimplantation mouse embryos.

Mouse zygotes and early cleavage-stage embryos are sensitive to increased osmolarity. However, development can occur at higher osmolarities if any of a number of organic compounds are present. One of the most effective of these is glycine. We have found that the amount of glycine accumulated by embryos during in vitro culture from the zygote to two-cell stage depends on the osmolarity of the medium, with significantly more glycine accumulated at 310 or 340 mOsM than at 250 mOsM. The accumulated glycine is largely retained in a freely diffusible form, as it can be released via a swelling-activated pathway in two-cell embryos. Increased glycine accumulation does not seem to depend on an increase in its rate of transport. The transport rate is not higher in two-cell embryos that have been cultured from zygotes in hypertonic vs. normal medium, and hypertonicity only slightly stimulates transport in zygotes. Our results indicate that glycine functions as an organic osmolyte in early mouse embryos.

Evidence that the conformation of unliganded human plasminogen is maintained via an intramolecular interaction between the lysine-binding site of kringle 5 and the N-terminal peptide.

Human Glu-plasminogen adopts at least three conformations that provide a means for regulating the specificity of its activation in vivo. It has been proposed previously that the closed (alpha) conformation of human Glu-plasminogen is maintained through physical interaction of the kringle 5 domain and a lysine residue within the N-terminal peptide (NTP). To examine this hypothesis, site-directed mutagenesis was used to generate variant proteins containing substitutions either for aspartic acid residues within the anionic centre of the kringle 5 domain or for conserved lysine residues within the NTP. Size-exclusion HPLC and rates of plasminogen activation by urokinase-type plasminogen activator were used to determine the conformational states of these variants. Variants with substitutions within the kringle 5 lysine-binding site demonstrated extended conformations, as did variants with alanine substitutions for Lys50 and Lys62. In contrast, molecules in which NTP residues Lys20 or Lys33 were replaced were shown to adopt closed conformations. We conclude that the lysine-binding site of kringle 5 is involved in maintaining the closed conformation of human Glu-plasminogen via an interaction with the NTP, probably through Lys50 and/or Lys62. These conclusions advance the current model for the initial stages of fibrinolysis during which fibrin is thought to compete with the NTP for the kringle 5 lysine-binding site.

Culture with Matrigel inhibits development of mouse zygotes.

PURPOSE: It was reported that Matrigel improved hatching of mouse blastocysts produced in vitro from F1 hybrid-derived zygotes. We investigated whether Matrigel would be similarly beneficial with outbred strain-derived embryos, which exhibit a "two-cell" block similar to the developmental blocks of other species. METHODS: Mouse embryo development was assessed with or without Matrigel in KSOM medium, which supports the development of blocking strain zygotes in vitro, and in human tubal fluid (HTF) medium, which normally does not but which is used for human IVF. RESULTS: Matrigel severely inhibited the development of zygotes to blastocysts in KSOM and did not improve culture in HTF. There was no effect on development from the two-cell stage. We were not able to replicate the previous finding of Matrigel's beneficial effect on hatching of F1-derived zygotes. CONCLUSIONS: Matrigel may be a deleterious addition to embryo culture or coculture systems.

Organic osmolytes and embryos: substrates of the Gly and beta transport systems protect mouse zygotes against the effects of raised osmolarity.

Mouse embryo development is identically inhibited by raised osmolarity, whether produced by added NaCl or raffinose, demonstrating that high osmolarity is itself detrimental to embryos. In the face of increased osmolarity, cells in the brain and kidney, and likely many other cells, accumulate nonperturbing organic osmolytes in their cytoplasm. In the presence of any of a number of organic compounds that were proven or probable substrates of either the Gly or the beta transport systems, mouse embryo development in vitro was protected from raised osmolarity. Zygotes developed past the "2-cell block," and with most Gly or beta substrates, to the blastocyst stage. The most effective osmoprotectants were glycine, glutamine, betaine, proline, beta-alanine, and hypotaurine; several others were partially effective. A model Gly substrate, glycine, was effective at a much lower concentration (EC50 = 50 microM) than was a model beta substrate, beta-alanine (EC50 = 1.3 mM). The protective effect of these two compounds was additive, indicating a common mode of action. The various effective compounds tested do not all share metabolic pathways or other such properties in common. Thus, it is likely that cleavage-stage mouse embryos utilize them, in large part, as organic osmolytes.

Age-related losses of cognitive function and motor skills in mice are associated with oxidative protein damage in the brain.

The hypothesis that age-associated impairment of cognitive and motor functions is due to oxidative molecular damage was tested in the mouse. In a blind study, senescent mice (aged 22 months) were subjected to a battery of behavioral tests for motor and cognitive functions and subsequently assayed for oxidative molecular damage as assessed by protein carbonyl concentration in different regions of the brain. The degree of age-related impairment in each mouse was determined by comparison to a reference group of young mice (aged 4 months) tested concurrently on the behavioral battery. The age-related loss of ability to perform a spatial swim maze task was found to be positively correlated with oxidative molecular damage in the cerebral cortex, whereas age-related loss of motor coordination was correlated with oxidative molecular damage within the cerebellum. These results support the view that oxidative stress is a causal factor in brain senescence. Furthermore, the findings suggest that age-related declines of cognitive and motor performance progress independently, and involve oxidative molecular damage within different regions of the brain.

Substitution of arginine 719 for glutamic acid in human plasminogen substantially reduces its affinity for streptokinase.

In isolation human plasminogen possesses no enzymatic activity, yet upon formation of an equimolar complex with the bacterial protein streptokinase, it acquires a plasminogen activator function. The region(s) of plasminogen and of streptokinase which mediate complex formation has (have) not been previously published. Here it is reported that a single-residue substitution (Arg719-->Glu) in the serine protease domain of full-length Glu-plasminogen substantially reduces its affinity for streptokinase. The plasminogen variant displays no other significant differences from the wild-type molecule with respect to activation by two-chain urokinase-type plasminogen activator, recognition by monoclonal antibodies, or ability to undergo conformational change. It is concluded that Arg719 in human plasminogen is an important determinant of the streptokinase binding site, although further sites are likely to contribute both to the affinity of plasminogen for streptokinase and to mechanisms by which the active site is formed within the complex.

Plasminogen mutants activated by thrombin. Potential thrombus-selective thrombolytic agents.

Plasmin, the enzyme responsible for degradation of fibrin in blood clots and thus thrombolysis, is normally formed when its zymogen plasminogen is activated by cleavage of the Arg561-Val562 bond by specific plasminogen activators. We have altered the activation characteristics of plasminogen by substituting the P3, P2, and P1' cleavage site residues with sequences from thrombin-cleavable proteins to produce a novel thrombolytic agent which instead is activated by the blood clotting system. Plasminogen variants with thrombin cleavage sites from fibrinogen, the thrombin receptor, factor XIII, and factor XI were cleaved by thrombin with times to 50% cleavage of 28 h, 2.5 h, 5.7 min, and 3 min, respectively. In vitro clot lysis studies have shown that a variant in which the P3-P1' residues of plasminogen were substituted by the P7-P1' residues (Thr363-Ile370) from factor XI (T51) was sufficiently rapidly cleaved by thrombin to be activated by the endogenous thrombin produced by the coagulation cascade, resulting in rapid clot dissolution. Thrombin-activatable plasminogen therefore has the capacity to short circuit the physiological hemostatic mechanisms and produce fibrinolytic activity localized to the site of thrombin formation, that is, at the thrombus itself. The novel activation mechanism combined with the natural long circulating half-life of plasminogen gives this type of thrombolytic agent the potential for thrombus-selective plasmin generation and an extended duration of action.

The cure for employee malaise--motivation.

Although working conditions, hours, pay, and advancement opportunities are better now than in the 1950s--the "golden age" of American business--today's workers are significantly less satisfied. Why? The authors believe the cause of this malaise is lack of motivation. This article examines several techniques to cure employee malaise and discusses the long-term benefits of these techniques, which include empowerment, recognition, career development, the Pygmalion effect, incentives, and rewards. By making a commitment to these motivational techniques, managers will boost the morale and enthusiasm of their employees and their organization. This motivational process is not quick and easy; developing your employees is an ongoing process.

The expression of hybrid HIV:Ty virus-like particles in yeast.

The yeast retrotransposon, Ty, encodes a set of proteins that are assembled into virus-like particles, Ty-VLPs (refs 1, 2). These proteins include Ty-VLP structural proteins, a protease that mediates cleavage of primary translation products and a reverse transcriptase. The major structural components of Ty-VLPs are proteolytic products of the primary translation product, p1 (ref. 3). We have recently shown that protein p1 alone can form Ty-VLPs (ref. 3). Here we demonstrate that p1 fusion proteins, comprising most of p1 and part of human immunodeficiency virus (HIV) protein gp120, form hybrid HIV:Ty-VLPs. These hybrid particles provide a rapid means of preparing and evaluating HIV antigens for a variety of immunological purposes.

Production of monoclonal antibodies recognising N-ethylmaleimide during attempted generation of monoclonal antibodies to human type I procollagen.

Mice were immunised for the production of monoclonal antibodies to human procollagen (I) using antigen purified from fibroblast conditioned medium. The procedure for procollagen (I) preparation included the addition of proteinase inhibitors N-ethylmaleimide, phenylmethylsulphonyl fluoride and ethylenediaminetetraacetic acid to prevent damage by proteolytic cleavage. Four of the five monoclonal antibodies subsequently produced were found to recognise the thiol proteinase inhibitor N-ethylmaleimide but not type I procollagen prepared in the absence of N-ethylmaleimide. One of these monoclonal antibodies was examined further and shown to recognise beta-galactosidase after it had been reacted with N-ethylmaleimide. As far as we are aware this is the first time that monoclonal antibodies have been produced which recognise N-ethylmaleimide. Our findings indicate an unexpected reaction between procollagen and N-ethylmaleimide and prompt the suggestion that the use of N-ethylmaleimide in the purification of procollagen and other proteins should be reexamined.

The antiproliferative activity of interferons assayed by measuring growth medium color changes related to cell number.

A simple colorimetric assay for estimating cell numbers has been developed based on the observation that the indicator color in cell culture medium is proportional to viable cell number. The assay is performed in multiwell plates to take advantage of the rapid color measurement and computerized data handling capabilities of multiwell scanning spectrophotometers. Since no centrifugation or washing steps are involved, the technique is particularly useful for cells that grow in suspension, although it is equally applicable to monolayer cultures. The assay was developed to measure the antiproliferative activity of interferons on Daudi lymphoblastoid cells but could equally well be applied to other cell growth inhibition or stimulation assays.

Monoclonal antibodies to human beta-interferon produced by adoptive transfer in irradiated mice.

Three murine anti-human beta-interferon (IFN-beta) monoclonal antibodies have been isolated following adoptive transfer of immune spleen cells. Adoptive transfer was used to increase the specific efficiency of the fusion. These antibodies have been used to define two epitopes on IFN-beta; the antiviral, antiproliferative, and immunomodulatory effects of IFN-beta are associated with one of these epitopes.

Activity of SC33428, a novel bishydrazone-bridged derivative of 4-demethoxydaunorubicin, against experimental tumors in mice.

SC33428, a novel bishydrazone-bridged analogue of 4-demethoxydaunorubicin, has been tested for antitumor activity in a range of experimental mouse tumor systems and has been found to be active when administered i.p., i.v., or p.o. When compared to Adriamycin, SC33428 was 4 times more potent and had antitumor activity which was superior against i.p. or i.v. L1210 leukemia, similar against i.p. P388 leukemia and i.v. Lewis lung carcinoma, and inferior against i.p. B16 melanoma. When compared to 4-demethoxydaunorubicin, SC33428 was 4 times less potent but a much more effective antitumor agent when given i.p. against i.p. L1210, P388, and B16 tumors. However, when given i.v. or p.o., the two compounds had similar potency and efficacy against systemic P388 and L1210 leukemias.

Biological properties of human interferon beta 1 synthesized in recombinant bacteria.

Human fibroblast interferon, designated IFN-beta 1, has been produced in E. coli by direct expression of the cloned cDNA coding for the mature polypeptide. Bacterial lysates from recombinant cultures contain a polypeptide with an apparent molecular weight of 17,500 that corresponds in size to the unglycosylated IFN-beta 1 molecule. The latter could be specifically immunoprecipitated by antibodies to purified natural IFN-beta and could inhibit the replication of Herpes simplex virus types 1 and 2 in many different cell lines. Like the natural fibroblast IFN-beta, the bacterial IFN-beta 1 was active in many human cell lines, less active in a monkey cell line and inactive in rabbit and mouse fibroblasts. The antibody titre required to neutralise the anti-herpes activity of both IFN preparations was similar suggesting that they have the same specific activities. Similarly, the bacterial IFN-beta 1 was equally active in inhibiting the proliferation of Daudi cells grown in culture. Bacterial IFN-beta 1 was also capable of enhancing natural killer cell activity and antibody-dependent cellular cytotoxicity in vitro. Thus, IFN-beta 1 produced in recombinant bacteria displays a large range of biological properties ascribed to the natural fibroblast IFN-beta molecule.

Variations in the contribution of induced interferon and adjuvanticity to the antiviral effect of different polyinosinic acid . polycytidylic acid formulations in mice infected with encephalomyocarditis virus.

Treatment of mice with polyinosinic acid . polycytidylic acid [poly(I) . poly(C)] 6 h before infection and once daily on days 1, 2, 3, 4, 7, and 10 after infection with encephalomyocarditis virus was found to confer no additional protection as compared with a single treatment 6 h before infection. When complexed with a colloid formed between carboxymethylcellulose and polylysine, poly(I) . poly(C) conferred significant additional protection with the multiple treatment regimen compared with a single treatment of 6 h before infection. The additional antiviral activity of multiple treatments could not be entirely attributed to interferon induction by the complexed form of poly(I) . poly(C), because free and complexed poly(I) . poly(C) both caused hyporesponsiveness to interferon induction after multiple treatments. However, mice protected against encephalomyocarditis virus infection by multiple treatments with the colloidal complex form of poly(I) . poly(C) showed a significant increase in resistance to reinfection, and this was attributable to adjuvant effects of the colloidal complex form of poly(I) . poly(C). The contribution of interferon induction and adjuvanticity of the poly(I) . poly(C) formulations varied with the times of treatment relative to infection, the dose of polynucleotide material, and the virus dose.

The antiviral activity of ribosomal polynucleotides against encephalomyocarditis virus infection of mice.

Intraperitoneal administration of ribosomal RNA (rRNA) was found to protect mice against subsequent lethal infection by encephalomyocarditis (EMC) virus without induction of detectable amounts of circulating interferon. The nature of this effect was examined in terms of the types of natural polyribonucleotides which could afford such protection. rRNA prepared from E. coli was slightly more effective than chicken liver rRNA which was, in turn, more effective than yeast rRNA. 5S ribosomal RNA was not effective, whereas the slightly smaller 4S transfer RNA was as good as E. coli rRNA, suggesting that molecular size is not the sole criterion for the protective effect. The separated 16S and 23S E. coli rRNAs where each as effective as the unfractionated RNA. Anti-viral activity was lost after complete hydrolysis with alkali and nucleoside monophosphates were also inactive. Digestion of rRNA with pancreatic ribonuclease greatly decreased its antiviral activity whereas digestion with T1 ribonuclease had no effect indicating that fairly short oligonucleotides, but not of random nucleotide sequence, are active components in the protection of mice against infection by EMC virus. In vitro, no antiviral effect against EMC virus infection was observed in treatment of L cells under various conditions.