Two-stage Assay Research Articles

The partial thromboplastin time: defining an era in coagulation

The partial thromboplastin time: defining an era in coagulation

Abciximab inhibits procoagulant activity but not the release reaction upon collagen- or clot-adherent platelets

In addition to inhibiting platelet (plt) aggregation abciximab, a glycoprotein (GP) IIb/IIIa antagonist, reduces coagulation in blood or platelet-rich plasma. We assessed the effects of abciximab (10 micro g mL(-1)) on adhesion-dependent procoagulant activity (PCA) of plt upon: (i) collagen to model initial adhesion; or (ii) plasma clot or fibrin to model a preformed thrombus with retained thrombin activity. In a two-stage assay gel-filtered plt (GFP) first adhered on collagen, plasma clot, or fibrin, and plt activation was traced with platelet factor 4 (PF 4) release. Second, PCA was measured on adherent plt (i) by soluble prothrombin fragments (F1 + 2); and (ii) chromogenically by adding defibrinated plasma and thromboplastin. Abciximab inhibited aggregation upon collagen-adherent plt both in the absence and presence of plasma. In contrast, without plasma abciximab enhanced plt deposition to fibrin surfaces depending on thrombin generation and fibrin polymerization. However, abciximab reduced PCA and generation of F1 + 2 on adherent plt surface-independently by 35%, whereas PF 4 release persisted. Also, a GP Ib inhibitor, mAb SZ2, attenuated PCA by 40% alone, and by 65% together with abciximab, leaving 35% of PCA unaltered. Abciximab decreased generation of new thrombin on both collagen- and clot-adherent plt. However, abciximab did not inhibit alpha-granule release, suggesting distinct pathways for PCA and release reaction. Deposition of isolated plt on clots in the presence of abciximab was dependent on thrombin and polymerizing plt-derived fibrin(ogen). Due to local consumption of natural anticoagulants adjacent to a preformed thrombus the antithrombotic effect of abciximab benefits from additional inhibition of thrombin.

塩素処理水の染色体異常誘発性・形質転換誘発性の変化過程と強変異原物質ＭＸの指標性

Changes in the toxicity in chlorinated water after chlorine addition were examined. For toxicity evaluation, the chromosomal aberration test and the transformation test were conducted as indexes of initiation activity and promotion activity, respectively, in the carcinogenesis process. Activity inducing chromosomal aberrations in chlorinated Lake Biwa water gradually decreased over time after chlorination. In contrast, activity inducing transformations determined by the two-stage assay gradually increased. Thus, toxicity that decreases or increases is present in chlorinated water. Furthermore, activity inducing transformations determined by the non-two-stage assay gradually decreased over time. This direction of change is opposite to that of activity inducing transformations determined by the two-stage assay and is consistent with that of activity inducing chromosomal aberrations. The drastic decrease in initiation activity detected as activity inducing chromosomal aberrations could be the main cause for the decrease in activity inducing transformations determined by the non-two-stage assay (an index of the sum of initiation and promotion activity). MX change was quantitatively consistent with those of activity inducing chromosomal aberrations and transformations determined by the non-two-stage assay. On the other hand, directions of changes in concentrations of typical by-products such as chloroform were consistent only with that of activity inducing transformations determined by the two-stage assay. Findings of this study suggest that MX is appropriate as an index for comparing the carcinogenicity of tap water near and far from a water purification plant.

The molecular basis of hemophilia A: genotype-phenotype relationships and inhibitor development.

The molecular basis of hemophilia A has been extensively studied over the last two decades, and this analysis of the factor VIII (FVIII) gene has rendered it one of the most studied of all human genes. A wide range of different mutation types has been identified that includes the novel intrachromosomal inversions involving regions in introns 1 and 22 of the FVIII gene as well as many mutation types found in other genetic diseases, including large and small deletions and insertions, and point mutations resulting in nonsense, missense, and splice site mutations. Inhibitory antibodies that develop in a proportion of patients with hemophilia A following replacement therapy are now known to correlate with FVIII mutation type and location. This correlation is demonstrated, and a potential algorithm for predicting inhibitor development in newly diagnosed patients is presented. Many patients with mild hemophilia A have a discrepancy between the levels of FVIII:C determined by the one-stage and two-stage assays. The molecular basis of the discrepancy is explored. This article thus highlights both the molecular basis of hemophilia and some of the additional information that can be gained from determination of the mutation responsible for hemophilia in affected patients.

Hemophilia A Mutations within the Factor VIII A2-A3 Subunit Interface Destabilize Factor VIIIa and Cause One-stage/ Two-stage Activity Discrepancy

Thrombin-activated factor VIII (FVIIIa) is a heterotrimer with the A2 subunit in a weak ionic interaction with the A1 and A3-C1-C2 subunits. Dissociation of the A2 subunit correlates with inactivation of FVIIIa. A homology model (Blood 89:2413, 1997) of the triplicated A domains of factor VIII (FVIII) predicts a pseudo-threefold axis at the tightly packed hydrophobic core with several interdomain interactions. These lie at the interface of A1-A2, A2-A3 and A1-A3. We have previously demonstrated that hemophilia A mutations (R531H, A284E, S289L) within the predicted A1-A2 and A1-A3 interface disrupt potential intersubunit hydrogen bonds and have the molecular phenotype of increased rate of inactivation of FVIIIa due to increased rate of A2 subunit dissociation. Patients with these mutations exhibit a clinical phenotype where the FVIII activity by one-stage(1-st) assay is at least two-fold higher than by two-stage(2-st) assay. We have now also explored mutations within the predicted A2-A3 interface (N694I, R698W and R698L) that also have the phenotype of 1-st/2-st activity discrepancy. These mutations exhibit the same molecular mechanism of increased instability of FVIIIa as those mutations described along the A1-A2 and A1-A3 interfaces. This suggests that the entire tightly packed hydrophobic core within the predicted pseudo-threefold axis contributes to stabilization of FVIIIa.

Changes of Activity Inducing chromosomal aberrations and Transformations of Chlorinated Humic Acid

The change of the toxicity of chlorinated water after chlorine injection was examined. For the measurement of toxicity, chromosomal aberration test and transforming test were carried out as indexes to initiating activity and to promoting activity in the carcinogenesis process, respectively. Activity inducing chromosomal aberrations of chlorinated humic acid gradually decreased with time after chlorination. In contrast, activity inducing transformations measured by the two-stage assay gradually increased. Thus, the toxicity that decreases or increases is present in chlorinated water. Furthermore, activity inducing transformations measured by the non-two-stage assay gradually decreased. This direction of change was reverse to that of activity inducing transformations by the two-stage assay and consistent with that of activity inducing chromosomal aberrations. It is speculated that the main reason of decreasing activity inducing transformations by the non-two-stage assay is because initiating activity detected as activity inducing chromosomal aberrations in chlorinated water decreases drastically. Directions of changes of total organic halogen and carbonyl group were qualitatively consistent with that of activity inducing chromosomal aberrations. Directions of changes of chloroform and dichloroacetic acid were qualitatively consistent with that of activity inducing transformations by the two-stage assay. Findings of this study suggest that further research is necessary to compare carcinogenicity of tap water near water purification plant and distant tap water.

Assay discrepancy in mild haemophilia A due to a factor VIII missense mutation (Asn694Ile) in a large Danish family.

Factor VIII gene analysis in a large consanguinous Danish family comprising 24 affected males and four homozygously affected females revealed an Asn694Ile mutation within the A2 domain. The factor VIII gene mutation led to a mild haemophilia A phenotype with factor VIII function displaying discordance between one-stage clotting and chromogenic two-stage assays. In one-stage assays, values ranged from 0.05 to 0.30 IU/ml (males) and from 0.19 to 0.29 IU/ml (homozygous affected females), whereas the chromogenic two-stage assay produced values of around only 50% of the one-stage result [0. 02-0.12 IU/ml (males); 0.06-0.10 IU/ml (females)]. The differences are suggested to be caused by the effect of the mutation on the active cleaved form of the factor (F)VIII protein. As the original amino acid (Asn) is conserved in all known FVIII A2 sequences, but not in ceruloplasmin, we suggest that Asn694 is involved in an A2-specific functional role. Examination of a homology model of the A domains predicts that the Asn694Ile mutation (i) results in the loss of two potential hydrogen-bonding interactions and (ii) hampers the integration of the bulky side-chain of Ile into the A2 domain core, probably causing an altered stability and/or folding of the protein. Interestingly, the disease in this Danish family was originally proposed to be von Willebrand-Jürgens disease. However, the current study rules out the co-existence of either von Willebrand's disease or the presence of the Normandy variant of von Willebrand factor (type 2N).

Issues with the Assay of Factor VIII Activity in Plasma and Factor VIII Concentrates

A review of the literature suggests that assays accurate for the determination of factor VIII in plasma samples may not necessarily retain this accuracy when used for the determination of factor VIII in high-purity factor VII concentrates such as Hemofil M. Review of assay data suggests that it is imperative to obtain maximal activation of the factor VIII in the sample with thrombin when using an assay system of isolated coagulation factors such as the two-stage assay or the various chromogenic substrate assays. Based on a combination of ease and reproducibility of performance and correlation of in vivo and in vitro measurements. it is recommended that the one-stage activated partial thromboplastin time performed with plasma from an individual with severe hemophilia A be used for the measurement of factor VIII potency. Chromogenic substrate assays can be used if care is taken to assure optimal activation of factor VIII by thrombin in the assay and the presence of sufficient factor IXa, phospholipid and calcium ions to stabilize factor VIIIa during the assay process.

Visible Light Irradiation of [60]Fullerene Causes Killing and Initiation of Transformation in Balb/3T3 Cells

Cell transformation in vitro is a model of carcinogenesis in vivo. Two-stage transformation assay increases the sensitivity of cells to chemicals and permits detection of carcinogens acting as initiating agents. [60]Fullerene (C60) was cytotoxic in BALB/3T3 cells when it was irradiated by visible light, but not without light irradiation. Under conditions when C60 was cytotoxic, it acted as an initiating agent for cell transformation, but it did not act as a complete transforming agent. the initiating activity of visible-light-irradiated C60 was statistically significant in a modified two-stage transformation assay including a procedure for replating cells treated by C60 and light, but it was equivocal in the standard two-stage transformation assay.

Mild Hemophilia A Caused by Increased Rate of Factor VIII A2 Subunit Dissociation: Evidence for Nonproteolytic Inactivation of Factor VIIIa In Vivo

Approximately 5% of hemophilia A patients have normal amounts of a dysfunctional factor VIII (FVIII) protein and are termed cross-reacting material (CRM)-positive. FVIII is a heterodimer (domain structure A1-A2-B/A3-C1-C2) that requires thrombin cleavage to elicit procoagulant activity. Thrombin-activated FVIII is a heterotrimer with the A2 subunit (amino acid residues 373 to 740) in a weak ionic interaction with the A1 and A3-C1-C2 subunits. Dissociation of the A2 subunit correlates with inactivation of FVIII. Recently, a phenotype of CRM-positive hemophilia A patients has been characterized whose plasma displays a discrepancy between their FVIII activities, where the one-stage clotting assay displays greater activity than the two-stage clotting assay. One example is a missense mutation whereARG531 has been substituted by HIS531. An FVIII cDNA construct was prepared containing theARG531HIS mutation and the protein was expressed in COS-1 monkey cells by transient DNA transfection. Metabolic labeling with [35S]-methionine demonstrated that ARG531HIS was synthesized at an equal rate compared with FVIII wild-type (WT) but had slightly reduced antigen in the conditioned medium, suggesting a modest secretion defect. A time course of structural cleavage of ARG531HISdemonstrated identical thrombin cleavage sites and rates of proteolysis as FVIII WT. Similar to the patient phenotypes,ARG531HIS had discrepant activity as measured by a one-stage activated partial thromboplastin time (aPTT) clotting assay (36% ± 9.6% of FVIII WT) and a variation of the two-stage assay using a chromogenic substrate (COAMATIC; 19% ± 6.9% of FVIII WT). Partially purified FVIII WT and ARG531HISproteins were subjected to functional activation by incubation with thrombin. ARG531HIS demonstrated significantly reduced peak activity and was completely inactivated after 30 seconds, whereas FVIII WT retained activity until 2.5 minutes after activation. Because the ARG531HIS missense mutation predicts a charge change to the A2 subunit, we hypothesized that theARG531HIS A2 subunit could be subject to more rapid dissociation from the heterotrimer. The rate of A2 dissociation, using an optical biosensor, was determined to be fourfold faster forARG531HIS compared with FVIII WT. Because the two-stage assay involves a preincubation phase before assay measurement, an increased rate of A2 dissociation would result in an increased rate of inactivation and reduced specific activity.

Mild Hemophilia A Caused by Increased Rate of Factor VIII A2 Subunit Dissociation: Evidence for Nonproteolytic Inactivation of Factor VIIIa In Vivo

AbstractApproximately 5% of hemophilia A patients have normal amounts of a dysfunctional factor VIII (FVIII) protein and are termed cross-reacting material (CRM)-positive. FVIII is a heterodimer (domain structure A1-A2-B/A3-C1-C2) that requires thrombin cleavage to elicit procoagulant activity. Thrombin-activated FVIII is a heterotrimer with the A2 subunit (amino acid residues 373 to 740) in a weak ionic interaction with the A1 and A3-C1-C2 subunits. Dissociation of the A2 subunit correlates with inactivation of FVIII. Recently, a phenotype of CRM-positive hemophilia A patients has been characterized whose plasma displays a discrepancy between their FVIII activities, where the one-stage clotting assay displays greater activity than the two-stage clotting assay. One example is a missense mutation whereARG531 has been substituted by HIS531. An FVIII cDNA construct was prepared containing theARG531HIS mutation and the protein was expressed in COS-1 monkey cells by transient DNA transfection. Metabolic labeling with [35S]-methionine demonstrated that ARG531HIS was synthesized at an equal rate compared with FVIII wild-type (WT) but had slightly reduced antigen in the conditioned medium, suggesting a modest secretion defect. A time course of structural cleavage of ARG531HISdemonstrated identical thrombin cleavage sites and rates of proteolysis as FVIII WT. Similar to the patient phenotypes,ARG531HIS had discrepant activity as measured by a one-stage activated partial thromboplastin time (aPTT) clotting assay (36% ± 9.6% of FVIII WT) and a variation of the two-stage assay using a chromogenic substrate (COAMATIC; 19% ± 6.9% of FVIII WT). Partially purified FVIII WT and ARG531HISproteins were subjected to functional activation by incubation with thrombin. ARG531HIS demonstrated significantly reduced peak activity and was completely inactivated after 30 seconds, whereas FVIII WT retained activity until 2.5 minutes after activation. Because the ARG531HIS missense mutation predicts a charge change to the A2 subunit, we hypothesized that theARG531HIS A2 subunit could be subject to more rapid dissociation from the heterotrimer. The rate of A2 dissociation, using an optical biosensor, was determined to be fourfold faster forARG531HIS compared with FVIII WT. Because the two-stage assay involves a preincubation phase before assay measurement, an increased rate of A2 dissociation would result in an increased rate of inactivation and reduced specific activity.

ADP-ribosylation factor 1 transiently activates high-affinity adaptor protein complex AP-1 binding sites on Golgi membranes.

Association of the Golgi-specific adaptor protein complex 1 (AP-1) with the membrane is a prerequisite for clathrin coat assembly on the trans-Golgi network (TGN). The AP-1 adaptor is efficiently recruited from cytosol onto the TGN by myristoylated ADP-ribosylation factor 1 (ARF1) in the presence of the poorly hydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). Substituting GTP for GTPgammaS, however, results in only poor AP-1 binding. Here we show that both AP-1 and clathrin can be recruited efficiently onto the TGN in the presence of GTP when cytosol is supplemented with ARF1. Optimal recruitment occurs at 4 microM ARF1 and with 1 mM GTP. The AP-1 recruited by ARF1.GTP is released from the Golgi membrane by treatment with 1 M Tris-HCl (pH 7) or upon reincubation at 37 degreesC, whereas AP-1 recruited with GTPgammaS or by a constitutively active point mutant, ARF1(Q71L), remains membrane bound after either treatment. An incubation performed with added ARF1, GTP, and AlFn, used to block ARF GTPase-activating protein activity, results in membrane-associated AP-1, which is largely insensitive to Tris extraction. Thus, ARF1. GTP hydrolysis results in lower-affinity binding of AP-1 to the TGN. Using two-stage assays in which ARF1.GTP first primes the Golgi membrane at 37 degreesC, followed by AP-1 binding on ice, we find that the high-affinity nucleating sites generated in the priming stage are rapidly lost. In addition, the AP-1 bound to primed Golgi membranes during a second-stage incubation on ice is fully sensitive to Tris extraction, indicating that the priming stage has passed the ARF1.GTP hydrolysis point. Thus, hydrolysis of ARF1.GTP at the priming sites can occur even before AP-1 binding. Our finding that purified clathrin-coated vesicles contain little ARF1 supports the concept that ARF1 functions in the coat assembly process rather than during the vesicle-uncoating step. We conclude that ARF1 is a limiting factor in the GTP-stimulated recruitment of AP-1 in vitro and that it appears to function in a stoichiometric manner to generate high-affinity AP-1 binding sites that have a relatively short half-life.

The presence of anti-neutrophil antibodies reflects clinical and genetic heterogeneity within inflammatory bowel disease.

A detailed investigation of the relationship between anti-neutrophil cytoplasmic antibodies (ANCA) status, HLA genotype, and clinical patterns of inflammatory bowel disease was carried out, involving 236 European patients resident in the United Kingdom [120 had ulcerative colitis (UC), 116 had Crohn's disease (CD)]. ANCA status was determined on coded plasma samples in Los Angeles using a two-stage assay [fixed neutrophil enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence], and HLA genotyping was carried out by polymerase chain reaction. The results provide evidence that ANCA reflect clinical and genetic heterogeneity within the inflammatory bowel diseases. In the UC patients, 78.3% were ANCA positive [64.2 perinuclear (pANCA)], but only 46.5% CD patients were ANCA positive (19.3% pANCA). Furthermore, mean ELISA binding was significantly lower in CD (14.5% +/- 18.8% versus 40.5% +/- 41.0% in UC, p = 2.31 x 10(-9)). Only 15 CD samples, all from patients with colonic disease, displayed ELISA > 20%; and the six CD patients with highest ELISA binding had clinical features very similar to ulcerative colitis. Moreover, in UC, significant relationships between ANCA status and genotype were noted. Thus, 92.7% of patients with the DR3 DQ2 TNF2 haplotype were ANCA positive [p = 0.03 versus DR3 DQ2 TNF2-negative patients (73.9%)]. ELISA binding was increased in DR3 DQ2 TNF2-positive patients (56.0 versus 35.7%, p = 0.02). In this population of UC, ANCA was not associated with DR2, DR4, or clinical pattern. These data emphasize the many factors that need to be considered in genetic marker studies in inflammatory bowel disease. Extensive disease heterogeneity, ethnicity, and methodological differences in ANCA detection are all pertinent.

Can cell-free enzymes replace rumen micro-organisms to model energy and protein suppty?

AbstractNew ruminant feeding systems being developed in Europe and North America have greatly increased the need for rapid measurement of rate and extent of ruminal digestion of complex carbohydrates and proteins in individual foods. Application of these systems is lagging because of a lack of reliable kinetic digestion data. This has increased interest in using cell-free enzymes to assess ruminal breakdown of carbohydrates and proteins. The simplest kinetic model describing ruminal digestion divides carbohydrates and proteins into three fractions: A (that already digested and/ or that soluble), B (that digestible) and C (that completely indigestible). Fractions A, B and C sum to 1·0; proportion of digestible fraction B that is degraded is given by: [kd/ (kd+ kp], where kpand kdare, respectively, rates of ruminal passage and digestion. Some models divide fraction B into two or three subfractions while others include a digestion lag. Fully successful, cell-free enzyme systems would yield accurate estimates of digestion fractions and rates and, where appropriate, lag time. As a minimum, results with enzymes should be well correlated to in situ extents of digestion. In vivo total tract digestibility of organic matter (OM) and energy was predicted accurately when food samples were pre-treated with acid-pepsin or neutral-detergent solution, followed by treatment with cellulase from Trichoderma viride (T. reesei). Pepsin-cellulase and neutral detergent-cellulase methods have predicted in vivo digestibility more precisely than Tilley and Terry methods in some cases. Two-stage cellulase assays have given good results with many forages and, when including an amylase treatment, with mixed foods; OM digestibility of straws was not well predicted. Many different cell-free proteases have been tested to estimate ruminal protein degradability. Generally, effectiveness of proteases was assessed by correlating proportions of food nitrogen solubilized after specific incubation times with extents of in situ protein degradation. The broad specificity protease fromStreptomyces griseushas been used most extensively for this purpose in incubations at pH 8·0. However, somewhat better correlations have been reported for ficin, bromelain, papain and neutral proteases of fungal and bacterial origin in incubations at pH more similar to the rumen. Prior treatment with amylases improved correlations for concentrate foodstuffs. As yet, cell-free proteases have not accurately predicted rates and extents of protein degradation observed in ruminal in vitro and in situ systems.

Inhibition of tumor promoter activity toward mouse fibroblasts and their in vitro transformation by tissue inhibitor of metalloproteinases-1 (TIMP-1).

Tissue inhibitor of metalloproteinases-1 (TIMP-1), a natural inhibitor of matrix metalloproteinases (MMPs), is known to inhibit invasion and metastasis of tumor cells. In the present study we examined anti-tumor promoter activity of TIMP-1 and its effect on in vitro cell transformation using BALB/3T3 cells in low serum culture medium. In the dye transfer assay the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) continuously blocked gap-junctional intercellular communication (GJIC) of BALB/3T3 cells in confluent phase. TIMP-1 did not prevent transient inhibition of GJIC induced by TPA, but it quickly restored the reduced GJIC level to the control level. The recovery of GJIC was dependent on the concentration of TIMP-1 from 1 to 1000 ng/ml. In an in vitro two-stage transformation assay in which BALB/3T3 cells were treated with 0.5 microg/ml N-metyl-N'-nitro-N-nitrosoguanidine as initiator and 100 ng/ml TPA as promoter, TIMP-1 at concentrations > 10 ng/ml inhibited the focus formation of transformed cells by approximately 60%. TIMP-2 and a synthetic MMP inhibitor showed a similar inhibitory activity on in vitro cell transformation. Furthermore, zymographyic analysis showed that TPA treatment of BALB/3T3 cells induced secretion of gelatinase B and stromelysin-1 into the culture medium. These results indicate that TIMP-1 and TIMP-2 have inhibitory activity on in vitro transformation of cells. It seems likely that TPA-inducible MMPs are involved in carcinogenesis and TIMPs have a protective role against carcinogenesis in vivo.

Mutations in a subgroup of patients with mild haemophilia A and a familial discrepancy between the one-stage and two-stage factor VIII:C methods.

A subgroup of patients with haemophilia A who have a familial discrepancy between the one-stage and two-stage factor VIII:C results has previously been described. These patients show factor VIII:C levels by one-stage assay that are 2-7-fold higher than their two-stage results. We have studied 10 such families and identified six different mutations in the factor VIII gene in this group. The chemical cleavage method and DNA sequencing was used to identify mutations in factor VIII gene fragments generated by reverse transcription and PCR. All available family members were tested to confirm the presence of the mutation in affected individuals. These patients were found to have one of six single point substitutions causing a missense mutation and alteration to one codon in exons 7, 11, 14 or 18. The mutations comprise three that have not previously been described (Ala284Glu. Arg698Leu. Leu1932Phe) and three that have been previously described (Ser289Leu, Arg531His, Arg698Trp). Alterations to the amino acid composition of the A1, A2 and A3 domains of factor VIII are predicted by these molecular studies. In contrast, a control group of 23 mild haemophilia families with equivalent factor VIII:C results by one-stage and two-stage assays did not have any of the above mutations. Detailed studies in seven of these latter families identified four mutations affecting the A3, C1 and C2 domains of factor VIII. These findings suggest a genetic basis to the unusual factor VIII phenotype but do not explain the mechanism of the discrepant factor VIII activity.

Potency measurement of factor VIII in recombinant concentrates.

In preliminary studies on the stability of recombinant factor VIII (rFVIII) concentrates post reconstitution, a rise in potency to 200% of labelled values was observed in concentrates stored at 22 °C over 24 h. This was observed in potency estimates by the one-stage clotting, but not the two-stage clotting or chromogenic assays, and was not observed for intermediate-purity product derived from plasma (IPVIII). Use of human serum albumin (HSA), rather than the usual bovine material (BSA), to dilute product for the stability study abolished the rise in potency. Incorporating purified von Willebrand Factor (VWF) in the diluent buffer abolished the rise observed in BSA. A similar rise was observed upon incubating rFVIII in the presence of 10(-4) u of thrombin per mL in HSA buffer. Potency estimates using the HSA in the dilution buffer resulted in severe underpotency in relation to the label claim when using the two-stage clotting and the chromogenic assays, but not the one-stage clotting assay. Predilution in severe haemophilic plasma restored potency levels to those claimed. We conclude that (i) commercial preparations of BSA may be unsuitable for inclusion in buffers for rFVIII studies; (ii) FVIII in rFVIII concentrates is exquisitely sensitive to activation by thrombin, presumably as a result of the lack of VWF; (iii) accurate potency estimation in the two-stage assay systems requires VWF in the assay system.

Asbestos promotes morphological transformation and elevates expression of a gene family invariably induced by tumor promoters in C3H/10T1/2 cells.

The murine proliferin gene family, which has been shown to respond consistently to tumor promoters and other cellular pro-oxidant agents in C3H/10T1/2 cells, was used to monitor responses after treatment of these cell cultures with toxic, pro-oxidant asbestos fibres. Proliferin mRNA levels were increased by amosite, crocidolite or chrysotile asbestos fibres, especially in the presence of fresh serum and at low cell densities. Promotion of morphological transformation was confirmed in two-stage focus formation assays using crocidolite at a fibre density that induced proliferin expression. Asbestos-induced gene expression was inhibited by millimolar levels of N-acetylcysteine (NAC), supporting a linkage between: (i) induced oxidant stress that was sufficient to promote morphological transformation; (ii) induction of proliferin expression. Other anti-oxidant compounds (dithiothreitol and pyrrolidine dithiocarbamate) or enzymes (superoxide dismutase and catalase) did not inhibit induced expression. Non-fibrous powders (titanium dioxide, quartz or silica gel) were also effective inducers of proliferin mRNA accumulation. Latex beads and activated charcoal were effective at higher particle densities, implying that ubiquitous particle-induced surface membrane effects can lead to an NAC-reversible step necessary for proliferin induction. The results showed that asbestos resembled all other promoters of morphological transformation in C3H/10T1/2 cells in that an antioxidant-sensitive induction of the proliferin gene family occurred following treatment.

Tri-n-butyltin chloride promotes morphological transformation and induces proliferin expression in C3H10T1/2 cells.

Transcripts from the murine gene family proliferin, which are increased by a wide assortment of chemical promoters of C3H10T1/2 cell morphological transformation, were shown to be induced by tri-n-butyltin chloride at concentrations above 50 nM. Two-stage transformation assays, with 3-methylcholanthrene as inducer and tri-n-butyltin chloride as promoter, were performed to determine if promotion of morphological transformation and proliferin induction were properties shared by this compound. Tri-n-butyltin chloride synergistically enhanced focus formation at concentrations ranging from 20 to 75 nM. Di-n-butyltin dichloride, n-butyltin trichloride and tin (II) chloride, but not tin (IV) chloride, were also effective inducers of proliferin. Changes in patterns of TPA-inducible, secreted proteins, including those likely to be proliferin, were detected following organotin treatment of confluent monolayers. Tri-n-butyltin chloride resembles other agents active as promoters in C3H10T1/2 two-stage transformation assays by possessing an ability to induce proliferin expression.

Antipromoting effects of 5-lipoxygenase inhibitors, 3-nitro-2,4,6- trihydroxybenzamide derivatives, on TPA-promoted transformation in BALB 3T3 cells.

In order to discover the role in the promotion process of 5-lipoxygenase (5-LO), a key enzyme in the arachidonate cascade, the antipromoting effects of the 5-LO inhibiting 3-nitro-2,4,6-trihydroxybenzamide (NTB) derivatives were studied in a two-stage transformation assay system using BALB 3T3 cells. All compounds inhibited TPA-promoted transformation in a dose-dependent manner. Most of them achieved a 70-80% inhibition. Good correlations were observed between the inhibition of TPA-promoted transformation and that of 5-LO. These results indicate that 5-LO plays an important role in the promotion stage of the transformation of BALB 3T3 cells.