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Haemophilia A is the most common inherited bleeding disorder, caused by a deficiency in coagulation factor VIII (FVIII). Current treatment of haemophilia A is based on repeated infusions of plasma-derived FVIII concentrate or of recombinant FVIII, which may be exposed to plasma-derived material of human or animal origin used in its tissue culture production process. We review epidemiological and experimental studies relevant to blood infectivity in the transmissible spongiform encephalopathies (TSEs, or 'prion' diseases), and evaluate the hypothetical risk of TSE transmission through treatment with plasma-derived or recombinant FVIII.

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Habit surveys were carried out around four licensed nuclear sites to identify people who collect foodstuffs from the wild (so-called 'free foods'). In total, around 800 collectors were readily identified, most of whom collected more than one free food. The data indicated that estimates of higher than average doses could reasonably be based on the three foodstuffs of most importance. Foods were selected for further study on the basis of either the number of collectors or the amount consumed. The radionuclides of interest were identified using published information on the discharges from each site. The resultant average and higher than average doses were estimated using the site-specific habit data. For all sites, doses from the consumption of free foods were low and of no radiological importance. Assessments based solely on data for cultivated foods would not therefore have underestimated radiological impact significantly. However, given the wide utilisation of free foods found in this study, for rigorous assessments it would be prudent to take account of the consumption of foods from the wild.

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A new process for the production of intramuscular immunoglobulin products is described which includes viral inactivation through solvent-detergent treatment. Removal of solvent-detergent was accomplished by precipitation, filtration and diafiltration. Process-scale preparations had appropriate antibody potency levels, and improved IgG integrity relative to traditional IGIM products. Moreover, acceptable results were obtained in all in vitro and in vivo pre-clinical toxicology testing, as well as clinical evaluation. Scaled-down experiments demonstrated that the new process provides effective viral inactivation. Taken together, these results indicate that the new products should have the same efficacy of the previous IGIM products albeit with safety and processing improvements.

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Tryptophan enantiomers have been separated by zwitterion pair chromatography using L-leucine-L-leucine-L-leucine peptide as the zwitterion pairing agent. The peptide ligand is adsorbed onto an octadecylsilane support with excess ligand present in bulk solution. This article examines the roles of the hydrophobic matrix and the mobile phase components on tryptophan enantiomer binding and resolution. Capacity factors and enantioselectivites are given for both hydrophobic and hydrophilic matrices using mobile phases containing Leu-Leu-Leu peptide and/or salt. A decrease in selectivity upon the addition of mobile phase salt suggests that quadrupolar ion-pairing contributes to chiral recognition. Results indicate that binding is significantly reduced and separation is not achieved when Leu-Leu-Leu is coupled onto cross-linked or polymerized hydrophilic resins as well as onto macroporous polystyrene resin. However, resin-immobilized Leu-Leu-Asp-Leu-Leu-Leu, Leu-Leu-Glu-Leu-Leu-Leu, and Leu-Leu-Leu-Glu-Leu-Leu peptides, with ion-pairing sites designed to mimic the Leu-Leu-Leu-saturated C18 support, also do not resolve tryptophan enantiomers. This suggests the Leu-Leu-Leu structure is critical for enantiomer resolution. Because D- and L-tryptophan are separated in the absence of bulk Leu-Leu-Leu, chiral discrimination is believed to occur at the surface of the octadecylsilane support.

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BACKGROUND AND OBJECTIVES: Alpha-proteinase inhibitor (PI) protects the lungs from proteolytic damage caused by elastase and can be used to treat congenital emphysema. We describe an improved method of purification of alpha 1 PI from redissolved fraction IV-1 paste. MATERIALS AND METHODS: The process used dimethylaminoethyl anion exchange chromatography, sulfopropyl cation exchange chromatography, virus inactivation by dry heat, and tri-n-butyl-phosphate/cholate treatment, followed by a second strong cation exchange chromatography. Optimizations of loading conditions for ion exchange chromatography at small scale (20-60 ml of suspension) are described. Virus inactivation was adjusted to provide the best yield of alpha 1 PI consistent with effective inactivation. The process has been effectively scaled up. RESULTS: The final product was approximately 90% pure by SDS-PAGE, with a 60-70% yield from starting fraction IV-1 paste. The process has been characterized by methods including nonreduced SDS-PAGE, alpha 1 PI inhibition assay, and biuret protein assay. CONCLUSION: The method described is an effective way of preparing large quantities of alpha 1 PI from fractionated plasma.

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A novel chromatographic process for purification of alpha 1 proteinase inhibitor (alpha 1-PI) from Cohn fraction IV-1 paste is described. This process has been successfully scaled up to 50-1 columns. It involves DEAE chromatography, sulfopropyl (S) cation chromatography, tri-n-butyl phosphate (TNBP)-cholate treatment, a second S cation chromatography, freeze-drying and dry-heat. The process has been optimized for purity, yield, lipid removal, chemical usage and water consumption. Filtration after TNBP-cholate treatment plays a key role in ensuring a low lipid content in the final product. Pre-equilibration with high salt buffer is necessary to reduce the water consumption significantly during the ion-exchange chromatography equilibration step. The final product is approximately 95% pure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a 64% to 70% yield from IV-1 paste.

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The chromatographic purification of vWF (von Willebrand Factor) from human plasma represents a challenge because it consists of multimers with molecular weights ranging from 0.5 to 10 million Daltons. Phage peptide library screening yielded a lead peptide (RLRSFY) that interacts with vWF. Conservative substitutions of terminal residues of the lead peptide led to a second peptide, RVRSFY, which was more efficient in the affinity chromatographic purification of vWF from protein mixtures. Adsorption isotherm measurements indicated multiple interactions between vWF and the immobilized peptide RVRSFY. Increases in peptide density on the chromatographic supports resulted in stronger association constants and higher maximum protein binding capacities. When the peptide density was lower than 32 mg/mL, there was no measurable interaction between vWF and immobilized peptide RVRSFY in HEPES buffer containing 0.5 M NaCl at pH 7. An increase in peptide density from 32 to 60 mg/mL increased the association constants from 0.9 x 10(6) to 2 x 10(6) (M-1). Divalent salts (calcium and magnesium chloride) were used to elute the retained vWF with 82.5% of the activity recovered. The interactions between vWF and the immobilized peptide RVRSFY are dominated by ionic attractions and also involve hydrophobic interactions at close contact. Finally, the purification of vWF from crude material PEG filtrate of a cryoprecipitate of human plasma is demonstrated using affinity chromatography with immobilized N-acetyl-RVRSFYK.

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We have developed a new resin for peptide synthesis that can be used to synthesize and evaluate directly combinatorial peptide libraries for binding target proteins. Fidelity of the peptide synthesis using this hydrophilic resin is comparable to polystyrene-based resins. Peptide libraries synthesized on this resin were probed by a two color PEptide Library Immunostaining Chromatographic ANalysis (PELICAN) technique for sequences binding the serine protease Factor IX zymogen. This PELICAN technique readily distinguishes between beads interacting with the reagents for target detection (blue beads) from those beads specific for the target protein itself (red beads). Validation of the PELICAN technique, as well as purification of Factor IX from plasma, is demonstrated utilizing this resin.

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We describe an improved method for large-scale purification of antithrombin III (AT-III) from human plasma involving heparin affinity chromatography of redissolved fraction IV-1 paste, viral inactivation by heating, followed by a second heparin affinity column. The characteristics of a new heparin affinity resin and the ability to extrapolate process behavior from small-scale (20 ml) to large-scale (40 liter) columns are described. This supports the use of the small-scale column for process optimization and validation studies in compliance with current regulatory requirements for biological products. The process has been characterized by analytical techniques including sodium dodecyl sulfate (SDS), reducing SDS, and nondenaturing polyacrylamide gel electrophoresis; laser desorption time-of-flight mass spectroscopy, and electrospray mass spectroscopy. These results demonstrate that greater than 95% of the protein in the final products is AT-III, which is greater than 95% active as defined by thrombin inhibition.

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A 96-well microtiter enzyme-linked immunosorbent assay (ELISA) for protein tyrosine kinases has been developed. This assay uses one of several substrates that are phosphorylated by tyrosine kinase, an antibody to phosphotyrosine, and a peroxidase-linked second antibody. Color development is monitored by a change in absorbance at 450 nm and is dependent upon time, enzyme, ATP, and substrate concentrations. Specificity of the ELISA for phosphotyrosine was shown by inhibition of binding of the anti-phosphotyrosine antibody with phenyl phosphate. Results obtained in the ELISA compared favorably with those obtained by direct phosphorylation of the substrate with [32P]ATP. Staurosporine and K252a, protein kinase inhibitors, showed titratable inhibition of tyrosine kinase activity. This assay is a rapid, nonradioactive alternative to traditional methodology and is also amenable to automation.

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Our strategy for preventing the transmission of Chagas' disease during blood transfusion is discussed. In addition, the possibility that the Peru, Sonya, Tulahuen and Y strains of Trypanosoma cruzi show varying sensitivities to a series of amphiphilic cationic drugs in vitro at 4 degrees C was investigated using a microscope lysis test. All 21 drugs tested at a concentration of 10(-3) M lysed Sonya bloodstream trypomastigotes, but Peru, Tulahuen and Y strains were affected by 17, 17 and 11 drugs, respectively. All four strains were most sensitive to the acridines; acranil, aminacrine and mepacrine. Although some variation was seen in their responses to certain drugs, no one strain was particularly insensitive to the series as a whole. The effects of gentian violet, maprotiline and mepacrine on the infectivity of Sonya trypomastigotes following incubation at 4 degrees C for 24 h were evaluated. Mepacrine, at a concentration of 2.5 X 10(-4) M greatly decreased the viability of trypomastigotes, while 10(-3) M concentrations of both maprotiline, mepacrine, and gentian violet (at low parasite densities only) apparently abolished all infectivity. Although the compounds we tested did not show a significant improvement over gentian violet, the compound currently used in some blood banks, other existing amphiphilic cationic drugs could be of use in preventing the transmission of Chagas' disease during blood transfusion.

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Glycosomes, purified from trypomastigote forms of Trypanosoma brucei, contained all the enzymes necessary to convert glucose to alpha-glycerophosphate and 3-phosphoglycerate. The multienzyme reaction which produces 2 alpha-glycerophosphate, 2 ADP, and 2 NAD+ from 1 glucose, 2 ATP, and 2 NADH was studied spectrophotometrically. Intact glycosomes, suspended with 5.6 mM alpha-glycerophosphate and 2 mM ADP, produced ATP inside the glycosomes for glucose phosphorylation at a rate of 0.7 mumol/min/mg protein, so confirming the feasibility of producing ATP from alpha-glycerophosphate and ADP catalyzed by glycosomal glycerol kinase, and coupling this ATP production to the ATP-requiring stages of glycolysis. No evidence was found for direct channeling of the ATP generated by glycerol kinase and either hexokinase or phosphofructose kinase in glycosomal enzyme complexes cross-linked by dimethyl suberimidate treatment of intact glycosomes prior to solubilization of their membrane. Compartmentation of glycolytic intermediates, enzymes, and ATP inside isolated glycosomes was demonstrated by their inaccessibility to exogenous enzymes. We conclude that the compartmentation of the glycosome and the efficient production of ATP in the glycosome from whole cell concentrations of sn-glycerol 3-phosphate and ADP account for the observed whole cell production of equimolar glycerol from glucose with net ATP synthesis by T. brucei under anaerobic conditions.

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About 200 clinically used amphiphilic cationic drugs have been shown to be active in vitro against Trypanosoma cruzi at concentrations of less than or equal to 1 mM. Activity against epimastigote and trypomastigote forms was similar, and in both cases the most potent drugs were litracene, maprotiline, thioproperazine, and the acridines: acranil, aminacrine, and mepacrine. Fluorescence microscopy demonstrated that epimastigotes rapidly accumulate acridines initially in discrete subcellular organelles. The amount of drug incorporated during 15 min of incubation was sufficient to produce subsequent lysis of both trypomastigotes and epimastigotes within 24 hr at 4 C. Trypanocidal activity was dependent on the extracellular pH (optimum greater than or equal to 8) and drug exposure time, but was independent of red blood cell density, serum dilution, and temperature (4 to 37 C). Despite their trypanocidal activity, amphiphilic cationic drugs appear to have no significant effect on the energy state of red blood cells at a concentration of 1 mM. These drugs have a possible role in the prevention of Chagas' disease by blood transfusion.

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The effects of the hydroxynaphthoquinone BW58C on some metabolite levels and the flux of H14CO3 through the de novo pyrimidine biosynthetic pathway of intact Plasmodium falciparum have been studied in vitro using HPLC techniques. 800 nM BW58C appeared to have no significant effect on the energy status of isolated P. falciparum, but at 0.1 nM it caused a dramatic decrease in the concentrations of pyrimidine nucleotides, specifically UTP, during 256 min of incubation. Although about one hour was required to achieve a significant decrease in pyrimidine nucleotide concentrations, a much more rapid inhibition of the flux of H14CO3 through the de novo pathway was found upon addition of 0.1 nM BW58C. This inhibition caused about a 10 fold increase in the radioactivity of carbamoyl-aspartate over a 64 min period, and an overall increase in the concentration of this metabolite of about 3 fold during 256 min of incubation. These effects of BW58C against P. falciparum in vitro are discussed in terms of inhibition of de novo pyrimidine biosynthesis at the site of dihydroorotate dehydrogenase.

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The pathways leading to purine and pyrimidine nucleotide production in members of the family Trypanosomatidae are discussed with special emphasis on data relating to pathogenic species published from 1974 to 1983 inclusive. Trypanosomes and leishmania in general lack a de novo purine biosynthetic pathway, but have a multiplicity of possible routes for purine salvage. In contrast, pyrimidine nucleotides can be produced by either de novo or salvage pathways. The properties of these pathways in trypanosomatids are compared and contrasted with those of their hosts.

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A rapid in vitro test system has shown that many drugs which possess a product licensed for use in man are also active at a concentration of less than 1mM against the blood forms of Trypanosoma cruzi. 62 of these are structurally related amphiphilic cationic drugs which completely lyse the trypomastigotes at 4 degrees C within 24 hr, yet most leave the erythrocytes intact. Three polyene and two anthracycline antibiotics were also found to be selectively trypanocidal under the same conditions.

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The orotate phosphoribosyltransferase of the epimastigote form of Trypanosoma cruzi was studied in its particulate state in preparations derived from glycosomes. Maximum activity was observed at pH 9. There was little activity in the absence of Mg2+; optimum [Mg2+] was related to [5'-phosphoribosyl-alpha-1-pyrophosphate]; Mn2+ could partially substitute for it. Kinetic analyses ruled out a substituted mechanism and suggested instead that the mechanism may be sequential. The apparent Km orotate was 2 microM; that for 5'-phosphoribosyl-alpha-1-pyrophosphate was 8 microM. The enzyme could not use uracil as substrate and was apparently not regulated by naturally-occurring nucleotides. It was, however, sensitive to inhibition by a wide range of pyrimidine analogues, the most active of which was 5-fluoroorotate. These inhibitors were as effective against the enzyme activity of intact glycosomes as broken preparations. This observation, when considered with an apparent lack of latency, suggests that the enzyme is located on the outside of the glycosome. The product of its reaction, orotidine 5'-phosphate, did not exchange readily with exogenous orotidine 5'-phosphate, suggesting that it is channeled directly to orotidine 5'-phosphate decarboxylase, the next enzyme in the pathway.

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All six enzymes of pyrimidine biosynthesis de novo have been detected in homogenates of the culture promastigote form of Leishmania mexicana amazonensis, the blood trypomastigote form of Trypanosoma brucei and the culture epimastigote, blood trypomastigote and intracellular amastigote forms of Trypanosoma cruzi. Dihydroorotate dehydrogenase is mitochondrial in mammals, but the isofunctional enzyme, dihydroorotate oxidase was found to be cytoplasmic, whereas orotate phosphoribosyltransferase and orotidine-5'-phosphate decarboxylase, which are cytoplasmic in mammals, were found to be particulate. Analysis by isopycnic sedimentation in sucrose showed that both particulate enzymes co-sedimented with glycosomal-(microbody-)marker enzymes such as hexokinase. Electron microscopy indicated that fractions containing these activities consisted essentially only of microbodies. It is concluded therefore that these enzymes are associated with glycosomes. Kinetic studies with intact glycosomal preparations suggested that there was no membrane barrier between 5-phosphoribose 1-pyrophosphate (P-Rib-PP) and orotate phosphoribosyltransferase, indicating either that the active site of this enzyme is probably on the outside of the glycosome or that the glycosome may have an efficient transport site for P-Rib-PP. Not all the UMP salvage enzymes assayed were detected. No uridine kinase activity was found in any of the species investigated, suggesting that uridine salvage might be routed via a uridine phosphorylase and uracil phosphoribosyltransferase. In agreement with this suggestion, these latter activities were detected in all organisms tested except the intracellular amastigote form of T. cruzi, where uracil phosphoribosyltransferase appeared absent. All the UMP salvage enzymes investigated occurred in cytoplamic fractions.

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Studies measuring the glycolytic intermediate and adenine nucleotide concentrations in Trypanosoma brucei metabolising glucose either aerobically or under conditions where glycerol-3-phosphate oxidase is inactive have shown the following: 1. Inhibition with 0.5 mM salicylhydroxamic acid (SHAM) accurately simulates anaerobic conditions in T. brucei; 2. On inhibition of respiring cells with 0.5 mM SHAM, the concentrations of most glycolytic intermediates decrease; they decrease further as the concentration of glycerol, an end product, increases. Only the concentration of sn-glycerol-3-phosphate is increased. This increase depends upon the method of preparation but is independent of time and glycerol concentration. 3. Glycerol formation from sn-glycerol-3-phosphate is coupled to the phosphorylation of another compound. The results of these studies are consistent with this compound being ADP; 4, The degree of inhibition of the anaerobic metabolism of glucose exerted by glycerol varies with the sn-glycerol-3-phosphate concentration, implying that the effect of glycerol is at the site of sn-glycerol-3-phosphate: ADP transphosphorylation.

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