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The effect of chemical inactivation of bovine viral diarrhea virus with beta-propiolactone and binary ethyleneimine on plasma proteins and coagulation factors.

In the present study chemical inactivation of bovine viral diarrhea virus (BVDV), as a substitute of hepatitis C virus was studied in human plasma pool. Beta-propiolactone (BPL), binary ethyleneimine (BEI) and chlorhexidine (CHX) were assessed. Treatment of virus-spiked human plasma with 0.025% BPL reduced virus infectivity titer to undetectable levels within 2 h, whereas BEI treatment (1 mM) showed a slower kinetic of inactivation, attaining a complete virus inactivation within 8 h of incubation. In contrast, CHX treatment at the adopted dose level (0.41mM) showed a limited virucidal capacity with a residual live virus titer after 24 h. BPL and BEI treatments reduced the recovery of labile plasma coagulation factors activity (V and VIII), while the activity of other coagulation factors (VII, IX and XI) was mildly decreased. Agarose gel electrophoresis of plasma proteins showed that albumin concentration is not affected, while gamma-globulin is slightly reduced by BPL and BEI treatment. Plasma fibrinogen level was modestly reduced by BPL treatment, while it remained unchanged by BEI treatment. This demonstrates the potential and safety use of BPL and BEI in BVDV inactivation in human plasma pool without affecting significantly the coagulant activity of important blood coagulation factors and the levels of plasma major protein fractions.

Animals↗

[Genetic analysis of spontaneous and 6-N-hydroxylaminopurine and propiolactone induced Adp+ mutants in Saccharomyces yeasts].

652 spontaneous and 6-N-hydroxylaminopurine and propiolactone-induced mutants were obtained in yeast. 598 of them were LYS2 mutants. Detailed genetic analysis of the mutants was performed, including analysis of growth pattern on lysineless medium, suppressibility by nonsense suppressors of three types and localization on the recombination map of the LYS2 gene. Mutants induced by different agents were different for all these criteria, except for distribution among the map regions.

2-Aminoadipic Acid↗

Quantitative assays for evaluation of HTLV-III inactivation procedures: tri(N-butyl)phosphate:sodium cholate and beta-propiolactone.

Human T-lymphotropic retrovirus type III (HTLV-III) can be quantitatively assayed for infectivity by inoculation of serial dilutions into cultures of the H-9 cell line and testing for reverse transcriptase in the culture supernatants. Sequential harvests revealed that 14 days of incubation of cultures fed twice weekly was sufficient to reveal maximal titers. Stocks prepared from unconcentrated H9:HTLV-IIIb supernatants have contained from 10(4.5) to 10(6.0) (TCID50)/ml. Stocks prepared by 100-fold concentration of such fluids by pelleting or by polyethylene glycol precipitation followed by pelleting onto sucrose cushions contained 10(6.0)-10(6.5) TCID50/ml. Preliminary studies are under way to utilize this system for evaluation of sterilization processes which can be applied to blood derivatives. Exposure of HTLV-III suspended in Factor VIII preparations to 0.3% tri(n-butyl)phosphate-0.2% sodium cholate resulted in inactivation of greater than or equal to 10(4.5) TCID50 in 2.5 h at 27 degrees C. Exposure of HTLV-III suspended in 4 g of gamma-globulin per 100 ml to 0.14% beta-propiolactone for 4 h at room temperature at pH 8.0 inactivated greater than or equal to 10(4.5) TCID50. However, exposure to gamma-globulin alone inactivated about 99% of HTLV-III infectivity.

Cell Line↗

[Mutability of LYS2 gene in diploid Saccharomyces yeasts. II. Frequency of mutants induced by 6-N-hydroxylaminopurine and propiolactone].

Chemical mutagens 6-N-hydroxylaminopurine (HAP) and propiolactone (PRO) induce Lys2 mutants with high frequency in diploid yeast Saccharomyces cerevisiae. HAP induces such mutants even in tetraploid strains. The genetic analysis of mutants was performed. It is shown that PRO induces mutants by means of "mutation-mitotic segregation" mechanism, while HAP induces mutants through novel mechanism "both allele mutation". Manifestation of such mechanism is the null fertility after meiosis of diploid mutants induced by HAP.

Adenine↗

Inhibition of beta-propiolactone-induced neoplasia of the forestomach and large bowel by 4-mercaptobenzene sulfonate in mice and rats.

Studies have been initiated to find compounds that can trap direct-acting carcinogens within the lumen of the gastrointestinal tract and thus prevent these carcinogens from attacking tissues of the host. Sodium 4-mercaptobenzene sulfonate (4-MBSNa) is a potent nucleophile and was found to react rapidly in vitro with the direct-acting carcinogen beta-propiolactone (BPL). In further investigations 4-MBSNa was shown to inhibit mutagenesis resulting from exposure of Salmonella typhimurium strain TA-100 to BPL and a second direct-acting carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine. Subsequent experiments were performed to determine if 4-MBSNa would inhibit BPL-induced carcinogenesis in vivo. In the first of these, 4-MBSNa was administered by p.o. intubation to female A/J mice 5 min before p.o. administration of BPL. Under these conditions inhibition of carcinogenesis of the forestomach occurred. In a second experiment, 4-MBSNa was given by rectal intubation 5 min before BPL also administered intrarectally. Administration of BPL intrarectally produced adenomatous polyps of the large intestine. The occurrence of these neoplasms was inhibited by the prior administration of 4-MBSNa. The data presented show that 4-MBSNa has the capacity to trap direct-acting carcinogens and to inhibit the occurrence of BPL-induced neoplasia.

Animals↗

Virus safety of beta-propiolactone treated plasma preparations. Clinical experiences.

Sterilization of human plasma with beta-propiolactone and UV-irradiation (cold sterilization) has been shown to be effective for a number of common pathogenic viruses. We have published data on the hepatitis safety of cold sterilized factor IX concentrates (PPSB) in healthy volunteers. This study has now been extended to include 6 virgin hemophilia B patients, who have been treated with cold sterilized PPSB for a period of up to 5 years. None of these volunteers or patients exhibited clinical symptoms or laboratory data indicating the transmission of either viral hepatitis (B or NANB) or acquired immunodeficiency syndrome (AIDS). Hyperimmunoglobulin (HIg) preparations from cold sterilized plasma, offering protection against hepatitis B and NANB are also safe regarding HTLV-III virus transmission, even though these preparations may contain HTLV-III antibody titers up to 1:1000.

Acquired Immunodeficiency Syndrome↗

Placental transfer of class G immunoglobulins treated with beta-propiolactone (beta-PL) for intravenous application--a case report.

Pregnant patients receive immunoglobulin preparations for the prevention of connatal viral infections of the newborn in the case of impending or manifest maternal infections. In the past, class G immunoglobulin preparations could only be given intramuscularly because of possible antigen independent complement activation by the Fc-part of the IgG molecule when administered intravenously. The I. M. application results in a delay in reaching maximum serum levels, and the levels are much lower compared to I. V. application. Today, I. V. tolerable IgG preparations exist because methods have been developed either to remove or to modify the IgG Fc-part. As the Fc-part is necessary for the placental transport of antibodies, the above mentioned manipulation can drastically impair the transport. In this study, an anti-HBs seronegative pregnant patient received an anti-HBs IgG preparation (greater than or equal to 500 IU anti-HBs) intravenously. These IgG molecules are rendered I. V. tolerable by modification of the Fc-part with beta-propiolactone (beta-PL). At delivery, the newborn's serum anti-HBs concentration was 13 mIU per ml, the mother's 35 mIU per ml. This result clearly demonstrates that IgG molecules which have been modified by treatment with beta-PL are transported from the circulation of the mother across the placenta to the fetus. The role of intravenously tolerable hyperimmunoglobulins with special respect to beta-PL treated preparations for the prevention of connatal infections is discussed.

Adult↗

Concentration-dependent mutation by alkylating agents in human lymphoblasts and Salmonella typhimurium: N-methyl-N-nitrosourethane and beta-propiolactone.

The toxic and mutagenic effects of the alkylating agents N-methyl-N-nitrosourethane (MNUT) and beta-propiolactone (BPL) were quantitatively measured in human lymphoblasts and Salmonella typhimurium. Forward mutation to 6-thioguanine resistance was measured in the human lymphoblasts, and forward mutation to 8-azaguanine resistance was measured in the bacterial cells after equigenerational (1.5 doubling times) exposures. In both systems, the induced mutant fraction rose linearly as a function of concentration for BPL and was biphasic for MNUT. The responses of the two assay systems to eight alkylating agents were compared. The exposure of the cells to each alkylating agent was calculated as exposure concentration multiplied by the time of exposure, and allowance was made for the decomposition of the alkylating agents during exposure (integral exposure). Human cells were 2.5--13 times more sensitive than was S. typhimurium to the alkylating agents methyl methanesulfonate, ethyl methanesulfonate, propyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, methylnitrosourea, and MNUT. S. typhimurium cells were three times more sensitive to butyl methanesulfonate and 25 times more sensitive to BPL than were human cells.

Alkylating Agents↗

[Principles of selective inactivation of the viral genome. III. Kinetics of the inactivation of bacteriophage MS2 infectivity by beta-propiolactone].

It is shown that the action of beta-propiolactone on the bacteriophage MS2 under the wide range of conditions (pH, temperature, initial concentration of the reagent) the survival curves could be accurately described, taking into account the reagent consumption during inactivation. The requirements for accurate description of the infectivity inactivation of viruses by the action of any chemical agent were formulated. That allowed rational development of a principal stage in preparation of the killed antiviral vaccines--inactivation of the virus infectivity to the necessary extent by the action of chemical agents.

Coliphages↗

Inactivation of hepatitis B and non-A, non-B viruses by combined use of Tween 80, beta-propiolactone, and ultraviolet irradiation.

To assess the sterilization efficacy of a combined Tween 80, beta-propiolactone and ultraviolet irradiation procedure applied to a F VIII preparation to which an estimated 10(5.9) chimpanzee infectious doses (CID50) of hepatitis B virus had been added per ml, two chimpanzees were inoculated with 10 ml each of treated and untreated preparations. The untreated preparation, which was obtained from donors with normal alanine aminotransferase (ALT) levels, induced non-A, non-B hepatitis in both recipient animals, and delayed hepatitis B infection in one of these. Neither animal receiving the treated preparation developed either type of hepatitis. When subsequently challenged with the untreated material, both of the latter animals developed non-A, non-B and hepatitis B infection, proving their susceptibility to both types of infection. It was concluded that the combined procedure inactivated an estimated 10(6.9) CID50 of hepatitis B virus and an unknown quantity of a non-A, non-B virus. The finding of non-A, non-B virus infectivity in a pooled F VIII preparation despite careful ALT screening of plasma donors emphasizes the necessity of subjecting such preparations to sterilization procedures.

Animals↗

[Beta-propiolactone as a sterilizing agent in the manufacture of intravenous immunoglobulin preparations].

It was demonstrated with phi X174, phi e and x phages that the beta-propiolactone step, which eliminated the anticomplementary activity of the intravenous immunoglobulin (IgG) preparation. Intraglobin, inactivates more than 10(6) viruses/ml. This means additional safety with respect to those viral diseases for which specific and sensitive diagnostic methods are not available at present.

Bacteriophages↗

Long-term performance of beta-propiolactone-treated nonviable homograft for aortic valve replacement and right ventricular outflow tract reconstruction.

The long-term performance of beta-propiolactone-treated homografts was clearly different between operative survivors of aortic valve replacement (n = 27) and those of right ventricular outflow tract reconstruction (n = 11). The reoperation-free survival rate at 20 years was 14.8 +/- 6.8 (standard error)% in the former and 77.8 +/- 13.9% in the latter. Even a nonviable aortic homograft can be favorably accepted as a material for right ventricular outflow tract reconstruction in contrast to the aortic position.

Adult↗

Enzymic and immunochemical properties of lysozyme. XIII. Accurate delineation of the reactive site around the disulfide 6-127 by immunochemical study of beta-propiolactone lysozyme derivative and of synthetic disulfide peptides.

In previous reports from this laboratory it was shown that an antigenic reactive site resides around the sequences 6-13 and 126-128 linked by the disulfide 6-127. The present work provides a strong support for the location of the reactive site by an independent approach. It also determines accurately the boundaries of the reactive site. 1. The two methionine residues in lysozyme were carboxyethylated by reaction with beta-propiolactone. The electrophoretically homogeneous derivative had no other modified amino acids and showed no conformational changes, relative to native lysozyme, as determined by ORD and CD measurements. However, it exhibited a slight increase in disulfide reducibility relative to native lysozyme and its lytic activity was about half that of native lysozyme, probably as a result of the slight conformational change. On the other hand, the antigenic reactivity of the derivative was equal to that of native lysozyme with several goat and rabbit antisera to lysozyem. It was therefore concluded that methionines 12 and 105 were not parts of antigenic reactive sites in native lysozyme. 2. Eleven peptides, corresponding to various sequences on the two sides of the disulfide 6-127 (i.e. two groups of peptides) were synthesized, purified and characterized. One group (A) of peptides comprised sequences 3-14, 5-14, 6-14, 5-13, 5-12 and an analog of sequence 5-14 in which methionine 12 is replaced by glycine. The second group (B) of peptides comprised sequences 125-129, 125-128, 126-128, 127-128, and 125-127. From groups A and B, nine disulfide-containing peptides (see Fig. 2) were synthesized, purified, characterized and their immunochemical interactions with antisera to native lysozyme studied. Towards each of the antisera studied here, Phe-3, Gly-4, Arg-5, Arg-125 and Leu-129 were not essential parts of the reactive site. On the other hand, Arg-14, Lys-13, Gly-126 and with some antisera Arg-128 were each critical for the reactivity of the site. Peptides from group A alone or group B alone did not inhibit the reaction of lysozyme with its antisera, confirming our previous findings that the integrity of the disulfide bond is essential for bringing the two distant (in sequence) parts of the site together. Finally, replacement of Met-12 by glycine did not influence the immunochemical reactivity of the site, confirming the above conclusion that neither of the two methionine residues takes part in interaction of lysozyme with its antibodies. An accurate delineation of the antigenic reactive site is, therefore derived here and its shape in the three-dimensional structure of native lysozyme is described.

Amino Acid Sequence↗

Intermolecular linking and fragmentation of DNA by beta-propiolactone, a monoalkylating carcinogen.

Brief exposure to beta-propiolactone (BPL) increases the sedimentation rate of purified Escherichia coli DNA in neutral and alkaline sucrose gradients. However, when electrophoresed in polyacrylamide-agarose gels, this BPL-treated DNA moves ahead of the control. Longer incubation with BPL gives rise to two new fractions, the first one sedimenting as a heterogeneous material of 6-8S, and the second one of very high sedimentation velocity. In acrylamide-agarose gels, the first fraction is again recovered in the 6-8S area, while the second fraction does not enter the gel at all. The DNA at this stage is hyperchromic in ultraviolet light suggesting that as much as 20% may be denaturated. Coliphage lambda DNA treated briefly with BPL and spread in a protein monolayer appears under the electron microscope as a rigid, extended molecule, up to 15% longer than the control DNA, and usually in compact, folded configurations suggesting intramolecular linking. After longer exposure, localized denaturation associated with single-strand breaks is observed. The single-stranded "whiskers" then interact with other DNA molecules, creating highly complex branched networks of single- and multi-stranded DNA. The possible relevance of these observations to the mechanisms involved in carcinogenesis and mutagenesis is considered.

Alkylating Agents↗

In vitro reaction of beta-propiolactone and gamma-butyrolactone with glutathione and cysteine.

The in vitro reaction of the carcinogenic beta-propiolactone (BPL) and the non-carcinogenic gamma-butyrolactone (GBL) with glutathione (GSH) and cysteine (CySH) was examined spectrophotometrically. The characteristic absorbance of thioesters in the region of 233 nm appeared when BPL and GSH reacted but not when BPL and CySH reacted or when GBL was mixed with GSH or CySH. The absorbance disappeared when hydroxylamine was added. The results are in striking contrast to the notion that all carcinogenic lactones alkylate nucleophiles and that all inactive lactones acylate nucleophiles. The reaction between BPL and GSH produced about equal quantities of thioester and thioether, which could be separated chromatographically.

Binding Sites↗

Reaction of beta-propiolactone with amino acids and its specificity for methionine.

1. The reactions of beta-propiolactone with amino acids were investigated under various conditions of pH and temperature to find those under which the reagent acted with specificity. 2. At pH9.0 and 22 degrees , after 15min. of reaction, at least 85% of each amino acid had reacted, methionine and cystine being the most reactive. 3. At pH7.0 and 22 degrees most amino acids reacted; methionine, cystine and histidine reacted almost entirely, and proline and lysine to a significantly smaller extent. 4. At pH3.0 and 22 degrees further specificity was obtained; methionine and cystine were the only reactive amino acids. 5. Reaction at pH3.0 and 0 degrees was specific for methionine; it was the only amino acid modified even after 145hr. of reaction.

Amino Acids↗

beta-Propiolactone Vapor Decontamination.

Although beta-propiolactone (BPL) is an effective vapor-phase decontaminant for enclosed areas, some problems have been encountered in its use. Adequate air circulation during BPL dissemination could eliminate most of these problems. It is recommended that, when decontaminating the ordinary building or laboratory, the amount of BPL sprayed be changed from the previously suggested 1 gal/16,000 cubic ft of space to 1 gal/25,000 cubic ft. The use of aqueous BPL solutions and thermal-type generators is not recommended.

Journal Article↗