Characterization of Missense Mutations in Factor VIII That Lead to Abnormal N-Linked Glycosylation

Abstract Most secreted proteins are glycosylated on the asparagine (N) residue with the consensus sequence N-X-S/T(X≠Proline).Coagulation factor VIII (FVIII) is heavily N-linked glycosylated with 5 consensus sites outside the B domain. However, the roles of these glycans are not well understood. Meanwhile, missense mutations which could create additional N-linked glycosylation sites have largely not been characterized in hemophilia A patients. In this study we first expressed individual domains of FVIII and determined that the A2, Cand C2 domains are efficiently secreted. The A1(N42,N239), A3 (N1810)and C1 (N2118)domains are glycosylated, whereas N582 in the A2 domain is not glycosylated. Only one hemophilia A missense mutation, S241C in the A1 domain, was found to abolish the consensus sequence for N-linked glycosylation at N239. We confirmed that the S241C mutant lost one glycan and became unstable inside cells. We also tested the other three glycosylation sites and found that elimination of the N-linked glycan at N2118 (N2118Q mutation) impaired the secretion of the C domain. This defect could not be rescued by adding another N-linked glycan (at N2062) in the C1 domain, indicating that the N2118 glycan is specifically required for the secretion of the C domain. We next searched the CHAMP F8 Mutation Database and the FVIII Variant Database and identified 19 missense mutations that potentially create an ectopic glycosylation site.These mutations are located in A1, A2, A3 and C1 domains, but none in the C2 domain. Only two mutations (I566T and M1772T) have previously been characterized.We found that all but one (I2071T) of these mutations gained an additional N-linked glycan. We further studied missense mutations in the A2 (A469T, A469S, I566T, M614T and G701S) and the C domain (W2062S, I2071T and D2131N) because these domains are secreted in cell culture. Whereas secretion of I566T, W2062S and D2131N mutants was comparable to their wild-type counterparts, secretion of other mutants decreased to 5%-30% of WT (P<0.05). Mutants that secreted into culture media nevertheless have low FVIII activity (<2%), indicating that these mutations cause cross reactive material positive hemophilia A. The consequences of additional N-linked glycan were further investigated using the A2 domain mutants, since this domain is normally unglycosylated. After treating with tunicamycin to block the N-linked glycosylation process in the endoplasmic reticulum (ER),the secretion of A2 domain with I566T andG701Smutants, which had relatively high secretion levels, decreased significantly. On the other hand, removing the additional glycan boosted the secretion of A469S and A469T, two low-secretion mutants.Tunicamycin treatment had no effect on another low secretion mutant,M614T.These results suggest that amino acid substitution in I566T andG701Smutationsis detrimental to the proper folding of the protein and the additional N-glycan plays a stabilization role. On the other hand, additional N-glycan plays a destabilization role in A469S and A469T mutations, contributing to disruption of folding in these mutants. For theM614Tmutation,the amino acid substitution alone is likely sufficient todestroy the protein folding. We also studied interactions of abnormally glycosylated mutants with ER chaperones.All the mutants with low secretion levels significantly induced expression of GRP78 to 1.5-2.0 folds(P<0.05), while mutants that maintain higher secretion levels did not affect GRP78 expression. The low secretion mutants also had increased binding to GRP78 and calreticulin, but not to calnexin.Therefore ER chaperones play a key role in the ER quality control of FVIII mutants. In conclusion, our results indicate that the effects of abnormal N-linked glycosylation on FVIII folding and secretionvary widely, from detrimental to beneficial. The impact of a particular glycan is likely determined by the location and the underlying amino acid change caused by the mutation. Disclosures No relevant conflicts of interest to declare.

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Direct Comparison of Bioengineered Factor VIII Expression Constructs: Towards an Optimal Transgene for Gene Therapy of Hemophilia A.

Blood ◽

10.1182/blood.v108.11.5477.5477 ◽

2006 ◽

Vol 108 (11) ◽

pp. 5477-5477

Author(s):

Kerry L. Titus ◽

Paul Lee ◽

H. Trent Spencer ◽

Christopher Doering

Keyword(s):

Gene Therapy ◽

Amino Acid ◽

Factor Viii ◽

Hemophilia A ◽

Double Mutation ◽

Specific Amino Acid ◽

Stable Transfectants ◽

Glycosylation Sites ◽

Fviii Activity ◽

A2 Domain

Abstract A major obstacle for gene therapy of hemophilia A is the achievement of adequate factor VIII (fVIII) expression. Bioengineering strategies have targeted specific sequences within human fVIII that are thought to be responsible for its generally poor expression. Specific amino acid substitutions, L303E/F309S herein referred to as double mutation (DM), function to decrease fVIII binding to BiP, a resident ER chaperone, which results in increased fVIII secretion (Swaroop, Moussalli et al. 1997). Furthermore, addition of 6 N-linked glycosylation sites, designated 226/N6, located within the human B domain also increases human fVIII expression (Miao, Sirachainan et al. 2004). We previously demonstrated that porcine and certain hybrid human/porcine fVIII constructs are expressed at 10 – 14-fold greater levels than human fVIII (Doering, Healey et al. 2002; Doering, Healey et al. 2004). The aim of the current study was to directly compare various fVIII expression constructs in order to determine an optimal transgene for gene therapy applications. The following fVIII constructs were generated: human B-domain-deleted fVIII (HBDD-fVIII), HBDD-fVIII with a 14 amino acid linker between the A2 domain and the activation peptide (HSQ-fVIII), porcine fVIII containing a 24 amino acid linker (HEP-fVIII), hybrid human/porcine-fVIII which has porcine A1 and A3 domains (HP47), and modified HBDD, HSQ and HEP-fVIII constructs containing DM and/or 226/N6. Each construct was transiently transfected into BHK-M cells, and fVIII production between 48 – 72 hrs post-transfection was measured using a one-stage clotting assay. Under these conditions, the addition of the DM and 226/N6 significantly increased fVIII expression for HBDD (P = 0.003), though not for HSQ. Addition of DM or 226/N6 alone did not significantly increase the expression of either human fVIII construct, and furthermore, the addition of DM to HEP-fVIII decreased its expression 98%. HEP-fVIII was expressed at 8-fold or greater levels than any of the other human constructs. Next, ~25 stably transfected BHK-M clones were isolated following transfection with each of the fVIII expression constructs and the rate of fVIII production for each clone was determined. Several clones did not express detectable fVIII activity (<0.01 units/mL) and were excluded from the analysis. Approximately 14% of the total number of clones were excluded, ranging from 0 – 42% for the different constructs. HEP-DM-fVIII was the exception, where 82% of the clones had activity <0.01 units/mL. Mean HEP-fVIII expression was 3.93 ± 3.22 units/mL/24 hr (n = 19) (Figure 1), and HP47 was similarly expressed at 3.21 ± 2.31 units/mL/24 hr (n = 19). All of the HSQ-based constructs and HBDD-DM/226/N6 showed similar mean expression levels (0.28 ± 0.03 units/mL/24 hr) and were significantly higher than HBDD and HBDD-DM, which had a mean of 0.12 ± 0.01 units/mL. In the current study, we provide experimental evidence that the expression of HEP-fVIII and HP47 is superior to other bioengineered fVIII expression constructs, which should eliminate the expression barrier that has hampered the clinical translation of gene therapy for hemophilia A. Figure 1: Stable Transfectants Figure 1:. Stable Transfectants

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Most Factor VIII B Domain Missense Mutations Are Unlikely to Be Causative Mutations for Hemophilia A: Implications for Factor VIII Genetic Analysis

Blood ◽

10.1182/blood.v112.11.513.513 ◽

2008 ◽

Vol 112 (11) ◽

pp. 513-513

Author(s):

Kyoichi Ogata ◽

Steven W. Pipe

Keyword(s):

Amino Acid ◽

Factor Viii ◽

Hemophilia A ◽

Coagulation Factor ◽

Causative Mutation ◽

Factor V ◽

Missense Mutations ◽

Severe Hemophilia ◽

Single Chain

Abstract Hemophilia A results from the quantitative or qualitative deficiency of coagulation factor VIII (FVIII). FVIII is synthesized as a single-chain polypeptide of approximately 280 kDa with the domain structure A1-A2-B-A3-C1-C2. Whereas the A and C domains exhibit ~40% amino acid identity to each other and to the A and C domains of coagulation factor V, the B domain is not homologous to any known protein and is dispensable for FVIII cofactor activity. Missense mutations in the FVIII B domain have been described in patients with variable phenotypes of hemophilia A. According to the NCBI SNPs (single nucleotide polymorphism) database, 22 SNPs are reported within FVIII, 11 of which occur within the B domain. FVIII B domain variant D1241E has been reported as a missense mutation associated with mild or severe hemophilia A, yet this mutation is also present in the NCBI SNPs database. We hypothesize that D1241E and most other reported B domain missense mutations are not the causative mutation for hemophilia A in these patients but represent SNPs or otherwise non-pathologic mutations. To investigate this, we analyzed 7 B domain missense mutations that were previously found in hemophilia A patients (T751S, V993L, H1047Y, D1241E, T1353A, P1641L and S1669L). Comparative analysis showed that the amino acids at these positions are not conserved in all species and in some cases, the amino acid substitution reported in hemophilia patients is represented in the native sequence in other species. Analysis with PolyPhen Software showed that only H1047Y mutation was considered as “possibly damaging”, while the others were considered as “benign”. To investigate this further, we constructed seven plasmid vectors containing these B domain missense mutations. The synthesis and secretion of FVIII wild-type (WT) and these seven mutants were compared after transient DNA transfection into COS-1 monkey cells in vitro. Analysis of the FVIII clotting activity and antigen levels in the conditioned medium demonstrated that all mutants had FVIII activity and antigen levels similar to FVIII WT. Further, FVIII WT, H1047Y and D1241E mutants were introduced into a FVIII exon 16 knock-out mouse model of hemophilia A by hydrodynamic tailvein injection in vivo. The mouse plasma was analyzed at 24 hrs for activity and antigen expression. Mutants H1047Y and D1241E expressed at 211 mU/mL and 224 mU/mL activity with FVIII antigen levels of 97 ng/mL and 118 ng/mL, respectively, similar to FVIII WT. These results suggested that H1047Y and D1241E mutants did not lead to impairments in secretion or functional activity. We conclude that most missense mutations within the FVIII B domain would be unlikely to lead to severe hemophilia A and that the majority of such missense mutations represent polymorphisms or non-pathologic mutations. Investigators should search for additional potentially causative mutations elsewhere within the FVIII gene when B domain missense mutations are identified.

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The cDNA and derived amino acid sequence of porcine factor VIII

Blood ◽

10.1182/blood.v88.11.4209.bloodjournal88114209 ◽

1996 ◽

Vol 88 (11) ◽

pp. 4209-4214 ◽

Cited By ~ 26

Author(s):

JF Healey ◽

IM Lubin ◽

P Lollar

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Factor Viii ◽

Untranslated Region ◽

Hemophilia A ◽

Sequence Determination ◽

C2 Domains ◽

Protein Alignments ◽

A2 Domain

The cDNA corresponding to 137 bp of the 5′ untranslated region, the signal peptide, and the A1, A3, C1, and C2 domains of porcine factor VIII (fVIII) have been cloned and sequenced. Along with previously determined sequences of the porcine fVIII B domain and the A2 domain, this completes the sequence determination of the cDNA corresponding to the translated protein. Alignments of the derived amino acid sequence of porcine fVIII with human and murine fVIII indicate that the A1, A2, A3, C1, and C2 domains are more conserved than the B domains or the proteolytic cleavage peptides corresponding to residues 337–372 and 1649–1689. The knowledge of the porcine fVIII cDNA may be useful to understand functional and immunological differences between human and porcine fVIII and may lead to improved fVIII replacement products for hemophilia. A patients through the development of recombinant porcine fVIII or hybrid human/porcine fVIII derivatives.

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The Molecular Basis for Cross-Reacting Material–Positive Hemophilia A Due to Missense Mutations Within the A2-Domain of Factor VIII

Blood ◽

10.1182/blood.v91.2.538 ◽

1998 ◽

Vol 91 (2) ◽

pp. 538-548 ◽

Cited By ~ 31

Author(s):

Kagehiro Amano ◽

Rita Sarkar ◽

Susan Pemberton ◽

Geoffrey Kemball-Cook ◽

Haig H. Kazazian ◽

...

Keyword(s):

Factor Viii ◽

Hemophilia A ◽

Specific Activity ◽

Genetic Alterations ◽

Published Data ◽

Missense Mutations ◽

Wild Type ◽

Factor Ixa ◽

Fviii Activity ◽

A2 Domain

Abstract Factor VIII (FVIII) is the protein defective in the bleeding disorder hemophilia A. Approximately 5% of hemophilia A patients have normal amounts of a dysfunctional FVIII protein and are termed cross-reacting material (CRM)-positive. The majority of genetic alterations that result in CRM-positive hemophilia A are missense mutations within the A2-domain. To determine the mechanistic basis of the genetic defects within the A2-domain for FVIII function we constructed six mutations within the FVIII cDNA that were previously found in five CRM-positive hemophilia A patients (R527W, S558F, I566T, V634A, and V634M) and one CRM-reduced hemophilia A patient (DeltaF652/3). The specific activity for each mutant secreted into the conditioned medium from transiently transfected COS-1 cells correlated with published data for the patients plasma-derived FVIII, confirming the basis of the genetic defect. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of immunoprecipitated FVIII protein radiolabeled in COS-1 cells showed that all CRM-positive mutant proteins were synthesized and secreted into the medium at rates similar to wild-type FVIII. The majority of the DeltaF652/3 mutant was defective in secretion and was degraded within the cell. All mutant FVIII proteins were susceptible to thrombin cleavage, and the A2-domain fragment from the I566T mutant had a reduced mobility because of use of an introduced potential N-linked glycosylation site that was confirmed by N-glycanase digestion. To evaluate interaction of FVIII with factor IXa, we performed an inhibition assay using a synthetic peptide corresponding to FVIII residues 558 to 565, previously shown to be a factor IXa interaction site. The concentration of peptide required for 50% inhibition of FVIII activity (IC50) was reduced for the I566T (800 μmol/L) and the S558F (960 μmol/L) mutants compared with wild-type FVIII (>2,000 μmol/L). N-glycanase digestion increased I566T mutant FVIII activity and increased its IC50 for the peptide (1,400 μmol/L). In comparison to S558F, a more conservative mutant (S558A) had a sixfold increased specific activity that also correlated with an increased IC50 for the peptide. These results provided support that the defects in the I566T and S558F FVIII molecules are caused by steric hindrance for interaction with factor IXa.

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The cDNA and derived amino acid sequence of porcine factor VIII

Blood ◽

10.1182/blood.v88.11.4209.4209 ◽

1996 ◽

Vol 88 (11) ◽

pp. 4209-4214 ◽

Cited By ~ 4

Author(s):

JF Healey ◽

IM Lubin ◽

P Lollar

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Factor Viii ◽

Untranslated Region ◽

Hemophilia A ◽

Sequence Determination ◽

C2 Domains ◽

Protein Alignments ◽

A2 Domain

Abstract The cDNA corresponding to 137 bp of the 5′ untranslated region, the signal peptide, and the A1, A3, C1, and C2 domains of porcine factor VIII (fVIII) have been cloned and sequenced. Along with previously determined sequences of the porcine fVIII B domain and the A2 domain, this completes the sequence determination of the cDNA corresponding to the translated protein. Alignments of the derived amino acid sequence of porcine fVIII with human and murine fVIII indicate that the A1, A2, A3, C1, and C2 domains are more conserved than the B domains or the proteolytic cleavage peptides corresponding to residues 337–372 and 1649–1689. The knowledge of the porcine fVIII cDNA may be useful to understand functional and immunological differences between human and porcine fVIII and may lead to improved fVIII replacement products for hemophilia. A patients through the development of recombinant porcine fVIII or hybrid human/porcine fVIII derivatives.

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The Molecular Basis for Cross-Reacting Material–Positive Hemophilia A Due to Missense Mutations Within the A2-Domain of Factor VIII

Blood ◽

10.1182/blood.v91.2.538.538_538_548 ◽

1998 ◽

Vol 91 (2) ◽

pp. 538-548 ◽

Cited By ~ 2

Author(s):

Kagehiro Amano ◽

Rita Sarkar ◽

Susan Pemberton ◽

Geoffrey Kemball-Cook ◽

Haig H. Kazazian ◽

...

Keyword(s):

Factor Viii ◽

Hemophilia A ◽

Specific Activity ◽

Genetic Alterations ◽

Published Data ◽

Missense Mutations ◽

Wild Type ◽

Factor Ixa ◽

Fviii Activity ◽

A2 Domain

Factor VIII (FVIII) is the protein defective in the bleeding disorder hemophilia A. Approximately 5% of hemophilia A patients have normal amounts of a dysfunctional FVIII protein and are termed cross-reacting material (CRM)-positive. The majority of genetic alterations that result in CRM-positive hemophilia A are missense mutations within the A2-domain. To determine the mechanistic basis of the genetic defects within the A2-domain for FVIII function we constructed six mutations within the FVIII cDNA that were previously found in five CRM-positive hemophilia A patients (R527W, S558F, I566T, V634A, and V634M) and one CRM-reduced hemophilia A patient (DeltaF652/3). The specific activity for each mutant secreted into the conditioned medium from transiently transfected COS-1 cells correlated with published data for the patients plasma-derived FVIII, confirming the basis of the genetic defect. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of immunoprecipitated FVIII protein radiolabeled in COS-1 cells showed that all CRM-positive mutant proteins were synthesized and secreted into the medium at rates similar to wild-type FVIII. The majority of the DeltaF652/3 mutant was defective in secretion and was degraded within the cell. All mutant FVIII proteins were susceptible to thrombin cleavage, and the A2-domain fragment from the I566T mutant had a reduced mobility because of use of an introduced potential N-linked glycosylation site that was confirmed by N-glycanase digestion. To evaluate interaction of FVIII with factor IXa, we performed an inhibition assay using a synthetic peptide corresponding to FVIII residues 558 to 565, previously shown to be a factor IXa interaction site. The concentration of peptide required for 50% inhibition of FVIII activity (IC50) was reduced for the I566T (800 μmol/L) and the S558F (960 μmol/L) mutants compared with wild-type FVIII (>2,000 μmol/L). N-glycanase digestion increased I566T mutant FVIII activity and increased its IC50 for the peptide (1,400 μmol/L). In comparison to S558F, a more conservative mutant (S558A) had a sixfold increased specific activity that also correlated with an increased IC50 for the peptide. These results provided support that the defects in the I566T and S558F FVIII molecules are caused by steric hindrance for interaction with factor IXa.

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Oxytocin analogues with non-coded amino acid residues in position 8: [8-Neopentylglycine]oxytocin and [8-cycloleucine]oxytocin

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19872317 ◽

1987 ◽

Vol 52 (9) ◽

pp. 2317-2325 ◽

Cited By ~ 5

Author(s):

Jan Hlaváček ◽

Jan Pospíšek ◽

Jiřina Slaninová ◽

Walter Y. Chan ◽

Victor J. Hruby

Keyword(s):

Amino Acid ◽

Solid Phase Synthesis ◽

Solid Phase ◽

The Other ◽

Amino Acid Residues ◽

Phase Synthesis ◽

Other Hand ◽

Fragment Condensation

[8-Neopentylglycine]oxytocin (II) and [8-cycloleucine]oxytocin (III) were prepared by a combination of solid-phase synthesis and fragment condensation. Both analogues exhibited decreased uterotonic potency in vitro, each being about 15-30% that of oxytocin. Analogue II also displayed similarly decreased uterotonic potency in vivo and galactogogic potency. On the other hand, analogue III exhibited almost the same potency as oxytocin in the uterotonic assay in vivo and in the galactogogic assay.

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Comparison of the Potency of 8-L-Arginine, 8-D-Arginine and 8-D-Homoarginine Vasopressin Analogs with Substituted Phenylalanine in Position 2

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19941439 ◽

1994 ◽

Vol 59 (6) ◽

pp. 1439-1450 ◽

Cited By ~ 2

Author(s):

Miroslava Žertová ◽

Jiřina Slaninová ◽

Zdenko Procházka

Keyword(s):

Amino Acid ◽

Solid Phase Synthesis ◽

Solid Phase ◽

Basic Amino Acid ◽

The Other ◽

Side Chain ◽

Inhibitory Potency ◽

Phase Synthesis ◽

Other Hand ◽

Order Of Magnitude

An analysis of the uterotonic potencies of all analogs having substituted L- or D-tyrosine or -phenylalanine in position 2 and L-arginine, D-arginine or D-homoarginine in position 8 was made. The series of analogs already published was completed by the solid phase synthesis of ten new analogs having L- or D-Phe, L- or D-Phe(2-Et), L- or D-Phe(2,4,6-triMe) or D-Tyr(Me) in position 2 and either L- or D-arginine in position 8. All newly synthesized analogs were found to be uterotonic inhibitors. Deamination increases both the agonistic and antagonistic potency. In the case of phenylalanine analogs the change of configuration from L to D in position 2 enhances the uterotonic inhibition for more than 1 order of magnitude. The L to D change in position 8 enhances the inhibitory potency negligibly. Prolongation of the side chain of the D-basic amino acid in position 8 seems to decrease slightly the inhibitory potency if there is L-substituted amino acid in position 2. On the other hand there is a tendency to the increase of the inhibitory potency if there is D-substituted amino acid in position 2.

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Polysomal organization in growing and resting cultures and its relation to protein synthesis in Tetrahymena

Journal of Cell Science ◽

10.1242/jcs.59.1.121 ◽

1983 ◽

Vol 59 (1) ◽

pp. 121-131

Author(s):

P. Isberner ◽

G. Cleffmann

Keyword(s):

Protein Synthesis ◽

Amino Acid ◽

Growth Conditions ◽

The Other ◽

Protein Accumulation ◽

Resting Cells ◽

Amino Acid Incorporation ◽

Total Rna ◽

Acid Incorporation ◽

Other Hand

Cytosol from Tetrahymena cells growing at different rates was isolated and separated by centrifugation into polysomal and non-polysomal fractions. The RNAs of either fraction were separated chromatographically into poly(A)+ RNA and poly(A)-RNA. It was found that in resting cultures the total RNA per cell is only about half of that of rapidly growing cultures. All fractions of RNA were reduced proportionally. Thus, the percentage of polysomally bound total RNA (70% of cytosol RNA) and polysomally bound poly(A)+ RNA (72% of cytosol poly(A)+ RNA) is the same in growing and resting cultures. Differences, however, were found in the polysomal structure. Polysomes from resting cultures contained significantly fewer ribosomes. The amounts of RNA bound to polysomes were related to the rate of protein synthesis under different growth conditions. The decrease in cellular RNA corresponded well with the reduction in amino acid incorporation in resting cells. The rate of protein accumulation in resting cells, on the other hand, was considerably less, suggesting that polypeptides in resting cultures are less stable.

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Radioprotective and Radiosensitizing Effects of Sulfur-Containing Amino Acid Derivatives on Mice

Zeitschrift für Naturforschung C ◽

10.1515/znc-1980-9-1012 ◽

1980 ◽

Vol 35 (9-10) ◽

pp. 726-728 ◽

Cited By ~ 1

Author(s):

Akihisa Nishimura ◽

Michinobu Hashimoto ◽

Katsunobu Konno ◽

Yasuhiko Ohta ◽

Satoshi Tahara ◽

...

Keyword(s):

Body Weight ◽

Amino Acid ◽

Radioprotective Effect ◽

The Other ◽

Amino Acid Derivatives ◽

Survival Ratio ◽

Other Hand ◽

Hydantoin Derivatives ◽

Γ Rays ◽

Sulfur Containing

Both protection and sensitization of Mice C57BL against 60Co γ-rays by sulfur-containing amino acid derivatives - S-alkyl-L-cysteines, S-alkyl-2-methyl-DL-cysteines and their hydantoin derivatives, and sulfoxides of these compounds - were examined. DL-5-Allylthiomethyl-5-methylhydantoin (150 mg/kg body weight) had a remarkable radioprotective effect. The survival ratio was 4.33 or above two times as much as that of L-cysteine. On the other hand, its sulfoxide had a radiosensitizing effects; survival ratio, 0.333.

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