• Toxicological Research
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• v.14 no.3
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• pp.415-425
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• 1998

A recombinant human erythropoietin (rHuEPO) was administered intravenously at dosage levels of 0, 100, 500, and 2500IU/kg/day for a period of 3 weeks. There were no observed clinical signs and deaths related to treatment in all groups tested. Decreases in body weight gain and food consumption were observed only in males of 2,5000IU/kg group after 2 weeks. In hematological parameters, erythrocyte content, hematocrit values and hemoglobin concentration were dose- dependently increased in rHuEPO treated groups. The ratio between kidney weight and whole body weight was significantly increased in females of 500 and 2,500IU/kg groups. The spleen weight was also increased in both sexes of 500 and 2,500IU/kg groups. However, the absolute weight change of other organs was not observed. In histopathological examinations, the renal tubular basophilia was observed only in males and females of 2,500IU/kg groups. From these results, it is concluded that the no-observed adverse effect level (NOAEL) of rHuEPO is 100 IU/kg in rats in the present study.

• Biomolecules & Therapeutics
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• v.6 no.2
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• pp.171-181
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• 1998

DA-3585, a biosynthetic recombinant human erythropoietin has been developed as a treatment for anemia associated with chronic renal failure in Dong-A pharmaceutical Co. Ltd. This study was carried out to assess its acute and subacute toxicities in rabbits. DA-3585 was intravenously administered to rabbits at dose levels of 6250, 12500 or 25000 lU/kg for single dose toxicity study and at dose levels of 100, 500 or 2500 lU/kg/day for 4-week repeated dose toxicity study. In the acute toxicity study, dose up to 25000 lU/kg had no adverse effect on the behavior or body weight gain. Pathological examinations revealed no abnormal gross lesions related to DA-3585. In the subacute toxicity study, all animals survived until termination of treatment. DA-3585 had no influence on clinical signs, food and water intake or on body weight changes. Hematological examination showed increases in the number of RBC, hemoglobin contents and hematocrit values with a dose dependent manner in the animals treated with DA-3585. Histopathological examination revealed erythroid hyperplasia in the bone marrow and extramedullary hematopoiesis in the liver. The changes detected in the hematological and histopathological examination presumably represent exaggerated pharmacological effects of erythropoietin. The NOAEL (no-observed-adverse-effect-level) of DA-3585 was estimated to be 100 lU/kg/ day under this study condition.

• Journal of The Korean Dental Society of Anesthesiology
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• v.12 no.1
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• pp.25-31
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• 2012

We report a case of 15 year-old Jehovah's Witness patient with mild anemia who underwent a successful orthognathic two-jaw surgery. Jehovah's Witness patients refuse transfusion of blood or blood products even in life threatening situations. The use of recombinant human erythropoietin and iron supplement increased hemoglobin during preoperative period. Intraoperatively, meticulous surgical hemostasis, acute normovolemic hemodilution and induced hypotension enabled the completion of the operation without the use of blood products.

• Journal of Life Science
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• v.27 no.6
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• pp.632-639
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• 2017

Mature haptoglobin (Hp) is a plasma glycoprotein and acts as an antioxidant by scavenging cell-free hemoglobin (Hb). Prohaptoglobin (proHp) is an unprocessed Hp precursor which is present a little in circulation. However, the biological function of proHp remains unknown. To investigate the structural and functional differences between proHp and Hp, we prepared recombinant proHp isoforms and compared their sialic acid content and Hb-binding capacity with those of mature isoforms. When proHp samples were analyzed by Western blot under non-reducing conditions, proHp1 was detected as one band of approximately 130 kDa and proHp2 as multiple bands >200 kDa, in the manner of mature Hp1-1 and Hp2-2, respectively. On the native polyacrylamide gel under non-reducing and non-denaturing conditions, both proHp isoforms migrated more slowly than their mature Hp counterparts. In addition, the lectin-based ELISA assay demonstrated that the content of sialic acid in proHp1 and proHp2 was much less than in Hp1-1 and Hp2-2. The Hb-binding capacity of proHp was also lower than those of mature Hp. These findings indicate that proHp and Hp are similar in the size and polymerization pattern, but different in sialic acid content and Hb-binding activity. It suggests precursor proHp may exert different functions in circulation than does mature Hp.

• Biomolecules & Therapeutics
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• v.10 no.1
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• pp.59-69
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• 2002

Recently, recombinant human erythropoietin (rHu-EPO) has been used to treat various types of anemia. YHB216 is a new rHu-EPO developed by Yuhan Research Institute. In this study, we investigated the single dose and 4-week repeated dose toxicity of YHB216 in Beagle dogs. In the single dose toxicity study, YHB216 was administered intravenously at single dose levels of 0 and 25,000 IU/kg to dogs (2 dogs/sex/group). There were no treament-related changes in survivals, clinical signs, body weight gain, hematological values, blood chemical values, and necropsy finding during experimental period. In the repeated dose toxicity study, YHB216 was administered intravenously to dogs for 4 weeks at the dose levels of 0, 100, 500, and 2,500IU／kg (3 dogs/sex／group). There were no toxicologically significant changes in clinical signs, body weights, food and water consumptions, ophthalmoscopy, urinalysis and blood chemistry. There were increased values of red blood cell, hemoglobin, and hematocrit at all treated groups. Spleen revealed increased weight and extramedullary hematopoiesis at 500 IU/kg or more. These changes are all considered to be Pharmacology-related effects and were recovered after 4-week recovery period. From these results, it is concluded that LD50 value was above 25,000 IU/kg in the single dose toxicity study of YHB216 in dogs and the no observed adverse effect level (NOAEL) was 100 IU/kg day in the repeated dose toxicity study of YHB216 in dogs.

• Journal of Microbiology and Biotechnology
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• v.27 no.1
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• pp.92-100
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• 2017

Acetoin (AC) is a volatile platform compound with various potential industrial applications. AC contains two stereoisomeric forms: (3S)-AC and (3R)-AC. Optically pure AC is an important potential intermediate and widely used as a precursor to synthesize novel optically active materials. In this study, chiral (3R)-AC production from meso-2,3-butanediol (meso-2,3-BD) was obtained using recombinant Escherichia coli cells co-expressing meso-2,3-butanediol dehydrogenase (meso-2,3-BDH), NADH oxidase (NOX), and hemoglobin protein (VHB) from Serratia sp. T241, Lactobacillus brevis, and Vitreoscilla, respectively. The new biocatalyst of E. coli/pET-mbdh-nox-vgb was developed and the bioconversion conditions were optimized. Under the optimal conditions, 86.74 g/l of (3R)-AC with the productivity of 3.61 g/l/h and the stereoisomeric purity of 97.89% was achieved from 93.73 g/l meso-2,3-BD using the whole-cell biocatalyst. The yield and productivity were new records for (3R)-AC production. The results exhibit the industrial potential for (3R)-AC production via whole-cell biocatalysis.

• Toxicological Research
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• v.12 no.2
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• pp.309-318
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• 1996

This study was conducted to investigate the subacute intravenous toxicity of rHu-EPO in Sprague-Dawley rats. rHu-EPO was daily administered to male and female rats for 30 days with different dose levels(100, 500, 2,500 unit/ kg). After the administration period of 30 days, 5 males and 5females rats per each dose group were assigned for recovery period of additional 30 days. There were no clinical signs compared with control group but slight decrease in spontaneous motor activities and locomotions were observed at high dose groups of males and females. In males, feed consumption was reduced at 500 unit/kg and body weight gain was retarded. In the administration sites of tail, focal congestion was observed in a few animals treated with rHu-EPO. No deaths were occurred during the course of study. In hematological examination. a significant increase of hemoglobin and hematocrit was observed in the males and females rats receiving high dose of rHu-EPO. rHu-EPO administration for 30 days showed a marked decrease in glucose concentration. At the highest dose groups, there was a significant incerase in the weights of spleens in both sexes. but this was considered to be related to its pharmacological activity. These results indicate that rHu-EPO does not induce any significant toxic effect on Sprague-Dawley rats for 30 days.

• BMB Reports
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• v.38 no.1
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• pp.115-119
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• 2005

Replacement of valine by tryptophan or tyrosine at position $\alpha$96 of the $\alpha$ chain ($\alpha$96Val), located in the ${\alpha}_1{\beta}_2$ subunit interface of hemoglobin leads to low oxygen affinity hemoglobin, and has been suggested to be due to the extra stability introduced by an aromatic amino acid at the $\alpha$96 position. The characteristic of aromatic amino acid substitution at the $\alpha$96 of hemoglobin has been further investigated by producing double mutant r Hb ($\alpha$42Tyr$\rightarrow$ Phe, $\alpha$96Val$\rightarrow$Trp). r Hb ($\alpha$42Tyr$\rightarrow$Phe) is known to exhibit almost no cooperativity in binding oxygen, and possesses high oxygen affinity due to the disruption of the hydrogen bond between $\alpha$42Tyr and $\beta$99Asp in the ${\alpha}_1{\beta}_2$ subunit interface of deoxy Hb A. The second mutation, $\alpha$96Val$\rightarrow$Trp, may compensate the functional defects of r Hb ($\alpha$42Tyr$\rightarrow$Phe), if the stability due to the introduction of trypophan at the $\alpha$96 position is strong enough to overcome the defect of r Hb ($\alpha$42Tyr$\rightarrow$Phe). Double mutant r Hb ($\alpha$42Tyr$\rightarrow$Phe, $\alpha$96Val$\rightarrow$Trp) exhibited almost no cooperativity in binding oxygen and possessed high oxygen affinity, similarly to that of r Hb ($\alpha$42Tyr$\rightarrow$Phe). $^1$H NMR spectroscopic data of r Hb ($\alpha$42Tyr$\rightarrow$Phe, $\alpha$96Val$\rightarrow$Trp) also showed a very unstable deoxy-quaternary structure. The present investigation has demonstrated that the presence of the crucible hydrogen bond between $\alpha$42Tyr and $\beta$99Asp is essential for the novel oxygen binding properties of deoxy Hb ($\alpha$96Val$\rightarrow$Trp).

• Parasites, Hosts and Diseases
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• v.56 no.5
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• pp.409-418
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• 2018

Acanthamoeba spp. are free-living protozoa that are opportunistic pathogens for humans. Cysteine proteases of Acanthamoeba have been partially characterized, but their biochemical and functional properties are not clearly understood yet. In this study, we isolated a gene encoding cysteine protease of A. castellanii (AcCP) and its biochemical and functional properties were analyzed. Sequence analysis of AcCP suggests that this enzyme is a typical cathepsin L family cysteine protease, which shares similar structural characteristics with other cathepsin L-like enzymes. The recombinant AcCP showed enzymatic activity in acidic conditions with an optimum at pH 4.0. The recombinant enzyme effectively hydrolyzed human proteins including hemoglobin, albumin, immunoglobuins A and G, and fibronectin at acidic pH. AcCP mainly localized in lysosomal compartment and its expression was observed in both trophozoites and cysts. AcCP was also identified in cultured medium of A. castellanii. Considering to lysosomal localization, secretion or release by trophozoites and continuous expression in trophozoites and cysts, the enzyme could be a multifunctional enzyme that plays important biological functions for nutrition, development and pathogenicity of A. castellanii. These results also imply that AcCP can be a promising target for development of chemotherapeutic drug for Acanthamoeba infections.

• Toxicological Research
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• v.22 no.1
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• pp.61-67
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• 2006

Changes in hematology and serum biochemistry after treatment of recombinant human erythropoietin (rHuEPO, HM10760) were screened in 4 male cynomolgus monkeys (Macaca fascicularis). Four monkeys, composed of a treatment group of HM10760 and a positive control group of $Aranesp^(R}$, were subcutaneously administered at same dose of $100{\mu}g/kg$. Both groups did not show any change in body weights and food consumption for 4 weeks compared with those of pretreatment. Both groups did not show any change in total leukocyte count (WBC) and platelet count, while both groups showed increased platelet distribution width (PDW) percentage in HM10760 group during a period from day 5 to day 59 and in $Aranesp^(R}$ group during a period from day 9 to day 26. Both groups showed increases in red blood cells (RBC), hemoglobin (HGB), and hematocrit (HCT) approximately 10 days after treatment compared with those of pretreatment (day 0). The increased levels of RBC, HGB, and HCT were much higher in HM10760 than in $Aranesp^(R}$ by the increases of $3.2%{\sim}12.5%$ for RBC, $3.8%{\sim}17.1%$ for HCT, and $1.85%{\sim}11%$ for HGB. Both groups showed increases in red cells distribution width (RDW) and reticulocyte (RET) compared with those of pretreatment, showing the highest peak from day 9. The increased level of RET lasted up to day 14 in $Aranesp^(R}$ group, while it lasted up to day 23 in HM10760 group. The increased level of RDW lasted up to day 59, it was much higher in HM10760 by the increase of $10.1%{\sim}17.6%$ than in $Aranesp^(R}$ group. In serum biochemistry, both groups showed a decrease in chloride level compared with those of pretreatment. These findings indicated that HM10760 increased RBC, HGB, HCT, RDW, and RET compared with those of pretreatment, and the increased levels were much higher in HM10760 than in $Aranesp^(R}$.