Antimicrobial peptide AR-23 derivatives with high endosomal disrupting ability enhance poly(l-lysine)-mediated gene transfer.

BACKGROUND: pH-sensitive peptides are a relatively new strategy for conquering the poor endosomal release of cationic polymer-mediated transfection. Modification of antimicrobial peptides by exchanging positively-charged residues with negatively-charged glutamic acid residues (Glu) greatly improves its lytic activity at the endosomal pH, which could improve cationic polymer-mediated transfection. METHODS: In the present study, we investigated the effect of the number of Glu substituted for positively-charged residues on the endosomal escape activity of AR-23 and the ability of mutated AR-23 with respect to enhancing cationic polymer-mediated transfection. Three analogs were synthesized by replacing the positively-charged residues in the AR-23 sequence with Glu one-by-one. RESULTS: The pH-sensitive lysis ability of the peptides, the effect of peptides on the physicochemical characteristics, the intracellular trafficking, the transfection efficiency and the cytotoxicity of the polyplexes were determined. Increased lytic activity of peptides was observed with the increased number of Glu replacement in the AR-23 sequence at acidic pH. The number of Glu substituted for positively-charged residues of AR-23 dramatically affects its lysis ability at neutral pH. Triple-Glu substitution in the AR-23 sequence greatly improved poly(l-lysine)-mediated gene transfection efficiency at the same time as maintaining low cytotoxicity. CONCLUSIONS: The results indicate that replacement of positively-charged residues with sufficient Glu residues may be considered as a method for designing pH-sensitive peptides, which could be applied as potential enhancers for improving cationic polymer-mediated transfection.

Overexpression of heparanase in mice promoted megakaryopoiesis.

Heparanase, an endo-glucuronidase that specifically cleaves heparan sulfate (HS), is upregulated in several pathological conditions. In this study, we aimed to find a correlation of heparanase expression and platelets production. In the transgenic mice overexpressing human heparanase (Hpa-tg), hematological analysis of blood samples revealed a significantly higher number of platelets in comparison with wild-type (Ctr) mice, while no significant difference was found in leukocytes and red blood cell number between the two groups. Total number of thiazole orange positive platelets was increased in Hpa-tg vs. Ctr blood, reflecting a higher rate of platelets production. Concomitantly, megakaryocytes from Hpa-tg mice produced more and shorter HS fragments that were shed into the medium. Further, thrombopoietin (TPO) level was elevated in the liver and plasma of Hpa-tg mice. Together, the data indicate that heparanase expression promoted megakaryopoiesis, which may be through upregulated expression of TPO and direct effect of released HS fragments expressed in the megakaryocytes.

Design of pH-sensitive peptides from natural antimicrobial peptides for enhancing polyethylenimine-mediated gene transfection.

BACKGROUND: Poor endosomal release is a major barrier of polyplex-mediated gene transfection. Antimicrobial peptides (AMPs) are commonly used to improve polyethylenimine (PEI)-mediated gene transfection by increasing endosomal release. In the present study, we designed novel pH-sensitive peptides that highly enhance transfection efficiency compared to their parent peptides. METHODS: Two analogues of melittin (Mel) and RV-23 (RV) were synthesized by replacing the positively-charged residues in their sequences with glutamic acid residues. The pH-sensitive lysis ability of the peptides, the effect of the peptides on physicochemical characteristics, the intracellular trafficking, the transfection efficiency, and the cytotoxicity of the polyplexes were determined. RESULTS: The acidic peptides showed pH-sensitive lytic activity. The hemolytic activity of acidic peptides at pH 5.0 was higher than that at pH 7.4. The incorporation of acidic peptides did not affect the DNA binding ability of PEI but affected the physicochemical characteristics of the PEI/DNA polyplexes, which may be beneficial for endosomal release and gene transfection. The incorporation of acidic peptides into PEI/DNA polyplexes enhanced the PEI-mediated transfection efficiency corresponding to up to 42-fold higher luciferase activity compared to that of PEI alone. CONCLUSIONS: The results of the present study indicate that replacement of positively-charged residues with glutamic acid residues in the AMP sequence yields pH-sensitive peptides, which enhance the transfection efficiency of PEI/DNA polyplexes in various cell lines.

Mechanistic and functional aspects of the interaction of AR-23 with mammalian cell membrane and improvement of branched polyethylenimine-mediated gene transfection.

BACKGROUND: Previous studies have suggested that reducing the positive charge of melittin could increase endosomal release activity and improve branched polyethylenimine (BPEI)-mediated transfection. AR-23 is a melittin-related peptide from Rana tagoi, which shows 81% sequence identity with melittin but has less positively-charged residues than melittin. The present study aimed to investigate the mechanistic and functional aspects of the interaction of AR-23 with mammalian cells and thus improve BPEI-mediated gene transfection. METHODS: AR23 and two AR-23 analogs (AR-20 without positively-charged residues and AR-26 with the same positively-charged residues as melittin) were analyzed. Circular dichroism (CD) spectrometry was used to analyze the secondary structures of the peptides. Peptide-induced depolarization of cell membrane, the membrane-lytic activity of the peptides, and their potency with respect to enhancing the cellular uptake of calcein were evaluated. The physicochemical characters of complexes were measured and the effect of the peptides on BPEI-mediated transfection was determined. RESULTS: The CD spectra results indicated that a positive charge in AR-23 played a crucial role in maintaining the α-helical conformation, whereas an extra positive charge could not increase α-helical formation. AR-23 displayed a similar depolarization ability to melittin. However, AR-23 showed a lower membrane lytic activity under physiological conditions and a higher lytic activity at endosomal pH than melittin and AR-26, which possess more positive charges. Compared to melittin and AR-26, AR-23, with a higher endosomal escaping activity, resulted in a higher enhancement of BPEI-mediated gene transfection, as well as the maintainance of a lower cytotoxicity. CONCLUSIONS: We suggest that AR-23 may be considered as a potential enhancer for improving the transfection efficiency of cationic polymers.

Circulating mitochondrial DNA is associated with anemia in newly diagnosed hematologic malignancies.

Recent reports discovered that red blood cells (RBCs) could scavenge cell-free mitochondrial DNA (mtDNA), which drives the accelerated erythrophagocytosis and innate immune activation characterized by anemia and inflammatory cytokine production. However, the clinical value of the circulating mtDNA copy number alterations in hematologic malignancies is poorly understood. Our data showed that in comparison to healthy group, the patients group had significantly higher mtDNA and histone H4 levels. Moreover, we observed that RBC-bound mtDNA and histone H4 were negatively correlated with hemoglobin in patients. In addition, cytokines and chemokines levels in patients differed significantly from normal controls (21 higher, 7 lower). Our study suggested that both circulating mtDNA and histone H4 were associated with anemia in hematologic malignancies, which helps to further understand the potential mechanism of anemia development in patients with hematologic malignancies. This information may play a vital role in the specific therapeutic interventions for leukemia in the future.

Truncated peptides from melittin and its analog with high lytic activity at endosomal pH enhance branched polyethylenimine-mediated gene transfection.

BACKGROUND: Melittin is a commonly used cell-penetrating peptide (CPP) for improving branched polyethylenimine (BPEI)-mediated gene transfection. However, its application is limited owing to the cytotoxicity generated by the lytic activity at neutral pH. In the present study, we report two truncated peptides from melittin and florae with improved transfection efficiency. METHODS: Two truncated peptides consisting of 1-20 residues of melittin (MT20) and florae (FL20) were synthesized. Circular dichroism (CD) spectrometry was used to analyze the secondary structures of the peptides. The membrane-lytic activity of the peptides and their potency in enhancing cellular uptake of calcein were evaluated. The peptides and BPEI mixtures were mixed with plasmid DNA to prepare peptide/BPEI/DNA complexes. The physicochemical characters of complexes were measured and the effect of the peptides on BPEI-mediated transfection was determined. RESULTS: CD analysis and structure observation showed that the truncated peptides have α-helical conformation, which was necessary for penetrating activity. The truncated peptides exhibited several advantages than their parent peptides: (i) they showed higher hemolytic potency in acidic pH but lower lytic activity than their parent peptides in neutral pH; (ii) enhanced calcein efficiently release from both early and late endosome; (iii) they did not affect the DNA-binding affinity of BPEI and the physicochemical characteristics of BPEI/DNA complexes. Moreover, the peptides could increase BPEI-mediated transfection efficiency in different cell lines (293FT, B16F10 and CHO-K1) by simply mixing with BPEI, without causing cytotoxicity. CONCLUSIONS: The results obtained in the present study indicate that the truncated peptides with higher endosomal disrupting activity were better enhancers for increasing transfection efficiency.

Reduction-degradable linear cationic polymers as gene carriers prepared by Cu(I)-catalyzed azide-alkyne cycloaddition.

Linear reduction-degradable cationic polymers with different secondary amine densities (S2 and S3) and their nonreducible counterparts (C2 and C3) were synthesized by Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) step-growth polymerization of the dialkyne-oligoamine monomers and the diazide monomers. These polymers were studied with a goal of developing a set of new gene carriers. The buffering capacity and DNA binding ability of these polymers were evaluated by acid-base titration, gel retardation, and ethidium bromide (EB) exclusion assay. The polymers with lower amine density exhibit a weaker DNA-binding ability but a stronger buffering capacity in the range of pH 5.1 and 7.4. Particle size and zeta-potential measurements demonstrate that the polymers with higher amine density condense pDNA to form polyplexes with smaller sizes, while the disulfide bond in the backbone shows a negative effect on the condensing capability of the polymers, resulting in the formation of polyplexes with large size and nearly neutral surface. The reduction-sensitive polyplexes formed by polymer S2 or S3 can be disrupted by dithiothreitol (DTT) to release free DNA, which has been proven by the combination of gel retardation, EB exclusion assay, particles sizing, and zeta potential measurements. Cell viability measurements by MTT assay demonstrate that the reduction-degradable polymers (S2 and S3) have little cytotoxicity while the nonreducible polymers (C2 and C3) show obvious cytotoxicity, in particular, at high N/P ratios. In vitro transfection efficiencies of these polymers were evaluated using EGFP and luciferase plasmids as the reporter genes. Polymers S3 and S2 show much higher efficiencies than the nonreducible polymers C3 and C2 in the absence of 10% serum; unexpectedly, the lowest transfection efficiency has been observed for polymer S3 in the presence of serum.

Apelin­13 alleviates diabetic nephropathy by enhancing nitric oxide production and suppressing kidney tissue fibrosis.

Diabetes is a serious metabolic disease, and the kidney damage induced by diabetes also seriously affects the survival of patients. Apelin is a molecule that plays a crucial role in lipid metabolism, and recent studies have revealed that apelin­13, a subtype of apelin, plays an important role in regulating blood glucose levels. However, the role of apelin­13 in diabetic nephropathy remains unclear. In the present study, a rat model of diabetic nephropathy was constructed by the injection of streptozocin (STZ). During this process, these rats were injected with apelin­13. The blood glucose, urine protein and insulin levels were determined weekly. Next, the expression of angiotensin domain type 1 receptor­associated protein (APJ), endothelial nitric oxide synthase (eNOS), E­cadherin and α­smooth muscle actin (α­SMA) in the kidney tissues was determined with western blotting. Then, the endothelial cells of glomerular vessels were cultured with high glucose medium. These cells were treated with apelin­13 for 24 h. Finally, cell viability of these cells and the expression of APJ, eNOS, E­cadherin and α­SMA in these cells were determined with western blotting. As a result, treatment of apelin­13 induced the lower levels of blood glucose and urine protein. In addition, application of apelin­13 promoted the production of insulin and alleviated the insulin resistance. Treatment with apelin­13 promoted the expression of APJ, eNOS and E­cadherin while it suppressed the expression of α­SMA in kidney tissues of rats and endothelial cells of glomerular vessels. Furthermore, application of apelin­13 also promoted the cell viability of these cells. In conclusion, apelin­13 relieved diabetic nephropathy by promoting the production of nitric oxide (NO) and alleviating the fibrosis of kidney tissues.

Heparanase Facilitates PMA-Induced Megakaryocytic Differentiation in K562 Cells via Interleukin 6/STAT3 Pathway.

Heparanase (HPSE) is an endo-ß-D-glucuronidase that cleaves heparan sulfate and hence participates in remodeling of the extracellular matrix, leading to release of cytokines that are immobilized by binding to heparan sulfate proteoglycans (HSPGs), and consequently activating signaling pathways. This function of HPSE is correlated to its expression level that is normally very low in majority of the tissues. Exceptionally, human platelets express high level of HPSE, suggesting a unique physiological role in this cell. Using K562 cell line, we found a progressive increase of HPSE during the megakaryocytic differentiation. Analysis of a series of megakaryocytic differentiation-related heparin-binding proteins (HBPs) in the cell culture medium revealed an exclusive positive correlation between the level of interleukin 6 (IL-6) and HPSE expression. IL-6 modulated megakaryocytic differentiation through activation of STAT3. Further, we demonstrated that overexpression of HPSE potentiates megakaryocytic differentiation, whereas elimination of HPSE led to a delayed differentiation. This function of HPSE is associated with its activity, as overexpression of inactive HPSE had no effect on IL-6 production and megakaryocytic differentiation. The role of HPSE is further supported by the observation in an umbilical cord blood CD34+ cells megakaryocytic differentiation model. Our data propose a novel role for HPSE in platelets production by a HPSE/IL-6/STAT3 positive feedback loop that specifically regulates megakaryocytes maturation.

B to O erythrocyte conversion by the recombinant alpha-galactosidase.

BACKGROUND: Human group O red blood cells have great benefit in specialized transfusion areas such as armed conflict and natural calamity. The group B antigen differs structurally from group O antigen only by the addition of one terminal alpha-linked galactose residue. In this study we aimed to remove the terminal galactose from group B red blood cell to get group O red blood cell. METHODS: alpha-galactosidase cDNA was cloned by RT-PCR from Catimor coffee beans grown on Hainan Island of China. The vector for alpha-galactosidase cDNA expression was constructed and transferred into Pichia pastoris cells by electroporation. The transgenic cells were cloned by fermentation and the recombinant alpha-galactosidase was purified by ion exchange chromatography. After studying the biochemical characters of alpha-galactosidase, we have used it in converting human erythrocytes from group B to group O. RESULTS: The purity of recombinant alpha-galactosidase was higher than 96%, which was thought to be suitable for the use of blood conversion. Enzymatically converted human group O red blood cells (ECHORBC) exhibited membrane integrity, metabolic integrity, normal cell deformation and morphology. There were no coagulation between ECHORBC and any group of human blood. The ECHORBC will keep normal structure and function for a period of 21 days at 4 degrees C in monoammoniumphosphate nutrient solution. Experiments with Rhesus monkeys and gibbons showed that transfusion of enzymatically converted erythrocytes was safe. CONCLUSION: ECHORBC can be easily obtained from group B red blood cell by alpha-galactosidase digestion. This study suggests that ECHORBC could be transfused to patients safely and efficiently.

The acid-sensing ion channel, ASIC2, promotes invasion and metastasis of colorectal cancer under acidosis by activating the calcineurin/NFAT1 axis.

BACKGROUND: The tumor acidic microenvironment, a common biochemical event in solid tumors, offers evolutional advantage for tumors cells and even enhances their aggressive phenotype. However, little is known about the molecular mechanism underlying the acidic microenvironment-induced invasion and metastasis. METHODS: We examined the expression of the acid-sending ion channel (ASIC) family members after acidic exposure using RT-PCR and immunofluoresence. Gene manipulation was applied to reveal the potential of ASIC2 on invasion, proliferation, colony formation of colorectal cancer (CRC). We assessed the in vivo tumor growth by subcutaneous transplantation and metastasis by spleen xenografts. Chromatin immunoprecipitation-sequencing was used to uncover the binding sites of NFAT1. Finally, we examined the expression of ASIC2 in CRC tissues using immunohistochemistry. RESULTS: Acidic exposure led to up-regulation of the acid-sensing ion channel, ASIC2, in colorectal cancer (CRC) cells. ASIC2 overexpression in CRC cell lines, SW480 and HCT116, significantly enhanced cell proliferation in vitro and in vivo, while ASIC2 knockdown had the reverse effect. Importantly, ASIC2 promoted CRC cell invasion under acidosis in vitro and liver metastasis in vivo. Mechanistically, ASIC2 activated the calcineurin/NFAT1 signaling pathway under acidosis. Inhibition of the calcineurin/NFAT pathway by cyclosporine A (CsA) profoundly attenuated ASIC2-induced invasion under acidosis. ChIP-seq assay revealed that the nuclear factor, NFAT1, binds to genes clustered in pathways involved in Rho GTPase signaling and calcium signaling. Furthermore, immunohistochemistry showed that ASIC2 expression is increased in CRC samples compared to that in adjacent tissues, and ASIC2 expression correlates with T-stage, distant metastasis, recurrence, and poor prognosis. CONCLUSION: ASIC2 promotes metastasis of CRC cells by activating the calcineurin/NFAT1 pathway under acidosis and high expression of ASIC2 predicts poor outcomes of patients with CRC.

Gemcitabine-induced heparanase promotes aggressiveness of pancreatic cancer cells via activating EGFR signaling.

Pancreatic cancer (PC), characterized by aggressive local invasion and metastasis, is one of the most malignant cancers. Gemcitabine is currently used as the standard drug for the treatment of advanced and metastatic PC, but with limited efficacy. In this study, we demonstrated that gemcitabine increased the expression of heparanase (HPA1), the only known mammalian endoglycosidase capable of cleaving heparan sulfate, both in vitro and in vivo. Furthermore, overexpression of HPA1 in PC cell lines enhanced proliferation and invasion, accompanied with elevated phosphorylation of EGFR. In addition, we showed that the NF-κB pathway mediated the gemcitabine-induced HPA1 expression. Importantly, we found that an HPA1 inhibitor attenuated gemcitabine-induced invasion of PC cells. Finally, we showed that HPA1 was of negative prognostic value for PC patients. Taken together, our results demonstrated that gemcitabine-induced HPA1 promotes proliferation and invasion of PC cells through activating EGFR, implying that HPA1 may serve as promising therapeutic target in the treatment of PC.

Heparanase expression upregulates platelet adhesion activity and thrombogenicity.

Heparanase is an endo-glucuronidase that specifically cleaves heparan sulfate (HS) and heparin polysaccharides. The enzyme is expressed at low levels in normal tissues, but is often upregulated under pathological conditions such as cancer and inflammation. Normal human platelets express exceptionally high levels of heparanase, but the functional consequences of this feature remain unknown. We investigated functional roles of heparanase by comparing the properties of platelets expressing high (Hpa-tg) or low (Ctr) levels of heparanase. Upon activation, Hpa-tg platelets exhibited a much stronger adhesion activity as compared to Ctr platelets, likely contributing to a higher thrombotic activity in a carotid thrombosis model. Furthermore, we found concomitant upregulated expression of both heparanase and CD62P (P-selectin) upon activation of mouse and human platelets. As platelets play important roles in tumor metastasis, these findings indicate contribution of the platelet heparanase to hyper-thrombotic conditions often seen in patients with metastatic cancer.

RV-23, a Melittin-Related Peptide with Cell-Selective Antibacterial Activity and High Hemocompatibility.

RV-23 is a melittin-related antibacterial peptide (MRP) with lower cytotoxicity than either melittin or AR-23, another MRP. The aim of this study was to explore the mechanism of RV- 23's antibacterial selectivity and its hemocompatibility. The results showed that all the peptides exhibited lytic activity against Staphylococcus aureus and Escherichia coli, with RV-23 showing the highest potency. Moreover, RV-23 had lower cytotoxicity than melittin or AR-23 at their minimal inhibitory concentration. In addition, CD experiments showed that melittin, RV-23, and AR-23 all had a typical α-helical structure, and RV-23 had the lowest α-helix content. The structural information showed that RV-23 has the lowest hydrophobicity and highest hydrophobic moment. Because hydrophobicity and α-helix content are believed to correlate with hemolysis, the results indicate that the selective lytic activity against bacteria of RV-23 may be due to its low hydrophobicity and α-helicity, which lead to low cytotoxicity without affecting antibacterial activity. Furthermore, RV-23 did not affect the structure and function of blood components such as red blood cells, platelets, albumin, and the blood coagulation system. In conclusion, RV-23 is a cell-selective antibacterial peptide with high hemocompatibility due to its unique structure.

Design of an α-helical antimicrobial peptide with improved cell-selective and potent anti-biofilm activity.

AR-23 is a melittin-related peptide with 23 residues. Like melittin, its high α-helical amphipathic structure results in strong bactericidal activity and cytotoxicity. In this study, a series of AR-23 analogues with low amphipathicity were designed by substitution of Ala1, Ala8 and Ile17 with positively charged residues (Arg or Lys) to study the effect of positively charged residue distribution on the biological viability of the antimicrobial peptide. Substitution of Ile17 on the nonpolar face with positively charged Lys dramatically altered the hydrophobicity, amphipathicity, helicity and the membrane-penetrating activity against human cells as well as the haemolytic activity of the peptide. However, substitution on the polar face only slightly affected the peptide biophysical properties and biological activity. The results indicate that the position rather than the number of positively charged residue affects the biophysical properties and selectivity of the peptide. Of all the analogues, A(A1R, A8R, I17K), a peptide with Ala1-Arg, Ala8-Arg and Ile17-Lys substitutions, exhibited similar bactericidal activity and anti-biofilm activity to AR-23 but had much lower haemolytic activity and cytotoxicity against mammalian cells compared with AR-23. Therefore, the findings reported here provide a rationalization for peptide design and optimization, which will be useful for the future development of antimicrobial agents.

Evaluation of group A1B erythrocytes converted to type as group O: studies of markers of function and compatibility.

BACKGROUND: Enzymatic conversion of blood group A1B red blood cells (RBC) to group O RBC (ECO) was achieved by combined treatment with α-galactosidase and α-N-acetylgalactosaminidase. The aim of this study was to evaluate the function and safety of these A1B-ECO RBC in vitro. MATERIALS AND METHODS: A 20% packed volume of A1B RBC was treated with enzymes in 250 mM glycine buffer, pH 6.8. The efficiency of the conversion of A and B antigen was evaluated by traditional typing in test tubes, gel column agglutination technology and fluorescence-activated cell sorting (FACS) analysis. The physiological and metabolic parameters of native and ECO RBC were compared, including osmotic fragility, erythrocyte deformation index, levels of 2,3-diphosphoglycerate, ATP, methaemoglobin, free Na(+), and free K(+). The morphology of native and ECO RBC was observed by scanning electron microscopy. Residual α-galactosidase or α-N-acetylgalactosaminidase in A1B-ECO RBC was detected by double-antibody sandwich ELISA method. Manual cross-matching was applied to ensure blood compatibility. RESULTS: The RBC agglutination tests and FACS results showed that A1B RBC were efficiently converted to O RBC. Functional analysis suggested that the conversion process had little impact on the physiological and metabolic parameters of the RBC. The residual amounts of either α-galactosidase or α-N-acetylgalactosaminidase in the A1B-ECO RBC were less than 10 ng/mL of packed RBC. About 18% of group B and 55% of group O sera reacted with the A1B-ECO RBC in a sensitive gel column cross-matching test. DISCUSSION: The conversion process does not appear to affect the morphological, physiological or metabolic parameters of A1B-ECO RBC. However, the A1B-ECO RBC still reacted with some antigens. More research on group O and B sera, which may partly reflect the complexity of group A1 the safety of A1B-ECO RBC is necessary before the application of these RBC in clinical transfusion.

[Expression of mRNA of matrix metalloproteinase and tissue inhibitor of matrix metalloproteinase after cardiopulmonary resuscitation for asphyxial cardiac arrest in rat].

OBJECTIVE: To study the change in mRNA expression of matrix metalloproteinase (MMPs) and tissue inhibitor-1 of matrix metalloproteinase (TIMP-1) in brain after cardiopulmonary resuscitation (CPR) for asphyxial cardiac arrest in rat. METHODS: The animal model of cardiac arrest was reproduced by clamping endotracheal tube at the expiration. Eighty male SD rats were randomly divided into two groups: control group and resuscitation group, and they were again divided into 0, 0.5, 3, 6, 9 hours subgroups (n=8). Evans blue content and mRNA expressions of MMPs and TIMP-1 in the brain after CPR were determined respectively. RESULTS: The mRNA expression of MMP-9 and TIMP-1 was up-regulated 3 hours after restoration of spontaneous circulation (ROSC). At 6 hours after ROSC, they were markedly high, and the ratio of MMP-9/TIMP-1 was higher too. The mRNA expression of MMP-2 showed no significant change at 9 hours after ROSC. CONCLUSION: The mRNA expression of MMP-9 and TIMP-1 and MMP-9/TIMP-1 are increased at early stage after CPR, but the mRNA expression of MMP-2 shows no significant change.

Quantification of α-Gal Antigen Removal in the Porcine Dermal Tissue by α-Galactosidase.

The α-Gal (Galα1,3-Galß1-4GlcNAc-R) epitope, the major xenoantigen, is the first barrier in a porcine-to-man tissue and organ xenotransplantation. The elimination or reduction of the α-Gal epitopes is therefore an important step for a successful xenotransplantation. The present study is to evaluate the α-Gal elimination in the porcine skin with α-galactosidase treatment, and to assess two methods (immunohistochemistry and inhibition ELISA) that may be used in quality control for quantifying the extent of the α-Gal elimination. Enzymatic cleavage in a single-step process is extremely efficient and affordable at eliminating the α-Gal epitope even in a tissue as dense as the porcine dermis. The cost of enzymatic cleavage is found to be less than US$7 for a 10 × 10 cm piece of porcine skin (0.5 mm thick) or about US$140 for 100 g of 3-dimensional soft tissues. After enzymatic cleavage, the α-Gal-positive immunostaining was essentially undetectable in enzyme-treated porcine skin. The inhibition rate constant of the monoclonal anti-Gal antibody M86 binding to α-Gal-bovine serum albumin in ELISA was reduced from 15.0 ± 4.3 (n = 10) to 6.1 ± 2.6 (n = 7) after enzyme treatment, in comparison to 4.4 ± 1.8 (n = 9) background inhibition of decellularized human skin (the ultimate negative control), which demonstrates ∼ 84% elimination of α-Gal epitopes in treated porcine skin. To examine the suitability of two detection methods for the routine quality control application, comparative studies were made with control and enzyme-treated porcine skin, porcine skin from the α-Gal knockout animal, as well as decellularized human skin. The data show that the traditional immunohistochemistry and, to a less extent, the inhibition ELISA with further modifications can be used as quality control tools in the production and selection of biocompatible bioprosthetic devices. The biological evaluation of enzyme-treated porcine skin is ongoing with a small animal model and a nonhuman primate model.

[Alanine solution as enzyme reaction buffer used in A to O blood group conversion].

The aim of this study was to investigate the effect of alanine solution as α-N-acetylgalactosaminidase enzyme reaction buffer on the enzymatic activity of A antigen. The binding ability of α-N-acetylgalactosaminidase with RBC in different reaction buffer such as alanine solution, glycine solution, normal saline (0.9% NaCl), PBS, PCS was detected by Western blot. The results showed that the efficiency of A to O conversion in alanine solution was similar to that in glycine solution, and Western blot confirmed that most of enzymes blinded with RBC in glycine or alanine solution, but few enzymes blinded with RBC in PBS, PCS or normal saline. The evidences indicated that binding of enzyme with RBC was a key element for A to O blood group conversion, while the binding ability of α-N-acetylgalactosaminidase with RBC in alanine or glycine solution was similar. It is concluded that alanine solution can be used as enzyme reaction buffer in A to O blood group conversion. In this buffer, the α-N-acetylgalactosaminidase is closely blinded with RBC and α-N-acetylgalactosaminidase plays efficient enzymatic activity of A antigen.

Glucose buffer is suitable for blood group conversion with α-N acetylgalactosaminidase and α-galactosidase.

BACKGROUND: It is well known that the buffer plays a key role in the enzymatic reaction involved in blood group conversion. In previous study, we showed that a glycine buffer is suitable for A to O or B to O blood group conversion. In this study, we investigated the use of 5% glucose and other buffers for A to O or B to O blood group conversion by α-N-acetylgalactosaminidase or α-galactosidase. MATERIALS AND METHODS: We compared the binding ability of α-N-acetylgalactosaminidase/α-galactosidase with red blood cells (RBC) in different reaction buffers, such as normal saline, phosphate-buffered saline (PBS), a disodium hydrogen phosphate-based buffer (PCS), and 5% commercial glucose solution. The doses of enzymes necessary for the A/B to O conversion in different reaction buffers were determined and compared. The enzymes' ability to bind to RBC was evaluated by western blotting, and routine blood typing and fluorescence activated cell sorting was used to evaluate B/A to O conversion efficiency. RESULTS: The A to O conversion efficiency in glucose buffer was similar to that in glycine buffer with the same dose (>0.06 mg/mL pRBC). B to O conversion efficiency in glucose buffer was also similar to that in glycine buffer with the same dose (>0.005 mg/mL pRBC). Most enzymes could bind with RBC in glycine or glucose buffer, but few enzymes could bind with RBC in PBS, PCS, or normal saline. CONCLUSION: These results indicate that 5% glucose solution provides a suitable condition for enzymolysis, especially for enzymes combining with RBC. Meanwhile, the conversion efficiency of A/B to O was similar in glucose buffer and glycine buffer. Moreover, 5% glucose solution has been used for years in venous transfusion, it is safe for humans and its cost is lower. Our results do, therefore, suggest that 5% glucose solution could become a novel suitable buffer for A/B to O blood group conversion.