In vivo inhibition of aldehyde dehydrogenase by disulfiram

Disulfiram (DSF) has found extensive use in the aversion therapy treatment of recovering alcoholics. Although it is known to irreversibly inhibit hepatic aldehyde dehydrogenase (ALDH), the specific mechanism of in vivo inhibition of the enzyme by the drug has not yet been determined. In this report, we demonstrate a novel, but simple and rapid method for structurally characterizing in vivo derived protein-drug adducts by linking on-line sample processing to HPLC-electrospray ionization mass spectrometry (HPLC-MS) and HPLC-tandem mass spectrometry (HPLC-MS/MS). Employing this approach, rats were administered DSF, and their liver mitochondria were isolated and solubilized. Both native and in vivo DSF-treated mitochondrial ALDH (rmALDH) were purified in one-step with an affinity cartridge. The in vivo DSF-treated rmALDH showed 77% inhibition in enzyme activity as compared to that of the control. Subsequently, the control and DSF-inhibited rmALDH were both subjected to HPLC-MS analyses. We were able to detect two adducts on DSF-inhibited rmALDH as indicated by the mass increases of approximately 71 and approximately 100 Da. To unequivocally determine the site and structure of these adducts, on-line pepsin digestion-HPLC-MS and HPLC-MS/MS were performed. We observed two new peptides at MH(+)=973.7 and 1001.8 in the pepsin digestion of DSF-inhibited enzyme. These two peptides were subsequently subjected to HPLC-MS/MS for sequence determination. Both peptides possessed the sequence FNQGQC(301)C(302)C(303), derived from the enzyme active site region, and were modified at Cys(302) by N-ethylcarbamoyl (+71 Da) and N-diethylcarbamoyl (+99 Da) adducts. These findings indicated that N-dealkylation may be an important step in DSF metabolism, and that the inhibition of ALDH occurred by carbamoylation caused by one of the DSF metabolites, most likely S-methyl-N,N-diethylthiocarbamoyl sulfoxide (MeDTC-SO).     

Overview — In vitro inhibition of aldehyde dehydrogenase by disulfiram and metabolites

Disulfiram (DSF) has found extensive use in the aversion therapy treatment of recovering alcoholics. It is known that DSF or a metabolite irreversibly inhibits aldehyde dehydrogenase (ALDH). However, the actual mechanism of inhibition is still not known. In this work we describe the in vitro interactions of DSF, as well as a principal metabolite S-methyl-N,N-diethylthiocarbamoyl sulfoxide (MeDTC-SO), with both recombinant rat liver mitochondrial monomeric ALDH (rmALDH) and homotetrameric rmALDH. We show that DSF directly inhibits rmALDH (IC₅₀=36.4 μM) by inducing the formation of an intramolecular disulfide bond. We also demonstrate by HPLC-MS analysis of a Glu-C digest of DSF-treated rmALDH that the intramolecular disulfide bridge formed involves two of the three cysteines located at the active site of the enzyme. Using a combination of HPLC-MS and HPLC-MS/MS, we further show that the electrophilic metabolite MeDTC-SO also inhibits rmALDH (IC₅₀=4.62 μM). We isolate and identify a carbamoylated peptide at Cys₃₀₂ with the sequence FNQGQC₃₀₁C₃₀₂C₃₀₃. Hence we show that MeDTC-SO exhibits its inhibitory effect by covalently modifying the -SH side-chain of Cys₃₀₂, present at the active site rmALDH. Finally we show using SEC-MS that both DSF and MeDTC-SO do not prevent formation of the homotetramer of rmALDH, but inhibit the enzyme by acting directly at the active site of specific monomers of rmALDH.     

Overview--in vitro inhibition of aldehyde dehydrogenase by disulfiram and metabolites

Disulfiram (DSF) has found extensive use in the aversion therapy treatment of recovering alcoholics. It is known that DSF or a metabolite irreversibly inhibits aldehyde dehydrogenase (ALDH). However, the actual mechanism of inhibition is still not known. In this work we describe the in vitro interactions of DSF, as well as a principal metabolite S-methyl-N,N-diethylthiocarbamoyl sulfoxide (MeDTC-SO), with both recombinant rat liver mitochondrial monomeric ALDH (rmALDH) and homotetrameric rmALDH. We show that DSF directly inhibits rmALDH (IC(50)=36.4 microM) by inducing the formation of an intramolecular disulfide bond. We also demonstrate by HPLC-MS analysis of a Glu-C digest of DSF-treated rmALDH that the intramolecular disulfide bridge formed involves two of the three cysteines located at the active site of the enzyme. Using a combination of HPLC-MS and HPLC-MS/MS, we further show that the electrophilic metabolite MeDTC-SO also inhibits rmALDH (IC(50)=4.62 microM). We isolate and identify a carbamoylated peptide at Cys(302) with the sequence FNQGQC(301)C(302)C(303). Hence we show that MeDTC-SO exhibits its inhibitory effect by covalently modifying the -SH side-chain of Cys(302), present at the active site rmALDH. Finally we show using SEC-MS that both DSF and MeDTC-SO do not prevent formation of the homotetramer of rmALDH, but inhibit the enzyme by acting directly at the active site of specific monomers of rmALDH.     

Effects of Angiotensin I-Converting Enzyme Inhibitory Substances Derived from Indonesian Dried-salted fish on Blood Pressure of Rats

Indonesian dried-salted fish (DSF) was produced from skipjack tuna by soaking the flesh in 15% NaCl (DSF I) or 25% NaCl (DSF II). The DSFs were then hydrolyzed by trypsin, chymotrypsin, Pronase E, and pepsin. Angiotensin I-converting enzyme (ACE) inhibitory activity was measured. The pepsin digest showed the highest inhibitory activity (IC₅₀; 0.63 mg protein/ml). DSF II hydrolysate had higher inhibitory activity than that in DSF I. A three-month storage period of DSF gave higher ACE-inhibitory activity than that of 6 months. An oral administration of pepsin hydrolysate significantly decreased the blood pressure of rats. From the purification steps, at least 4 inhibitor peptides were found. The amino acid sequences of the peptides were Val-Ala-Trp-Lys-Leu, Trp-Ser-Lys-Val-Val-Leu, Ser-Lys-Val-Pro-Pro, and Cys-Trp-Leu-Pro-Val-Tyr, with an IC₅₀ value of 31.97, 156.28, 74.22, and 22.20 μM, respectively.     

Purification and identification of novel angiotensin-I converting enzyme (ACE) inhibitory peptides from cultured marine microalgae (Nannochloropsis oculata) protein hydrolysate

Isolation of bioactive compounds and commercialization of marine microalgae sources are interesting targets in future marine biotechnology. Cultured biomass of the marine microalga, Nannochloropsis oculata, was used to purify angiotensin-I converting enzyme (ACE) inhibitory peptides using proteases including pepsin, trypsin, α-chymotrypsin, papain, alcalase, and neutrase. The pepsin hydrolysate exhibited the highest ACE inhibitory activity, compared to the other hydrolysates and then was separated into three fractions (F1, F2, and F3) using Sephadex G-25 gel filtration column chromatography. First fraction (F1) showed the highest ACE inhibitory activity and it was further purified into two fractions (F1-1 and F1-2) using reverse-phase high-performance liquid chromatography. The IC₅₀ value of purified ACE inhibitory peptides were 123 and 173 μM and identified as novel peptides, Gly-Met-Asn-Asn-Leu-Thr-Pro (GMNNLTP; MW, 728 Da) and Leu-Glu-Gln (LEQ; MW, 369 Da), respectively. In addition, nitric oxide production level (%) was significantly increased by the purified peptide (Gly-Met-Asn-Asn-Leu-Thr-Pro) compared to the purified peptide (Leu-Glu-Gln) and other treated pepsin hydrolysate fractions on human umbilical vein endothelial cells (HUVECs). Cell viability assay showed no cytotoxicity on HUVECs with the treated purified peptides and fractions. These results suggest that the isolated peptides from cultured marine microalga, N. oculata protein sources may have potentiality to use commercially as ACE inhibitory agents in functional food industry.     

Using chemical derivatization and mass spectrometric analysis to characterize the post-translationally modified Staphylococcus aureus surface protein G

The Staphylococcus aureus surface protein G (SasG) is an important mediator of biofilm formation in virulent S. aureus strains. A detailed analysis of its primary sequence has not been reported to date. SasG is highly abundant in the cell wall of the vancomycin-intermediate S. aureus strain HIP5827, and was purified and subjected to sequence analysis by MS. Data from MALDI-TOF and LC-MS/MS experiments confirmed the predicted N-terminal signal peptide cleavage site at residue A₅₁ and the C-terminal cell wall anchor site at residue T₁₀₈₆. The protein was also derivatized with N-succinimidyloxycarbonyl-methyl-tris(2,4,6-trimethoxyphenyl) phosphonium bromide (TMPP-Ac-OSu) to assess the presence of additional N-terminal sites of mature SasG. TMPP-derivatized SasG peptides featured m/z peaks with a 572 Da mass increase over the equivalent underivatized peptides. Multiple N-terminal peptides, all of which were observed in the 150 amino acid segment following the signal peptide cleavage at the residue A₅₁, were characterized from MS and MS/MS data, suggesting a series of successive N-terminal truncations of SasG. A strategy combining TMPP derivatization, multiple enzyme digestions to generate overlapping peptides and detailed MS analysis will be useful to determine and understand functional implications of PTMs in bacterial cell wall-anchored proteins, which are frequently involved in the modulation of virulence-associated bacterial surface properties.     

ALDH1A1诱导肺腺癌细胞发生上皮-间质转化及化疗耐药

目的:探索醛脱氢酶1A1(aldehyde dehydrogenase 1A1,ALDH1A1)在肺腺癌细胞(lung adenocarcinoma cell,LAC)化疗耐药中的作用及机制,为肺癌临床治疗和新型药物的研发提供实验依据。方法:采用慢病毒载体构建ALDH1A1高表达肺腺癌细胞模型,并通过流式细胞术和western blot技术对该细胞模型进行验证。通过CCK8法检测ALDH1A1高表达肺腺癌细胞对肺癌治疗药物顺铂(cisplatin,DDP)、紫杉醇(paclitaxcel)、厄洛替尼(erlotinib)和吉非替尼(gefitinib)的耐药性。通过检测肿瘤干细胞(cancer stem cell,CSC)分子标志物、上皮-间质转化(Epithelial-Mesenchymal Transition,EMT)分子标志物及细胞迁移能力探讨ALDH1A1高表达对肺腺癌细胞的干性和EMT特征的影响。双硫仑(disulfiram,DSF)是ALDH的抑制剂,我们通过CCK8法和transwell细胞迁移实验探究DSF对肺腺癌细胞体外生长和迁移能力的影响,体内实验探究DSF和厄洛替尼联合用药对HCC827-ALDH1A1细胞皮下异种移植瘤生长的影响。结果:ALDH1A1高表达诱导肺腺癌细胞对厄洛替尼、吉非替尼、紫杉醇和顺铂产生不同程度的耐药,干细胞标志物CD44、CD133蛋白表达上调,EMT间充质标志物vimentin蛋白表达上调,transwell实验结果显示ALDH1A1高表达肺腺癌细胞的迁移能力增强,使用ALDH靶向抑制剂DSF能选择性抑制ALDH1A1高表达肺腺癌细胞所增高的迁移能力并克服HCC827-ALDH1A1细胞皮下异种移植瘤的生长,延缓体内耐药。结论:ALDH1A1能诱导肺腺癌细胞对多种抗肺癌药物产生耐药并发生干细胞样转化,靶向抑制ALDH酶活性可克服由ALDH1A1高表达所产生的耐药,为肺癌的临床治疗提供新的思路。     

Formation of acetaldehyde-derived DNA adducts due to alcohol exposure

Epidemiological studies have identified chronic alcohol consumption as a significant risk factor for cancers of the upper aerodigestive tract, including the oral cavity, pharynx, larynx and esophagus, and for cancer of the liver. Ingested ethanol is mainly oxidized by the enzymes alcohol dehydrogenase (ADH), cytochrome P-450 2E1 (CYP2E1), and catalase to form acetaldehyde, which is subsequently oxidized by aldehyde dehydrogenase 2 (ALDH2) to produce acetate. Polymorphisms of the genes which encode enzymes for ethanol metabolism affect the ethanol/acetaldehyde oxidizing capacity. ADH1B*2 allele (ADH1B, one of the enzyme in ADH family) is commonly observed in Asian population, has much higher enzymatic activity than ADH1B*1 allele. Otherwise, approximately 40% of Japanese have single nucleotide polymorphisms (SNPs) of the ALDH2 gene. The ALDH2 *2 allele encodes a protein with an amino acid change from glutamate to lysine (derived from the ALDH2*1 allele) and devoid of enzymatic activity. Neither the homozygote (ALDH2*2/*2) nor heterozygote (ALDH2*1/*2) is able to metabolize acetaldehyde promptly. Acetaldehyde is a genotoxic compound that reacts with DNA to form primarily a Schiff base N²-ethylidene-2′-deoxyguanosine (N²-ethylidene-dG) adduct, which may be converted by reducing agents to N²-ethyl-2′-deoxyguanosine (N²-ethyl-dG) in vivo, and strongly blocked translesion DNA synthesis. Several studies have demonstrated a relationship between ALDH2 genotypes and the development of certain types of cancer. On the other hand, the drinking of alcohol induces the expression of CYP2E1, resulting in an increase in reactive oxygen species (ROS) and oxidative DNA damage. This review covers the combined effects of alcohol and ALDH2 polymorphisms on cancer risk. Studies show that ALDH2*1/*2 heterozygotes who habitually consume alcohol have higher rates of cancer than ALDH2*1/*1 homozygotes. Moreover, they support that chronic alcohol consumption contributes to formation of various DNA adducts. Although some DNA adducts formation is demonstrated to be an initiation step of carcinogenesis, it is still unclear that whether these alcohol-related DNA adducts are true factors or initiators of cancer. Future studies are needed to better characterize and to validate the roles of these DNA adducts in human study.     

Antioxidant peptides isolated from the marine rotifer,Brachionus rotundiformis

Protein derived from the rotifer Brachionus rotundiformis was hydrolyzed using different proteases (Alcalase, α-chymotrypsin, Neutrase, papain, pepsin and trypsin) for production of antioxidant peptide. Antioxidant activities of hydrolysates were evaluated using DPPH radical scavenging activity. Peptic hydrolysate exhibited the highest antioxidative activity compared to other hydrolysates. To identify antioxidant peptides, peptic hydrolysate was purified using consecutive chromatographic methods, and antioxidant peptides were identified to be Leu-Leu-Gly-Pro-Gly-Leu-Thr-Asn-His-Ala (1076 Da), and Asp-Leu-Gly-Leu-Gly-Leu-Pro-Gly-Ala-His (1033 Da) by Q-TOF ESI mass spectroscopy. EC₅₀ values of purified peptides were 189.8 and 167.7 μM, respectively. Antioxidant activities of peptides purified from the rotifer protein hydrolysate were evaluated, with results showing that peptides significantly quenched free radicals.     

Characterization of tyrosinase inhibitory constituents from the aerial parts of Humulus japonicus using LC-MS/MS coupled online assay

In the screening of natural products for the development as cosmetic ingredients, the EtOAc-soluble fraction of Humulus japonicus showed tyrosinase inhibitory activity. HPLC-MS/MS coupled online tyrosinase assay of EtOAc-soluble fraction of H. japonicus characterized the twenty-eight constituents including two unknown ones and their tyrosinase inhibitory activity. Fractionation of H. japonicus using various chromatographic techniques yielded thirty-eight compounds. The chemical structures of isolated compounds were identified by spectroscopic analysis. As characterized by HPLC-MS/MS analysis, we isolated twenty-four predicted compounds and further identified two unknown ones, named humulusides A (1) and B (2). Additional ten compounds were also identified by purification. Tyrosinase inhibitory activity of isolated compounds were evaluated, which was closely correlated with the results from HPLC-MS/MS coupled online tyrosinase assay. Consistent with predicted data, two major compounds, trans-N-coumaroyltyramine (14) and cis-N-coumaroyltyramine (15) showed tyrosinase inhibition with IC₅₀ values of 40.6 and 36.4?μM. Taken together, H. japonicus is suggested as whitening ingredient in cosmetic products. In addition, HPLC-MS/MS coupled tyrosinase assay is powerful tool for predicting active compounds with short time and limited amounts, although identification of new compounds and verification of predicted data are also needs to be demonstrated by further experiment.     

The disulfiram metabolites S-methyl-N,N-diethyldithiocarbamoyl sulfoxide and S-methyl-N,N-diethylthiocarbamoyl sulfone irreversibly inactivate betaine aldehyde dehydrogenase from Pseudomonas aeruginosa, both in vitro and in situ, and arrest bacterial growth

Betaine aldehyde dehydrogenase from the human opportunistic pathogen Pseudomonas aeruginosa (PaBADH) catalyzes the irreversible, NAD(P)⁺-dependent oxidation of betaine aldehyde, producing glycine betaine, an osmoprotectant. PaBADH participates in the catabolism of choline and likely in the defense against the osmotic and oxidative stresses to which the bacterium is exposed when infecting human tissues. Given that choline or choline precursors are abundant in infected tissues, PaBADH is a potential drug target because its inhibition will lead to the build up of the toxic betaine aldehyde inside bacterial cells. We tested the thiol reagents, disulfiram (DSF) and five DSF metabolites—diethyldithiocarbamic acid (DDC), S-methyl-N,N-diethyldithiocarbamoyl sulfoxide (MeDDTC-SO) and sulfone (MeDDTC-SO₂), and S-methyl-N,N-diethylthiocarbamoyl sulfoxide (MeDTC-SO) and sulfone (MeDTC-SO₂)—as inhibitors of PaBADH and P. aeruginosa growth. As in vitro PaBADH inhibitors, their order of potency was: MeDDTC-SO₂ > DSF > MeDTC-SO₂ > MeDDTC-SO > MeDTC-SO. DDC did not inactivate the enzyme. PaBADH inactivation by DSF metabolites (i) was not affected by NAD(P)⁺, (ii) could not be reverted by dithiothreitol, and (iii) did not affect the quaternary structure of the enzyme. Of the DSF metabolites tested, MeDTC-SO₂ and MeDDTC-SO produced significant in situ PaBADH inactivation and arrest of P. aeruginosa growth in choline containing media, in which the expression of PaBADH is induced. They had no effect in media lacking choline, indicating that PaBADH is their main intracellular target, and that arrest of growth is due to accumulation of betaine aldehyde. The in vitro and in situ kinetics of enzyme inactivation by these two compounds were very similar, indicating no restriction on their uptake by the cells. MeDDTC-SO₂ and DSF have no inhibitory effects in situ, probably because their high reactivity towards intracellular nonessential thiols causes their depletion. Our results support that PaBADH is a promising target to treat P. aeruginosa infections, and that some DSF metabolites might be of help in this aim.     

Free radical scavenging and angiotensin-I converting enzyme inhibitory peptides from Pacific cod (Gadus macrocephalus) skin gelatin

Potent antioxidative peptides were purified from Pacific cod (Gadus macrocephalus) skin gelatin using alcalase, neutrase, papain, trypsin, pepsin, and α-chymotrypsin. Among them, the papain hydrolysate exhibited the highest antioxidant activity. Therefore, it was further purified and obtained two peptides with amino acid sequences of Thr-Cys-Ser-Pro (388 Da) and Thr-Gly-Gly-Gly-Asn-Val (485.5 Da). The antioxidant activity of the purified peptides was performed by electron spin resonance technique. Moreover, their intracellular free radical scavenging activity using 2′,7′-dichlorofluorescin diacetate and the protective effect against oxidation-induced DNA damage were evaluated in mouse macrophages (RAW 264.7 cells). Furthermore, both peptides have shown potential angiotensin-I converting enzyme inhibitory effect. The present study demonstrated that the peptides derived from Pacific cod (G. macrocephalus) skin gelatin could be used in the food industry as functional ingredients with potent antioxidative and antihypertensive benefits.     

Purification and characterization of grass carp mitochondrial aldehyde dehydrogenase

The molecular biology and enzymology of aldehyde dehydrogenase (ALDH) have been extensively investigated. However, most of the studies have been confined to the mammalian forms, while the sub-mammalian vertebrate ALDHs are relatively unexplored. In the present investigation, an ALDH was purified from the hepatopancreas of grass carp (Ctenopharygodon idellus) by affinity chromatographies on α-cyanocinnamate-Sepharose and Affi-gel Blue agarose. The 800-fold purified enzyme had a specific activity of 4.46 U/mg toward the oxidation of acetaldehyde at pH 9.5. It had a subunit molecular weight of 55 000. Isoelectric focusing showed a single band with a pI of 5.3. N-terminal amino acid sequencing of 30 residues revealed a positional identity of ∼70% with mammalian mitochondrial ALDH2. The kinetic properties of grass carp ALDH resembled those of mammalian ALDH2. The optimal pH for the oxidation of acetaldehyde was 9.5. The K_m values for acetaldehyde were 0.36 and 0.31 μM at pH 7.5 and 9.5, respectively. Grass carp ALDH also possessed esterase activity which could be activated in the presence of NAD⁺.     

ACE Inhibitory and Antioxidant Activities of Novel Peptides from <Emphasis Type="Italic">Scorpaena notata</Emphasis> By-product Protein Hydrolysate

This study describes the isolation of angiotensin I converting enzyme and antioxidative peptides from head protein hydrolysate of red scorpionfish (Scorpaena notata) prepared by treatment with a protease from the fungus Penicillium digitatum. After ultrafiltration, three peptides were isolated by a two-step procedure: size exclusion chromatography on a Toyopearl HW-40 followed by reversed-phase high performance liquid chromatography (RP-HPLC) with a high purification yield of 2.22 mg of peptide/g of initial protein. Active peptides were then identified by nanoscale liquid chromatography coupled to tandem mass spectrometry (nanoLC/MS–MS), corresponding to the following sequences: Gln–Gln–Pro–His–Ser–Arg–Ser–Lys–Gly–Phe–Pro–Gly–Pro (1424.724 Da), Gly–Gln–Lys–Ser–Val–Pro–Glu–Val–Arg (1000.565 Da) and Val–Glu–Gly–Lys–Ser–Pro–Asn–Val (830.448 Da). Peptides D-I, E-I and F-I showed high angiotensin-I converting enzyme inhibitory activity with an IC₅₀ values of 0.98, 1.69 and 1.44 µM, respectively as well as a synergistic antioxidant activity between the different fractions. Thus, we have demonstrated that underutilized wastes can be valorized by production of peptides that can be used as potential therapeutic compounds active against oxidative stress and hypertension.     

Production and Isolation of Levan by Use of Levansucrase Immobilized on the Ceramic Support SM-10

The complete amino acid sequence of rye seed chitinase-a (RSC-a) has been analyzed. RSC-a was cleaved with cyanogen bromide and the resulting three fragments, CB1, CB2, and CB3, were separated by gel filtration. The amino acids of the N-terminal fragment CB1 were sequenced by analyzing the peptides produced by digestion with trypsin, lysylendopeptidase, or pepsin of reduced S-carboxymethyl ated or S-aminoethylated CB1. The sequences of fragments CB2 and CB3 were established by sequencing the tryptic peptides from reduced S-carboxymethylated CB2 and CB3, and by aligning them with the sequence of rye seed chitinase-c (RSC-c) to maximize sequence homology. The complete amino acid sequence of RSC-a was established by connecting these three fragments.

RSC-a consists of 302 amino acid residues including hydroxyproline residues, and has a molecular mass of 31,722 Da. RSC-a is basic protein with a cysteine-rich amino terminal domain, indicating that this enzyme belongs to class I chitinases. The amino acid sequence of RSC-a showed that the sequence from Gly60 to C-terminal Ala302 in this enzyme corresponds to that of RSC-c belonging to class II chitinases with 92% identity, and that RSC-a has high similarity to other plant class I chitinases but a longer hinge region and an extra disulfide bond.     

Discrimination of in vitro and in vivo digestion products of meat proteins from pork,beef, chicken,and fish

In vitro digestion products of proteins were compared among beef, pork, chicken, and fish. Gastric and jejunal contents from the rats fed these meat proteins were also compared. Cooked pork, beef, chicken, and fish were homogenized and incubated with pepsin alone or followed by trypsin. The digestion products with molecular weights of less than 3000 Da were identified with MALDI‐TOF‐MS and nano‐LC‐MS/MS. Gastric and jejunal contents obtained from the rats fed the four meat proteins for 7 days were also analyzed. After pepsin digestion, pork, and beef samples had a greater number of fragments in similarity than chicken and fish samples, but the in vitro digestibility was the greatest (p < 0.05) for pork and the smallest for beef samples. After trypsin digestion, the species differences were less pronounced (p > 0.05). A total of 822 and 659 peptides were identified from the in vitro and in vivo digestion products, respectively. Our results could interpret for the differences in physiological functions after the ingestion of different species of meat.     

Purification and Characterization of Antioxidant Peptides from Leukocyte Extract of Crocodylus siamensis

Antioxidant peptides were isolated from the leukocyte extract of the Siamese crocodile, Crocodylus siamensis. Crocodile leukocyte was extracted by a combination of methods including freeze-thawing, acetic acid extraction and homogenization. The peptides in the leukocyte extract were purified by anion exchange chromatography and reversed phase-high performance liquid chromatography. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay was used to evaluate the antioxidant activity of the elution peaks at each purification step. As a result, there were two purified peptides exhibiting strong antioxidant activity in reducing free radicals on DPPH molecules. The amino acid sequences of these peptides were determined by LC-MS/MS as TDVLGLPAK (912.5 Da) and DPNAALPAGPR (1,148.6 Da), and their IC₅₀ values were 153.4 and 95.7 μM, respectively. The results of this study therefore indicate that leukocyte extract of C. siamensis contains peptides with antioxidant activity which could be used as a novel antioxidant.     

Gemcitabine response in pancreatic adenocarcinoma cells is synergistically enhanced by dithiocarbamate derivatives

Pancreatic adenocarcinoma is a common malignancy that remains refractory to all available therapies, including the gold standard drug gemcitabine (GEM). We investigated the effect of the combination of GEM and each of the ionophore compounds pyrrolidine dithiocarbamate (PDTC) and disulfiram [DSF; 1-(diethylthiocarbamoyldisulfanyl)-N,N-diethylmethanethioamide] on p53^?/? pancreatic adenocarcinoma cell growth. PDTC or DSF synergistically inhibited cell proliferation when used in combination with GEM by inducing apoptotic cell death. This effect was associated with an increased mitochondrial O₂^?? production and was further enhanced by zinc ions. Basal levels of mitochondrial O₂^?? or manganese superoxide dismutase (MnSOD) strictly correlated with the IC₅₀ for GEM or the percentage of synergism. Thus, the most relevant values of the antiproliferative synergism were obtained in GEM-resistant pancreatic adenocarcinoma cell lines. Interestingly, the GEM-sensitive T3M4 cells transfected with MnSOD expression vector showed mitochondrial O₂^?? and IC₅₀ for GEM similar to those of resistant cell lines. In vivo experiments performed on nude mice xenotransplanted with the GEM-resistant PaCa44 cell line showed that only the combined treatment with GEM and DSF/Zn completely inhibited the growth of the tumoral masses. These results and the consideration that DSF is already used in clinics strongly support the GEM and DSF/Zn combination as a new approach to overcoming pancreatic cancer resistance to standard chemotherapy.     

N-Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

N-Glycosylation can modulate enzyme structure and function. In this study, we identified two pepsin-resistant histidine acid phosphatase (HAP) phytases from Yersinia kristensenii (YkAPPA) and Yersinia rohdei (YrAPPA), each having an N-glycosylation motif, and one pepsin-sensitive HAP phytase from Yersinia enterocolitica (YeAPPA) that lacked an N-glycosylation site. Site-directed mutagenesis was employed to construct mutants by altering the N-glycosylation status of each enzyme, and the mutant and wild-type enzymes were expressed in Pichia pastoris for biochemical characterization. Compared with those of the N-glycosylation site deletion mutants and N-deglycosylated enzymes, all N-glycosylated counterparts exhibited enhanced pepsin resistance. Introduction of the N-glycosylation site into YeAPPA as YkAPPA and YrAPPA conferred pepsin resistance, shifted the pH optimum (0.5 and 1.5 pH units downward, respectively) and improved stability at acidic pH (83.2 and 98.8% residual activities at pH 2.0 for 1 h). Replacing the pepsin cleavage sites L197 and L396 in the immediate vicinity of the N-glycosylation motifs of YkAPPA and YrAPPA with V promoted their resistance to pepsin digestion when produced in Escherichia coli but had no effect on the pepsin resistance of N-glycosylated enzymes produced in P. pastoris. Thus, N-glycosylation may improve pepsin resistance by enhancing the stability at acidic pH and reducing pepsin''s accessibility to peptic cleavage sites. This study provides a strategy, namely, the manipulation of N-glycosylation, for improvement of phytase properties for use in animal feed.