二氧化氯杀菌机理研究进展

二氧化氯是一种安全、高效的食品和饮用水消毒剂,可以氧化酪氨酸、色氨酸和半胱氨酸等氨基酸使蛋白质变性。本文综述了二氧化氯与重要生物分子的作用以及二氧化氯在个体水平上对微生物的致死靶点等方面的研究进展,并指出二氧化氯的杀菌机理目前仍然存在较大的争议。     

呼吸抑制在二氧化氯杀菌中的作用

【目的】二氧化氯是一种高效低毒的食品和饮用水消毒剂,其杀菌机理至今还不甚明了。本文的目的是研究二氧化氯对白色念珠菌呼吸功能的抑制作用及其与杀菌效应之间的对应性。【方法】超显微结构观察、流式细胞仪检测、耗氧率的测定以及平板培养。【结果】二氧化氯对白色念珠菌的线粒体的结构和外形没有明显的损伤作用,但是线粒体的跨膜电位会随着二氧化氯剂量的增加而逐渐崩溃;有氧呼吸的抑制程度与菌体的死亡率保持正相关但并不相等,各种作用条件下呼吸的抑制率始终显著低于菌体死亡率;二氧化氯作用后用厌氧培养和好氧培养两种方法所检测的死亡率没有明显区别。【结论】实验结果说明了二氧化氯对真核细胞的细胞器有明显的损伤作用并与死亡率呈正相关,但呼吸抑制可能不是细菌死亡的首要原因。     

Studies on the Reaction of Dipeptides with Glyoxal

The reaction of various dipeptides with glyoxal at 100°C, pH 5.0 was studied. Carbon dioxide, ammonia, amino acids and aldehydes were detected from the reaction solutions. Besides, a series of new pyrazinones—2-(3′-alkyl-2′-oxo-pyrazin-1′-yl)alkyl acid—were isolated, and their chemical structures were confirmed by UV, IR, MS and NMR spectra. These pyrazinones seem to play a role in the browning of the reaction.

In the reaction with glyoxal, acetaldehyde and glucose, reactivity of peptides was proved to be much higher than that of amino acids.

The reaction mechanism of dipeptides with glyoxal was also proposed.     

Choice of peptide and peptide length for the generation of antibodies reactive with the intact protein

N-terminal peptides of bovine ribonuclease (RNase) of 20, 13 and 7 amino acid residues were isolated by reversed-phase high-performance liquid chromatography (HPLC). Antibodies were raised in mice against these peptides coupled to bovine serum albumin (BSA). It was shown that antibodies against the peptides reacted with the intact protein and that the immune response decreased with decreasing size of peptide. In order to obtain a satisfactory reaction with the intact protein, the peptide immunogen should be longer than 7 amino acids.     

The use of monoclonal antibodies to study the structure and function of cytochrome f.

Monoclonal antibodies (MAbs) were prepared against native cytochrome f (cyt f) isolated from turnip leaves. The two MAbs obtained, designated MAb-JB2 and MAb-ED4, were Western blot positive to purified turnip cytochrome f and also reacted with inside-out (ISO) but not right-side-out (RSO) spinach thylakoid membranes. MAb-ED4 reacted with a covalent adduct formed by crosslinking cyt f and plastocyanin (PC), whereas MAb-JB2 did not. In contrast, MAb-JB2 reacted with the isolated cyt b6/f complex but MAb-ED4 did not. These results indicate that MAb-JB2 binds to cyt f at or near the PC binding site on f, whereas MAb-ED4 binds to a portion of cyt f which is not exposed in the cyt b6/f complex. The location of the epitopes in the primary sequence of cyt f was determined by trypsin hydrolysis, HPLC separation of tryptic peptides, and ELISA identification of the purified peptides. The molecular weights of the purified peptides, determined by gel exclusion chromatography, were found to be 5040 and 3130 Da for MAb-JB2 and MAb-ED4, respectively. Amino acid sequencing showed that the first eight amino acids of the MAb-ED4 positive peptide were L-D-Q-P-L-T-S-N. These results suggest that the 3130-Da peptide has 28 amino acids extending from Leu 223 to Arg 250. This peptide is located on the N-terminal (lumen) side of the postulated membrane-spanning sequence. The first eight amino acids of the MAb-JB2-positive peptide were N-I-L-V-I-G-P-V. This sequence and the peptide molecular weight indicate that the epitope for MAb-JB2 is located within a 44-amino acid peptide extending from Asn 111 to Arg 154.     

The structure of l-CB3, a cyanogen bromide fragment from the central portion of the l chain of rat collagen. The tryptic peptides from skin and dentin collagens

The mixture of peptides released by tryptic hydrolysis of the collagen CNBr peptide, αl-CB3, has been resolved by ion-exchange chromatography. The resultant eleven tryptic peptides ranged in size from 3 to 46 amino acids and accounted for all the amino acids of the parent CNBr peptide. Two of the lysines in αl-CB3 from rat dentin collagen were shown to be hydroxylated to a substantial degree by isolation of the appropriate hydroxylysine-containing tryptic peptides. An analysis of the tryptic peptides indicated that αl-CB3 from dentin collagen is identical in structure to that from skin collagen, if hydroxylysine and hydroxyproline are considered equivalent to lysine and proline, respectively.     

Neuropsychological and biochemical investigations in heterozygotes for phenylketonuria during ingestion of high dose aspartame (a sweetener containing phenylalanine)

Aspartame, a high intensity sweetener, is used extensively worldwide in over 5,000 products. Upon ingestion, aspartame is completely metabolized to two amino acids and methanol (approximately 50% phenylalanine, 40% aspartic acid, and 10% methanol). The effects of aspartame on cognitive function, electroencephalograms (EEGs) and biochemical parameters were evaluated in 48 adult (21 men, 27 women) heterozygotes for phenylketonuria (PKUH). PKUH subjects whose carrier status had been proven by DNA analysis ingested aspartame (either 15 or 45 mg/kg/day) and placebo for 12 weeks on each treatment using a randomized, doubleblind, placebo-controlled, crossover study. A computerized battery of neuropsychological tests was administered at baseline weeks -2 and -1, and during treatment at weeks 6, 12, 18, and 24. Samples for plasma amino acids and urinary organic acids were also collected during these visits. EEGs were evaluated by conventional and spectral analysis at baseline week -1 and treatment weeks 12 and 24. The results of the neuropsychological tests demonstrated that aspartame had no effect on cognitive function. Plasma phenylalanine significantly increased, within the normal range for PKUH, at 1 and 3 h following the morning dose of aspartame in the group receiving the 45 mg/kg per day dose only. There were no significant differences in the conventional or spectral EEG analyses, urinary organic acid concentrations, and adverse experiences when aspartame was compared with placebo. This study reaffirms the safety of aspartame in PKUH and refutes the speculation that aspartame affects cognitive performance, EEGs, and urinary organic acids.     

Rapid robust separation of hydroxyproline and proline.

The presence of hydroxyproline, and the determination of the ratio of the secondary amino acids proline to hydroxyproline, in amino acid hydrolysates specifically identifies collagen and collagen peptides. o-Phthalaldehyde, and then 9-fluorenylmethyl chloroformate, were used to carry out sequential prederivatization of amino acid hydrolysates in an in-line high-performance liquid chromatography sample loop. After derivatization, reversed-phase high-performance liquid chromatography with a C-18 ODS Hypersil cartridge column was used to resolve the hydroxyproline and proline from all primary amino acids, with resolution and detection of hydroxyproline and proline within 2.0 and 2.8 min, respectively, at concentrations in the range of picomoles per microliter of derivatized amino acid. The assay has a turnaround time of 10.75 min.     

Hydrolysis technology of biomass waste to produce amino acids in sub-critical water

Hydrolysis of biomass waste (such as fish waste, chicken waste, hair and feather) to produce amino acids was studied in sub-critical water, with reaction temperatures from 180 to 320 degrees C and reaction pressures from 3 to 30 MPa. The product of amino acid was determined by Amino Acid Analyzer (BioLC), and 18 kinds of amino acid were obtained. The results show that the controlling of reaction atmosphere, pressure, temperature and time of hydrolysis is very important to obtain high yield of amino acid; most of amino acids reached maximum yield at reaction temperature range of 200-290 degrees C and reaction time range of 5-20 min. There are obvious changes of amino acids yield at reaction pressures of 6-16 MPa and reaction temperature around 260 degrees C, owing to the homogeneousness of the first two phases of water in the formation of vapor and liquid. There are different yields of the same amino acid in different reaction atmospheres (e.g. air, carbon dioxide and nitrogen).     

Temporin/VesCP (T/V)-like antibiotic peptides, derived from frogs and wasps, induce migration of human monocytes and neutrophils

Summary  Several naturally occurring antimicrobial peptides, from mammals and insects, have previously been shown to be chemotactic for human inflammatory cells. Based on this evidence, ten synthetic analogs of naturally occurring antibiotic peptides from the skin secretions of three species of Ranid frogs and the venom of one species of Vespid wasp (i.e., T/V-like peptides) were tested for their abilities to induce migration of human neutrophils and monocytes. These included temporin A (TA fromRana temporaria), temporin 1P (T1P fromR. pipens), ranateurin 6 (Rana-6 fromR. catesbeiana)], three TA analogs [all D-amino acids (D-TA), reversed sequence (Rev-TA), and Pro3→Gly (G3-TA)], two frog skin-related T/V-like peptide consensus sequences (I4S10-Con and I4G10-Con), VesCP-M (VCP-M fromVespa mandarinia), and a hybrid peptide composed of portions of the insect antibiotic peptide, cecropin A (CA), and TA (CATA). TA, T1P, Rana-6, VCP-M, G3-TA, I4S10-Con, I4G10-Con, and CATA all induced cell migration at micromolar concentrations. D-TA and Rev-TA did not induce cell migration, suggesting that this process involves a chiral interaction, such as receptor binding, and also depends on the order of amino acids within TA. The results demonstrate, for the first time, that certain T/V-like antibiotic peptides are capable of inducing chemotaxis of human phagocytes and suggest that these peptides are multifunctional molecules with antimicrobial, hemolytic, and chemotactic capabilities.     

Temporin/VesCP (T/V)-like antibiotic peptides,derived from frogs and wasps,induce migration of human monocytes and neutrophils

Summary Several naturally occurring antimicrobial peptides, from mammals and insects, have previously been shown to be chemotactic for human inflammatory cells. Based on this evidence, ten synthetic analogs of naturally occurring antibiotic peptides from the skin secretions of three species of Ranid frogs and the venom of one species of Vespid wasp (i.e., T/V-like peptides) were tested for their abilities to induce migration of human neutrophils and monocytes. These included temporin A (TA fromRana temporaria), temporin 1P (T1P fromR. pipens), ranateurin 6 (Rana-6 fromR. catesbeiana)], three TA analogs [all D-amino acids (D-TA), reversed sequence (Rev-TA), and Pro3→Gly (G3-TA)], two frog skin-related T/V-like peptide consensus sequences (I4S10-Con and I4G10-Con), VesCP-M (VCP-M fromVespa mandarinia), and a hybrid peptide composed of portions of the insect antibiotic peptide, cecropin A (CA), and TA (CATA). TA, T1P, Rana-6, VCP-M, G3-TA, I4S10-Con, I4G10-Con, and CATA all induced cell migration at micromolar concentrations. D-TA and Rev-TA did not induce cell migration, suggesting that this process involves a chiral interaction, such as receptor binding, and also depends on the order of amino acids within TA. The results demonstrate, for the first time, that certain T/V-like antibiotic peptides are capable of inducing chemotaxis of human phagocytes and suggest that these peptides are multifunctional molecules with antimicrobial, hemolytic, and chemotactic capabilities.     

Reactions between amino acid compounds and phenols during oxidation

Summary About 30 per cent of organic soil nitrogen can be hydrolized with HCl to amino acids; about 30 per cent is nonhydrolizable. In contrast to this high content of amino acid nitrogen is the small availability of the nitrogen to micro-organisms. In light of the theory proposing a reaction between the -amino group of amino acids or peptides and quinones formed during oxidation of lignin degradation products or other phenolic compound, different types of phenols were oxidized by phenolases in presence of amino acid compounds.It could be shown that the reaction of binding of nitrogen started at pH values higher than 6.5, and that only such phenols reacted which had no methoxylated hydroxyl groups. The reaction of some phenols during oxidation in presence of amino acids was accompanied by deamination and decarboxylation of the latter.The reaction products of phenols with amino acids were stable against hydrolysis. Using peptides it was found that all amino acids, except the N-terminal which is bound to oxidized phenols, could be hydrolyzed normally.With serum albumin it could be shown that there is a reaction with the amino group of the N-terminal amino acid and also with the -amino group of lysine residues with phenols during oxidation. The reacted protein seemed to be degraded normally with a protease ofBacillus subtilis.Guest Scientist as Fulbright Research Scholar from the Agronomy Department of the Iowa State University, Ames, Iowa, U.S.A.     

Studies of the chemical composition of a healing skin wound in rats, and of the concentrations of some constituents of tissues distant from the healing wound

1. A skin lesion was made in rats by dorsal incision and the insertion of a polythene tube. 2. Over a period of 25 days after wounding, assays were performed for ascorbic acid, DNA, hydroxyproline, methionine, tryptophan, tyrosine and free amino acids in the lesion tissue. 3. The neutral-salt-soluble proteins of the lesion tissue were fractionated on DEAE-Sephadex, with the separation of fibrinogen and γ-globulin from a serum protein fraction. 4. Over a period of 20 days after wounding, in wounded rats and in controls, assays were conducted for: ascorbic acid in lens and liver, hydroxyproline, soluble protein, methionine and water in muscle and tendon, and free amino acids in muscle. 5. Relative to controls there was a decrease in lens and liver ascorbic acid, a rise in tendon hydroxyproline, a rise in muscle free amino acids, a fall in muscle protein and a rise in tendon and muscle water.     

Reactivity of anti-human milk fat globule antibodies with synthetic peptides

The nucleotide sequence of partial cDNA clones coding for the core protein of a human polymorphic epithelial mucin has recently been obtained, this mucin consists of a highly conserved 60 bp tandem repeat and the amino acids commonly found are PDTRPAPGSTAPPAHGVTSA. We synthesized three peptides, 1) P1.24 containing the 20 amino acids and four amino acids (PDTR) of the adjoining repeat; 2) P1.15 consisting of the first fifteen (PDTRPAPGSTAPPAH) and P1.09 the second nine amino acids (GVTSAPDTR) of peptide P1.24. The reactivities of the synthetic peptides with mAb known to react with breast cancer (BC1, BC2, BC3, HMFG-1, 3E1.2, and RCC-1) were studied. The synthetic peptide, P1.24, corresponding to the antigenic sequence predicted from the tandem repeat reacted with antibodies BC1, BC2, and BC3 (known to react with human milk mucin and mucin expressed in breast cancer) and the antibody HMFG-1 which was used to select the cDNA clones. In addition, the epitopes recognized by BC1, BC2, and BC3 appear to be in the same region of the molecule represented by their reactions with the nine amino acids in peptide P1.09 (GVTSAPDTR). By contrast, other antibodies such as 3E1.2 which reacts only weakly with components of human milk, and RCC-1 that detects a low Mr component (95 kDa) in breast cancer, had no specific reaction with the synthetic peptides, indicating that their epitopes are distinct from those of BC1, BC2, BC3, and HMFG-1. Inasmuch as the antibodies HMFG-1, BC1, BC2, and BC3 react with the fully processed milk mucin, it is likely that some of the peptide is exposed, even in the fully glycosylated molecule. Identification of the different epitopes could lead to the development of "second generation" mAb with enhanced specificity for breast carcinoma using the appropriate synthetic peptides as immunogens.     

The composition of the cyst wall of the beet cyst-nematode Heterodera schachtii

1. Cyst walls of the beet cyst-nematode (Heterodera schachtii Schmidt) were obtained by sieving a suspension of crushed cysts; about 15mg of dried cyst walls was obtained from 1000 cysts. 2. The cyst walls contained 68% protein calculated from nitrogen content. Glutamic acid, glycine, proline and hydroxyproline made up about 54% by weight of the amino acids obtained on acid hydrolysis. 3. Minor constituents of the cyst wall were hexosamine (3.3%), lipid (6%), carbohydrate (2%) and phenols (2%). The hexosamine was identified as galactosamine. 4. The cyst walls contained inorganic material (ash 17%), most of which was extractable with EDTA, but not with water. Major inorganic components were calcium and phosphorus (1.7% and 1.5% respectively, by weight). Carbon dioxide (about 1% by weight) was liberated from the cyst walls on acidification. 5. The cyst walls of H. schachtii and the potato cyst-nematode (Heterodera rostochiensis) contained different amounts of the same amino acids. They also differed in their inorganic content and in the nature of the hexosamine present.     

Amphipathic control of the 3(10)-/alpha-helix equilibrium in synthetic peptides.

A series of short, amphipathic peptides incorporating 80% C(alpha),C(alpha)-disubstituted glycines has been prepared to investigate amphipathicity as a helix-stabilizing effect. The peptides were designed to adopt 3(10)- or alpha-helices based on amphipathic design of the primary sequence. Characterization by circular dichroism spectroscopy in various media (1 : 1 acetonitrile/water; 9 : 1 acetonitrile/water; 9 : 1 acetonitrile/TFE; 25 mM SDS micelles in water) indicates that the peptides selectively adopt their designed conformation in micellar environments. We speculate that steric effects from ith and ith + 3 residues interactions may destabilize the 3(10)-helix in peptides containing amino acids with large side-chains, as with 1-aminocyclohexane-1-carboxylic acid (Ac(6)c). This problem may be overcome by alternating large and small amino acids in the ith and ith + 3 residues, which are staggered in the 3(10)-helix.     

Identification of the amino acids of human serum albumin involved in the reaction with the naproxen acyl coenzyme A thioester using liquid chromatography combined with fluorescence and mass spectrometric detection

Xenobiotic carboxylic acids, that via their metabolites covalently modify proteins, have been associated with serious side effects in man. Such reactive metabolites may be acyl glucuronides or alternatively, the corresponding acyl-CoA thioesters. In this study, the reaction of a model xenobiotic acyl-CoA, the naproxen-CoA, with human serum albumin (HSA), was characterized by high-performance liquid chromatography employing fluorescence and mass spectrometric detection. One mM naproxen-CoA was incubated for 6h with HSA (0.45 mM) at 37 degrees C in a 0.1M phosphate buffer (pH 7.4). The tryptic digest of the reduced and alkylated protein was analyzed in order to identify the amino acids in the sequence that were covalently modified with naproxen. Fluorescent peptides, that represented naproxen-modified peptides, were characterized using HPLC-MS-MS and HPLC-MS in zoom scan mode, which provided information on the structure and the charge of the modified peptides. The naproxen-CoA reacted predominantly with lysine 199, lysine 541, and lysine 351, which was in agreement with the binding pattern that has previously been reported for the reactive acyl glucuronides and their reaction with HSA.     

Identification of a major hepatitis B core antigen (HBcAg) determinant by using synthetic peptides and monoclonal antibodies

To map the location of hepatitis B core and e Ag (HBcAg and HBeAg) on the hepatitis B virus core particle, we produced and analyzed four synthetic peptides which correspond to the most hydrophilic regions of the core P22 protein. Each peptide was tested in an ELISA for the ability to inhibit the binding between rHBcAg or rHBeAg and either polyclonal or monoclonal anti-HBc or anti-HBe antibodies. The former comprised 20 antisera positive for anti-HBc (anti-HBs and anti-HBe negative) and five antisera positive for anti-HBe and anti-HBc; the latter included three anti-HBc mAb developed in independent laboratories: G6F5, C51B10, and F8, as well as two anti-HBe mAb, E2 and E6. These experiments revealed the presence of a major HBcAg epitope expressed on C3, a peptide which covers amino acids 107-118 and reacted with all polyclonal and monoclonal antibodies tested. Another peptide, C2, sequence 73-85, reacted with 26% of human antisera but none of the anti-HBc mAb. None of the peptides seemed to express HBeAg activity because they do not cause any significant inhibition of the HBeAg/anti-HBe reaction. These data indicate the expression of an immunodominant HBcAg determinant on a linear dodecapeptide and argue against a strict conformation dependency of this Ag.     

Pitfalls in using sodium hypochlorite as a seed disinfectant in C incorporation studies

Seeds sterilized with sodium hypochlorite (NaOCl) retained sufficient amounts to interfere with studies of amino acid metabolism of the sterilized seeds during germination. Repeated washing in water did not remove NaOCl completely. However, soaking the seeds for 10 min in 0.01 n HCl removed NaOCl completely, without reducing germinability.Residual NaOCl reacted with the amino acids and reduced their concentrations in the incubation media. This reaction resulted in high production of CO(2) and low uptake of amino acids by the seeds. Decarboxylation of the amino acids occurred in the incubation medium outside the seed, was independent of the presence of seeds in the reaction, and therefore was not related to amino acid metabolism by the seeds. Effects of NaOCl on uptake, incorporation, and CO(2) production from indoleacetic acid were similar to those of the amino acids studied.     

Separation of 17 DL-amino acids and chiral sequential analysis of peptides by reversed-phase liquid chromatography after labeling with R(-)-4- (3-isothiocyanatopyrrolidin-1-yl)-7-(N, N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole

Seventeen DL-amino acids labeled with a fluorescent chiral labeling reagent, R(-)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N, N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole (R(-)-DBD-PyNCS), were separated by reversed-phase chromatography and detected fluorometrically at 550 nm (excitation at 460 nm). The reagent reacted with amino functional group in dl-amino acids under basic medium. The thiocarbamoyl derivatives were converted to thiohydantoin via thiazolinone in trifluoroacetic acid (TFA) solution. The epimerization ratios during the reaction of the cyclization were less than 37% in all dl-amino acids tested. The resulting thiohydantoin derivatives of individual dl-amino acids were completely separated with isocratic elutions using acidic mobile phase involving 0.1% TFA. The separations of the thiohydantoins yielded from acidic, basic, neutral, hydroxyl, and aromatic amino acids were good enough for the identification of dl-amino acid. The method using the reagent was adopted to identification of dl-amino acid sequences in eight peptides. The separation and identification of the thiohydantoin derivatives liberated from the peptides labeled were performed by the isocratic elutions. The applicability of the proposed procedure to sequential analysis of peptide was demonstrated with [D-Ala(2)]-leucine enkephalin, [D-Ala(2)]-deltorphin II, d-Phe-Met-Arg-Phe-amide, and Phe-D-Met-Arg-Phe-amide. D-Ala, D-Phe, and D-Met in the peptides were positively identified with the proposed procedures. [L-Ala(2)]-leucine enkephalin, beta-lipotropin, Asp-Ser-Asp-Pro-Arg, and Pro-Asp-Val-Asp-His-Val-Phe-Leu-Arg-Phe-amide were also analyzed as the references without D-amino acid.