Nucleotide sequence of the streptococcal pyrogenic exotoxin type B gene and relationship between the toxin and the streptococcal proteinase precursor.

The streptococcal pyrogenic exotoxin (SPE) type B-encoding structural gene, speB, was subcloned from a 4.5-kilobase streptococcal DNA insert onto a 2.4-kilobase insert, which was then sequenced. Studies indicated that a 1,194-base-pair open reading frame encoded a 398-amino-acid protein. Removal of the putative signal peptide resulted in a mature protein with 371 residues (molecular weight, 40,314), which was subsequently proteolyzed to yield a 253-residue breakdown product (molecular weight, 27,588). This processing was confirmed by amino-terminal sequencing of both the 40,314-molecular-weight protein and the breakdown product. Monte Carlo analysis indicated that SPE B was relatively dissimilar to other members of the pyrogenic toxin family that also includes SPEs A and C, toxic shock syndrome toxin 1, and the staphylococcal enterotoxins. Comparison with the published amino acid sequence of streptococcal proteinase precursor as well as DNA hybridization experiments indicated that SPE B is a variant of this protein even though the particular gene sequenced did not encode a proteolytically active molecule.     

Activation of murine T cells by streptococcal pyrogenic exotoxin type A. Requirement for MHC class II molecules on accessory cells and identification of V beta elements in T cell receptor of toxin-reactive T cells

We investigated the mechanisms of murine T cell activation by streptococcal pyrogenic exotoxin type A (SPE A), focusing on the role of MHC class II molecules on accessory cells (AC) and V beta usage in alpha beta TCR of SPE A-reactive T cells in comparison with staphylococcal enterotoxin B-reactive T cells. L cells transfected with I-Ab genes functioned as effective AC for SPE A-induced responses by C57BL/6 T cells, proliferation, and IL-2 production, but control L cells were not effective AC. Anti-I-Ab mAb inhibited the SPE A-induced responses. Staphylococcal enterotoxin B-induced C57BL/6 T cell blasts were composed of cells bearing V beta 3, members of the V beta 8 family, and V beta 11. Most of the SPE A-induced T cell blasts (about 80%) bore V beta 8.2. mAb reactive to V beta 8.2 markedly inhibited SPE A-induced T cell responses. Apparently, SPE A activates mainly T cells bearing V beta 8.2 in physical association with MHC class II molecules expressed on AC. We also discuss the pathogenic activities of SPE A in relation to toxic shock syndrome.     

Crystal structure of a papain-fold protein without the catalytic residue: a novel member in the cysteine proteinase family

A 31kDa cysteine protease, SPE31, was isolated from the seeds of a legume plant, Pachyrizhus erosus. The protein was purified, crystallized and the 3D structure solved using molecular replacement. The cDNA was obtained by RT PCR followed by amplification using mRNA isolated from the seeds of the legume plant as a template. Analysis of the cDNA sequence and the 3D structure indicated the protein to belong to the papain family. Detailed analysis of the structure revealed an unusual replacement of the conserved catalytic Cys with Gly. Replacement of another conserved residue Ala/Gly by a Phe sterically blocks the access of the substrate to the active site. A polyethyleneglycol molecule and a natural peptide fragment were bound to the surface of the active site. Asn159 was found to be glycosylated. The SPE31 cDNA sequence shares several features with P34, a protein found in soybeans, that is implicated in plant defense mechanisms as an elicitor receptor binding to syringolide. P34 has also been shown to interact with vegetative storage proteins and NADH-dependent hydroxypyruvate reductase. These roles suggest that SPE31 and P34 form a unique subfamily within the papain family. The crystal structure of SPE31 complexed with a natural peptide ligand reveals a unique active site architecture. In addition, the clear evidence of glycosylated Asn159 provides useful information towards understanding the functional mechanism of SPE31/P34.     

Cloning and characterization of the gene, speC, for pyrogenic exotoxin type C from Streptococcus pyogenes

Summary The structural gene of streptococcal pyrogenic exotoxin type C (SPE C) was cloned from the chromosome of Streptococcus pyogenes strain T18P into Escherichia coli using pBR328 as the vector plasmid. Subcloning enabled the localization of the gene (speC) to a 1.7 kb fragment. Partially purified E. coli-derived SPE C and purified streptococcal-derived SPE C, were shown to have the same molecular weight (23 800) and biological activities. A DNA probe, prepared from cloned speC, cross-hybridized with the structural genes of SPE A and SPE B indicating relatedness at the nucleotide level. The speC-derived probe also hybridized to a fragment of CS112 bacteriophage DNA containing the phage attachment site.     

Systematic and comprehensive strategy for reducing matrix effects in LC/MS/MS analyses

A systematic, comprehensive strategy that optimizes sample preparation and chromatography to minimize matrix effects in bioanalytical LC/MS/MS assays was developed. Comparisons were made among several sample preparation methods, including protein precipitation (PPT), liquid-liquid extraction (LLE), pure cation exchange solid-phase extraction (SPE), reversed-phase SPE and mixed-mode SPE. The influence of mobile phase pH and gradient duration on the selectivity and sensitivity for both matrix components and basic analytes was investigated. Matrix effects and overall sensitivity and resolution between UPLC technology and HPLC were compared. The amount of specific matrix components, or class of matrix components, was measured in the sample preparation extracts by LC/MS/MS with electrospray ionization (ESI) using both precursor ion scanning mode and multiple reaction monitoring (MRM). PPT is the least effective sample preparation technique, often resulting in significant matrix effects due to the presence of many residual matrix components. Reversed-phase and pure cation exchange SPE methods resulted in cleaner extracts and reduced matrix effects compared to PPT. The cleanest extracts, however, were produced with polymeric mixed-mode SPE (both reversed-phase and ion exchange retention mechanisms). These mixed-mode sorbents dramatically reduced the levels of residual matrix components from biological samples, leading to significant reduction in matrix effects. LLE also provided clean final extracts. However, analyte recovery, particularly for polar analytes, was very low. Mobile phase pH was manipulated to alter the retention of basic compounds relative to phospholipids, whose retention tends to be relatively independent of pH. In addition to the expected resolution, speed and sensitivity benefits of UPLC technology, a paired t-test demonstrated a statistically significant improvement with respect to matrix effects when this technology was chosen over traditional HPLC. The combination of polymeric mixed-mode SPE, the appropriate mobile phase pH and UPLC technology provides significant advantages for reducing matrix effects resulting from plasma matrix components and in improving the ruggedness and sensitivity of bioanalytical methods.     

Inclusion complex-based solid-phase extraction of steroidal compounds with entrapped beta-cyclodextrin polymer

Although the hydrophobic interaction-based solid-phase extraction (SPE) has been widely used, the extraction yields of steroids including androgens, estrogens, and corticoids were slightly different along with the physical and chemical properties of each molecule. A new SPE technique based on the formation of an inclusion complex with beta-cyclodextrin (betaCD) has been achieved for comprehensive sample purification in mass spectrometric analysis of 45 endogenous or synthetic androgens, 11 endogenous estrogens, and 21 corticoids. A copolymer of betaCD with epichlorohydrin was prepared by a cross-linking reaction followed by entrapment with 0.3M CaCl(2) to yield an improved SPE sorbent and the hydrolyzed urine samples were applied for purification. Steroidal compounds tested on the entrapped betaCD polymer were extracted with tetrahydrofuran and the overall recoveries ranged from 82% to 112% for 77 steroids in urine. Especially, the hydroxylated estrogens showed an excellent binding capacity (96-116% recovery) to betaCD through hydrogen bonding between their phenolic hydroxyl and exterior hydroxyl groups. A comparison between SPE methods with betaCD and Oasis HLB as a conventional cartridge showed that the extraction efficiency of polar steroids was significantly increased in the betaCD experiment, which has no connection with different polarity of steroid molecules. Due to its multi-functional mechanism derived from molecular inclusion and chemical interactions, this new SPE sorbent resulted in better selectivity and extraction efficiency than that obtained using the conventionally used hydrophobicity-based SPE method.     

Salvia plebeia extract inhibits the inflammatory response in human rheumatoid synovial fibroblasts and a murine model of arthritis

Salvia plebeia R. Br. has been used to treat a variety of inflammatory diseases and as an antioxidant in many countries, including Korea and China. In this study, we investigated the effects of S. plebeia extract (SPE) on inflammatory arthritis and the underlying mechanisms of action. We used a collagen-induced arthritis (CIA) mouse model. TNF-α-stimulated rheumatoid arthritis (RA) synovial fibroblasts were used to elucidate the underlying mechanisms of action. Oral administration of SPE improved the clinical arthritis score, footpad thickness, and histologic changes, as well as serum IgG1 and IgG2a levels. SPE administration inhibited Th1/Th2/Th17 phenotype CD4⁺ T lymphocyte expansion in inguinal lymph node and expression of inflammatory mediators such as cytokines, MMP-1, and MMP-3 in the ankle joint tissue. SPE significantly suppressed the expression of cytokines and MMP-1 by down-regulating NF-κB, Akt, and mitogen-activated protein kinases in RA synovial fibroblasts. Taken together, these results indicate that SPE is therapeutically efficacious against chronic inflammatory arthritis, suggesting that SPE is a candidate for treating RA.     

Macrophage hyperreactivity to endotoxin induced by streptococcal pyrogenic exotoxin in rabbits

Pretreatment of rabbits with streptococcal pyrogenic exotoxin (SPE) resulted in an enhancement of their febrile response to subsequent endotoxin challenge. This suggested that SPE may enhance the macrophage capacity to respond to endotoxin in vivo to produce an endogenous pyrogen. It was also demonstrated that peritoneal macrophages derived from SPE-treated rabbits exhibited hyperreactivity to endotoxin in vitro as assessed by endotoxin-induced increase in glucose consumption. These data indicate that SPE has the ability to enhance macrophage reactivity to endotoxin.     

In vitro generation of K562 killers in human T-lymphocyte subsets

Group A streptococcal pyrogenic exotoxin (SPE) is a potent modulator of the immune system when used experimentally in mice. Typically, a late burst of plaque-forming cells (PFC) follows an early suppression of the antibody response in appropriately immunized and SPE-treated mice or their spleen cells in vitro. This altered response to antigen caused by SPE is termed a deregulated antibody response. The site of action of SPE was studied by use of cellular reconstruction and complementation experiments using the separated subpopulations of immunocytes which are required for full expression of mouse spleen PFC responses to sheep erythrocytes or to trinitrophenylated (TNP) rabbit erythrocytes in vitro. The SPE site was thus localized to the T-cell subpopulation. Recently SPE has been purified to a very high degree, making it possible to ascertain that SPE alone generates the deregulation of the immune system as described before and to limit the role of nondefined components of cruder preparations of SPE. A purified horse anti-scarlet fever antitoxin which recognizes highly purified SPE as being homogeneous also recognized a single component of crude SPE by agar-gel analysis. A rabbit anti-SPE immunoglobulin raised against crude SPE and absorbed with killed, strain NY5, Group A streptococci recognized the pure SPE and a major component of the homologous crude SPE similarly. Both of these antisera neutralized the capacity of SPE to deregulate the in vitro PFC response to TNP almost completely. A third antiserum raised in rabbits against a NY5 Group A streptococcal whole cell vaccine recognized a different component of crude SPE and totally failed to recognize pure SPE. This antiserum also recognized a purified Group A streptococcal peptidoglycan as being related to components contained in the crude SPE preparation. This antiserum, however, totally failed to neutralize the capacity of SPE to deregulate the PFC response to TNP. These results show that SPE-A is the active component of cruder preparations of SPE which deregulates PFC responses.     

Simultaneous determination of nitrendipine and hydrochlorothiazide in spontaneously hypertensive rat plasma using HPLC with on-line solid-phase extraction

A HPLC method with on-line solid phase extraction (SPE) and DAD detection was developed for the simultaneous determination of nitrendipine and hydrochlorothiazide in spontaneously hypertensive rat (SHR) plasma. Plasma samples (100 μL) were injected directly onto a CAPCELL MF C(8) SPE column. High-abundance proteins and most matrixes in plasma were removed by on-line SPE technology, while nitrendipine and hydrochlorothiazide trapped on the SPE column were effectively separated on a C(18) analytical column. The column temperature was maintained at 20°C. The optimal detection wavelength was 237 nm for NTDP and 271 nm for HCTZ. The total analytical run time was 34 min. The proposed method was linear over the range 5-500 ng mL(-1) for nitrendipine and 10-1000 ng mL(-1) for hydrochlorothiazide. The lower limit of detection (LLOD) was 0.5 and 0.6 ng mL(-1) for nitrendipine and hydrochlorothiazide, respectively. The sensitivity and precision of the method were within acceptable limits during validation period. The method was successfully used to investigate the pharmacokinetic characteristics of nitrendipine and hydrochlorothiazide in spontaneously hypertensive rats.     

Serum proteins in the leopard seal, Hydrurga leptonyx, in Prydz Bay, Eastern Antarctica and the coast of NSW, Australia

Blood protein analysis including total serum protein and albumin by chemical methods, fibrinogen estimation and serum protein electrophoresis (SPE) was performed on the leopard seal, Hydrurga leptonyx. The most commonly observed SPE pattern was eight fractions designated albumin, alpha(1a), alpha(1b), alpha(2a), alpha(2b), beta(1), beta(2) and gamma-globulin. Significantly higher total serum protein and albumin concentrations, as determined by chemical methods, and significantly higher alpha(2)-globulin concentrations, determined by SPE, were seen in free-ranging male seals compared to females, whilst significantly higher beta-globulin concentrations were seen in female seals. Season of sampling influenced fibrinogen and beta(2)-globulin concentrations, whereas there were no significant differences in any protein concentrations with moult status. Qualitative comparison of SPE traces of leopard seals in Antarctica with "sick" individuals in NSW, Australia revealed obvious differences, as did quantitative comparison of protein concentrations where differences in alpha(1), alpha(2), beta(1), beta(2), and gamma-globulin concentrations were seen. These findings suggest that SPE is a useful tool for investigating serum proteins in the leopard seal, with applications for the investigation of "sick" individuals and the assessment of variation in homeostasis. This technique could also be used to identify the presence of environmental stressors, subclinical disease and physiological variation within specific seal populations.     

Streptococcal pyrogenic exotoxin type A (scarlet fever toxin) is related to Staphylococcus aureus enterotoxin B

Summary The nucleotide sequence of the gene encoding group A streptococcal pyrogenic exotoxin type A (SPE A) was determined by the dideoxy chain termination method. The first 30 residues of the translation product represented a hydrophobic signal peptide. The mature protein was 220 amino acids in length and had a molecular weight of 25,805. It has significant protein sequence homology with Staphylococcus aureus enterotoxin B but not with other proteins in the Dayhoff library.     

cDNA cloning,functional expression and antifungal activities of a dimeric plant defensin SPE10 from <Emphasis Type="Italic">Pachyrrhizus erosus</Emphasis> seeds

SPE10 is an antifungal protein isolated from the seeds of Pachyrrhizus erosus. cDNA encoding a 47 amino acid peptide was cloned by RT-PCR and the gene sequence proved SPE10 to be a new member of plant defensin family. The synthetic cDNA with codons preferred in yeast was cloned into the pPIC9 plasmid directly in-frame with the secretion signal -mating factor, and highly expressed in methylotrophic Pichia pastoris. Activity assays showed the recombinant SPE10 inhibited specifically the growth of several pathogenic fungi as native SPE10. Circular dichroism and fluorescence spectroscopy analysis indicated that the native and recombinant protein should have same folding, though there are eight cystein residues in the sequence. Several evidence suggested SPE10 should be the first dimeric plant defensin reported so far.Nucleotide sequence data reported in this paper are available in the DDBJ/EMBL/GenBank database under accession number AY679170     

早发型重度子痫前期患者血清ADMA、INH-A与胎儿生长受限的关系研究

摘要 目的：探讨早发型重度子痫前期（SPE）患者血清非对称性二甲基精氨酸（ADMA）、抑制素A（INH-A）与胎儿生长受限（FGR）的关系。方法：选取2021年1月～2023年1月于海南医学院第一附属医院分娩的110例早发型SPE患者为观察组,另选取同期健康孕妇100例为对照组,根据是否合并FGR将早发型SPE患者分为FGR组46例和非FGR组64例。比较观察组与对照组血清ADMA、INH-A水平。采用多因素Logistic回归分析早发型SPE患者合并FGR的影响因素,受试者工作特征曲线（ROC）分析血清ADMA、INH-A水平对早发型SPE患者合并FGR的预测效能。结果：与对照组比较,观察组血清ADMA、INH-A水平升高（P＜0.05）。与非FGR组比较,FGR组ADMA、INH-A、收缩压、舒张压、血肌酐、24 h尿蛋白定量、脐动脉血流收缩期末流速/舒张期末流速比值（S/D）水平升高,而孕前体质指数（BMI）降低（P＜0.05）。多因素Logistic回归分析显示,24 h尿蛋白定量、动脉血流S/D、ADMA和INH-A升高为早发型SPE患者合并FGR的独立危险因素（P＜0.05）。ROC分析显示,血清ADMA、INH-A水平联合预测早发型SPE患者合并FGR的曲线下面积大于单独预测。结论：早发型SPE患者血清ADMA、INH-A水平异常升高,且参与FGR的发生、发展,此外,血清ADMA、INH-A水平联合对早发型SPE患者合并FGR的预测效能较高。     

Isolation of a new superantigen with potent mitogenic activity to murine T cells from Streptococcus pyogenes

Abstract A mitogenic substance on murine lymphocytes was detected in the culture supernate of Streptococcus pyogenes type 12 strain. This substance had a molecular weight of 28 000 and p I 9.2, and was designated as S. pyogenes mitogen (SPM). The proliferative response of C3H/HeN spleen cells began at 1 ng ml⁻¹ and reached a maximal response at 100 ng ml⁻¹ of SPM for 4 days culture. Anti-Thy 1.2 mAb and complement-treated spleen cells abrogated the proliferative response to any dose of SPM. Although the anti-major histocompatibility complex class I mAbs had no blocking effect on proliferation by SPM, this proliferation was substantially inhibited by the addition of either anti-I-A or anti-I-E mAb, and complete inhibition was produced by the addition of both mAbs. Fixed antigen-presenting cells still induced T cell proliferation by SPM. A significant expansion of T cells bearing Vβ13 T-cell receptor was observed up to 73% among the Thy1.2⁺ cells in cultures stimulated with SPM, indicating expansion in a Vβ-specific manner. Immunoblotting of IEF-separated proteins showed that anti-streptococcal pyrogenic exotoxin (SPE) C reacted with a protein of p I 6.9 and anti-SPEB did not show any reactivity. SPEA was reported to expand Vβ8.1 and 8.2 bearing murine T cells, and SPM did not. SPM also exhibited potent mitogenic activity on human T cells and Vβ21⁺ T cells were selectively expanded. These results lead to the conclusion that SPM was neither SPEA, B nor C, but a new protein belonging to a group of streptococcal superantigens with activity on not only human but also murine lymphocytes.     

白念珠菌SPE1基因高表达菌株构建及鉴定

目的构建白念珠菌SPEl基因高表达菌株。方法将白念珠菌．SPEl基因的ORF置于高表达质粒载体pCaE—xP的MErF3启动子后面,构建pCaEXP—SPEJ的高表达质粒,然后采用醋酸锂转染法将高表达质粒转染白念珠菌RMl000中,在SD—ura’met—cys-选择性固体培养基上筛选阳性克隆,抽取基因组进行PCR验证,将验证为阳性转染子的菌落采用RealTimeRT．PCR方法进行SPE1基因转录水平的表达验证。结果通过酶切鉴定pCaEXP—SPEl高表达质粒构建正确;通过PCR验证表明SPEl基因整合到亲本菌中的RPl0位点;通过RealTimeRT—PCR方法筛选出sPEJ基因在转录水平高表达的菌株。结论利用高表达质粒载体pCaEXP通过基因同源重组等方法正确构建SPEl基因高表达的白念珠菌。     

Stabilization of Nrf2 protein by D3T provides protection against ethanol-induced apoptosis in PC12 cells

Previous studies have demonstrated that maternal ethanol exposure induces a moderate increase in Nrf2 protein expression in mouse embryos. Pretreatment with the Nrf2 inducer, 3H-1, 2-dithiole-3-thione (D3T), significantly increases the Nrf2 protein levels and prevents apoptosis in ethanol-exposed embryos. The present study, using PC12 cells, was designed to determine whether increased Nrf2 stability is a mechanism by which D3T enhances Nrf2 activation and subsequent antioxidant protection. Ethanol and D3T treatment resulted in a significant accumulation of Nrf2 protein in PC 12 cells. CHX chase analysis has shown that ethanol treatment delayed the degradation of Nrf2 protein in PC12 cells. A significantly greater decrease in Nrf2 protein degradation was observed in the cells treated with D3T alone or with both ethanol and D3T. In addition, D3T treatment significantly reduced ethanol-induced apoptosis. These results demonstrate that the stabilization of Nrf2 protein by D3T confers protection against ethanol-induced apoptosis.     

Ion suppression effects in liquid chromatography-electrospray-ionisation transport-region collision induced dissociation mass spectrometry with different serum extraction methods for systematic toxicological analysis with mass spectra libraries

Ion suppression effects during electrospray-ionsation mass spectrometry (ESI-MS) caused by different sample preparation procedures for serum were investigated. This topic is of importance for systematic toxicological analysis for which LC-ESI-MS has been developed with transport-region collision-induced dissociation (ECI-CID) and mass spectra library searching. With continuous postcolumn infusion of two test compounds-codeine and glafenine-the ion suppression effects of extracted biological matrix obtained after a standard liquid-liquid extraction, a mixed-mode solid-phase extraction (SPE) method, a protein precipitation method and a combination of precipitation with polymer-based mixed-mode SPE have been investigated. Extracted ion chromatograms of codeine ([M+H](+), m/z 300) and glafenine ([M-H](-), m/z 371) were used for monitoring ion suppression. Severe ion suppression effects for codeine and glafenine were detected in positive and in negative ionisation modes, respectively, in the LC-front peak after serum clean-up with SPE (acid/neutral fraction) and protein precipitation as well as with protein precipitation combined with SPE. Less ion suppression of codeine in positive mode was found with liquid-liquid extraction of serum samples. No ion suppression was detected with the second fraction of the mixed-mode SPE (using RP-C(8) and cation-exchange phase) in both ionisation modes. All suppression effects were caused by polar and unretained matrix components, which were present after extraction and/or protein precipitation. However, no specific ion suppression was seen after elution of the polar LC-front throughout the whole gradient. It could be demonstrated, that ion suppression is not generally present at any retention time when using reversed-phase HPLC with rather long gradient programs, but may play an important role in case of high-throughput LC-MS analysis, when the analyte is not separated from the LC-front, or in flow injection analysis without chromatographic separation.     

Growth-related expression of a double-stranded RNA-dependent protein kinase in 3T3 cells

Cultured mouse 3T3-F442A and 3T3-C2 fibroblasts exhibit a transient double-stranded RNA (dsRNA)-dependent phosphorylation of a 67,000-dalton protein (67K) without prior treatment with interferon (IFN). This phosphoprotein is similar but not identical to the dsRNA-dependent eukaryotic initiation factor-2 (eIF-2) alpha protein kinase (dsI), which regulates protein synthesis in rabbit reticulocytes. We have studied the relationship between cell growth and phosphorylation of the 67K protein (designated 3T3-dsRNA-dependent eIF-2 alpha kinase). A low level of dsRNA-dependent phosphorylation of 3T3-dsI was detectable in extracts prepared from cells not treated with IFN and grown at a low cell density. The phosphorylation of dsI and the phosphorylation of a 38K protein identified as the alpha-subunit (38K) of 3T3-eIF-2 (eIF-2 alpha) occurred concomitantly; the levels of these phosphorylations confluent and thereafter decreased markedly. Treatment of cells with IFN at all stages of growth resulted in an increase in phosphorylation of dsI. 3T3-F442A and 3T3-C2 fibroblasts were found to produce and secrete IFN at levels sufficient to induce an elevated dsI activity.