Appearance of a stage-specific immunodominant glycoprotein in encysting Entamoeba invadens

The appearance of cyst-specific proteins in encysting Entamoeba invadens and their immunogenicity were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting using an axenic encystation system in vitro. A rabbit antiserum against trophozoites of E. invadens reacted with a number of proteins of cysts after 1–4 days of incubation. Thus, a number of cyst proteins remained antigenically unchanged as common antigens of the two forms after transformation from trophozoites to cysts. A rabbit antiserum against cysts also reacted with the trophozoite proteins as well as the cyst proteins. The most interesting result was that the rabbit anticyst serum reacted predominantly with an 88-kDa protein of cysts after 1 day of incubation. The 88-kDa protein reacted with the anticyst serum absorbed with trophozoite proteins and was thus cyst-specific. The reactivity of the 88-kDa protein of cysts with the absorbed anticyst serum decreased as encystation proceeded. When soluble and particulate fractions prepared from cysts after 1 day of incubation were examined by electrophoresis and immunoblotting, the 88-kDa protein that had reacted with the absorbed anticyst serum was found to be present in the particulate fraction, which was rich in cell-wall fragments, and stained with periodic acid-Schiff 's reagent, indicating that it is a glycoprotein. The results indicate that encystation is accompanied by appearance of the cyst-specific 88-kDa glycoprotein, which is immunodominant and most abundantly expressed in cysts after 1 day of incubation and appears to be associated with the cyst wall. Received: 31 May 1999 / Accepted: 20 July 1999     

Pneumocystis carinii pneumonia. An approach to rapid laboratory diagnosis.

Laboratory procedures used to establish the diagnosis of Pneumocystis carinii pneumonia were evaluated using an experimental murine model. Touch preparations and suspension smears were prepared from lung tissue know to contain Pneumocystis cysts. These preparations were stained by a variety of methods known to demonstrate either cyst forms or sporozoites and trophozoites. Suspension smears proved to be superior to touch preparations in terms of cyst content and homogeneity of staining. Also, methods that stain cyst forms were superior to those that stain sporozoites and trophozoites for location and identification of organisms. The authors believe that suspension smears prepared from lung tissue and stained with toluidine blue O should be examined initially as a rapid screening method for Pneumocystis cysts. When the results of this initial screen are negative or inconclusive, additional suspension smears stainded by the modified Gomori methenamine silver nitrate method should be examined, pending availability of histologic sections.     

幽门螺杆菌L型分子生物学性状的研究

为了探讨幽门螺杆菌L(HP—L)型随着形态学的变异其生物学性状是否发生了改变,作者对HP细菌型和L型作了SDS-PAGE、免疫印迹以及DNA酶切图谱分析。SDS—PAGE结果显示HP—L型分子量为6.9×10~4,6.1×10~4的菌体蛋白含量减少,而分子量为1.25×10~4,5.8×10~4,1.3×10~4菌体蛋白含量增加。免疫印迹显示分子量为1.8×10~4,3×10~4,3.4×10~4蛋白条带基本一致,但L型缺失一些弱反应的条带。用限制性核酸内切酶HindⅢ消化染色体DNA,进行琼脂糖凝胶电泳后显示HP—L型和细菌型的酶切图谱基本相同,无肉眼可测出的差别。     

Giardia cyst wall-specific carbohydrate: evidence for the presence of galactosamine

Gas chromatographic (GC), mass spectrometric (MS), lectin binding and enzymatic analyses of the carbohydrates from Giardia cyst walls, intact cysts and trophozoites were performed to investigate the carbohydrate composition of Giardia cyst walls and to test the hypothesis that the Giardia cyst wall is composed largely of chitin. Galactosamine, verified by MS, was present in Giardia cyst walls and intact cysts (ca. 47 nmol 10(-6) cysts). Since not even trace amounts of it were detected in trophozoites by either GC or lectin binding, galactosamine is hypothesized to be a cyst wall-specific amino hexose. Based on the putative binding affinity of Phaseolus limensis lectin, galactosamine may be present in cyst walls as N-acetylgalactosamine. Neither glucosamine nor sialic acid were detected in as much as 11 mg dry weight of cysts, cyst walls, or trophozoites. Glucose, the most abundant carbohydrate, and ribose were detected in Giardia cysts and trophozoites. Galactose (ca. 10 nmol 10(-6) cysts) was detected in cysts but not in trophozoites. The lack of detectable levels of (1) glucosamine in cyst wall hydrolysates, (2) cyst staining by Calcofluor M2R, (3) endogenous chitinase activity and (4) N-acetylglucosamine when cysts served as a substrate for exogenous chitinase suggests that the Giardia cyst wall is not composed largely of chitin as previously reported. beta-N-Acetylgalactosaminidase, EC 3.2.1.32, activity was detected in cysts and trophozoites and represents the first carbohydrate splitting hydrolase detected in Giardia.     

猪囊尾蚴抗原的免疫化学研究

应用琼脂双扩散(DID)、免疫电泳(IEP)、聚丙烯酰胺凝胶电泳(PAGE)和十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)对猪囊尾蚴的各部分抗原,即完整幼虫、囊液、头节和囊壁抗原进行分析。实验结果表明,4种抗原间存在相同的抗原成分。囊液中含有糖蛋白和脂蛋白。囊液、头节和囊壁蛋白质的主要分子量均在100KDa以下。囊液与头节有3条分子量相同的主带(60、46、38KDa),与囊壁只有1条分子量(46KDa)相同主带。从免疫学和生物化学角度初步探索了囊液、头节和囊壁蛋白质之间的相互关系及主要蛋白质组分。     

Seasonal differences in ram seminal plasma revealed by partition in an aqueous two-phase system

Seminal plasma plays an important role in maturation of spermatozoa through hormonal, enzymatic and surface-modifying events. We have previously shown that adsorption of seminal plasma proteins (SPPs) to the sperm cell surface partially restores the functional characteristics of damaged spermatozoa, reproducing those of live cells. In the present report, we investigate the hypothesis that seasonal differences in seminal plasma could affect its ability to recover membrane integrity of cold-shocked sperm. The effect of seminal plasma proteins, obtained in breeding (bsSPPs) and non-breeding (nbsSPPs) season, on cold-shocked ram spermatozoa previously freed from seminal plasma, was analysed by centrifugal counter-current distribution (CCCD) in an aqueous two-phase system as well as membrane integrity determination by fluorescence markers. Cold-shock treatment greatly lowered cell viability in both breeding and non-breeding season spermatozoa. The cold-shocked sperm viability obtained was approximately 20%. The loss of heterogeneity and the decrease in viability revealed by CCCD analysis was reversed by the addition of increasing amounts of bsSPP, which induced restoration of the surface characteristics of viable-like spermatozoa, as well as an increase in the number of recovered viable sperm. However, this restoring effect was much lower when nbsSPPs were added, even in a sixfold higher concentration than used with bsSPPs. Incubation of cold-shocked cells with both kinds of proteins performed in both seasonal periods, showed that the recovering effect was related to the season when the plasma sample was obtained rather than to the semen season. The addition of bsSPPs to cold-shocked sperm accounted for a nearly 50% reversion for both studied breeding seasons. However, the reversion percentages obtained with nbsSPPs were significantly lower (P<0.05) than those found with bsSPPs in both studied seasonal periods. This different reversion capacity of bsSPPs and nbsSPPs was related to a different protein composition, as revealed by comparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The bands of 20, 21, 24, 36 and 67 kDa of the bsSP sample profile decreased in winter-spring SP, and were even less intensely stained in summer SP. Densitometric analysis of the stained gel patterns allows automatic comparison among the separated bands, and revealed an important decrease in the content of several bands. The 21.5 kDa band showed the highest decrease, lowering to 14% in June-August plasma with respect to the value obtained in September-December plasma.     

Molecular interactions during pregnancy: Identification of cyclophilin A from human decidual and placental tissue in the first trimester of pregnancy

The protein patterns of tissue homogenates from human deciduaand placenta of first trimester pregnancies were investigated.Particular attention was paid to the low molecular weight componentsof these tissues, since substantial evidence has accumulatedthat some of these smaller proteins show a characteristic cyclicand pregnancy expression. Two specific bands were purified fromhomogenates of first trimester decidua and placenta using gelfiltration and anion exchange chromatography. These bands wereseparated by gel electrophoresis and blotting onto polyvinylidendifluoridemembrane. Partial amino acid sequencing of both proteins revealedsequences identical to human cyclophilin A. One protein wassequenced V-N-P-T-V-F-F-D-l-A-V-D-G-E-P-L-G-R-(X)-S-F-E-L-F-A-D-K-V-Pand identified as the 17 kDa isoform of cyclophilin A. The otherprotein was sequenced V-N-P-T-V-F-F-D-I-A and identified asthe 18 kDa isoform of cyclophilin A.     

Characterization of cell wall proteins from yeast and mycelial cells of Candida albicans by labelling with biotin: comparison with other techniques.

Candida albicans ATCC 26555 blastoconidia and blastoconidia bearing germ tubes were metabolically labelled by incubating the cells with 14C-labelled protein hydrolysate and were subsequently tagged with biotin. Double-labelled (radioactive and biotinylated) cell wall proteins and glycoproteins were extracted from intact cells of both growth forms by treatment with 2-mercaptoethanol (beta ME) and with beta-glucanases (Zymolyase) after treatment with beta ME. The beta ME- and Zymolyase-extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotted (immunoblotted) to nitrocellulose paper. Polyacrylamide gels were stained with Coomassie blue and processed for fluorography. Western blot analysis was performed either with peroxidase conjugated-concanavalin A (ConA) or Extravidin. Blotted proteins were also reacted with polyclonal antibodies and monoclonal antibodies against mannoprotein components from mycelial cell walls of the ATCC 26555 strain. Labelling with biotin allowed identification of a complex array of cell wall protein and glycoprotein components within a very wide molecular mass range (from 650 to 13 kDa). These appeared to be genuine cell wall components. Biotinylated high-molecular-mass glycoproteins that were not stained with Coomassie blue or that appeared as poorly resolved polydisperse bands by indirect ConA-peroxidase staining of Western blots were detected as sharply defined bands following reaction with the Extravidin-peroxidase conjugate. Biotinylated molecules retained unaltered reactivities against ConA, polyclonal antibodies, and monoclonal antibodies.     

An immunoblot for detection of Taenia saginata cysticercosis

Control measures to prevent human infections with the food-borne zoonotic helminth Taenia saginata are currently based on meat inspection, which shows rather low diagnostic sensitivity. To develop an immunoblot for detection of T. saginata-infected cattle, crude proteins of T. saginata cysts were extracted and separated with SDS-PAGE. The cyst antigens showed ten protein bands ranging from 260 to 14 kDa. T. saginata cyst proteins 260, 150, 130, 67, 60, 55, 50, and 23 kDa were immunoreactive with known positive sera of T. saginata-infected cattle but cross-reacted with sera from Echinocccus granulosus-infected ruminants. By contrast, 14- and 18-kDa cyst proteins reacted specifically with T. saginata-positive sera and thus might be potential candidates for the development of a T. saginata-specific immunoassay. Proteins of E. granulosus cysts and Taenia hydatigena cysts were also extracted and separated with SDS-PAGE. E. granulosus cysts revealed 11 protein bands ranging from 260 to 23 kDa. E. granulosus protein 60 kDa was immunoreactive with E. granulosus-positive sera only. The cyst of T. hydatigena showed 11 protein bands ranging from 290 to 14 kDa. The protein band 35 kDa showed cross-reaction with positive sera from both T. saginata- and E. granulosus-infected animals. A protein of 67 kDa was present in all three tested cestode species and was the major antigenic protein detected by sera of T. saginata- and E. granulosus-infected animals. Therefore, this protein represents a potential vaccine candidate against both cysticercosis and cystic echinococcosis in cattle.     

A simple protocol for the isolation of proteorhodopsins of the dinoflagellate Oxyrrhis marina

For the first time, native proteorhodopsins of the marine dinoflagellate Oxyrrhis marina were isolated. Total cell membrane fractions were minced in a bead beater and solubilized with the detergent Triton X-100. Subsequent sucrose density gradient centrifugation resulted in three or four red-colored bands. Nonsolubilized, but still red colored, membranes sedimented at the bottom. For each of these bands, absorbance maxima were registered at approximately 514–516 nm with shoulders toward shorter wavelengths (470–490 nm). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the uppermost band represented free retinal chromophore, as it contained no protein. The other bands were almost pure proteorhodopsin fractions as the banding patterns showed one major protein of 25 kDa. Tryptic, in-gel digestion of the 25 kDa proteins and of faint protein bands above and below 25 kDa was followed by mass spectrometry, confirming these protein bands to consist, nearly exclusively, proteorhodopsins. Only single peptides of few other proteins were detected. In total, at least seven predicted proteorhodopsin protein sequences were experimentally verified.     

贾第虫研究──Ⅰ．西蒙氏贾第虫（Giardia simoni）滋养体和包囊对实验动物的感染

作者从自然感染西蒙氏贾第虫（Ｇｉａｒｄｉａｓｉｍｏｎｉ）的金黄地鼠肠道内和粪便中分离、收集滋养体和包囊，分别经口感染大鼠、小鼠、豚鼠和兔子。结果表明兔子和脉鼠均不感染，大鼠、小鼠均不同程度地感染该虫，且４周龄大鼠比８周龄大鼠更易感（Ｐ＜０．０５），而在小鼠中成年鼠与老年鼠对西蒙氏贾第虫的敏感性没有差异（Ｐ＞０．０５）。滋养体在感染动物的肠道内主要分布于十二指肠前段，中前段和中段。同时观察到结肠和直肠内有包囊，这表明该虫在大鼠、小鼠体内完成了其生活史。作者还对感染前后的滋养体作了蛋白银和铁苏木精染色比较，二者在形态上完全相同。     

Recognition of protein apparently specific to odontogenic keratocyst fluids.

Separate antisera were raised against keratocyst, dentigerous cyst, and radicular cyst fluids and used to analyse a range of fluids from cysts of known type. Samples were subjected to crossed immunoelectrophoresis into homologous antiserum through an intermediate gel containing antibody to whole human serum to screen out serum derived components. A major antigen, designated X, which seems to be of epithelial origin but is not a keratin, was identified in keratocyst fluids. X resolves as two bands on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) with molecular weights of 81 K and 89 K and its major antigenic epitope is associated with disulphide bonds. Of the cysts studied to date, antigen X has been found consistently and exclusively in fluids from keratocysts; its presence and detection is independent of total soluble protein concentration and thus offers real potential as a reliable marker for preoperative diagnosis.     

Electrophoretic analysis of erythrocyte membrane proteins and glycoproteins from different species

The erythrocyte membrane proteins and glycoproteins of man, rat, mouse, sheep and dog were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), using a discontinuous buffer system. Considerable similarities between the species were observed in the pattern of protein bands seen when gels were stained with Coomassie Blue. Equivalents to human Bands 1, 2, 3, 4.2, 5 and 8 appeared to be present in the rat, mouse, sheep and dog, and Band 4.1 was identified as a closely spaced doublet in all species except the rat.When RBC membranes were stained with periodic acid-Schiff (PAS) after SDS-PAGE analysis, glycoproteins equivalent to human glycophorins were identified in all the species studied. However, in contrast to the overall similarity of the protein patterns, the number, relative staining intensity and apparent molecular masses of the PAS-stained bands differed between species.The silver stain was assessed in the detection of RBC membrane proteins in polyacrylamide gels, and found to be more sensitive than Coomassie Blue. The technique also stained many of the PAS-positive glycophorins as diffuse orange zones, which could be distinguished from the darker protein bands by their differential colouration.In view of the interspecies variation in the glycophorins after SDS-PAGE, it is suggested that, unlike the membrane proteins, their functions do not require a conserved structure.     

Comparison of immunoblot analyses of spherule-endospore-phase extracellular protein and mycelial-phase antigen of Coccidioides immitis.

The extracellular proteins produced by Coccidioides immitis during growth of the spherule-endospore-phase and mycelial-phase antigen (coccidioidin) were studied by polyacrylamide gel electrophoresis followed by immunoblot analysis to detect specific serologic function. Filtrates obtained from 28- and 120-h growth of the spherule-endospore phase were compared with each other and with coccidioidin by using negative, immunoglobulin M (IgM) precipitin-positive, or complement fixation-positive pooled and single human sera followed by peroxidase-labeled anti-human IgA, IgE, IgG, or IgM (heavy chain specific) or peroxidase-labeled concanavalin A to detect the reaction. A total of 35 bands was seen in the stained gels. Different patterns were noted among the two spherule-endospore preparations and unheated and heated coccidioidin. At least 15 electrophoretically separate antigens were detected with positive serum ranging in approximate molecular weight (Mr) from 100,000 to 18,000. Most were clustered between 45 and 60 kilodaltons (kDa). Common bands were noted at 48 and 18 kDa. At least one band at 48 kDa was strongly reactive with complement fixation-positive serum demonstrated by reaction with anti-IgG and anti-IgE. In contrast, doublet bands in the 50- to 65-kDa area were highly reactive with IgM precipitin-positive serum detected by anti-IgM. IgM antibodies present in both positive sera reacted with a band at 46 kDa which was not reactive with IgG. Heating the antigens altered the reactivity of many of the antigens, including the 48-kDa band, but not the 46-kDa band.     

Biochemical and immunological characterization of major surface antigens ofSarcocystis muris andS. suicanis cyst merozoites

Surface proteins ofSarcocystis cyst merozoites were labeled by biotinylation or radioiodination and identified on Western blots after sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). The major labeled proteins ofS. muris andS. suicanis have relative molecular masses of 31 and 33 kDa, respectively. Immunoblots performed with the 31-kDa protein and sera of experimentally infected mice or with monoclonal antibodies toS. muris revealed that this protein is immunogenic. Indirect fluorescent antibody tests (IFAT) executed usingSarcocystis cyst merozoites and polyclonal monospecific antibodies obtained from rabbits immunized with homogeneous major surface antigens gave additional evidence for the localization of the identified antigens in the pellicle. Analysis ofS. muris andS. suicanis proteins by two-dimensional gel electrophoresis revealed multiple isoelectric forms.Supported by the Deutsche Forschungsgemeinschaft and dedicated to Prof. Dr. Johannes Eckert (Zürich) on the occasion of his 60th birthday     

Glycoproteins composed of major surface immunodeterminants of Pneumocystis carinii.

Pneumocystis carinii is a pathogen which causes fatal pneumonia in patients with acquired immune deficiency syndrome. To facilitate the basic study of P. carinii, we analyzed the major surface proteins by immunochemical and biochemical methods. The major protein components of both cysts (resting form) and trophozoites (vegetative form) are part of a group of proteins called P115 with apparent masses of 105 to 120 kilodaltons. They represent an unusually large portion of the total proteins of this organism. The purified proteins exhibited six isoelectric variants when analyzed by two-dimensional gel electrophoresis. A monoclonal antibody raised against cysts recognized all six variants and reacted with epitopes that were located in the cell wall, thereby indicating that P115 is an immunoreactive surface component. Data are presented that the isoelectric variants contain identical or closely related protein components and that they are mannose-rich glycoproteins. Deglycosylated P115 migrates primarily as a single more acidic protein in two-dimensional gels, suggesting that the isoelectric variants may be due primarily to differences in glycosylation. The majority of sera tested from humans with diagnosed pneumocystosis reacted strongly with the P115 proteins.     

MDCK cysts: An in vitro model of epithelial cyst formation and growth

Summary MDCK cells dissociated from monolayer culture were either dispersed within medium-hydrated collagen gel or seeded atop a collagen substrate which was immediately overlaid with collagen gel. Individual cells exhibited clonal growth in three dimensions to form spherical cysts in which a simple epithelium surrounded a fluid-filled lumen. The cells of MDCK cysts were polarized with apical surface bordering the lumen. MDCK cysts increased in diameter with continued culture. Maximum cyst size was dependent on seeding density and was influenced by medium composition. MDCK cysts could be isolated from the collagen substrate by digestion with collagenase. Also, collagen gel could be dissected from the cyst wall to give unrestricted access to regions of the basolateral cell surface. This novel method of renal cell culture provides a study system to model the influence of the extracellular matrix on kidney epithelial cell structure and function. It also offers an in vitro model of general application to the study of epithelial cyst formation and growth.     

Analysis of proteins in urinary tract stones and urine of urolithic patients

In order to investigate the mechanism of urinary tract stone formation, we analyzed protein components in urine and the stone. Urinary proteins of healthy subjects and urolithic patients as well as protein components urinary tract stone of the urolithic patients were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoretic patterns of urinary proteins of the patients differed from those of healthy subjects after separating protein patterns into those larger than 66kDa or smaller than 30kDa. Protein constituents of urinary tract stone were mainly separated into 18 bands ranging from 26.8 to 143 kDa. Major bands among these 18 bands differed among stones from different patients. On western blotting, the developed intensities of Tamm-Horsfall protein (THP) were fainter than those of healthy subjects. Whereas intensities of albumin (ALB) were stronger than those of healthy subjects. Moreover, blotting patterns of THP of the patients on non-reducing SDS-PAGE were obviously broad. Thus, we suggest that analysis of fractionated urinary proteins or protein components of urinary tract stone may provide a tool for monitoring the prognosis or relapse in the patients.     

Identification and localization of cyst-specific antigens of Giardia lamblia.

We induced Giardia lamblia trophozoites to encyst in vitro by exposure to conditions which are specific to the human small intestinal milieu. We now show that encystation entails the appearance of two new groups of antigens detected in Western blots by rabbit antiserum against cysts which had been purified from human feces. A heterodisperse group of lower-molecular-mass antigens (approximately 21 to 39 kilodaltons) was expressed relatively early (less than 19 h) in encystation. In contrast, discrete bands at approximately 66, 78, 92, and 103 kilodaltons only appeared after 24 h of incubation under conditions which lead to production of large numbers of water-resistant cysts. We also describe for the first time the appearance of prominent cytoplasmic vesicles, which were the earliest morphologic change in encysting trophozoites observable by light microscopy. Early in encystation, cyst wall antigens were concentrated in these vesicles, as shown by immunocytochemistry, suggesting that the vesicles function in export of cyst wall constituents.