Physicochemical and immunochemical properties of a thermo-labile antigen (TLA a) from yeast cell surface

The physicochemical and immunochemical properties of a thermo-labile antigen (TLA a) which is located on the cell surface of Saccharomyces cerevisiae were studied. The sedimentation constant (S20,W) and molecular weight (sedimentation equilibrium method) were 6.26S and 68,800, respectively. The circular dichroic (CD) spectrum of TLA a had negative maxima at 210 and 221 nm, indicating the presence of alpha-structure of a polypeptide chain. The molar ratio of antibody to antigen which gave maximum precipitation was 2.7. Approximately 50% of the antigenic activity of heat-denatured TLA a was recovered when denatured molecules were dissolved in 6 M guanidine hydrochloride followed by 25-fold dilution with H2O. The amount of TLA a existing on the yeast cell surface was estimated to be 37.5 micrograms per 10.5 mg of fresh cells, corresponding to 0.36% by weight of the fresh yeast.     

Localization of immuno-analogues of erythrocyte protein 4.1 and spectrin in epidermis of psoriasis vulgaris

The presence and localization of immuno-analogues of human erythrocyte protein 4.1 and spectrin were examined in the epidermis of psoriasis vulgaris. Immunoblot analysis with antibodies against human erythrocyte protein 4.1 revealed that psoriatic epidermis contains a 4.1-like protein of 80 kDa, and also minor immunoreactive polypeptides, including a 45-kDa polypeptide. The 45-kDa band was not detected in non-lesional epidermis. Lesional epidermis of psoriasis contains spectrin-like proteins of 240 kDa. Analysis with immunofluorescence microscopy revealed that 4.1-like proteins were detected mainly in the cytoplasm of the suprabasal cells in lesional epidermis and in the peripheral cytoplasm of the basal cells in non-lesional epidermis. On the other hand, spectrin-like proteins were localized to the peripheral cytoplasm of basal keratinocytes in both lesional and non-lesional psoriatic epidermis. The present results indicate that proteins related to protein 4.1 and spectrin are consistently detected within epidermal cells of psoriasis, a chronic skin disease characterized by epidermal hyperplasia; the expression and distribution of protein 4.1 in lesional epidermis of psoriasis differs from that in non-lesional epidermis. These membrane skeletal proteins may be of significance in the hyperproliferative epidermis of psoriasis.     

Purification and Properties of a Thermo-labile Antigen from Baker’s Yeast Cells

A thermo-labile antigen (TLA) on the yeast cell surface was isolated from a yeast cell autolyzate and purified to a homogeneous state by chromatography on an immunoadsorbent affinity column. The molecular weight of TLA was about 1.45 x 10⁵ on SDS-polyacrylamide gel electrophoresis and about 1.5 x l0⁵ on gel chromatography on Sephadex G-200. The TLA contained 74.5% protein and 25.5% sugar. It was characterized by high contents of glycine, glutamic acid, serine and aspartic acid. Half-cystine, methionine, histidine and arginine were not found. The sugar moiety was composed of galactose, mannose, N-acetylglucosamine and fucose. The antigenic determinant of TLA was distinct from that of cell wall mannan in the Ouchterlony immunodiffusion test. No precipitin line against anti-TLA serum was observed, when TLA was heated at 90°C for 10 min. Oxidation with periodate had little effect on antigenicity, but digestion with Pronase or treatment with protein denaturants resulted in loss of the antigenicity. These results suggest that the protein moiety plays an important role as the antigenic determinant of TLA. Moreover, the antiserum specific to TLA agglutinated fresh yeast cells, and the distribution of TLA was apparent on the yeast cell surface by immunofluorescence staining. These findings suggest that TLA molecules were exposed on the outer surface of the yeast cell wall.     

Colorimetric determination of fructose for the high-throughput microtiter plate assay of glucose isomerase

A colorimetric method for the reducing monosaccharide determination is optimized for the assay of glucose isomerase, which converts glucose (Glc) to fructose (Fru). Test solution was mixed with 20-fold volume of the 50 mM Na₂SiO₃, 600 mM Na₂MoO₄, and 0.95 M HCl aqueous solution (pH 4.5), in which a yellow molybdosilicate species was formed. The mixture was kept at 70 °C for 30 min. Test solution containing 10 mM level Fru gave a remarkable blue reaction mixture, in which the Mo(VI) species was reduced by Fru to form a blue molybdosilicate species. The blueness increased with the Fru concentration. Glc cannot render the reaction mixture blue as strong as Fru. Thus, the colorimetric method can be used advantageously for the determination of 10 mM level Fru in the Glc isomerase reaction mixture, even in the presence of 100 mM level Glc, and has been applied successfully to the microtiter plate assay of the enzyme.     

Epitope Mapping for Monoclonal Antibody Reveals the Activation Mechanism for αVβ3 Integrin

Epitopes for a panel of anti-αVβ3 monoclonal antibodies (mAbs) were investigated to explore the activation mechanism of αVβ3 integrin. Experiments utilizing αV/αIIb domain-swapping chimeras revealed that among the nine mAbs tested, five recognized the ligand-binding β-propeller domain and four recognized the thigh domain, which is the upper leg of the αV chain. Interestingly, the four mAbs included function-blocking as well as non-functional mAbs, although they bound at a distance from the ligand-binding site. The epitopes for these four mAbs were further determined using human-to-mouse αV chimeras. Among the four, P3G8 recognized an amino acid residue, Ser-528, located on the side of the thigh domain, while AMF-7, M9, and P2W7 all recognized a common epitope, Ser-462, that was located close to the α-genu, where integrin makes a sharp bend in the crystal structure. Fibrinogen binding studies for cells expressing wild-type αVβ3 confirmed that AMF-7, M9, and P2W7 were inhibitory, while P3G8 was non-functional. However, these mAbs were all unable to block binding when αVβ3 was constrained in its extended conformation. These results suggest that AMF-7, M9, and P2W7 block ligand binding allosterically by stabilizing the angle of the bend in the bent conformation. Thus, a switchblade-like movement of the integrin leg is indispensable for the affinity regulation of αVβ3 integrin.     

Normal Lung Quantification in Usual Interstitial Pneumonia Pattern: The Impact of Threshold-based Volumetric CT Analysis for the Staging of Idiopathic Pulmonary Fibrosis

BackgroundAlthough several computer-aided computed tomography (CT) analysis methods have been reported to objectively assess the disease severity and progression of idiopathic pulmonary fibrosis (IPF), it is unclear which method is most practical. A universal severity classification system has not yet been adopted for IPF.ObjectiveThe purpose of this study was to test the correlation between quantitative-CT indices and lung physiology variables and to determine the ability of such indices to predict disease severity in IPF.MethodsA total of 27 IPF patients showing radiological UIP pattern on high-resolution (HR) CT were retrospectively enrolled. Staging of IPF was performed according to two classification systems: the Japanese and GAP (gender, age, and physiology) staging systems. CT images were assessed using a commercially available CT imaging analysis workstation, and the whole-lung mean CT value (MCT), the normally attenuated lung volume as defined from −950 HU to −701 Hounsfield unit (NL), the volume of the whole lung (WL), and the percentage of NL to WL (NL%), were calculated.ResultsCT indices (MCT, WL, and NL) closely correlated with lung physiology variables. Among them, NL strongly correlated with forced vital capacity (FVC) (r = 0.92, P <0.0001). NL% showed a large area under the receiver operating characteristic curve for detecting patients in the moderate or advanced stages of IPF. Multivariable logistic regression analyses showed that NL% is significantly more useful than the percentages of predicted FVC and predicted diffusing capacity of the lungs for carbon monoxide (Japanese stage II/III/IV [odds ratio, 0.73; 95% confidence intervals (CI), 0.48 to 0.92; P < 0.01]; III/IV [odds ratio. 0.80; 95% CI 0.59 to 0.96; P < 0.01]; GAP stage II/III [odds ratio, 0.79; 95% CI, 0.56 to 0.97; P < 0.05]).ConclusionThe measurement of NL% by threshold-based volumetric CT analysis may help improve IPF staging.     

Sensitive determination of aspirin and its metabolites in plasma by LC-UV using on-line solid-phase extraction with methylcellulose-immobilized anion-exchange restricted access media

We describe a sensitive determination of aspirin (ASA) and its three metabolites (salicylic acid [SA], 2,3-dihydroxybenzoic acid [2,3-DHBA], and 2,5-dihydroxybenzoic acid [gentisic acid (GA)]) in rat plasma. Analysis was carried out by on-line solid-phase extraction (SPE) using a methylcellulose-immobilized-strong anion-exchanger (MC-SAX), followed by liquid chromatography (LC) coupled with UV detection. The lower limits of quantitation for ASA and SA were 60 ng/mL in 100 microL of plasma, respectively. This method was validated with respect to intra- and inter-day precision, accuracy, and linearity up to concentrations of 20,000 ng/mL for ASA, SA, 2,3-DHBA and gentisic acid, respectively. The method was successfully applied to an analysis of the pharmacokinetics of ASA and SA in rats.     

Identification and characterization of the UL56 gene product of herpes simplex virus type 2

The UL56 gene product of herpes simplex virus (HSV) has been shown to play an important role in viral pathogenicity. However, the properties and functions of the UL56 protein are little understood. We raised rabbit polyclonal antisera specific for the UL56 protein of HSV type 2 (HSV-2) and examined its expression and properties. The gene product was identified as three polypeptides with apparent molecular masses ranging from 32 to 35 kDa in HSV-2-infected cells, and at least one species was phosphorylated. Studies of their origins showed that the UL56 protein of HSV-2 is also translated from the upstream in-frame methionine codon that is not present in the HSV-1 genome. Synthesis was first detected at 6 h postinfection and was not abolished by the viral DNA synthesis inhibitor phosphonoacetic acid. Indirect immunofluorescence studies revealed that the UL56 protein localized to both the Golgi apparatus and cytoplasmic vesicles in HSV-2-infected and single UL56-expressing cells. Deletion mutant analysis showed that the C-terminal hydrophobic region of the protein was required for association with the cytoplasmic membrane and that the N-terminal proline-rich region was important for its translocation to the Golgi apparatus and cytoplasmic vesicles. Moreover, the results of protease digestion assays and sucrose gradient fractionation strongly suggested that UL56 is a tail-anchored type II membrane protein associated with lipid rafts. We thus hypothesized that the UL56 protein, as a tail-anchored type II membrane protein, may be involved in vesicular trafficking in HSV-2-infected cells.     

Existence of cerebroside in Saccharomyces kluyveri and its related species

Sphingolipids are ubiquitous compounds derived from ceramide that consist of a sphingoid long-chain base with a 2-amino group amide linked to fatty acid and are present in the membranes of many organisms. As a principal sphingolipid, Saccharomyces cerevisiae contains a free ceramide and its inositol-phosphorylated derivatives (acidic types) but not a neutral glycosylated ceramide, glucosylceramide (cerebroside), which usually appears in eukaryotic cells. When 31 strains accepted in the genera Saccharomyces, Torulaspora, Zygosaccharomyces, and Kluyveromyces were analyzed for sphingolipids, cerebrosides were found in S. kluyveri, Z. cidri, Z. fermentati, K. lactis, K. thermotolerans, and K. waltii. The cerebrosides of S. kluyveri and K. lactis included 9-methyl 4-trans, 8-trans-sphingadienine and its putative metabolic intermediates. A unique characteristic of S. kluyveri was the presence of a trihydroxy sphingoid base, which rarely occurs in fungal cerebrosides. A polymerase chain reaction with primers targeted to the glucosylceramide synthase gene of other microorganisms amplified the fragments of the expected size from S. kluyveri and K. lactis and further extended to the adjacent regions. The presumed protein of S. kluyveri had 54.4% similarity to that of K. lactis, higher than the glucosylceramide synthases from Candida albicans, Pichia pastoris, and other organisms. From these observations, the divergence of S. kluyveri from the lineage of K. lactis in their evolution is discussed.     

Uncoupling of gate and fence functions of MDCK cells by the actin-depolymerizing reagent mycalolide B

The tight junction serves as a paracellular gate to seal the paracellular space of apposing cells and as a molecular fence to prevent diffusion of membrane proteins and lipids in epithelial cells. Although involvement of the actin cytoskeleton has been considered to be important in these two functions, it remains to be elucidated whether both functions are regulated in a coupled manner or differentially by actin. Treatment of highly polarized MDCK cells with mycalolide B (MB), a recently developed actin-depolymerizing reagent, induced a decrease of transepithelial resistance in a dose- and time-dependent manner with reversibility when the reagent was washed out. Changes in cytoskeletal actin, such as a reduction of cortical actin, irregularity of stress fibers, and punctated actin aggregates, were observed after MB treatment. However, the fence function, as studied by diffusion of apically labeled sphingomyelin/BSA complex, remained intact in the MB-treated MDCK cells. Localization of junctional molecules and apical marker proteins such as E-cadherin, ZO-1, and 114-kDa protein was shown to be unaffected. Furthermore, freeze-fracture study showed apparent tight junction strands. Collectively, MB treatment abolished the paracellular gate but not the fence function of MDCK cells, suggesting that cytoskeletal actin may play differential roles in the gate and fence functions of the tight junction.