Candida albicans and Candida parapsilosis Rapidly Up-Regulate Galectin-3 Secretion by Human Gingival Epithelial Cells

Galectin-3 is a β-galactoside-binding C-type lectin that plays an important role in innate immunity. The purpose of this study was to determine whether Candida albicans and Candida parapsilosis up-regulate galectin-3 secretion by human gingival epithelial cells and gingival fibroblasts. Ca9-22, a human gingival epithelial cell line, and human gingival fibroblasts were incubated in the presence or absence of C. albicans or C. parapsilosis without serum. Levels of secreted human galectin-3 in culture supernatants were measured by enzyme-linked immunosorbent assay. We also pretreated Ca9-22 cells with cytochalasin D (an actin polymerization inhibitor), ALLN (a calpain inhibitor) and LY294002 [a phosphatidylinositol-3 kinase (PI3K) inhibitor] to determine whether the up-regulation of galectin-3 secretion was mediated by cytoskeletal changes, protease activity, or PI3K signaling. Galectin-3 secretion was significantly and rapidly up-regulated by live C. albicans and C. parapsilosis, as well as heat-killed C. albicans. In addition, cytochalasin D, LY294002 and ALLN did not inhibit the up-regulation in galectin-3 secretion. These results suggest that both live and heat-killed C. albicans and C. parapsilosis may increase the activity of the innate immune system and invasion by other microorganisms via up-regulation of galectin-3 secretion.     

Secretion of Mucor rennin, a fungal aspartic protease of Mucor pusillus, by recombinant yeast cells

Summary The aspartic protease gene of a zygomycete fungus Mucor pusillus was expressed in Saccharomyces cerevisiae under the control of the yeast GAL7 promoter. A putative preproenzyme with an NH₂-terminal extension of 66 amino acids directed by the gene was processed in yeast cells and the mature enzyme, whose NH₂-terminus was identical to that of the Mucor enzyme, was efficiently secreted into the medium at a concentration exceeding 150 mg/l. The enzyme secreted from the recombinant yeast was more glycosylated than the native Mucor enzyme but its enzymatic properties were almost identical with those of the native enzyme, which has been used as a milk coagulant in cheese manufacture.     

Changes in membrane constituents and chemiluminescence in vitamin E-deficient red blood cells induced by the xanthine oxidase reaction

The oxidation of vitamin E-deficient rat red blood cells (RBCs) induced by the hypoxanthine-xanthine oxidase (HX-XOD) system has been performed in an aqueous suspension. The generation of chemiluminescence and the accumulation of thiobarbituric acid-reactive substances (TBARS) were observed initially and were followed by hemolysis. Interestingly, the total counts of chemiluminescence were closely related to the amount of TBARS. The predominant change of membrane proteins induced by the reaction was the depletion of spectrin bands in gel electrophoresis. When RBC ghosts were oxidized with HX-XOD, the sulfhydryl (SH) groups of membrane proteins decreased at an early stage of the incubation, which was coincident with the above protein alteration. Membrane alpha-tocopherol suppressed not only the formation of TBARS but also chemiluminescence and hemolysis; nevertheless, it did not inhibit the protein damage and the loss of SH groups. Moreover, it was concluded that the chemiluminescence observed during the oxidation of RBC membranes was associated mainly with the peroxidation of lipids and only to a minor extent with the oxidation of proteins.     

Primary photoprocesses of phytochrome. Picosecond fluorescence kinetics of oat and pea phytochromes

The primary photoprocesses of etiolated oat and pea phytochromes (Pr forms) are diffusion-modulated by the microscopic viscosity within the chromophore pocket. The chromophore pocket is preferentially accessible to glycerol but not to Ficoll. Glycerol preferentially retarded the rate (rate constant ca. 1-2 X 10(10) s-1) of the initial reaction from the Qy excited state of phytochrome, whereas it increased the long fluorescence lifetime (nanosecond) component that can be attributed to either an emitting intermediate or to modified/conformationally heterogeneous phytochrome populations. The picosecond time-resolved fluorescence spectra of different phytochrome preparations (i.e., full-length vs 6/10-kDa NH2-terminus truncated forms of phytochromes from monocot and dicot plants) revealed no significant differences. The spectra in the picosecond time scale showed no spectral shifts, but at longer time scales of up to approximately 1.90 ns, significant blue spectral shifts were observed. The shifts were more in the truncated than in the full-length pea phytochrome. Comparison of the fluorescence decay data and the picosecond time-resolved fluorescence spectra suggests differences in conformational flexibility/heterogeneity among the preparations of the monocot vs dicot phytochromes and the full-length native vs the amino terminus truncated phytochromes.     

Detection of point mutation in the tyrosinase gene of a Japanese albino patient by a direct sequencing of amplified DNA

Summary Enzymatic DNA amplification and direct DNA sequencing were used to detect a mutation in the tyrosinase gene of an albino patient. Single-base change could be detected by direct sequencing. This base change (G to A) is thought to result in an amino acid change (Arg to Gln) in tyrosinase of the patient.     

Functional and phenotypic analyses of interleukin 2-activated tumor-infiltrating lymphocytes

The tumor-infiltrating lymphocytes (TILs) were cultured with interleukin 2 (IL-2) to induce the activated killer cells possessing autologous tumor-killing activity, and analysed their cell surface phenotypes and assessed anti-tumor killing activity. Furthermore, the activated TILs were transferred into 7 patients adoptively resulting in complete remission in a patient with pancreatic cancer and partial remission in another patient with gastric cancer.The cytotoxic activities of activated TILs at 3 weeks-incubation was 72 ± 15, 42 ± 26, 27 ± 21 and 25 ± 15% against K562, Daudi, KATO-III and autologous tumor, respectively. The negative selection method, indicated that the killer cells recognizing autologous tumor cells consisted of CD4- or CD8-positive T lymphocytes and CD16- or CD56-positive natural killer cells. The activated TILs could not only lyse cultured tumor cell lines, but also autologous tumor cells.     

Isolation and Characterization of a Photosystem II Core Complex Depleted in the 43 kDa-Chlorophyll-Binding Subunit

The photosystem II core complex purified from digitonin extractsof spinach chloroplasts was resolved into two chlorophyll-proteincomplexes by digitonin polyacrylamide gel electrophoresis aftertreatment with 1 M potassium thiocyanate. One of the chlorophyll-proteincomplexes resolved consisted of 47, 32, 30 and 9 kDa polypeptidesand the other was complementally composed of only the 43 kDapolypeptide. The former complex was highly active in the photoreductionof 2, 6-dichlorophenol indophenol by 1,5-diphenylcarbazide andretained all of the components responsible for the electrontransport from the secondary electron donor (Z) to the primaryelectron acceptor (Q_A). EPR signal II_fast and II_slow were alsopreserved in this complex although their hyperfine structureswere largely modified. The complex was estimated to contain1.8 molecules of plastoquinone A as well as 1.5, 3.7 and 3.9molecules of cytochrome b559, pheophytin and ß-carotene,respectively, per Q_A. These results indicate that potassiumthiocyanate specifically removes the 43 kDa polypeptide fromthe PS II core complex leaving the electron transport systemin an almost intact state. (Received June 17, 1987; Accepted October 23, 1987)     

Change in Gene Expression in Radish Cotyledons during Dark-Induced Senescence

Cotyledons detached from light-grown radish (Raphanus sativusL. cv. Comet) seedlings were used as a model system to studythe changes in nuclear gene expression during dark-induced senescenceof green leaves. Polyadenylated RNA was prepared from the cotyledonsat different times and then translated in a wheat germ system.Approximately 1,000 different polypeptides of the translationproducts were separated from each other by two-dimensional gelelectrophoresis. As judged from the density of autoradiographicspots of the translation products, the induction of senescenceby dark treatment involved an increase in 26 species, a decreasein 11 species, and a temporary increase and subsequent decreasein 8 species of translatable mRNA. A similar pattern of changein protein synthesis was also observed in the dark-treated cotyledonswhen the cotyledons were pulse-labeled with ³⁵S-methionine andthe soluble proteins separated by two-dimensional gel electrophoresis,though the polypeptide pattern on the gel did not coincide exactlywith those of the cell-free translation products. These findingsstrongly suggest that the process of leaf senescence is notsimply a passive and gradual death of the tissue, but involvesa drastic and sequential response of the cells to environmentalstimuli with respect to the gene expression of the cells. (Received July 21, 1987; Accepted September 30, 1987)     

The promotive effect of interleukin 4 with interleukin 2 in the proliferation of tumor-infiltrating lymphocytes from patients with malignant tumor

In adoptive immunotherapy, the number of effector cells is one of the major factors relating to the therapeutic efficacy. We demonstrated that tumor-infiltrating lymphocytes (TILs) were stimulated to proliferate by incubation with interleukin 2 (IL-2) plus interleukin 4 (IL-4). TILs cultured with IL-2 plus IL-4 increased 3.1-fold more than TILs cultured with IL-2 alone. However, IL-4 did not alter the cytotoxic activity of TILs against autologous tumor cells and established tumor cell lines. It is suggested that IL-2 receptor is related to the mechanism of the proliferation of activated TILs cultured by combination with IL-2 and IL-4. Thus, the combination of IL-2 and IL-4 may increase the efficacy of adoptive immunotherapy using activated TILs.