Lobeline production by hairy root culture of Lobelia inflata L.

Hairy roots were obtained following inoculation of the stems of Lobelia inflata L. with Agrobacterium rhizogenes strain ATCC 15834. These hairy roots contained agropine and mannopine. In addition, lobeline was detected by HPLC and confirmed by mass spectrometry. Various media were tested for the growth of hairy roots as well as for the content of lobeline in hairy roots. The growth rate of hairy roots cultured in Nitsch and Nitsch's medium was approximately one third of those cultured in other media. The lobeline content of hairy roots (18–54 g/g dry weight) cultured in these media was the same order of magnitude compared with that of roots of L. inflata (24 g/g dry weight) cultivated in pots. The hairy roots cultured in Nitsch and Nitsch's medium were morphologically different from those cultured in other media.Abbreviations MS medium Murashige and Skoog's medium - 1/2 MS medium one-half strength of the standard Murashige and Skoog's medium - B5 medium Gamborg's B5 medium - NN medium Nitsch and Nitsch's medium - FW fresh weight - DW dry weight     

Alterations of human erythrocyte membrane fluidity by oxygen-derived free radicals and calcium

Two possible reasons for the structural alterations of cell membranes caused by free radicals are lipid peroxidation and an increase in the intracellular calcium ion concentration. To characterize the alterations in membrane molecular dynamics caused by oxygen-derived free radicals and calcium, human erythrocytes were spin-labeled with 5-doxyl stearic acid, and alterations in membrane fluidity were quantified by electron spin resonance oxidase (0.07 U/mL) decreased membrane fluidity, and the addition of superoxide dismutase and catalase inhibited the effect on membrane fluidity of the hypoxanthine-xanthine oxidase system. Hydrogen peroxide (0.1 and 1 nM) also decreased membrane fluidity and caused alterations to erythrocyte morphology. In addition, a decrease in membrane fluidity was observed in erythrocytes incubated with 2.8 mM CaCl₂. On the other hand, incubation of erythrocytes with calcium-free solution decreased the changes in membrane fluidity caused by hydrogen peroxide.

These results suggest that changes in membrane fluidity are directly due to lipid peroxidation and are indirectly the result of increased intracellular calcium concentration. We support the hypothesis that alterations of the biophysical properties of membranes caused by free radicals play an important role in cell injury, and that the accumulation of calcium amplifies the damge to membranes weakened by free radicals.     

Seed coat morphology and evolution in Celtidaceae and Ulmaceae (Urticales)

The seed coat surface morphology of Celtidaceae and Ulmaceae (Urticales) indicates a significant evolutionary diversity.Celtis, Chaetachme andPteroceltis (Celtidaceae) have a unique sculpturing with many crateriform holes; such holes occasionally sparsely occur in seeds ofAphananthe, Gironniera (Celtidaceae) andPlanera (Ulmaceae), but not in those of the nine remaining genera of the two families. The perforated seed coat further occurs in at least some genera of all other urticalean families. A pattern of its occurrence in families and genera suggest that the perforation represents a common archaic feature of all Urticales, rather than a feature derived many times independently within the order. The seed coat of Celtidaceae and Ulmaceae seems to have lately lost the holes probably by a neotenic evolution: one or more times within Celtidaceae, and one time in an ancestral line leading to all Ulmaceae. The derived reticulate seed coat surface sculpturing, which is shared byGironniera (Celtidaceae) and some Ulmaceae, is probably the result of parallel evolution. On the basis of evidence from seed coat morphology and other sources, close relationships ofLozanella, Parasponia andTrema within Celtidaceae, as well as variously distinct positions ofAmpelocera, Aphananthe andGironniera, are also discussed.     

Antimicrobial activities of polyether compounds of dinoflagellate origins

Twelve polyether compounds originating from dinoflagellates were tested for growth-inhibiting activities againstAspergillus niger, Penicillium funiculosum, Candida rugosa, Escherichia coli, Bacillus megaterium andStaphylococcus aureus by a paper disc method. These polyethers represent six groups of different skeletons and originate from three species;Prorocentrum lima, Dinophysis fortii andGambierdiscus toxicus. Potent antifungal activities were observed with okadaic acid and its two congeners, desulfated yessotoxin, and ciguatoxin but not with okadaic acid esters, prorocentrolide, pectenotoxin-1, yessotoxin, maitotoxin, and desulphated maitotoxin. The antifungal activities and mouse lethalities of the polyethers were markedly affected by slight modification of their structures. Antibacterial potency of the tested compounds was not significant.     

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.     

Peri-operative immunotherapy with OK-432

Pre- and postoperative intradermal administration of OK-432 enhanced the SU-PS skin reaction in patients with gastric cancer, but failed to prevent a fall in the NK activity induced by the operation.The change in NK activity was not associated with a change in the proportion of Leu 7-positive cells, but was related to Leu 11a-positive cells. Intradermal injection of OK-432 increased the proportion of Leu 7-positive cells in the patients in whom they accounted for less than 20% of lymphocyte population. The case was the same with Leu 11a-positive cells.Intravenous injection of OK-432 tended to increase suppressor-inducer T cells (CD4⁺2HA⁺ cells), B cells and Leu 7-positive cells. Particularly, the proportions of OK-M₁-positive cells and MHC class II antigen-positive cells increased in all patients. Immunotherapy with OK-432 given intravenously at a dose of 0.1 KE appeared to be safe because no side effects were essentially observed.     

Studies on detection of Candida antigen in the sera of mice inoculated orally with Candida albicans

Summary The authors succeeded in establishing a murine model of systemic candidiasis being disseminated from the primary gastrointestinal lesions caused by oral inoculation of Candida albicans. Using this model, an attempt was made for detecting the Candida antigen by enzyme-linked immunosorbent assay using avidin-biotin (AB-ELISA) from the serum of infected mice.Gastrointestinal candidiasis was formed in all of the 20 mice treated with the drugs (antibiotics, antineoplastic agents, hydrocortisone, etc.) and inoculated orally with C. albicans. Fourteen of these mice suffered from submucosal candidiasis, and C. albicans was cultured from the visceral organs in 12 of them. The assay by AB-ELISA was able to detect 1.0 ng/ml Candida mannan in the mouse serum. The Candida antigen was detected in the sera of 11 of the 14 mice with submucosal candidiasis. However, the antigen could not be detected in the sera of the 6 mice with intramucosal candidiasis.The assay by AB-ELISA is more sensitive and specific for the diagnosis of systemic candidiasis than other serological assays.     

Analysis of restriction profiles of Mitochondrial DNA from Sporothrix schenckii and related fungi

Restriction profiles by HaeIII of mitochondrial DNA were studied for classification and distinction of Sporothrix schenckii (100 strains), S. schenckii var. luriei (1), S. curviconia (1), S. inflata (7), Ceratocystis stenoceras (17) and C. minor (7). These 6 species showed unique restriction profiles which could be discriminated from each other. S. schenckii was further separable into 11 types, S. inflata into 4 types, C. stenoceras into 4 types and C. minor into 7 types based on restriction profile heterogeneity.     

Epidermal growth factor (EGF)-induced morphological changes in the basement membrane of chick embryonic skin

Summary The effect of epidermal growth factor (EGF) on the basement membrane structure of chick embryonic skin cultured in a chemically defined medium (BGJb) containing 20 mM hydrocortisone, and EGF at 10, 50, or 100 ng/ml supplemented with 5% delipidized fetal calf serum, was examined by electron microscopy. During development of the epidermis in vitro, EGF (100 ng/ml) caused striking changes to occur in the basement membrane structure and in the keratinization process. The basement membrane frequently became discontinuous with many gaps apparent in section, and occasionally became folded following detachment from the basal surface of the epidermis and protruded into the underlying dermis. In the basal and intermediate cells of EGF-treated epidermis, tonofilament bundles were decreased in number, while desmosomes and hemidesmosomes revealed no significant changes in morphology.     

Induction of tumor growth inhibitory factor (TGIF) in human mononuclear cells by OK-432, a streptococcal preparation

Summary A tumor growth inhibitory factor (TGIF) was induced in the culture supernatant from mixed culture of human peripheral blood mononuclear cells (PBMC) and a streptococcal preparation, OK-432, in vitro. The activity generated in the supernatant increased in a time-dependent fashion and first appeared 6 h after the initiation of culture, reaching its maximum around 48 h. The TGIF was cytostatic against seven of ten human tumor targets, but not against three murine tumor targets. Tumor cell growth was inhibited by a transient contact, i.e., 1 h, with TGIF. The TGIF was produced by lymphocytes but not by monocytes, because the activity was usually enhanced by elimination of plastic-adherent cells from the original PBMC fraction. The TGIF was relatively stable against heating at 56° C for 30 min, but the activity was totally destroyed after heating at 70° C for 5 min. The molecular weight of TGIF was estimated to be about 43×10³ daltons by gel filtration. No interferon (IFN) activity was detected in the TGIF-positive fractions obtained by gel filtration, and the TGIF-positive fractions did not inhibit the growth of tumor necrosis factor (TNF)-sensitive mouse L929 cells. The TGIF activity was not significantly affected in neutralizing tests using specific antibodies against human IFN and TNF. The OK-432 was administered i.p. for management of cancer patients with malignant ascites. Ascites-derived mononuclear cells (ASMC) were obtained before and 3 to 5 days after OK-432 injection. The ASMC obtained after the injection produced TGIF in vitro in the absence of OK-432; the preinjection ASMC showed no such production. A positive correlation was found between TGIF-producing activity by ASMC and the effect of OK-432 injection on ascites volume. These results indicate that TGIF is induced in mononuclear cells by OK-432 not only in vitro but also in vivo and plays an important role in inhibition of tumor growth in cancer patients.