Verbascoside production by plant cell cultures

Verbascoside was found to be produced in all calli derived from eleven species that contained the compound in their leaves. Cell suspension cultures were also established in three species, i.e., Leucosceptrum japonicum f. barbinerve, Syringa josikaea, and Sy. vulgaris, all of which were found to produce verbascoside at more than 1 g/l. Of the three species, suspension cultures of L. japonicum f. barbinerve showed rapid growth and the highest yield of verbascoside (1.89 g/l). In these cultures, the effects of major salt concentration in B5 medium on cell growth and verbascoside production were examined. Maximum cell growth and maximum verbascoside production were both achieved by reducing the major salt concentration to half that of the original medium.     

Mechanism of melatonin action

Melatonin transduces the effect of photoperiod on the neuroendocrine system. Synthesis of melatonin in the pineal gland is well described, but the location of its target(s) and the mechanism of its action are little known. In attempt to localize melatonin target(s), the presence of high affinity binding sites in rat brain was determined. Such sites were detected in discrete brain areas, including the hypothalamus and anterior pituitary. Subcellular analysis indicated these binding sites were on plasma membranes, which suggests that melatonin modulates cell functions through intracellular second messengers. The effects of melatonin on second messengers were studied using the neonatal anterior pituitary, in which melatonin is known to inhibit the LHRH-induced release of LH. Studies on the effects of melatonin on second messenger indicated [corrected] that melatonin inhibits accumulation of cAMP and cGMP as well as synthesis of diacylglycerol and release of arachidonic acid. Time-course analysis indicates that inhibition by melatonin of the LHRH-induced release of LH increases following long preincubation. Since the effect of melatonin on LHRH-induced release of LH is prevented by dibutyryl cAMP, we conclude that melatonin might act by inhibiting production of cAMP.     

Neutrophils-induced increase of adenosine triphosphate depletion in rat neonatal cardiac myocytes with impaired energy metabolism.

Isolated, cultured rat neonatal cardiac myocytes were placed in medium supplemented with mitochondrial respiratory inhibitor potassium cyanide which caused a rapid adenosine triphosphate (ATP) depletion. These myocytes with the impaired energy metabolism ("hypoxia-like state") were exposed to unstimulated human neutrophils. Effect of human neutrophils on the myocytes in the "hypoxia-like state" was quantified as a total change in the amount of ATP in cardiac cells. After 5 hours of incubation of neutrophils with the myocytes in the "hypoxia-like state" an additional decrease (of 50 per cent) in ATP content was observed. Since catalase (which destroys hydrogen peroxide) prevented the further decline in ATP level in the myocytes with impaired energy metabolism, it seem that hydrogen peroxide and possibly their products are responsible for this effect. These results suggest that unstimulated human neutrophils after activation by the contact with injured cardiac cells caused further decrease of ATP level in target cells.     

Whole blood superoxide anion generation and efficiency of some erythrocyte antioxidant systems during recombinant human erythropoietin therapy of uremic anemia

Six chronic uremic patients on regular hemodialysis treatment were given recombinant human erythropoietin (r-huEPO) in a dose of 50 U/kg of body weight intravenously thrice weekly for 14 weeks. Following r-huEPO therapy, unstimulated whole blood superoxide anion ( ₂) generation did not change significantly, while opsonized zymosan-stimulated whole blood ₂ generation increased. At the same time, erythrocyte superoxide dismutase and, in particular, glutathione peroxidase activities were found to be reducing with concomitant lowering of erythrocyte malonydialdehyde (MDA) concentrations and increase in plasma MDA concentrations.     

Bone morphogenetic protein 4 (BMP-4), a member of the TGF-beta family, in early embryos of Xenopus laevis: analysis of mesoderm inducing activity

We have screened a Xenopus ovary cDNA library using a synthetic oligonucleotide derived from that part of the inhibin beta A sequence, which is highly conserved within the TGF-beta family. Out of several clones yielding autoradiographic signals four turned out to represent Xenopus counterparts to the human bone morphogenetic protein 4 (BMP-4). Each two of the four sequences are nearly identical and probably account for different alleles whereas the two pairs showing 5% divergence may have arisen by genome duplication in this tetraploid species. The amino acid sequence of the Xenopus protein is 80% homologous to the human sequence showing no single exchange within the last 100 amino acids at the C-terminus. This region, which constitutes the main part of the mature, biologically active protein, also exhibits substantial homologies to other representatives of the TGF-beta family, especially to the Drosophila DPPC protein. Transfection of COS-1 cells with the Xenopus BMP-4 sequence under control of the CMV-promoter leads to the secretion of a protein which exhibits mesoderm inducing activity when tested with animal cap explants from Xenopus blastula stage embryos.     

Biodegradation of toxic and environmental pollutants.

Organic chemicals that are toxic to humans and to the environment can be transformed and metabolized by a variety of microorganisms. Such chemicals include trichloroethylene, chloroform, carbon tetrachloride, toluene, phenols, chlorinated phenols, polychlorinated biphenyls and polyaromatic hydrocarbons. This review focuses on some of the most important recent developments in the biodegradation of these toxic chemicals. Depending on the compound and the organism, the extent of our understanding ranges from the molecular level to the conceptual.     

Informed consent: study of quality of information given to participants in a clinical trial.

OBJECTIVE--To determine whether the participants in a clinical trial had perceived adequate information about the trial according to the guidelines of the Declaration of Helsinki. DESIGN--About 18 months after the end of a gynaecological clinical trial the participants received a questionnaire by post, which focused on the quality of the information given to them before entering the trial. Neither researchers nor participants were aware in advance that the trial would become the subject of this follow up investigation. SETTING--Eight different centres in Sweden. SUBJECTS--43 women out of the 53 who completed the trial (mean (range) age 23 (16 to 35) years) returned the questionnaire. MAIN OUTCOME MEASURES--Adequacy of the information (based on requirements of the Declaration of Helsinki) to enable the following: understanding of the aims of the study; awareness of what participation meant; and awareness of the possibility of withdrawing from participation at any time. Motives for agreeing to participate, and a subjective evaluation of the given information were also recorded. RESULTS--All but one of the participants had been aware that they were taking part in a research project. Five women stated that they had not been aware that a second laparoscopy was performed only for research reasons. Seven women reported that they had not been aware of the meaning of participating in the project and 17 that they had had no information about the possibility of withdrawing from the study whenever they wanted. In the subjective rating 22 women considered the information given as good or very good. There was a systematic variation in the quality of the given information among the eight centres. CONCLUSION--Although all but one of the participants had been aware that they were taking part in a clinical trial, the quality of the information understood and recalled by participants varied, and in many cases clearly did not meet the guidelines of the Declaration of Helsinki. Variations among centres in participants'' perception of information suggest that deficiencies in perception may be caused by informers rather than the participants.     

Mechanisms for activation and subsequent removal of cytosolic Ca2+ in bradykinin-stimulated neuronal and glial cell lines

Mechanisms for activation and for removal of cytosolic Ca2+ after stimulation with bradykinin were investigated in two neural cell lines by measuring cytosolic Ca2+ activity and 45Ca2+ fluxes. In the neuronal (neuroblastoma x glioma hybrid) and in the glial (rat glioma) cell lines, the transient, bradykinin-induced rise in cytosolic Ca2+ activity (determined by fura-2 or indo-1 fluorescence) was blocked by a bradykinin B2 receptor antagonist. Ca2+ ionophores (ionomycin and 4-Br-A23187) caused a comparable transient rise in cytosolic Ca2+ activity. After addition of ionophores, the Ca2+ response to bradykinin was reduced or completely blocked in both cell lines. At the concentrations used, the ionophores primarily depleted intracellular Ca2+ stores and prevented refilling of the stores. Thus, the bradykinin-induced rise of cytosolic Ca2+ activity seems to be mostly due to Ca2+ release from internal stores. In the neuronal but not in the glial cell line, a brief stimulation by bradykinin of 45Ca2+ uptake was followed by a long-lasting inhibition below control values. Thus, in the neuronal cells bradykinin presumably blocks Ca2+ channels by a readily reversible, pertussis toxin-insensitive mechanism. Excess cytosolic Ca2+ of the bradykinin-stimulated cells is mostly not resequestered into the internal Ca2+ pool accessible to bradykinin, but is mainly extruded through the plasma membrane, as indicated by (i) stimulation of 45Ca2+ release by bradykinin, (ii) quick reduction by bradykinin of cellular 45Ca2+ content of cells preequilibrated with 45Ca2+, and (iii) diminution of the ionophore-inducible Ca2+ response after the addition of bradykinin.     

Properties of purified squalene-hopene cyclase from Bacillus acidocaldarius

The squalene-hopene cyclase from Bacillus acidocaldarius cytoplasmic membrane, was purified to homogeneity by solubilization with Triton X-100, chromatography on DEAE-cellulose, phenyl Sepharose and two gel-filtration columns. The enzyme monomer had a molecular mass of 75 kDa. The sequence of the first 23 amino acids was determined by Edman degradation. The enzyme activity was efficiently inhibited by n-alkyldimethylammonium halides with alkyl chain lengths between 12 and 18 C atoms. Inhibition was also observed with (5-hydroxycarvacryl)trimethylammonium chloride 1-piperidine carboxylate, dodecyldimethylamine N-oxide, azasqualene and farnesol. Competitive inhibition with dodecyltrimethylammonium bromide, (5-hydroxycarvacryl)trimethylammonium chloride 1-piperidine carboxylate and dodecyldimethylamine N-oxide was demonstrated by Lineweaver-Burk plots.