Organellar DNA restriction fragment length polymorphism (RFLP) and nuclear random amplified polymorphic DNA (RAPD) analyses of morphotypes of Gracilaria (Gracilariales,Rhodophyta) from Chile

The extreme phenotypic variability recognized among the species of Gracilaria has highlighted the need for the application of refined methods to help solve taxa identifications. In Chile, there still exists uncertainty about the exact number of Gracilaria species. Our investigations are centered on DNA analyses of morphotypes collected from different geographical locations, namely Lenga and Isla Santa María, Region VIII (36°00 S to 38°00 S), and Maullín, Region X (39°30 S to 43°40 S). These two regions of Chile are considered as areas of confluence of G. chilensis, G. verrucosa, and a species of Gracilariopsis. In this study four morphotypes, from a natural bed located in Maullín, were analyzed for RFLP of plastid DNA and the results compared with data of four morphotypes from a bed in Lenga. The DNA banding patterns from each enzyme digest were identical irrespective of morphotypes and/or locations. In an attempt to unravel the nature of the morphological differences found among Lenga and Maullín morphotypes, RAPD analyses of nuclear DNA were also performed; however, no polymorphism has been found yet. Therefore, the data of this study, as well as concurrent data from preliminary interfertility tests, suggest that all morphotypes belong to a single taxon, Gracilaria chilensis.Departamento de Botánica, Facultad de Ciencias Naturales y Oceanográficas     

Mechanical effects of ET-1 in cardiomyocytes isolated from normal and heart-failed rabbits

Endothelin (ET-1) is found at elevated concentrations in the plasma of patients with heart failure and in animal models of cardiomyopathy. The peptide is a potent positive inotropic agent, the effects of which are mediated by increases in cytosolic Ca²⁺ in cardiomyocytes. The object of this study was to investigate at the cellular level, the actions of ET-1 on contractile function and on Ca²⁺ currents in heart-failed ventricular myocardium. Male New Zealand White rabbits (8 wks) were treated with twice weekly injections of epirubicin (4 mg/kg/wk, n=7) or with saline (n=7) for 6 wks, followed by a washout period of 2 wks. Ventricular cardiomyocytes were isolated from rabbit hearts using Langendorff perfusion with collagenase; contractile function was examined using a video microscopy method, and L-type Ca²⁺ currents were recorded using a whole-cell patch-clamp technique. ET-1 produced a concentration-dependent increase in contractile response (% increase from basal value) to a maximum at 1 nM ET-1 of 69 ± 11% (mean ± S.D.) in control cardiomyocytes and 33 ± 6% in heart-failed cells. However, there was no significant change in the EC₅₀ obtained with ET-1 for healthy (0.31 ± 0.1 nM) and for failed cardiomyocytes (0.24 ± 0.1 nM). The effects of ET-1 on L-type Ca²⁺ channels were similar with a peak amplitude at 1 nM ET-1 of –3.26 ± 0.8 in control cardiomyocytes and –3.32 ± 0.9 nA in heart-failed cells. The attenuation of the contractile response to ET-1 in heart-failed cells may reflect a desensitization of ET receptors as a consequence of elevated circulating levels of ET and was not reflected by alteration of transmembrane Ca²⁺ conductance. It is probable, therefore, that multiple signalling pathways are involved in the actions of ET on ventricular myocardium.Recipient of Servier Investigator Award     

Occurrence of heavy metals,sodium, calcium,and potassium in human hair,teeth, and nails

Heavy metals in biological samples: nails, teeth, and hair were examined during 1991–1993. Investigations of biological samples (hairn=249 samples, teethn=145, nailsn=80 samples) were provided for inhabitants of selected towns in Beskid Śląski. The towns are small mountain towns in southern Poland: Wista, Szczyrk, Istebna, Koniaków, and Jaworzynka. The analysis of ANOVA and MANOVA variances were used for biological samples in the context of age, sex, and type of samples for 12 elements (Pb, Cd, Cr, Zn, Fe, Cu, Mn, Ni, Co, Ca, Na, and K). The matrix correlation and cluster analysis were applied to explain the behavior of metals in human hair, teeth, and nails.     

AtJ1, a mitochondrial homologue of theEscherichia coli DnaJ protein

The nucleotide sequence of a cDNA clone fromArabidopsis thaliana ecotype Columbia was determined, and the corresponding amino sequence deduced. The open reading frame encodes a protein, AtJ1, of 368 residues with a molecular mass of 41 471 Da and an isoelectric point of 9.2. The predicted sequence contains regions homologous to the J- and cysteine-rich domains ofEscherichia coli DnaJ, but the glycine/phenylalanine-rich region is not present. Based upon Southern analysis,Arabidopsis appears to have a singleatJ1 structural gene. A single species of mRNA, of 1.5 kb, was detected whenArabidopsis poly(A)⁺ RNA was hybridized with theatJ1 cDNA. The function ofatJ1 was tested by complementation of adnaJ deletion mutant ofE. coli, allowing growth in minimal medium at 44°C. The AtJ1 protein was expressed inE. coli as a fusion with the maltose binding protein. This fusion protein was purified by amylose affinity chromatography, then cleaved by digestion with the activated factor X protease. The recombinant AtJ1 protein was purified to electrophoretic homogeneity.In vitro, recombinant AtJ1 stimulated the ATPase activity of bothE. coli DnaK and maize endosperm cytoplasmic Stress70. The deduced amino acid sequence of AtJ1 contains a potential mitochondrial targeting sequence at the N-terminus. Radioactive recombinant AtJ1 was synthesized inE. coli and purified. When the labeled protein was incubated with intact pea cotyledon mitochondria, it was imported and proteolytically processed in a reaction that depended upon an energized mitochondrial membrane.Abbreviations MBP maltose binding protein - PCR polymerase chain reaction - Stress70c the cytosolic member of the 70 kDA family of stress-related proteins     

NADP+-isocitrate dehydrogenase in germinating cucumber cotyledons: Purification and characterization of a cytosolic isoenzyme

The activity of NADP⁺-dependent isocitrate dehydrogenase (ICDH, EC 1.1.1.42) was investigated during the post-germinative growth of cucumber ( Cucumis sativus L. cv. Marketmore) seedlings. Isoelectric focusing showed the presence of several isoenzymes, two of which represented 70–80% of the total NADP⁺-ICDH activity in cotyledons of seedlings grown in the dark. They had pI values between 4.8 and 5.8. The isoenzyme with higher pI was purified to homogeneity by hydrophobic interaction, affinity, hydroxylapatite and anion exchange chromatography. The purified isoenzyme is a dimeric protein, consisting of two apparently identical 43-kDa subunits. It is specific for NADP⁺, inhibited by ATP and by 2-oxoglutarate, whereas it is not inhibited by citrate, succinate, and glyoxylate. The data indicate that NADP⁺-ICDH from cucumber is structurally similar to ICDHs from other plants, but it shows some peculiar biochemical characteristics.     

Rapid reactions of spruce cells to elicitors released from the ectomycorrhizal fungus Hebeloma crustuliniforme, and inactivation of these elicitors by extracellular spruce cell enzymes

Elicitors released from hyphae or cell walls of the ectomycorrhizal fungus Hebeloma crustuliniforme (Bull. ex Fries.) Quél. induced in suspension-cultured cells of Picea abies (L.) Karst. a set of fast reactions: (i) an immediate efflux of Cl^– into the medium, followed by a K⁺ efflux; (ii) an influx of Ca²⁺ (measured as accumulation of ⁴⁵Ca²⁺ in the cells); (iii) a phosphorylation of a 63-kDa protein and dephosphorylation of a 65-kDa protein (detectable by 4 min after elicitor application); (iv) an alkalinization of the medium, and (v) a transient synthesis of H₂O₂. The removal of extracellular Ca²⁺ by EGTA delayed the elicitor-induced alkalinization. A further reduction of this response could be achieved by TMB-8 an inhibitor of Ca²⁺ release from intracellular stores. Moreover, the inhibition of protein kinase activity by staurosporine prevented the extracellular alkalinization completely. However, the effectiveness of the elicitors in inducing the extracellular alkalinization was strongly impaired by constitutively secreted enzymes of spruce cells which cleaved the elicitors to inactive fragments. It is suggested that in ectomycorrhizae the efficacy of elicitors released from fungal cell walls is controlled by apoplastic enzymes of the host; the plant itself is able to reduce the activity of fungal elicitors on their way through the plant cell wall. But those elicitors which finally reach the plasma membrane of host cells induce reactions that are similar to the early defense reactions in plant-pathogen interactions.Abbreviations DW dry weight - FW fresh weight - TMB-8 3,4,5 trimethoxybenzoic acid 8-(diethylamino)-octyl ester We thank Prof. M. Zenk (Universität München, Germany) for providing spruce cell cultures, and Dr. I. Kottke (Universität Tübingen, Germany) for isolates of Hebeloma crustuliniforme Tü 704. We are also thankful to Dr. W. Mayer (Universität Tübingen) for valuble discussions. This work was supported by Deutsche Forschungsgemeinschaft. B. Zitterell-Haid was financed by Graduiertenkolleg Interaktion in Waldökosystemen (supported by Deutsche Forschungsgemeinschaft) and G. Hebe by a scholarship of the Landesgraduiertenförderungsgesetz.     

Liver and intestinal fatty acid binding proteins in control and TGFβ1 gene targeted deficient mice

The effect of transforming growth factor beta-1 (TGF1) expression on fatty acid binding proteins was examined in control and two strains of gene targeted TGF1-deficient mice. Homozygous TGF1-deficient 129 × CF-1, expressing multifocal inflammatory syndrome, had 25% less liver fatty acid binding protein (L-FABP) when compared to control mice. The decrease in L-FABP expression was not due to multifocal inflammatory syndrome since homozygous TGF1-deficient/immunodeficient C3H mice on a SLID background had 36% lower liver L-FABP than controls. This effect was developmentally related and specific to liver, but not the proximal intestine, where L-FABP is also expressed. Finally, the proximal intestine also expresses intestinal-FABP (1-FABP) which decreased 3-fold in the TGF1-deficient/immunodeficient C3H mice only. Thus, TGF1 appears to regulate the expression of L-FABP and I-FABP in the liver and the proximal intestine, respectively.Abbreviations L-FABP liver fatty acid binding protein - I-FABP intestinal fatty acid binding protein - TGF1 transforming growth factor beta-1 - TNF- tumor necrosis factor- - MIP- macrophage inflammatory protein- - PMSF phenylmethyl sulfonyl fluoride - PBS phosphate buffered saline     

Evaluation of hydroxyl radical-scavenging property of garlic

While antibiotics are broadly used in dental and medical therapy, little attention has been directed towards the potential toxic side effects of antibiotics on tissue regeneration. Here we examined the effect of a quinolone antibiotic, pefloxacin (Rhone Poulenc) on rat parotid gland responses to chronic isoproterenol treatment. Groups of rats received injections of isoproterenol to induce glandular growth, saline (controls), pefloxacin, or isoproterenol and pefloxacin in combination. Parotid gland weight decreased significantly after pefloxacin treatment for 7 days as well as inhibiting glandular enlargement provoked by isoproterenol. The same trend was observed for the rates of DNA synthesis, with the incorporation of [³H]-thymidine in isoproterenol/pefloxacin-treated rats reduced to 49% of isoproterenol treatment alone levels. Saline-treated animals were 42% of the rate of [³H]-thymidine incorporation into DNA observed in isoproterenol treated rats. While isoproterenol treatment increased steady-state mRNA levels for fos, jun, myc, src, c-erbB-2, ras and topo II, inclusion of pefloxacin with the isoproterenol regimen blocked these increases. Pefloxacin treatment by itself did not alter proto-oncogene mRNA levels in the parotid gland. Glandular amylase activity was decreased in the pefloxacin treated group, while the combination of isoproterenol with pefloxacin did not decrease glandular amylase levels to the extent of that observed with -agonist treatment alone. In acute experiments, pefloxacin significantly decreased the volume of saliva secreted by the parotid gland. These results suggest that quinolone-based antibiotics disturb the secretory function of the parotid gland and can inhibit cell proliferation and regeneration. (Mol Cell Biochem 165: 55–63, 1996)