Inositol tetrakisphosphates as second messengers induce Ca(++)-dependent chloride currents in Xenopus laevis oocytes.

Microinjection of inositol 1,3,4,5-tetrakisphosphate or inositol 1,4,5-trisphosphate induced distinct chloride membrane currents in defolliculated Xenopus laevis oocytes. To decide whether these Cl(-)-currents were due to the injected compounds or their metabolic products, [3H]Ins(1,3,4,5)P4 or [3H]Ins(1,4,5)P3 were injected into oocytes and their metabolites were analyzed by HPLC. Our results indicate that Ins(1,3,4,5)P4 itself or its metabolite Ins(1,3,4,6)P4 is able to induce Cl(-)-membrane currents, most likely by increasing the cytosolic Ca(++)-concentration.     

A combined study of aggregation, membrane affinity and pore activity of natural and modified melittin

The pore activity of melittin and several chemically modified derivatives has been investigated using conductance measurements on planar lipid bilayers and marker release from small unilamellar vesicles. The modifications included N-terminal formylation, acetylation, succinylation and modification of the tryptophan residue. All of the compounds showed bilayer permeabilizing properties, though quantitative differences were evident. These comprised changes in the voltage dependence of the conductance, in the single-pore kinetics, in the concentration of aqueous peptide required to induce a given pore activity and in the apparent 'molecularity' reflected by the power law of its concentration dependence. A strong tendency for disrupting bilayers was not always correlated with strong pore activity. For a better understanding of these results, measurements of pore activity were complemented by studying the aggregation behavior in solution and the water-membrane partition equilibrium. Modifications of charged residues gave rise to significant changes in the aggregation properties, had virtually no influence on the partition coefficient. The latter decreased strongly, however, as a result of tryptophan modification. Analysis of the isotherms was consistent with the assumption that the arginine residues in melittin do not contribute very much to charge accumulation at the immediate membrane/water interface.     

Multiple shoot production from seedling explants of slash pine (Pinus elliottii,Engelm.)

Hypocotylary explants obtained from 30- to 40-day-old slash pine (Pinus elliottii, Engelm.) seedlings treated with 6-benzylaminopurine produced multiple buds that eventually elongated into axillary shoots. The explants were pulse treated (45-s dip) with 6-benzylaminopurine (22.2, 111, 222 M) plus a control and cultured on three different basal media containing activated charcoal (0.5% w/v). Hormonal concentration and basal medium were compared for the number and size of axillary shoots induced after 12 and 29 days. The greatest number of axillary shoots was produced by explants that were pulse treated with 111 M 6-benzylaminopurine and cultured on Gresshoff and Doy medium. The axillary shoots were fewer in number per explant than shoots previously reported resulting from hormonally induced advantitious buds of slash pine, but the axillary shoots developed more rapidly.Abbreviations BA 6-benzylaminopurine - DMSO dimethyl sulfoxide - PAR photosynthetically active radiation     

Ecology ofVibrio vulnificus in Galveston Bay oysters,suspended particulate matter,sediment and seawater: Detection by monoclonal antibody — immunoassay — most probable number procedures

Summary Oysters, suspended particulate matter (SPM), sediment and seawater samples were collected from West Galveston Bay, Texas over a 16-month period and analyzed for the presence ofVibrio vulnificus, a naturally-occurring human marine pathogen. Detection and enumeration ofV. vulnificus was performed using a species-specific monoclonal antibody (mAb FRBT37) in an enzyme immunoassay (EIA)-most probable number (MPN) procedure capable of detecting as few as 2000 target organisms.V. vulnificus was not detected in seawater, oyster or SPM samples during the cold weather months, but was detected at low levels in several sediment samples during this time period. Increased levels of the organism were first observed in early spring in the sediment, and then in SPM and oysters. The major increase inV. vulnificus occurred only after the seawater temperature had increased above 20°C and the winter-spring rainfall had lowered the salinity below 16. The highestV. vulnificus levels at each site were associated with suspended particulate matter. These results are consistent with the hypothesis that (1)V. vulnificus over-winters in a floc zone present at the sediment-water interface, (2) is resuspended into the water column in early spring following changes in climatic conditions, (3) colonizes the surfaces of zooplankton which are also blooming during early spring and (4) are ingested by oysters during their normal feeding process.     

Determination of clozapine and its major metabolites in human serum using automated solid-phase extraction and subsequent isocratic high-performance liquid chromatography with ultraviolet detection

An isocratic high-performance liquid chromatographic (HPLC) method with ultraviolet detection is described for the quantification of the atypical neuroleptic clozapine and its major metabolites, N-desmethylclozapine and clozapine N-oxide, in human serum or plasma. The method included automated solid-phase extraction on C₁₈ reversed-phase material. Clozapine and its metabolites were separated by HPLC on a C₁₈ ODS Hypersil analytical column (5 μm particle size; 250 mm × 4.6 mm I.D.) using an acetonitrile—water (40:60, v/v) eluent buffered with 0.4% (v/v) N,N,N′,N′-tetramethylethylenediamine and acetic acid to pH 6.5. Imipramine served as internal standard. After extraction of 1 ml of serum or plasma, as little as 5 ng/ml of clozapine and 10 or 20 ng/ml of the metabolites were detectable. Linearity was found for drug concentrations between 5 and 2000 ng/ml as indicated by correlation coefficients of 0.998 to 0.985. The intra- and inter-assay coefficients of variation ranged between 1 and 20%. Interferences with other psychotropic drugs such as benzodiazepines, antidepressants or neuroleptics were negligible. In all samples, collected from schizophrenic patients who had been treated with daily oral doses of 75–400 mg of clozapine, the drug and its major metabolite, N-desmethylclozapine, could be detected, while the concentrations of clozapine N-oxide were below 20 ng/ml in three of sixteen patients. Using the method described here, data regarding relations between therapeutic or toxic effects and drug blood levels or metabolism may be collected in clinical practice to improve the therapeutic efficacy of clozapine drug treatment.     

Dielectric probe of the electric-field-sensitive peptide alamethicin

Dielectric constant and loss of alamethicin in solvents of various degrees of lipophilicity (namely mixtures of n-octanol and dioxane) have been measured at frequencies from 5 kHz to 50 MHz. By means of a conventional Cole-Cole approach, dielectric properties were evaluated to obtain information about the structural and dipolar properties of the peptide in view of its function as a voltage-dependent pore former in membranes. The results for a pure octanol solvent (together with an apparent molecular weight determined by ultracentrifugation) indicate the existence of primarily monomeric particles of quite elongated shape and of comparatively high dipole moment. Adding dioxane was found to yield considerable aggregation and a decrease of polarity.     

Purification and structural properties of adenylylated and deadenylylated glutamine synthetase from Rhodopseudomonas sphaeroides

Glutamine synthetase (GS) of Rhodopseudomonas sphaeroides is regulated by adenylylation and deadenylylation. The extent of adenylylation/deadenylylation of the enzyme in cell free extracts was influenced by inorganic phosphate (P _i), -ketoglutarate, ATP and other nucleotides. While P _i and -ketoglutarate stimulated deadenylylation, ATP and other nucleotides enhanced adenylylation of the GS. By using proper combinations of the effectors and incubation conditions, any desired adenylylation state of GS could be adjusted in vitro. The enzyme was purified to electrophoretic homogenity by three steps including affinity chromatography on 5-AMP-Sepharose. Adenylylated and deadenylylated enzyme showed different UV-spectra and isoelectric points. The native enzyme had a molecular weight of 600,000, deadenylylated subunits of 50,000±1,000. Electron microscopic investigations revealed a dodecameric arrangement of subunits in two hexameric planes.     

Condensation of chromatin into chromosomes preserves an open configuration but alters the DNase I hypersensitive cleavage sites of the transcribed gene

DNase I was used to probe the molecular organization of the chicken ovalbumin (OV) gene and glyceraldehyde 3-phosphate dehydrogenase (GPD) gene in interphase nuclei and in metaphase chromosomes of cultured chicken lymphoblastoid cells (MSB-1 line). The OV gene was not transcribed in this cell line, whereas the GPD gene was constitutively expressed. The GPD gene was more sensitive to DNase I digestion than the OV gene in both interphase nuclei and metaphase chromosomes, as determined by Southern blotting and liquid hybridization techniques. In addition, we observed DNase I hypersensitive sites around the 5' region of the GPD gene. These hypersensitive sites were not always at the same locations between the interphase nuclei and metaphase chromosomes. Our results suggest that chromatin condensation and decondensation during cell cycle alters nuclease hypersensitive cleavage sites.     

Taxonomic relations between archaebacteria including 6 novel genera examined by cross hybridization of DNAs and 16S rRNAs

DNAs from 16 species of archaebacteria including 6 novel isolates were hybridized with 16S rRNAs from 7 species representing different orders or groups of the urkingdom of archaebacteria. The yields, normalized for the number of genes per microgram of DNA, and the temperature stabilities of all hybrids were determined and related to each other. A taxonomic tree constructed from such fractional stability data reveals the same major divisions as that derived from comparative cataloging of 16S rRNA sequences. The extreme halophiles appear however as a distinct order besides the three known divisions of methanogens. The methanogens, the halophiles and Thermoplasma form one of two clearly recognizable branches of the archaebacterial urkingdom. The order represented by Sulfolobus and the related novel order Thermoproteales form the other branch. Three novel genera, Thermoproteus, Desulfurococcus and the "stiff filaments" represent three families of this order. The extremely thermophilic methanogen Methanothermus fervidus belongs to the Methanobacteriales. SN1, a methanogen from Italy, appears as another species of the genus Methanococcus. Another novel methanogen, M3, represents a genus or family of the order Methanomicrobiales.