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.     

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.     

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.     

Hell-und Dunkeladaptation der Augen vonPieris brassicae L. (Lepidoptera)

Summary The compound eyes ofPieris brassicae L. have a tiered retina. During light and dark adaptation, ultrastructural changes have been observed throughout the length of the ommatidia in the latero-ventral region of the eyes. These changes have been quantitated by mapping at distinct levels of the ommatidia, and plotted as histograms. Both in visual cells and secondary pigment cells and at the attachment region between crystalline cone and rhabdome such ultrastructural changes have been found to be correlated to the state of adaptation.Distal and proximal photoreceptor cells show different adaptation mechanisms. Whereas the distal cells show a clear pupil mechanism in their distal parts, there is only very little horizontal movement of pigment granules in the proximal cells. In the proximal cells, multivesicular bodies (MVB) are always abundant, while in the distal cells their number is small and increases slightly during light adaptation. In the proximal cells light adaptation causes pigment granules, located in the distal process, to move proximally. Increasing the light intensity from 160 to 1600 W/cm² results in more intense migration of pigments.In the secondary pigment cells, a slight but significant distal movement of pigment granules is observed at high light intensity. If continued this condition causes the granules to aggregate in the vicinity of the apical cell membrane, and to move up to the distal inflated extensions of the distal processes formed by these cells. In dark adapted eyes, these processes are nearly devoid of pigment and the pigment granules beneath the apical membrane disperse. In addition to these structural changes, there is a tendency for retinal movements at the attachment from crystalline cone to rhabdome. — The various adaptation mechanisms are not equally well developed in different regions of the compound eye.

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Hell-und Dunkeladaptation der Augen vonPieris brassicae L. (Lepidoptera)

Zusammenfassung Die Retina vonPieris brassicae L. ist mehrreihig. Erstmals wurden feinstrukturelle Veränderungen während der Hell und Dunkeladaptation über die gesamte Länge der Ommatidien des latero-ventralen Augenbereichs anhand von Kartierungen in vergleichbaren Höhen der Ommatidien untersucht und in Histogrammen wiedergegeben. — Sowohl in den Sehzellen als auch Nebenpigmentzellen und am Übergang von Kristallkegel zum Rhabdom wurden feinstrukturelle Veränderungen in Korrelation mit der Adaptation gefunden.Die Adaptation erfolgt bei distalen und proximalen Sehzellen jeweils auf andere Art. Während die distalen Sehzellen in ihrem distalsten Bereich sehr gut die Pupillenreaktion zeigen, adaptieren die proximalen Sehzellen nur geringfügig mit horizontaler Pigmentwanderung. Auch die Anzahl der multivesikulären Körper (MVB), die in den proximalen Sehzellen immer groß ist, steigt bei Helladaptation (HA) nur in den distalen Sehzellen etwas an. In den proximalen Sehzellen wandern die Pigmentgranula bei HA geringfügig aus dem distalen Fortsatz dieser Sehzellen proximalwärts. Intensitätssteigerung auf das 10fache (von 160 auf 1600W/cm²) bewirkt eine Verstärkung der genannten Pigmentwanderungs-Reaktionen in den Sehzellen.Die Granula der Nebenpigmentzellen wandern bei HA mit starker Intensität etwas distalwärts. — Bei starker langer HA häufen sich diese Granula unter der apikalen Membran dieser Nebenpigmentzellen und wandern bis in die distalen kleinen Erweiterungen der distalen Fortsätze dieser Zellen. Bei Dunkeladaptation (DA) sind diese Fortsätze nahezu frei von Pigment; unter der apikalen Zellmembran verteilen sich die Pigmente locker. Außerdem besteht am Übergang von Kristallkegel zu Rhabdom die Tendenz zur Retinomotorik. — In den verschiedenen Augenbereichen erfolgen die genannten Adaptationsreaktionen unterschiedlich gut.

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Mit Unterstützung der Deutschen Forschungsgemeinschaft und der Stiftung Volkswagenwerk

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Herrn Prof. Dr. Kurt Hamdorf (Bochum) danken wir für kritische Diskussion und Fräulein Althaus für die graphischen Darstellungen     

The α-keto acids of branched-chain amino acids: Simplified derivatization for physiological samples and complete separation as quinoxalinols by packed column gas chromatography

By use of a relatively new mixed stationary phase, complete separation of the branched-chain α-keto acids as O-trimethylsilyl-quinoxalinol derivatives is achieved within 10 min by packed column gas chromatography. Precise quantification of less than 5 nmol of α-keto acids in biological samples is possible. In small aqueous samples the α-keto acids are directly derivatized without prior purification. Plasma need only be deproteinized by perchlorate and neutralized before derivatization. Average relative precision for determination of the three main branched-chain α-keto acids is ± 5.8%.     

Observations on the Leydig cells in the male East African spring hare (Pedetes surdaster larvalis).

The morphology of Leydig cells of the testis of sexually mature and sexually immature spring hares was studied. The cytoplasm of the Leydig of cells the sexually immature spring hares was packed with large lipid droplets leaving little space for the other organelles. Smooth endoplasmic reticulum was poorly developed and occasionally formed concentric layers of fenestrated cisterns around the large lipid droplets. The Leydig of cells the sexually mature spring hares were almost devoid of lipid droplets and their cytoplasm was occupied by abundant tubular smooth endoplasmic reticulum. Cells which shared characteristics with both immature Leydig cells and undifferentiated mesenchymal cells were observed in the limiting membrane of the seminiferous tubulus. These Leydig-like cells may play a role in the differentiation of Leydig cells in the spring hare.     

Interference with glycosylation of glycoproteins. Inhibition of formation of lipid-linked oligosaccharides in vivo.

Influenza-virus-infected cells were labelled with radioactive sugars and extracted to give fractions containing lipid-linked oligosaccharides and glycoproteins. The oligosaccharides linked to lipid were of the 'high-mannose' type and contained glucose. In the glycoprotein fraction, radioactivity was associated with virus proteins and found to occur predominantly in the 'high-mannose' type of glycopeptides. In the presence of the inhibitors 2-deoxy-D-glucose, 2-deoxy-2-amino-D-glucose (glucosamine), 2-deoxy-2-fluoro-D-glucose and 2-deoxy-2-fluoro-D-mannose incorporation of radiolabelled sugars into lipid- and protein-linked oligosaccharides was decreased. Kinetic analysis showed that the inhibitors affected first the assembly of lipid-linked oligosaccharides and then protein glycosylation after a lag period. During inhibition by deoxyglucose and the fluoro sugars lipid-linked oligosaccharides were formed that contained oligosaccharides of decreased molecular weight. No such aberrant forms were found during inhibition by glucosamine. In the case of inhibition by deoxyglucose it was shown that the aberrant oligosaccharides were not transferred to protein. Inhibition of formation of lipid-linked oligosaccharides by deoxyglucose and fluoro sugars was antagonized by mannose, in which case oligosaccharides of normal molecular weight were formed. The inhibition by glucosamine was reversed by its removal from the medium. The reversible effects of these inhibitors exemplify their usefulness as tools in the study of glycosylation processes.