Sensitivity to dihydropyridines, ω-conotoxin and noradrenaline reveals multiple high-voltage-activated Ca2+ channels in rat insulinoma and human pancreatic β-cells

High-voltage-activated (HVA) Ba²⁺ currents of rat insulinoma (RINm5F) and human pancreatic -cells were tested for their sensitivity to dihydropyridines (DHPs), -conotoxin (-CgTx) and noradrenaline. In RINm5F cells, block of HVA currents by nimodipine, nitrendipine and nifedipine was voltage- and dose-dependent (apparent K _D<37 nM) and largely incomplete even at saturating doses of DHPs (mean 53%, at 10 M and 0 mV). Analysis of slow tail currents in Bay K 8644-treated cells indicated the existence of Bay K 8644-insensitive channels that turned on at slightly more positive voltages and deactivated more quickly than Bay K 8644-modified channels. DHP Ca²⁺ agonists and antagonists in human -cells had similar features to RINm5F cells except that DHP block was more pronounced (76%, at 10 M and 0 mV) and Bay K 8644 action was more effective, suggesting a higher density of L-type Ca²⁺ channels in these cells. In RINm5F cells, but not in human -cells, DHP-resistant currents were sensitive to -CgTx. The toxin depressed 10–20% of the DHP-resistant currents sparing a residual current (25–35%) with similar voltage-dependent characteristics and Ca²⁺/Ba²⁺ permeability. Noradrenaline (10 M) exhibited different actions on the various HVA current components: (1) it prolonged the activation kinetics of -CgTx-sensitive currents, (2) it depressed by about 20% the size of DHP-sensitive currents, and (3) it had little or no effects on the residual DHP- and -CgTx-resistant current although intracellularly applied guanosine 5-O-(3-thiotriphosphate) (GTP--S) prolonged its activation time course. The first action was clearly voltage-dependent and most evident in RINm5F cells that displayed neuronal-like processes. The second was observed more frequently, was voltage-independent and fully blocked by saturating doses of nifedipine (10 M). Both actions were prevented by intracellular perfusion with guanosine 5-O-(2-thiodiphosphate) (GDP--S). Our data suggest that beside a majority of L-type channels, RINm5F and human pancreatic -cells may express a variable fraction of DHP-insensitive channels that may be involved in the control of insulin secretion during -cell activity.     

Differential effects of prednisolone and indomethacin on zymosaninduced inflammation in a modified murine tissue-chamber model

A tissue-chamber model of inflammation in mice has been modified and used to investigate the kinetics of zymosan-induced inflammatory mediators such as tumour necrosis factor (TNF), interleukin-1 (IL-1) and prostaglandin E₂ (PGE₂). In addition, the influx of polymorphonuclear leukocytes (PMN) into the chamber fluid and the granuloma surrounding the chamber was measured by myeloperoxidase (MPO) activity using a new microtitre plate assay. TNF and IL-1 reached peak concentrations at 3 and 6 h respectively after zymosan injection. Intermediate high concentrations of IL-1 were observed until the end of the experiment at 72 h, but TNF concentrations decreased from 24 h to biologically insignificant values. In contrast, exudate PGE₂ and MPO activity increased up to 24 h after zymosan injection and remained high until 72 h. At 6h after zymosan challenge, oral pre-treatment with prednisolone (3 to 30mg/kg) dose-dependently reduced TNF, IL-1 and PGE₂ concentrations while indomethacin (0.3 to 3 mg/kg) significantly attenuated PGE₂, slightly enhanced TNF and had no effect on IL-1 concentrations in the exudate. Both drugs had similar potencies against exudate and tissue MPO activities. Prednisolone inhibited IL-1 at 72 h post-zymosan. Indomethacin was more potent than prednisolone against PGE₂ (ID₅₀ of <0.3 versus 0.6mg/kg). The data obtained confirm the usefulness and reliability of this model in evaluating the effects of anti-inflammatory agents on inflammatory mediators induced by zymosan.     

Comparison of membrane properties of the cell body and the initial part of the axon of phasic motoneurones in the spinal cord of the cat

Summary Membrane properties of the cell body and the initial part of the axon of cat phasic motoneurones were analyzed.The mean input resistance was 9.1 M in the axon and 2.0 M in the cell body. A great scattering of values, however, was observed in the axons.The passive membrane time constants were 0.35 msec ( m) in the axon and 3.2 msec ( m) and 0.63 msec ( ₁) in the soma. A close electrotonic coupling between the soma and the axon was shown by means of synaptic potentials.In the cell body the threshold for excitation was 1.46 times higher than in the axon (14.2 mV and 9.7 mV respectively).Stimulating with current pulses an S-shaped spike inactivation curve was found in the axon. At threshold current intensity, spike inactivation reached 37.2%. In the cell body either no or only slight inactivation (7%) occurred at rheobase.During suprathreshold current stimulation the axon discharged only a few spikes, whereas the soma fired steadily. The current-frequency curve of the soma consisted of two linear ranges and, in addition, a logarithmic range quite similar to the logarithmic frequency curve of the axon was observed.The differences of the discharge behaviour of the cell body and the axon were discussed.This research was supported by the Sonderforschungsbereich 38, Membranforschun.     

Genetic characterization of an α-specific gene responsible for sexual agglutinability in Saccharomyces cerevisiae: mapping and gene dose effect

Summary A recessive ag1 mutation leads to specific defect in sexual agglutinability specifically in cells of the yeast Saccharomyces cerevisiae. The cryptopleurine resistance gene cry^R 1, closely linked to the mating type locus, was used to select / strains which emerged from / strains by mitotic nonreciprocal recombination, to genetically analyse ag1, since ag1 is expressed only in mating type. The ag1 gene was found to be linked to the centromere tightly, to met3 at 4.4 cM, and to ilv3 at 12 cM on chromosome X. Sexual agglutinability of cells was shown to be dependent on the dose of the AG1 gene, using / isogenic strains carrying AG1/AG1, AG1/ag1 or ag1/ag1. The sst2-1 mutation did not suppress the ag1 mutation. Based on these results, function of the AG1 gene is discussed.Abbreviations cM centimorgan - FDS first division segregation - NPD nonparental ditype - PD parental ditype - SDS second division segregation - TT tetratype     

Particular RNA primer from growth medium differentially stimulates in vitro DNA synthesis and in vivo cell growth of Neurospora crassa and its slime mutant

Summary Purine rich small RNA-primer molecules (about 10–12 nucleotides), secreted into the growth medium of 3-h germinated conidia of N. crassa, strongly stimulated a concentration-dependent in vitro DNA synthesis of N. crassa slime mutant as well as DNAs from the human cancer cells but did not affect that from normal cells. These RNA-primer molecules stimulated also in vivo cell growth of N. crassa slime mutant, but not of the N. crassa wild type. Our studies suggest that DNAs from the slime mutant of N. crassa as well as DNAs from human cancerous cells provide increased sites for enhanced in vitro and in vivo replication of DNAs. RNA-primer molecules can be hydrolyzed by T1 RNase but not by pancreatic RNase.     

Evidence for a direct relationship between mitochondrial genome organization and regeneration ability in hexaploid wheat somatic tissue cultures

Summary Embryogenic and non-embryogenic long-term callus cultures of hexaploid wheat exhibit differences in the organization of their mitochondrial genome. Embryogenic and non-embryogenic fractions of callus cultures initiated from immature embryos of the wheat cultivar Chinese Spring have been isolated and subsequently subcultured. DNA-DNA hybridization experiments using labelled cloned wheat mitochondrial DNA fragments have shown that the mitochondrial DNA organization of embryogenic subcultures derived from embryogenic parts of Chinese Spring calli is closely related to that of the initial Chinese Spring calli, while non-embryogenic subcultures derived from non-embryogenic fragments of Chinese Spring calli exhibit a mitochondrial DNA organization similar to that found in non-embryogenic calli derived from cultivar Aquila. In addition, somatic tissue cultures initiated from three other non-embryogenic wheat cultivars (Talent, Thésée and Capitole) display mitochondrial DNA arrangements similar to those found in cultivar Aquila. These results strongly suggest that, in wheat callus cultures, a particular mitochondrial genome organization is correlated with the ability of cultured cells to regenerate whole plants.Abbreviations mtDNA mitochondrial DNA - ctDNA chloroplast DNA - rRNA ribosomal RNA - kb kilobase pair - cv cultivar - 2,4-D 2,4-dichlorophenoxyacetic acid     

Mechanisms of antagonistic action of internal Ca2+ on serotonin-induced potentiation of Ca2+ currents in Helix neurones

The influence of internal Ca²⁺ ions has been investigated during intracellular perfusion of isolated neurones from pedal ganglia of Helix pomatia in which serotonin (5-HT) induces a cyclic-adenosine-monophosphate-(cAMP)-dependent enhancement of high-threshold Ca²⁺ current (I _Ca). Internal free Ca²⁺ ([Ca²⁺]_i) was varied between 0.01 and 10 M by addition of Ca²⁺-EGTA [ethylenebis(oxonitrilo)tetraacetate] buffer. Elevation of [Ca²⁺]_i depressed the 5-HT effect. The dose/ effect curve for the Ca²⁺ blockade had a biphasic character and could be described by the sum of two Langmuir's isotherms for tetramolecular binding with dissociation constants K _d1=0.063 M and K _d2=1 M. Addition of calmodulin (CM) antagonists (50 M trifluoperazine or 50 M chlorpromazine), phosphodiesterase (PDE) antagonists [100 M isobutylmethylxanthine (IBMX) or 5 mM theophylline] and protein phosphatase antagonists [2 M okadaic acid (OA)] in the perfusion solution caused anticalcium action and modified the Ca²⁺ binding isotherm. Using the effect of OA and IBMX, two components of the total Ca²⁺ inhibition were separated and evaluated. In the presence of one of these blockers tetramolecular curves with K _d1=0.04 M and K _d2=0.69 M were obtained describing the activation of the retained unblocked enzyme — PDE or calcineurin (CN) correspondingly. The sum of these isotherms gave a biphasic curve similar to that in control. Leupeptin (100 M), a blocker of Ca²⁺-dependent proteases did not influence the amplitude of 5-HT effect, indicating that channel proteolysis is not involved in the depression. Our findings show that the molecular mechanism of Ca²⁺-induced suppression of the cAMP-dependent upregulation of Ca²⁺ channels is due to involvement of two Ca²⁺-CM-dependent enzymes: PDE reducing the cAMP level, and CN causing channel dephosphorylation. No other processes are involved in the investigated phenomenon at a Ca²⁺ concentration of less than or equal to 10 M.     

Mechanism of modulation of single sodium channels from skeletal muscle by the β 1-subunit from rat brain

We studied the molecular mechanism of the rat skeletal muscle -subunit (_I) gating kinetics modulation by the brain ₁-subunit by heterologous expression of single sodium channels from _I and ₁ in Xenopus laevis oocytes. Coexpression of ₁ reduced mean open time at –10 mV to 21% when compared to channels expressed by _I alone. Channels formed by _I exerted multiple openings per depolarization, which occurred in bursts, in contrast to the channels formed by the _I/₁ complex that opened in average only once per depolarizing voltage pulse. Macroscopic current decay (mcd), as evidenced by reconstructed open probability vs. time , was greatly accelerated by ₁, closely resembling mcd of sodium currents from native skeletal muscle. Generally was larger for channels expressed from the pure _I subunit.From our single channel data we conclude that ₁ accelerates the inactivation process of the sodium channel complex.     

Mechanisms underlying facilitation by dopamine of ATP-activated currents in rat pheochromocytoma cells

Mechanisms underlying facilitation by dopamine of extracellular adenosine 5-triphosphate (ATP)-activated current were investigated in rat pheochromocytoma PC12 cells using the whole-cell voltage-clamp techniques. Dopamine (10 and 100 M) augmented the peak amplitude of an inward current elicited by ATP (3–100 M). The activation time course of the ATP-evoked current was accelerated by dopamine; the presence of 10 M dopamine shifted the dependence of activation rate constants on the concentration of ATP toward a lower concentration range two fold. Dopamine also accelerated the inactivation and the deactivation, which was determined from the current decay upon washout of ATP. Intracellular mediators responsible for the dopamine-induced facilitation was estimated by loading various compounds in patch pipettes. Facilitation was not observed when K-252a (1 M), a protein kinase inhibitor, was included in the intracellular solution. In addition, facilitation was also attenuated by intracellular adenosine 5-O-(thiotriphosphate)tetralithium salt (ATPS (1 mM) or --methylene ATP (1 mM). Inclusion of adenosine 3, 5-cyclic monophosphate sodium salt (cAMP, 100 M), guanosine 3,5-cyclic monophosphate sodium salt (cGMP, 100 M), 12-O-tetradecanoylphorbol-13-acetate (TPA, 1 M) or phorbol-12,13-dibutylate (1 M) in the intracellular solution did not affect the facilitation. Guanonsine 5-O-(thiotriphosphate)tetralithium salt (GTPS, 500 M) or guanosine 5-O(2-thiodiphosphate)-trilithium salt (GDPS, 500 M) did not modify the facilitation either. The results suggest that dopamine augments the ATP-activated inward current by facilitating association of ATP to its binding site, and that the augmentation may be mediated through some protein kinase which is different from cyclic-nucleotide-dependent protein kinases or protein kinase C.     

Presynaptic and postsynaptic effects of inhibitory drugs on the crayfish neuromuscular junction

Summary -aminobutyric acid (GABA) and related drugs reduce the size of the excitatory postsynaptic potential (e.p.s.p.) in the crayfish neuromuscular preparation. The effective inhibitory concentrations of these drugs were compared and the sites of action—postsynaptic and presynaptic—were determined.GABA, -amino--hydroxy-butyric acid, guanidino acetic acid, -alanine and others were found to increase the membrane conductance of the muscle fibers. These drugs also shift the membrane potential in the same direction as the inhibitory postsynaptic potential. It is concluded that this group of drugs imitates the postsynaptic action of the inhibitory transmitter substance.-guanidino-propionic acid (GP), -guanidino-butyric acid and others did not affect the membrane conductance of the muscle fibers. These drugs therefore do not inhibit through the conductance type of postsynaptic inhibition.Both groups of drugs mentioned above (GABA and GP) did reduce the amount of transmitter released from the excitatory nerve terminal per stimulus, they thus have a presynaptic inhibitory effect similar to neural inhibition. This was shown by analysis of the quantum content of the excitatory postsynaptic potential: The inhibitory drugs reduced the number of quanta liberated per stimulus, the size of the quantum remaining constant.Although GP has no direct effects on the membrane resistance of the muscle fiber, it reduces the inhibitory postsynaptic potential and diminishes the action of applied GABA on the membrane conductance. GABA and GP seem to compete for the inhibitory receptor site on the muscle membrane.

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Zusammenfassung -amino-Buttersäure (GABA) und verwandte Substanzen verkleinern das postsynaptische erregende Potential (e.p.s.p.) des Nerv-Muskel-Präparates des Krebses. Die wirksamen hemmenden Konzentrationen dieser Drogen wurden verglichen, und es wurde versucht, Art und Ort der Hemmungswirkungen — postsynaptisch oder präsynaptisch — festzustellen.GABA, -amino--hydroxy-Buttersäure, guanidino-Essigsäure, -Alanin und andere erhöhten die Leitfähigkeit der Muskelzellmembran. Bei Gabe dieser Drogen verschiebt sich auch das Membranpotential in derselben Richtung wie das hemmende postsynaptische Potential. Folglich hat diese Gruppe von Drogen dieselbe Wirkung auf die Muskel-Zell-Membran wie der hemmende neurale Überträgerstoff.-guanidino-Propionsäure (GP), -guanidino-Buttersäure und andere Guanidinosäuren haben keinen Einfluß auf die Membranleitfähigkeit der Muskelzellen. Der hemmende Effekt dieser Drogen ist also nicht vom Typ der postsynaptischen Leitfähigkeitshemmung.Beide oben erwähnten Gruppen von Substanzen (GABA und GP) setzen die Menge des pro Reiz von der erregenden Nervenendigung freigesetzten Überträgerstoffes herab, sie hemmen also präsynaptisch ebenso wie die neurale Hemmung. Diese präsynaptische Hemmung wurde bewiesen durch Analyse des Quantengehalts der erregenden postsynaptischen Potentiale: Die hemmenden Drogen verkleinerten die Zahl der pro Reiz ausgeschütteten Quanten, ohne die Größe des Quantums zu verändern.Obgleich GP keine direkten Effekte auf den Membranwiderstand der Muskelfasern hat, verkleinert es das postsynaptische hemmende Potential und setzt die Wirkung von GABA auf die Leitfähigkeit der Muskelzellmembran herab. GABA und GP verdrängen sich gegenseitig am Hemmungs-Receptor der Muskelmembran, so daß GP den Effekt von GABA kompetitiv hemmt.

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This work was supported by the Deutsche Forschungsgemeinschaft.     

Expression of estrogen receptors-α and -β in primary breast neoplasms and tumors exposed to neoadjuvant hormonal therapy

We studied the content and expression of mRNA for estrogen receptors receptors- and - in breast tumors before and after 3-month neoadjuvant hormone therapy with antiestrogen tamoxifen and/or aromatase inhibitors. Expression of estrogen receptors- and - was most often detected in ER⁺PR⁺ tumors and most significantly decreased in these neoplasms after exemestane therapy. Immunocytochemical and radioligand assays showed that tamoxifen and anastrozole have little effect on the number of estrogen receptors- The number of progesterone receptors in tumors decreased by the end of anastrozole therapy. Estrogen receptors- were immunocytochemically revealed in 50% primary breast tumors. Anastrozole slightly decreased, while tamoxifen increased the incidence of these receptors. Interruption of signaling through estrogen receptors and suppression of estrogen biosynthesis had different effects on the receptor status of neoplasms and distribution of estrogen receptors- and -.Translated from Byulleten Eksperimentalnoi Biologii i Meditsiny, Vol. 138, No. 11, pp. 559–562, November, 2004     

Heterogeneity of Procoagulant Activity and Cytokine Release in Subpopulations of Alveolar Macrophages and Monocytes

We have studied the expression of tissue factor (TF) and fibrinopeptide A (FPA) generation as well as the release capacity of TNF-, IL-1, and IL-6 in density-defined subpopulations of alveolar macrophages (AM) and monocytes (Mo). TF was equally expressed on all AM subpopulations and Mo, while the FPA-forming capacity was at the same level in low density AM as in Mo and was significantly (P < 0.05) higher in low density AM than in high density AM. The lipopolysaccharide (LPS)-induced release of TNF- was higher (P < 0.05) in high density AM than in low density AM and in Mo. IL-1 release was undetectable in unstimulated AM and in LPS-stimulated low density AM, while the LPS-induced IL-1 release in high density AM was low compared to the levels demonstrated in Mo. LPS-stimulated IL-6 release was not distinctively different in the AM subpopulations and Mo. The presented study showed that FPA generation and LPS-stimulated TNF- release were dependent on the density (i.e., maturity) of AM. This implies that a skewed distribution of AM subpopulations induced by disease processes may profoundly influence the inflammatory reactions, including extravascular activation of coagulation.     

Molecular organisation of an A mating type factor of the basidiomycete fungus Coprinus cinereus

Summary The A3 and A3 genes, which together constitute the A42 mating type factor of Coprinus cinereus, were isolated from a cosmid genomic library by walking 50 kb, a map distance of 0.5 units, from the closely linked metabolic gene pab-1. Cosmid clones having A gene function were identified by transformation into compatible A6 (22) and A5 (11) host cells where either 3 or 3 was expected to elicit the A factor — regulated development of unfused clamp cells. DNAs were digested with various enzymes before transformation in order to identify the smallest fragments containing an active 3 or 3 gene. Two non-overlapping fragments were identified as containing the 3 and 3 genes respectively. Southern hybridisation analyses showed that these two cloned genes had no detectable sequence homology, and that there was little or no hybridisation to the and gene alleles that constitute the A5 and A6 factors. 3 and 3 were shown to be less than 2.0 kb apart and embedded in a DNA sequence extending over 9.0 kb which was unique to our A42 strain and may contain a third A factor gene.     

Foraging strategies ofDrosophila melanogaster: A chromosomal analysis

Two larval foraging strategies inDrosophila melanogaster were identified, rover and sitter. Rovers traverse a large area while feeding whereas sitters cover a small area. The difference between rovers and sitters was analyzed genetically by chromosomal substitutions between isogenic stocks. Differences in larval locomotor behavior (crawling behavior) can be attributed to the second chromosome, the rover strategy being dominant over the sitter strategy. Differences in feeding rate (shoveling behavior) are affected additively by both the second and third chromosomes. Natural populations ofDrosophila larvae were sampled three times over a 2-month period; rovers and sitters were at constant frequencies in these populations. The two foraging strategies are discussed in the light of resource utilization in environments where food is distributed continuously or discontinuously.     

Effect of a series of 1-alkyl ether lipids on inhibition of phospholipase A2 activity and PAF responses

Several 1-alkyl ether lipids were studied for their ability to inhibit PLA₂ and antagonize PAF responses. Studies with synthetic micellar substrate (1-stearyl-2-arachidonyl phosphocholine), at concentrations ranging from 0.02 to 1000M, demonstrate that CL 118326 inhibits porcine pancreatic PLA₂ in vitro. As the substrate concentration increases, there is a dose-dependent increase in the IC₅₀ value (IC₅₀ ranges: 1.6–84.6g/ml or 2.6–137M). CL 118326 inhibits mammalian pancreatic PLA₂, but not snake or bee venom PLA₂. CL 118326 inhibits thrombin (IC₅₀ =7.9M), but not Na arachidonate- (IC₅₀ > 100M) induced platelet aggregation, indicative of inhibition of cellular PLA₂. CL 118326 inhibits other PLA₂-dependent processes such as antigen-induced leukotriene (LTC₄) release (IC₅₀=2.3g/ml or 3.8M) and histamine release (IC₅₀=1.4g/ml or 2.2M) in basophil-enriched WBCs. Intradermal coinjection of CL 118326 (10g) with PLA₂ into guinea pig skin inhibits pancreatic PLA₂-induced increase in vascular permeability and leakage, but not snake or bee venom PLA₂-induced leakage. CL 118326 shows no PAF-like agonist activity in stimulating rabbit platelet-rich plasma. It inhibits PAF-induced aggregation (IC₅₀=5.8M), but not ADP-induced aggregation. CL 118326 has greater efficacy as a PLA₂ inhibitor than as a PAF antagonist since the IC₅₀-substrate concentration ratio for PLA₂ inhibition is <- 1.0 at substrate concentrations of 10–1000M while the IC₅₀-agonist ratio for PAF antagonism is > 100. Results for four other compounds related to CL 118326 are also presented.     

Gene activation by copy transposition in mating-type switching of a homothallic fission yeast

Summary Mating-type switching in homothallic clones of the fission yeast, Schizosaccharomyces pombe, appears to follow the same route as previously found for mutations from homothallism to heterothallic strains. A copy of mat2-P is transposed to and inserted at mat1, where it functionally replaces the mat1-M allele, and only the mat1 segment is expressed (!) to determine the actual mating type: mat1-M(!) mat2-P = = mat1-P(!) mat2-P. This phenomenon has hitherto been concealed by the high switch-back rate from to observed in homothallic wild-type strains. It only becomes apparent in the presence of mutant switching genes, which retard the rates of mating-type interconversion and temporarily freeze one or the other state of gene activation at the mat1 segment. Mutations to lowered rates of switching are found to map both inside and outside the mating-type locus. While the internal mutations of this kind exert their effect autonomously in the cis-configuration, the unlinked mutations are recessive to their wild-type alleles.We dedicate this paper to Carsten Bresch. The authors first met in the most stimulating scientific environment C. B. had created at the Southwest Center for Advanced Studies.     

Intraspecific variation in the structural organization and redundancy of chloroplast ribosomal DNA cistrons in Euglena gracilis

Summary Chloroplast DNAs from six different laboratory collections of Euglena gracilis strain Z and var. bacillaris were analyzed with restriction endonucleases EcoRI and Bam HI. The most variable portion of the organelle genome is the region containing the ribosomal cistrons. Intraspecific differences occur in both ribosomal DNA cistron number (one or three) and structural organization among those strains designated as strain Z and bacillaris. One culture previously designated as Z is most likely bacillaris.     

Characterization and localization of α-connectin (titin 1): An elastic protein isolated from rabbit skeletal muscle

Summary A simplified procedure to isolate-connectin (titin 1, TI), a gigantic elastic protein, from rabbit skeletal muscle is described. A rapid column chromatography step to concentrate-connectin is introduced. Separation of-connectin from-connectin is introduced. Separation of-connectin from-connectin (titin 2, TII) in the presence of 4 M urea at pH 7.0 did not cause any change in the secondary structure of-connectin as judged by circular dichroic spectra. Ultraviolet absorption spectra and the amino acid composition of-connectin (MW, approximately 3×10⁶) were similar to those of its proteolytic product,-connectin (MW, approximately 2×10⁶). Circular dichroic spectra suggested that both- and-connectin consist of 60%-sheet and 30%-turn. It thus appears that the whole elastic filament of connectin has a folded-strand structure. Proteolysis of-connectin by calpain resulted in formation of-connectin and smaller peptides. The-connectin interacted with both myosin and actin filaments similarly to-connectin. Polyclonal antibodies raised against 1200 kDa peptides obtained from aged rabbit skeletal myofibrils reacted with-connectin (titin 1, TI) but only weakly with-connectin (titin 2, TII) in rabbit skeletal muscle. Immunoelectron microscopy and indirect immunofluorescence microscopy revealed that the antibodies bound at the Z-line and at the epitope regions in the I-band near the binding site of a monoclonal antibody SMI whose position depends on sarcomere length. It thus appears that-connectin extends from the edge of M-line to the above epitope region in the I-band.     

Candida albicans induces the release of inflammatory mediators from human peripheral blood monocytes

Candida albicans (C. albicans) is a major nosocomial pathogen. We examined arachidonic acid (AA) and cytokine production by monocytes stimulated with C. albicans. [¹⁴C]-AA labeled monocytes released 8.9 ±2.3% of the incorporated AA following stimulation with live C. albicans (C. albicans: monocyte of 161) (P=0.0002). Prior studies indicate that soluble-mannans and-glucans antagonize mannose and-glucan receptors, respectively. Preincubation of monocytes with-mannan (100g/ml) caused 45.8 ±5.7% inhibition of [¹⁴C]-AA release, whereas-glucan (100g/ml) yielded 43.7 ±6.0% inhibition (P<0.05 for each compared to control). Additionally, monocytes stimulated with C. albicans also released interleukin-1 (IL-1), tumor necrosis factor- (TNF), interleukin-6 (IL-6) and interleukin-8 (IL-8). However, a-mannan or-glucan failed to inhibit IL-1 release. These data indicate that C. albicans induces monocytes to release AA and inflammatory cytokines. Furthermore, AA, but not cytokine liberation, is partially mediated by a-mannan and-glucan components of the fungus.     

Roles of inositol trisphosphate and protein kinase C in the spontaneous outward current modulated by calcium release in rabbit portal vein

We examined the effects of heparin, guanosine nucleotides, protein kinase C (PKC) modulators, such as phorbol 12,13-dibutylate (PDBu) and H-7 on Ca²⁺-dependent K⁺ currents in smooth muscle cells of the rabbit portal vein using the whole-cell patch-clamp technique, to explore the effects of PKC on the oscillatory outward current (I _oo). Neomycin (30 M), an inhibitor of phospholipase C, and intracellular applications of heparin (10 g/ml) and guanosine 5-O-(2-thiodiphosphate) (GDP[S]; 1 mM) partly but consistently inhibited the generation of I _oo, whereas a higher concentration of heparin (100 g/ml) transiently enhanced then suppressed the generation of I _oo. Inhibition of I _oo generation by heparin was more powerful at the holding potential of + 20 mV than at –20 mV. Inositol 1,4,5-trisphosphate (InsP ₃; 30 M) continuously generated I _oo at holding potentials more positive than –60 mV. Noradrenaline (10 M) and caffeine (3–20 mM) transiently augmented, then reduced the generation of I _oo. Heparin (10 g/ml) completely inhibited responses induced by InsP ₃ and noradrenaline, but not those induced by caffeine. Intracellular application of guanosine 5-triphosphate (GTP; 200 M) or low concentrations of guanosine 5-O-(3-thiotriphosphate) (GTP[S]; 3 M) continuously augmented the generation of I _oo. High concentrations of GTP[S] (10 M) transiently augmented, then inhibited I _oo. Neither GTP[S] nor noradrenaline induced the transient augmentation or the subsequent inhibition of I _oo when applied in the presence of GDP[S] (1 mM), neomycin (30 M) or heparin (10 g/ml). PDBu (0.1 M) reduced the generation of I _oo but failed to produce an outward current following application of caffeine (3–5 mM). This action of PDBu was inhibited by pretreatment with H-7 (20 M). In the presence of H-7, GTP[S] continuously enhanced the generation of I _oo. The suppression of the generation of I _oo during application of noradrenaline (10 M) was reduced by pretreatment with H-7. Thus both InsP₃ and protein kinase C contribute to the generation of I _oo in smooth muscle cells of the rabbit portal vein and heparin is not a specific InsP ₃ antagonist on the InsP ₃-induced Ca²⁺-release channel (PIRC). InsP ₃ opens PIRC and protein kinase C may deplete the stored Ca²⁺ by either inhibiting the reuptake of Ca²⁺ or by enhancement of the releasing actions of InsP ₃.