Barium- and calcium-permeable channels open at negative membrane potentials in rat ventricular myocytes

Summary Ca²⁺- and Ba²⁺-permeable channel activity from adult rat ventricular myocytes, spontaneously appeared in the three single-channel recording configurations: cell-attached, and excised inside-out or outside-out membrane patches. Single-channel activity was recorded at steady-state applied membrane potentials including the entire range of physiologic values, and displayed no rundown in excised patches. This activity occurred in irregular bursts separated by quiescent periods of 5 to 20 min in cell-attached membrane patches, whereas in excised patch experiments, this period was reduced to 2 to 10 min. During activity, a variety of kinetic behaviors could be observed with more or less complex gating patterns. Three conductance levels: 22, 45 and 78 pS were routinely observed in the same excised membrane patch, sometimes combining to give a larger level. These channels were significantly permeable to divalent cations and showed little or no permeability to potassium or sodium ions. The inorganic blockers of voltage-gated Ca channels, cobalt (2mm), cadmium (0.5mm) or nickel (3mm), had no apparent effect on these spontaneous unitary currents carried by barium ions. Under 10^–5 m bay K 8644 or nitrendipine, the activity was clearly increased in about half of the tested excised inside-out membrane patches. Both dihydropyridines enhanced openings of the larger conductance level, which was only very occasionally seen under control conditions. When the single-channel activity became sustained under 5×10^–6 m Bay K 8644, it was possible to calculate the mean unitary current at different membrane potentials and show that the mean current value increased with membrane potential.     

Presymptomatic testing for Huntington's disease

Summary Presymptomatic testing for Huntington's disease (HD) is possible through the use of restriction fragment length polymorphisms (RFLPs) at the closely linked D4S10 locus. Recombination between the HD and D4S10 loci will occur in 4%–5% of meioses, and is a well-recognised complication of predictive testing. Recombination between RFLPs within the D4S10 locus is a rare event and can usually be ignored. We report a case where such an intra-locus recombination frustrated attempts to predict the chance of a high-risk individual inheriting the HD gene.     

Role for [corrected] Agrobacterium tumefaciens ChvA protein in export of beta-1,2-glucan.

Functional chvA and chvB genes are required for attachment of Agrobacterium tumefaciens to plant cells, an early step in crown gall tumor formation. Strains defective in these loci do not secrete normal amounts of cyclic beta-1,2-glucan. Whereas chvB is required for beta-1,2-glucan synthesis, the role of chvA in glucan synthesis or export has not been clearly defined. We found that cultures of chvA mutants contained as much neutral beta-1,2-glucan in the cell pellets as did the wild type, with no detectable accumulation of glucan in the culture supernatant. The cytoplasm of chvA mutant cells contained over three times more soluble beta-1,2-glucan than did the cytoplasm of the wild-type parent. Unlike the wild type, chvA mutants contained no detectable periplasmic glucan. The amino acid sequence of chvA is highly homologous to the sequences of bacterial and eucaryotic export proteins, as observed previously in the case of ndvA, a rhizobial homolog of chvA. Strong sequence homology within this family of export proteins is concentrated in the carboxy-terminal portions of the proteins, but placement of consensus ATP-binding sites, internal signal sequences, and hydrophobic domains are conserved over their entire lengths. These data suggest a model for beta-1,2-glucan synthesis in A. tumefaciens in which glucan is synthesized inside the inner membrane with the participation of ChvB and transported across the inner membrane with the participation of ChvA.     

Cartilage contains mixed fibrils of collagen types II, IX, and XI

The distribution of collagen XI in fibril fragments from 17-d chick embryo sternal cartilage was determined by immunoelectron microscopy using specific polyclonal antibodies. The protein was distributed throughout the fibril fragments but was antigenically masked due to the tight packing of collagen molecules and could be identified only at sites where the fibril structure was partially disrupted. Collagens II and IX were also distributed uniformly along fibrils but, in contrast to collagen XI, were accessible to the antibodies in intact fibrils. Therefore, cartilage fibrils are heterotypically assembled from collagens II, IX, and XI. This implies that collagen XI is an integral component of the cartilage fibrillar network and homogeneously distributed throughout the tissue. This was confirmed by immunofluorescence.     

Reinstatement of Microtubule Arrays from Cortical Nucleating Sites in Stomatal Complexes of Lolium rigidum Following Depolymerization of Microtubules by Oryzalin and High Pressure

Immunofluorescence visualization of microtubule (MT) arraysin stomatal complexes of Lolium rigidum shows that disassemblyof the arrays can be successfully achieved using oryzalin orhigh pressure treatments. Under conditions allowing for MT recovery,MTs reappear within an hour after oryzalin or within 5 min afterhigh pressure treatment. During recovery guard mother cells(GMCs) nucleate MTs at sites distributed randomly in the cellcortex. Even after 22 h of recovery the MTs are not arrangedinto any configuration found in untreated tissue. This inabilityto reorganize their MTs after treatment makes GMCs more sensitiveto the loss of MTs than are other cells of the leaf. In guardcells (GCs) MTs reappear around the pore at the junction ofthe periclinal and ventral walls. They subsequently appear throughoutmost of the cell cortex and the majority of stomatal complexesrecover fully organized MT arrays indistinguishable from thosein untreated cells. The results support and extend ultrastructuraland immunofluorescence observations that suggest that MTs inGCs of developing stomata are nucleated in the cell cortex. ²Present address: Department of Biology, The University of SouthwesternLouisiana, Lafayette, Louisiana 70504-2451, U.S.A. (Received April 24, 1990; Accepted July 7, 1990)     

Characterisation of chloride transport at the tonoplast of higher plants using a chloride-sensitive fluorescent probe

The characteristics of Cl^– transport in isolated tonoplast vesicles from red-beet (Beta vulgaris L.) storage tissue have been investigated using the Cl^–-sensitive fluorescent probe, 6-methoxy-1-(3-sulfonatopropyl)-quinolinium (SPQ). The imposition of (inside) positive diffusion potentials, generated with K⁺ and valinomycin, increased the initial rate of Cl^– transport, demonstrating that Cl^– could be electrically driven into the vesicles. Chloride influx was unaffected by SO ₄ ^2- , but was competitively blocked by NO ₃ ^– , indicating that both Cl^– and NO ₃ ^– may be transported by the same porter. In some preparations, increases in free-Ca²⁺ concentration from 10^–8 to 10^–5 mol·dm^–3 caused a significant decrease in Cl^– influx, which may indicate that cytosolic Ca²⁺ concentration has a role in controlling Cl^– fluxes at the tonoplast. However, this effect was only seen in about 50% of membrane preparations and some doubt remains over its physiological significance. A range of compounds known to block anion transport in other systems was tested, and some partially blocked Cl^– transport. However, many of these inhibitors interfered with SPQ fluorescence and so only irreversible effects could be tested. The results are discussed in the context of recent advances made using the patch-clamp technique on isolated vacuoles.Abbreviations and Symbols BTP 1,3-bis[tris(hydroxymethyl)-methylamino]propane - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - membrane potential - pH pH gradient - SPQ 6-methoxy-1-(3-sulfonatopropyl)quinolinium - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl] glycine     

Bradykinin Stimulates Phosphoinositide Hydrolysis and Mobilization of Arachidonic Acid in Dorsal Root Ganglion Neurons

Cultures of fetal rat dorsal root ganglion neurons (7 days in culture) were prelabeled with myo-[3H]inositol or [3H]arachidonic acid for 24 h and stimulated with 10 microM bradykinin for time intervals of 5-300 s. The incubation was terminated by addition of 5% perchloric acid to extract inositol phosphates or organic solvent to extract lipids. Inositol phosphates were resolved by anion-exchange HPLC; lipids were resolved by TLC. Bradykinin stimulation resulted in a 10-fold increased accumulation of inositol 1,4,5-trisphosphate (IP3) and inositol bisphosphate (IP2) (fivefold) by 5 s. The increase in IP3 was transient (half maximal by 1 min), whereas stimulated IP2 levels were sustained for several minutes. Even longer term increases were observed in inositol monophosphate. Stimulation also resulted in a threefold increase in arachidonic acid which was preceded by transient increases in diacylglycerol (twofold) and arachidonoyl-monoacylglycerol (threefold). The temporal lag in the accumulation of arachidonic acid with respect to diglyceride and monoglyceride suggested the involvement of di- and monoglyceride lipases in arachidonic acid mobilization. A role for phospholipase A2 is also possible, because pretreatment of cultures with quinacrine partially blocked arachidonic acid release. Bradykinin-stimulated arachidonic acid release was decreased in the presence of calcium channel blockers nifedipine or verapamil (50 microM), or EDTA (2.5 mM). The role of calcium was verified further in that accumulation of phosphatidic acid, diacylglycerol, and arachidonic acid was maximally stimulated by treatment with the calcium ionophore A23187 (20 microM).     

Substrate stereospecificity of phosphatidylinositol-specific phospholipase C from Bacillus cereus examined using the resolved enantiomers of synthetic myo-inositol 1-(4-nitrophenyl phosphate).

The substrate stereospecificity of phosphatidylinositol-specific phospholipase C from Bacillus cereus is examined using the resolved optical isomers of synthetic myo-inositol 1-(4-nitrophenyl phosphate), a chromogenic substrate for the phospholipase. The synthetic route employs mild acid-labile protecting groups and separation of the substituted myo-inositol enantiomers as the (-)-camphanyl ester diastereomers. Measurements of the initial rates of cleavage of the D and L enantiomers of the nitrophenyl substrate by phosphatidylinositol-specific phospholipase C from B. cereus show that this enzyme is essentially stereospecific for the D enantiomer. Under identical conditions, the rate of cleavage of the L isomer is less than 0.2% of that observed for the D isomer. The same is observed for the highly homologous enzyme from Bacillus thuringiensis. There is no measurable inhibition by the L enantiomer of the B. cereus enzyme acting on the D enantiomer, even when the molar ratio of L:D is 5, indicating that binding of the L enantiomer to the phospholipase is negligible. Thus, the enzyme active site is exquisitely sensitive to the stereochemistry of the myo-inositol group of the substrate.