Antibodies to junctional sarcoplasmic reticulum proteins: probes for the Ca2+-release channel.

The junctional face membrane plays a key role in excitation-contraction coupling in skeletal muscle. A protein of 350 kDa, tentatively identified as a component of the junctional feet, connects transverse tubules to terminal cisternae of sarcoplasmic reticulum [Kawamoto, Brunschwig, Kim & Caswell (1986) J. Cell Biol. 103, 1405-1414]. The membrane topology and protein composition of sarcoplasmic reticulum Ca2+-release channels of rabbit skeletal muscle were investigated using an immunological approach, with anti-(junctional face membrane) and anti-(350 kDa protein) polyclonal antibodies. Upon preincubation of the terminal cisternae with anti-(junctional face membrane) antibodies, Ca2+-ATPase and Ca2+-loading activities were not affected, whereas anti-(350 kDa protein) antibodies stimulated Ca2+-ATPase activity by 25% and inhibited Ca2+-loading activity by 50% (at an antibody/terminal cisternae protein ratio of 1:1). Specific photolabelling of terminal cisternae proteins with [14C]doxorubicin was prevented by both anti-(junctional face membrane) and anti-(350 kDa protein) antibodies. Stimulation of Ca2+ release by doxorubicin was prevented by both anti-(junctional face membrane) and anti-(350 kDa protein) antibodies. Half-maximal inhibition was obtained at an antibody/terminal cisternae protein ratio of 1:1. Kinetic measurements of Ca2+ release indicated that anti-(350 kDa protein) antibodies prevented Ca2+-induced Ca2+ release, whereas the ATP-stimulation and the inhibition by Mg2+ were not affected. These results suggest that: (i) Ca2+- and doxorubicin-induced Ca2+ release is mediated by Ca2+ channels which are selectively localized in the junctional face membrane; (ii) the 350 kDa protein is a component of the Ca2+-release channel in native terminal cisternae vesicles; and (iii) the Ca2+-activating site of the channel is separate from other allosteric sites.     

Low density lipoprotein and apoprotein B induce increases in inositol trisphosphate and cytosolic free Ca2+ via pertussis toxin-sensitive GTP-binding protein in vascular smooth muscle cells

Low density lipoprotein (LDL), a major cholesterol-carrying lipoprotein in the plasma, binds to its receptor through apoprotein B (Apo-B). The addition of LDL and Apo-B induced rapid (5 s), but transient increase in the inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) level with K0.5 values of 1.1 and 0.07 microgram/ml, accompanied by increases of cytosolic free Ca2+ concentration [( Ca2+]i), in vascular smooth muscle cells (VSMC). The increases by LDL and Apo-B were both reduced by pretreatment of the VSMC with pertussis toxin. The early change in Ins-1,4,5-P3 involving a GTP-binding protein may function as an initial signal for the action of LDL in VSMC.     

Comparison of the inhibitions of proliferation of normal and psoriatic fibroblasts by 1 alpha,25-dihydroxyvitamin D3 and synthetic analogues of vitamin D3 with an oxygen atom in their side chain

The inhibitory effects of 1 alpha,25-(OH)2D3 and synthetic oxa-derivatives of vitamin D3 on growth of normal and psoriatic fibroblasts in culture were compared. Proliferation of normal fibroblasts was strongly inhibited by these new compounds in the following order: 22-oxa-1 alpha,25-(OH)2D3 greater than 22-oxa-1 alpha-(OH)D3 greater than 1 alpha,25-(OH)2D3 greater than 20-oxa-1 alpha,25-(OH)2D3. 22-Oxa-1 alpha,25-(OH)2D3 was about 10-times more inhibitory than 1 alpha,25-(OH)2D3. Proliferation of psoriatic fibroblasts was not inhibited by 1 alpha,25-(OH)2D3 at concentrations of up to 10(-6) M, but was suppressed by 10(-8)-10(-6) M 22-oxa-1 alpha,25-(OH)2D3 and 10(-6) M 22-oxa-1 alpha-(OH)D3. These results suggest that oxa-derivatives of vitamin D3, especially 22-oxa-1 alpha,25-(OH)2D3, should be useful in further studies on the cause and treatment of psoriasis.     

Selective growth of a mutant in continuous culture of Bacillus caldolyticus for production of α-amylase

Summary In a continuous culture of Bacillus caldolyticus strain SP, which requires maltose as an inducer for production of -amylase in batch culture, a predominant mutant strain M1 which produced high amounts of -amylase in the absence of maltose in batch culture, developed. The change of cell population from strain SP to strain M1 in maltose-casitone medium was linear with time in the transient state after the change from batch to continuous culture at a dilution rate of 0.17 h^-1, and was completed in about 11 generations of bacterial growth. The dilution rate effect of continuous culture on -amylase activity was almost the same with both strains SP and M1. The maximum -amylase activity of 380 units/ml was observed at an intermediate dilution rate that was 11.5 times higher than -amylase activity at the end of a batch culture using the same medium. It was deduced that the enhancement of -amylase production in continuous culture was attributed partly to the predominant growth of a mutant strain with higher -amylase productivity.     

Sequence analysis of alpha 1(VI) and alpha 2(VI) chains of human type VI collagen reveals internal triplication of globular domains similar to the A domains of von Willebrand factor and two alpha 2(VI) chain variants that differ in the carboxy terminus.

Amino acid sequences of human collagen alpha 1(VI) and alpha 2(VI) chains were completed by cDNA sequencing and Edman degradation demonstrating that the mature polypeptides contain 1009 and 998 amino acid residues respectively. In addition, they contain small signal peptide sequences. Both chains show 31% identity in the N-terminal (approximately 235 residues) and C-terminal (approximately 430 residues) globular domains which are connected by a triple helical segment (335-336 residues). Internal alignment of the globular sequences indicates a repetitive 200-residue structure (15-23% identity) occurring three times (N1, C1, C2) in each chain. These repeating subdomains are connected to each other and to the triple helix by short (15-30 residues) cysteine-rich segments. The globular domains possess several N-glycosylation sites but no cell-binding RGD sequences, which are exclusively found in the triple helical segment. Sequencing of alpha 2(VI) cDNA clones revealed two variant chains with a distinct C2 subdomain and 3' non-coding region. The repetitive segments C1, C2 and, to a lesser extent, N1 show significant identity (15-18%) to the collagen-binding A domains of von Willebrand factor (vWF) and they are also similar to some integrin receptors, complement components and a cartilage matrix protein. Since the globular domains of collagen VI come into close contact with triple helical segments during the formation of tissue microfibrils it suggests that the globular domains bind to collagenous structures in a manner similar to the binding of vWF to collagen I.     

Developmental changes in galactosyltransferase activity in the rat small intestine

During postnatal development, UDP-Gal: GlcNAc(beta 1-4)-galactosyltransferase (4 beta-GT) and UDP-Gal:GalNAc(beta 1-3)-galactosyltransferase (3 beta-GT) activities were increased by 17- and 24-fold, respectively, in the rat small intestine. The injection of cortisone into suckling rats resulted in precocious induction of distal 4 beta- and 3 beta-GT activities by 2.7- and 1.8-fold, respectively. Injection of phorbol-12-myristate-13-acetate (PMA) resulted in precocious induction of distal 3 beta-GT by 2.7-fold. These results suggest that intestinal galactosyltransferase activities are under developmental regulation and can be modified by cortisone and PMA.     

Effect of cytoskeletal geometry on intracellular diffusion.

A method is presented for determining the retardation of diffusion of particles inside cells owing to cytoskeletal barriers. The cytoskeletal meshwork is treated as a repeating periodic two-dimensional or three-dimensional lattice composed of elements of given size, shape, and spacing. We derive an analytic expression for the diffusion coefficient relative to that of the cytosol. This expression is evaluated by solving numerically an appropriate boundary-value problem for the Laplace equation. For the two-dimensional case, e.g., diffusion in a membrane, the results are quantitatively similar to those obtained by Saxton (1987. Biophys. J. 52:989-997) using Monte Carlo methods. The three-dimensional results are quantitatively similar to experimental results reported by Luby-Phelps et al. (1987. Proc. Natl. Acad. Sci. USA. 84:4910-4913) for the diffusion of dextran and Ficoll particles in Swiss 3T3 cells. By accounting for geometrical factors, these results allow one to assess the relative contributions of geometrical hindrance and of binding to the cytoskeletal lattice from measurements of intracellular diffusion coefficients of proteins.     

Lysophosphatidylcholine metabolism to 1,2-diacylglycerol in lymphoblasts: involvement of a phosphatidylcholine-hydrolyzing phospholipase C

We have previously described the chemoattraction of lymphoblasts by lysophosphatidylcholine [Hoffman, R. D., et al. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3285-3289]. In studying the mechanism of chemoattraction it was found that lysophosphatidylcholine was metabolized to 1,2-diacylglycerol by the lymphoblastic cell line 6C3HED. One route of metabolism involves the acylation of lysophosphatidylcholine to phosphatidylcholine with subsequent hydrolysis to 1,2-diacylglycerol and phosphocholine by the action of phospholipase C. The increase in cellular 1,2-diacylglycerol was established by metabolic experiments using [14C]glycerol-labeled lysophosphatidylcholine and by mass measurements of 1,2-diacylglycerol. The presence of a phosphatidylcholine-hydrolyzing phospholipase C was confirmed in 6C3HED cell homogenates. In intact cells, lysophosphatidylcholine induced a pattern of protein phosphorylation similar to those of 1,2-dioctanoylglycerol and phorbol 12-myristate 13-acetate, two known activators of protein kinase C. This pathway of lysophosphatidylcholine metabolism, which involves a phosphatidylcholine-hydrolyzing phospholipase C, may be important in the activation of protein kinase C independent of inositol phospholipid hydrolysis.     

Comparison of effects of a potassium channel opener BRL34915, a specific potassium ionophore valinomycin and calcium channel blockers on endothelin-induced vascular contraction

The effects of a potassium (K+) channel opener BRL34915 and a specific K+ ionophore valinomycin on vasoconstriction induced by endothelin (ET) were compared with those of calcium (Ca2+) channel blockers, nicardipine and verapamil, using helical strips from rat thoracic aorta. ET induced potent and persistent contraction in control solution and similar but smaller contraction in Ca2+-free solution. BRL34915 and valinomycin inhibited the ET-induced contraction dose-dependently in control solution, but not in Ca2+-free solution. The ET-induced contraction was also inhibited by nicardipine and verapamil, though less strongly. On the other hand, high K+ (35 mM)-induced vasoconstriction was strongly inhibited by nicardipine and verapamil, but not by BRL34915 or valinomycin. These results support the idea that the extracellular Ca2+-dependent component of the ET-induced contraction may be mediated by Ca2+ influx by a route other than voltage-dependent Ca2+-channels.