Subcellular origin of the oxalate- or inorganic phosphate-stimulated Ca2+ transport by smooth muscle microsomes: revisitation of the old problem by a new approach using saponin

Saponin, a cell-skinning reagent which perforates the cell membrane via its specific interaction with plasmalemmal cholesterol, was used to identify the subcellular origin of ATP-dependent Ca2+ accumulation in the presence and absence of inorganic phosphate and oxalate by microsomal fractions isolated from rat vas deferens and dog aorta. The purified plasma membranes from rat gastric fundus muscle, which elicit the stimulation of ATP-dependent Ca2+ accumulation by inorganic phosphate but not by oxalate, were used as a control reference. Saponin at concentrations effective for skinning smooth muscle fibres (10-50 micrograms/ml) inhibited Ca2+ binding in the absence of ATP to a similar extent in all fractions, but the inhibition of ATP-dependent Ca2+ accumulation was more pronounced in dog aorta microsomes and rat gastric fundus muscle plasma membranes than in rat vas deferens microsomes. The resistance of phosphate- and oxalate-stimulated ATP-dependent Ca2+ accumulation to inhibition by saponin was much greater in rat vas deferens than in dog aorta microsomes. Our results suggest that phosphate- and oxalate-stimulated ATP-dependent Ca2+ accumulation also occurs in plasma membrane vesicles isolated from smooth muscle and is by no means an unique property of endoplasmic reticulum.     

Immunocytochemical detection of a murine leukemia virus-related nuclear antigen in mouse oocytes and early embryos.

The expression of murine leukemia virus (MuLV)-related antigens in oocytes and early embryos of Swiss mice was studied by the unlabeled antibody peroxidase-antiperoxidase method using an antiserum to the major core protein, p30, of AKR MuLV. This procedure resulted in an intense staining, at antibody dilutions up to 1:500, of the germinal vesicles of oocytes and the interphase nuclei of early embryos. Nuclear staining was restricted to a specific period of oocyte growth and embryo development. It developed gradually in oocytes having reached one third to one half the full-grown size and persisted until meiotic maturation. In early embryos, nuclear staining was present from the one-cell stage to the morula, but disappeared during transition from morula to blastocyst and was not seen in expanded blastocysts. Nuclear staining was abolished by absorption of the anti-p30 serum with detergent-disrupted virions of Gross(A), AKR, Kirsten and Moloney MuLV, and Kirsten MSV(MuLV), but not with the Friend and Rauscher strains of MuLV, the xenotropic BALB:virus 2, a mouse tropic and an amphotropic clone of wild mouse MuLV, or with FeLV and RSV. On the basis of these results, it is suggested that the nuclear antigen (termed germinal vesicle antigen) reacting with the anti-p30 serum is the product of a cellular gene having a normal function in early embryonic development, and that sequences related to this gene are incorporated into the genome of AKR-type MuLV.     

Confirmation of the single-step membrane filtration procedure for estimating Pseudomonas aeruginosa densities in water.

The efficiency of two procedures, membrane filtration and most probable number, to resuscitate and enumerate Pseudomonas aeruginosa have been compared at two temperatures and varying incubation periods. Data indicate that the membrane filtration procedure using mPA or mPA medium B is more efficient than the most-probable-number procedure in estimating P. aeruginosa populations. It was also found that the specificity of the membrane filtration procedure was such that 92 to 99% of the colonies counted as P. aeruginosa were confirmed, whereas only 2.7 to 10% of the nontypical colonies were confirmed as P. aeruginosa. Furthermore, the data indicate that mPA medium B combined with a 3- to 4-day incubation period at 4.15 degrees C is slightly more specific than mPA medium and is a valid single-step procedure for the resuscitation and enumeration of P. aeruginosa from water or sewage effluent.     

Modulation of auxin-binding proteins in cell suspensions

Summary Cultured cell lines from carrot (Daucus carota L.) with little or no embryogenic potential were examined for the auxin-binding capacity of their membranes. The lines belonged to different classes: (a) wild-type lines kept in culture for different periods (the longer the period, the lower being their embryogenic potential); (b) variants, isolated after mutagenesis, showing normal growth but a lack of embryogenic response; (c) auxin-resistant lines, isolated as colonies on solid media containing 45 M 2,4-d; (d) a previously described tumorous line (E9) isolated because of its resistance to hypomethylating drugs. All of these lines showed alterations in auxin-induced, auxin-binding capacity (modulation), i.e. in the non-embryogenic lines the addition of auxin increased the auxinbinding capacity to a very small degree, or removal of the hormone did not produce the proper decrease in that capacity, or both defects could be simultaneously present. Both types of defects were shown to be correctable: after treatments designed to increase the amplitude of modulation, embryogenic capacity was restored in a number of lines.     

A spin-label study of the effects of drugs on calcium release from isolated sarcoplasmic reticulum vesicles

The effects of caffeine, thymol, and procaine on calcium release from fragmented sarcoplasmic reticulum (FSR) from rabbit skeletal white muscle were investigated by the spin label method at the organellar level. Two thiol-directed spin labels, 4-maleimide-2,2,6,6-tetramethylpiperidinooxyl and 4-(2-iodoacetamide)-2,2,6,6-tetramethylpiperidinooxyl, were used for the labeling of SR proteins. The ratio (W/S) of the weakly (W) and strongly (S) immobilized ESR signals was measured for the maleimide and iodoacetamide labeled FSR. The two labels gave different W/S values, which means that the two labels report conformational changes at different loci of SR proteins. The dependences of the W/S ratios on the concentration of the drugs showed that conformational changes of SR proteins induced by these drugs are not the same. From measurements of the distribution of 5-doxyldecanoic acid methylester between the lipid and water phases, it was found that the conformational changes of the SR proteins caused by thymol or procaine induced a disorder in local regions of the phospholipid bilayers of FSR, while such disordering was not induced by caffeine. On the other hand, caffeine and thymol showed definite effects on calcium release from FSR, while procaine did not. These results indicate that the effects of the drugs on the protein conformations can be well characterized at the organellar level by means of the spin label technique and that some specific changes in the conformations of SR proteins are necessary for calcium release from FSR.     

Patterns of early and late discharges in somatotopically identified precentral neurons in awake monkeys in response to somatic inputs.

Extracellular recordings were made of single neurons in precentral cortex of awake monkeys. These neurons were somatotopically identified with respect to their responses to inputs from single joints or their somatic surround. Many of these neurons exhibited early (less than 50 ms) and late (greater than 50 ms) discharges in response to flexion or extension torques delivered about the wrist. With the monkey in a mode requiring opposition to the injected torque, all responsive neurons showed a parallel excitatory or inhibitory modification in the early and late discharges. This was true both for cells identified as wrist (flexion-extension) neurons and those identified as nonwrist (flexion-extension) neurons. These findings indicate that the reflex and voluntary components of percentral discharge invariably show a congruent functional response to a torque disturbance, for this particular instruction set.     

Evidence for widespread genomic methylation in the migratory locust, Locusta migratoria (Orthoptera: Acrididae)

The importance of DNA methylation in mammalian and plant systems is well established. In recent years there has been renewed interest in DNA methylation in insects. Accumulating evidence, both from mammals and insects, points towards an emerging role for DNA methylation in the regulation of phenotypic plasticity. The migratory locust (Locusta migratoria) is a model organism for the study of phenotypic plasticity. Despite this, there is little information available about the degree to which the genome is methylated in this species and genes encoding methylation machinery have not been previously identified. We therefore undertook an initial investigation to establish the presence of a functional DNA methylation system in L. migratoria. We found that the migratory locust possesses genes that putatively encode methylation machinery (DNA methyltransferases and a methyl-binding domain protein) and exhibits genomic methylation, some of which appears to be localised to repetitive regions of the genome. We have also identified a distinct group of genes within the L. migratoria genome that appear to have been historically methylated and show some possible functional differentiation. These results will facilitate more detailed research into the functional significance of DNA methylation in locusts.     

Partial characterization of type X collagen from bovine growth-plate cartilage. Evidence that type X collagen is processed in vivo

Sequential extraction of bovine growth-plate cartilage with 4 M guanidinium chloride and pepsin was used to identify the intact and pepsinized forms respectively of type X collagen. This collagen occurs predominantly as the processed [alpha 1(X)]3 form in vivo, although the procollagen [pro alpha 1(X)]3 form can also be detected. The bovine pro alpha 1(X) and alpha 1(X) chains have Mr values identical to the corresponding chick species (Mr 59,000 and 49,000). However, the pepsinized alpha 1(X)p chains (Mr 47,000) are larger than those of the chick (Mr 45,000), and the bovine collagen type X is further distinguished by being disulphide-bonded within the triple-helical domain.     

Specific and nonspecific effects of nucleotides on hormone-induced phosphoinositide turnover in permeabilized human pituitary tumour cells (Flow 9000)

Previous studies have shown that agonist-induced inositol phosphate formation in the human embryonic pituitary cell line Flow 9000 is regulated by guanine nucleotides, and it is likely that a guanine nucleotide-binding protein is involved in coupling receptors to phosphoinositidase C (PIC) [(1986) Biochem.Soc.Trans. 14, 1135-1136]. We have now tested the specificity of various nucleotides in regulating PIC activity in the absence or presence of the hormone cholecystokinin (CCK-8) in saponin-permeabilized [3H]inositol-labelled Flow 9000 cells. We found that all nucleotides tested (i.e. CTP, UTP, ITP, TTP, GTP, GppNHp, GTP[S], ATP, AppNHp and ATP[S]) stimulated total [3H]inositol phosphate ([3H]IP) formation in a dose-dependent manner with similar potency and efficacy. However, only guanine nucleotides significantly enhanced CCK-8 stimulation of [3H]IP production. These results indicate a physiological role for guanine nucleotides in regulating hormone-induced phosphoinositide turnover. In addition, the effects of nucleotides on calcium-dependent PIC activity are discussed.