Correlation between changes in the membrane organization and susceptibility to phospholipase C attack induced by ATP depletion of rat erythrocytes

About 20 and 43% of the total membrane phospholipids are hydrolized in fresh rat erythrocytes by treatment with phospholipase C (Bacillus cereus), or both sphingomyelinase and phospholipase C, respectively, without causing cell lysis. Treatment of ATP-depleted cells with phospholipase C alone results in 50% hydrolysis and extensive lysis. Depletion of ATP causes a marked increase in the aggregation of intramembranous particles accompanied by a similar increase in the smooth area between the particle clusters as revealed by the freeze-etch technique. Such changes are not induced by extensive phospholipid hydrolysis in absence of cell lysis in fresh cells.Based on these and additional data, it is suggested that the membrane phospholipid organization can be divided into 3 types: phospholipids exposed to phospholipase C; phospholipids protected against phospholipase C by presence of sphingomyelin; phospholipids which can be exposed following alteration of the proteinlipid interactions. Such alterations which might be induced by a variety of means, including ATP depletion, might result in clustering of intramembranous particles and increase of the free lipid bilayer phase of the membrane.     

Changes in plasma gonadotropin concentrations and urethral closure pressure in the bitch during the 12 months following ovariectomy

Urinary incontinence due to acquired urethral sphincter incompetence is a common side effect of spaying, for which the underlying cause remains unknown. Spaying not only results in a significant reduction in the urethral closure pressure within 1 year but also in an increase in the plasma gonadotropin concentrations. To investigate the possible link between the post-ovariectomy changes in plasma gonadotropins and in urethral closure pressure, gonadotropin and urodynamic measurements were performed in 10 Beagle bitches before and for a period of 1 year after spaying. Plasma gonadotropin concentrations rose quickly after ovariectomy and peak levels were seen within 3-5 weeks, followed by a sharp drop until week 10. A steady increase was observed subsequently until week 42, when a plateau was reached. One year after spaying, the mean FSH concentration was 75.3 +/- 32.1 ng/ml, a 17-fold increase, and the LH was 8.3 +/- 3.8 ng/ml, an eightfold increase over the pre-spaying values. Ten months after spaying, the mean urethral closure pressure (9.7 cm H2O) was significantly reduced when compared to the mean pre-operative value of 15.4 cm H2O. However, there was no clear relationship between the gonadotropin concentrations and the urethral closure pressure. From these results it seems unlikely that chronically elevated gonadotropins are the underlying cause for reduced urethral closure pressure after spaying resulting in urinary incontinence.     

The effect of GnRH analogs on urinary incontinence after ablation of the ovaries in dogs

After removal of the ovaries approximately 20% of dogs develop urinary incontinence. Removal of the gonads results in estrogen deficiency and chronic elevation in the production and secretion of FSH and LH. The gonadotrophins may directly or indirectly, adversely affect the sphincter function of the urethra. Estrogen replacement therapy and treatment with sympathomimetics, such as ephedrine or phenylpropanolamine (PPA), are effective only in some of the affected dogs, and many of these subsequently become nonresponsive. Since the role of the elevated gonadotrophins has not been elucidated, we used depot preparations of GnRH analogues to down-regulate gonadotrophins once or twice in 13 ovariectomized (ovx), incontinent dogs, which were either refractory to alpha-adrenergics (n=11) or in which alpha-adrenergics were contraindicated (n=2). Dogs were treated with leuprolide, deslorelin, buserelin or triptorelin. In 7 dogs treatments with GnRH analogues alone (n=11) resulted in continence for 50-738 days (mean 247). In all dogs except one, where GnRH treatments did not resolve the incontinence completely, additional treatment with phenylpropanolamine was successful. With additional treatment of phenylpropanolamine complete continence was restored for 21-367 days (mean 159). All treatments caused long-term reduction of circulating FSH and LH concentrations to very low or undetectable levels. No adverse effects of treatments were observed.     

Apyrases (nucleoside triphosphate-diphosphohydrolases) play a key role in growth control in Arabidopsis

Expression of two Arabidopsis (Arabidopsis thaliana) apyrase (nucleoside triphosphate-diphosphohydrolase) genes with high similarity, APY1 and APY2, was analyzed during seedling development and under different light treatments using beta-glucuronidase fusion constructs with the promoters of both genes. As evaluated by beta-glucuronidase staining and independently confirmed by other methods, the highest expression of both apyrases was in rapidly growing tissues and/or tissues that accumulate high auxin levels. Red-light treatment of etiolated seedlings suppressed the protein and message level of both apyrases at least as rapidly as it inhibited hypocotyl growth. Adult apy1 and apy2 single mutants had near-normal growth, but apy1apy2 double-knockout plants were dwarf, due primarily to reduced cell elongation. Pollen tubes and etiolated hypocotyls overexpressing an apyrase had faster growth rates than wild-type plants. Growing pollen tubes released ATP into the growth medium and suppression of apyrase activity by antiapyrase antibodies or by inhibitors simultaneously increased medium ATP levels and inhibited pollen tube growth. These results imply that APY1 and APY2, like their homologs in animals, act to reduce the concentration of extracellular nucleotides, and that this function is important for the regulation of growth in Arabidopsis.     

Urodynamic parameters and plasma LH/FSH in spayed Beagle bitches before and 8 weeks after GnRH depot analogue treatment

The pathophysiology of urinary incontinence due to spaying remains unknown. Incontinent bitches can be treated successfully with depot preparations of GnRH-analogues and there are differences in plasma gonadotropin levels between continent and incontinent spayed bitches. It is therefore assumed that the supraordinated hormones, GnRH, FSH, and/or LH, have an effect on the urodynamic parameters. In this study, the potential influence of these hormones on the lower urinary tract was investigated by measuring urethral pressure profiles and cystometry. Simultaneously, plasma concentrations in 10 spayed Beagle bitches were determined 5 weeks prior to and 8 weeks after treatment with the GnRH analogue leuprolide. Within 1 week of GnRH analogue administration, plasma FSH and LH levels decreased from 72.5 and 7.7 to 7.75 and 0.72ng/mL, respectively. These plasma gonadotropin levels correspond with those of intact bitches during anoestrus. Urethral pressure profiles indicated that the treatment had no significant effect on maximum urethral closure pressure, functional and total length of the urethra, or area of the closure pressure curve. The data obtained by cystometry regarding mean bladder threshold volume showed a significant increase from 109 to 172mL. The improvement in bladder function after the application of GnRH-application is presumably a direct effect of the GnRH as a relationship between the plasma gonadotropin levels and the urodynamic parameters could not demonstrated.     

Innervation of developing intrafusal muscle fibers in the rat

The chronology of development of spindle neural elements was examined by electron microscopy in fetal and neonatal rats. The three types of intrafusal muscle fiber of spindles from the soleus muscle acquired sensory and motor innervation in the same sequence as they formed--bag2, bag1, and chain. Both the primary and secondary afferents contacted developing spindles before day 20 of gestation. Sensory endings were present on myoblasts, myotubes, and myofibers in all intrafusal bundles regardless of age. The basic features of the sensory innervation--first-order branching of the parent axon, separation of the primary and secondary sensory regions, and location of both primary and secondary endings beneath the basal lamina of the intrafusal fibers--were all established by the fourth postnatal day. Cross-terminals, sensory terminals shared by more than one intrafusal fiber, were more numerous at all developmental stages than in mature spindles. No afferents to immature spindles were supernumerary, and no sensory axons appeared to retract from terminations on intrafusal fibers. The earliest motor axons contacted spindles on the 20th day of gestation or shortly afterward. More motor axons supplied the immature spindles, and a greater number of axon terminals were visible at immature intrafusal motor endings than in adult spindles; hence, retraction of supernumerary motor axons accompanies maturation of the fusimotor system analogous to that observed during the maturation of the skeletomotor system. Motor endings were observed only on the relatively mature myofibers; intrafusal myoblasts and myotubes lacked motor innervation in all age groups. This independence of the early stages of intrafusal fiber assembly from motor innervation may reflect a special inherent myogenic potential of intrafusal myotubes or may stem from the innervation of spindles by sensory axons.     

Light regulation of the abundance of mRNA encoding a nucleolin-like protein localized in the nucleoli of pea nuclei.

A cDNA encoding a nucleolar protein was selected from a pea (Pisum sativum) plumule library, cloned, and sequenced. The translated sequence of the cDNA has significant percent identity to Xenopus laevis nucleolin (31%), the alfalfa (Medicago sativa) nucleolin homolog (66%), and the yeast (Saccharomyces cerevisiae) nucleolin homolog (NSR1) (28%). It also has sequence patterns in its primary structure that are characteristic of all nucleolins, including an N-terminal acidic motif, RNA recognition motifs, and a C-terminal Gly- and Arg-rich domain. By immunoblot analysis, the polyclonal antibodies used to select the cDNA bind selectively to a 90-kD protein in purified pea nuclei and nucleoli and to an 88-kD protein in extracts of Escherichia coli expressing the cDNA. In immunolocalization assays of pea plumule cells, the antibodies stained primarily a region surrounding the fibrillar center of nucleoli, where animal nucleolins are typically found. Southern analysis indicated that the pea nucleolin-like protein is encoded by a single gene, and northern analysis showed that the labeled cDNA binds to a single band of RNA, approximately the same size and the cDNA. After irradiation of etiolated pea seedlings by red light, the mRNA level in plumules decreased during the 1st hour and then increased to a peak of six times the 0-h level at 12 h. Far-red light reversed this effect of red light, and the mRNA accumulation from red/far-red light irradiation was equal to that found in the dark control. This indicates that phytochrome may regulate the expression of this gene.     

Plasmid segregation into minicells is associated with membrane attachment and independent of plasmid replication.

The role of plasmid replication in the segregation of plasmids into Escherichia coli minicells was investigated with temperature-sensitive replication mutants derived from E. coli plasmids ColE1 and pSC101. For as long as six generations of growth, at permissive or nonpermissive temperatures (when greater than 80% of plasmid replication was inhibited), the same amount of previously 3H-labeled plasmid DNA segregated into minicells. Density gradient separations of wild-type and temperature-sensitive plasmid DNA from both replicons segregated into the minicells showed that about 20 to 25% was stably associated with the minicell membrane at both temperatures. Electron microscopy showed this DNA to consist of circular plasmid molecules attached to the minicell membrane. These combined findings suggest that segregation of plasmids into minicells and their association with the minicell membrane are interrelated and independent of plasmid replication.     

1-Deoxy-D-xylulose-5-phosphate synthase, a limiting enzyme for plastidic isoprenoid biosynthesis in plants

The initial step of the plastidic 2C-methyl-D-erythritol 4-phosphate (MEP) pathway that produces isopentenyl diphosphate is catalyzed by 1-deoxy-d-xylulose-5-phosphate synthase. To investigate whether or not 1-deoxy-d-xylulose-5-phosphate synthase catalyzes a limiting step in the MEP pathway in plants, we produced transgenic Arabidopsis plants that over- or underexpress this enzyme. Compared with non-transgenic wild-type plants, the transgenic plants accumulate different levels of various isoprenoids such as chlorophylls, tocopherols, carotenoids, abscisic acid, and gibberellins. Phenotypically, the transgenic plants had slight alterations in growth and germination rates. Because the levels of several plastidic isoprenoids correlate with changes in 1-deoxy-D-xylulose-5-phosphate synthase levels, we conclude that this enzyme catalyzes one of the rate-limiting steps of the MEP biosynthetic pathway. Furthermore, since the product of the MEP pathway is isopentenyl diphosphate, our results suggest that in plastids the pool of isopentenyl diphosphate is limiting to isprenoid production.     

Effect of a long acting GnRH analogue or placebo on plasma LH/FSH, urethral pressure profiles and clinical signs of urinary incontinence due to Sphincter mechanism incompetence in bitches

In 23 bitches with urinary incontinence due to spaying, the effect of treatment with a long-acting formulation of leuprolide acetate on frequency of incontinence, plasma gonadotropin levels and urodynamic parameters was evaluated. In addition, the clinical effect was compared with that of treatment with alpha-adrenergics. Before treatment, the dogs' incontinent episodes occurred, on average, 4 times per day on up to 6 days per week. In the pre-trial after therapy with phenylpropanolamine (n=23) the episodes of incontinence decreased by 92%, in the double-blind study 5 weeks after GnRH-analogue (n=11) by 71%; and by 28% after the placebo (n=12). By the end of the study, nine of twenty-two leuprolide treated bitches responded completely to treatment and were continent for periods lasting 70-575 days after treatment. In another 10 dogs, response to therapy was partial and the frequency of incontinence was reduced by at least 50%. After therapy with placebo, one bitch had no episodes of incontinence for 412 days. Treatment with the GnRH-analogue significantly decreased the plasma gonadotropin levels but there was no correlation between the effect on gonadotropin levels and response to treatment. Treatment with leuprolide or placebo had no effect on urethral closure pressure regardless of the response to treatment. The hypothesis that the change of the plasma gonadotropin levels after spaying is the cause of reduced urethral closure function was not supported by the results of this study. A possible direct effect of GnRH-analogues on the bladder is discussed. Long acting GnRH analogues appear to be a well-tolerated alternative for urinary incontinence treatment, but they appear to be less effective than the alpha-adrenergics.