The effect of sacral nerve stimulation on uterine activity: a pilot study.

INTRODUCTION: Sacral nerve stimulation (SNS) is a treatment for consecutive therapy resistant faecal incontinence or constipation. Little is known about the effects of SNS on uterocervical function. Therefore, it is advised to turn off the stimulator during pregnancy or to wait with permanent implantation of the stimulator until family completion. Diagnostic ultrasound provides an, non-invasive, opportunity to study various aspects of uterine activity. The purpose of this pilot study was to assess the influence of SNS on endometrial waves of the nonpregnant uterus by ultrasound recordings. METHOD: Six patients with an implanted SNS were included. Ultrasound recordings were performed with the stimulator turned off and in three stimulation frequencies. Uterine activity is described as wave frequency and wave direction. RESULTS: All three premenopausal patients showed endometrial activity with the stimulator turned off. This activity was maintained with the stimulator turned on in two patients, but disappeared in one patient. All three postmenopausal patients had no endometrial activity with the stimulator turned off. In one patient there was activity with the stimulator turned on at a frequency of 21 Hz. CONCLUSION: We have shown some effect of SNS on uterine activity. In premenopausal women we discovered that SNS seems to exhibit no effect or an inhibitory effect rather than an excitatory effect on uterine activity. Based on the preliminary results of this study, we can not recommend any guidelines for SNS usage during conception and pregnancy. A larger study in premenopausal women with SNS is needed to investigate the significance of these changes.

[Cystocele and perineal ultrasound : Comparison of reliability and patient satisfaction]. / Zystozele und perinealer Ultraschall : Vergleich von Reliabilität und Patientenzufriedenheit.

BACKGROUND: Pelvic organ prolapse is a common medical finding. The use of perineal ultrasound for diagnosis of cystoceles is gaining in importance. OBJECTIVES: The purpose of this work was to test whether perineal ultrasound can be used to diagnose a cystocele before surgery and for follow-up examination. Furthermore, patient satisfaction during speculum examination and perineal ultrasound was compared. MATERIALS AND METHODS: 33 women with cystocele were examined before and after anterior colporrhaphy. Symptoms and satisfaction were documented with questionnaires. RESULTS: Ultrasound measurements of both examiners were correlated before and after colporrhaphy. Also, the degree of cystocele and ultrasound were correlated during Valsalva after surgery. There was no clear relation between typical symptoms of the cystocele and ultrasound measurements. The patient's comfort is higher during ultrasound than during speculum examination (r = 0.45; p = 0.04. t = 4,418; p < 0.01). CONCLUSION: The results of the perineal ultrasound are reproducible before and after colporrhaphy. Patients prefer ultrasound to the speculum examination. A sonographic scale of the cystocele would extend the use of perineal ultrasound.

Properties and subunit composition of RNA polymerase II from plant cell cultures.

The purification of DNA-dependent RNA polymerase II (EC 2.7.7.6) from plant cell cultures of Petroselinum (parsley) is described. The procedure during which enzyme I is eliminated includes initial precipitation with (NH4)2SO4, an ultracentrifugation step, gel filtration on Sepharose 4B, chromatography on DEAE-cellulose, DNA-agarose and DEAE-Sephadex. The enzyme purified almost to homogeneity exhibits maximal activity with denatured DNA, and is activated preferentially by Mn2+; alpha-amanitin acts as a strong inhibitor. Electrophoresis of the enzyme in the presence of dodecylsulphate indicates that it is composed of seven subunits with mol. wts of 200 000, 180 000, 140 000, 43 000, 26 000, 25 000 and 16 000. The results of molecular weight and molar ratio determinations suggest that Petroselinum RNA polymerase II may exist in two active forms differing only in the composition of their high molecular weight subunits.

Long-term safety and efficacy of zidovudine in patients with advanced human immunodeficiency virus disease. Zidovudine Epidemiology Study Group.

An epidemiologic study was initiated in 1987 to evaluate the long-term safety and efficacy of zidovudine in patients with advanced human immunodeficiency virus disease. Data from 886 patients with the acquired immunodeficiency syndrome (AIDS) or AIDS-related complex and CD4+ lymphocyte count less than 0.25 x 10(9)/L are reported. Eighteen-month survival was 67% for the cohort. Pretreatment factors associated with increased survival time included index diagnosis of AIDS-related complex, hematocrit of 0.35 or greater, CD4+ lymphocyte count of 0.15 x 10(9)/L or greater, high functional status, and time from diagnosis of AIDS to treatment of less than 60 days. By proportional hazards analysis, development of serious anemia was the most significant factor associated with early death. Receiving zidovudine for a high proportion of time significantly improved chances of survival even if anemia developed. Serious leukopenia occurring in 37% and serious anemia occurring in 32% of patients. Nonhematologic adverse events were uncommon and no previously unreported adverse events were seen.

Iron chelation.

Adequate iron chelation in thalassaemia has resulted in a striking improvement in survival, with a reduction of cardiac mortality at age 15 years from 14-3%, and a predicted survival at age 36 years of 85%. Long term desferrioxamine (DF) therapy in thalassaemic children should be started between 2-4 years of age. In addition to daily 8-12 h subcutaneous infusions, intermittent high dose (9-16 g) i.v. supplementation over 24-48 h may be given on the occasion of blood transfusions. In established myocardiopathy continuous i.v. DF infusion at 100-125 mg/kg/d may result in improved myocardial function. In addition, there is considerable current interest in the use of DF in conditions unrelated to iron overload by preventing the formation of free-radicals in inflammatory reactions, or by S-phase inhibition of cell proliferation. Although at present highly experimental, this novel approach may have important implications for the management of patients with inflammatory conditions and perhaps in the control of protozoal infections. Over the last decade several hundred candidate compounds have been studied in cell cultures and in animal models and a number of orally effective iron chelators have been identified, all of which are superior to DF in their in vivo iron chelating effect. Although we do not yet have a new drug which is immediately available for replacing DF in clinical practice, significant progress has already been made, and some of the most promising candidate drugs are currently undergoing extensive toxicity tests in anticipation of their development for large-scale clinical use.

Protein kinase C mu selectively activates the mitogen-activated protein kinase (MAPK) p42 pathway.

Here we show that human protein kinase C mu (PKC mu) activates the mitogen-activated protein kinase (MAPK). Transient expression of constitutive active PKC mu leads to an activation of Raf-1 kinase as demonstrated by in vitro phosphorylation of MAPK. PKC mu enhances transcriptional activity of a basal thymidine kinase promotor containing serum response elements (SREs) as shown by luciferase reporter gene assays. SRE driven gene activation by PKC mu is triggered by the Elk-1 ternary complex factor. PKC mu-mediated activation of SRE driven transcription can be inhibited by the MEK1 inhibitor PD98059. In contrast to the activation of the p42/ERK1 MAPK cascade, transient expression of constitutive active PKC mu does neither affect c-jun N-terminal kinase nor p38 MAPK.

In vitro activation and substrates of recombinant, baculovirus expressed human protein kinase C mu.

To study enzymatic activity and activation conditions of the recently identified novel protein kinase C mu (PKC mu) subtype, epitope tagged PKC mu was propagated in the baculovirus expression system and was purified to homogeneity. PKC mu displays high affinity phorbol ester binding (Kd=7 nM) resulting in enhanced phosphatidylserine-dependent kinase activity. From various lipid second messengers known to activate PKCs only diacylglycerol and PtdIns-4,5-P2, were found to promote PKC mu kinase activity. Two peptides derived from the glycogen synthase, GS-peptide and syntide 2, were found to be phosphorylated efficiently in vitro. MARCKS (myristoylated alanine-rich C-kinase substrate) served as an in vitro substrate for PKC mu too. However, in contrast to other PKCs, a peptide derived from the MARCKS phosphorylation domain is phosphorylated only at serine 156, and not at serines 152 and 163, implicating a differential regulation by PKC mu.

Bruton's tyrosine kinase (Btk) associates with protein kinase C mu.

Bruton's tyrosine kinase (Btk) is considered an essential signal transducer in B-cells. Mutational defects are associated with a severe immunodeficiency syndrome, X-chromosome linked agammaglobulinemia (XLA). Here we show by coimmunoprecipitation that a member of the protein kinase C (PKC) family, PKCmu, is constitutively associated with Btk. Neither antigen receptor (Ig) crosslinking nor stimulation of B-cells with phorbol ester or H(2)O(2) affected Btk/PKCmu interaction. GST precipitation analysis revealed association of the Btk pleckstrin/Tec homology domain with PKCmu. Transient overexpression of PKCmu deletion mutants as well as expression of selected PKCmu domains in 293T cells revealed that both the kinase domain and the regulatory C1 region are independently capable of binding to the Btk PH-TH domain. These data show the existence of a PKCmu/Btk complex in vivo and identify two PKCmu domains that participate in Btk interaction.

Intrauterine elimination of pyridoxal 5'-phosphate in full-term and preterm infants.

This study addressed the intrauterine elimination of pyridoxal 5'-phosphate (PLP) in 15 preterm and 31 full-term infants, thereby providing estimates of fetal vitamin consumption as well as maternal vitamin requirements during pregnancy. Elimination was calculated as the difference in the plasma PLP concentration between umbilical vein and umbilical artery times the umbilical plasma flow. Plasma flow in the umbilical vein was calculated from pulsed Doppler ultrasonographic determination of blood flow and from the hematocrit value. Plasma PLP concentrations were assayed in maternal and umbilical veins and the umbilical artery; PLP concentrations were similar in preterm and full-term infants (P > 0.05). In both groups of infants the PLP concentration in the umbilical vein (preterm: 100.3 nmol/L; full-term: 63.9 nmol/L) was ninefold higher than in maternal circulation (P < 0.001). In full-term infants, PLP concentrations in maternal and umbilical veins correlated weakly (r = 0.358, P < 0.05), but no significant correlation was found in the preterm group (P > 0.05). The arteriovenous concentration gradient of PLP in cord vessels was higher in preterm infants (15.0 nmol/L) than in full-term infants (2.1 nmol/L), but the difference between groups was not significant (P > 0.05). Preterm infants eliminated 1.7 nmol PLP.kg-1.min-1 in utero, whereas full-term infants eliminated 0.2 nmol PLP.kg-1.min-1 (P < 0.05). The significantly higher plasma flow in preterm infants (116 mL.min-1.kg-1) compared with full-term infants (78 mL.min-1.kg-1) contributed to the higher PLP elimination in preterm infants.

A rapid and sensitive ELISA for serum ferritin employing a fluorogenic substrate.

A fluorescent enzyme-linked immunosorbent assay is described for the rapid measurement of serum ferritin. Increased sensitivity was achieved by using 4-methyl-umbelliferyl-beta-D-galactopyranoside as the substrate for beta-galactosidase coupled to the purified antiferritin antibody. Further enhancement of the specific antigen-antibody reaction was attained by the addition of 4% polyethylene glycol 6000 to the antiferritin-beta-galactosidase conjugate. The procedure is performed in microELISA plates. These modifications of the method permit the measurement of serum ferritin at concentrations ranging from 0.25 to 50 microgram/liter with a coefficient of variation of 8% or less. The entire procedure is performed at ambient temperature and is completed within one working day. The cost of the assay is less than 10% of the immunoradiometric assay for serum ferritin.

Primary structures of Sm31/32 diagnostic proteins of Schistosoma mansoni and their identification as proteases.

We have constructed cDNA clones containing the complete nucleotide sequences coding for two highly antigenic Schistosoma mansoni adult worm proteins, Sm31 and Sm32. The predicted amino acid sequence of Sm31 shows significant homology to mouse, rat and human cathepsin B. The nucleotide sequence of Sm32 is identical to that reported by others for S. mansoni "haemoglobinase'. The different nucleotide sequences demonstrate the existence of two different proteolytic enzymes, both of which are synthesised in the form of precursor molecules. Structural homology of the schistosome cathepsin B to the mammalian ones indicates that the mature protein is processed from a propeptide. The calculated molecular weight of haemoglobinase of 47,000 suggests that post-translational processing is also involved in generating an active protease.

Expression of diagnostic 31/32 kilodalton proteins of Schistosoma mansoni as fusions with bacteriophage MS2 polymerase.

An expression plasmid pEx34b was used to synthesise parts of the 31/32 kDa Schistosoma mansoni antigens as fusions with the amino terminus of the phage MS2 polymerase. Purified MS2-schistosome fusion proteins reacted specifically in an enzyme-linked immunosorbent assay with sera from S. mansoni-infected patients. The observation that the majority of human sera tested recognised schistosome-specific epitopes, but not the MS2 polymerase fragment, suggests that the fusion proteins are useful for the immunodiagnosis of schistosomiasis and might be incorporated in a serological test system based on recombinant antigens.

Syntheses of iron bis(pyridoxal isonicotinoylhydrazone)s and the in vivo iron-removal properties of some pyridoxal derivatives.

Pyridoxal isonicotinoylhydrazone (PINH; 1) and its isomeric O-acetates (E and Z) were synthesized and complexed with ferrous ions to afford the hitherto unisolated chelates iron(II) bis(pyridoxal isonicotinoylhydrazone)s (11) and iron(II) bis(O-acetylpyridoxal isonicotinoylhydrazone)s (12). The analytical and spectroscopic data of the new coordination compounds are presented. In addition, a series of imino derivatives of pyridoxal of structures 2-3 and 5-10 have been prepared and tested in vivo as chelators of storage iron, and the cumulative net excretion of radioiron in urine and in feces was estimated. This study reestablishes that PINH is a potent iron chelator in vivo comparable in efficiency with parenteral desferrioxamine (DF) and indicates that it requires further attention.

Inhibition of calcium accumulation by the sarcoplasmic reticulum: a putative mechanism for the cardiotoxicity of adriamycin.

The aim of this study was to examine the effect of adriamycin (ADR) on calcium accumulation by the sarcoplasmic reticulum (SR). Chemical skinning of cultured rat myocardial cells compromised the barrier function of the cell membrane and thus permitted direct exposure of mitochondrial and non-mitochondrial sites to ADR. In the presence of ATP, and sodium azide, mitochondrial calcium accumulation was negligible. Furthermore, it has previously been shown that non-mitochondrial calcium accumulation is mediated mainly by the SR under these conditions. Incubation with 10 microM ADR for 2 hr reduced the level of calcium accumulation by the SR by 50%. A similar effect was obtained after 24 hr incubation with 1 microM ADR. The addition of ferric iron to the culture medium further reduced the level of calcium accumulation. Neither vitamin E nor beta-carotene affected calcium accumulation by the SR. These results suggest that ADR interferes with the calcium accumulation activity of the SR and that ferric iron potentiates this effect.

Pathophysiology of iron overload.

In thalassemia, iron overload is the joint outcome of excessive iron absorption and transfusional siderosis. While iron absorption is limited by a physiologic ceiling of about 3 mg/d, plasma iron turnover in thalassemia may be 10 to 15 times normal, caused by the wasteful, ineffective erythropoiesis of an enormously expanded erythroid marrow. This outpouring of catabolic iron exceeds the iron-binding capacity of transferrin and appears in plasma as non-transferrin-plasma iron (NTPI). The toxicity of NTPI is much higher than of transferrin-iron as judged by its ability to promote hydroxyl radical formation resulting in peroxidative damage to membrane lipids and proteins. In the heart, this results in impaired function of the mitochrondrial respiratory chain and abnormal energy metabolism manifested clinically in fatal hemosiderotic cardiomyopathy. Ascorbate increases the efficacy of iron chelators by expanding the intracellular chelatable iron pool, but, at suboptimal concentrations is a pro-oxidant, enhancing the catalytic effect of iron in free radical formation. NTPI is removed by i.v. DFO in a biphasic manner and reappears rapidly upon cessation of DFO, lending support to the continuous, rather than intermittent, use of chelators. Unlike DFO and other hexadentate chelators, bidentate chelators such as L1 may produce incomplete intermediate iron complexes at suboptimal drug concentrations.

Human beta-casomorphin-8 immunoreactive material in the plasma of women during pregnancy and after delivery.

A method for the extraction of human beta-casomorphin-8 immunoreactive material from human plasma and a radioimmunoassay for its determination in the plasma extracts were developed. Blood was collected from 34 men, from 35 non-pregnant women, from 35 pregnant women and from 138 women after delivery and plasma extracts were assayed for the presence of human beta-casomorphin-8 immunoreactive materials. No human beta-casomorphin-8 immunoreactive material was detected in the plasma of men or non-pregnant women, whereas such material was found in the plasma of 26 out of 35 pregnant women and in the plasma of 100 out of 138 women after parturition. Material collected from women after delivery was characterized by gel filtration and high-performance liquid chromatography (HPLC) and was found to be of different composition in various individuals; its components, one of which coeluted with human beta-casein from the HPLC column, have apparently higher molecular weights than human beta-casomorphin-8. Some of these compounds seem to be very stable against enzymatic degradation at 37 degrees C in human plasma, whereas human beta-casomorphin-8 proved to be degraded very fast under identical conditions. A physiological significance of mammary products of the beta-casomorphin type during pregnancy or after parturition is suggested.

The role of iron and iron chelators in anthracycline cardiotoxicity.

The redox cycling of anthracyclines promotes the formation of free radicals which are believed to play a central role in their cardiotoxicity. A number of observations indicate that the mechanism of the antineoplastic effect of anthracyclines is independent of their cardiotoxic effect and that it may be possible to prevent toxicity without interfering with therapeutic effect. Iron plays an important role in anthracycline toxicity by promoting the conversion of superoxide into highly toxic hydroxyl radicals through the Haber-Weiss reaction. Conversely, iron deprivation by its high-affinity binding to iron chelating compounds may inhibit anthracycline toxicity by interfering with free radical formation. ICRF-187, a bispiperazonedione which is hydrolyzed intracellularly into a bidentate chelator resembling EDTA, is able to decrease adriamycin-induced free hydroxyl radical formation and to prevent the development of clinical cardiac toxicity in patients receiving long-term anthracycline therapy. Our studies in rat heart cell cultures have shown that iron overload aggravates anthracycline toxicity and that this interaction can be prevented by prior iron chelating treatment. Since iron overload caused by multiple blood transfusions and bone marrow failure is a common condition in patients requiring anthracycline therapy, these observations may have significant clinical implications to the prevention of anthracycline cardiotoxicity.

Cell populations of tendon: a simplified method for isolation of synovial cells and internal fibroblasts: confirmation of origin and biologic properties.

Tendons transmit the force of muscle contraction to bone to effect limb movement. Special structural and biological properties of tendon have developed to facilitate force transmission. The tendon has a complex organization of cells surrounding the collagen bundles inside tendon as well as at the tendon surface. Internal cells may act to maintain the bulk of the collagen in tendon. External cells in the epitenon may provide lubrication for tendon gliding. To develop better understanding of these processes and the roles the cell populations play, we isolated cells from the surface and interior of tendon and studied them in vitro. Flexor tendons from 8-week-old white Leghorn chickens were separated into two distinct cell populations: the outer synovial cells and the fibroblasts more internal in tendon. These cell populations were discernible by their locations in the intact tendon, determined by sequential enzymatic and physical release from their substrata. Initially, some cells eluted in Hanks' salt solution (HSS) (population 1); then synovial cells were released after a 2-min treatment with 0.5% collagenase (population 2). Next, a population of synovial cells was released in high yield by treatment with 0.25% trypsin (step III, population 3). Step III, population 3 cells were used as synovial cells (SCs). Next, a population of SCs and fibroblasts were released by scraping with a rubber policeman (population 4). Subsequently, fibroblasts were released after incubation with 0.5% collagenase (population 5). A more direct procedure (procedure 2) to isolate the synovial and internal tendon cells involved treatment in 0.5% collagenase followed by sedimentation at 900 g. Cells that sedimented were largely fibroblasts, whereas the cells that remained at the top of the tube were largely SCs. Cells designated as SCs, isolated by procedure 2, most likely contained surface cells from epitenon and internal interfascicular cells from endotenon and paratenon. Surface tendon cells separated by sequential enzymatic and physical release from their substrata (by procedure 1) had all the following characteristics: distinct subpopulations of cells based on morphology; presence of cytoplasmic, lipid-containing vesicles; decreased sensitivity to trypsin; and reduced generation time as compared with that of internal fibroblasts. Conversely, the internal fibroblasts (IFs) appeared to represent a more uniform population based on morphological characteristics.

Tendon synovial cells secrete fibronectin in vivo and in vitro.

The chemistry and cell biology of the tendon have been largely overlooked due to the emphasis on collagen, the principle structural component of the tendon. The tendon must not only transmit the force of muscle contraction to bone to effect movement, but it must also glide simultaneously over extratendonous tissues. Fibronectin is classified as a cell attachment molecule that induces cell spreading and adhesion to substratum. The external surface of intact avian flexor tendon stained positively with antibody to cellular fibronectin. However, if the surface synovial cells were first removed with collagenase, no positive reaction with antifibronectin antibody was detected. Analysis of immunologically stained frozen sections of tendon also revealed fibronectin at the tendon synovium, but little was associated with cells internal in tendon. The staining pattern with isolated, cultured synovial cells and fibroblasts from the tendon interior substantiated the histological observations. Analysis of polyacrylamide gel profiles of 35S-methionine-labeled proteins synthesized by synovial cells and internal fibroblasts indicated that fibronectin was synthesized principally by synovial cells. Fibronectin at the tendon surface may play a role in cell attachment to prevent cell removal by the friction of gliding. Alternatively, fibronectin, with its binding sites for hyaluronic acid and collagen, may act as a complex for boundary lubrication.

Strain profiles for circular cell culture plates containing flexible surfaces employed to mechanically deform cells in vitro.

Cells in the body are constantly subjected to cyclic mechanical deformation involving tension, compression, or shear strain or all three. A mechanical loading system which deforms cultured cells in vitro was analyzed in order to quantify the deformation or strain to which the cells are subjected. The dynamic system utilizes vacuum pressure to deform a circular silicone rubber substrate on which cells are cultured. These thick circular growth surfaces or plates are formed in the bottoms of the wells of 6-well culture plates. An axisymmetric model was formulated and analyzed using rectangular hyperelastic elements in a finite element analysis (FEA) software package. The thick circular plate has some disadvantages such as difficulty in observing cells and a nonhomogeneous strain profile which is maximum at the periphery and minimal at the center. A thinner circular surface (a thin plate) was also investigated in order to provide a more homogeneous strain profile. The radial strain on the thick circular plate, as determined by FEA, was nonlinear with a peak strain value of 0.30 (vacuum pressure of 22 kPa) about three-quarters of the distance from the center to the edge. In contrast, the radial strain of the thin circular plate was moderately constant across the surface. The circumferential strain for both of these models was less than the radial strain except for the center where they are equal. Avian tendon cells were cultured on the surface of a thick plate and exposed to cyclic strains for 24 h at a rate of 0.17 Hz and observed for cellular alignment.(ABSTRACT TRUNCATED AT 250 WORDS)