Dreams reflect nocturnal cognitive processes: Early-night dreams are more continuous with waking life, and late-night dreams are more emotional and hyperassociative.

Contributions of specific sleep stages to cognitive processes are increasingly understood. Non-REM sleep is particularly implicated in episodic memory consolidation, whilst REM sleep preferentially consolidates and regulates emotional information, and gives rise to creativity and insight. Dream content reflects these processes: non-REM dreams are more likely to picture episodic memories, whereas REM dreams are more emotional and bizarre. However, across-the-night differences in the memory sources of dream content, as opposed to sleep stage differences, are less well understood. In the present study, 68 participants were awoken from sleep in the early and late night and recorded their dreams and waking-life activities. Early-night dreams were more clearly relatable to (or continuous with) waking life than late-night dreams. Late-night dreams were more emotional-important, more time orientation varied, and more hyperassociative, than early-night dreams. These dream content differences may underlie the mental content that accompanies sleep processes like memory consolidation, emotion-processing, and creativity.

Do animals dream?

The understanding of biological functions of sleep has improved recently, including an understanding of the deep evolutionary roots of sleep among animals. However, dreaming as an element of sleep may be particularly difficult to address in non-human animals because in humans dreaming involves a non-wakeful form of awareness typically identified through verbal report. Here, we argue that parallels that exist between the phenomenology, physiology, and sleep behaviors during human dreaming provide an avenue to investigate dreaming in non-human animals. We review three alternative measurements of human dreaming - neural correlates of dreaming, 'replay' of newly-acquired memories, and dream-enacting behaviors - and consider how these may be applied to non-human animal models. We suggest that while animals close in brain structure to humans (such as mammals and birds) may be optimal models for the first two of these measurements, cephalopods, especially octopuses, may be particularly good candidates for the third.

The dream-lag effect: Selective processing of personally significant events during Rapid Eye Movement sleep, but not during Slow Wave Sleep.

Incorporation of details from waking life events into Rapid Eye Movement (REM) sleep dreams has been found to be highest on the night after, and then 5-7 nights after events (termed, respectively, the day-residue and dream-lag effects). In experiment 1, 44 participants kept a daily log for 10 days, reporting major daily activities (MDAs), personally significant events (PSEs), and major concerns (MCs). Dream reports were collected from REM and Slow Wave Sleep (SWS) in the laboratory, or from REM sleep at home. The dream-lag effect was found for the incorporation of PSEs into REM dreams collected at home, but not for MDAs or MCs. No dream-lag effect was found for SWS dreams, or for REM dreams collected in the lab after SWS awakenings earlier in the night. In experiment 2, the 44 participants recorded reports of their spontaneously recalled home dreams over the 10 nights following the instrumental awakenings night, which thus acted as a controlled stimulus with two salience levels, high (sleep lab) and low (home awakenings). The dream-lag effect was found for the incorporation into home dreams of references to the experience of being in the sleep laboratory, but only for participants who had reported concerns beforehand about being in the sleep laboratory. The delayed incorporation of events from daily life into dreams has been proposed to reflect REM sleep-dependent memory consolidation. However, an alternative emotion processing or emotional impact of events account, distinct from memory consolidation, is supported by the finding that SWS dreams do not evidence the dream-lag effect.

Replication of genetic loci for ages at menarche and menopause in the multi-ethnic Population Architecture using Genomics and Epidemiology (PAGE) study.

STUDY QUESTION: Do genetic associations identified in genome-wide association studies (GWAS) of age at menarche (AM) and age at natural menopause (ANM) replicate in women of diverse race/ancestry from the Population Architecture using Genomics and Epidemiology (PAGE) Study? SUMMARY ANSWER: We replicated GWAS reproductive trait single nucleotide polymorphisms (SNPs) in our European descent population and found that many SNPs were also associated with AM and ANM in populations of diverse ancestry. WHAT IS KNOWN ALREADY: Menarche and menopause mark the reproductive lifespan in women and are important risk factors for chronic diseases including obesity, cardiovascular disease and cancer. Both events are believed to be influenced by environmental and genetic factors, and vary in populations differing by genetic ancestry and geography. Most genetic variants associated with these traits have been identified in GWAS of European-descent populations. STUDY DESIGN, SIZE, DURATION: A total of 42 251 women of diverse ancestry from PAGE were included in cross-sectional analyses of AM and ANM. MATERIALS, SETTING, METHODS: SNPs previously associated with ANM (n = 5 SNPs) and AM (n = 3 SNPs) in GWAS were genotyped in American Indians, African Americans, Asians, European Americans, Hispanics and Native Hawaiians. To test SNP associations with ANM or AM, we used linear regression models stratified by race/ethnicity and PAGE sub-study. Results were then combined in race-specific fixed effect meta-analyses for each outcome. For replication and generalization analyses, significance was defined at P < 0.01 for ANM analyses and P < 0.017 for AM analyses. MAIN RESULTS AND THE ROLE OF CHANCE: We replicated findings for AM SNPs in the LIN28B locus and an intergenic region on 9q31 in European Americans. The LIN28B SNPs (rs314277 and rs314280) were also significantly associated with AM in Asians, but not in other race/ethnicity groups. Linkage disequilibrium (LD) patterns at this locus varied widely among the ancestral groups. With the exception of an intergenic SNP at 13q34, all ANM SNPs replicated in European Americans. Three were significantly associated with ANM in other race/ethnicity populations: rs2153157 (6p24.2/SYCP2L), rs365132 (5q35/UIMC1) and rs16991615 (20p12.3/MCM8). While rs1172822 (19q13/BRSK1) was not significant in the populations of non-European descent, effect sizes showed similar trends. LIMITATIONS, REASONS FOR CAUTION: Lack of association for the GWAS SNPs in the non-European American groups may be due to differences in locus LD patterns between these groups and the European-descent populations included in the GWAS discovery studies; and in some cases, lower power may also contribute to non-significant findings. WIDER IMPLICATIONS OF THE FINDINGS: The discovery of genetic variants associated with the reproductive traits provides an important opportunity to elucidate the biological mechanisms involved with normal variation and disorders of menarche and menopause. In this study we replicated most, but not all reported SNPs in European descent populations and examined the epidemiologic architecture of these early reported variants, describing their generalizability and effect size across differing ancestral populations. Such data will be increasingly important for prioritizing GWAS SNPs for follow-up in fine-mapping and resequencing studies, as well as in translational research.

Production, purification, and crystallization of human interleukin-1 beta converting enzyme derived from an Escherichia coli expression system.

Interleukin-1 beta converting enzyme (ICE) is a cysteine protease that catalyzes the conversion of the inactive precursor form of IL-1 beta to an active mature form. The mature form of IL-1 beta is involved in mediating inflammatory responses and in the progression of autoimmune diseases. We recently reported on the production of active human ICE in insect cells using the baculovirus expression system (Wang XM et al., 1994, Gene 145:273-277). Because the levels of expression achieved with this system were limiting for the purpose of performing detailed biochemical and biophysical studies, we examined the production of ICE in Escherichia coli. By using a tac promoter-based expression system and fusion to thioredoxin we were able to recover high levels of active ICE protein. The expressed protein, which was distributed between the soluble and insoluble fractions, was purified to homogeneity from both fractions using a combination of classical and affinity chromatography. Comparisons of ICE derived from both fractions indicated that they were comparable in their specific activities, subunit composition, and sensitivities to specific ICE inhibitors. The combined yields of ICE obtained from the soluble and insoluble fractions was close to 1 mg/L of induced culture. Recombinant human ICE was crystallized in the presence of a specific ICE inhibitor in a form suitable for X-ray crystallographic analysis. This readily available source of ICE will facilitate the further characterization of this novel and important protease.

Production of active human interleukin-1 beta-converting enzyme in a baculovirus expression system.

The cDNA coding for the precursor form of human interleukin-1 beta-converting enzyme (proICE) was expressed in Spodoptera frugiperda (Sf9) insect cells using a baculovirus expression system. The 45-kDa recombinant protein was further processed to several smaller forms of 32, 24, 20, 13 and 10 kDa. Active recombinant ICE derived from the baculovirus expression system (bvICE) was found to be present in soluble lysates of insect cells as an associated heterodimer consisting of 10- and 20-kDa subunits. The activity of bvICE was determined by conversion of precursor interleukin-1 beta (preIL-1 beta) to the mature form (mIL-1 beta) and via site-specific cleavage of a decapeptide which spans the ICE cleavage site in preIL-1 beta. The bvICE system was inhibited by an ICE inhibitor to the same extent as native ICE from the monocytic cell line THP-1. Expression of an active-site mutant (Cys285 to Ser) of proICE in insect cells resulted in the accumulation of partially processed (32-kDa) ICE. The availability of a facile expression system will permit further characterization of the biochemical properties and processing pathway of this unique protease.

Two-phase partitioning bioreactors in fermentation technology.

The two-phase partitioning bioreactor concept appears to have a great potential in enhancing the productivity of many bioprocesses. The proper selection of an organic solvent is the key to successful application of this approach in industrial practice. The integration of fermentation and a primary product separation step has a positive impact on the productivity of many fermentation processes. The controlled substrate delivery from the organic to the aqueous phase opens a new area of application of this strategy to biodegradation of xenobiotics. In this review, the most recent advances in the application of two-liquid phase partitioning bioreactors for product or substrate partitioning are discussed. Modeling and performance optimization studies related to those bioreactor systems are also reviewed.

Effects of Plasmodium vinckei hemozoin on the production of oxygen radicals and nitrogen oxides in murine macrophages.

When murine peritoneal macrophages were loaded in vitro with Plasmodium vinckei hemozoin and stimulated with opsonized zymosan for 90 min or with lipopolysaccharide and/or murine interferon-gamma for 24 hr, significant decreases in the production of oxygen radicals and nitrogen oxides, respectively, could be detected by comparison with macrophages without hemozoin. Moreover, nonradioactive in situ hybridization and immunohistologic analysis in liver sections of P. vinckei-infected mice with more than 60% parasitemia showed that liver cells were still expressing considerable levels of inducible nitric oxide synthase in the late phase of murine malaria, but most of the liver macrophages presenting accumulation of malaria pigment were negative in this analysis. These results further indicate that malaria pigment accumulation may be responsible for toxicity and impairment of macrophage functions during murine malaria.

Rosiglitazone in the treatment of type 2 diabetes mellitus: a critical review.

OBJECTIVE: This article reviews the pharmacology, pharmacokinetics, clinical efficacy, adverse effects, drug interactions, and dosing of rosiglitazone, the second thiazolidinedione approved for the treatment of type 2 diabetes mellitus. METHODS: Background information for this article was obtained from searches of MEDLINE , Iowa Drug Information Service, and International Pharmaceutical Abstracts, as well as from data on file with the manufacturer of rosiglitazone. RESULTS: Rosiglitazone is indicated for use alone or in combination with metformin or sulfonylureas for the maintenance of glycemic control in patients with type 2 diabetes mellitus. Rather than stimulation of insulin secretion, rosiglitazone's primary mechanism of action is sensitization of tissues to insulin through activation of the peroxisome proliferator-activated receptor gamma and increasing expression of the glucose transporter-4 receptor. Rosiglitazone is administered orally, is absorbed almost completely, and is 99.8% bound to plasma proteins. The majority of a dose is metabolized by the cytochrome P-450 2C8 isozyme, with the inactive metabolites excreted primarily in the urine. Four to 8 mg/d of rosiglitazone given alone or in combination with metformin, sulfonylureas, or insulin has produced reductions in baseline fasting plasma glucose and glycosylated hemoglobin in studies of up to 1 year's duration. Common adverse effects (occurring in > or = 5.0% of patients) include upper respiratory tract infection, injury, and headache. Edema, weight gain, and increased low-density lipoprotein cholesterol concentrations have also been observed. It is recommended that rosiglitazone be avoided in patients with alanine aminotransferase levels >2.5 times normal. No clinically relevant drug interactions have been documented with rosiglitazone to date. The initial starting daily dose of rosiglitazone is 4 mg in single or divided doses, without regard to meals, to a maximum of 8 mg. CONCLUSIONS: No direct comparative trials of the efficacy and safety of rosiglitazone versus those of the other available thiazolidinedione, pioglitazone, have yet been performed. The role of rosiglitazone as a single agent and in combination with other antidiabetic agents remains to be clarified as additional comparative and long-term data become available.

Antithrombotic and hemostatic capacity of factor Xa versus thrombin inhibitors in models of venous and arteriovenous thrombosis.

Thrombin plays a central role in venous and arterial thrombosis. We utilized two different rabbit models of in vivo thrombosis to investigate the effect of inhibitors of thrombin generation and thrombin activity. The agents tested were specific inhibitors of factor Xa (fXa) [N2-[(phenylmethyl)sulfonyl]-D-arginyl-N-[(1S)-4-[(aminoiminomethyl++ +)a mino]-1-(2-thiazolylcarbonyl)butyl]-glycinamide (C921-78)] and thrombin [D-phenylalanyl-N-[4-[(aminoiminomethyl)amino]-1-(chloroacetyl)but yl]-L-prolinamide (PPACK)], as well as drugs that affect both thrombin and fXa, unfractionated and low molecular weight (enoxaparin) heparin. The agents administered as constant intravenous infusion were evaluated for antithrombotic efficacy in anesthetized rabbits. All four agents were capable of dose dependent inhibition of thrombosis in venous and arteriovenous thrombosis models. However, due to the more aggressive nature of thrombotic stimulation in the arteriovenous shunt model, complete cessation of thrombus growth was not achieved for any of the agents at the doses tested. Comparison between the agents focused on the differences in extension of coagulation parameters (activated partial thromboplastin time, prothrombin time, thrombin clotting time), changes in hematological parameters, and extension of rabbit cuticle bleeding time at doses required to produce maximum inhibition in the thrombosis models. In the venous thrombosis model at the maximally effective dose, C921-78 had minimal extension of ex vivo clotting parameters, while enoxaparin and unfractionated heparin demonstrated a two to sevenfold increase in activated partial thromboplastin times, and PPACK had a threefold extension of thrombin clotting times. In addition, unlike the other three agents, which exhibited no significant changes in hematological parameters, PPACK demonstrated dose dependent thrombocytopenia. A standardized cuticle bleeding time was used as a measure of perturbation of hemostasis. The agents were evaluated for significant increases in bleeding time at doses up to eight times that needed to completely inhibit venous thrombus formation. Unfractionated heparin displayed a significant bleeding time effect at the dose required to inhibit venous thrombosis (100 u/kg+2 u/kg/min). Enoxaparin and PPACK caused significant bleeding time extensions at four times the fully efficacious venous dose (800 u/kg+8 u/kg/min and 30 microg/kg/min). By contrast, C921-78 did not significantly increase bleeding time even at eight times the maximally effective dose (240 microg/kg+7.2 microg/kg/min). Our results demonstrate that specific inhibition of fXa can be utilized to derive potent antithrombotic activity without disrupting extravascular hemostasis.

Hemozoin differentially modulates the production of interleukin 6 and tumor necrosis factor in murine malaria.

When murine peritoneal macrophages were loaded in vitro with Plasmodium vinckei hemozoin and stimulated for 24 hours with interferon-gamma and/or Escherichia coli lipopolysaccharide, the production of interleukin 6 (IL-6) was drastically reduced, whereas the secretion of tumor necrosis factor (TNF) was increased. In addition, non-radioactive in situ hybridizations in spleen sections of P. vinckei infected mice showed more TNF than IL-6 gene expression in the red pulp around hemozoin accumulation. These results provide evidence that IL-6 and TNF are differentially modulated by hemozoin and that subsequently, the secretion of IL-6 seems to be independent of the TNF production during murine malaria.

Reactive extraction for downstream separation of 1,3-propanediol.

The downstream separation of 1,3-propanediol from dilute aqueous solution was studied. A process combining reversible reaction of 1, 3-propanediol with acetaldehyde to 2-methyl-1,3-dioxane and a simultaneous extraction of the product by organic solvent appears to be technically feasible and attractive. The dioxane yield was 91-92%, the overall conversion of 1,3-propanediol was ca. 98%, and recovery of dioxane into the organic extractant was 75%.

Effect of anticoagulants and antiplatelet agents on platelet-mediated thrombin generation.

The present study was designed to determine the mechanism by which and extent to which antagonists of glycoprotein IIbIIIa (GPIIbIIIa or alpha beta ) or activated factor X (FXa) activity block tissue factor-initiated thrombin generation by prothrombinase complexes assembled on the surface of activated platelets. In the presence of high concentrations of GPIIbIIIa antagonists, which eliminate platelet aggregation but not activation, there is still a substantial amount of thrombin produced. In contrast, specific antagonists of the coagulation cascade lead to abolition of both thrombin generation and platelet aggregation. In addition, inhibitors with similar inhibitory activity (Ki) against purified human FXa require a much broader range of concentrations (a variation of 10 000-fold or more) to reduce the amount of thrombin produced in a platelet-rich plasma assay. At the doses tested, inhibitors with greater potency in prevention of thrombin production in the platelet-rich plasma assay were effective in vivo antithrombotics in an animal model system, whereas a lower potency compound did not reduce thrombus mass. Therefore, inhibition of FXa within platelet bound prothrombinase rather than inhibition of purified FXa in solution may be a better predictor of antithrombotic efficacy. In addition, all the studied anticoagulants fared better than the antiplatelet agents in reducing thrombin generation.

Isolation and studies of the mutagenic activity in the Ames test of flavonoids naturally occurring in medical herbs.

Quercetin, rhamnetin, isohamnetin, apigenin and luteolin were isolated from medicinal herbs: Erigeron canadensis L., Anthyllis vulneraria L. and Pyrola chloranta L. The mutagenicity of these naturally occurring flavonoids was tested by the Ames method with S. typhimurium strains TA1535, TA1538, TA97, TA98, TA100 and TA102 in the presence and absence of metabolic activation. Of the above flavonoids only quercetin and rhamnetin revealed mutagenic activity in the Ames test. Quercetin induced point mutations in strains TA97, TA98, TA100 and TA102 of S. typhimurium. The presence of S9 rat liver microsome fraction markedly enhanced the mutagenic activity of quercetin in these strains. Rhamnetin appeared to be a much weaker mutagen in the Ames test. The compound induced mutations in strains TA97, TA98 and TA100 of S. typhimurium but only in the presence of metabolic activation. Comparison of the structure of the studied flavonoids with their mutagenic activity indicates that the mutagenicity of flavonoids is dependent on the presence of hydroxyl groups in the 3' and 4' positions of the B ring, and that the presence of a free hydroxy or methoxy group in the 7 position of the A ring also probably contributes to the appearance of mutagenic activity of flavonoids in the Ames test. It also appeared that the presence of methoxy groups, particularly in the B ring of the flavonoid molecule, markedly decreases the mutagenic activity of the compound.

Precise determination of uranium in pure uranium and uranium compounds by constant-current coulomeric titration.

A procedure is described for very precise determination of uranium in high-purity uranium and uranium compounds. Uranium(VI) is reduced in a concentrated hydrochloric acid solution by metallic aluminium in the presence of cadmium ions to uranium(III). This is oxidized to uranium(IV) by protons on addition of an excess of orthophosphoric acid, and then oxidized to uranium(VI) by adding a weighed quantity of potassium dichromate in small excess. The excess of potassium dichromate is determined by constant-current coulometry. The coefficient of variation does not exceed 0.003%.

Atorvastatin: a hydroxymethylglutaryl-coenzyme A reductase inhibitor.

The pharmacology, pharmacokinetics, clinical efficacy, adverse effects, interactions, and formulary considerations of atorvastatin relative to other hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) are discussed. Atorvastatin calcium, a synthetic stereoisomer of a pentasubstituted pyrrole, prevents the conversion of HMG-CoA by competitive and selective inhibition of HMG-CoA reductase. This limits cholesterol formation. Atorvastatin undergoes extensive first-pass metabolism; the first-pass effect is saturable at higher doses. Time to maximum plasma concentration ranges from one to four hours. The plasma elimination half-life is considerably longer than for other statins. Like other statins, atorvastatin reduces low-density-lipoprotein cholesterol (LDL-C) and total cholesterol in patients with hypercholesterolemia. However, the reductions achieved with atorvastatin exceed those for other statins. Atorvastatin recipients are more likely to achieve LDL-C goals and to do so more quickly. Atorvastatin also moderately reduces triglyceride levels in patients with hypertriglyceridemia and may play a role in the management of familial hypercholesterolemia. Adequate lipid control with atorvastatin monotherapy may preclude the need for combination drug therapy in some patients. The adverse effects of atorvastatin include mild gastrointestinal disturbances, increased liver enzyme levels, and myalgia. Drug interactions involving atorvastatin can be expected to parallel those of other statins metabolized via CYP3A4. Atorvastatin has become a popular addition to hospital formularies, even though formal pharmacoeconomic analyses are lacking. Atorvastatin effectively reduces blood lipids and may offer some advantages over other statins, but more studies are needed to clarify its optimal role in pharmacotherapy.

Raloxifene hydrochloride.

The pharmacology, pharmacokinetics, clinical efficacy, adverse effects, and therapeutic role of raloxifene hydrochloride are reviewed. Raloxifene is a selective estrogen-receptor modulator (SERM) that has been approved for use in the prevention and treatment of osteoporosis in postmenopausal women. A SERM interacts with estrogen receptors, functioning as an agonist in some tissues and an antagonist in other tissues. Because of their unique pharmacologic properties, these agents can achieve the desired effects of estrogen without the possible stimulatory effects on the breasts or uterus. Raloxifene is rapidly absorbed from the gastrointestinal tract and undergoes extensive first-pass glucuronidation. Approximately 60% of a dose is absorbed; however, absolute bioavailability is only 2%. The volume of distribution is 2348 L/kg for a single oral dose of 30-150 mg, and the elimination half-life averages 32.5 hours. In clinical trials in postmenopausal women, raloxifene had an estrogen-like effect on bone turnover and increased bone mineral density. It reduced the risk of fractures in women with osteoporosis. Raloxifene also seemed to reduce the risk of breast cancer and positively influenced blood lipid markers of cardiovascular disease. Raloxifene is generally well tolerated; the most common adverse effects are hot flashes and leg cramps. A serious adverse effect is venous thromboembolism. The recommended dosage is 60 mg/day orally without regard to meals. Ultimately, it will be information on cardiovascular or breast cancer benefits that will determine the future role of raloxifene. Raloxifene is an alternative to traditional hormone replacement therapy for the prevention and treatment of osteoporosis in selected postmenopausal women. More study is needed to verify possible benefits related to heart disease and breast cancer.

Mental rotation and real-world wayfinding.

Sex differences in mental rotation skills are a robust finding in small-scale laboratory-based studies of spatial cognition. There is almost no evidence in the literature, however, relating these skills to performance on spatial tasks in large-scale, real-world activities such as navigating in a new city or in the woods. This study investigates the connections between mental rotation skills as measured by the Vandenburg-Kuse Mental Rotations test and the performance of college students (n=211) navigating a 6-km orienteering course. The results indicate that mental rotation skills are significantly correlated with wayfinding performance on an orienteering task. The findings also replicate sex differences in spatial ability as found in laboratory-scale studies. However, the findings complicate the discussion of mental rotation skills and sex because women often performed as well as men despite having lower mean test scores. This suggests that mental rotation ability may not be as necessary for some women's wayfinding as it is for men's navigation.

[Aminopeptidases activity in the amniotic fluid in women in labor with EPH-gestosis]. / Aktywnosc aminopeptydazowa w plynie owodniowym rodzacych z EPH-gestoza.

The thesis of this work was to compare the aminopeptidases activity in the amniotic fluid obtained during the physiological labor and during the labor of pregnant with EPH-gestosis, in presence of the beta-naphtlamidic L-amino-acids (alanine, leucine, phenylalanine, tyrosine, histidine, cysteine) chromogenic substrates. It was assumed the 3-7 times increase in the aminopeptidases activity counted to the proteins from the labored with EPH-gestosis comparing to the labored in physiological labor. Among the used substrates the highest activity in both groups of labored women was measured in presence of substrates with exact amino-acids in this order: L-Ala > L-Leu > L-Phe > L-Tyr > L-His > L-Cys.

[Activity of aminopeptidase and inter-alpha-trypsin inhibitor in serum of women in physiological labor]. / Aktywnosc aminopeptydazowa i stezenie inter-alfa-inhibitora trypsyny w surowicy krwi kobiet w okresach porodu fizjologicznego.

Aminopeptidase activity was measured towards L-alanine and L-leucine naphtylamides in the blood serum obtained from the non-pregnant women and from women during the stages of the physiological delivery. In the same samples the concentration of inter-alpha-trypsin inhibitor (alpha ITI) was measured. The highest aminopeptidase activity was observed in the stage II, and it was statistically significant comparing to the stage I, whereas the statistically significant increase in the alpha ITI concentration was noticed in stage I in primiparae and in stage I in multiparae.