Effect of rapamycin on lysosomal accumulation in a CRISPR/Cas9-based cellular model of VPS13A deficiency.

VPS13A is a lipid transfer protein localized at different membrane contact sites between organelles, and mutations in the corresponding gene produce a rare neurodegenerative disease called chorea-acanthocytosis (ChAc). Previous studies showed that VPS13A depletion in HeLa cells results in an accumulation of endosomal and lysosomal markers, suggesting a defect in lysosomal degradation capacity leading to partial autophagic dysfunction. Our goal was to determine whether compounds that modulate the endo-lysosomal pathway could be beneficial in the treatment of ChAc. To test this hypothesis, we first generated a KO model using CRISPR/Cas9 to study the consequences of the absence of VPS13A in HeLa cells. We found that inactivation of VPS13A impairs cell growth, which precludes the use of isolated clones due to the undesirable selection of edited clones with residual protein expression. Therefore, we optimized the use of pool cells obtained shortly after transfection with CRISPR/Cas9 components. These cells are a mixture of wild-type and edited cells that allow a comparative analysis of phenotypes and avoids the selection of clones with residual level of VPS13A expression after long-term growth. Consistent with previous observations by siRNA inactivation, VPS13A inactivation by CRISPR/Cas9 resulted in accumulation of the endo-lysosomal markers RAB7A and LAMP1. Notably, we observed that rapamycin partially suppressed the difference in lysosome accumulation between VPS13A KO and WT cells, suggesting that modulation of the autophagic and lysosomal pathway could be a therapeutic target in the treatment of ChAc.

Short communication: Test for nonpregnancy in dairy cows based on plasma progesterone concentrations before and after timed artificial insemination.

Timed artificial insemination (AI) programs have increased reproductive efficiency in dairy herds. A low timed AI pregnancy per AI is partially explained by cows that fail to respond optimally to the series of treatments that are designed to synchronize ovulation for AI. We hypothesized that testing cows for plasma progesterone concentrations during a timed AI protocol could be used as an early diagnostic test for nonpregnancy. Lactating Holstein cows (n=160) in 2 confinement-style dairies were used. Cows were treated with Presynch Ovsynch 56 for timed AI. Concentrations of progesterone in plasma were measured at -3, 0, 7, and 25 d relative to timed AI. Progesterone data were analyzed and receiver operating characteristic curves were generated by using logistic regression. The area under the receiver operating curves for a progesterone test for nonpregnancy on d -3 (PGF2α), 0 (AI), 7, and 25 d relative to timed AI were 0.68, 0.52, 0.55, and 0.89, respectively. The cutpoints and sensitivity (respectively) for the progesterone test were 0.51ng/mL (lower=nonpregnant) and 28.2% for the day of PGF2α, 0.43ng/mL (greater=nonpregnant) and 17.9% for the day of AI, 1.82ng/mL (lower=nonpregnant) and 23.1% for 7 d after AI, and 2.67ng/mL (lower=nonpregnant) and 76.0% for 25 d after AI. The false positive rate was less than 5% for all tests. Analysis of a second data set from a published study gave approximately the same cutpoints and sensitivity. When both studies were combined, approximately 20% of nonpregnant cows could be identified with a single test that was done before or shortly after AI with a false positive rate of less than 5%. When 2 and 3 tests were applied sequentially, the sensitivity for identifying nonpregnant cows increased from 38.4 to 50.5%. The pregnancy per AI for those cows that met the established progesterone criteria was approximately 3 to 4 times greater than those that failed to meet the criteria. The conclusions were that cows destined to be nonpregnant after timed AI can be identified before or shortly after AI. Testing for nonpregnancy before or shortly after AI may have utility with respect to eliminating a nonproductive AI (cows identified before AI) or shortening the time to reinsemination (cows identified by 1 wk after AI).

Follicular populations and luteal function in dairy heifers treated with a controlled internal drug release insert for 14 days as a method to synchronize the estrous cycle before prostaglandin F2α treatment and artificial insemination.

Progesterone-containing controlled internal drug release (CIDR) inserts are used to synchronize the estrous cycle before PGF2α is administered for timed AI (14dCIDR-PGF2α program). The program, initially designed for beef cattle, was recently shown to be efficacious in dairy heifers. We hypothesized that the 14-d CIDR treatment would synchronize the estrous cycle in dairy heifers and result in a uniformly sized corpus luteum (CL) and largest follicle (LF) at the time of PGF2α treatment. Holstein (n=110) or Holstein × Guernsey (n=4) dairy heifers were assigned to 2 treatments: (1) 14dCIDR-PGF2α [CIDR in for 14 d, CIDR out for 16d, PGF2α and AI after observed estrus (n=57)] or (2) control [PGF2α and AI after observed estrus (n=57)]. Regardless of treatment, additional PGF2α injections were administered at 14-d intervals to heifers that were not seen in estrus. Ovarian ultrasonography and blood sampling were done on d 0 (CIDR administered), 14 (day CIDR removed), 19 (5d after CIDR removed), 30 (PGF2α administered), and 44 (second PGF2α dose administered to heifers that were not detected in estrus after the first PGF2α). Compared with control (untreated), more CIDR-treated heifers were categorized as having a small CL (≤ 9.9 mm) and large LF (15.0-19.9 mm) on d 14 (CIDR removal) and, as expected, a greater percentage of CIDR-treated heifers were in estrus during the 5d after the CIDR removal compared with control heifers (75.4 vs. 22.8%, respectively). On d 19, the CIDR-treated heifers had apparently ovulated based on disappearance of LF and appearance of small CL. On d 30 (PGF2α administration), 89% of 14dCIDR-PGF2α heifers had CL that were ≥ 20 mm in diameter compared with 55% for control. Presence of larger CL on d 30 was associated with greater concentrations of plasma progesterone in 14dCIDR-PGF2α compared with control (10.5 ± 0.5 vs. 5.0 ± 0.6 ng/mL, respectively). The percentages of heifers with LF in the smallest category (≤ 9.9 mm) tended to be less (5.3 vs. 16.6%) and the percentage of heifers with LF in the medium-size category (10.0 to 14.9 mm) tended to be greater (84.2 vs. 69.1%) for 14dCIDR-PGF2α versus control, respectively, on d 30. More heifers were detected in estrus within 5d after the first PGF2α (86.0 vs. 56.1%) and conception rate to AI using sexed semen tended to be greater (61.2% vs. 40.6%) for 14dCIDR-PGF2α compared with control (respectively). Treating dairy heifers with a CIDR for 14 d was an effective method to synchronize an estrous cycle before PGF2α was administered.

Short communication: Glucose infusion into early postpartum cows defines an upper physiological set point for blood glucose and causes rapid and reversible changes in blood hormones and metabolites.

Low blood glucose concentrations after calving are associated with infertility in postpartum dairy cows perhaps because glucose is a master regulator of hormones and metabolites that control reproductive processes. The hypothesis was that low blood glucose postpartum is caused by inadequate glucose entry rate relative to whole-body demand as opposed to the alternative possibility that postpartum cows have a lower regulatory set point for blood glucose. Eight early postpartum (10 to 25 d) dairy cows (5 Holstein and 3 Guernsey) were jugular catheterized. During the first 24 h, cows were infused with physiological saline at 83.3 mL/h. After 24 h, the infusion solution was switched to 50% dextrose that was infused at a rate of 41.7 mL/h (total daily glucose dose=500 g). On d 3 and d 4, the rate of glucose infusion was increased to 83.3 mL/h (daily dose=1,000 g) and 125 mL/h (daily dose=1,500 g), respectively. On d 5, physiological saline was infused at 83.3 mL/h. Blood was sampled hourly through a second jugular catheter (contralateral side) and analyzed for glucose, nonesterified fatty acids, ß-hydroxybutyrate, insulin-like growth factor 1, and insulin. Blood glucose concentrations on d 1 (saline infusion) averaged 53.4±1.7 mg/dL. Blood glucose concentrations increased on d 2 when cows were infused with 500 g/d and increased further on d 3 when cows were infused with 1,000g of glucose/d. Increasing the infusion rate to 1,500 g/d on d 4 did not cause a further increase in blood glucose concentrations. Based on a segmented regression analysis, the upper physiological set point for blood glucose was 72.1 mg/dL. Both insulin and insulin-like growth factor 1 concentrations increased in response to glucose infusion and decreased when cows were infused with saline on d 5. Serum nonesterified fatty acids and ß-hydroxybutyrate concentrations decreased in response to glucose infusion and rebounded upward on d 5 (saline infusion). In conclusion, early postpartum cows had circulating blood glucose concentrations that were well below the upper set point defined in this study (72.1 mg/dL). Infusing approximately 1,000 g of glucose daily increased blood glucose to the physiological set point and rapidly changed the hormonal and metabolic profile that typifies postpartum cows. The inability of the early postpartum cow to achieve an adequate entry rate for glucose relative to whole-body demand is a possible mechanism that links postpartum physiology and nutrition to reproduction in dairy cows.

Reproduction in grazing dairy cows treated with 14-day controlled internal drug release for presynchronization before timed artificial insemination compared with artificial insemination after observed estrus.

Progesterone-releasing (controlled internal drug release, CIDR) devices inserted for 14 d are used to presynchronize the estrous cycle for timed artificial insemination (TAI) in beef heifers (14-d CIDR-PGF(2α) program). The objective was to test a similar program in dairy cows by measuring first-service conception rates (FSCR), pregnancy rates after 2 AI, and time to pregnancy compared with a control (AI after observed estrus). Postpartum cows (Holstein, Jersey, or crossbred; n=1,363) from 4 grazing dairy farms were assigned to 1 of 2 programs: 14dCIDR_TAI [CIDR in for 14 d, CIDR out, PGF(2α) injection at 19 d after CIDR removal, GnRH injection 56 h later, and then TAI 16 h later; n=737] or control [AI after observed estrus; reproductive program with PGF(2α) (cycling cows) and CIDR (noncycling cows) to synchronize estrus with the start of the breeding season; n=626]. Body condition was scored (1 to 5; thin to fat) at the start of the trial. The interval from the start of the breeding period (final PGF(2α) injection of either program) to first AI was shorter for 14dCIDR_TAI compared with the control (3.0±0.2 vs. 5.3±0.2 d; mean ± SEM) but 14dCIDR_TAI cows had lesser FSCR than controls (48 vs. 61%). Farm affected FSCR (50, 51, 67, and 58% for farms 1 to 4). The BCS affected FSCR (50, 55, and 62% for BCS=2, 2.5, and 3, respectively). Cows that either calved the year before (carryover) or that calved early in the calving season had greater FSCR than cows that calved later in the calving season (55, 61, and 42%, respectively). The percentage of cows pregnant to AI (first and second inseminations within 31-d breeding season) was similar for 14dCIDR_TAI and control (64 vs. 70%) cows, but farm (64, 62, 80, and 69%) and time of calving (70, 76, and 56%: carryover, early, and late, respectively) affected the percentage. Survival analyses showed an initial advantage for 14dCIDR_TAI (more cows inseminated and more pregnancies achieved early in the breeding season) that was not maintained over time. Conclusions were that the 14dCIDR_TAI program achieved acceptable FSCR (48%) and overall AI pregnancy rates (64%), but did not surpass a control program that used AI after observed estrus (61 and 70%, respectively).

Short communication: presynchronization for timed artificial insemination in grazing dairy cows by using progesterone for 14 days with or without prostaglandin F2α at the time of progesterone withdrawal.

Progesterone-containing devices can be inserted intravaginally for 14 d to presynchronize the estrous cycle for timed artificial insemination (TAI) in beef heifers ("14-day CIDR-PG" or "Show-Me-Synch" program). The progesterone treatment is effective for presynchronization because cattle develop a persistent dominant follicle during treatment that ovulates within 3 d after progesterone removal. The subsequent estrous cycle can be effectively used for a TAI program. Some cattle will retain a functional corpus luteum (CL) for the entire 14-d treatment period and will not be synchronized effectively because the interval to ovulation depends on the lifespan of their existing CL. The objective was to test the effect of a luteolytic dose of PGF(2α) at progesterone removal for improving synchrony of estrus after treatment and increasing conception rate to a subsequent TAI in dairy cows. Postpartum cows (n = 1,021) from 2 grazing dairy herds were assigned to 1 of 2 presynchronization programs that used a controlled internal drug releasing (CIDR) device containing progesterone: 14dCIDR (CIDR in, 14 d, CIDR out; n = 523) or 14dCIDR+PGF(2α) (CIDR in, 14 d, CIDR out, and PGF(2α); n = 498). Cows were body condition scored (BCS; 1 to 5, thin to fat) and tail painted at CIDR removal. Paint score (PS) was recorded after CIDR removal [PS = 0 (all paint removed, indication of estrus), PS = 3 (paint partially removed), or PS = 5 (no paint removed; indication of no estrus)]. At 19 d after CIDR removal, all cows were treated with PGF(2α), 56 h later treated with GnRH, and then 16 h later were TAI. Treating cows with PGF(2α) at CIDR removal increased the percentage with PS = 0 within 5 d (58.1% vs. 68.9%; 14dCIDR vs. 14dCIDR+PGF(2α)). We found no effect of treatment, however, on conception rate at TAI (41.1% vs. 43.6%; respectively). The TAI conception rate increased with increasing BCS and was greater for cows that had PS = 0 within 5 d after CIDR removal. In summary, treating cows with PGF(2α) at CIDR removal increased the percentage of cows with all tail paint removed but did not increase percentage of pregnant cows after TAI.

Update of the PANCCO clinical practice guidelines for the treatment of ulcerative colitis in the adult population.

Ulcerative colitis (US) is a chronic disease of unknown etiology. It is incurable and its clinical course is intermittent, characterized by periods of remission and relapse. The prevalence and incidence of the disease has been increasing worldwide. The update presented herein includes the participation of healthcare professionals, decision-makers, and a representative of the patients, all of whom declared their conflicts of interest. Answerable clinical questions were formulated, and the outcomes were graded. The information search was conducted on the Medline/PubMed, Embase, Epistemonikos, and LILACS databases, and covered grey literature sources, as well. The search was updated on November 30, 2020, with no restrictions regarding date or language. The Grading of Recommendations Assessment, Development and Evaluation (GRADE) classification system was implemented to establish the strength of the recommendation and quality of evidence. A formal consensus was developed, based on the RAND/UCLA methodology and the document was peer reviewed. The short version of the Clinical Practice Guidelines for the Treatment of Ulcerative Colitis in the Adult Population is presented herein, together with the supporting evidence and respective recommendations. In mild-to-moderate UC, budesonide MMX is an option when treatment with 5-ASA fails, and before using systemic steroids. In moderate-to-severe UC, infliximab, adalimumab, vedolizumab, ustekinumab, and tofacitinib can be used as first-line therapy. If there is anti-TNF therapy failure, ustekinumab and tofacitinib provide the best results. In patients with antibiotic-refractory pouchitis, anti-TNFs are the treatment of choice.

International Liaison Committee on Resuscitation: COVID-19 consensus on science, treatment recommendations and task force insights.

Consensus on Science and Treatment recommendations aim to balance the benefits of early resuscitation with the potential for harm to care providers during the COVID-19 pandemic. Chest compressions and cardiopulmonary resuscitation have the potential to generate aerosols. During the current COVID-19 pandemic lay rescuers should consider compressions and public-access defibrillation. Lay rescuers who are willing, trained and able to do so, should consider providing rescue breaths to infants and children in addition to chest compressions. Healthcare professionals should use personal protective equipment for aerosol generating procedures during resuscitation and may consider defibrillation before donning personal protective equipment for aerosol generating procedures.

Role of age, bowel function and parity on anorectocele pathogenesis according to cinedefecography and anal manometry evaluation.

OBJECTIVE: The study aimed to verify the role of parity, age and bowel function in the pathogenesis of anorectocele. METHOD: A cross-sectional study was conducted regarding age, obstetrical history, Cleveland Clinic Constipation Score (CCCS), cinedefecography and anal manometry findings. Forty-five adult women complaining of obstructed defecation were evaluated; the median age was 46 years and median CCCS, 13. Fifteen patients were nulliparous and 23 multiparous (median parity 2). Eighteen had a history of episiotomy, fourteen delivered large babies and two had forceps-assisted delivery. Statistical analysis was performed using Spearman's correlation test and Fisher's exact test. RESULTS: Anal hypertonia was found in 14 (31.1%) patients, anal hypotonia in eight (17.8%), anismus in 13 (28.9%) and anorectoceles in 34 (75.6%) [median size 2.8 cm (0-6.4)]. There were no correlations between anorectocele and anal hypertonia (P = 0.7171), anismus (P = 0.4666), parity comparing nulliparous and multiparous patients (P = 1.000), episiotomy (P = 1.0000), forceps assistance (P = 1.0000), delivery of a large baby (P = 1.0000) anal resting pressure (P = 0.0883), anal voluntary pressure (P = 0.7327), parity (P = 0.4987) and age (P = 0.8603). There were correlations between anorectocele and the CCCS (P = 0.0082) and anal hypotonia (P = 0.0141). CONCLUSION: Anorectocele is not correlated with parity, age, episiotomy, delivery of a large baby and anismus. It was more frequent in patients with severe constipation and less common in patients with anal hypotonia.

"All citizens of the world can save a life" - The World Restart a Heart (WRAH) initiative starts in 2018.

"All citizens of the world can save a life". With these words, the International Liaison Committee on Resuscitation (ILCOR) is launching the first global initiative - World Restart a Heart (WRAH) - to increase public awareness and therefore the rates of bystander cardiopulmonary resuscitation (CPR) for victims of cardiac arrest. In most of the cases, it takes too long for the emergency services to arrive on scene after the victim's collapse. Thus, the most effective way to increase survival and favourable outcome in cardiac arrest by two- to fourfold is early CPR by lay bystanders and by "first responders". Lay bystander resuscitation rates, however, differ significantly across the world, ranging from 5 to 80%. If all countries could have high lay bystander resuscitation rates, this would help to save hundreds of thousands of lives every year. In order to achieve this goal, all seven ILCOR councils have agreed to participate in WRAH 2018. Besides schoolchildren education in CPR ("KIDS SAVE LIVES"), many other initiatives have already been developed in different parts of the world. ILCOR is keen for the WRAH initiative to be as inclusive as possible, and that it should happen every year on 16 October or as close to that day as possible. Besides recommending CPR training for children and adults, it is hoped that a unified global message will enable our policy makers to take action to address the inequalities in patient survival around the world.

Chemotaxis to cAMP and slug migration in Dictyostelium both depend on migA, a BTB protein.

Chemotaxis in natural aggregation territories and in a chamber with an imposed gradient of cyclic AMP (cAMP) was found to be defective in a mutant strain of Dictyostelium discoideum that forms slugs unable to migrate. This strain was selected from a population of cells mutagenized by random insertion of plasmids facilitated by introduction of restriction enzyme (a method termed restriction enzyme-mediated integration). We picked this strain because it formed small misshapen fruiting bodies. After isolation of portions of the gene as regions flanking the inserted plasmid, we were able to regenerate the original genetic defect in a fresh host and show that it is responsible for the developmental defects. Transformation of this recapitulated mutant strain with a construct carrying the full-length migA gene and its upstream regulatory region rescued the defects. The sequence of the full-length gene revealed that it encodes a novel protein with a BTB domain near the N terminus that may be involved in protein-protein interactions. The migA gene is expressed at low levels in all cells during aggregation and then appears to be restricted to prestalk cells as a consequence of rapid turnover in prespore cells. Although migA- cells have a dramatically reduced chemotactic index to cAMP and an abnormal pattern of aggregation in natural waves of cAMP, they are completely normal in size, shape, and ability to translocate in the absence of any chemotactic signal. They respond behaviorally to the rapid addition of high levels of cAMP in a manner indicative of intact circuitry connecting receptor occupancy to restructuring of the cytoskeleton. Actin polymerization in response to cAMP is also normal in the mutant cells. The defects at both the aggregation and slug stage are cell autonomous. The MigA protein therefore is necessary for efficiently assessing chemical gradients, and its absence results in defective chemotaxis and slug migration.

Trypanosoma cruzi infection and/or administration of the nonsteroidal anti-inflammatory nimesulide increase the number of colonic crypts overexpressing metallothioneins in rat colon carcinogenesis.

Trypanosoma cruzi infection and nonsteroidal anti-inflammatory drugs inhibit colorectal carcinogenesis by mechanisms not completely known and metallothionein proteins (MTs) may be involved in this process. Sixty-six male Wistar rats weighing 90 to 120 g were randomly divided into seven groups (GI to GVII). GI, GII and GIII animals were subcutaneously infected with 200,000 trypomastigote forms of the Y strain of T. cruzi. After 8 weeks, GI, GII, GIV, and GVI were injected with one weekly subcutaneous dose of 12 mg/kg dimethylhydrazine for 4 weeks. In sequence, GI, GIV and GV were treated with nimesulide (10 mg/kg per dose, five times per week for 8 weeks). Groups I, III, IV, and VI had 12 animals, and each of the other groups had 6 animals. All the animals were euthanized 8 weeks after the last dimethylhydrazine injection. The colons were fixed and processed for MT immunohistochemistry. The index of MT-overexpressing colonic crypts (MTEC) was estimated as the percentage of MT-stained crypts in relation to the total number of crypts scored. Five hundred crypts per animal were scored. Data were analyzed by the Kruskal-Wallis test followed by the Dunn test. There was an increase in MTEC index in the groups either infected with T. cruzi or treated with nimesulide or both infected and treated when compared to control (401, 809, and 1011%, respectively). We suggest that the increased formation of MTEC may be related to the protection against carcinogenesis provided both by T. cruzi infection and nimesulide.

Dictyostelium discoideum: a model system for differentiation and patterning.

In Dictyostelium, development begins with the aggregation of free living amoebae, which soon become organized into a relatively simple organism with a few different cell types. Coordinated cell type differentiation and morphogenesis lead to a final fruiting body that allows the dispersal of spores. The study of these processes is having increasing impact on our understanding of general developmental mechanisms. The availability of biochemical and molecular genetics techniques has allowed the discovery of complex signaling networks which are essential for Dictyostelium development and are also conserved in other organisms. The levels of cAMP (both intracellular and extracellular) play essential roles in every stage of Dictyostelium development, regulating many different signal transduction pathways. Two-component systems, involving histidine kinases and response regulators, have been found to regulate intracellular cAMP levels and PKA during terminal differentiation. The sequence of the Dictyostelium genome is expected to be completed in less than two years. Nevertheless, the available sequences that are already being released, together with the results of expressed sequence tags (ESTs), are providing invaluable tools to identify new and interesting genes for further functional analysis. Global expression studies, using DNA microarrays in synchronous development to study temporal changes in gene expression, are presently being developed. In the near future, the application of this type of technology to the complete set of Dictyostelium genes (approximately 10,000) will facilitate the discovery of the effects of mutation of components of the signaling networks that regulate Dictyostelium development on changes in gene expression.

The bond force constant and bulk modulus of small fullerenes using density functional theory and finite element analysis.

Dedicated bond force constant and bulk modulus of C n fullerenes (n = 20, 28, 36, 50, 60) are computed using density functional theory (DFT). DFT predicts bond force constants of 611, 648, 675, 686, and 691 N/m, for C20, C28, C36, C50, and C60, respectively, indicating that the bond force constant increases for larger fullerenes. The bulk modulus predicted by DFT increases with decreased fullerene diameter, from 0.874 TPa for C60 to 1.830 TPa for C20. The bond force constants predicted by DFT are then used as an input for finite element analysis (FEA) of the fullerenes, considered as spatial frames in structural models where the bond stiffness is represented by the DFT-computed bond force constant. In agreement with DFT, FEA predicts that smaller fullerenes are stiffer, and underestimates the bulk modulus with respect to DFT. The difference between the FEA and DFT predictions of the bulk modulus decreases as the size of the fullerene increases, from 20.9% difference for C20 to only 4% difference for C60. Thus, it is concluded that knowing the appropriate bond force constant, FEA can be used as a plausible approximation to model the elastic behavior of small fullerenes.

The effect of upright tilt on nifedipine-induced natriuresis.

Calcium channel blockers are antihypertensive agents with diuretic actions. Yet edema occurs in some patients receiving long-term treatment with these drugs. As with other vasodilators, stimulation for fluid retention could result from systemic vasodilation. We speculated that the upright posture could enhance sodium retention. To test this hypothesis, we studied the effect of upright tilt in 10 patients before and after the oral administration of 20 mg nifedipine. Before nifedipine upright tilt caused a 41% drop in the sodium excretion rate, from 0.27 +/- 0.04 to 0.16 +/- 0.03 meq/min (p less than 0.05). Fractional sodium excretion decreased by 46%, from 2.4 +/- 0.5 to 1.3 +/- 0.3% (p less than 0.01). Urinary volume and renal plasma flow also decreased (p less than 0.05). Plasma renin activity (PRA) rose by 46% (p less than 0.005). With the patients in the supine posture nifedipine increased the sodium excretion rate to 0.49 +/- 0.09 meq/min (p less than 0.05). Fractional sodium excretion was 3.1 +/- 0.6 meq/min (p = 0.2). The natriuresis took place despite a fall in mean blood pressure and a significant rise in PRA (up 115% from prenifedipine supine values, p less than 0.005). Renal plasma flow also increased (p less than 0.01). The upright tilt caused a reversal of the nifedipine-induced natriuresis. The sodium excretion rate dropped to 0.23 +/- 0.05 meq/min and fractional sodium excretion to 1.3 +/- 0.2% (both not different from control). This drop in natriuresis occurred while mean blood pressure was at its lowest and PRA was 254% above the initial levels (p less than 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of an Artemia franciscana retropseudogene containing part of the last exons of the sarco/endoplasmic reticulum Ca-ATPase-encoding SERCA gene.

A genomic clone has been isolated which contains sequences highly homologous to part of exon 14 and exons 15, 16 and 17 of the Artemia franciscana sarco-endoplasmic reticulum Ca-ATPase(SERCA)-encoding gene, but none of the introns. The homologous region extends to the 3' end of the mRNA, although the poly(A) tail is not present. The structure of this clone suggests that it represents a 5'-end-truncated retropseudogene (r psi).

Transferrin gene expression in the mammary gland of the rat. The enhancing effect of 17 beta-oestradiol on the level of RNA is tissue-specific.

We have investigated the physiological factors which regulate transferrin gene expression in the mammary gland of the rat. Our studies by dot blot analysis have demonstrated that multiple doses of 17 beta-oestradiol (OE2; 0.5 mg/kg per day for 3 days) elicit a specific 3.5-fold increase in the transferrin mRNA levels in the mammary glands of virgin rats. The hormonal action of OE2 in mammary tissue was specific for the transferrin gene, as judged by hybridization with beta-actin cDNA. The accumulation of transferrin mRNA induced by OE2 treatment was similar to the developmentally regulated expression of the gene observed during the reproductive cycle. The steady-state level of mammary transferrin mRNA increased by up to 4.5-fold at day 21 of lactation, when compared with virgin and pregnant rats. Our results show that the pattern of transferrin gene expression is different in mouse and rat mammary glands. The specific response of the transferrin gene to OE2 was not found in the liver or in the uterus. In the uterus alone, OE2 produced a significant increase in the content of nucleic acids and also induced the accumulation of transferrin and beta-actin mRNAs. We have detected for the first time an induction of transferrin gene expression in the mammary gland in response to OE2, and these results support the view that the pattern of transferrin gene multi-modulated expression is tissue- and species-specific.

Tissue-specific expression of two Artemia franciscana sarco/endoplasmic reticulum Ca-ATPase isoforms.

The sarco/endoplasmic reticulum Ca-ATPase (SERCA) gene from Artemia franciscana is transcribed into two mRNAs that code for two different enzyme isoforms. We investigated the tissue-specific expression of each mRNA by in situ hybridization of larval tissue sections. One of the isoforms is expressed in the muscle fibers of the appendages. The other isoform is generally expressed throughout all tissues of the larvae. The tissue distribution of these two isoforms is very similar to the one described for the two homologous isoforms generated from the vertebrate SERCA 2 gene, and shows the evolutionarily conserved nature of their tissue-specific expression.

In situ hybridization analyses of Na, K-ATPase alpha-subunit expression during early larval development of Artemia franciscana.

The spatial pattern of expression of the mRNA encoded by the Na,K-ATPase alpha-subunit cDNA clone pArATNa136 was determined by in situ hybridization of first, second, and third instar Artemia franciscana larvae. This mRNA was expressed at high levels in the salt gland, the antennal gland, and the end of the midgut, which are the three main osmoregulatory organs in Artemia at these stages of development. The pattern of expression was similar at the three stages of development analyzed, although the level of expression increased during development, especially in the salt and antennal glands. The expression of the mRNA coding for another Na, K-ATPase alpha-subunit isoform, the proposed alpha 2-isoform, was also determined and was shown to be limited to the salt gland. These results suggest that the clone pArATNa136 codes for the biochemically defined alpha 1-isoform of the Na,K-ATPase alpha-subunit and reinforce the importance of this isoform in osmoregulation at the three larval stages studied. The alpha 2-isoform may also be involved in osmoregulation during the first stages of larval development.

Effects of the selective inhibition of platelet thromboxane synthesis on the platelet-subendothelium interaction.

1. Drugs that inhibit TxA(2) synthesis are used to reduce platelet aggregation. The aim of this study was to compare the effects of a cyclo-oxygenase (COX) inhibitor (acetylsalicylic acid, ASA), a thromboxane synthetase (TxS) inhibitor (dazoxiben) and a dual TxS inhibitor and TxA(2) receptor blocker (DT-TX 30) on platelet aggregation and the platelet-subendothelium interaction in flow conditions. 2. The techniques used in this in vitro study were platelet aggregometry in whole blood, and measurement of platelet thromboxane B(2) and prostaglandin E(2) production and leucocyte production of 6-keto-PGF(1alpha). The platelet-subendothelium interaction was evaluated in rabbit aorta subendothelium preparations exposed to flowing blood at a shear stress of 800 s(-1). Morphometric methods were used to calculate the percentage of subendothelium occupied by platelets. 3. The 50% inhibitory concentration (IC(50)) of DT-TX 30 in whole blood was in the range of 10(-7) micro M (induced with collagen or arachidonic acid) to 10(-5) micro M (induced with thrombin) or 10(-4) (induced with ADP). IC(50) values under all experimental conditions were lower with DT-TX 30 than with ASA. For thromboxane B(2) the IC(50) were: ASA 0.84+/-0.05 micro M, dazoxiben 765+/-54 micro M, DT-TX 30 8.54+/-0.60 micro M. Prostaglandin E(2) was inhibited only by ASA (IC(50) 1.21+/-0.08 micro M). Leucocyte 6-keto-PGF(1alpha) was inhibited by ASA (IC(50) 6.58+/-0.76 micro M) and increased by dazoxiben and DT-TX 30. The greatest reduction in percentage subendothelial surface occupied by platelets after blood perfusion was seen after treatment with DT-TX 30 in the range of concentrations that inhibited collagen-induced platelet aggregation (control group: 31.20+/-3.8%, DT-TX 30 at 0.1 micro M: 10.71+/-0.55%, at 1.0 micro M: 6.53+/-0.44%, at 5.0 micro M; 1.48+/-0.07%). All three drugs reduced thrombus formation, although ASA (unlike dazoxiben or DT-TX 30) increased the percentage surface occupied by adhesions. 4. In conclusion, the effect of specific blockage of TxS together with blockage of membrane receptors for TxA(2) can surpass the effect of ASA in inhibiting the platelet-subendothelium interaction in flow conditions.