Pregnancy and fetal characteristics after transfer of vitrified in vivo and cloned bovine embryos

This study was conducted to examine pregnancy progression and fetal characteristics following transfer of vitrified bovine nuclear transfer versus in vivo-derived embryos. Nuclear transfer (NT) was conducted using cumulus cells collected from an elite Holstein-Friesian dairy cow. Expanding and hatching blastocysts on Day 7 were vitrified using liquid nitrogen surface vitrification. Day 7 in vivo embryos, produced using standard superovulation procedures applied to Holstein-Friesian heifers (n=6), were vitrified in the same way. Following warming, embryos were transferred to synchronized recipients (NT: n=65 recipients; Vivo: n=20 recipients). Pregnancies were monitored by ultrasound scanning on Days 25, 45 and 75 and a sample of animals were slaughtered at each time point to recover the fetus/placenta for further analyses. Significantly more animals remained pregnant after transfer of in vivo-derived embryos than NT embryos at all time points: Day 25 (95.0 versus 67.7%, P<0.05), Day 45 (92.8 versus 49.1%, P<0.01) and Day 75 (70.0 versus 20.8%, P<0.0). There was no significant difference (P=0.10) in the weight of the conceptus on Day 25 from NT transfers (1.14+/-0.23 g, n=8) versus in vivo transfers (0.75+/-0.19 g, n=8). On Day 45, there was no significant difference in the weight of either fetus (P=0.393) or membranes (P=0.167) between NT embryos (fetus: 2.76+/-0.40, n=12; membranes: 59.0+/-10.0, n=11) or in vivo-derived embryos (fetus: 2.60+/-0.15, n=6; membranes: 41.8+/-5.2, n=4). However, on Day 75 the weight of the fetus and several of the major organs were heavier from NT embryos. These data suggest that morphological abnormalities involving the fetus and the placenta of cloned pregnancies are manifested after Day 45.     

Anandamide levels in human female reproductive tissues: Solid-phase extraction and measurement by ultraperformance liquid chromatography tandem mass spectrometry

Anandamide (N-arachidonoylethanolamide), a bioactive lipid, is reported to play a role in pregnancy maintenance and parturition. Our aims were to (1) evaluate AEA levels at the human maternal:fetal interface and (2) validate the use of solid-phase extraction of AEA from tissues. AEA was analyzed in cord and maternal blood, amniotic fluid, placenta, and fetal membranes collected during Caesarean section (n = 14). Extraction efficiencies were 42 and 36% for the placenta and the fetal membranes, respectively. Tissue AEA was quantified using an isotope-dilution method and UPLC-ESI-MS/MS giving intra- and inter-day variability for tissues spiked with 0.2, 1, and 5 pmol/g AEA of less than 12%. Accuracy for these spiked samples was between 95% and 103% for fetal membranes and between 99% and 114% for placenta. Mean AEA concentrations were 2.72 ± 1.04 pmol/g for placenta and 1.19 ± 0.68 pmol/g for fetal membranes, and 0.93 ± 0.28, 0.88 ± 0.33, 0.77 ± 0.30, and 0.06 ± 0.04 nM for maternal, umbilical vein, and umbilical artery plasma and amniotic fluid. Higher AEA concentrations were found in placenta compared to fetal membranes (P < 0.0001), in umbilical vein compared with umbilical artery (P = 0.0015), and in plasma from maternal circulation compared with umbilical artery (P = 0.0152). The relevance of these changes in AEA concentrations at the maternal:fetal interface requires further investigation.     

Occurrence of fumonisins (B<Subscript>1</Subscript>, B<Subscript>2</Subscript>, B<Subscript>3</Subscript>) in maize-based food and feed samples from Indonesia

A survey to evaluate the contamination level of total fumonisins in maize-based foodstuffs, maize and feed from Indonesia is described. The analyses were carried out by enzyme-linked immunosorbent assay (ELISA). Samples were collected from local retail stores around Yogyakarta, Indonesia between February and May 2001. The 101 samples were classified into six categories, i.e. industrially-produced food (n=24), products of small food manufacturers (n=17), maize flour (n=4), maize for food (n=9), maize for feed (n17), and formulated feed (n30). Control of the method showed that the detection limit was 8.7 μg/kg and repeatability is shown by relative standard deviation (RSD) of analyses of contaminated maize (n=5) of 10 %. Results of analyses indicate that 80 samples analysed were contaminated over a large range from 10.0-3307 pg/kg, and the concentration of fumonisins depended on the type of sample. Of four samples of maize flour, none were contaminated (below detection limit). Of 24 samples of industrially produced food, 14 were contaminated in the range 22.8 - 105 μg/kg and 18 of 19 food samples from small manufacturers were contaminated ranging from 12.9 to 234 μg/kg. The highest contamination was observed in maize samples: six of ten samples of maize for food were contaminated between 68.0 - 2471 μg/kg and 16 of 17 samples for feed contained fumonisins over a large range from 17.6 to 3306 μg/kg.     

Selenium levels in related biological samples: human placenta, maternal and umbilical cord blood, hair and nails.

A study on selenium levels has been carried out in human placenta, maternal and umbilical cord blood, hair and nails of a group of 50 mothers and in the hair of the newborns. The determinations were perfomed by electrothermal atomic absorption spectrometry. The selenium concentration obtained for each sample type was as follows: For the human placenta the values obtained were between 0.56 and 1.06 microg/g (mean +/- standard deviation: 0.81 +/- 0.02 microg/g). The levels for the umbilical cord blood were 51.1-104.2 microg/l (76.3 +/- 6.5 microg/l). For the maternal blood the values measured were between 57.3 and 117.9 microg/l (90.0 +/- 15.2 microg/l), and for hair and nails were 0.22-1.5 microg/g (0.60 +/- 0.37 microg/g) and 0.46-1.57 microg/g (0.90 +/- 0.27 microg/g), respectively. For the hair of the newborns the values obtained were between 0.40 and 2.53 microg/g (1.04 +/- 0.48 microg/g). The effect of different variables as age, habitat, nutritional index or gestation age of the mothers on the selenium concentration in the samples was studied. The influence of the habitat is significant with a confidence level of 95% for the selenium concentration in maternal blood and umbilical cord blood samples. The influence of the mothers' age is significant with a confidence level of 95% for the selenium concentration in the umbilical cord blood samples. For the placenta samples, the effect of the nutritional index is significant with a confidence level of 95%. There is a positive correlation between samples of umbilical cord blood and the newborns' hair, between placenta and umbilical cord, and between cord blood and maternal blood.     

Development of a validated capillary electrophoresis method for enantiomeric purity control and quality control of levocetirizine in a pharmaceutical formulation

A chiral capillary electrophoresis method has been developed for the quantification of 0.1% of the enantiomeric impurity (dextrocetirizine) in levocetirizine and determination of both in pharmaceuticals using sulfated-β-cyclodextrins (CDs) as chiral selector. Several parameters affecting the separation were studied such as the type and concentration of chiral selectors, buffer composition and pH, organic modifier, mixtures of two CDs in a dual system, voltage, and temperature. The optimal separation conditions were obtained using a 50 mM tetraborate buffer (pH 8.2) containing 1% (w/v) sulfated-β-CDs on a fused-silica capillary. Under these conditions, the resolution of two enantiomers was higher than 3. To validate the method, the stability of the solutions, robustness (two level half fraction factorial design for 5 factors using 19 experiments [2(n-1)+3]), precision, linearity (dextrocetirizine 0.25-2.5 μg/ml, R(2) = 0.9994, y = 0.0375x + 0.0008; levocetirizine 15-100 μg/ml, R(2) = 0.9996, y = 0.0213x + 0.0339), limit of detection (0.075 μg/ml, 0.03% m/m), limit of quantification (0.25 μg/ml, 0.1% m/m), accuracy (dextrocetirizine 84-109%, levocetirizine 97.3-103.1%), filter effect, and different CD batches were examined. The validated method was further applied to bulk drug and tablets of levocetirizine.     

Determination of Selenium in Serum Samples of Preterm Newborn Infants with Bronchopulmonary Dysplasia Using a Validated Hydride Generation System

Bronchopulmonary dysplasia (BPD), also known as chronic lung disease, is one of the most challenging complications in premature newborn infants. Selenium plays a role in antioxidant system by protecting cell membranes and neutralizing the deleterious effects of free radicals. The aim of this study was to determine the relationship between selenium concentration and incidence of bronchopulmonary dysplasia using a validated analytical method. Umbilical cord blood and blood samples 30 days after the birth were collected from 38 preterm newborn infants with gestation age of 32 weeks or less, and the separated serums were kept at -70°C until analysis time. Selenium concentration of serum was determined using an atomic absorption spectrophotometer. The method was validated on the basis of standard validation techniques. The analytical method was linear in the range of 1 to 500 μg/L with the limit of detection of 0.4 μg/L. Samples were collected from 38 infants whose gestation age was 32 weeks or less. The blood samples were collected from the umbilical cord blood at birth in 19 cases. In 25 cases, blood samples were collected 1 month after birth. Of the 15 patients diagnosed with BPD, 10 were boys (p = 0.02). The mean serum selenium concentration was not different at birth between patients with and without BPD, but it was significantly lower at 30 days after birth in patients with BPD (38.5 ± 14.1vs. 45.4 ± 18.7 μg/L, p = 0.02). Preterm newborn infants with BPD had lower serum selenium concentrations 1 month after birth.     

Ultrasonographic and histological characterization of the placenta of somatic nuclear transfer-derived pregnancies in dairy cattle

The high incidence of pregnancy loss and prenatal morbidity and mortality in cloned animals may be due to placental insufficiency, thereby compromising fetal survival. Our objective was to characterize morphological changes in fetal membranes of cloned bovine pregnancies. Two groups of cows with cloned fetuses, produced by two cloning techniques, a commercial group (n=16) and a hand-made group (n=4), and control fetuses derived from traditional embryo transfer (n=6) or AI (n=6), were compared at various stages of gestation (Days 80, 120, 150, 180, 210, and 240; Day 0=estrus). Thickness and shape of the amniotic membrane, placentome shape and length, umbilical cord shape and diameter, and fetal fluid echodensities were assessed by ultrasonography, and the placenta was evaluated histologically. Only eight (40%) of cloned pregnancies reached term and seven calves (35%) were alive at birth. Both placentome length and umbilical cord diameter were larger (P<0.05) in clones than in normal fetuses at all stages of gestation. Amniotic membrane abnormalities (Day 120) including focal edema and the presence of a series of nodules were detected in 38% of the clones and were always accompanied by hyper-echodense spikes or irregularities (detected ultrasonographically) around the umbilical cord. Histopathology revealed degenerate inflammatory cells, edematous chorioallantoic membranes, and decreased epithelial thickness. We inferred that these morphological anomalies of placentomes compromised fetal development, and we concluded that ultrasonographic monitoring of pregnancies enabled characterization of changes in the placentae and may be useful to assess fetal well-being.     

Distribution of CA 125 in placental tissues

The presence of the tumor marker CA 125 was studied in different compartments of the human placenta. Levels of CA 125 in the cytosol of chorionic villi ranged from 27-17100 U/g (median 560 U/g). In the placental amnion and chorion concentrations ranged from 175-29000 U/g, median 1060 U/g and were not statistically different. In the umbilical cord values were significantly lower (range 44-7600 U/g; median 180 U/g). Maternal serum probes were above the upper limit of normal in all cases (range 48-500 U/ml; median 131 U/ml). Immunohistochemistry detected CA 125 exclusively within the amniotic cells of the placenta and the umbilical cord. This might be because CA 125 fixes more to insoluble structures in the amnion or because of contamination of chorionic villi with the underlying decidua.     

The role of endogenous estrogens in the maturation process of the bovine placenta

The plasma estrogen and progesterone concentrations of 19 pregnant cows (average duration of pregnancy 266.0 +/- 2.3 d at the start of the study) were determined daily from Day 6 pre partum to Day 1 post partum. Parturition was induced in all cows by administration of 10 mg i.m. flumethasone. Values were centered around the delivery date (Day 0) following either induced normal calving (n = 3) or surgical delivery (n = 16). In animals showing spontaneous expulsion of the fetal membranes (Group 1, n = 6) the average total estrogen concentration increased significantly from Day 6 until Day 1 before parturition (1329.2 +/- 317.9 to 3719.8 +/- 951.2 pg/ml in total estrogens). A marked decrease was observed on Day 1 post partum (459.4 +/- 344.2 pg/ml). In comparison with Group 1, animals showing either a delayed or partial expulsion of the fetal membranes, or in which the placenta could be withdrawn 16 h after calving (Group 2, n = 5), had consistently lower total estrogen concentrations between Day 6 (595.4 +/- 174.8 pg/ml) and Day 1 (1884.3 +/- 565.1 pg/ml) before parturition. The estrogen values of the cows with retained placenta (Group 3, n = 8) from Days 6 to 0 pre partum were significantly lower than those of Group 2. Total estrogen concentrations of the three groups 1 d post partum did not differ significantly. It is generally recognized that estrogens play an important role in the maturation process of the placentomes. Our investigation demonstrates that not only is the magnitude of the prepartum rise in estrogens of great influence of the maturation process but the duration of this rise is likewise important. These two factors are vital for a normal expulsion of the fetal membranes.     

Transplacental transfer of medetomidine and ketamine in pregnant ewes

The extent of placental transfer of medetomidine and ketamine is unknown in pregnant ewes. Date-mated singleton (n = 8) and twin (n = 8) pregnant merino cross ewes were anaesthetized for Caesarean delivery of preterm lamb fetuses. A combination of medetomidine (20 μg/kg) and ketamine (10 mg/kg) was administered by intravenous injection and surgery performed immediately thereafter. Blood samples were collected from the ewe at one, five and 10 min after intravenous injection and from the umbilical vein of the fetus at delivery. Non-pregnant ewes were also anaesthetized (n = 8). There was no difference in the plasma concentration of medetomidine or ketamine when comparing singleton and twin ewes or pregnant and non-pregnant ewes for the short duration of the study. Fetal plasma concentrations of each drug were comparable to the maternal concentrations at the same time. We conclude that both drugs cross the placenta readily and provide anaesthesia and analgesia for the fetus when it is delivered.     

The measurement of the placenta, umbilical cord and components of amniotic fluid in beagle dogs]

The body weight of newborns, the weight of placenta, the length of umbilical cord and the content of urobilinogen, gulcose, protein, pH, keton body in amniotic fluid were investigated with samples from 16 litters of beagles. The mean body weight of newborn, weight of placenta and length of umbilical cord were 258.0 +/- 44.6 g (85 samples), 48.5 +/- 8.5 g (85 samples) and 11.0 +/- 2.7 cm (52 samples), respectively without any sex difference. The significant correlation were recognized between the weight of placenta and the body weight of newborn, between the length of umbilical cord and the body weight of newborn and between the weight of placenta and the length of umbical cord (P less than 0.01).     

血清瘦素和胰岛素与犊牛初生重相关性

本实验旨在研究分娩奶牛(Bos taurus)、犊牛和脐静脉血瘦素、胰岛素之间的相关性及与犊牛初生重的相关性,为探究瘦素、胰岛素对犊牛初生重的影响机理提供理论数据。实验选取规模化养殖场正常分娩奶牛54头,按犊牛初生重划分为A组(≤40 kg)9头、B组(40~45 kg)25头、C组(≥45 kg)20头共3组,分别采集分娩奶牛、犊牛和脐静脉血,ELISA法检测血清瘦素、胰岛素含量。多组间采用单因素方差分析和双变量Pearson分析瘦素和胰岛素在各部位静脉血中表达的相关性及与犊牛初生重的相关性。结果表明:(1)奶牛静脉血瘦素、胰岛素含量极显著高于犊牛静脉血和脐静脉血(P0.01),奶牛静脉血瘦素、胰岛素含量与犊牛静脉血和脐静脉血瘦素、胰岛素相关性均不显著(P0.05);(2)瘦素、胰岛素含量在犊牛静脉血与脐静脉血之间均差异不显著(P0.05),犊牛静脉血瘦素、胰岛素含量与脐静脉血瘦素、胰岛素均分别呈极显著正相关(P0.01);(3)奶牛静脉血和脐静脉血中瘦素与胰岛素含量均呈显著正相关(P0.05),而犊牛静脉血中瘦素与胰岛素相关性不显著(P0.05);(4)犊牛初生重与奶牛静脉血瘦素、胰岛素含量均不相关(P0.05),但与犊牛静脉血和脐静脉血瘦素含量显著正相关(P0.05),与犊牛静脉血和脐静脉血胰岛素含量极显著正相关(P0.01);(5)奶牛静脉血、犊牛静脉血和脐静脉血瘦素和胰岛素含量在公犊牛与母犊牛间均无显著差异(P0.05)。可见,奶牛静脉血、犊牛静脉血和脐静脉血均有瘦素、胰岛素表达,且其含量奶牛静脉血显著高于犊牛静脉血和脐静脉血。奶牛静脉血瘦素、胰岛素含量与犊牛初生重相关性不显著,而犊牛静脉血和脐静脉血瘦素、胰岛素含量与犊牛初生重显著正相关性。     

Direct determination by high-performance liquid chromatography of sn-2 monopalmitin after enzymatic lipase hydrolysis

An alternative method to determine the sn-2 monopalmitin in infant formulas was developed and validated. This method offers many advantages over the traditional methods. It follows the official method in the first steps, purification of the fat or oil through an alumina column, and subsequently the triglycerides are incubated with pancreatic lipase in order to obtain the sn-2 monoglycerides. In traditional methods the sn-2 monoglycerides are separated by preparative thin-layer chromatography and then, the 2-monoglycerides are converted into the corresponding fatty acid methyl esters and analysed by gas chromatography. In our method, separation, quantification and identification of the sn-2 monoglycerides were achieved by high-performance liquid chromatography with evaporative light-scattering detection. The detection limit (0.19 μg), quantification limit (0.38 μg), linearity range (r=0.999, range 1–200 μg) and precision (SD=1.10) show the suitability of the proposed method. This method is faster, cheaper and simple and does not consume large quantities of reagents and materials.     

Prostaglandin E(2) 9-keto reductase activity in bovine retained and not retained placenta

Prostaglandin E(2) 9-keto reductase (9-KPR) activity shifts reversibly PGE(2) into PGF(2 alpha) and may be responsible for the control of prostaglandins (PGs) levels in, among others, placental tissues. The retention of fetal membranes in cows is the postpartum disorder where the disturbances in PGs metabolism have been reported. It has been argued whether these disturbances are due to alterations in 9-KPR activity. In this study, the activity of the enzyme was determined in maternal and fetal bovine placental tissues which were divided into 6 groups as follows: (A) caesarian section before term without retained fetal membranes (n=10), (B) caesarian section before term with retained fetal membranes (n=10), (C) caesarian section at term without retained fetal membranes (n=12), (D) caesarian section at term with retained fetal membranes (n=12), (E) spontaneous delivery at term without retained fetal membranes (n=12), (F) spontaneous delivery at term with retained fetal membranes (n=12). The enzyme activity was measured spectrophotometrically and expressed in nanokatals (nkat) per protein content. The activity increased towards parturition and was significantly higher in maternal than in fetal part of placenta in all groups examined. The significantly higher values in retained than in not retained placental tissues were observed in the samples examined. The present results indicate that the disturbances in 9-KPR activity in bovine retained placenta exist but their reasons still require further experiments.     

Rate of disappearance of glycerol from plasma of fetal and newborn sheep

The disappearance of glycerol from plasma was studied after a single intravenous injection to estimate its volume of distribution (Vdist), plasma clearance rate, and rate constant for irreversible loss (kd). Studies were repeated before and after birth of the lamb to test whether loss of the placenta could account for rapidly increasing plasma concentrations in the newborn. The disappearance of glycerol was closely described by a double-exponential model in each instance. In fetal sheep Vdist averaged 0.41 +/- 0.15 (SD) 1/kg fetal wt (n = 15). This volume decreased to 0.33 +/- 0.11 l/kg (n = 8) soon after functionally removing the placenta (by snaring the umbilical cord and maintaining the fetus with intrauterine ventilation), but the change was not significant. In newborn lambs 1-3 days of age, Vdist averaged 0.45 +/- 0.11 l/kg (n = 5, NS). Plasma clearance rate also did not change significantly, averaging 7.9 +/- 2.9, 7.9 +/- 3.8, and 9.0 +/- 5.9 ml.min-1.kg-1 in the fetus, after simulated birth, and in the newborn lamb, respectively, kd also was not altered measurably and averaged 0.020 +/- 0.006, 0.024 +/- 0.007, and 0.019 +/- 0.007 min-1 during the same time periods. Similar results were obtained by using three widely different amounts of infused glycerol. The results indicate that removal of glycerol does not depend on placental function to an appreciable extent. It is concluded that plasma glycerol concentration reflects principally glycerol turnover and, hence, lipolysis before and after birth.     

Detection of endothelin-like immunoreactivity in epithelium and fibroblasts of the human umbilical cord

We studied tissue sections of freshly obtained full-term and premature human umbilical cords using polyclonal antibody to endothelin and immunocytochemistry. Endothelin immunoreactivity was detected in the cytoplasm of epithelial cells and primitive fibroblasts, but not in the endothelial cells of both full-term and premature umbilical cords. Immunoelectron microscopy using indirect immunogold staining technique localized endothelin immunoreactivity to the cytoplasm of the epithelial cells and fibroblasts but not confined to any particular structures. No endothelin immunoreactivity was detected in the nucleus or on the cell membrane. Pre-absorption tests with synthetic endothelin-1, -2, and -3 independently established that the immunoreactivity represented endothelin-1 and -2, but not -3. The presence of endothelin-1 and -2-like immunoreactive materials in epithelial cells and fibroblasts of human umbilical cord suggests a role of endothelin in parturition.     

Extraction and determination of opium alkaloids in urine samples using dispersive liquid-liquid microextraction followed by high-performance liquid chromatography

A simple, rapid and sensitive method based on dispersive liquid-liquid microextraction (DLLME) combined with high-performance liquid chromatography-ultraviolet detection (HPLC-UV) was used to determine opium alkaloids in urine samples. Some effective parameters on extraction were studied and optimized. Under the optimum conditions, enrichment factors and recoveries for different opiates are in the range of 63.0-104.5 and 31.5-52.2%, respectively. The calibration graphs are linear in the range of 0.50-500 μg L(-1) and limit of detections (LODs) are in the range of 0.2-10 μg L(-1). The relative standard deviations (RSDs) for 200 μg L(-1) of morphine, codeine and thebaine, 5.0 μg L(-1) of papaverine and 10.0 μg L(-1) of noscapine in diluted urine sample are in the range of 2.8-6.1% (n=7). The relative recoveries of urine samples spiked with alkaloids are 84.3-106.0%. The obtained results show that DLLME combined with HPLC-UV is a fast and simple method for the determination of opium alkaloids in urine samples.     

The pulmonary pharmacology of [4-methoxy-N1-(4-trans-nitrooxycyclohexyl)-N3-(3-pyridinylmethyl)-1,3-benzenedicarboxamide] (2NTX-99), an anti-atherotrombotic compound with therapeutic potential in pathological conditions that target lung vasculature

The pharmacological activity of 2NTX-99 ([4-methoxy-N1-(4-trans-nitrooxycyclohexyl)-N3-(3-pyridinylmethyl)-1,3-benzenedicarboxamide]) was investigated in vitro in the intact, rat pulmonary vasculature and in guinea pig airways. Rat lungs were perfused at constant flow and changes in vascular tone recorded. Challenge with the TXA? analogue 9,11-dideoxy-9α11α-methanoepoxy ProstaglandinF? (U46619, 0.5 μM) increased vessel tone (32.48±1.5 vs 13.13±0.56 mmHg; n=12). 2NTX-99 (0.1-100 μM; n=5), caused a concentration-dependent relaxation, prevented by 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, 10 μM, n=4), an inhibitor of soluble guanylate cyclase. Acetylcholine (0.1-10 μM; n=3) and a reference NO-donor, isosorbide-5-mononitrate (5-100 μM; n=4), were ineffective. Intraluminal perfusion of washed human platelets (2 × 10? cells/ml) increased intravascular pressure after challenge with arachidonic acid (AA, 2 μM; n=5), an increase abolished by acetylsalicylic acid and significantly reduced by 2NTX-99 (40 μM; n=5). TXB? in the lung perfusate was detected after platelet activation, 2NTX-99 inhibited TXA? synthesis (6.45±0.6 and 1.10±0.2 ng/ml, respectively). 2NTX-99 did not alter central or peripheral airway responsiveness to Histamine (0.001-300 μM; n=6), U46619 (0.001-3 μM, n=3) or LTD? (1 pM-1 μM; n=6). 2NTX-99 vasodilates the pulmonary vasculature via the release of nitric oxide (NO) and reduces intraluminal, AA-induced, TXA? formation. The combined activity of 2NTX-99 as an NO-donor and a TXA?-synthesis inhibitor provides strong support for its potential therapeutic use in pathologies of the pulmonary vascular bed (e.g. pulmonary hypertension).     

Fingerprinting of gelatinase subtypes for different topographic regions on non-retaining placenta of Holstein cows

The contribution of matrix metalloproteinases (MMP) to timely discharge of the placenta from bovine uterus at parturition is yet inconclusive, partly because of the presence of multiple MMP forms in situ. In the current study, the expression of different gelatinase subtypes on non-retaining placentas of Holstein cows was fingerprinted by using gelatin zymography. Different topographic regions on the placenta were measured separately, including the placentome-like structure and the fetal and maternal sides of interplacentomal placenta, all sampled from the central and peripheral areas of the placenta, respectively. The spontaneously ruptured umbilical cords were cross-sectioned as fetus end, middle and placenta end also for separate measurement. Body fluids including blood samples from the parturient cows, their neonatal calves and umbilical cord, as well as fetal fluids and the first colostrum were measured concomitantly. Results showed multiple forms of gelatinases subtypes in the placenta tissues and body fluids, including neutrophil gelatinase-associated lipocalin (NGAL)-MMP-9 complex, both the latent and active forms of MMP-2 and MMP-9; of them, the latent forms were much more abundantly and frequently expressed than the active forms. NGAL-MMP-9 complex was more prevalently present in the body fluids than in the placenta tissues. No distinguishable pattern of the expression of any gelatinase subtype was observed among the placentome-like structure, interplacentomal placenta and umbilical cord, or between fetal and maternal sides. Nonetheless, for interplacentomal placenta, proMMP-9 expression was higher in the central than in the peripheral area. In addition, proMMP-2 expression was higher in the rupture end (fetus end) than the placenta end of the umbilical cord. In conclusion, the current validated gelatin zymography detected a gradient proMMP-9 expression on the non-retaining placenta of cows in reverse to the proximity to the umbilical insertion point, and a gradient proMMP-2 expression on a section of the umbilical cord in reverse to the proximity to the rupture site, suggesting roles played by gelatinases in normal discharge of the placenta at term.