Reactivity of human placental chorionic plate vessels from pregnancies complicated by intrauterine growth restriction (IUGR)

A successful pregnancy is dependent on liberal placental perfusion via the maternal and fetal circulations. Doppler waveform analyses of umbilical arteries suggest increased resistance to flow in the fetoplacental circulation of pregnancies complicated by intrauterine growth restriction (IUGR). Neither the site nor the mediators responsible for this altered vascular reactivity are known, to date. In placentas in normal pregnancy, reduced oxygenation promotes contraction of the in vitro-perfused placental cotyledon and modulates agonist-induced contraction of chorionic plate arteries and veins. Placental oxygenation has also been suggested to be reduced in IUGR. We tested the hypothesis that oxygen tension could directly modify placental chorionic plate vessel vasoreactivity in IUGR. Small arteries and veins from the chorionic plate were dissected from biopsies from placentas of pregnancies complicated by IUGR and were studied using parallel wire myography. Vasoconstriction at 20%, 7%, and 2% oxygen was assessed utilizing the thromboxane mimetic U46619. Experiments were also performed in the presence of 4-aminopyridine (4AP), a blocker of voltage-gated potassium channels. Increased oxygenation reduced venous vasoconstriction but did not modify arterial vasoconstriction. 4AP increased basal tone in arteries and veins. We suggest that venoconstriction in response to hypoxia may provide a mechanism for increased fetoplacental vascular resistance associated with IUGR.     

Coronary artery reactivity in human vessels: some questions and some answers

Spasm of a conduit coronary artery, converting it into a major resistance vessel impeding myocardial blood flow, may have severe short- or long-term effects on cardiac rhythm and systolic ejection of blood. It is now clear that human coronary arteries in vitro contract to acetylcholine but that relaxation is the only response observed in dog coronary vessels. Acetylcholine is as powerful a constrictor of human coronary arteries, in terms of tension induced, as 5-hydroxytryptamine (5-HT) or histamine and is a substantially more powerful constrictor than norepinephrine. Field stimulation of coronary artery strips caused a vasoconstriction that was partially antagonized by atropine (3.45 X 10(-6) M). An enhanced reactivity of the epicardial arteries of cardiac and older patients to several agonists was also observed and appears to provide a background against which a number of vasoactive agents might induce spasm. Coronary tissue from cardiac patients also contains stores of 5-HT and histamine, and the histamine levels are substantially increased above the values in vessels from noncardiac patients. Coronary artery spasm or contraction probably can be initiated by diverse intrinsic and extrinsic influences, including autonomic discharge from either the parasympathetic or sympathetic nervous system or from histamine or 5-HT, and probably no one agent or entity is causative in all cases.     

Preliminary X-ray diffraction analysis of human chorionic gonadotropin

Hexagonal bipyramidal crystals of deglycosylated human chorionic gonadotropin have been grown using the method of vapor diffusion against ammonium sulfate. These crystals grow to nearly 0.4 mm along each axis, diffract to better than 3.5-A resolution and are relatively stable to irradiation. The crystals belong to the hexagonal space group P6(1)22 or enantiomer, and have unit cell parameters a = b = 88.7 A and c = 177.3 A.     

Proteomic analysis of human vessels: application to atherosclerotic plaques

Atherosclerosis is a chronic disease that affects medium and large arteries. This process originates from the interaction between cells of the arterial wall, lipoproteins and inflammatory cells, leading to the development of complex lesions or plaques that protrude into the arterial lumen. Plaque rupture and thrombosis result in acute clinical complications such as myocardial infarction and stroke. Owing to the heterogeneous cellular composition of the plaques, a proteomic analysis of the whole lesion is not appropriate. Therefore, we have studied the proteins secreted by human carotid atherosclerotic plaques, obtained by endarterectomy. Normal artery segments and different regions of the surgical pieces (noncomplicated plaque, complicated plaque with thrombus) were cultured in protein-free medium and the secreted proteins (supernatants) analyzed by two-dimensional gel electrophoresis. Normal artery segments secreted a moderate number of proteins (42 spots). However in the two-dimensional (2-D) gels (pH 3-10) of segments bearing a plaque, the number of spots increased markedly (154). The number of spots also increased (202) in the 2-D gels of artery segments with a ruptured plaque and thrombus. Thus, the more complicated the lesion, the higher the number of secreted proteins, suggesting the production of specific proteins relating to the complexity of the atherosclerotic lesion.     

Neurokinin B causes concentration-dependent relaxation of isolated human placental resistance vessels

Placental neurokinin B (NKB) was recently identified as the causative agent in preeclampsia, a condition characterized by increased maternal and feto-placental vascular resistance. We hypothesized that NKB should constrict placental resistance vessels. Placentas were obtained from normotensive pregnancies. Immediately after delivery, stem villous arteries (300 microm diameter, 1.2 mm long) were dissected from macroscopically normal tissue in cold HEPES-physiological salt solution (PSS), mounted on a wire myograph system, and bathed in HEPES-PSS at 37 degrees C. After determination of the passive-tension internal circumference characteristics, the arteries were set to 90% of the internal circumference they would have under a normal physiological transmural pressure. Cumulative concentration-response curves were constructed for NKB (1 x 10(-12) to 1 x 10(-5) mol/l). Since there was no constrictive response to NKB, cumulative constrictive concentration-response curves were constructed to the thromboxane A(2) mimetic U46619 (1 x 10(-9) to 1 x 10(-5) mol/l). The vessels were then pre-constricted to 80% of maximal response and exposed to cumulative concentrations of NKB (1 x 10(-12) to 1 x 10(-6) mol/l). NKB caused a concentration-dependent relaxation (Maximal response NKB, 51+/-5%, n=5; time control, 12+/-6%, n=4; P<0.05). Removal of the endothelium did not alter the vasodilatory response to NKB. We conclude that, contrary to our hypothesis, NKB causes an endothelium-independent relaxation of the placental resistance vessels. We propose that NKB plays a role in the maintenance of high placental blood flow in normal pregnancy.     

Reconstructing paleoecology and paleoenvironmental variables using factor analysis and regression: some limitations

We test the success of Principal Components, Factor and Regression Analysis at recovering environmental signals using numerical experiments in which we control species environmental responses, the environmental conditions and the sampling scheme used for calibration. We use two general conditions, one in which sampling of a continental margin for benthic foraminiferal assemblages is done in a standard grid and the driving environmental variables are correlated to one another, and the other where sampling is done so that the environmental variables are uncorrelated. The first condition mimics many studies in the literature. We find that where the controlling environmental variables are correlated, Principal Components/Factor Analysis yield factors that reflect the common variance (correlation) of those variables. Since this common variance is largely a product of the sampling scheme, the factors extracted do not reliably present true species ecologic behavior. This behavior cannot be accurately diagnosed and faulty interpretations may lead to substantial error when using factor coefficients to reconstruct conditions in the past. When the sampling scheme is constructed so that the controlling environmental variables for the calibration data set are uncorrelated the factor patterns will reflect these variables more accurately. Species responses can be more successfully interpreted from the Principal Components/Factor Analysis structure matrices. Additionally, regression analysis can successfully extract the independent environmental signals from the biotic data set. However, matrix closure is a confounding effect in all our numerical results as it distorts species' abundances and spatial distribution in the calibration data set. Our results show clearly that a knowledge of the controlling environmental variables, and the correlations among these variables over a study area, is essential for the successful application of multivariate techniques for paleoenvironmental reconstruction.     

Screening of some variables influencing the results of embryo transfer in the ewe. Part II: two-day-old embryos

Each of sixty Rasa aragonesa ewes received two embryos on Days 2-3 of the estrous cycle (Day 0=estrus) from 27 donors of the same breed that were superovulated with pFSH. The influence of several variables on fertility and prolificacy after transfer was studied by discriminant analysis. Our results showed that the main variables contributing to higher fertility were: in the donor-recipient couple, degree of estrus synchrony between them (better if donors were in estrus before recipients); in recipients, interval from FGA withdrawal to estrus onset, prolificacy in the previous lambing, age (all, better if inferior to the mean) and interval from the previous lambing (better if superior to the mean); in donors, ovulation rate (better if lower than the mean); and in embryos, developmental stage (better if superior to the mean). Likewise, the main variables contributing to higher prolificacy were: in donors, body condition score (better if higher than the mean) and weight (better if inferior to the mean); and in recipients, plasma progesterone concentration at transfer (better if inferior to the mean). The percentage of ewes correctly classified as lambing or not was 71.7% (P<0.01), and 72.5% of the ewes were correctly classified as having one or two lambs (P<0.05). Whether the criteria we have found for optimum results after transfer are applicable or not to conditions other than ours will need to be confirmed.     

Cryopreservation of isolated blood vessels

Canine saphenous veins were immersed in fetal calf serum (FCS) containing various cryoprotective agents, slowly frozen and stored for several weeks at subzero temperatures. Pharmacological investigations of frozen/thawed tissues revealed considerable attenuation of the contractile force of frozen stored veins as compared to unfrozen veins. The best recovery after thawing of frozen stored canine veins was obtained on tissues which had been frozen slowly to -70 degrees C and stored in liquid nitrogen while being immersed in FCS containing 1.8 mol/l dimethyl sulfoxide (DMSO). Though the maximum response to noradranline of helical strips prepared from these veins was diminished to about 60% the evidence suggests that there may be a very good preservation of the main biochemical properties, such as monoamine oxidase activity, endogenous prostaglandin synthesis and neuronal uptake mechanism in veins stored under these conditions. The same method of cryopreservation was applied to store samples of human veins. Comparison of the pD2 values for various agonists and of the blocking activities of various antagonists of both 5-HT receptors and alpha-adrenoceptors yielded an excellent correlation between the parameters determined on frozen/thawed and unfrozen human veins. It is concluded that freezing isolated blood vessels may be considered an effective means of preserving and storing vascular tissues for pharmacological investigations.     

Contractile and dilatory action of neuropeptides on isolated human mesenteric blood vessels

Neuropeptide Y (NPY), substance P (SP), vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP), lysyl-bradykinin, somatostatin, Met- and Leu-enkephalin were tested for their smooth muscle activity in isolated human mesenteric arteries and veins. Only NPY regularly contracted both arteries and veins. Alpha-adrenergic and 5-HT2 antagonists did not affect the response. Somatostatin contracted the veins, but not the arteries, in a variable but concentration-dependent way. The other neuropeptides were without contractile effect. CGRP, bradykinin, and SP regularly dilated, in a concentration-dependent way, both arteries and veins precontracted with prostaglandin F2 alpha or uridine triphosphate. CGRP and bradykinin were the most potent dilators. VIP and somatostatin usually caused a moderate dilatation in the arteries, whereas in the veins, somatostatin was without dilatory effect and the VIP-induced dilatation was irregular. In both types of vessels Met-enkephalin seldom gave any significant dilatation, and no response occurred in the presence of Leu-enkephalin or NPY. The SP-antagonist (D-Arg, D-Trp, Leu)-SP (spantide) caused a dextal shift of the concentration-response curves for SP, in the case of the arteries also including a reduced maximum effect.     

Human chorionic gonadotropin increases chromatin solubility in isolated bovine and human luteal nuclei

We have previously demonstrated that bovine and human luteal nuclei contain human chorionic gonadotropin/luteinizing hormone (hCG/LH) receptors and that these gonadotropins can directly stimulate nuclear membrane enzyme activity (nucleoside triphosphatase) involved in messenger ribonucleic acid (mRNA) transport from the nucleus to the cytoplasm. The present studies were undertaken to investigate the effect or hCG on chromatin solubility, reflecting perhaps synthesis and transport of RNA, in isolated bovine and human luteal nuclei. hCG increased chromatin solubility in a concentration-dependent manner. This hCG effect is either blocked or substantially reduced by the addition of hCG antiserum; denatured hCG had no effect and cyclic adenosine 3',5'-monophosphate could not mimic the hCG response. hCG had no effect on chromatin solubility in bovine liver or kidney nuclei and hormones other than hCG, human LH, or the beta subunit of hCG had no effect on chromatin solubility in bovine luteal nuclei, demonstrating the tissue and hormone specificity of the response. These findings further strengthen the concept of direct gonadotropin regulation of nuclear functions of luteal cells.     

Gonadotropin-releasing hormone effects on placental hormones during gestation: I. Alpha-human chorionic gonadotropin, human chorionic gonadotropin and human chorionic somatomammotropin

The release of alpha-human chorionic gonadotropin (alpha hCG), gonadotropin human chorionic gonadotropin (hCG) and human chorionic somatomammotropin (hCS) in vitro from placentas of different gestational ages was studied. In addition, the effect of gonadotropin-releasing hormone (GnRH) on these hormonal releases, as related to the gestational age of the placenta cultured and the dose of GnRH, was determined. The basal release of alpha hCG and hCG was greatest at 9-13 wk of gestation (1000-1500 ng/mg and 250-350 ng/mg, respectively). Lowest release rates were at term (28 ng/mg and 20 ng/mg, respectively). Hormonal release declined with extended culture, except from the cultures of 13- and 15-wk placentas, in which the initially high release continued throughout the 8 days of culture. The initial release of hCS was low at 6 wk, increased to maximum rates by 15 wk, and was similar to the initial rate of release at term. Gonadotropin-releasing hormone stimulated the release of alpha hCG and hCG most dramatically in cultures of 16-wk and 17-wk placentas, where as much as a 400- and 250-fold increase, respectively, on Day 6 of culture was observed (p less than 0.0001). In term placenta cultures after 6 days in vitro, a 20-fold stimulation of alpha hCG and a 10-fold increase of hCG was effected by GnRH (p less than 0.001). The largest responses of alpha hCG and hCG to GnRH were observed when estrogen levels were low. Dose-related responses were observed in some placentas, yet in some instances, maximal effects were attained with all doses utilized in these studies (0.2 to 50 micrograms/ml). These data demonstrate that human placentas of different gestational ages have varying hormonogenic capabilities in vitro. The data also establish that synthetic GnRH is capable of stimulating alpha hCG and hCG production, but the degree and pattern of response to GnRH stimulation are related to the gestational age of the placental tissue and its time in culture. The most responsive period to exogenous GnRH stimulation of alpha hCG and hCG release was on Days 5 and 6 of culture, when basal estrogen release was very low. These data support the hypothesis that hCG release might be controlled by a chorionic GnRH stimulation and suggest that local steroid levels may modulate the hCG response to GnRH stimulation.     

Immunoaffinity extraction and tandem mass spectrometric analysis of human chorionic gonadotropin in doping analysis

A confirmatory and quantitative HPLC-tandem mass spectrometry (MS-MS) method for human chorionic gonadotropin hormone (hCG) at concentrations as low as 5 IU/l following immunoaffinity extraction of the glycoprotein from urine was developed. The extraction method involved retention of urinary hCG in the immunoaffinity column via specific antigen-antibody interaction. A variety of eluents were then used to quantitatively elute hCG from the immunoaffinity column. Qualitative and quantitative analysis of hCG were undertaken using MS-MS by identifying the amino acid sequence of the marker peptide betaT5 obtained from hCG by tryptic digestion and the peak areas of three product ions b(6)(+), b(9)(+) and y(11)(+), respectively.     

Immunobiology of human chorionic gonadotropin

A comprehensive review of the immunobiology of human chorionic gonadotropin (hCG), including the structure of both alpha and beta chains, immunogenicity of various segments and epitopes of each, secretion and function of the hormone, determinants of receptor recognition, and finally, clinical studies of possible contraceptive beta-hCG-based vaccines, is presented. hCG is composed of 2 glycosylated peptides. The alpha subunit is identical to that found in hLH, hFSH and hTSH. The beta subunit, which is limiting in the sense that it is secreted in smaller amounts, defines the biological activity of hCG. hCG is secreted throughout pregnancy from 170 hours after fertilization to a peak at 8-10 weeks of and is essential for maintenance of early pregnancy by progesterone secreted by the corpus luteum. Although native hCG evokes antibodies, they cross react with LH, so such a vaccine would not be useful for contraception. Beta-hCG has been purified and also produced by monoclonal antibodies, and shown to produce antibodies and infertility in baboons. Phase I clinical trials of immunologically purified beta-hCG complexed to tetanus toxoid were conducted on 63 women in an international study in the mid-1970s, but results were mixed in terms of antibody titer and duration. New vaccines have been designed based on more sophisticated adjuvants, beta- hCG-terminal peptides, and polyvalent vaccines and are being tested in 4 Phase I trials currently, sponsored by the Population Council, the Indian government-sponsored program, and the WHO.     

Pulsed-field gel electrophoretic analysis and some characteristics of Staphylococcus aureus isolated from retail foods and human hands

This study investigates whether there is a predominant Staphylococcus aureus strain in retail foods and healthy human hands, and examines the relationship between pulsed-field gel electrophoresis (PFGE) banding patterns and the S. aureus characteristics of staphylococcal enterotoxin (SE) type, coagulase type, and β-lactamase activity. Ninety-four strains of S. aureus isolated from retail foods and healthy human hands were analyzed by PFGE. Several strains isolated from the same shop or a chain store showed identical patterns, indicating that the origins of these strains were identical. After excluding these strains showing identical patterns, 54 strains were used for the PFGE analysis. No spread of a particular clone in the environment surrounding the food was apparent. The PFGE analysis of these 54 strains was classified in 6 lineages (L1-L6). There was no relationship between the PFGE banding pattern and coagulase type or SE type. Eleven (84.6%) of the 13 isolates in PFGE banding pattern L5 did not produce β-lactamase, suggesting that the production of β-lactamase influenced a specific PFGE banding pattern.     

Screening of some variables influencing the results of embryo transfer in the ewe. I. Five-day-old embryos

Rasa Aragonesa ewes (n = 89) received 2 embryos on Day 6 of the estrous cycle (Day 0 = estrus) from 46 donors of the same breed that had been superovulated with FSH-p. The influence of several variables on fertility and prolificacy after transfer was studied by discriminant analysis. Our results showed that the main variables that contributed to a high fertility rate were the degree of synchrony (better outcome if donors come into estrus later than the recipients); Fluorogestone acetate (FGA) to estrus interval and interval from previous lambing in the recipients, ovulation rate of the donors and recipients (better if superior to the mean); prolificacy of recipients in the previous lambing; and difference in developmental stage of the pair of transferred embryos (better if inferior to the mean). The main variables affecting prolificacy were the ovulation rate and weight of the donors and progesterone concentrations of the recipients (better if lower than the mean); age of the donors and difference in progesterone concentrations between donors minus those of the recipients (better if higher than the mean). The percentage of ewes correctly classified into lambing or not lambing status was 73% (P < 0.001) and that of the ewes correctly classified as lambing 1 or 2 lambs was 81.8% (P < 0.001). Whether or not the criteria we have established for optimum transfer results are applicable to conditions other than our own still needs to be confirmed.     

Preparation and analysis of the common urinary forms of human chorionic gonadotropin

Human chorionic gonadotropin (hCG) is the hormone of pregnancy and forms the basis of all pregnancy tests as well as diagnostic assays for a variety of pathological states including certain types of cancers and some diseases of pregnancy and genetic abnormalities. In recent years, the discovery of the diagnostic utility of measurement of the free subunits and fragments of the hormone, especially in urine, has proven of special use for diagnosis of very early pregnancy loss, an important phenomenon related to infertility, as well as part of screening programs for Down Syndrome and gynecological cancers. This article summarizes existing and new methods for the preparation of hCG, its subunits, and the beta core fragment from urinary sources. The methods for proper analyses of these materials are also described to enable investigators to prepare and analyze these materials in various quantities in their own laboratories.