Elevated expression of KiSS-1 in placenta of preeclampsia and its effect on trophoblast

The expression of KiSS-1, MMP-9 and MMP-2 mRNAs and proteins was studied in placentas of women with preeclampsia (PE, n=47) and women of normal pregnancy (NP; n=30). In addition, KiSS-1 mRNA expression as well as cell growth, proliferation and invasion were examined in JAR cells (human trophoblast cell line) transfected with pcDNA3-KiSS-1vector. Expression of KiSS-1 mRNA and protein was higher (p<0.05) in women with PE compared with that of NP women. In contrast, expression of MMP-9 and MMP-2 was lower (p<0.05) in PE than in NP women. KiSS-1 mRNA was detected in JAR cells successfully transfected with pcDNA3-KiSS-1 gene (JAR-K1, JAR-K2, JAR-K3). KiSS-1 mRNA was not detected in JAR cells transfected with pcDNA3 gene (JAR-P1, JAR-P2) and non-transfected JAR cells. No difference (p>0.05) was observed in cell growth among these three cell types. Invasion ability was significantly lower (p<0.01) in JAR-K1, JAR-K2 and JAR-K3 cells compared to JAR-P cells and non-transfected JAR cells. Overexpression of KiSS-1 and insufficient expression of MMP-9 and MMP-2 in placenta were demonstrated in women with PE. The data suggests that KiSS-1 gene plays an important role in inhibiting trophoblast invasion during placental development.     

Elevated Expression of KiSS-1 in Placenta of Chinese Women with Early-Onset Preeclampsia

Preeclampsia (PE) is a heterogeneous syndrome affecting 2% to 8% of all pregnancies and is the world’s leading cause of fetal and maternal morbidity and mortality. In many cases of PE, shallow trophoblast invasion results in inappropriate maternal spiral artery remodeling and impaired placental function. Multiple genes have been implicated in trophoblast invasion, among which are KiSS-1 and GPR54. The gene product of KiSS-1 is metastin, which is a ligand for the receptor GPR54. Both metastin and GPR54 are expressed in the placenta of normal pregnancy and have been implicated in modulating trophoblast invasion through inhibiting migration of trophoblast cells. We have previously reported that the expression level of KiSS-1 was higher in trophoblasts from women with preeclampsia as compared to normal controls. Here, using quantitative RT-PCR, Western blot analysis and immunohistochemistry, we extend our analysis to demonstrate that elevated KiSS-1 expression occurs only in early-onset preeclampsia (ePE) and not late-onset preeclampsia (lPE). However, no difference in the expression levels of GPR54 is observed between ePE, lPE, and normal controls. Further, we show that KiSS-1 expression is also increased in placenta of intrauterine death and birth asphyxia in comparison to normal newborns of ePE and lPE. Our findings suggest that aberrant upregulation of KiSS-1 expression may contribute to the underlying mechanism of ePE as well as birth asphyxia.     

An altered pattern of circulating apolipoprotein E3 isoforms is implicated in preeclampsia

Preeclampsia is a common pregnancy complication that is an important cause of preterm birth and fetal growth restriction. Because there is no diagnostic test yet available for preeclampsia, we used a proteomic approach to identify novel serum/plasma biomarkers for this condition. We conducted case control studies comparing nulliparous women who developed preeclampsia at 36-38 weeks of gestation with healthy nulliparous women matched by gestational age at sampling. Serum/plasma was depleted of six abundant proteins and analyzed by two-dimensional gel electrophoresis (n = 12 per group) and difference gel electrophoresis (n = 12 per group). Differences in abundance of protein spots were detected by univariate and multivariate statistical analyses. Proteins were identified by mass spectrometry and expression of selected proteins was validated by immunoblotting. Proteins whose concentrations were selectively associated with preeclampsia included apolipoprotein E (apoE), apoC-II, complement factor C3c, fibrinogen, transthyretin, and complement factor H-related protein 2. An increase in a deglycosylated isoform of apoE3 and concomitantly decreased amounts of one apoE3 glycoisoform were identified in preeclamptic plasma and confirmed by immunoblotting. Altered production of these preeclampsia-related apoE3 isoforms might impair reverse cholesterol transport, contributing to arterial damage. These findings point to a novel mechanistic link between preeclampsia and subsequent cardiovascular disease.     

A Role for Nrf2 in Redox Signalling of the Invasive Extravillous Trophoblast in Severe Early Onset IUGR Associated with Preeclampsia

Background

Preeclampsia (PE) is characterized by increased lipid oxidation and diminished antioxidant capacity, while intrauterine growth restriction (IUGR) is characterized by impaired invasion of the extravillous trophoblast. Vascular endothelial growth factor (VEGF) has been reported to be altered in preeclampsia. A relationship between VEGF and nuclear factor erythroid 2-related factor-2 (Nrf2) has been shown in vitro, where VEGF prevents oxidative damage via activation of the Nrf2 pathway. In this study the expression of Nrf2, VEGF and 4-hydroxynonenal (4-HNE), was determined in interstitial and endovascular/intramural extravillous trophoblast (EVT) in normal pregnancies and those complicated by severe early onset IUGR associated with preeclampsia IUGR/PE.

Materials and Methods

Full-thickness uterine tissues derived from caesarean hysterectomies performed in 5 healthy normotensive women delivering term infants and 6 women with severe early onset IUGR with preeclampsia (29–34 weeks gestation) were analyzed. Interstitial and endovascular extravillous trophoblast were quantified after immunohistochemical staining of paraffin sections using antibodies against Nrf2, 4-HNE, VEGF, and cytokeratin 7.

Results

Uterine tissues from women suffering from severe early onset IUGR/PE were characterized by reduced invasion of extravillous trophoblast into the endometrial and myometrial segments of spiral arteries in the placental bed. Extravillous trophoblast showed an increased cytoplasmic expression of Nrf2 and 4-HNE in IUGR/PE cases. The increased expression of Nrf2 in cases of IUGR/PE was associated with decreased expression of VEGF in these cells compared to controls.

Conclusion

Our data suggests that besides villous cytotrophoblast, also the extravillous trophoblast is a source of Nrf2-dependent genes. VEGF deficiency may cause higher oxidative stress in extravillous trophoblast in cases with IUGR/PE. The resulting reduced basal defence against oxidative stress and the higher vulnerability to oxidative damage may play a role in the limited trophoblast invasion into spiral arteries in cases suffering from severe early onset IUGR/PE.     

Maternal TLR4 and NOD2 gene variants, pro-inflammatory phenotype and susceptibility to early-onset preeclampsia and HELLP syndrome

Background

Altered maternal inflammatory responses play a role in the development of preeclampsia and the hemolysis, elevated liver enzymes and low platelets (HELLP) syndrome. We examined whether allelic variants of the innate immune receptors Toll-like receptor 4 (TLR4) and nucleotide-binding oligomerization domain 2 (NOD2), that impair the inflammatory response to endotoxin, are related to preeclampsia and HELLP syndrome.

Methods and Findings

We determined five common mutations in TLR4 (D299G and T399I) and NOD2 (R702W, G908R and L1007fs) in 340 primiparous women with a history of early-onset preeclampsia, of whom 177 women developed HELLP syndrome and in 113 women with a history of only uneventful pregnancies as controls. In addition, we assessed plasma levels of pro-inflammatory biomarkers C-reactive protein, interleukin-6, soluble intercellular adhesion molecule-1, fibrinogen and von Willebrand factor in a subset of 214 women included at least six months after delivery. After adjustment for maternal age and chronic hypertension, attenuating allelic variants of TLR4 were more common in women with a history of early-onset preeclampsia than in controls (OR 2.9 [95% CI 1.2–6.7]). Highest frequencies for TLR4 variants were observed in women who developed HELLP syndrome (adjusted OR 4.1 [95% CI 1.7–9.8]). In addition, high levels of interleukin-6 and fibrinogen were associated with a history of early-onset preeclampsia. Combined positivity for any of the TLR4 and NOD2 allelic variants and high levels of interleukin-6 was 6.9-fold more common in women with a history of early-onset preeclampsia (95% CI 2.1–23.2) compared to controls.

Conclusions

We observed an association of common TLR4 and NOD2 gene variants, and pro-inflammatory phenotype with a history of early-onset preeclampsia and HELLP syndrome. These findings suggest involvement of the maternal innate immune system in severe hypertensive disorders of pregnancy.     

Proteome analysis reveals elevated serum levels of clusterin in patients with preeclampsia

Preeclampsia is a pregnancy-specific syndrome and a major cause of maternal mortality. The pathophysiology of preeclampsia is unknown, and no proteome analysis of preeclampsia has been reported. We sought to identify proteins associated with preeclampsia using a proteomic technique and performed two-dimensional electrophoresis (2-DE) on sera from six patients with preeclampsia and six normal pregnant women, followed by comparison of the SYPRO Ruby-stained 2-DE profiles. A group of overexpressed spots was identified in the limited study set. Overexpressed spots were identified as clusterin by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) followed by peptide mass fingerprinting, a protein database search, and Western blot analysis. Additionally, sera of 80 preeclamptic women and 80 normal pregnant women were processed by immunoassay methods to confirm changes in clusterin concentrations quantitatively. Immunoassays showed that clusterin levels in the 80 preeclamptic women were significantly higher than those in the 80 controls (mean +/- SD; 1.62 +/- 0.46 times reference level in preeclamptic women vs. 1.30 +/- 0.46 times reference level in controls, P < 0.001). Proteomic analysis of serum proteins is a promising tool for studying preeclampsia pathophysiology and identifying proteins associated with preeclampsia.     

Human placental transthyretin in fetal growth restriction in combination with preeclampsia and the HELLP syndrome

Fetal growth restriction is a serious, still poorly understood pregnancy-related pathology often associated with preeclampsia. Recent studies speculate on the role of human transthyretin, a carrier protein for thyroxin and retinol binding protein, in the etiology of both pregnancy pathologies. Objective was to investigate the localization and abundance of transthyretin (TTR) in placentas of pregnancies suffering from fetal growth restriction with and without preeclampsia and HELLP. This was a retrospective case control study on human paraffin-embedded placentas from pregnancies with a gestational age at delivery between the 24th and 34th week of gestation. 16 placentas were included in this study, 11 cases and 5 from normotensive pregnancies as controls. Cases were divided into three groups: four from early onset idiopathic intrauterine growth restriction (IUGR), four from early-onset severe preeclampsia (PE), and three from early-onset IUGR with preeclampsia plus HELLP syndrome. Distribution and abundance of TTR were investigated by means of immunohistochemistry. Semi quantitative analysis of TTR staining of placental sections revealed that TTR was mostly expressed in the villous trophoblast covering placental villi. Only weak staining of TTR in villous stroma could be detected. The comparison of placentas revealed that in pure IUGR and severe PE there is a much stronger TTR reactivity compared to controls and cases with IUGR?+?PE?+?HELLP. Concluding, the study showed that TTR is dysregulated in cases of IUGR and severe early onset preeclampsia. Interestingly, TTR expression is not affected in cases with HELLP syndrome that reveal the same staining intensities as age-matched controls.     

血脂代谢指标及血清维生素A、E水平与子痫前期的相关性分析

目的:探讨血脂代谢指标及血清维生素A、E水平与子痫前期的相关性。方法:选取2016年12月至2017年12月期间来我院产检及住院分娩的722例妊娠妇女,选取94例子痫前期的妊娠妇女作为A组,其中轻度子痫前期32例作为A1组,重度子痫前期62例作为A2组,并从剩余的628例正常妊娠者中选取126例自愿参与本研究的妊娠妇女作为B组。收集并记录妊娠妇女的临床指标,包括入院时的孕周、孕次、产次、流产次数、血脂代谢指标、血清维生素A、E水平,分析血脂代谢指标、血清维生素A、E水平与子痫前期的相关性。结果:三组孕妇总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白(LDL)、高密度脂蛋白(HDL)、载脂蛋白A(ApoA)、载脂蛋白B(ApoB)、维生素A、维生素E水平整体比较差异均具有统计学意义(均P0.05);A1组、A2组的TC、TG、LDL、HDL、ApoA、ApoB、维生素A、维生素E水平与B组比较,差异均有统计学意义(均P0.05),A2组TG、LDL高于A1组,而维生素A、维生素E水平低于A1组,差异均有统计学意义(均P0.05),Spearman秩相关分析结果显示,TC、TG、LDL、ApoB水平与子痫前期呈正相关(r=0.214,0.432,0.517,0.226,P=0.012,0.008,0.005,0.012),HDL、ApoA、维生素A、维生素E水平与子痫前期呈负相关(r=-0.282,-0.357,-0.539,-1.217,P=0.010,0.009,0.003,0.000)。结论:血脂代谢指标、维生素A、维生素E水平在子痫前期孕妇中表达异常,且这些指标与子痫前期密切相关,应重视妊娠期孕妇的血脂代谢指标、维生素A、维生素E水平的监测,并控制其水平,从而有效防治子痫前期。     

Quantitative determination of apolipoprotein A-I in high-density lipoproteins and 'free' apolipoprotein A-I by two-dimensional agarose gel lipoprotein-'rocket' immunoelectrophoresis of human serum

A method has been developed for quantitative analysis of 'free' apolipoprotein A-I and apolipoprotein A-I associated with high-density lipoprotein (HDL) in serum. The method utilizes the difference between the rate of electrophoretic migration of apolipoprotein A-I associated with HDL (alpha) and 'free' apolipoprotein A-I (pre-beta) in agarose gel. Apolipoprotein A-I is subsequently quantitated by electrophoresis in a second dimensional gel containing anti-apolipoprotein A-I antibodies. Using this method all apolipoprotein A-I of normal fasting serum was found associated with HDL (n = 16). By contrast, 'free' apolipoprotein A-I accounted for up to 12% of the total in the serum of patients with isolated hypertriglyceridemia (n = 8) or mixed hyperlipoproteinemia (n = 8). Between 30 and 35% of 'free' apolipoprotein A-I was found in one patient afflicted with the apolipoprotein C-II deficiency syndrome. Also, 'free' apolipoprotein A-I could be detected in normal postabsorptive serum. 30 and 90 min following heparin-enhanced lipolysis 'free' apolipoprotein A-I accounted for 23 and 20%, respectively, of the total apolipoprotein A-I of serum. Apolipoprotein A-I associated with HDL remained unaltered. It appears, therefore, that 'free' apolipoprotein A-I is liberated from triglyceride-rich lipoproteins during lipolysis.     

Elevated maternal IL-16 levels, enhanced IL-16 expressions in endothelium and leukocytes, and increased IL-16 production by placental trophoblasts in women with preeclampsia

Cytokine IL-16 plays an important role in innate immune responses. However, little information is available about IL-16 function in human pregnancy. In this study, we collected maternal blood samples from 125 pregnant women between 26 and 41 wk of gestation, 63 from normal pregnant women and 62 from women with preeclampsia (PE). Serum IL-16C levels were measured by ELISA. We also examined IL-16C and IL-16N immunostaining in maternal vessels and protein expression in leukocytes from normal and PE pregnant women. In addition, IL-16C production by placental trophoblasts was also determined. Our results showed that IL-16C levels were significantly higher in severe PE than in mild PE and normal pregnant controls, 515 +/- 58 vs 287 +/- 46 (p < 0.05) and 163 +/- 9 pg/ml (p < 0.01), respectively, indicating that increased IL-16 levels in PE is associated with the severity of the disease. There was no difference for the IL-16C levels in normal pregnant women throughout the third trimester. The correlation of maternal IL-16C levels with labor and body mass index was also analyzed. IL-16C levels were neither associated with labor nor associated with body mass index. Moreover, increased IL-16C immunostaining in maternal vessel endothelium and enhanced IL-16C protein expression in leukocytes were observed in PE. We also found that IL-16C production was increased by trophoblasts from PE placentas. Our study demonstrated up-regulation of the IL-16 profile in both the maternal and the placental systems in PE, suggesting that IL-16 could be an important cytokine engaged in the altered immune system and exaggerated inflammatory response in PE syndrome.     

High-end arteriolar resistance limits uterine artery blood flow and restricts fetal growth in preeclampsia and gestational hypertension at high altitude

The reduction in infant birth weight and increased frequency of preeclampsia (PE) in high-altitude residents have been attributed to greater placental hypoxia, smaller uterine artery (UA) diameter, and lower UA blood flow (Q(UA)). This cross-sectional case-control study determined UA, common iliac (CI), and external iliac (EI) arterial blood flow in Andeans residing at 3,600-4,100 m, who were either nonpregnant (NP, n = 23), or experiencing normotensive pregnancies (NORM; n = 155), preeclampsia (PE, n = 20), or gestational hypertension (GH, n = 12). Pregnancy enlarged UA diameter to ~0.62 cm in all groups, but indices of end-arteriolar vascular resistance were higher in PE or GH than in NORM. Q(UA) was lower in early-onset (≤34 wk) PE or GH than in NORM, but was normal in late-onset (>34 wk) illness. Left Q(UA) was consistently greater than right in NORM, but the pattern reversed in PE. Although Q(CI) and Q(EI) were higher in PE and GH than NORM, the fraction of Q(CI) distributed to the UA was reduced 2- to 3-fold. Women with early-onset PE delivered preterm, and 43% had stillborn small for gestational age (SGA) babies. Those with GH and late-onset PE delivered at term but had higher frequencies of SGA babies (GH=50%, PE=46% vs. NORM=15%, both P < 0.01). Birth weight was strongly associated with reduced Q(UA) (R(2) = 0.80, P < 0.01), as were disease severity and adverse fetal outcomes. We concluded that high end-arteriolar resistance, not smaller UA diameter, limited Q(UA) and restricted fetal growth in PE and GH. These are, to our knowledge, the first quantitative measurements of Q(UA) and pelvic blood flow in early- vs. late-onset PE in high-altitude residents.     

Restriction fragment length polymorphism of the apolipoprotein A-I gene among inhabitants of Novosibirsk]

Restriction fragments' length polymorphism in the region of apolipoprotein A-I (apo A-I) gene was investigated in Novosibirsk (Siberia, USSR) population. Correlation between PstI apo A-I alleles (2,2 kb-P1; 3,3 kb-P2) and total cholesterol, triglycerides, high density polyproteins cholesterol, and apolipoprotein A-I level was analysed. A tendency to increase in cholesterol index of atherogenicity and to decrease in high density lipoproteins cholesterol as well as apolipoprotein A-I level was shown to occur for P1P2 genotype patients.     

Influence of lipid environment on insulin binding in cultured hepatoma cells

Three mouse monoclonal antibodies (Mabs) to human apo A-I were produced using apolipoprotein A-I or HDL3 as immunogens. These monoclonal antibodies, 2G11, 4A12 and 4B11, were characterized for their reactivity with isolated apolipoprotein A-I and HDL in solution. The immunoblotting patterns of the HDL3 two-dimensional electrophoresis show that these three monoclonal antibodies reacted with all the polymorphic forms of apolipoprotein A-I. Cotitration experiments indicated that they correspond to three distinct epitopes. In order to locate these three antigenic determinants on the isolated apolipoprotein A-I, the reactivity of the three monoclonal antibodies has been studied on CNBr-cleaved apolipoprotein A-I. The monoclonal antibodies 2G11 and 4A12 addressed to the amino (CNBr 1) and carboxy (CNBr 4) terminal segments, respectively. In comparison with the monoclonal antibodies characterized by Weech et al. ((1985) Biochim. Biophys. Acta 835, 390-401), monoclonal antibody 4A12 is the only one described in the literature which is specific of the carboxy terminal segment of apolipoprotein A-I. Monoclonal antibody 4B11 does not react with any CNBr fragment, its binding is temperature dependent, it could be directed to a conformational epitope. Relative differences were demonstrated in the expression of the three epitopes in HDL subfractions isolated by density gradient ultracentrifugation. According to Curtiss and Edgington ((1985) J. Biol. Chem. 260, 2982-2993) our results indicate the existence of an immunochemical heterogeneity in the organization of apolipoprotein A-I at the surface of HDL particles as well as in the soluble form of apolipoprotein A-I.     

Isolation and characterization of human apolipoprotein A-I Fukuoka (110 Glu----Lys). A novel apolipoprotein variant

A novel genetic variant of apolipoprotein(apo) A-I Fukuoka, has been identified in a Japanese family. This variant has a relative charge of +2 compared to normal apolipoprotein A-I (A-I4), on the isoelectric focusing gels and the same molecular mass and immunologic characteristics as normal apolipoprotein A-I. This variant, transmitted as an autosomal co-dominant inheritance was purified by preparative Immobiline isoelectric focusing. Sequence analysis after cleavage with lysyl endopeptidase and CNBr, followed by high-performance liquid chromatography revealed a single substitution of lysine at position 110, instead of the usual glutamic acid. This mutant apolipoprotein A-I has much the same potential as to activate lecithin-cholesterol acyltransferase.     

The role of apolipoprotein A-I and apolipoprotein A-II in high-density lipoprotein binding to human adipocyte plasma membranes

Adipocyte plasma membranes purified from omental fat tissue biopsies of massively obese subjects possess specific binding sites for high-density lipoprotein (HDL3). This binding was independent of apolipoprotein E as HDL3 isolated from plasma of an apolipoprotein E-deficient individual was bound to a level comparable to that of normal HDL3. To examine the importance of apolipoprotein A-I, the major HDL3 apolipoprotein, in the specific binding of HDL3 to human adipocytes, HDL3 modified to contain varying proportions of apolipoproteins A-I and A-II was prepared by incubating normal HDL3 particles with different amounts of purified apolipoprotein A-II. As the apolipoproteins A-I-to-A-II ratio in HDL3 decreased, the binding of these particles to adipocyte plasma membranes was reduced. Compared to control HDL3, a 92 +/- 3.1% reduction (mean +/- S.E., n = 3) in maximum binding capacity was observed along with an increased binding affinity for HDL3 particles in which almost all of the apolipoprotein A-I had been replaced by A-II. The uptake of HDL cholesteryl ester by intact adipocytes as monitored by [3H]cholesteryl ether labeled HDL3, was also significantly reduced (about 35% reduction, P less than 0.005) by substituting apolipoprotein A-II for A-I in HDL3. These data suggest that HDL binding to human adipocyte membranes is mediated primarily by apolipoprotein A-I and that optimal delivery of cholesteryl ester from HDL to human adipocytes is also dependent on apolipoprotein A-I.     

Decreased expression of MMP-9 in CD8+ cells in placenta with severe preeclampsia

We compared the number of CD4-positive (CD4⁺) and CD8-positive (CD8⁺) cells in severe and non-severe preeclampsia (PE), and in normal pregnancy. We also evaluated the expression of matrix metalloproteinase 9 (MMP-9) in CD4⁺ and CD8⁺ cells. Immunohistochemistry for CD4⁺ and CD8⁺ was performed on the decidua basalis of 15 severe and 13 non-severe PE women and compared to decidual tissue of 19 normal pregnancies (control group). Co-expression of MMP-9 with CD8⁺ and CD4⁺ cells was determined by double immunofluorescence staining. The median number of CD8⁺ cells/mm² was significantly lower for the severe PE group than for the normal pregnancy group, as was the number of CD4⁺ cells and MMP-9⁺CD8⁺ cells. No statistical difference was found between the non-severe PE group and the normal pregnancy group. The significant decrease of CD4⁺, CD8⁺ and MMP-9⁺CD8⁺ cells at the fetal-maternal interface only in the severe PE group suggests that immunological disorders play a role in the pathophysiology of severe PE.     

Anal sphincteric neurogenic injury in asymptomatic nulliparous women and fecal incontinence

While anal sphincter neurogenic injury documented by needle electromyography (EMG) has been implicated to cause fecal incontinence (FI), most studies have been uncontrolled. Normal values and the effects of age on anal sphincter motor unit potentials (MUP) are ill defined. The functional significance of anal sphincter neurogenic injury in FI is unclear. Anal pressures and EMG were assessed in 20 asymptomatic nulliparous women (age, 38 ± 5 yr; mean ± SE) and 20 women with FI (54 ± 3 yr). A computerized program quantified MUP duration and phases. These parameters and MUP recruitment were also semiquantitatively assessed by experienced electromyographers in real time. Increasing age was associated with longer and more polyphasic MUP in nulliparous women by quantitative analysis. A higher proportion of FI patients had prolonged (1 control, 7 patients, P = 0.04) and polyphasic MUP (2 controls, 9 patients, P = 0.03) at rest but not during squeeze. Semiquantitative analyses identified neurogenic or muscle injury in the anal sphincter (11 patients) and other lumbosacral muscles (4 patients). There was substantial agreement between quantitative and semiquantitative analyses (κ statistic 0.63 ± 95% CI: 0.32-0.96). Anal resting and squeeze pressures were lower (P ≤ 0.01) in FI than controls. Anal sphincter neurogenic or muscle injury assessed by needle EMG was associated (P = 0.01) with weaker squeeze pressures (83 ± 10 mmHg vs. 154 ± 30 mmHg) and explained 19% (P = 0.01) of the variation in squeeze pressure. Anal sphincter MUP are longer and more polyphasic in older than younger nulliparous women. Women with FI have more severe neurogenic or muscle anal sphincter injury, which is associated with lower squeeze pressures.     

Changes in phospholipids and free fatty acids in the brains of mice preconditioned by hypoxia.

Changes in the composition and contents of phospholipids and free fatty acids were observed and compared in three groups: (A) unpreconditoned normal controls, (B) exposure to 1 run of hypoxia and (C) exposure to 4 runs of hypoxia. In group B, the content of phosphatidyl ethanolamine (PE), phosphatidyl serine (PS) and free fatty acids (FFAs) increased significantly and the content of phosphatidyl choline (PC) and sphingomyelin (SM) decreased significantly. While in group C the content of PE, PS, PC and FFAs changed significantly when compared with that of group B, all phospholipid (except SM) and FFA contents tended to decrease to the level of group A. No new FFA was seen in the brain homogenates in any of the three groups. These results suggest that the changes in the content of mouse brain phospholipids and FFAs may be adaptive and involved in the animals' tolerance to hypoxia.     

Radioimmunoassay of human high density lipoprotein apo-protein A-1.

A double antibody radioimmunoassay technique was developed for the measurement of apolipoprotein A-I, the major apoprotein of human high density lipoproteins. Apolipoprotein A-I was prepared from human delipidated high density lipoprotein (d equal to 1.085-1.210) by gel filtration and ion-exchange chromatography. Purified apolipoprotein A-I antibodies were obtained by means of apolipoprotein A-I immunoadsorbent. Apolipoprotein A-I was radiolabeled with 125-I by the iodine monochloride technique. 65-80% of 125 I-labeled apolipoprotein A-I could be bound by the different apolipoprotein A-I antibodies, and more than 95% of the 125-I-labeled apolipoprotein A-I was displaced by unlabeled apolipoprotein A-I. The immunoassay was found to be sensitive for the detection of about 10 ng of apolipoprotein A-I in the incubation mixture, and accurate with a variability of only 3-5% (S.E.M.). This technique enables the quantitation of apolipoprotein A-I in whole plasma or high density lipoprotein without the need of delipidation. The quantitation of apolipoprotein A-I in high density lipoprotein was found similar to that obtained by gel filtration technique. The displacement capacity of the different lipoproteins and apoproteins in comparison to unlabeled apolipoprotein A-I was: very low density lipoprotein, 1.8%; low density lipoprotein, 2.6%; high density lipoprotein, 68%; apolipoprotein B, non-detectable; apolipoprotein C, 0.5%; and apolipoprotein A-II, 4%. The distribution of immunoassayable apolipoprotein A-I among the different plasma lipoproteins was as follows: smaller than 1% in very low density lipoprotein and low density lipoprotein; 50% in high density lipoprotein, and 50% in lipoprotein fraction of density greater than 1.21 g/ml. The amount of apolipoprotein A-I in the latter fraction was found to be related to the number of centrifugations.