Egtazic Acid Research Articles

Ca2+ affects the hyphal differentiation to sclerotia formation of Athelia rolfsii.

RNA-Sequencing (RNA-Seq) and transcriptomic analyses have become powerful tools to study the developmental stages of fungal structures scuh as sclerotia. While RNA-Seq experiments have been set up for many important sclerotia- and microsclerotia-forming fungi, it has not been implemented to study Athelia rolfsii, which is one of the earliest fungi used in literature to uncover the roles of reactive oxygen species (ROS) in stimulating sclerotia formation. This study applied RNA-Seq to profile gene expression in four developmental stages of A. rolfsii sclerotia. Surprisingly, gene ontology and expression patterns suggested that most ROS-scavenging genes were not up-regulated in the stages from hyphal differentiation to the initial sclerotia stage. Using antioxidant and oxidant-amended culture assay, the results suggested none of the ascorbic acid, dithiothreitol (DTT), H2O2, or superoxide dismutase inhibitors [diethyldithiocarbamate (DETC), NaN3, and sodium dodecyl sulfate] affected the sclerotia number. Instead, only glutathione reduced the sclerotia number. Because glutathione has also been suggested to facilitate Ca2+ influx, therefore, glutathione culture assays with the combination of CaCl2, Ca2+-chelator egtazic acid, DETC, and H2O2 were tested on A. rolfsii, as well as two other fungi (Sclerotinia sclerotiorum and Macrophomina phaseolina) for comparison. Although the addition of CaCl2 caused sclerotia or microsclerotia reduction for all three fungi, the CaCl2-ROS interaction was only observed for S. sclerotiorum and M. phaseolina, but not A. rolfsi. Collectively, this study not only pointed out a conserved function of Ca2+ in suppressing fungal sclerotia and microsclerotia formation but also highlighted sclerotia formation of A. rolfsii being only sensitive to Ca2+ and independent of ROS stimuli.IMPORTANCEManagement for plant diseases caused by soil-borne fungal pathogens is challenging because many soil-borne fungal pathogens form sclerotia for long-term survival. Advanced understanding of the molecular and cellular mechanisms of sclerotia formation may provide novel insights to prevent these fungal residues in fields. This study discovered that Ca2+ acts as a negative signal cue to suppress sclerotia and microsclerotia formation in three economically important fungal pathogens. Moreover, the southern blight fungus Athelia rolfsii appears to be only regulated by Ca2+ but not reactive oxygen species. Accordingly, A. rolfsii can be a useful system for studying the detailed mechanism of Ca2+, and the applicability of Ca2+ in reducing sclerotia could be further assessed for disease management.

Small-Caliber Vascular Grafts Engineered from Decellularized Leaves and Cross-Linked Gelatin.

Despite advances in vascular replacement and repair, fabricating small-diameter vascular grafts with low thrombogenicity and appropriate tissue mechanics remains a challenge. A wide range of platforms have been developed to use plant-derived scaffolds for various applications. Unlike animal tissue, plants are primarily composed of cellulose which can offer a promising, nonthrombogenic alternative capable of promoting cell attachment and redirecting blood flow. By taking advantage of the biocompatibility and mechanical properties of cellulose, we developed small-diameter vascular grafts using decellularized leatherleaf viburnum and cross-linked gelatin. Different terrestrial plant leaves (leatherleaf, spinach, and parsley) were decellularized with sodium dodecyl sulfate, egtazic acid and/or Tergitol, followed by a bleach and Triton X-100 clearing solution, and then evaluated for decellularization efficiency, mechanical integrity, and recellularization potential. Hematoxylin and eosin staining and DNA quantification revealed successful removal of cells in all leatherleaf conditions. Methods of 3D graft fabrication were evaluated, and leatherleaf scaffolds maintained suitable tensile and rupture strength properties. 2D scaffolds and 3D grafts were seeded with rat endothelial cells. Cells remained viable for over 14 days with cell densities comparable to other natural and synthetic scaffolds. This study demonstrates the potential of cost effective and readily available decellularized plants to generate small-diameter vascular grafts capable of recellularization and with suitable mechanical properties. Impact statement Due to the prevalence of coronary heart disease in the United States, small-caliber vascular grafts for coronary bypass surgery are in high demand. We evaluate decellularized plant leaves as potential candidates for small-diameter vascular grafts with appropriate mechanical properties and recellularization potential.

Chelator-Based Parameterization of the 12-6-4 Lennard-Jones Molecular Mechanics Potential for More Realistic Metal Ion-Protein Interactions.

Metal ions are associated with a variety of proteins and play critical roles in a wide range of biochemical processes. There are multiple ways to study and quantify protein–metal ion interactions, including molecular dynamics simulations. Recently, the AMBER molecular mechanics forcefield was modified to include a 12-6-4 Lennard-Jones potential, which allows for a better description of nonbonded terms through the additional pairwise Cij coefficients. Here, we demonstrate a method of generating Cij parameters that allows parametrization of specific metal ion-ligating groups in order to tune binding energies computed by thermodynamic integration. The new Cij coefficients were tested on a series of chelators: ethylenediaminetetraacetic acid, nitrilotriacetic acid, egtazic acid, and the EF1 loop peptides from the proteins lanmodulin and calmodulin. The new parameters show significant improvements in computed binding energies relative to existing force fields and produce coordination numbers and ion-oxygen distances that are in good agreement with experimental values. This parametrization method should be extensible to a range of other systems and could be readily adapted to tune properties other than binding energies.

Evaluation of biohydrogen production potential of fragmented sugar industry biosludge using ultrasonication coupled with egtazic acid

In this study, the influence of ultrasonication (USN) combined with egtazic acid fragmentation method on sugar industry waste (SWS) sludge was studied for improving biohydrogen production. Initially, USN method was proceeded by varying power and fragmentation time. At an optimal power (115 W) and fragmentation time (30 min), USN method demanded 23,000 kJ/kg TS specific energy for achieving 15.2% complex organics solubilization rate (COR). To further upsurge of SWS sludge solubilization, USN assisted with egtazic acid (USNEG) method was performed by varying fragmentation time and egtazic acid dosage at an optimal power (115 W). USNEG method has achieved more COR (23%) at minimum specific energy of 11,500 kJ/kg TS (fragmentation time: 15 min and EGTA dosage: 0.03 g/g TS). Then, biohydrogen potential (BHP) assay was investigated in which USNEG method has obtained higher biohydrogen production. Also, energy analysis revealed that USNEG method exhibited an energy ratio of 1.78.

From an assessment of multiple chelators, clodronate has potential for use in continuous chelation.

To identify chelators which when mixed with sodium hypochlorite (NaOCl) are stable, exhibiting minimal loss of free available chlorine (FAC) over 80min and to further investigate potential mixtures by assessing FAC over 18h and the capacity to remove smear layer. 0.25molL-1 EDTA (10%), 0.25molL-1 EGTA (egtazic acid), 0.25molL-1 CDTA (cyclohexanediaminetetraacetic acid), 0.25molL-1 DTPA (pentetic acid), 0.5molL-1 ATMP (aminotri(methylene phosphonic acid)) and 1molL-1 HPAA, (hydroxyphosphonoacetic acid), all at alkaline pH, were mixed equally with 5% NaOCl. 0.5molL-1 alkaline clodronate and 0.5molL-1 Na4 etidronate (15%) were mixed equally with 10% NaOCl. For all mixtures, the pH and temperature were measured over 80min and additionally for the clodronate mixture over 18h. Iodometric titration was used to measure the FAC of all mixtures except for HPAA. The following were compared with respect to their ability to remove smear layer: 1molL-1 clodronate+10% NaOCl, 0.5molL-1 clodronate+10% NaOCl, 1molL-1 etidronate+10% NaOCl, 0.5molL-1 clodronate+10% NaOCl and the sequences 5% NaOCl/17% EDTA/5% NaOCl and 5% NaOCl/17% EDTA. The area fraction occupied by open dentinal tubules as a percentage of the total area (% AF) from scanning electron microscopy micrographs was calculated using Image J. The results were statistically analysed with alpha set at 0.05. Compared to its control, the mixture 0.5molL-1 clodronate+10% NaOCl lost no FAC over 18h (P>0.05). The FAC of 0.25molL-1 CDTA mixed with 5% NaOCl fell to 96%, 92%, 75% and 4.9% at 20, 40, 60 and 80min, respectively. Temperature rises were observed in all cases except in the etidronate and clodronate mixtures. Only in the clodronate mixture did the pH remain above pH 12 for the whole experiment. Although smear layer was removed, the % AF in 1molL-1 clodronate+10% NaOCl, 0.5molL-1 clodronate+10% NaOCl, 1molL-1 etidronate+10% NaOCl was less than for 0.5molL-1 etidronate+10% NaOCl and 5% NaOCl/17% EDTA/5% NaOCl and 5% NaOCl/17% EDTA. Alkaline 0.5molL-1 clodronate mixed equally with 10% NaOCl has potential for use in continuous chelation, based on this assessment of stability and smear layer removal. Further research is needed to establish its efficacy and safety.

Biofilm inactivation by synergistic treatment of atmospheric pressure plasma and chelating agents

In this study, we investigated the bacterial biofilm reduction by combined treatment of atmospheric pressure plasma (APP) and chelating agents. Many of hospital acquired infections (HAI) are related to biofilm infections. APP and chelating agents were reported to be effective in bacterial biofilm inactivation and eradication. It is believed that chelating agents disrupt the biofilm formation through bonding metal ions, while APP inactivates bacteria mainly through reactive species [1–3]. In our study, we used a surface micro-discharge (SMD) driven by sinusoidal power input of 2 kHz and peak to peak voltage of 9 kV to treat Escherichia coli (E. coli), Enterococcus faecalis (E. faecalis), and Staphylococcus capitis (S. capitis) biofilm on 316 L stainless steel and hopkins rod lens glass plates in combination with chelating agents of trisodium citrate (TSC), ethylenediaminetetraacetic acid (EDTA), egtazic acid (EGTA), and alizarin. Bacterial biofilm reduction was measured by means of colony count assay and BacTiter-Glo cell viability assay. The results of colony count assay showed that combined treatment of EDTA and TSC with plasma has synergistic effects on all three bacterial biofilms, while EGTA only on E. coli and none for alizarin. Experiments of BacTiter-Glo cell viability assay indicate that EDTA, EGTA, and TSC has synergistic effects on all three biofilms while also none for alizarin.

Resveratrol and genistein inhibition of rat isolated gastrointestinal contractions and related mechanisms.

To investigate the effects and underlying mechanisms of resveratrol and genistein on contractile responses of rat gastrointestinal smooth muscle. Isolated strips of gastrointestinal smooth muscle from Spraque-Dawley rats were suspended in organ baths containing Kreb's solution, and the contractility of smooth muscles was measured before and after incubation with resveratrol and genistein, and the related mechanisms were studied by co-incubation with various inhibitors. Resveratrol and genistein dose-dependently decreased the resting tension, and also reduced the mean contractile amplitude of gastrointestinal smooth muscle. Estrogen receptor blockades (ICI 182780 and tamoxifen) failed to alter the inhibitory effects induced by resveratrol and genistein. However, their effects were attenuated by inhibitions of α-adrenergic receptor (phentolamine), nitric oxide synthase (levorotatory-NG-nitroarginine), ATP-sensitive potassium channels (glibenclamide), and cyclic adenosine monophosphate (SQ22536). In high K(+)/Ca(2+)-free Kreb's solution containing 0.01 mmol/L egtazic acid, resveratrol and genistein reduced the contractile responses of CaCl2, and shifted its cumulative concentration-response curves rightward. Resveratrol and genistein relax gastrointestinal smooth muscle via α-adrenergic receptors, nitric oxide and cyclic adenosine monophosphate pathways, ATP-sensitive potassium channels, and inhibition of L-type Ca(2+) channels.

Inhibitory effects of genistein and resveratrol on guinea pig gallbladder contractility in vitro

To observe and compare the effects of phytoestrogen genistein, resveratrol and 17beta-estradiol on the tonic contraction and the phasic contraction of isolated gallbladder muscle strips and to study the underlying mechanisms. Isolated strips of gallbladder muscle from guinea pigs were suspended in organ baths containing Kreb's solution, and the contractilities of strips were measured before and after incubation with genistein, resveratrol and 17beta-estradiol respectively. Similar to 17beta-estradiol, genistein and resveratrol could dose-dependently inhibit the phasic contractile activities, they decreased the mean contractile amplitude and the contractile frequencies of gallbladder muscle strips, and also produced a marked reduction in resting tone. The blocker of estrogen receptor ICI 182780 failed to alter the inhibitory effects induced by genistein and resveratrol, but potassium bisperoxo (1, 10 phenanthroline) oxovanadate bpV (phen), a potent protein tyrosine phosphatase inhibitor, markedly attenuated the inhibitory effects induced by genistein and resveratrol. In calcium-free Kreb's solution containing 0.01 mmol/L egtazic acid (EGTA), genistein and resveratrol inhibited the first phasic contraction induced by acetylcholine (ACh), but did not affect the second contraction induced by CaCl(2). In addition, genistein, resveratrol and 17beta-estradiol also could reduce the contractile responses of ACh and KCl, and shift their cumulative concentration-response curves rightward. Phytoestrogen genistein and resveratrol can directly inhibit the contractile activity of isolated gallbladder muscle both at rest and in response to stimulation. The mechanisms responsible for the inhibitory effects probably due mainly to inhibition of tyrosine kinase, Ca(2+) influx through potential-dependent calcium channels (PDCs) and Ca(2+) release from sarcoplasmic reticulum (SR), but were not related to the estrogen receptors.

ALTERED INDUCIBLE NITRIC OXIDE SYNTHASE EXPRESSION AND NITRIC OXIDE PRODUCTION IN THE BLADDER OF CATS WITH FELINE INTERSTITIAL CYSTITIS

Alterations in nitric oxide (NO) levels have been demonstrated in some humans with interstitial cystitis (IC) as well as in chemically induced animal models of cystitis. Thus, in the current study we investigated whether inducible NO synthase (iNOS) mediated NO production is altered in the bladder of cats with a naturally occurring model of IC termed feline IC (FIC). We examined iNOS expression using Western immunoblotting and baseline NO production using an NO microsensor from smooth muscle and mucosal bladder strips in 9 healthy cats and 6 diagnosed with FIC. There was a significant increase in baseline NO production in cats with FIC compared with that in healthy cats in smooth muscle and mucosal strips. This production was not ablated in the absence of extracellular Ca (100 microM egtazic acid) or following incubation with the calmodulin antagonist trifluoroperazine (20 microM), indicating iNOS mediated Ca independent NO production. Release was significantly decreased following incubation with the NOS antagonist L-NAME (N-nitro-L-arginine methyl ester) (100 microM). Furthermore, immunoblotting revealed a trend toward increased iNOS expression in smooth muscle and mucosal strips from FIC cats but not from healthy cats. In light of previous findings that the barrier property of the urothelial surface is disrupted in FIC and iNOS mediated increase in NO alters barrier function in other types of epithelium our findings suggest that iNOS dependent NO production may have a role in epithelial barrier dysfunction in FIC.

Ca2+ participates in alpha1B-adrenoceptor-mediated cAMP response in HEK293 cells

To investigate the alpha1B-adrenoceptor (alpha1B-AR)-mediated cAMP response and underlying mechanisms in HEK293 cells. Full-length cDNA encoding alpha1B-AR was transfected into HEK293 cells using the calcium phosphate precipitation method, and alpha1B-AR expression and cAMP accumulation were determined by using the saturation radioligand binding assay and ion-exchange chromatography, respectively. Under agonist stimulation, alpha1B-AR mediated cAMP synthesis in HEK293 cells, and blockade by PLC-PKC or tyrosine kinase did not reduce cAMP accumulation induced by NE. Pretreatment with pertussis toxin (PTX) had little effect on basal cAMP accumulation as well as norepinephrine (NE)-stimulated cAMP accumulation. In addition, pretreatment with cholera toxin (CTX) neither mimicked nor blocked the effect induced by NE. The extracellular Ca2+ chelator egtazic acid (EGTA), nonselective Ca2+ channel blocker CdCl2 and calmodulin (CaM) inhibitor W-7 significantly reduced NE-induced cAMP accumulation from 1.59%+/-0.47% to 1.00%+/-0.31%, 0.78%+/-0.23%, and 0.90%+/-0.40%, respectively. By coupling with a PTX-insensitive G protein, alpha1BAR promotes Ca2+ influx via receptor-dependent Ca2+ channels, then Ca2+ is linked to CaM to form a Ca2+-CaM complex, which stimulates adenylyl cyclase (AC), thereby increasing the cAMP production in HEK293 cell lines.

The protective role of cellular glutathione peroxidase against trauma-induced mitochondrial dysfunction in the mouse brain

Reactive oxygen species are believed to participate in the pathogenesis of traumatic brain injury (TBI). To evaluate the role of cellular glutathione peroxidase (Gpx1), a selenium-containing enzyme functioning in reduction of hydrogen peroxide and alkyl hyperoxides, in protecting animals against TBI, a line of Gpx1 transgenic mice was generated. Overexpression of Gpx1 was found in many organs including the brain of the transgenic mice. This line of transgenic mice and knockout mice deficient in Gpx1 were used in a model of controlled cortical impact injury and the efficiency of oxidative phosphorylation in brain mitochondria was determined. Although a 2-mm depth of mechanical impact caused a drastic decrease in NAD-linked electron transfer activities and energy-coupling capacities in brain mitochondria of nontransgenic mice, the decrease in mitochondrial function was completely prevented by overexpression of Gpx1 in Gpx1 transgenic mice. In addition, a 1-mm deformation depth hardly affected brain mitochondrial function in wild-type (Gpx1 +/+) mice, yet resulted in a significant decrease in mitochondrial bioenergetic capacity in brains of homozygous Gpx1 knockout (Gpx1 −/−) mice. Further experiments showed that inclusion of calcium chelator egtazic acid in measurement of mitochondrial respiration could completely restore the efficiency of mitochondrial respiration in injured brains of nontransgenic mice and Gpx1 −/− mice, suggesting that the observed mitochondrial dysfunction is a direct result of increase in mitochondrion-associated calcium, which is secondary to the increased oxidative stress. These studies not only establish the role of Gpx1 in preventing mitochondrial dysfunction in mouse brain after TBI, but also suggest the species of reactive oxygen responsible for this event.

Magnesium sulfate inhibits the oxytocin-induced production of inositol 1,4,5-trisphosphate in cultured human myometrial cells

Objective: The purpose of this study was to determine the effects of magnesium sulfate on inositol trisphosphate production and the mechanism of these effects. Study Design: Myometrium was obtained at the time of cesarean delivery from women before labor at term. Inositol trisphosphate was measured in the primary myometrial cell cultures after stimulation with oxytocin, sodium fluoride, or Bay K 8644 with or without preincubation with magnesium sulfate or nifedipine. Experiments were performed in either calcium-containing or calcium-free medium that contained egtazic acid and after preincubation with the intracellular calcium chelator BAPTA-acetoxymethylester. Inositol trisphosphate production was measured by radioreceptor assay. In separate experiments, changes in intracellular calcium concentrations ([Ca2+]i) were measured with the use of Fura-2 and spectrophotofluorometry. Results: Oxytocin, sodium fluoride, and Bay K 8644 increased inositol trisphosphate production 2- to 4-fold. Preincubation with magnesium sulfate (3 × 10−3 mol/L) for ≥5 minutes decreased oxytocin-, sodium fluoride-, and Bay K 8644-induced inositol trisphosphate production in either calcium-containing or calcium-free media. Preincubation with BAPTA-acetoxymethylester decreased oxytocin-stimulated inositol trisphosphate production by 78% in calcium-containing media and completely prevented the oxytocin response in calcium-free media. Magnesium sulfate decreased inositol trisphosphate production in calcium-containing media but had no additional effect in calcium-free media. Oxytocin and Bay K 8644 increased [Ca2+]i in either calcium-containing or calcium-free media, and magnesium sulfate reduced this in both cases. Conclusion: Magnesium sulfate appears to inhibit phosphatidylinositol-4, 5-bisphosphate-specific phospholipase C activity and subsequent calcium release in cultured myometrial cells by a direct effect on phospholipase C. (Am J Obstet Gynecol 2002;187:419-24.)

Left ventricular diastolic dysfunction during demand ischemia: rigor underlies increased stiffness without calcium-mediated tension. Amelioration by glycolytic substrate

OBJECTIVESThe goal of this study was to determine the subcellular mechanism(s) underlying increased left ventricular (LV) diastolic chamber stiffness (DCS) during angina (demand ischemia).BACKGROUNDIncreased DCS may result from increased diastolic myocyte calcium concentration and/or rigor. Therefore, we assessed the effects of direct alterations of both calcium-activated tension and high-energy phosphates on increased DCS.METHODSDemand ischemia was reproduced in isolated, isovolumic, red-cell perfused rabbit hearts by imposing low-flow ischemia and pacing tachycardia. This resulted in increased DCS. Interventions were performed after LV end-diastolic pressure had increased approximately 7 mm Hg. Initially, to determine the effects of altered calcium concentration or myofilament calcium responsiveness, hearts received either: 1) 5 or 14 mmol/L calcium chloride; 2) 8 mmol/L egtazic acid; 3) 5 mmol/L butane-dione-monoxime (BDM); or 4) 50 mmol/L ammonium chloride (NH4Cl). Then, to assess the contribution of decreased high-energy phosphate supply, hearts received 5) glucose (25 mmol/L) and insulin (400 μU/ml).RESULTS1) Calcium chloride, 5 and 14 mmol/L, increased LV systolic pressure by 42% and 70%, respectively (p < 0.001), indicating increased calcium-activated tension, but did not further increase DCS, implying intact diastolic calcium resequestration. 2) Egtazic acid reduced LV systolic pressure by 30% (p < 0.001), indicating reduced intracellular calcium, but failed to reduce increased DCS. 3) Butane-dione-monoxime and NH4Cl chloride affected contractile function (i.e., a calcium-driven force) but did not alter increased DCS. 4) Glucose and insulin, which increase high-energy phosphates during ischemia, reduced increased DCS by 50% (p < 0.001).CONCLUSIONSIncreased DCS during demand ischemia was insensitive to maneuvers altering intracellular calcium concentration or myofilament calcium-responsiveness, that is, evidence against an etiology of calcium-activated tension. In contrast, increased glycolytic substrate ameliorated increased DCS, supporting a primary mechanism of rigor-bond formation.

Active and passive compliance of the fetal bovine bladder.

Others have shown that the fetal bovine bladder is relatively noncompliant. Previous studies on compliance of fetal bovine bladders have demonstrated that the youngest fetal bladders had lowest and the oldest fetal bladders (near full-term) had greatest compliance. Our study was designed to determine the level of participation of active tension in the compliance of fetal bladders during gestation. Fetal bovine bladders were obtained immediately after maternal harvest and crown-to-rump length was measured to determine gestational age. The fetus was inspected for genitourinary anomalies and the bladder was immediately placed in chilled M199 media. Strips (1 x 0.5 cm.) were excised from the anterior sagittal plane of the bladder and subjected to length-tension analysis in oxygenated Tyrode's buffer at 37C. Tension was measured using a force transducer and length was increased using a micropositioner. Compliance refers to the length-tension studies performed in normal Tyrode's solution and consists of a combination of active (smooth muscle tone) and passive properties. Passive compliance refers to length-tension studies performed after inactivation of bladder smooth muscle tone. Compliance with muscle tone intact was determined by incrementally stretching the strips to twice resting length in physiological buffer and then permitting them to return to resting length. Passive compliance with muscle tone ablated was determined in the same fashion after overnight incubation in calcium-free Tyrode's buffer in the presence of 5 mM. egtazic acid and 10 mM. sodium azide. An exponential function was fit to the normalized length-tension curves, where the exponential coefficient (EC) is numerically inversely proportional to compliance. Passive compliance was greatest in the youngest bladders (EC = 0.5 in the first trimester) and gradually decreased with increasing fetal age (EC = 1.2 in the third trimester). Active compliance demonstrated the opposite pattern, since the younger bladders were more stiff (EC = 2.1 in the first and 1.6 in the third trimesters). These studies demonstrate that passive compliance is greatest in the youngest bladders and progressively decreases with gestation. However, active smooth muscle tone is greatest in the youngest bladders and decreases with gestation. Thus, high active smooth muscle tone in the youngest fetal bladders results in relatively poor compliance of the early stage fetal bladder.

Active and Passive Compliance of the Fetal Bovine Bladder

Purpose Others have shown that the fetal bovine bladder is relatively noncompliant. Previous studies on compliance of fetal bovine bladders have demonstrated that the youngest fetal bladders had lowest and the oldest fetal bladders (near full-term) had greatest compliance. Our study was designed to determine the level of participation of active tension in the compliance of fetal bladders during gestation. Materials and Methods Fetal bovine bladders were obtained immediately after maternal harvest and crown-to-rump length was measured to determine gestational age. The fetus was inspected for genitourinary anomalies and the bladder was immediately placed in chilled M199 media. Strips (1 × 0.5 cm.) were excised from the anterior sagittal plane of the bladder and subjected to length-tension analysis in oxygenated Tyrode's buffer at 37C. Tension was measured using a force transducer and length was increased using a micropositioner. Compliance refers to the length-tension studies performed in normal Tyrode's solution and consists of a combination of active (smooth muscle tone) and passive properties. Passive compliance refers to length-tension studies performed after inactivation of bladder smooth muscle tone. Compliance with muscle tone intact was determined by incrementally stretching the strips to twice resting length in physiological buffer and then permitting them to return to resting length. Passive compliance with muscle tone ablated was determined in the same fashion after overnight incubation in calcium-free Tyrode's buffer in the presence of 5 mM. egtazic acid and 10 mM. sodium azide. An exponential function was fit to the normalized length-tension curves, where the exponential coefficient (EC) is numerically inversely proportional to compliance. Results Passive compliance was greatest in the youngest bladders (EC = 0.5 in the first trimester) and gradually decreased with increasing fetal age (EC = 1.2 in the third trimester). Active compliance demonstrated the opposite pattern, since the younger bladders were more stiff (EC = 2.1 in the first and 1.6 in the third trimesters). Conclusions These studies demonstrate that passive compliance is greatest in the youngest bladders and progressively decreases with gestation. However, active smooth muscle tone is greatest in the youngest bladders and decreases with gestation. Thus, high active smooth muscle tone in the youngest fetal bladders results in relatively poor compliance of the early stage fetal bladder.

Ca binding of rat fibrinogen and fibrin(ogen) degradation products

The authors studied the binding of Ca to rat fibrinogen and plasmic fibrin(ogen) degradation products by means of equilibrium dialysis with special reference to the protective effect of Ca2+ in the plasmic degradation of fibrinogen. Direct binding studies demonstrate that rat fibrinogen and the plasmic degradation products D(cate) and D-dimer strongly bind Ca2+ and that this binding in D(cate) and D-dimer is correlated with a protective effect against further plasmic degradation. Chemicals/CAS: calcium, 7440-70-2; fibrinogen, 9001-32-5; Calcium, 7440-70-2; Egtazic Acid, 67-42-5; Fibrin Fibrinogen Degradation Products; Fibrinogen, 9001-32-5