scholarly journals A statherin and calcium enriched layer at the air interface of human parotid saliva

2005 ◽  
Vol 389 (1) ◽  
pp. 111-116 ◽  
Author(s):  
Gordon B. PROCTOR ◽  
Sawsan HAMDAN ◽  
Guy H. CARPENTER ◽  
Peter WILDE
Keyword(s):  
Surface Layer ◽  
Molecular Mass ◽  
Calcium Binding ◽  
Solid Phase ◽  
Salivary Protein ◽  
Major Protein ◽  
Parotid Saliva ◽  
Surface Rheology ◽  
Major Protein Band ◽  
The Mean

Parotid saliva placed in 35-mm-diameter tissue culture dishes developed increasing surface viscoelasticity at the interface with air. A surface layer became visible with time, and was collected and analysed by protein electrophoresis which indicated that a single protein (pI 4.2; molecular mass approx. 6 kDa) predominated. Western blot analysis demonstrated that the major protein band reacted with an antiserum directed against the C-terminal of the calcium-binding salivary protein statherin. Matrix-assisted laser-desorption ionization–time-of-flight MS analysis gave a molecular mass of 5380 Da for the protein, corresponding to the molecular mass of statherin. Staining of film protein in electrophoresis gels was compared with statherin synthesized on a solid phase, and the mean statherin content of film formed from 1 ml of parotid saliva was measured as 7 nmol. The mean calcium content of the surface layer was 250 nmol. Surface rheology was greatly decreased in the presence of EDTA, whereas surface tension of saliva was unaffected by calcium chelation, suggesting that protein accumulated at the surface was unaffected. The results suggest that a layer rich in statherin forms at the interface of saliva and air, and that the surface rheology developed is dependent upon protein interactions mediated by calcium. The surface layer may enhance the function of saliva as a protective layer on the mucosal surfaces and teeth.

scholarly journals Cloning and expression in Escherichia coli of a dog thyroid cDNA encoding a novel inositol 1,4,5-trisphosphate 5-phosphatase

1994 ◽  
Vol 300 (1) ◽  
pp. 85-90 ◽  
Author(s):  
B Verjans ◽  
F De Smedt ◽  
R Lecocq ◽  
V Vanweyenberg ◽  
C Moreau ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Mass ◽  
Bovine Brain ◽  
Cloning And Expression ◽  
Major Protein ◽  
Type I ◽  
Degenerate Primers ◽  
Calculated Molecular Mass ◽  
Major Protein Band ◽  
Inositol 1,4,5 Trisphosphate

In brain and many other tissues, type I inositol 1,4,5-trisphosphate (InsP3) 5-phosphatase is the major isoenzyme hydrolysing the calcium-mobilizing second messenger InsP3. This protein has been purified to apparent homogeneity from a crude soluble fraction of bovine brain, yielding a single major protein band with a molecular mass of 43 kDa after SDS/PAGE. This material was used to determine internal microsequences. A partial DNA sequence has been amplified by PCR by using degenerate primers deduced from two protein sequences (FKAKKYKKV and DENYKSQE). A cDNA clone (BVCT) was isolated by screening a dog thyroid cDNA library. The encoded protein of 412 amino acids has a calculated molecular mass of 47,681 Da. Peptide sequences generated from the bovine brain enzyme were found to be 96% conserved compared with the dog thyroid protein. When clone BVCT was expressed in Escherichia coli, the recombinant protein was shown to hydrolyse both InsP3 and inositol 1,3,4,5-tetrakisphosphate, with apparent Km values of 28 and 3 microM respectively. Enzyme activity was inhibited by EDTA and 2,3-bisphosphoglycerate, both inhibitors of native InsP3 5-phosphatase, but not by EGTA and LiCl, as previously shown for the bovine brain enzyme. Our data show the cloning of type I InsP3 5-phosphatase which, interestingly, does not share any significant sequence identity with the previously cloned type III isoenzyme.

scholarly journals Purification and characterization of rat epididymal-fluid α-d-mannosidase: similarities to sperm plasma-membrane α-d-mannosidase

1993 ◽  
Vol 290 (2) ◽  
pp. 427-436 ◽  
Author(s):  
D R P Tulsiani ◽  
M D Skudlarek ◽  
S K Nagdas ◽  
M C Orgebin-Crist
Keyword(s):  
Molecular Mass ◽  
Plasma Membranes ◽  
Antigenic Site ◽  
Protein Band ◽  
Soluble Form ◽  
Major Protein ◽  
Major Protein Band ◽  
Membrane Bound ◽  
Epididymal Fluid

We have previously reported the occurrence and partial characterization of a novel alpha-D-mannosidase activity on rat sperm plasma membranes [Tulsiani, Skudlarek and Orgebin-Crist (1989) J. Cell Biol. 109, 1257-1267]. Here, we report the presence of a similar alpha-D-mannosidase activity in a soluble form in rat epididymal fluid. The soluble enzyme was purified nearly 500-fold with 9-12% recovery to a state approaching homogeneity using: (1) (NH4)2SO4 precipitation; (2) affinity chromatography on immobilized mannan and D-mannosamine; (3) ion-exchange (DE-52) column chromatography; (4) molecular-sieve chromatography. The enzyme was eluted from the final column (Sephacryl S-400) at an apparent molecular mass of 460 kDa. When resolved by SDS/PAGE (under denaturing conditions), the enzyme showed a major protein band (115 kDa) and few very minor bands. The polyclonal antibody raised against the major protein band was found to cross-react with the alpha-D-mannosidase activity present in epididymal fluid (soluble) and detergent-solubilized spermatozoa from the rat and mouse. This result suggested that the soluble and membrane-bound enzyme activities shared a common antigenic site(s). The antibody was used to characterize further the alpha-D-mannosidase activity(ies) present in the rat epididymal fluid and rat sperm plasma membranes. Data from these studies show that the two forms are similar in (a) subunit molecular mass, (b) substrate specificity and (c) inhibitory effect of several sugars. These similarities suggest that the soluble and membrane-bound alpha-D-mannosidase activities are isoforms. Immunoprecipitation studies after solubilization of the testis and epididymal particulate fraction from sexually immature rats show that the testis (but not the epididymis) contains the immunoreactive alpha-D-mannosidase activity. This result and the fact that spermatozoa from the rat rete testis show alpha-D-mannosidase activity indicate that the sperm enzyme is synthesized in the testis during spermatogenesis.

Increased S100B Levels in Cannabis Use Disorder

2015 ◽  
Vol 22 (4) ◽  
pp. 177-180 ◽  
Author(s):  
Huseyin Bayazit ◽  
Erdinc Cicek ◽  
Salih Selek ◽  
Nurten Aksoy ◽  
I. Fatih Karababa ◽  
...  
Keyword(s):  
Calcium Binding ◽  
Neuronal Damage ◽  
Cannabis Use ◽  
S100b Protein ◽  
Control Group ◽  
Blood Levels ◽  
Cannabis Use Disorder ◽  
Protein Levels ◽  
S100 Calcium Binding Protein ◽  
The Mean

Background: It has been determined that cannabis has adverse effects on brain tissue, and that increased S100 calcium binding protein B (S100B) blood levels are markers of neuronal damage. Therefore, the aim of this study was to evaluate the S100B levels in cannabis use disorder. Method: Thirty-two patients with cannabis use disorder and 31 matched healthy controls were enrolled in this study. Appropriate blood samples were taken from the enrolled subjects, and the serum S100B protein levels were measured with an electrochemiluminescence immunoassay for the quantification of the protein. Findings: We found significantly increased S100B protein levels in patients with cannabis use disorder. The mean serum concentration of S100B was 0.081 ± 0.018 μg/l in patients with cannabis use disorder, and 0.069 ± 0.018 μg/l in the control group (p = 0.008). Interpretation: Our data suggest that elevated S100B protein levels might indicate neuronal damage in the brains of people with cannabis use disorder.

Dynamic computed tomography with low- and high-molecular-mass contrast agents to assess microvascular permeability modifications in a model of liver fibrosis

2002 ◽  
Vol 103 (2) ◽  
pp. 213-216 ◽  
Author(s):  
Roland MATERNE ◽  
Laurence ANNET ◽  
Stéphane DECHAMBRE ◽  
Christine SEMPOUX ◽  
Anne M. SMITH ◽  
...  
Keyword(s):  
Liver Fibrosis ◽  
Contrast Agents ◽  
Molecular Mass ◽  
Hepatic Fibrosis ◽  
Mean Transit Time ◽  
Distribution Volume ◽  
High Molecular Mass ◽  
Permeability Changes ◽  
Molecular Masses ◽  
The Mean

Interstitial collagen formation and transformation of the fenestrated hepatic sinusoids into continuous capillaries are major ultrastructural changes that occur in liver cirrhosis and fibrosis. These modifications lead to progressive restriction of blood–liver exchanges. The purpose of our study was to evaluate the permeability changes in a model of hepatic fibrosis by using dynamic computed tomography (CT) enhanced with contrast agents of different molecular masses. Dynamic single-section CT of the liver was performed after intravenous bolus administration of a low-molecular-mass contrast agent (iobitridol) and an experimental high-molecular-mass agent (P840) in normal control rabbits and in rabbits with hepatic fibrosis. Hepatic, aortic and portal venous time–density curves were fitted with a dual-input one-compartmental model to calculate the hepatic mean transit time and distribution volume of the contrast agents. In the rabbits with liver fibrosis, the mean transit time of the high-molecular-mass agent was shorter than that of the low-molecular-mass agent (10.0±1.8s and 12.0±1.2s respectively; P<0.05). The distribution volume accessible to the high-molecular-mass agent was also smaller (22.2±4.8% compared with 32.0±6.7%; P<0.01). In the normal rabbits, the mean transit times of the high- and low-molecular-mass agents did not differ significantly, and nor did their distribution volumes. Our results demonstrate decreased sinusoidal permeability for the high-molecular-mass agent P840 in a model of hepatic fibrosis. Non-invasive assessment of permeability changes in liver fibrosis can be performed with dynamic CT and contrast agents of different molecular masses.

A metalloproteinase extracellularly released byCrithidia deanei

2003 ◽  
Vol 49 (10) ◽  
pp. 625-632 ◽  
Author(s):  
Claudia Masini d'Avila-Levy ◽  
Rodrigo F Souza ◽  
Rosana C Gomes ◽  
Alane B Vermelho ◽  
Marta H Branquinha
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Residual Activity ◽  
Major Protein ◽  
Motile Cells ◽  
Sds Page ◽  
Triton X

Actively motile cells from a cured strain of Crithidia deanei released proteins in phosphate buffer (pH 7.4). The molecular mass of the released polypeptides, which included some proteinases, ranged from 19 to 116 kDa. One of the major protein bands was purified to homogeneity by a combination of anion-exchange and gel filtration chromatographs. The apparent molecular mass of this protein was estimated to be 62 kDa by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE). The incorporation of gelatin into SDS–PAGE showed that the purified protein presented proteolytic activity in a position corresponding to a molecular mass of 60 kDa. The enzyme was optimally active at 37 °C and pH 6.0 and showed 25% of residual activity at 28 °C for 30 min. The proteinase was inhibited by 1,10-phenanthroline and EDTA, showing that it belonged to the metalloproteinase class. A polyclonal antibody to the leishmanial gp63 reacted strongly with the released C. deanei protease. After Triton X-114 extraction, an enzyme similar to the purified metalloproteinase was detected in aqueous and detergent-rich phases. The detection of an extracellular metalloproteinase produced by C. deanei and some other Crithidia species suggests a potential role of this released enzyme in substrate degradation that may be relevant to the survival of trypanosomatids in the host.Key words: endosymbiont, trypanosomatid, extracellular, proteinase.

3D Numerical Simulation of Polydisperse Pressurized Gas-Solid Fluidized Bed

2011 ◽  
Author(s):  
P. Fede ◽  
O. Simonin ◽  
I. Ghouila
Keyword(s):  
Numerical Simulation ◽  
Fluidized Bed ◽  
Ethylene Polymerization ◽  
Solid Phase ◽  
Three Dimensional ◽  
Gas Velocity ◽  
Particle Segregation ◽  
3D Numerical Simulation ◽  
Model Predictions ◽  
The Mean

Three dimensional unsteady numerical simulations of dense pressurized polydisperse fluidized bed have been carried out. The geometry is a medium-scale industrial pilot for ethylene polymerization. The numerical simulation have been performed with a polydisperse collision model. The consistency of the polydisperse model predictions with the monodisperse ones is shown. The results show that the pressure distribution and the mean vertical gas velocity are not modified by polydispersion of the solid phase. In contrast, the solid particle species are not identically distributed in the fluidized bed indicating the presence of particle segregation.

Functional specificity of jejunal brush-border pteroylpolyglutamate hydrolase in pig

1991 ◽  
Vol 260 (6) ◽  
pp. G865-G872 ◽  
Author(s):  
C. J. Chandler ◽  
D. A. Harrison ◽  
C. A. Buffington ◽  
N. A. Santiago ◽  
C. H. Halsted
Keyword(s):  
Brush Border ◽  
Protein Band ◽  
Major Protein ◽  
Functional Specificity ◽  
Major Protein Band ◽  
Regional Location ◽  
Minor Protein ◽  
A Minor ◽  
Hydrolysis Of

To determine the functional specificity of intestinal brush-border pteroylpolyglutamate hydrolase (PPH), we compared the regional location of in vivo hydrolysis of pteroyltriglutamate (PteGlu3) with the location of activity and immunoreactivity of the enzyme in the pig. After in vivo incubations, PteGlu3 hydrolytic products were recovered from intestinal segments in the jejunum but not from the ileum. Brush-border PPH activity in fractionated mucosa was 10-fold greater in the jejunum than in the ileum, whereas the activity of intracellular PPH was increased in the distal ileum. Antibodies to purified brush-border PPH identified a major protein band at 120 kDa and a minor protein band at 195 kDa in solubilized jejunal brush border. Immunohistochemistry identified the enzyme only on the brush-border surface of the jejunum, whereas an immunoblot of solubilized brush-border membranes identified brush-border PPH in the jejunum but not in the ileum. The parallel of the regional location of in vivo hydrolysis of PteGlu3 with the location of brush-border PPH activity and immunoreactivity demonstrates the functional specificity of this enzyme in folate digestion.

scholarly journals Turbulence Adjustment and Scaling in an Offshore Convective Internal Boundary Layer: A CASPER Case Study

2020 ◽  
Vol 77 (5) ◽  
pp. 1661-1681
Author(s):  
Qingfang Jiang ◽  
Qing Wang ◽  
Shouping Wang ◽  
Saša Gaberšek
Keyword(s):  
Boundary Layer ◽  
Surface Layer ◽  
Kinetic Energy ◽  
Wind Shear ◽  
Vertical Wind Shear ◽  
Internal Boundary Layer ◽  
Turbulence Kinetic Energy ◽  
Internal Boundary ◽  
Field Observations ◽  
The Mean

Abstract The characteristics of a convective internal boundary layer (CIBL) documented offshore during the East Coast phase of the Coupled Air–Sea Processes and Electromagnetic Ducting Research (CASPER-EAST) field campaign has been examined using field observations, a coupled mesoscale model (i.e., Navy’s COAMPS) simulation, and a couple of surface-layer-resolving large-eddy simulations (LESs). The Lagrangian modeling approach has been adopted with the LES domain being advected from a cool and rough land surface to a warmer and smoother sea surface by the mean offshore winds in the CIBL. The surface fluxes from the LES control run are in reasonable agreement with field observations, and the general CIBL characteristics are consistent with previous studies. According to the LESs, in the nearshore adjustment zone (i.e., fetch &lt; 8 km), the low-level winds and surface friction velocity increase rapidly, and the mean wind profile and vertical velocity skewness in the surface layer deviate substantially from the Monin–Obukhov similarity theory (MOST) scaling. Farther offshore, the nondimensional vertical wind shear and scalar gradients and higher-order moments are consistent with the MOST scaling. An elevated turbulent layer is present immediately below the CIBL top, associated with the vertical wind shear across the CIBL top inversion. Episodic shear instability events occur with a time scale between 10 and 30 min, leading to the formation of elevated maxima in turbulence kinetic energy and momentum fluxes. During these events, the turbulence kinetic energy production exceeds the dissipation, suggesting that the CIBL remains in nonequilibrium.

A solid-phase radioimmunoassay for human corticosteroid binding globulin

1985 ◽  
Vol 104 (2) ◽  
pp. 259-267 ◽  
Author(s):  
P. A. Robinson ◽  
M. S. Langley ◽  
G. L. Hammond
Keyword(s):  
Solid Phase ◽  
Binding Capacity ◽  
High Serum ◽  
Protein L ◽  
Parallel Displacement ◽  
Standard Curve ◽  
Corticosteroid Binding Globulin ◽  
Assay Tube ◽  
The Mean

ABSTRACT A radioimmunoassay (RIA) for human corticosteroid binding globulin (CBG) has been developed using 125I-labelled CBG and a monospecific solid-phase CBG-antiserum (CBG-Ab-cellulose). In an RIA of serum CBG concentrations, pure CBG standards (1–100 ng protein) or samples (1 : 200) were incubated (16 h at 20 °C) with 125I-labelled CBG and CBG-Ab-cellulose. After addition of 2 ml 0·9% NaCl, the tubes were centrifuged, supernatants were aspirated and the 125I-labelled CBG bound to the CBG-Ab-cellulose pellet was counted. The specificity of the RIA was confirmed by parallel displacement curves for serial dilutions of male, female and pregnancy sera, as well as pure CBG standards. The mean ± s.d. recovery (99±8%) of pure CBG (1·6–25·0 ng) added to a diluted serum sample verified the accuracy of the method, and a good correlation (r = 0·97; n = 43) existed between serum CBG cortisol binding capacity (nmol/l) measurements and CBG concentrations (mg protein/l) measured by RIA. Intra- and interassay precisions (C.V.) at low to high serum CBG concentrations were <5% and <9% respectively. The mean ± s.d. serum CBG concentrations (mg protein/l) measured by the RIA were: 21·8±4·6 in boys (n = 12), 20·0±4·2 in girls (n = 9), 20·7±2·7 in men (n = 6), 20·5±2·9 in women (n = 6) and 47·1 ±10·5 in pregnant women (n = 5). The sensitivity of the standard curve used in the routine RIA of serum CBG was 1·0 ng CBG/assay tube, but this could be increased to 0·2 ng/assay tube by reducing the amount of CBG-Ab-cellulose used. The RIA is suitable for both clinical and research purposes, and will aid the identification of abnormal forms of CBG and facilitate studies of the regulation of CBG production in vitro. J. Endocr. (1985) 104, 259–267

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