scholarly journals Crystals of the Chlamydomonas reinhardtii cell wall: polymerization, depolymerization, and purification of glycoprotein monomers.

1986 ◽  
Vol 103 (2) ◽  
pp. 405-417 ◽  
Author(s):  
U W Goodenough ◽  
B Gebhart ◽  
R P Mecham ◽  
J E Heuser
Keyword(s):  
Cell Wall ◽  
Chlamydomonas Reinhardtii ◽  
Sodium Perchlorate ◽  
Living Cells ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Apparent Homogeneity ◽  
Transmission Electron

Two of the three major outer layers of the Chlamydomonas reinhardtii cell wall (W6 and W4) can be solubilized from living cells with sodium perchlorate or other chaotropes and will repolymerize in vitro to form milligram amounts of wall crystals. Conditions for optimal crystalization are presented, and conditions that fail to induce polymerization are exploited to maintain monomers in aqueous solution for ion-exchange chromatography. The four major glycoproteins of the complex (GP1, 1.5, 2, and 3) have in this way been purified to apparent homogeneity and have been characterized morphologically by transmission electron microscopy using the quick-freeze, deep-etch technique and by amino acid composition. Three of the four are hydroxyproline-rich species that copolymerize to form the W6 layer. The fourth (GP1.5) is a glycine-rich species that binds to the interior of the in vitro crystal; it is apparently equivalent to the granules within the W4 layer in situ.

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

Purification and characterization of prostate-specific antigen (PSA) complexed to alpha 1-antichymotrypsin: potential reference material for international standardization of PSA immunoassays

1995 ◽  
Vol 41 (9) ◽  
pp. 1273-1282 ◽  
Author(s):  
Z Chen ◽  
A Prestigiacomo ◽  
T A Stamey
Keyword(s):  
Molecular Mass ◽  
Prostate Specific Antigen ◽  
Specific Antigen ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Molar Ratio ◽  
Free Psa ◽  
Apparent Homogeneity ◽  
International Standardization

Abstract We describe for the first time a protocol to purify to apparent homogeneity an in vitro-prepared complex of prostate-specific antigen (PSA) and alpha 1-antichymotrypsin (ACT) by using a combination of gel filtration and ion-exchange chromatography. The purity of the PSA-ACT complex was confirmed by gel electrophoresis and Western blot. The PSA-ACT complex was stable in the pH range 6.0 to 7.8; it was also stable in various matrices, temperatures, and high concentrations of salt. Purification of the PSA-ACT complex was highly reproducible. An absorptivity of 0.99 L x g-1 x cm-1 at 280 nm was assigned to the PSA-ACT complex, based on amino acid analysis. Because PSA and ACT bind in a 1:1 molar ratio, we determined the molecular mass of the PSA-ACT complex as the mass encoded by the cDNA of ACT (plus 26% carbohydrate) plus the molecular mass of PSA (28,430 Da), which totals 89,280 Da. Using this material, we made two common calibrators, one of 100% PSA-ACT complex and one of 90% PSA-ACT complex plus 10% free PSA by volume (90:10 calibrator). Substitution of these calibrators for the manufacturers' calibrators in nine commercial immunoassays substantially reduced differences between immunoassays, especially for serum PSA values between 4 and 10 micrograms/L. The 90:10 calibrator is recommended as a universal calibrator for international standardization of PSA immunoassays.

False-negative results for urinary phenothiazines and imipramine in Forrest's qualitative assays.

1980 ◽  
Vol 26 (2) ◽  
pp. 345-347 ◽  
Author(s):  
G P James ◽  
M H DJang ◽  
H H Hamilton
Keyword(s):  
Ascorbic Acid ◽  
Ion Exchange ◽  
Exchange Resin ◽  
Anion Exchange Resin ◽  
False Negative ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Negative Results ◽  
False Negative Results

Abstract When a series of patients' urine samples supplemented in vitro with chlorpromazine or imipramine was assayed with the Forrest qualitative assays, we observed an occasional false-negative result, which we found was attributable to interference by ascorbic acid. It interferes with the reagent, not with the analytes, in both assays. We easily eliminated this interference with the phenothiazine test by using an anion-exchange resin. Eliminating the interference with the assay for imipramine, however, is more difficult; false-negative results can be obtained even after ion-exchange chromatography if the imipramine concentration is less than 50 mg/L.

Isolation of a spermatozoal immobilization factor from Staphylococcus aureus filtrates

2009 ◽  
Vol 55 (7) ◽  
pp. 874-878 ◽  
Author(s):  
Vijay Prabha ◽  
Tanushree Gupta ◽  
Siftjit Kaur ◽  
Navchetan Kaur ◽  
Sushila Kala ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Gel Permeation Chromatography ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Human Spermatozoa ◽  
Infertile Woman ◽  
Ammonium Sulfate Precipitation ◽  
Tail Region ◽  
Sperm Immobilization

Staphylococcus aureus isolated from the cervix of an infertile woman was found to cause complete immobilization of human spermatozoa in vitro. Only the cell culture and cell-free supernatant showed immobilization activity, indicating that the sperm immobilization factor might be released extracellularly by the organism because no activity was observed with the washed cells. Heat treatment of the supernatant at 60 °C for 10 min waived its immobilizing activity, indicating that the active component may be a protein. The bioactive molecule from the supernatant was purified to homogeneity by ammonium sulfate precipitation, gel permeation chromatography, and ion exchange chromatography. Sperm immobilization factor (SIF) was found to be an ~20 kDa protein. SIF at a concentration of 10 µg/mL was required to cause 100% immobilization of human spermatozoa after 30 min of incubation at 37 °C, whereas a concentration of 150 µg/mL caused immediate immobilization, and a concentration of 200 µg/mL resulted in instant loss of viability of human spermatozoa, observed by eosin–nigrosin staining. Scanning electron microscopy showed that the treatment of human spermatozoa with SIF caused multiple defects in the head, midpiece, neck, and tail region of human spermatozoa.

THE UPTAKE OF TESTOSTERONE AND ZINC IN VITRO BY THE HUMAN BENIGN HYPERTROPHIC PROSTATE

1973 ◽  
Vol 58 (3) ◽  
pp. 405-419 ◽  
Author(s):  
M. JOAN REED ◽  
S. R. STITCH
Keyword(s):  
Ion Exchange ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Zinc Binding ◽  
Supernatant Fraction ◽  
Low Molecular Weight ◽  
Molecular Weight Peak ◽  
Steroid Binding

SUMMARY The uptake of 65Zn and [1,2-3H]testosterone by minced tissue of human benign hypertrophic prostates and the subcellular distribution of radioactivity were examined. The nature of steroid and 65Zn binding by the cytosol (105000 g supernatant) fraction was investigated by gel filtration, ion-exchange chromatography and electrophoresis. It was found that steroid binding after incubation at 4°C was specific. One or two regions of steroid binding were observed after gel filtration of the cytosol using Sephadex G-200, depending upon incubation conditions. Binding of 65Zn was found in the low molecular weight peak after G-200 gel filtration. Equimolar CdCl2 and 65ZnCl2 were incubated with [1,2-3H]testosterone and minced tissue and the cytosol was subjected to gel filtration. Compared with control values, without CdCl2, reduction of 65Zn binding by about 50% occurred, while binding of 3H-labelled steroid was unaffected. Electrophoresis and ion-exchange chromatography showed that 65Zn and 3H-labelled steroid were bound to different proteins. A sample of the zinc-binding protein was prepared by ion-exchange chromatography and the homogeneity was checked by electrophoresis.

In situ degradability of organic matter, crude protein and cell wall of various tree forages

1992 ◽  
Vol 55 (1) ◽  
pp. 29-34 ◽  
Author(s):  
P. Kamatali ◽  
E. Teller ◽  
M. Vanbelle ◽  
G. Collignon ◽  
M. Foulon
Keyword(s):  
Cell Wall ◽  
Organic Matter ◽  
Leucaena Leucocephala ◽  
Crude Protein ◽  
In Vitro Digestibility ◽  
Sesbania Sesban ◽  
Holstein Friesian ◽  
Ruminal Degradation

AbstractLeaves of Leucaena leucocephala, Sesbania sesban and Calliandra callothyrsus were harvested in Rwanda from young shoots at 8 weeks after the first cutting. They were dried, ground and incubated at the same time in polyester bags in three non-lactating Holstein-Friesian cows each fitted with a ruminal cannula. The bags were removed at 0, 2, 4, 8, 24, 48, 72 and 144 h after the start of incubations. The different parameters characterizing extent and rate of ruminal degradation of organic matter (OM), neutral-detergent fibre (NDF), and crude protein (CP) were calculated. In vitro digestibility of residual protein after 24 h and 48 h incubation was also determined. Sesbania sesban had lowest cell wall contents and gave highest ruminal degradability for OM, NDF and CP. Leucaena leucocephala was degraded to a lesser extent, but its undegraded protein had a somewhat higher in vitro digestibility. In contrast, protein of Calliandra callothyrsus was poorly degraded and digested. The proportion and composition of cell wall could not explain these differences in digestion characteristics and other measurements, such as tannins, were incriminated. Increased ruminal incubation time augmented the extent of ruminal degradation and reduced in vitro digestibility of undegraded protein but did not affect the undigestible protein fraction.

scholarly journals Human β-Defensin 3 Inhibits Cell Wall Biosynthesis in Staphylococci

2010 ◽  
Vol 78 (6) ◽  
pp. 2793-2800 ◽  
Author(s):  
Vera Sass ◽  
Tanja Schneider ◽  
Miriam Wilmes ◽  
Christian Körner ◽  
Alessandro Tossi ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Wall ◽  
Host Defense ◽  
Transcriptional Response ◽  
Cell Wall Biosynthesis ◽  
Host Defense Peptides ◽  
Lipid Ii ◽  
Transmission Electron ◽  
Host Defense Peptide

ABSTRACT Human β-defensin 3 (hBD3) is a highly charged (+11) cationic host defense peptide, produced by epithelial cells and neutrophils. hBD3 retains antimicrobial activity against a broad range of pathogens, including multiresistant Staphylococcus aureus, even under high-salt conditions. Whereas antimicrobial host defense peptides are assumed to act by permeabilizing cell membranes, the transcriptional response pattern of hBD3-treated staphylococcal cells resembled that of vancomycin-treated cells (V. Sass, U. Pag, A. Tossi, G. Bierbaum, and H. G. Sahl, Int. J. Med. Microbiol. 298:619-633, 2008) and suggested that inhibition of cell wall biosynthesis is a major component of the killing process. hBD3-treated cells, inspected by transmission electron microscopy, showed localized protrusions of cytoplasmic contents, and analysis of the intracellular pool of nucleotide-activated cell wall precursors demonstrated accumulation of the final soluble precursor, UDP-MurNAc-pentapeptide. Accumulation is typically induced by antibiotics that inhibit membrane-bound steps of cell wall biosynthesis and also demonstrates that hBD3 does not impair the biosynthetic capacity of cells and does not cause gross leakage of small cytoplasmic compounds. In in vitro assays of individual membrane-associated cell wall biosynthesis reactions (MraY, MurG, FemX, and penicillin-binding protein 2 [PBP2]), hBD3 inhibited those enzymes which use the bactoprenol-bound cell wall building block lipid II as a substrate; quantitative analysis suggested that hBD3 may stoichiometrically bind to lipid II. We report that binding of hBD3 to defined, lipid II-rich sites of cell wall biosynthesis may lead to perturbation of the biosynthesis machinery, resulting in localized lesions in the cell wall as demonstrated by electron microscopy. The lesions may then allow for osmotic rupture of cells when defensins are tested under low-salt conditions.

scholarly journals Beta 2-glycoprotein I is a requirement for anticardiolipin antibodies binding to activated platelets: differences with lupus anticoagulants

Blood ◽  
1993 ◽  
Vol 81 (5) ◽  
pp. 1255-1262 ◽  
Author(s):  
W Shi ◽  
BH Chong ◽  
CN Chesterman
Keyword(s):  
Cross Reactivity ◽  
Anticardiolipin Antibodies ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Glycoprotein I ◽  
Activated Platelets ◽  
Major Interest ◽  
Thrombin Activation ◽  
Beta 2

Abstract Antiphospholipid (aPL) antibodies are of major interest not only because the lupus anticoagulant (LA) causes an inhibition of in vitro blood coagulation, but also because the presence of aPL antibodies confers a risk of thrombosis. The inhibition of in vitro phospholipid- dependent coagulation (LA) is thought to be caused by the binding of LA to procoagulant phospholipid surfaces, thus impeding the clotting process. Another class of aPL antibodies are those originally described to be directed against negatively charged phospholipids, in particular cardiolipin (ACA). ACA are usually directed against a complex antigen consisting of negatively charged phospholipid and a plasma protein, beta 2-glycoprotein I (beta 2-GPI). Further, there is antibody heterogeneity even within individual patients so that ACA and LA are separable using physicochemical techniques such as ion exchange chromatography and chromatofocusing. Using such techniques we have enriched Ig fractions for LA and ACA from two patient plasmas. The majority of Ig with LA activity had a pI of 7.2 to 7.3 whereas ACA had a pI of 5.0 to 5.2. Using these enriched fractions labeled with [125I]- iodine we have shown that LA binds to platelets in a specific and saturable manner. Binding is dependent on thrombin activation. [125I]- ACA behaves differently. Like LA, binding is specific and dependent on thrombin activation but in this case requires the presence of beta 2- GPI. ACA, in the presence of beta 2-GPI, competes for binding with LA suggesting the same or contiguous site. There is no cross-reactivity of these antibodies with GPIIb/IIIa and the most likely binding site is phospholipid. In neither case does LA nor ACA have an effect on thrombin-induced release of serotonin or beta-thromboglobulin nor do they affect platelet aggregation induced by a number of agonists. This antibody binding may play an etiological role in thrombocytopenia associated with aPL, but does not explain thrombosis on the basis of hyperaggregability or increased platelet release.

scholarly journals Adhesion of Hydroxyapatite Nanoparticles to Dental Materials under Oral Conditions

Scanning ◽  
2020 ◽  
Vol 2020 ◽  
pp. 1-12 ◽  
Author(s):  
Cíntia Mirela Guimarães Nobre ◽  
Norbert Pütz ◽  
Matthias Hannig
Keyword(s):  
Electron Microscopy ◽  
Dental Materials ◽  
Hydroxyapatite Nanoparticles ◽  
Pellicle Formation ◽  
Transmission Electron ◽  
Different Shapes ◽  
Oral Conditions ◽  
Dental Applications

Hydroxyapatite nanoparticles (nano-HAP) are receiving considerable attention for dental applications, and their adhesion to enamel is well established. However, there are no reports concerning the effects of HAP on other dental materials, and most of the studies in this field are based on in vitro designs, neglecting the salivary pellicle-apatite interactions. Thus, this in situ pilot study aims to evaluate the effects of three hydroxyapatite-based solutions and their interactions with different dental material surfaces under oral conditions. Hence, two volunteers carried intraoral splints with mounted samples from enamel and from three dental materials: titanium, ceramics, and polymethyl-methacrylate (PMMA). Three HAP watery solutions (5%) were prepared with different shapes and sizes of nano-HAP (HAP I, HAP II, HAP III). After 3 min of pellicle formation, 10 ml rinse was performed during 30 sec. Rinsing with water served as control. Samples were accessed immediately after rinsing, 30 min and 2 h after rinsing. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to characterize the particles, and SEM evaluated the pellicle-HAP interactions. SEM and TEM results showed a high variation in the size range of the particles applied. A heterogeneous HAP layer was present after 2 h on enamel, titanium, ceramics, and PMMA surfaces under oral conditions. Bridge-like structures were visible between the nano-HAP and the pellicle formed on enamel, titanium, and PMMA surfaces. In conclusion, nano-HAP can adhere not only to enamel but also to artificial dental surfaces under oral conditions. The experiment showed that the acquired pellicle act as a bridge between the nano-HAP and the materials’ surface.

scholarly journals Measurement and analysis of sarcomere length in rat cardiomyocytes in situ and in vitro

2010 ◽  
Vol 298 (5) ◽  
pp. H1616-H1625 ◽  
Author(s):  
G. Bub ◽  
P. Camelliti ◽  
C. Bollensdorff ◽  
D. J. Stuckey ◽  
G. Picton ◽  
...  
Keyword(s):  
Measurement Errors ◽  
Sarcomere Length ◽  
Ex Vivo ◽  
Living Cells ◽  
Perfused Heart ◽  
Experimental Conditions ◽  
Rat Cardiomyocytes ◽  
Two Photon

Sarcomere length (SL) is an important determinant and indicator of cardiac mechanical function; however, techniques for measuring SL in living, intact tissue are limited. Here, we present a technique that uses two-photon microscopy to directly image striations of living cells in cardioplegic conditions, both in situ (Langendorff-perfused rat hearts and ventricular tissue slices, stained with the fluorescent marker di-4-ANEPPS) and in vitro (acutely isolated rat ventricular myocytes). Software was developed to extract SL from two-photon fluorescence image sets while accounting for measurement errors associated with motion artifact in raster-scanned images and uncertainty of the cell angle relative to the imaging plane. Monte-Carlo simulations were used to guide analysis of SL measurements by determining error bounds as a function of measurement path length. The mode of the distribution of SL measurements in resting Langendorff-perfused heart is 1.95 μm ( n = 167 measurements from N = 11 hearts) after correction for tissue orientation, which was significantly greater than that in isolated cells (1.71 μm, n = 346, N = 9 isolations) or ventricular slice preparations (1.79 μm, n = 79, N = 3 hearts) under our experimental conditions. Furthermore, we find that edema in arrested Langendorff-perfused heart is associated with a mean SL increase; this occurs as a function of time ex vivo and correlates with tissue volume changes determined by magnetic resonance imaging. Our results highlight that the proposed method can be used to monitor SL in living cells and that different experimental models from the same species may display significantly different SL values under otherwise comparable conditions, which has implications for experiment design, as well as comparison and interpretation of data.

Sign in / Sign up

Export Citation Format

Share Document