COMPARATIVE PROLIFERATIVE KINETICS OF CELLS FROM NORMAL HUMAN EPIDERMIS AND BENIGN EPIDERMAL HYPERPLASIA (PSORIASIS) IN VITRO

1974 ◽  
Vol 7 (1) ◽  
pp. 69-76 ◽  
Author(s):  
D. P. Chopra ◽  
B. A. Flaxman
Keyword(s):  
Human Epidermis ◽  
Epidermal Hyperplasia ◽  
Normal Human ◽  
Kinetics Of

Cleavage of Recombinant and Plasma-Derived VWF by Human ADAMTS13

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1415-1415
Author(s):  
Hanspeter Rottensteiner ◽  
Katalin Varadi ◽  
Susanne Vejda ◽  
Hartmut J. Ehrlich ◽  
Friedrich Scheiflinger ◽  
...  
Keyword(s):  
Shear Stress ◽  
Human Plasma ◽  
Phase 1 ◽  
Dependent Manner ◽  
Normal Human ◽  
Typical Satellite ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Kinetics Of

Abstract Abstract 1415 A recombinant human CHO-expressed von Willebrand factor (rVWF) consisting of ultra-high molecular weight (UHMW) multimers resembles the VWF stored in Weibel-Palade bodies of endothelial cells. Once secreted into plasma, UHMW multimers are rapidly cleaved by ADAMTS13 and are usually missing in plasma-derived VWF (pdVWF). Here we analyzed in vitro whether the kinetics of cleavage of rVWF by ADAMTS13 is similar to that of pdVWF. The kinetics of ADAMTS13-mediated proteolysis of rVWF were explored under denaturing conditions (1.5 M urea) or under shear stress so as to expose the ADAMTS13 cleavage site of VWF. Multiple assays showed that rVWF was efficiently cleaved by ADAMTS13. UHMW multimers disappeared within seconds at physiological concentrations of ADAMTS13. Using lower concentrations of ADAMTS13 (10-30 mU/ml, equivalent to 1–3% of normal human plasma), UHMW were cleaved within 30 minutes. The typical satellite bands appeared very early in an ADAMTS13 dose-dependent manner. Virtually the same results were obtained when human plasma was used as a source for ADAMTS13. Although pdVWF differs from rVWF in its multimeric structure, the decrease in activity was similar for rVWF and pdVWF. Finally, the extent of ADAMTS13 cleavage was similar for rVWF and pdVWF when exposed to shear stress using a cone-plate viscometer. Our data clearly indicate that rVWF is a good substrate for ADAMTS13. Ongoing phase 1 studies demonstrated that rVWF is indeed processed by the protease when administered in humans with severe VWD. Disclosures: Rottensteiner: Baxter Innovations GmbH: Employment. Varadi:Baxter Innovations GmbH: Employment. Vejda:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment. Schwarz:Baxter Innovations GmbH: Employment. Turecek:Baxter Innovations GmbH: Employment.

scholarly journals Transformation ofLeishmania mexicanametacyclic promastigotes to amastigote-like forms mediated by binding of human C-reactive protein

Parasitology ◽  
2001 ◽  
Vol 122 (5) ◽  
pp. 521-529 ◽  
Author(s):  
A. BEE ◽  
F. J. CULLEY ◽  
I. S. ALKHALIFE ◽  
K. B. BODMAN-SMITH ◽  
J. G. RAYNES ◽  
...  
Keyword(s):  
Human Serum ◽  
Fluorescent Antibody ◽  
Antibody Test ◽  
Normal Serum ◽  
C Reactive Protein ◽  
Reactive Protein ◽  
Metacyclic Promastigotes ◽  
Normal Human ◽  
Kinetics Of

Infective metacyclic promastigote forms ofLeishmania mexicanaare introduced by the bite of sandfly vectors into their human hosts where they transform into the amastigote form. The kinetics of this process was examinedin vitroin response to different combinations of temperature (26 °C or 32 °C), pH (7.2 or 5.5), and exposure to human serum. Little transformation occurred at 26 °C/pH 7.2, intermediate levels at 26 °C/pH 5.5 and 32 °C/ pH 7.2, and the greatest response at 32 °C/pH 5.5. Transformation was stimulated by exposure to normal human serum, but was markedly reduced when serum previously incubated at 56 °C for 1 h was used (complement heat-inactivated). This stimulatory effect was reproduced by exposure to a single purified component of human serum, C-reactive protein (CRP). Binding of CRP to the whole surface ofL. mexicanametacyclic promastigotes, including the flagella, was demonstrated by an indirect fluorescent antibody test. The effect of purified CRP was dose dependent and occurred using normal serum concentrations. The stimulatory effect of whole serum was oblated by CRP depletion and restored by addition of purified CRP. The effects of cAMP analogues indicated that transformation could be mediated via an adenylate cyclase cascade.

In vitro reconstructed normal human epidermis expresses differentiation-related proteoglycans

2008 ◽  
Vol 51 (2) ◽  
pp. 135-138 ◽  
Author(s):  
Marek Haftek ◽  
Marie-Aude Le Bitoux ◽  
Sylvie Callejon ◽  
Alain Denis ◽  
Ingrid Pernet ◽  
...  
Keyword(s):  
Human Epidermis ◽  
Normal Human

scholarly journals Use of monospecific antisera and cRNA probes to localize the major changes in keratin expression during normal and abnormal epidermal differentiation.

1988 ◽  
Vol 107 (2) ◽  
pp. 427-446 ◽  
Author(s):  
A Stoler ◽  
R Kopan ◽  
M Duvic ◽  
E Fuchs
Keyword(s):  
Normal Skin ◽  
Terminal Differentiation ◽  
Epidermal Differentiation ◽  
Human Epidermis ◽  
Isolation And Characterization ◽  
Encoded Proteins ◽  
Normal Human ◽  
Monospecific Antisera

We report here the isolation and characterization of three antisera, each of which is specific for a single keratin from one of the three different pairs (K1/K10, K14/K5, K16/K6) that are differentially expressed in normal human epidermis and in epidermal diseases of hyperproliferation. We have used these antisera in conjunction with monospecific cRNA probes for epidermal keratin mRNAs to investigate pathways of differentiation in human epidermis and epidermal diseases in vivo and in epidermal cells cultured from normal skin and from squamous cell carcinomas in vitro. Specifically, our results suggest that: (a) the basal-specific keratin mRNAs are down-regulated upon commitment to terminal differentiation, but their encoded proteins are stable, and can be detected throughout the spinous layers; (b) the hyperproliferation-associated keratin mRNAs are expressed at a low level throughout normal epidermis when their encoded proteins are not expressed, but are synthesized at high levels in the suprabasal layers of hyperproliferating epidermis, coincident with the induced expression of the hyperproliferation-associated keratins in these cells; and (c) concomitantly with the induction of the hyperproliferation-associated keratins in the suprabasal layers of the epidermis is the down-regulation of the expression of the terminal differentiation-specific keratins. These data have important implications for our understanding of normal epidermal differentiation and the deviations from this process in the course of epidermal diseases of hyperproliferation.

Kinetics of Human VWF Cleavage by Human ADAMTS13: Comparison of Recombinant and Plasma-Derived VWF

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4092-4092
Author(s):  
Hanspeter Rottensteiner ◽  
Katalin Varadi ◽  
Susanne Vejda ◽  
Jutta Schreiner ◽  
Herbert Gritsch ◽  
...  
Keyword(s):  
Plasma Concentration ◽  
Human Plasma ◽  
Degradation Kinetics ◽  
Dependent Manner ◽  
Normal Human Plasma ◽  
Normal Plasma ◽  
Normal Human ◽  
Typical Satellite ◽  
Kinetics Of

Abstract A recombinant human CHO-expressed von Willebrand factor (rVWF) which consists of ultra-high molecular weight (UHMW) multimers resembles the VWF that is stored in Weibel-Palade bodies of endothelial cells. Once secreted into plasma, UHMW multimers are rapidly cleaved by ADAMTS13 and therefore are usually missing in VWF purified from plasma. We analyzed in vitro whether the cleavage of rVWF by ADAMTS13 is similar to the cleavage of plasma VWF by ADAMTS13, using a standard assay that depends on denaturing conditions (1.5 M urea) to expose the ADAMTS13 cleavage site of VWF. We explored the kinetics of ADAMTS13-mediated proteolysis of rVWF over time by exposing 1 VWF:Ag IU/ml of rVWF to various concentrations of recombinant and plasma-derived purified ADAMTS13, ranging from 4 mU/ml to 1 U/ml (corresponding to 0.4 to 100% of the normal human plasma concentration), and to ADAMTS13 present in normal human plasma and VWF-deficient plasma. The multimeric structure and function of VWF were analyzed by multiple assays to compare VWF before, during, and after proteolytic cleavage. In addition, the degradation kinetics of recombinant VWF were compared with those of a plasma-derived VWF product. Recombinant VWF was cleaved rapidly and with the same efficiency using recombinant or plasma-derived ADAMTS13. With 0.5 U/ml pADAMTS13 or rADAMTS13, VWF:RCo activity was below the detection limit of 0.17 IU/ml after 15 s. UHMW multimers disappeared within seconds at physiological concentrations of 0.5–1.0 U/ml ADAMTS13 (50–100% of the normal plasma concentration). Furthermore, UHMW were cleaved within 30 minutes with much lower concentrations of 10–30 mU/ml ADAMTS13 (1–3% of normal plasma concentration). The typical satellite bands appeared very early in an ADAMTS13 dose-dependent manner. Although plasma-derived VWF differs substantially from rVWF in its multimeric structure, the decrease in activity was similar for the recombinant and plasma-derived VWF. Specific cleavage of rVWF (3 IU VWF:Ag/ml) by rADAMTS13 (5 U/ml) was also demonstrated without urea under shear stress in the presence of platelets, detected by a monoclonal antibody (N10) that recognizes VWF only when cleaved by ADAMTS13. The combined data show that ADAMTS13 is able to readily cleave human rVWF even at low concentrations and that the UHMW multimeric fraction of human rVWF is removed within minutes by ADAMTS13 in vitro.

SKALP/elafin is an inducible proteinase inhibitor in human epidermal keratinocytes

1994 ◽  
Vol 107 (8) ◽  
pp. 2335-2342
Author(s):  
J.A. Alkemade ◽  
H.O. Molhuizen ◽  
M. Ponec ◽  
J.A. Kempenaar ◽  
P.L. Zeeuwen ◽  
...  
Keyword(s):  
Blot Analysis ◽  
Proteinase Inhibitor ◽  
Calf Serum ◽  
Human Epidermis ◽  
Psoriatic Skin ◽  
Epidermal Keratinocytes ◽  
Functional Assay ◽  
Normal Human

Skin-derived antileukoproteinase (SKALP), otherwise known as elafin, is a recently discovered epidermal proteinase inhibitor with specificity for polymorphonuclear leukocyte (PMN)-derived elastase and proteinase-3; in addition to the proteinase-inhibiting domain, SKALP contains several transglutaminase substrate motifs. SKALP is virtually absent in normal human epidermis but is found in a number of inflammatory skin diseases, including psoriasis. Here we report the induction and processing of SKALP in vivo and in vitro. SKALP expression in vivo could be demonstrated following injury in normal human epidermis, using histology, western blotting, northern blotting and a functional assay. In vitro, SKALP expression was studied in conventional submerged keratinocyte culture systems and in keratinocytes cultured in an air-liquid interface model. Induction of SKALP activity in epidermis could be measured as early as 16 hours after skin injury; immunohistological examination showed that SKALP expression was confined to the outer layers of the stratum spinosum and the stratum granulosum. Northern blot analysis revealed a 0.8 kb transcript, both in vivo (psoriatic skin, injured skin) and in vitro (cultured keratinocytes). Western blot analysis showed that the major SKALP form in vivo was a low molecular mass fragment, containing the antiproteinase domain. In all cultures that were positive for SKALP, larger (8-10 kDa) forms of SKALP, containing the N-terminal transglutaminase substrate motifs in addition to the antiproteinase domain, were found. SKALP expression in cultured cells was found to be dependent on the system used. In a submerged culture system, SKALP could be induced by fetal calf serum.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of pH and inhibitors on beta-amyloid fibril assembly

1996 ◽  
Vol 54 ◽  
pp. 752-753
Author(s):  
Beverly E. Maleeff ◽  
Timothy K. Hart ◽  
Stephen J. Wood ◽  
Ronald Wetzel
Keyword(s):  
Amyloid Fibril ◽  
Peptide Fragment ◽  
Hydrophobic Peptides ◽  
Amyloid Fibril Formation ◽  
Effects Of Ph ◽  
Severity Of The Disease ◽  
Kinetics Of ◽  
The Brain

Alzheimer's disease is characterized post-mortem in part by abnormal extracellular neuritic plaques found in brain tissue. There appears to be a correlation between the severity of Alzheimer's dementia in vivo and the number of plaques found in particular areas of the brain. These plaques are known to be the deposition sites of fibrils of the protein β-amyloid. It is thought that if the assembly of these plaques could be inhibited, the severity of the disease would be decreased. The peptide fragment Aβ, a precursor of the p-amyloid protein, has a 40 amino acid sequence, and has been shown to be toxic to neuronal cells in culture after an aging process of several days. This toxicity corresponds to the kinetics of in vitro amyloid fibril formation. In this study, we report the biochemical and ultrastructural effects of pH and the inhibitory agent hexadecyl-N-methylpiperidinium (HMP) bromide, one of a class of ionic micellar detergents known to be capable of solubilizing hydrophobic peptides, on the in vitro assembly of the peptide fragment Aβ.

Kinetics of Various 99mTc-Sn-Pyrophosphate Compounds in the Rat

1977 ◽  
Vol 16 (04) ◽  
pp. 157-162 ◽  
Author(s):  
C. Schümichen ◽  
B. Mackenbrock ◽  
G. Hoffmann
Keyword(s):  
Normal Saline ◽  
Half Life ◽  
Compound A ◽  
Short Half Life ◽  
Low Concentrations ◽  
The Stability ◽  
Kinetics Of ◽  
In Vitro Stability

SummaryThe bone-seeking 99mTc-Sn-pyrophosphate compound (compound A) was diluted both in vitro and in vivo and proved to be unstable both in vitro and in vivo. However, stability was much better in vivo than in vitro and thus the in vitro stability of compound A after dilution in various mediums could be followed up by a consecutive evaluation of the in vivo distribution in the rat. After dilution in neutral normal saline compound A is metastable and after a short half-life it is transformed into the other 99mTc-Sn-pyrophosphate compound A is metastable and after a short half-life in bone but in the kidneys. After dilution in normal saline of low pH and in buffering solutions the stability of compound A is increased. In human plasma compound A is relatively stable but not in plasma water. When compound B is formed in a buffering solution, uptake in the kidneys and excretion in urine is lowered and blood concentration increased.It is assumed that the association of protons to compound A will increase its stability at low concentrations while that to compound B will lead to a strong protein bond in plasma. It is concluded that compound A will not be stable in vivo because of a lack of stability in the extravascular space, and that the protein bond in plasma will be a measure of its in vivo stability.

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