[30] Purification of soluble N-ethylmaleimide-sensitive fusion attachment proteins from bovine brain microsomes

SummaryWe compared factor VII clotting activity (FVIIc) assays using different thromboplastins to determine which is the most sensitive for activated FVII (FVIIa) or for FVII antigen (FVIIag). FVIIc levels were measured using thromboplastins derived from bovine brain (FVIIc Bov), human placenta (FVIIc Hum), and rabbit brain (FVIIc Rab). FVIIa levels were measured by fluorogenic assays using human soluble tissue factor (rsTF) or bovine rsTF. We also measured FVII activity by an amidolytic assay (FVIIc:am Hum) using human thromboplastin and a chromogenic substrate for thrombin. FVIIag levels were determined by ELISA. In the FVIIa assay, the reaction time obtained from using bovine rsTF was shorter than that with human rsTF, suggesting that the interaction of plasma FVIIa with bovine rsTF was stronger than with human rsTF. The plasma FVIIa levels measured using human rsTF and bovine rsTF were almost the same (r=0.947, p<0.0001). Among the three FVIIc assays, FVIIc Bov had the strongest positive correlation with the plasma FVIIa level (r=0.886, p<0.000l), but had no correlation with FVIIag. An increase of 1 ng/ml in the plasma FVIIa level yielded a 27.9% increase of FVIIc Bov. Plasma FVIIc Hum and FVIIc:am Hum showed moderate correlations with both FVIIa (r=0.520, p<0.02 and r=0.569, p<0.01, respectively) and FVIIag (r=0.438, p<0.05 and r=0.468, p<0.05, respectively). FVIIc Rab had the lowest correlation with FVIIa (r=0.367, p<0.1), but had a moderate correlation with FVIIag (r=0.436, p<0.05). After in vitro cold activation, FVIIc Bov levels increased the most and FVIIc:am levels showed the least change. These findings indicate that consideration of the thromboplastin used for assay is necessary when assessing the clinical significance of FVII activity as a cardiovascular risk factor.

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THE LESS-POLAR METABOLITES PRODUCED BY INCUBATION OF TESTOSTERONE-4-14C WITH RAT AND BOVINE BRAIN

Acta Endocrinologica ◽

10.1530/acta.0.0610641 ◽

1969 ◽

Vol 61 (4) ◽

pp. 641-648 ◽

Cited By ~ 15

Author(s):

Leon J. Sholiton ◽

Emile E. Werk

Keyword(s):

Thin Layer ◽

Thin Layer Chromatography ◽

Paper Chromatography ◽

Specific Activity ◽

Bovine Brain ◽

Polar Fraction ◽

End Products ◽

Polar Metabolites ◽

Layer Chromatography ◽

Constant Specific Activity

ABSTRACT Rat and bovine brain have been incubated with testosterone-4-14C under standard conditions. With use of paper chromatography, the extracted metabolites were noted to fall into less-polar, iso-polar, and more polar fractions. The components of the less-polar fraction were separated by acetylation and thin-layer chromatography and the major end-products identified by recrystallization to constant specific activity or constant 3H/14C ratios. Androst-4-enedione and 5α-dihydrotestosterone were formed consistently under the conditions utilized. Trace amounts of other less-polar metabolites were noted occasionally.

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Mislocalization of Adherens Junction- Associated Proteins in a Patient with Darier Disease

SKIN The Journal of Cutaneous Medicine ◽

10.25251/skin.2.3.9 ◽

2018 ◽

Vol 2 (3) ◽

pp. 184-201

Author(s):

George D Glinos ◽

Irena Pastar ◽

Marjana Tomic-Canic ◽

Rivka C Stone

Keyword(s):

Adherens Junction ◽

Cellular Adhesion ◽

Calcium Flux ◽

Keratinocyte Differentiation ◽

Calcium Dependent ◽

Endoplasmic Reticulum Calcium ◽

Darier Disease ◽

Associated Proteins ◽

Attachment Proteins ◽

E Cadherin

Darier disease (DD) is an autosomal dominant keratinizing genodermatosis that manifests clinically with red-brown pruritic papules in a seborrheic distribution often in association with palmoplantar pits and dystrophic nail changes. It is caused by mutation in ATP2A2 which encodes a sarco/endoplasmic reticulum calcium ATPase isoform 2 (SERCA2) pump that regulates calcium flux. Consequent alteration of intracellular calcium homeostasis is thought to impair trafficking of cellular adhesion proteins and to lead to aberrant keratinocyte differentiation, contributing to the characteristic histopathologic features of acantholysis and dyskeratosis in DD, though the precise mechanisms are incompletely understood. Previous studies have identified defective localization of desmosomal attachment proteins in skin biopsies and cultured keratinocytes from DD patients, but reports of effects on adherens junction proteins (including calcium-dependent E-cadherin) are conflicting. Here we describe a case of DD presenting with characteristic clinical and histologic features in which we performed immunofluorescence staining of four adherens junction-associated proteins (E-cadherin, α-catenin, β-catenin, and vinculin). In lesional (acantholytic) DD skin, we identified loss of distinctive bright membranous staining that was present at the periphery of keratinocytes throughout the epidermis in the healthy skin of a matched donor. Perilesional (non-acantholytic) portions of DD skin partially recapitulated the normal phenotype. Our findings support a role for SERCA2 dysfunction in impaired assembly of adherens junctions, which together with defective desmosomes contribute to acantholysis in DD.

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Heat Processing of Edible Plants Grown in Korea Has Differential Effects on Their Antioxidant Capacity in Bovine Brain Homogenate

Preventive Nutrition and Food Science ◽

10.3746/jfn.2002.7.4.378 ◽

2002 ◽

Vol 7 (4) ◽

pp. 378-385 ◽

Cited By ~ 2

Author(s):

Sang-Hee Oh ◽

Dai-Eun Sok ◽

Kun-Jong Lee ◽

Mee-Ree Kim

Keyword(s):

Antioxidant Capacity ◽

Brain Homogenate ◽

Bovine Brain ◽

Heat Processing ◽

Edible Plants ◽

Differential Effects

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Identification of a Coronavirus Hemagglutinin-Esterase with a Substrate Specificity Different from Those of Influenza C Virus and Bovine Coronavirus

Journal of Virology ◽

10.1128/jvi.73.5.3737-3743.1999 ◽

1999 ◽

Vol 73 (5) ◽

pp. 3737-3743 ◽

Cited By ~ 46

Author(s):

Alfred Klausegger ◽

Birgit Strobl ◽

Gerhard Regl ◽

Alexandra Kaser ◽

Willem Luytjes ◽

...

Keyword(s):

Substrate Specificity ◽

Solid Phase ◽

Hepatitis Virus ◽

Bovine Brain ◽

Brain Extract ◽

Binding Assays ◽

Bovine Coronavirus ◽

Close Relationship ◽

Influenza C Virus ◽

Cloned Gene

ABSTRACT We have characterized the hemagglutinin-esterase (HE) of puffinosis virus (PV), a coronavirus closely related to mouse hepatitis virus (MHV). Analysis of the cloned gene revealed approximately 85% sequence identity to HE proteins of MHV and approximately 60% identity to the corresponding esterase of bovine coronavirus. The HE protein exhibited acetylesterase activity with synthetic substratesp-nitrophenyl acetate, α-naphthyl acetate, and 4-methylumbelliferyl acetate. In contrast to other viral esterases, no activity was detectable with natural substrates containing 9-O-acetylated sialic acids. Furthermore, PV esterase was unable to remove influenza C virus receptors from human erythrocytes, indicating a substrate specificity different from HEs of influenza C virus and bovine coronavirus. Solid-phase binding assays revealed that purified PV was unable to bind to sialic acid-containing glycoconjugates like bovine submaxillary mucin, mouse α1macroglobulin or bovine brain extract. Because of the close relationship to MHV, possible implications on the substrate specificity of MHV esterases are suggested.

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