Application of factor VII-Sepharose affinity chromatography in the purification of human tissue factor apoprotein

Coagulation factor VII covalently coupled to Sepharose proved to be an effective binding ligand for human tissue factor apoprotein, the specific cofactor of factor VII for the activation of factor X and IX. This interaction is completely calcium-dependent and the calcium ions cannot be replaced by magnesium or barium ions. The binding of the apoprotein to immobilized factor VII seems to be independent of the presence of phospholipid. When factor VII-Sepharose column chromatography is combined with a mild extraction procedure, tissue factor apoprotein could be purified approximately 40,000-fold from an acetone powder of human brain. SDS-PAA gel electrophoresis revealed that with this simple purification scheme human tissue factor apoprotein can be purified to apparent homogeneity and that the apoprotein migrates at a molecular weight of 47,000. The isolated human protein is heterogeneously glycosylated; the two different forms of the apoprotein function as cofactor of factor VII in the activation of both factor X and factor IX.     

Angle-dependent backscatter from the arterial wall

The anisotropic nature of intra-arterial echographic images is reported, and the source of this anisotropy is investigated using postmortem human iliac arteries. A 27 MHz transducer, mounted on an ultrasonic microscope, is used to quantify the angular dependence of the backscatter power versus the angle of incidence, and these results are correlated with histological findings. Besides the observed differences in the acoustic response of morphologically different tissues, significant variations in backscatter power are found in both media and internal elastic lamina due to variations in the angle of incidence. This angle dependence is caused by the dominant orientation of fibers in tissue layers and by the shape and size various scattering particles. The results indicate that long microscopic structures with one main orientation are responsible for the backscattered signal and that the angular-dependent response is related to the histologically determined orientation of these fibers. These results may have an impact on the assessment of intra-arterial echographic images.     

Galactosylated chitosan as a synthetic extracellular matrix for hepatocytes attachment

Galactose moiety as the hepatocyte anchorage was covalently coupled with chitosan for the development of synthetic extracellular matrix. Hepatocytes adhesion to galactosylated chitosan (GC)-coated polystyrene (PS) dish became as high as 94.7% after 2 h incubation whereas the hepatocytes adhesion to chitosan-coated PS dish was 69.1%, indication of galactose-specific recognition between GC molecules and asialoglycoprotein receptors of hepatocytes. The DNA synthesis of the hepatocytes adhered to GC-coated dish was increased in the presence of epidermal growth factor (EGF) at low concentration of GC (0.05 microg/ml) whereas the DNA synthesis of the hepatocytes adhered to GC-coated dish was decreased in the presence of EGF at high concentration of GC (5 microg/ml). The spreading shapes of the hepatocytes adhered to the surface in the presence of EGF at low concentration of GC (0.05 microg/ml) were enhanced than in the absence of EGF. The hepatocytes adhered to the surface at high concentration of GC (5 microg/ml) showed round shapes and exhibited many spheroid formation after 24 h in the presence of EGF.     

The terminal protein of a linear mitochondrial plasmid is encoded in the N-terminus of the DNA polymerase gene in white-rot fungus Pleurotus ostreatus

The gene structure and expression of the linear mitochondrial plasmids of the white-rot fungus Pleurotus ostreatus, pMLP1 and pMLP2, were analyzed. Cleavage by proteinase K and exonucleases indicated that the 5′ ends of pMLP1 and pMLP2 DNAs were associated with terminal proteins. Nucleotide sequencing of the entire pMLP1 DNA revealed that it consists of 9,879 bp with terminal inverted repeat (TIR) sequences of 381 bp. The end sequence of TIR in pMLP1 is 3′-CCCCC-5′, similar to those of Escherichia coli phage PRD1. The pMLP1 plasmid harbors two long open reading frames (ORF1 and ORF2) and at least one minor ORF (mORF1). The deduced product of ORF1 is homologous to RNA polymerases of yeast mitochondria and several bacteriophages, whereas that of ORF2 is homologous to the protein-primed DNA polymerases of family B type. The mORF1 encodes a highly basic protein, most likely a TIR-binding protein, with no apparent sequence homology in the database. Expression of the predicted gene products from pMLP1 in mitochondria was demonstrated by Western blot analysis using antibodies against various expressed regions of pMLP1 ORFs. A plasmid-free strain, generated by curing with ethidium bromide, did not express any of these gene products. Terminal proteins of 70 kDa (TP1) and 73 kDa (TP2) were identified from pMLP1 and pMLP2, respectively. Western blot analysis indicated that TP1 was generated from the N-terminal half of the full-length product of ORF2 encoding a putative DNA polymerase. Received: 9 February 2000 / Accepted: 18 July 2000     

Fibrinolytic activity in peripheral atherosclerosis in the elderly

Increased concentrations of plasminogen activator inhibitor type 1 (PAI-1) and of D-dimer have jointly been found in subjects with cardiovascular disease. To understand this apparent paradox of increased inhibition of fibrinolysis (high PAI-1) combined with increased fibrinolytic activity (high D-dimer), we examined the relation between D-dimer, PAI-1 and the activator of fibrinolysis, tissue type plasminogen activator (t-PA) in subjects with varying severity of peripheral atherosclerosis. In 325 subjects selected from the Rotterdam Study, a cohort of 7983 men and women aged 55 years and over, the ankle to brachial systolic blood pressure ratio, t-PA antigen and activity, PAI-1 antigen and D-dimer were measured. T-PA antigen and t-PA activity were, independent from each other, increased with degree of atherosclerosis; t-PA antigen increased with 3.5 ng/ml (SE 1.7, p = 0.04) and t-PA activity with 0.46 IU/ml (0.20, p = 0.02) per unit decrease in ankle to brachial pressure ratio (i.e. increase in atherosclerosis). PAI-1 antigen was not related to atherosclerosis. More marked atherosclerosis was associated with increased D-dimer, mainly in subgroups with PAI-1 antigen below 50 ng/ml, t-PA antigen below 10 ng/ml, or t-PA activity above 1.5 IU/ml. In contrast to current beliefs, we found that only a fraction of the variation of t-PA antigen was due to the variation in circulating PAI-1 antigen. A slight positive association was observed between t-PA antigen and D-dimer. PAI-1 and t-PA activity were not associated with D-dimer concentration. In conclusion, in subjects with peripheral atherosclerosis PAI-1 antigen is not increased, but low PAI-1 levels (and possibly also low levels of t-PA antigen and high levels of t-PA activity) appear to be required to increase circulating D-dimer. This suggests that increased D-dimer levels in subjects with atherosclerosis do not reflect increased inhibition, but rather reflect increased fibrinolysis.     

Familial progressive vestibulocochlear dysfunction caused by a COCH mutation (DFNA9)

OBJECTIVE: To describe the decline of vestibulocochlear function in a man with vestibulocochlear dysfunction caused by a Pro51Ser mutation within the COCH gene on chromosome 14q12-13 (DFNA9). METHODS: A follow-up of more than 15 years was performed in a single case. Clinical investigations were supplemented by oculomotor, vestibular, and auditory tests. RESULTS: A 50-year-old man had had progressive sensorineural hearing loss and dysequilibrium for 15 years; he had been asymptomatic at the age of 35 years. He suffered from instability in the dark, head movement-dependent oscillopsia, paroxysmal positional vertigo, and vertigo with and without nausea. Hearing impairment started unilaterally, predominantly in the high frequencies. He also reported tinnitus. Disease progressed to severe bilateral high-frequency hearing impairment and vestibular areflexia. Fluctuation of vestibulocochlear function was documented and mentioned by the patient. CONCLUSIONS: Our patient proved to suffer from an autosomal dominant vestibulocochlear disorder caused by a COCH gene mutation. The remarkable medical history has some features in common with Meniere disease; however, there are also different clinical and neurophysiological features. In the family, phenotypic variability is present.