Effect of plasmid size on transformation efficiency by electroporation of Escherichia coli DH5 alpha.
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Ampicillin Resistance↗
Biomedical subjects
D Meyer
Publications and source records attributed to D Meyer.
Comparison of functional assays for protein S: European collaborative study of patients with congenital and acquired deficiency.
Four functional assays for protein S were evaluated by 4 different laboratories, each center using its own method. The aim of this study was to compare these different assays and to establish a relationship with results of immunological assays of total and free protein S antigen and C4bBP. The same plasma samples were distributed to each center and tested in blind. In 47 normal subjects, there was no significant difference between the 4 functional assays, with mean values ranging from 93 to 100%. These values were in good agreement with those of free and total protein S antigen. In 34 patients with a quantitative congenital deficiency of protein S the mean values of protein S activity were decreased with the 4 assays, ranging from 25 to 40%. Free protein S antigen was reduced to a similar extent, whereas total antigen was either normal or decreased. The correlation of protein S activity with free protein S antigen was satisfactory for 3 methods, with coefficients of correlation varying from 0.84 to 0.92 whereas it was only 0.70 in one lab. When total protein S antigen was reduced, protein S activity was decreased in all the patients with the 4 assays. In contrast when total protein S antigen was normal an important overlap of protein S activity between normals and patients was observed in one lab with 12 patients misclassified. In 8 patients with a functional defect, results of protein S activity differed substantially according to the assay used and about half of these patients were misclassified.(ABSTRACT TRUNCATED AT 250 WORDS)
Administration, Oral↗
Solid-phase von Willebrand factor contains a conformationally active RGD motif that mediates endothelial cell adhesion through the alpha v beta 3 receptor.
The interaction of von Willebrand factor (vWF) with the alpha v beta 3 integrin of human umbilical vein endothelial cells is dependent on the RGD sequence present at residues 1744-1746 of the mature vWF subunit. We compared vWF and its two dimeric fragments, SpIII (residues 1-1365) and SpII (residues 1366-2050), as adhesion substrates. Solid-phase vWF and SpII supported endothelial cell adhesion, whereas SpIII, which contains the glycoprotein (GP) Ib binding domain, did not. Soluble SpII inhibited adhesion to immobilized ligands, whereas soluble vWF did not, suggesting that exposure of the cell attachment domain involves a conformational modification of vWF. Dendroaspin and albolabrin, two RGD-containing peptides of the disintegrin family, were potent inhibitors of cell adhesion to vWF (IC50 approximately 15 nmol/L). Complete inhibition of endothelial cell adhesion to vWF was obtained in the presence of F(ab')2 of monoclonal antibody 9 to vWF, which blocks vWF binding to platelet GPIIb/IIIa. In contrast, monoclonal antibody 713 to vWF, which blocks its binding to platelet GPIb, did not inhibit cell adhesion to vWF. These results indicate that endothelial cell adhesion to vWF is mediated by an RGD-dependent interaction with alpha v beta 3, but does not seem to involve a GPIb-like receptor, and show the importance of the conformation of the RGD sequence.
Amino Acid Sequence↗
Functional analysis of the Arg91Gln substitution in the factor VIII binding domain of von Willebrand factor demonstrates variable phenotypic expression.
An Arg91Gln substitution in the mature von Willebrand factor (vWF) has been associated with defective binding of vWF to factor VIII (FVIII). We studied four families with members initially classified as having type I von Willebrand disease (vWD) who were either homozygous or heterozygous for the Arg91Gln change. The first family was the original case described by Nishino et al. (1) where three members were homozygous for the Gln91 allele. They had a low FVIII coagulant activity:vWF antigen (VIIIC:vWFAg) ratio, from 0.29 to 0.44, and the ability of their plasma vWF to bind FVIII was markedly decreased. All the heterozygous members had normal vWF and FVIII levels but the capacity of their plasma vWF to bind FVIII was reduced and intermediate between the homozygous members and normals. The affected individual from the second family was heterozygous for the Gln91 allele and demonstrated a VIIIC:vWFAg ratio of 0.98. The FVIII binding assay confirmed the heterozygous status indicating that the moderately low levels of vWF were due to reduced expression of both alleles. The propositus from the third family was also heterozygous and had below normal levels of vWF as well as a low VIIIC:vWFAg ratio of 0.34; however, FVIII binding to her plasma vWF was similar to that of the homozygous individuals suggesting that Gln91-vWF was the major circulating form. Her daughter who has type I vWD inherited the allele without the Gln91 mutation indicating that the expression of this allele was indeed impaired. The heterozygous patient in the fourth family had a vWF level of 24 U/dl but an VIIIC:vWFAg ratio greater than 2.(ABSTRACT TRUNCATED AT 250 WORDS)
Alleles↗
Activation and substrate specificity of the human protein kinase C alpha and zeta isoenzymes.
Protein kinase C (PKC), a class of serine/threonine kinases activated by Ca2+ and/or phospholipids, is involved in a variety of cellular processes such as proliferation, differentiation and secretion. Nine members of the PKC gene family are known; these are differentially expressed in eukaryotic cells and can be divided into two sub-groups: the Ca(2+)-dependent (classical) PKC isoenzymes alpha, beta I, beta II and gamma, and the Ca(2+)-independent neoPKC isoenzymes delta, epsilon, zeta, eta and theta. A detailed biochemical characterisation of these PKC isoenzymes is one prerequisite for the elucidation of their distinct roles within cellular signal transduction. In this study, we report the cloning of a human PKC-zeta cDNA, its expression in recombinant baculovirus-infected insect cells and the partial purification of the PKC-zeta isoenzyme. In comparison to highly purified human PKC alpha, a representative of the classical PKC subgroup, purified PKC zeta was characterised with respect to activator requirement, substrate specificity, proteolytic activation and sensitivity towards PKC inhibitors. In contrast to PKC alpha, PKC zeta exhibits a constitutive kinase activity which is independent of Ca2+, phosphatidylserine and diacylglycerol. Arachidonic acid alone or a combination of gamma-linolenic acid and phosphatidylserine slightly enhance PKC zeta activity. In the presence of the classical PKC activators phosphatidylserine/diacylglycerol, PKC alpha phosphorylates a PKC-alpha pseudosubstrate-derived peptide, an epidermal-growth-factor-receptor-derived peptide, histone III-S and myelin basic protein to an equal extent, whilst PKC zeta phosphorylates only the PKC-alpha-derived peptide. However, arachidonic acid greatly diminishes PKC-alpha activity towards the epidermal-growth-factor-receptor-derived peptide, histone III-S and myelin basic protein, but enhances PKC-zeta activity towards the PKC-alpha-derived peptide. These results indicate a possible modulation of substrate specificity of these two PKC isoenzymes by (the binding of) different activators (to their regulatory domains). In the case of PKC zeta, this finding is strengthened by the fact that the epidermal growth factor receptor-derived peptide, which is not a substrate for the holoenzyme, is significantly phosphorylated by a protein fragment generated by limited proteolysis and comprising only the kinase domain. Furthermore, PKC zeta, in contrast to PKC alpha, is insensitive to PKC inhibitors known to interfere either with the regulatory or the catalytic domain and cannot be activated by phorbol ester treatment of NIH 3T3 cells or insect cells, overexpressing the respective PKC isoenzyme. The potential implications of these findings on the mechanism(s) of activation and the substrate specificity of PKC zeta are discussed.
Amino Acid Sequence↗
Treatment of severe venous thrombo-embolism with intravenous Hirudin (HBW 023): an open pilot study.
Recombinant Hirudin (rH) is an anticoagulant agent with a specific antithrombin activity independent of antithrombin III. We report the results of the first open pilot study on the curative treatment of acute venous thrombo-embolism (VTE) with rH (HBW 023) in ten patients. The dose of rH tested was 0.07 mg/kg (i.v. bolus) followed by 0.05 mg kg-1 h-1 (i.v. infusion) for 5 days, without activated partial thromboplastin time (APTT) adjustment. Within the trial, no death, VTE recurrence or major bleeding was observed; lung scan pulmonary vascular obstruction improved from 44 to 37%, whereas the venographic Marder score was unchanged; APTT ratio ranged between 1.2 and 2.8. The dose of rH assessed in this study seems to be safe and efficient in the treatment of acute VTE.
Aged↗
International survey of patients with von Willebrand disease and angiodysplasia.
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Angiodysplasia↗
Primary structure of the factor VIII binding domain of human, porcine and rabbit von Willebrand factor.
von Willebrand factor (vWF) binds to Factor VIII (FVIII) and the FVIII binding domain has been localized to the amino-terminal of the vWF subunit. The DNA sequence coding for part of the vWF precursor (provWF) including the FVIII binding domain has been compared in man, pig and rabbit. The sequenced fragment corresponds to nucleotides 2416 to 2886 of the human vWF cDNA and encodes for the last 41 amino acids of the propolypeptide, the cleavage site and the first 116 amino acids of the mature vWF subunit. The homology of the three deduced amino acids sequences is remarkable: 88% between porcine and human and 87% between rabbit and human sequences. Four contiguous amino acids are lacking in the rabbit propolypeptide (-10 to -7) when compared to the human and porcine sequences. The cleavage site of the propolypeptide is conserved in the three species as well as amino acids where mutations in the human gene lead to a binding defect of vWF to FVIII. The Asn-94 N-glycosylation site is present in the human and rabbit sequences but absent in the pig sequence.
Amino Acid Sequence↗
Triplet structure of von Willebrand factor reflects proteolytic degradation of high molecular weight multimers.
High molecular weight (HMW) and low molecular weight (LMW) forms of von Willebrand factor (vWF) were isolated from normal human plasma in the presence of protease inhibitors. HMW and LMW vWF preparations were subjected to reduction of interdimeric disulfide bridges under mild reducing conditions. Following sodium dodecyl sulfate electrophoresis in 3% agarose, the vWF bands were detected by immunoblotting with a polyclonal rabbit anti-vWF antiserum as well as with two monoclonal antibodies directed against epitopes located in the NH2-terminal (MAb 418) or in the COOH-terminal (MAb 9) region of the vWF subunit. Our results suggest that the slowest migrating band of the dimeric triplet set of LMW vWF represents an asymmetric structure composed of an intact subunit to which one NH2-terminal and one COOH-terminal fragment are linked by disulfide bridges. The intermediate band of the first triplet of LMW vWF strongly reacted with MAb 9 but not with MAb 418, indicating that it represents a dimer of COOH-terminal fragments. The fastest migrating band of the same triplet is apparently a dimer of the NH2-terminal fragments because it reacted with MAb 418 but not with MAb 9. Each next higher family of triplets seems to contain one more asymmetric fragment of dimeric size. These results are compatible with a model according to which LMW forms of vWF are derived from HMW vWF by proteolytic cleavage in the circulating blood.
Chromatography, Gel↗
The role of the 5'-flanking region in the cell-specific transcription of the human von Willebrand factor gene.
Transcriptional regulation of the human von Willebrand factor (vWF) gene was investigated in calf pulmonary artery endothelial (CPAE), HeLa, COS 7 and Hep G2 cells. Various lengths of flanking sequences extending up to 2123 bp 5' of the transcription initiation site and containing 19 bp of the first exon, were linked to the bacterial chloramphenicol acetyltransferase (CAT) gene and these constructs were assayed in transient transfection assays. Sequences up to 89 bp upstream of the cap site showed transcriptional activity in all cell types. Sequences between -147 and -419 bp markedly reduced CAT activity in CPAE cells and abolished it in other cell lines. A domain from -592 to -810 bp generated low levels of expression only in CPAE cells. This transcriptional activity was repressed with constructs containing 1041 to 1240 bp upstream of the cap site. Endothelial cell-specific transcription was restored by a construct that contained 1286 bp upstream of the cap site. The additional 46 bp upstream of the negative regulatory domain were within the 5' end of an inverse human Alu-family DNA repeat. RNAase-protection assays confirmed the correct transcriptional initiation. The sequence between -89 and -420 contained at least one negative regulatory element able to repress the CAT gene expression controlled by the heterologous thymidine kinase promoter in all cell types. A construct that included the sequence from -89 to -1286 bp increased the transcriptional activity directed by the thymidine kinase promoter only in CPAE cells. These results indicate that negative and positive elements in the 5'-flanking region interact to regulate vWF gene expression.
Animals↗
Treatment of choroidal melanoma with I-125 plaque.
PURPOSES: To evaluate efficacy of I-125 episcleral plaque therapy in patients with ocular melanoma and determine survival, eventual visual acuity, and complications. METHODS AND MATERIALS: Between July 1, 1984 and January 1, 1991, 144 patients with diagnosis of ocular melanoma were treated with high activity I-125 episcleral plaques. Tumor volumes ranged from 14 to 3449 mm3. Lesion size included small (n = 15; height < 5 mm, and/or largest basal diameter of 8-16 mm) and large (n = 45; height > 8 mm, and/or largest basal diameter > 16 mm). Apical doses ranged from 74.25 to 83.66 Gy with scleral doses ranging from 41 and 160 Gy. Follow-up has ranged from 25 to 90 months (Med = 46 months). RESULTS: Ocular survival was noted in 130/144. Reasons for enucleation included progressive tumor growth (n = 4), extrascleral extension (n = 4), or blind/painful eye (n = 6), 94 patients developed complications which included cataract (n = 43), optic neuropathy (n = 12), neovascular glaucoma (n = 8) and retinopathies (n = 31). Visual acuity testing pre-episcleral plaque therapy revealed 102 patients with 20/200 vision; at last follow-up 59 patients demonstrated visual acuity testing of 20/200 or better. CONCLUSION: The use of episcleral I-125 plaque therapy allows for safe and effective therapy in patients with ocular melanoma of various size depending on location and probable visual acuity outcome. A total apical dose of 75 Gy given at 60-65 cGy/hour provides durable local control with acceptable complication rates.
Adult↗
von Willebrand factor: structure and function.
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Amino Acid Sequence↗
Nucleotide substitutions at the -6 position in the promoter region of the factor IX gene result in different severity of hemophilia B Leyden: consequences for genetic counseling.
Mutations in the promoter region of the factor IX gene result in hemophilia B Leyden, which is characterized by considerable improvement in the disease after puberty. We have found that distinct nucleotide substitutions at the -6 position in the Leyden-specific (LS) region are associated with a different severity of hemophilia B. The proband (aged 2) from one family is a severe hemophiliac with factor IX activity (F.IXC) and antigen (F.IXAg) levels less than 1.0 U/dl. F.IXC and F.IXAg levels in two affected uncles are approximately 30% of normal levels. The LS region was targeted for analysis because the phenotypes suggested the inheritance of a factor IX Leyden gene. An abnormal TaqI digestion pattern was found in amplified DNA from the proband, and sequencing showed a G(-6) to C transversion that was linked to the disease in the family. In another family, two brothers (aged 8 and 9) suffer from mild hemophilia with F.IXC ranging from 7 to 10 U/dl and F.IXAg from 3 to 4 U/dl. They are the only documented members of the family with a bleeding tendency. Denaturing gradient gel electrophoresis on amplified fragments from one of the patient's genomic DNA corresponding to the 8 exons and flanking sequences of the factor IX gene suggested a defect only in a segment from the 5' region. This segment showed an altered TaqI digestion pattern, and sequencing demonstrated a G(-6) to A transition that was traced to the patient's mother and a grandmother. The different phenotypes associated with the G(-6) to A purine nucleotide transition compared with a G(-6) to C transversion provide evidence that this area is directly involved in the regulation of the human factor IX gene expression in vivo by binding of regulatory factors. The ability to predict that the conditions of a hemophilia B patient will improve with age has important implications for genetic counseling of the family. Therefore, the LS region should always be included when scanning the factor IX gene for mutations.
Adult↗
The c-ets-1 proto-oncogenes in Xenopus laevis: expression during oogenesis and embryogenesis.
We previously reported the cloning and sequencing of two cDNAs derived from the Xenopus laevis ets-1 gene (Stiegler et al., 1990). The Xl-ets-1a cDNA encodes a polypeptide highly homologous to known ets-1 proteins. The 3'-UTR contains two AATAAA polyadenylation signals together with three copies of the TTTTTAT sequence thought to confer a maturation-specific polyadenylation and implicated in the deadenylation of dormant mRNAs. Several transcripts with maternal characteristics were detected in oogenesis and early embryogenesis. A marked augmentation of the major transcript in the poly(A)+ fraction was detected at fertilization. Ets-1 transcripts were observed at constant levels during the cleavage stages but decreased abruptly at gastrulation, to reappear from neurulation to late embryogenesis. The possible contribution of 3'-UTR sequence elements to this behavior is discussed.
Adenosine↗
Xl-fli, the Xenopus homologue of the fli-1 gene, is expressed during embryogenesis in a restricted pattern evocative of neural crest cell distribution.
The Xenopus laevis fli cDNA, belonging to the ets family of transcription factors, was isolated from a library prepared from unfertilized eggs. It encodes a polypeptide with extensive homology to murine and human Fli proteins. The long 3'-untranslated region contains five nuclear polyadenylation signals and three cytoplasmic polyadenylation elements, as well as many A/T rich elements. Two polyadenylated transcripts appear at the early neurula and accumulate up to the tadpole stage. In situ hybridization reveals an expression in territories invaded by neural crest cells. In the head region, fli is expressed in the peri-ocular zone, in the branchial buds and at the level of the brain floor. In the trunk, a metamerized expression is detected in the dorsum. At a lower level, the tailbud and the peri-cardiac region also appear positive.
Animals↗
The response of rat liver perisinusoidal lipocytes to polypeptide growth regulator changes with their transdifferentiation into myofibroblast-like cells in culture.
During culture on uncoated plastic wells rat liver perisinusoidal lipocytes change their differentiated phenotype (transdifferentiate) within 1-2 weeks and obtain a myofibroblast-like phenotype (myofibroblast-like cells). This transdifferentiation was documented by morphologic (loss of fat droplets, flat cell shape, cytoplasmic extensions, expression of iso-alpha smooth muscle actin) and biochemical criteria (loss of retinyl-palmitate, enhanced matrix synthesis). Whereas transforming growth factor alpha (TGF alpha) stimulated and transforming growth factor beta (TGF beta 1) inhibited the proliferation of perisinusoidal lipocytes (early culture) these cytokines did not effect the growth of the myofibroblast-like cells. Opposite effects were obtained with platelet-derived growth factor (PDGF) which stimulated the growth of myofibroblast-like cells only. Insulin-like growth factor (IGF1) was mitogenic in both perisinusoidal lipocytes and myofibroblast-like cells, respectively. Furthermore, whereas the expression of the mRNAs of decorin and biglycan was stimulated by TGF beta 1 in perisinusoidal lipocytes, the synthesis of these mRNAs was stimulated in myofibroblast-like cells predominantly by TGF alpha. Similar effects of TGF alpha and TGF beta 1 have been observed on the glycosaminoglycan-([35S]sulfate incorporation) and proteoglycan level ([3H]leucin incorporation into decorin and biglycan). Neither IGF1 and PDGF stimulated glycosaminoglycan synthesis in perisinusoidal lipocytes or in myofibroblast-like cells. The results demonstrate that the effects of the polypeptide growth regulators TGF alpha, TGF beta 1 and PDGF depend on the cell phenotype (stage of cell activation/transdifferentiation) and may be completely different in perisinusoidal lipocytes and its transformed counterpart the myofibroblast-like cells.
Adipose Tissue↗
Small incision trabeculotomy combined with phacoemulsification and intraocular lens implantation.
Cataract surgeons frequently face the challenge of treating an open-angle glaucoma patient presenting for cataract surgery and intraocular lens implantation. It seems logical to treat both problems during the same surgical procedure if it can be done without jeopardizing the results of either. We present a short historic overview of the trabeculotomy procedure in adults, together with our technique using the new Fukasaku modification of the instrumentation (probes and forceps) and report 50 consecutive cases with a 100% three-month follow-up. The mean preoperative intraocular pressure was 19.3 mm Hg (range 16.0 to 37.0 mm Hg); three months after surgery it was 14.2 mm Hg (range 6.0 to 24.0 mm Hg). The mean preoperative pressure-reducing medications were 2.3 drugs per patient, and the postoperative mean was 0.7. Except for three cases of small postoperative hyphemas, no other complications were encountered during the study.
Cataract Extraction↗
Red cell collection by apheresis technology.
To determine the feasibility of collecting 2 units (450 mL) of red cells per donation by apheresis technology, apheresis red cell collections were compared to whole-blood donations. Forty blood donors were equally divided between the two study arms on the basis of gender and iron supplementation (650 mg ferrous gluconate/day vs. no supplementation). During the 1-year study period, the apheresis participants donated 450 mL of red cells three times, and the whole-blood donors gave 225 mL of red cells (1 unit of blood) on six occasions. There were no reported side effects during the 102 whole-blood donations, whereas symptoms were noted in 83 percent of the 59 apheresis procedures. The most common symptoms were numbness and tingling, which were relieved by a decrease in the plasma-return rate or by the administration of oral calcium supplements. Seven donors dropped out or were deferred during the study. Two whole-blood donors left with medical problems unrelated to the study, one apheresis donor and one whole-blood donor dropped out of the study because of excessive fatigue, and three non-iron-supplemented whole-blood donors had unacceptably low hematocrit levels. By the end of the study, 70 percent of the apheresis donors considered the procedure acceptable, 15 percent were undecided, and 15 percent thought it was not acceptable. As measures of iron balance, the serum ferritin and the red cell zinc protoporphyrin:heme ratios were significantly more abnormal in the non-iron-supplemented donors than in the iron-supplemented donors. However, there were no differences in iron balance according to the donation method.
Adolescent↗