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Biomedical subjects

G Falk

Publications and source records attributed to G Falk.

At least 19 recordsLinked to original sources
PubMed1999

A rise in intracellular Ca2+ underlies light adaptation in dogfish retinal 'on' bipolar cells.

1. This investigation was to determine the extent to which retinal 'on' bipolar cells contribute to the adaptive changes that occur with light, which enable the rod visual system to operate over a wide range of ambient light intensities, and to elucidate the underlying adaptive mechanism. 2. Whole-cell voltage clamp recordings were obtained from bipolar cells in dark-adapted dogfish retinal slices. Current responses to brief flashes and steps of light were analysed. 'On' bipolar cell inward current light responses are mediated by a metabotropic glutamate receptor linked to the control of a cGMP cascade, with cGMP opening cation channels. Outward current responses to light of 'off' bipolar cells are mediated by the closure of ionotropic glutamate receptor channels. 3. When Ca2+ buffer was omitted from the patch pipette solution, 'on' bipolar cells rapidly desensitized to steps of light as dim as one rhodopsin molecule bleached per rod per second (1 Rh* s-1), whereas 'off' bipolar cells did not desensitize. Responses of 'on' bipolar cells to flashes in the presence of dim backgrounds recovered after a delay, but with diminished sensitivity, i.e. the cells adapted. 4. With the Ca2+ chelator BAPTA in the patch pipette solution, step responses of 'on' bipolar cells were sustained and flash responses following steps showed rapid recovery. Buffering Ca2+ in the patch pipette solution to 1 microM prevented desensitization, whereas 50 microM free Ca2+ reduced the 'on' bipolar cell flash responses, suppressed inward dark current and decreased input conductance. 5. We conclude that a major component of adaptation of the visual system is due to a reduction in gain at the rod-'on' bipolar cell synapse as a result of Ca2+ loading of the dendrites when their cGMP-gated cation channels open with light.

Adaptation, Ocular↗
PubMed1998

Failure to achieve gene conversion with chimeric circular oligonucleotides: potentially misleading PCR artifacts observed.

Recently, a novel strategy for nucleotide exchange of target DNA using chimeric RNA/DNA oligonucleotides (CO) was reported. The CO can easily be transfected into cells, remain stable within the cells, and migrate to the nucleus. We have in this study used 42 similar constructs for targeting six different human and canine loci. A variety of cationic lipids, electroporation, and microinjection were used for transfection of the CO into lymphoblastoids, Huh7, HT 1080, and Jurkat cell lines, and canine primary fibroblasts and hepatocytes. However, no nucleotide exchange was detected in any of the targeted loci. Using PCR followed by restriction enzyme analysis, nucleotide exchange in approximately 2%-10% of the PCR products was observed during the first 3 days after transfection with CO-vWF-28S2 designed for repairing a mutation in the von Willebrand gene. Surprisingly, the observed exchange reverted after culturing the cells for a longer period of time (14 days). Furthermore, a positive indication of gene conversion (5%) was also obtained using an allele-specific PCR method for analysis of the PAI-1 gene. However, cloning of the PCR products revealed no nucleotide exchange. In our view, the most likely explanation is that the initial false positive result originates from a PCR artifact created by the CO itself. Our results imply that an independent method, that is, Southern blotting, must be used to verify an observed nucleotide exchange.

Animals↗
PubMed1997

Purification and characterisation of a plasminogen-binding protein from Haemophilus influenzae. Sequence determination reveals identity with aspartase.

Plasminogen binding proteins have been described both for Gram positive and Gram negative bacteria. In the present work we describe the purification and characterization of a plasminogen binding protein from Haemophilus influenzae (strain HI-23459). Bacteria were sonicated in order to solubilize plasminogen-binding proteins. The supernatant was subjected to affinity chromatography on plasminogen kringle-4 fragment bound to Sepharose 4B and subsequently processed by ion-exchange chromatography on DEAE-Sepharose CL-6B. Characterization of the protein by SDS-PAGE displayed a single band with a molecular mass of about 55,000, both prior to and after reduction. The purified protein stimulates tPA (tissue plasminogen activator) catalysed plasminogen activation by a factor of approximately 300, mainly due to a decrease in K(m). Antibodies were raised in rabbits and used in quantitative and qualitative analysis. However, using a FITC-conjugate we failed to demonstrate the presence of the purified protein on the surface of intact bacteria. The corresponding gene was isolated from a lambda EMBL3 phage library prepared from chromosomal DNA from the same H. influenzae strain, using an oligonucleotide probe based on the NH2-terminal amino acid sequence. An open reading frame corresponding to 472 amino acid was found. The amino acid sequence of the translated gene demonstrates 97% identity with the recently published sequence from aspartate ammonia lyase (aspartase) from H. influenzae. Enzymatic analysis of the purified protein revealed a high aspartase activity.

Amino Acid Sequence↗
PubMed1997

Search for mutations in the genes for coagulation factors V and VIII with a possible predisposition to activated protein C resistance.

A total of 74 non-pregnant women with a previous episode of thrombosis were investigated for activated protein C (APC) resistance in the aPTT-based and factor IXa-X-based assays and for the presence of mutations in all APC-cleavage sites in the heavy chains of factor V and factor VIII. DNA fragments were amplified with the polymerase chain reaction (PCR) and those encoding for the Arg-306 and Arg-506 (factor V) and for Arg-740 (factor VIII) cleavage sites were subjected to restriction enzyme analysis. DNA fragments of 29 selected patients corresponding to the Arg-306 and Arg-679 cleavage sites in factor V, and to the Arg-336 and Arg-562 cleavage sites in factor VIII were sequenced. APC resistance was found in 40 cases, using the aPTT-based assay and in 35, using the factor IXa-X-based assay (23 patients were APC resistant in both assays), whereas 22 individuals had a normal response to APC. Forty-three patients carried Arg-506 to Gln mutation in factor V. No other polymorphism or mutation was found in the genes for factors V or VIII in the vicinity of the APC cleavage sites. It was concluded that the difference in response to APC in the two assays may not be explained by the presence of mutations in the APC cleavage sites of factor V and factor VIII in this group of patients. The data do not exclude the presence of mutations elsewhere in the factor V or factor VIII genes.

Adult↗
PubMed1995

Studies on phospholipid antibodies, APC-resistance and associated mutation in the coagulation factor V gene.

The influence of antibodies against phospholipids (PLa) on APC response was investigated in 155 women with a history of thromboembolism and/or repeated foetal losses. PLa were determined as antibodies against cardiolipin (CLa) and phosphatidyl serine (PSa) and as lupus anticoagulant (LA) tested by dilute Russell's Viper Venom time and by the Textarin/Ecarin ratio. APC-response was studied by a clotting (aPTT-based) and by an amidolytic (factor IXa-X-based) assay. A reduced response to APC (APC-resistance) was found in 49% of 65 PLa-positive and in 13% of 90 PLa-negative samples (chi 2 = 23.9; p < 0.5 x 10(-4)). It was more common in the samples with LA, as compared to CLa+PSa positive (58% vs. 30%, not significant). The presence of the mutation causing Arg506-Gln substitution in coagulation factor V was investigated in 84 samples. The occurrence of the mutation in APC-resistant patients with CLa+PSa or with LA in one of the two assays was similar to those without PLa (84% and 100%, respectively). In the absence of APC resistance, the occurrence of the mutation was similar in the samples with and without PLa (14% vs. 11%). Samples with LA, determined by both tests used, comprised a special group where the frequency of the mutation in the APC resistant samples was significantly reduced (p < 0.01). In the latter samples, the pathogenic mechanism of APC resistance may be connected with the influence on phospholipid membranes.

Abortion, Habitual↗
PubMed1995

Technique and results of laparoscopic choledochotomy for the management of bile duct calculi.

Laparoscopic choledochotomy has been performed in 50 patients to remove common bile duct calculi demonstrated on routine operative cholangiography at the time of laparoscopic cholecystectomy. The patients ranged from 16 to 91 years old. One patient died, giving a mortality of 2%. At postoperative T-tube cholangiography, retained stones were demonstrated in three patients (6%) with all stones being removed using a choledochoscope via the T-tube track. Laparoscopic common bile duct exploration via a choledochotomy is a feasible and effective method to manage common bile duct calculi demonstrated during laparoscopic cholecystectomy.

Adolescent↗
PubMed1995

Thrombotic risk factors and oral contraception.

Eighty-one women with a history of thrombosis were classified into three groups: group I (n = 29), women in whom thrombosis developed during oral contraception; group II (n = 33), those who used oral contraceptives (OC) without complications but experienced vascular occlusion in other risk situations; group III (n = 19), women who never used OC. The level of antibodies to anionic phospholipids (PLa), a response to activated protein C (APC), and the presence for the mutation in the coagulation factor V gene causing APC resistance were studied. In the studied groups, APC resistance was present in 14% to 42% of patients. PLa were elevated in about half of APC-resistant patients. The incidence of APC resistance correlated with the recurrency of the thrombotic events within the groups. In most cases it was tightly connected to the mutation in the factor V gene. Women in whom thrombosis developed while they were taking OC (group I) differed from the others, having a remarkable disagreement between the lowest incidence of APC resistance and a relatively increased number of the mutation (14% vs 38%, p < 0.025). This finding suggested that the APC response is flexible. An influence of OC that predisposes a reduction in APC response is discussed.

Adolescent↗
PubMed1994

Characterization of the von Willebrand factor gene (VWF) in von Willebrand disease type III patients from 24 families of Swedish and Finnish origin.

Twenty-four patients with von Willebrand disease type III were screened for mutations in the von Willebrand factor (VWF) gene using the PCR technique, followed by direct sequencing. More than 250 kb of genomic DNA were sequenced, including the promoter and coding regions (52 exons) of the VWF gene from 24 patients. In addition to the previously reported mutations of a single cytosine deletion in exon 18 and the nonsense mutations in exons 28, 32, and 45, nine new mutations were detected: two nonsense mutations in exons 15 and 16, one allele with a thymidine insertion in exon 14, one allele with a cytosine insertion in exon 28, one 20-bp deletion in exon 15, one mutation in the donor splice site of exon 43, and three missense mutations in exons 28, 49, and 51. Forty-two mutant chromosomes were identified (42/48); 11 probands are homozygous for the mutations, and 8 are compound heterozygous. In addition, a new subfamily of the Alu sequence in the promoter region and 10 new polymorphisms were identified.

Alleles↗
PubMed1994

Behavior of different suture materials in the urinary bladder of the rabbit with special reference to wound healing, epithelization and crystallization.

Five urinary bladder incisions were performed in 16 rabbits and the defects closed by plain catgut 4-0, chromic catgut 4-0, polypropylene 4-0, polyglactin 910 4-0 and polyglactin 910 8-0 in the form of an all-layer suture, thereby obtaining a total of 80 suture sites. The running sutures in 8 animals were reviewed by light microscopy after 1, 2, 7 and 15 weeks, and in 8 additional animals after 3 days, 1, 2, 7 and 15 weeks by scanning electron microscopy. Moreover, we analyzed and compared the autopsy findings on gross inspection, the urinary sediments and urinary cultures of the individual animals. Scanning electron-microscopic evaluations showed that epithelization of the intraluminal suture portions can be accomplished after 3 days already. Reduced suture strength and rapid absorption of the suture material prevent later postoperative incrustations. Light-microscopic inspection of the segments of the bladder wall bearing this suture material suggests that a minor inflammatory tissue response around the suture material--with polyglactin 910 8-0 in particular--is more likely to enhance regeneration of the tunica muscularis and to prevent formation of cysts than seen with a long-standing highly inflammatory tissue response around the suture site of catgut threads. In the animal experiment, the polyglactin 910 suture has shown sufficient firmness and proved to be superior to any other suture material as regards the reaction to foreign bodies and inclination to incrustation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

The glutamate-receptor linked cGMP cascade of retinal on-bipolar cells is pertussis and cholera toxin-sensitive.

Whole-cell patch-clamp recordings were obtained from light-responsive on-bipolar cells in retinal slices of the dogfish. Inclusion of the A-subunit of pertussis toxin in the patch-pipette solution resulted in an increase in inward current and membrane conductance, and a block of light-evoked currents of on-bipolar cells. The opposite effect was obtained with the A-subunit of cholera toxin, which blocked light responses, and induced an outward current and a decrease in membrane conductance. These actions were NAD+ dependent. The results show that the G-protein(s) linking glutamate receptors to a cGMP cascade in on-bipolar cells possess sites which are ADP-ribosylated by pertussis and cholera toxins, with no homology to the adenylate cyclase system but possibly with a homology to transducin. Furthermore, inclusion of H-7, a kinase inhibitor in the patch-pipette solution, or of a non-hydrolysable ATP analogue (AMP-PNP) had no effect on light responses, membrane conductance or dark current of on-bipolar cells, suggesting that the components of this cGMP cascade are unlikely to be regulated by protein kinases.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

Properties of the cGMP-activated channel of retinal on-bipolar cells.

Whole-cell patch-clamp recordings were obtained from on-bipolar cells in, or isolated from, retinal slices prepared from dogfish retina. The properties of the cGMP-activated conductance of on-bipolar cells were compared with that of rod photoreceptors. The on-bipolar cell cGMP-activated channel was blocked by L-cis-diltiazem, a block which was strongly voltage dependent. However, this channel is not identical with that of photoreceptors. The location of the L-cis-diltiazem blocking site and its accessibility in the channel are not the same as in rods. The voltage dependence of block suggests that the blocking site, although near the intracellular side of the channel, is accessible to the positively charged form of L-cis-diltiazem only from the outward facing side of the channel. Furthermore, in contrast to rod channels, the conductance of the on-bipolar cell channels is unaltered by the removal of external divalent cations.

Aminobutyrates↗
PubMed1992

Nucleotide sequence of cDNA clones encoding the beta subunit of mitochondrial ATP synthase from the green alga Chlamydomonas reinhardtii: the precursor protein encoded by the cDNA contains both an N-terminal presequence and a C-terminal extension.

cDNA and genomic clones encoding the beta subunit of mitochondrial ATP synthase from Chlamydomonas reinhardtii have been isolated using heterologous DNA probes from the photosynthetic bacterium Rhodospirillum rubrum. The protein encoded by the cDNA is 79-83% identical to corresponding proteins from higher-plant and mammalian mitochondria, and 75% identical to the R. rubrum protein. It contains both an N-terminal presequence and a unique C-terminal extension. The presequence, which is the first mitochondrial presequence determined in C. reinhardtii, is similar in structure to mitochondrial presequences from other organisms. As chloroplast presequences from C. reinhardtii also share features with mitochondrial presequences from other organisms (L.-G. Franzén et al., FEBS Lett 260 (1990) 165-168), this raises interesting questions about protein targeting to chloroplasts and mitochondria in C. reinhardtii. The possibility that the C-terminal extension is involved in targeting the protein to the mitochondrion is discussed. Southern blot analysis indicates that the protein is encoded by a single-copy gene.

Amino Acid Sequence↗
PubMed1992

Retinal on-bipolar cells contain a nitric oxide-sensitive guanylate cyclase.

Retinal on-bipolar cells possess specialized glutamate receptors which are coupled via a G-protein to the control of a cyclic GMP (cGMP) cascade. Whole-cell voltage clamp recordings were obtained from light-responsive on-bipolar cells in retinal slices of the dogfish. Inclusion of nitroprusside in the patch-pipette solution induced effects in on-bipolar cells which were consistent with a rise in intracellular cGMP and thus stimulation of guanylate cyclase (GC) activity. Conversely, the soluble GC inhibitors, methylene blue and ferricyanide, induced effects consistent with a fall in intracellular cGMP. Activators of particulate GC had no effect. We conclude that cGMP synthesis in on-bipolar cells is catalysed by a NO-sensitive cyclase.

Animals↗
PubMed1992

Nonsense mutations of the von Willebrand factor gene in patients with von Willebrand disease type III and type I.

von Willebrand disease (vWD) is the most common inherited bleeding disorder in humans. The disease is caused by qualitative and quantitative abnormalities of the von Willebrand factor (vWF). Genomic DNA from 25 patients with vWD type III, the most severe form of the disease, was studied using PCR followed by restriction-enzyme analysis and direct sequencing of the products. Nonsense mutations (CGA----TGA) were detected in exons 28, 32, and 45 by screening of all the 11 CGA arginine codons of the vWF gene. Two patients were found to be homozygous and five heterozygous for the mutation. Both parents and some of the relatives of the homozygous patients carry the mutation. These are the first reported examples of homozygous point mutations associated with the severe form of vWD. In the three heterozygous probands, one of the parents carried the mutation and had vWD type I. Family studies including parents and family members with or without vWD type I indicated that these three heterozygous patients are likely to be compound heterozygous. Twenty-one individuals from these seven families with vWD type I were found to be heterozygous for the mutation.

Adolescent↗