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  • 1
    ISSN: 1432-1203
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract We have analyzed cDNA from a 46-year-old atypical neurofibromatosis type 2 (NF2) patient who had lumbar tumors, cataract and schwannomas of peripheral nerves but no vestibular schwannomas, and have identified a 163-bp deletion in the NF2 transcript. The deletion is predicted to remove 54, alter 15 and add four extra amino acids at the C-terminus of the NF2-gene product. The same deletion was found in her two daughters and in a 3-year-old grandson. Bilateral vestibular schwannomas were detected in the two asymptomatic daughters, whereas no abnormality was found in the grandson.
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  • 2
    ISSN: 1432-1203
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Since the identification of the NF2 tumor suppressor gene in 1993, various mutations have been found in NF2-related tumors and in lymphocytes from NF2 patients. Most of the reported mutations result in truncated gene products. Missense mutations affecting the tumor suppressor are rare. These missense mutations would provide valuable information for the understanding of the function of the tumor suppressor, since they should affect critical parts of the protein. In this study we describe a novel point mutation in exon 15 of the NF2 gene, which is found in lymphocyte DNA of two NF2 patients from one family. This mutation is expected to result in a substitution of Pro for Gln at codon 538. Though both of the two patients developed bilateral vestibular schwannomas, the first patient showed onset of the disease at the age of 31 years and presented with various central, peripheral and abdominal tumors, while the second patient showed later onset of clinical symptoms (at age 52 years) and presented with only two additional small spinal tumors.
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  • 3
    ISSN: 1432-1203
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder. It is caused by mutations in the NF1 gene, which comprises 60 exons and is located on chromosome 17q11.2. A total of 170 unrelated NF1 patients were screened for mutations in four exons by temperature-gradient gel electrophoresis. Preparatory work revealed the presence of a previously uncharacterized intron (19a) in what was previously designated exon 19; this allowed us to develop assays for genomic mutation screening in the newly defined exons 19a and 19b. Two novel NF1 mutations were detected: a single-base insertion in exon 19a creating a frameshift, and a second mutation affecting the splice donor site of intron 20 and leading to skipping of exon 20. A novel BsaBI polymorphism was identified in intron 19a.
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  • 4
    ISSN: 1432-1203
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Since the identification of the NF2 tumor suppressor gene in 1993, various mutations have been found in NF2-related tumors and in lymphocytes from NF2 patients. Most of the reported mutations result in truncated gene products. Missense mutations affecting the tumor suppressor are rare. These missense mutations would provide valuable information for the understanding of the function of the tumor suppressor, since they should affect critical parts of the protein. In this study we describe a novel point mutation in exon 15 of the NF2 gene, which is found in lymphocyte DNA of two NF2 patients from one family. This mutation is expected to result in a substitution of Pro for Gln at codon 538. Though both of the two patients developed bilateral vestibular schwannomas, the first patient showed onset of the disease at the age of 31 years and presented with various central, peripheral and abdominal tumors, while the second patient showed later onset of clinical symptoms (at age 52 years) and presented with only two additional small spinal tumors.
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  • 5
    ISSN: 1364-6753
    Keywords: Key words Neurofibromatosis 2 ; Schwannomatosis ; Allelic expression ; Polymorphism
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Neurofibromatosis type 2 (NF2) is a genetic disorder characterized by formation of multiple schwannomas and meningiomas due to inactivating mutations in the NF2 tumor suppressor gene on chromosome 22. We describe a polymorphism in the 3′ untranslated region of the NF2 gene that is informative in about one-third of individuals. This polymorphism permitted an assessment of the relative expression of NF2 transcripts in lymphoblastoid cell RNA from 22 unrelated NF2 patients heterozygous for a germline NF2 mutation, along with 6 schwannomatosis patients, and 14 unaffected controls. Unequal allelic expression (1.8- to 20-fold) was detected in 15 of the NF2 cases, but in none of the schwannomatosis or control individuals. Underexpression of the NF2 mutant allele was documented for all 6 nonsense or frameshift mutations, 3 of 6 splice mutations, and 1 of 4 missense mutations, which, unexpectedly, was shown to alter the NF2 transcript and create a premature stop codon. In contrast, equal expression or slight overexpression of NF2 mutant alleles was observed for 2 in-frame deletions, 2 splice alterations, and 3 missense mutations. In the remaining 5 cases, the allele representing the mutant transcript was not known. Thus, truncating NF2 mutations, which are the most frequent alterations in NF2 patients and NF2-associated tumors, were associated with underexpression of the mutant allele, whereas the less common in-frame alterations usually showed normal or slight overexpression of the mutant transcript.
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  • 6
    ISSN: 1432-1203
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
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  • 7
    ISSN: 1432-1203
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Neurofibromatosis 2 (NF2) is a clinically variable autosomal dominant disorder, caused by mutations in the NF2 tumor suppressor gene on chromosome 22q12, that predisposes to nervous system tumors and ocular abnormalities. To assess intrafamilial phenotypic variability, we performed mutation analysis and clinical assessment on two multigeneration NF2 families with five patients and seven asymptomatic first-degree relatives of patients. One family had a point mutation of agCC→ggCC at position 1447–2 at the exon 13/14 boundary predicted to lead to an altered splice acceptor sequence and exon deletion. The other family had an insertion of 2 base pairs (TC) at position 761 in exon 8, leading to a frameshift. Both mild and severe phenotypes occurred in each family, indicating that phenotypic variability in NF2 can be caused by factors other than NF2 mutations. Genetic counseling of NF2 families should include the possibility that presymptomatic NF2 mutation carriers can develop a different phenotype than previously diagnosed patients.
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  • 8
    ISSN: 1432-2013
    Keywords: Key words Troponin I ; Calcium sensitivity ; Cardiac muscle contraction ; Skinned fibers ; Site-directed mutagenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Using treatment with vanadate solutions, we extracted native cardiac troponin I and troponin C (cTnI and cTnC) from skinned fibers of porcine right ventricles. These proteins were replaced by exogenously supplied TnI and TnC isoforms, thereby restoring Ca2+-dependent regulation. Force then depended on the negative logarithm of Ca2+ concentration (pCa) in a sigmoidal manner, the pCa for 50% force development, pCa50, being about 5.5. For reconstitution we used fast-twitch rabbit skeletal muscle TnI and TnC (sTnI and sTnC), bovine cTnI and cTnC or recombinant sTnIs that were altered by site-directed mutagenesis. Incubation with TnI inhibited isometric tension in TnI-extracted fibers in the absence of Ca2+, but restoration of Ca2+ dependence required incubation with both TnI and TnC. Relaxation at low Ca2+ levels and the steepness of the force/pCa relation depended on the concentration of exogenously supplied TnI in the reconstitution solution (range 20–150 μM), while Ca2+ sensitivity, i.e. the pCa50, was dependent on the isoform, and also on the concentration of TnC in the reconstitution solution. At pH 6.7, skinned fibers reconstituted with optimal concentrations of sTnC and sTnI (120 μM and 150 μM, respectively) were more sensitive to Ca2+ than those reconstituted with cTnC and cTnI (difference in pCa50 approx. 0.2 units). Rabbit sTnI was cloned and expressed in Escherichia coli using a high yield expression plasmid. We introduced point mutations into the TnI inhibitory region comprising the sequence of the minimal common TnC/actin binding site (-G104-K-F-K-R-P-P-L-R-R-V-R115-). The four mutants produced by substitution of T for P110, G for P110, G for L111, and G for K105 were chosen, based on previous work with synthetic peptides showing that single amino acid substitution in this region diminished the capacity of these peptides to inhibit acto-S1 ATPase or contraction of skinned fibers. Therefore, all amino acid residues of the inhibitory region are thought to contribute to biological activity of TnI. However, each of the recombinant TnIs could substitute for endogenous TnI. In combination with exogenous TnC, Ca2+ dependence could be restored when gly110sTnI, thr110sTnI or gly111sTnI was used for reconstitution. The mutant gly105sTnI, on the other hand, reduced the ability of skinned fibers to relax at low Ca2+ concentrations and it caused an increase in Ca2+ sensitivity.
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  • 9
    ISSN: 1432-2013
    Keywords: Troponin I ; Calcium sensitivity ; Cardiac muscle contraction ; Skinned fibers ; Site-directed mutagenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Using treatment with vanadate solutions, we extracted native cardiac troponin I and troponin C (cTnI and cTnC) from skinned fibers of porcine right ventricles. These proteins were replaced by exogenously supplied TnI and TnC isoforms, thereby restoring Ca2+-dependent regulation. Force then depended on the negative logarithm of Ca2+ concentration (pCa) in a sigmoidal manner, the pCa for 50% force development, pCa50, being about 5.5. For reconstitution we used fast-twitch rabbit skeletal muscle TnI and TnC (sTnI and sTnC), bovine cTnI and cTnC or recombinant sTnIs that were altered by site-directed mutagenesis. Incubation with TnI inhibited isometric tension in TnI-extracted fibers in the absence of Ca+, but restoration of Ca2+ dependence required incubation with both TnI and TnC. Relaxation at low Ca2+ levels and the steepness of the force/pCa relation depended on the concentration of exogenously supplied TnI in the reconstitution solution (range 20–150 μM), while Ca2+ sensitivity, i.e. the pCa50, was dependent on the isoform, and also on the concentration of TnC in the reconstitution solution. At pH 6.7, skinned fibers reconstituted with optimal concentrations of sTnC and sTnI (120 μM and 150 μM, respectively) were more sensitive to Ca2+ than those reconstituted with cTnC and cTnI (difference in pCa50 approx. 0.2 units). Rabbit sTnI was cloned and expressed inEscherichia coli using a high yield expression plasmid. We introduced point mutations into the TnI inhibitory region comprising the sequence of the minimal common TnC/actin binding site (-G104-K-F-K-R-P-P-L-R-R-V-R115-). The four mutants produced by substitution of T for P110, G for P110, G for L111 and G for K105 were chosen, based on previous work with synthetic peptides showing that single amino acid substitution in this region diminished the capacity of these peptides to inhibit acto-Si, ATPase or contraction of skinned fibers. Therefore, all amino acid residues of the inhibitory region are thought to contribute to biological activity of TnI. However, each of the recombinant TnIs could substitute for endogenous TnI. In combination with exogenous TnC, Ca2+ dependence could be restored whengly110sTnI,thr110sTnI orgly111sTnI was used for reconstitution. The mutantgly105sTnI, on the other hand, reduced the ability of skinned fibers to relax at low Ca2+ concentrations and it caused an increase in Ca2+ sensitivity.
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  • 10
    ISSN: 1573-2657
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary When expressed in E. coli, skeletal muscle α-tropomyosin has an unacetylated N-terminus. Unacetylated α-tropomyosin lacks important functions; this is non-polymerizable and has a low affinity to actin. In the present work, in order to obtain fully functional recombinant α-tropomyosin, rabbit skeletal muscle α-tropomyosin (α-tropomyosinBV) has been expressed in baculovirus-infected insect cells. α-TropomyosinBV was not distinguishable from the authentic tropomyosin, not only in functional properties but also in blocked N-terminus. To know the N-terminus structure of α-tropomyosinBV, the N-terminal segment six amino acids long, MDAIKK, has been specifically and efficiently removed from α-tropomyosinBV by use of an immobilized proteolytic enzyme system based on E. coli cell bodies which carry the ompT gene product, a proteolytic enzyme localized on the outer cell wall of E. coli. The structure of recombinant α-tropomyosinBV was shown to be identical to the authentic protein by electrospray mass spectrometry and protein sequencing analysis. Additionally, electrospray mass spectometry indicated a single phosphorylation not only in α-but also β-tropomyosin chains in the rabbit skeletal muscle. The differentiated susceptibilities of potential ompT cleavage sites are indicative of a non-coiled-coil conformation of the N-terminus of α-tropomyosin.
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