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Biomedical subjects

M D Benson

Publications and source records attributed to M D Benson.

At least 91 records · Page 5Linked to original sources
PubMed1994

The Ile-84-->Ser amino acid substitution in transthyretin interferes with the interaction with plasma retinol-binding protein.

In plasma the thyroid hormone-binding protein transthyretin (TTR) forms a tight complex with the specific retinol carrier retinol-binding protein (RBP). The Ile-84-->Ser mutation and several other point mutations in TTR are associated with familial amyloidotic polyneuropathy, which is characterized by extracellular depositions of amyloid fibrils mainly consisting of mutated TTRs. The interactions with human RBP of recombinant human normal and Ser-84 TTRs were investigated by monitoring the fluorescence anisotropy of RBP-bound retinol. A nearly negligible affinity of the recombinant Ser-84 TTR for RBP was found. This result indicates the participation of a region on the outer surface of TTR that comprises Ile-84 in the recognition of RBP. In preliminary studies the Ser-84 TTR was the only one among several amyloidogenic variant TTRs to display negligible interaction with RBP. Therefore, in general a substantially altered binding of TTR to RBP is not associated with familial amyloidotic polyneuropathy. Instead, the altered binding of Ser-84 TTR to RBP appears to be responsible for an abnormal plasma transport of RBP. The recombinant normal TTR exhibits binding properties, in its interaction with human RBP, approximately similar to those of TTR purified from human plasma. Two independent and equivalent RBP binding sites on recombinant normal TTR are characterized by a dissociation constant of about 0.4 microM.

Humans↗
PubMed1994

Fibril formation from recombinant human serum amyloid A.

Three isotypes of human serum amyloid A (SAA), SAA1, SAA2 beta, and SAA4 were expressed at high levels in Escherichia coli (E. coli) using a pET vector expression system. Each SAA cDNA was ligated to the vector pET-21a(+) and transformed into E. coli, strain BL21(DE3)pLysS. Expression conditions required high concentrations of antibiotics in order to obtain a high ratio of synthesized SAA to total E. coli proteins. Each recombinant SAA (rSAA) was purified by molecular sieve chromatography followed by chromatofocusing or hydrophobic interaction chromatography. The yield of purified protein was 5-10 mg per 11 of culture. When subjected to in vitro fibril forming conditions, rSAA1 formed amyloid-like fibrils confirmed by Congo red staining and electron microscopy. In contrast, rSAA2 beta and rSAA4 showed negative Congo red staining and curvilinear or flattened fibrillar structures on electron microscopy. This suggests that SAA1 has greater potential for forming amyloid fibrils than either SAA2 beta or SAA4.

Amino Acid Sequence↗
PubMed1994

Hereditary renal amyloidosis with a novel variant fibrinogen.

Two families with hereditary renal amyloidosis were found to have a novel mutation in the fibrinogen A alpha chain gene. This form of amyloidosis is an autosomal dominant condition characterized by proteinuria, hypertension, and subsequent azotemia. DNAs of patients with amyloidosis were screened for a polymorphism in fibrinogen A alpha chain gene by single-strand conformation polymorphism analysis, and affected individuals from two kindreds were found to have a mutation. Both of these kindreds are American of Irish descent presenting with non-neuropathic, nephropathic amyloidosis in the fifth to the seventh decade of life. DNA sequencing showed a point mutation in the fibrinogen A alpha chain gene that is responsible for substitution of valine for glutamic acid at position 526. By restriction fragment length polymorphism analysis, 7 affected individuals and 14 asymptomatic individuals in these two kindreds were positive for the fibrinogen A alpha chain Val 526 gene. Fibrinogen was isolated from plasma of a heterozygous gene carrier and shown to contain approximately 50% variant fibrinogen. Discovery of this new mutation confirms the association between fibrinogen A alpha chain variant and hereditary renal amyloidosis and establishes a new biochemical subtype of amyloidosis.

Aged↗
PubMed1994

Threonine for alanine substitution at position 109 of transthyretin differentially alters human transthyretin's affinity for iodothyronines.

The heterozygous substitution of threonine for alanine at amino acid 109 of human transthyretin (TTR) increases its affinity for T4. We compared the affinity of recombinant wild-type (WT) and Thr109-TTRs for various iodothyronines in an attempt to elucidate how this mutation alters the T4-binding site. Homozygous WT and Thr109-TTRs were expressed recombinantly in Escherichia coli, and heterozygous Thr109-TTR was purified from plasma. The affinities of the iodothyronines for TTR were determined by measuring [125I]T4 bound by TTR in the presence of increasing concentrations of unlabeled iodothyronines. Homozygous Thr109-TTR bound T4 with an affinity slightly, but not significantly, greater than that of heterozygous Thr109-TTR. The affinity of Thr109-TTR for all iodothyronines was higher than that of WT TTR. However, the Thr109 mutation increased TTR's affinity for T4, Triac (triiodothyroacetic acid), and T3 to a greater extent than it did for Tetrac (tetraiodothyroacetic acid), EMD21388 (3',5'-dibromo-4',6'-dihydroxy-3-methylflavone), and dextro-T4. These data demonstrate that a subtle change in the structure of the T4-binding channel in TTR differentially alters the affinity of binding of various iodothyronines and suggests that site-directed mutagenesis of residues within the binding channel might clarify the relative importance of specific domains of this binding channel.

Alanine↗
PubMed1993

Characterization of amyloid fibril beta-peptide in familial Alzheimer's disease with APP717 mutations.

Amyloid fibrils were isolated from the brain tissue of two individuals who died with familial Alzheimer's disease, one with the phenylalanine 717 mutation in amyloid precursor protein (APP) and one with the isoleucine 717 APP mutation. After solubilization in guanidine hydrochloride and fractionation by sieve chromatography, low molecular weight fractions were treated with cyanogen bromide to generate the beta-peptide fragment starting after the methionine at position 35. Amino acid sequence analysis of all resultant peptides identified the peptide Val-Gly-Gly-Val-Val-Ile-Ala which represents residues 36-42 of the beta-amyloid peptide. No sequence beyond position 42 was found. These findings show that the amino acid substitution at position 717 is not incorporated into the amyloid deposits and suggests that the mutation may have metabolic affects which determine pathogenesis.

Alzheimer Disease↗
PubMed1993

Recombinant murine serum amyloid A from baculovirus-infected insect cells: purification and characterization.

Serum amyloid A (SAA) is an extremely sensitive acute-phase reactant and precursor to the subunit protein in reactive amyloid deposits. Although the mouse has long served as an informative experimental model, both the function of SAA and the pathogenic mechanism of amyloid formation remain unknown. The production of SAA by a heterologous system was pursued as means of generating readily-renewable amounts of SAA of defined sequence. Murine SAA2 has been expressed in and purified from baculovirus-infected insect cells. Using the transfer vector pBlueBac, SAA2 cDNA was cloned into baculovirus DNA such that expression was under the control of the polyhedrin promoter. Lysates prepared from infected cells contained three amyloid A-immunoreactive forms which accumulated intracellularly over a three day periods. The form having the lowest relative molecular mass, 12.5 kDa, co-migrated in SDS-polyacrylamide gels with the SAA2 present in murine acute-phase serum. Recombinant SAA2 was purified by Sepharose CL-6B chromatography followed by chromatofocusing between pH 8 and pH 5. Amino-terminal sequencing of the purified 12.5 kDa sample confirmed the first 20 residues of mature murine SAA2. After incubation with normal mouse serum, purified recombinant SAA2 fractionated exclusively with lipoprotein complexes, suggesting that it was bound to HDL. Based on this observation, we believe that recombinant SAA can serve as a suitable substitute for the native protein in physiologically relevant studies.

Amino Acid Sequence↗
PubMed1993

Apolipoprotein E epsilon 4 allele distributions in late-onset Alzheimer's disease and in other amyloid-forming diseases.

The frequency of the allele for apolipoprotein E type 4 (epsilon 4) is increased in late-onset familial and sporadic Alzheimer's disease (AD). We have examined epsilon 4 frequencies in four distinct, normal, elderly control groups and, most importantly, in patients with amyloid-forming diseases whose epsilon 4 distributions were not previously known (Creutzfeldt-Jakob disease, familial amyloidotic polyneuropathy, Down's syndrome). There were no differences between any of these controls and published control series, cementing the relevance of epsilon 4 for late-onset AD. The increase in late-onset AD was confirmed in two new series.

Age Factors↗
PubMed1993

The x-ray crystal structure refinements of normal human transthyretin and the amyloidogenic Val-30-->Met variant to 1.7-A resolution.

The x-ray crystal structures of normal human transthyretin (prealbumin) and the amyloidogenic Val-30-Met variant have been refined at 1.7-A resolution to R-values of 0.168 and 0.179, respectively, for 19,882 and 20,362 reflections (Fobs > 2.0 sigma). Standard deviations for stereochemical parameters are 0.018 and 0.022 A for bond distances, 0.030 and 0.038 A for angle distances, and 0.035 and 0.070 A for planar 1-4 distances. The newly refined normal structure shows improvement over the original structure of Blake and Swan (Blake, C. C. F., and Swan, I. D. A. (1971) J. Mol. Biol. 61, 217-224) in stereochemistry and in the conformation of the loop regions. Residues Arg-103, Thr-123, Asn-124, and Pro-125 have now been resolved, and residues 1-9 and 126-127 have been modeled with the aid of simulated annealing refinement. The functional form of transthyretin is a tetramer, having a cylindrical cavity which will bind thyroxine and an exterior binding site for the complex of retinol with retinol-binding protein. The monomer is a beta barrel flattened to become more like a sandwich with residue 30 in the interior. The methionyl for valyl substitution forces the beta sheets of the monomer as much as 1 A apart, resulting in a distortion of the thyroxine-binding cavity, in agreement with the independent observations that the Met-30 variant has low affinity for thyroxine.

Amino Acid Sequence↗
PubMed1993

X-ray crystal structure of the Ala-109-->Thr variant of human transthyretin which produces euthyroid hyperthyroxinemia.

The structure of the Ala-109-->Thr mutation of human transthyretin, a nonamyloidogenic variant with enhanced thyroxine binding, has been determined by x-ray diffraction to a resolution of 1.7 A. The model, including 175 solvent water molecules, has been refined by constrained least squares to an R-value of 0.157. The standard deviations for protein geometry are 0.016 A for bond distances, 0.5 degree for bond angles, 0.031 A for 1-4 distances, and 0.005 A for deviations of planar groups from their least squares plane. The estimated error in protein atomic coordinates is 0.12 A. Residue 109 extends inward between the two beta sheets which form the major component of the monomer, as does the side chain of residue 30 in the amyloidogenic Met-30 variant. Comparison of the Thr-109 structure with that of the normal shows that the extra atoms of the threonine fit into empty space between sheets and make no extensive changes to the molecular conformation. The substitution at 109 causes small local changes in the secondary structure of the A, G, and H strands resulting in a shift of residues 15-17, 108-110, and 117 in each monomer. The thyroxine-binding sites of the Thr-109 and Met-30 variants and of the normal protein are compared, and the results suggest that the variation in affinity for thyroxine between the three proteins may arise from differences in the size of the binding pocket.

Binding Sites↗
PubMed1993

Nonfatal amniotic fluid embolism. Three possible cases and a new clinical definition.

Three obstetrical patients who experienced cardiovascular collapse followed by disseminated intravascular coagulation were cared for by the author during residency training and 5 years of private practice. All patients survived. Their clinical courses were strongly reminiscent of those of patients described in the medical literature who ultimately died with the diagnosis of "amniotic fluid embolism." Paradoxically, the mere fact of survival is generally regarded as proof that a given individual did not have an amniotic fluid embolism. Proposed herein is a new clinical definition of amniotic fluid embolism syndrome that could apply to patients who survive as well as to those who die. With this definition in mind, the prevalence and prognosis of amniotic fluid embolism syndrome is reexamined. Finally, the traditional assumption that this syndrome is a result of amniotic fluid leakage into the maternal circulation is challenged. A new source is suggested and some new thoughts regarding treatment are provided.

Adult↗
PubMed1993

Hereditary renal amyloidosis associated with a mutant fibrinogen alpha-chain.

Three members of a family who died with renal amyloidosis were found to share a single nucleotide substitution in the fibrinogen alpha-chain gene. The predicted arginine to leucine mutation (Arg554Leu) was proven by amino acid sequence analysis of amyloid fibril protein isolated from postmortem kidney of an affected individual. Direct genomic DNA sequencing and restriction fragment length polymorphism analysis demonstrated that all three affected individuals had the guanine to thymine 4993 transversion. This is the first demonstration of hereditary amyloidosis associated with a variant fibrinogen alpha-chain. Variants of circulating fibrinogen may be the cause of a number of systemic amyloidoses with primarily renal involvement.

Adult↗
PubMed1993

A transthyretin variant (alanine 49) associated with familial amyloidotic polyneuropathy in a French family.

A transthyretin mutation was discovered in a French family with familial amyloidotic polyneuropathy originally described in 1983. The syndrome is of early onset (approximate age 35 to 40) with carpal tunnel syndrome. Death is from cardiac disease. By direct genomic DNA sequencing an A-->G mutation was found in the position corresponding to the first base of transthyretin codon 49. The predicted alanine for threonine substitution in the transthyretin protein was proven by amino acid sequencing of transthyretin isolated from the plasma of an affected subject. Since the DNA mutation does not result in the creation or abolition of a restriction endonuclease recognition site, a new DNA analysis technique was used in which site directed mutagenesis is used to create an RFLP when the introduced mutation is in proximity to the natural mutation. Using a 27 nucleotide mutagenesis primer in the PCR reaction, a new Bg1I site was created on amplification of the variant allele. Using this test, termed PCR-IMRA, four affected members of the family were shown to have the mutation.

Adult↗
PubMed1993

A transthyretin variant (alanine 71) associated with familial amyloidotic polyneuropathy in a French family.

A transthyretin (TTR) mutation is described in a 44 year old French woman from Caen who presented at the age of 40 with neuropathy in all four extremities, diarrhoea, and orthostatic hypotension. Her father died with a similar syndrome including vitreous opacities. A nerve biopsy from the proband showed amyloid deposits which stained with anti-transthyretin. Direct genomic DNA sequencing of TTR exon 3 showed both thymine and cytosine in the position corresponding to the second base of codon 71. This codes for a variant alanine (GCG) as well as the normal valine (GTG), indicating that the proband is heterozygous for the substitution. Since this substitution does not result in the creation or abolition of a restriction endonuclease recognition site, a new technique (PCR-IMRA) was used to create an RFLP. Using a 24 bp nucleotide mutagenesis primer in the PCR reaction, a new NspBII site is created on amplification of the variant allele. With this method a 170 bp TTR exon 3 PCR product was generated for both the normal and the variant allele. On digestion of the PCR product with NspBII, DNA from a heterozygous subject showed both the 170 bp undigested product from the normal allele and a 146 bp digestion product from the variant allele. By PCR-IMRA, two of five children of the proband were positive for the variant allele. This non-radioactive technique gives a rapid method for testing subjects at risk for this mutation.

Adult↗
PubMed1993

RFLP analysis for APP 717 mutations associated with Alzheimer's disease.

Familial Alzheimer's disease (FAD) has been shown to be associated with three distinct point mutations within the same codon of the amyloid precursor protein (APP) gene. The mutation identified in the Indiana kindred is a G-->T transversion at the first position of the codon for amino acid 717, resulting in a substitution of phenylalanine for valine in the APP protein. Screening of persons at risk for the APP Phe-717 mutation using a variation of the polymerase chain reaction identified nine positives among 34 tested. In addition, DNA from 145 FAD subjects were tested for the three known APP 717 mutations.

Alzheimer Disease↗
PubMed1993

Thyroxine interactions with transthyretin: a comparison of 10 different naturally occurring human transthyretin variants.

Transthyretin (TTR) is a tetrameric protein that transports 15-20% of circulating T4. Alterations in TTR structure can manifest clinically as familial amyloidotic polyneuropathy or euthyroid hyperthyroxinemia. We have measured the relative affinity for T4 of several variant TTR molecules in human plasma. We have compared control plasma to plasma from a patient heterozygous for a [Thr109]TTR mutation associated with a 3-fold increased affinity for T4 and to plasma from patients with familial amyloidotic polyneuropathy with different point mutations in TTR. Relative affinity was measured in an assay in which [125I]T4 bound by TTR was isolated by immunoprecipitation with an antibody specific for TTR. [Thr109]TTR displayed the highest affinity for T4, whereas homozygous [Met30]TTR did not bind appreciable amounts of [125I]T4. The rank order of affinity of the various TTR mutations for T4 was: Thr109 heterozygous > wild type = Ala60 heterozygous = Ile122 heterozygous > His58 heterozygous approximately Tyr77 heterozygous approximately Ser84 heterozygous approximately Ile122 homozygous approximately Met30 heterozygous >> Met30 homozygous TTR. Thus, different point mutations in TTR increase, decrease, or do not affect TTR's affinity for T4. The ability of TTR to form amyloid fibrils does not appear to be related to its affinity for T4. Further study is required to define the molecular basis of alterations in T4 binding induced by point mutations located along the TTR tetramer.

Amyloidosis↗
PubMed1992

Low cerebrospinal-fluid concentrations of soluble amyloid beta-protein precursor in hereditary Alzheimer's disease.

In Alzheimer's disease, deposits of amyloid beta-protein are apparently derived from intracellular processing of a large precursor protein. We have measured concentrations of this precursor in cerebrospinal fluid (CSF) from six members of a family affected by presenile Alzheimer's disease associated with a point mutation of the precursor gene. One gene carrier with clinical signs of the disorder had low CSF concentrations of the precursor, similar to those of three patients with sporadic Alzheimer's disease subsequently confirmed at necropsy. Two symptom-free gene carriers had CSF precursor concentrations similar to those of non-demented controls, though the value was lower in one, who had deficits revealed on neuropsychological testing, than in the other. These findings suggest that low concentrations of soluble amyloid precursor proteins in the CSF reflect the process that results in amyloid plaque formation and vascular deposition in Alzheimer's disease.

Adult↗
PubMed1992

Production and functional analysis of normal and variant recombinant human transthyretin proteins.

The most common form of hereditary systemic amyloidosis is familial amyloidotic polyneuropathy associated with single amino acid changes in the plasma protein, transthyretin. In addition, there are two variants of transthyretin (Ser6 and Thr109) not associated with familial amyloidotic polyneuropathy but with familial euthyroid hyperthyroxinemia, also an autosomal dominant disorder. In these autosomal dominant diseases, most affected individuals are heterozygous and therefore have hybrid forms of the tetrameric plasma transthyretin. In order to study the structure/function relationships of homozygous variant transthyretins, normal human transthyretin and five variant transthyretins (Gly6----Ser, Leu58----His, Thr60----Ala, Ile84----Ser, and Ala109----Thr) were produced in Escherichia coli using the expression vector, pCZ11, and site-directed mutagenesis. These recombinant transthyretin (r-TTR) proteins showed the correct size (14 kilodaltons) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis and self-associated into tetramers as determined by size exclusion chromatography. Recombinant normal, Ser6, and Ala60 r-TTRs had an affinity for thyroxine indistinguishable from normal human TTR purified from plasma, whereas His58 and Ser84 r-TTRs had significantly reduced affinity. On the other hand, Thr109 r-TTR had a much higher affinity, probably due to its position within the thyroxine-binding pocket. Expression of mutant transthyretins in E. coli provides the opportunity to study structure/function relationships and amyloid-forming capabilities induced by single amino acid substitutions in the transthyretin molecule.

Amino Acid Sequence↗