Batia Kaplan

Microextraction and purification techniques applicable to chemical characterization of amyloid proteins in minute amounts of tissue.

This article described micromethods useful for the extraction, purification, and amino acid sequencing of amyloid proteins contained in minute specimens obtained from patients with systemic forms of amyloidosis. We posit that these procedures can also be applied to the biochemical characterization of cerebral amyloid deposits. The selection of the techniques is dependent on the type of sample to be extracted (fresh or formalin fixed) as well as the amount of congophilic material present. Although amyloid proteins are isolated and purified more easily from fresh tissue, it must be noted that formalin-fixed specimens are available more readily for analysis due to the common diagnostic use of fine needle tissue biopsies and are therefore, important for both current and retrospective studies. Remarkably, despite the expected difficulties associated with formalin treatment we were able to extract and sequence amyloid proteins from fixed tissues presumably due to the resistance of amyloid to formalin cross-linking. Through the continued development of techniques for small-scale protein separation and application of highly sensitive microsequencing and mass spectral methods, exact identification of the protein contained in fibrillar amyloid deposits can be determined. Such information has therapeutic and prognostic relevance and can increase our understanding of the pathogenesis of amyloidosis.

Functional and morphological alterations in compound transgenic mice overexpreszing Cu/Zn superoxide dismutaze and amyloid precursor protein

Downs syndrome (DS), the phenotypic manifestation of trisomy 21, involves overexpression of chromosome 21‐encoded genes. The gene for amyloid precursor protein (APP), known to be involved in AD pathology, resides on chromosome 21 along with the gene for Cu/Zn superoxide dismutase (SOD1), a key enzyme in the metabolism of oxygen free radicals. We investigated the consequences of a combined increase in APP and SOD1, in a double‐transgenic (tg)‐APP–SOD1 mouse. These mice expressed severe impairment in learning, working and long‐term memory. Expression of long‐term potentiation in hippocampal slices was impaired in both tg‐SOD and tg‐APP–SOD mice, but not in tg‐APP mice, indicating that increased APP by itself did not affect in vitro synaptic plasticity. In tg‐APP–SOD mice, membrane‐bound high molecular weight APP species accumulated while APP cleavage products did not increase and levels of secreted APP were unchanged. Severe morphological damage, including lipofuscin accumulation and mitochondria abnormalities, were found in aged tg‐APP–SOD but not in the other mice. Thus, a combined elevation of the two chromosome 21 genes in tg‐APP–SOD mice induced age‐dependent alterations in morphological and behavioural functions.

Free light chain monomers in the diagnosis of multiple sclerosis

A new procedure of free light chain (FLC) analysis was developed to assist the diagnosis of multiple sclerosis (MS). In this procedure, Western blotting technique was used to analyze monomeric and dimeric FLCs in the cerebrospinal fluid (CSF) and serum of patients with MS and other neurological diseases. The intensity of immunoreactive FLC bands was quantified by a specially developed software. Analysis of the obtained monomer/dimer patterns of κ and λ type FLCs allowed the determination of the diagnostically useful FLC parameters. The combined use of three FLC indices accounting for monomeric FLC-κ level and κ/λ ratio values in the CSF and serum was found to be of promising diagnostic importance for differentiation of MS from other non-MS neurological diseases.

Immunoglobulin Free Light Chain Dimers in Human Diseases

Immunoglobulin free light chain (FLC) kappa (κ) and lambda (λ) isotypes exist mainly in monomeric and dimeric forms. Under pathological conditions, the level of FLCs as well as the structure of monomeric and dimeric FLCs and their dimerization properties might be significantly altered. The abnormally high fractions of dimeric FLCs were demonstrated in the serum of patients with multiple myeloma (MM) and primary systemic amyloidosis (AL), as well as in the serum of anephric patients. The presence of tetra- and trimolecular complexes formed due to dimer-dimer and dimer-monomer interactions was detected in the myeloma serum. Analysis of the amyloidogenic light chains demonstrated mutations within the dimer interface, thus raising the possibility that these mutations are responsible for amyloidogenicity. Increased κ monomer and dimer levels, as well as a high κ/λ monomer ratio, were typically found in the cerebrospinal fluid from patients with multiple sclerosis (MS). In many MS cases, the elevation of κ FLCs was accompanied by an abnormally high proportion of λ dimers. This review focuses on the disease-related changes of the structure and level of dimeric FLCs, and raises the questions regarding their formation, function, and role in the pathogenesis and diagnosis of human diseases.

Free light chains in plasma of patients with light chain amyloidosis and non‐amyloid light chain deposition disease. High proportion and heterogeneity of disulfide‐linked monoclonal free light chains as pathogenic features of amyloid disease

Immunoglobulin light chain amyloidosis (AL) and non‐amyloid light chain deposition disease (NALCDD) are different forms of protein aggregation disorders accompanied by a monoclonal gammopathy. Monoclonal free light chains (FLCs) are precursors of the pathological light chain tissue deposits that are fibrillar in AL and granular in NALCDD. However, direct biochemical examination of plasma FLC precursors, which would allow comparison and better understanding of these two diseases, is still lacking. In this study, we examined FLCs in plasma of patients with AL and NALCDD by employing separation on Sep‐PaK C18 cartridges, micro‐preparative electrophoresis, Western blotting and mass spectrometry. Comparative analysis of AL versus NALCDD and control plasma samples showed new evidence of increased level and heterogeneity of circulating disulfide‐bound FLC species in AL. In addition to full length monomers comprising the disulfide‐linked FLCs, the monoclonal disulfide‐bound FLC fragments were typically revealed in AL plasma. We hypothesized that enhanced disulfide binding of FLCs in AL interferes with their normal clearance and metabolism, which in turn might play a role in amyloid formation. The applied methods might be useful to diagnose or predict the pathological form of the disease and shed light on the mechanisms involved in light chain aggregation in tissues.

Immunochemical characterization of amyloid in diagnostic biopsy tissues

The heterogeneity of the amyloidoses requires the determination of the chemical type of amyloid protein deposited in tissues. in the present study of 15 diagnostic biopsy specimens classified as light chain amyloid, nonamyloid light chain disease, amyloid A and transthyretin amyloid by immunojluorescence microscopy, the residual frozen tissues were independently analyzed by immunochemical methods. The tissue deposits were extracted in aqueous 20% acetonitrile containing 0.1% trlfluoroacetic acid and the extracted material subjected to Western blotting. Similarly analyzed were 9 control amyloid negative and 6 amyloid positive tissues. From the previously characterized amyloid positive tissues, Western blotting of both isolated fibrils prepared by solubilization in distilled water and the material extracted in acetonitrile from the same specimens yielded identical low molecular weight proteins (>28kDa) immunoreactive with only one of the 4 antibodies tested, whereas the control amyloid negative tissue extra...

The molecular basis of reactive amyloidosis

Reactive amyloidosis is a disease occurring in patients suffering from chronic infections, inflammation, and certain malignant conditions that are characterized by a considerable elevation of the acute phase reactant serum amyloid A (SAA). It is defined by the presence of extracellular deposits of fibrillar material containing amyloid A (AA) as its main component. AA is an 8.5-kd protein structurally identical to the NH2-terminal of the acute phase reactant SAA. SAA consists of a group of evolutionally conserved amphipathic proteins, encoded by a large number of genes and produced abundantly during inflammation, all suggesting an important role, probably of a neutralizing (anti-inflammatory) nature. An analysis of various aspects of SAA provides no clues to the mechanism of amyloid production, its occurrence in only selected individuals, and its preferential relationship to one isotype of SAA. Until more data is available, the present view on AA amyloidogenesis remains hypothetical.

Isolation and Characterization of Amyloid Proteins Using Milligram Amounts of Amyloid - Containing Tissue

Abstract A new procedure is developed for extraction and purification of amyloid proteins from milligram amounts of amyloid-containing tissue. The procedure involves extraction of amyloid proteins with acidic agueous acetonitrile and their purification by size-exclusion high-performance liquid chromatography (HPLC). The molecular weight and type of isolated amyloid proteins were determined by using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), followed by immunoblotting technique. The developed procedure is more rapid and requires significantly smaller amount of tissue material (about 10 mg), comparing with the conventional amyloid isolation technique performed by using gram amounts of autopsy specimen. The techniques applied presently may be useful for precise determination of amyloid type in biopsies.

Preparative fractionation of amyloid proteins on a microgram scale by high-performance liquid chromatography and polyacrylamide gel electrophoresis

Preparative separation of amyloid proteins on a microgram scale is presented. Amyloid fibrils solubilized in aqueous 50% acetonitrile containing 0.1% trifluoroacetic acid, are fractionated by reverse-phase high-performance liquid chromatography. Fractionation of amyloids obtained from patients with familial Mediterranean fever allowed isolation of a protein identical with a conventionally isolated AA-protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis is used for preparative separation of AL-proteins. Two protein extraction procedures from Coomassie Blue stained gels are applied using elution in 0.1% sodium dodecyl sulfate containing buffer and 6 mol/l guanidine-HCl solution. The eluted proteins are concentrated and sodium dodecyl sulfate and dye are removed by acetonitrile precipitation of sample.

pH-dependent fibrillogenesis of a VkappaIII Bence Jones protein.

Disorders of immunoglobulin (Ig) synthesis that occur in malignant plasma‐cell proliferation may result in either granular (LCDD) or fibrillar (AL) tissue deposition of light‐chain monoclonal components. The structural features that govern the transition from soluble polypeptides to either fibrillar or granular conformational states remain undefined. Among the many factors presumed to play a role in these transitions the net charge of the molecule has been associated with folding conformation changes. The majority of the proteins involved in AL amyloidosis show acidic isoelectric points (pI 3.8–5.2), whereas most L chains with basic pIs deposit in granular patterns. In our studies a 12 kD VκIII fragment was purified as the main component of the fibrils isolated from myocardium and adipose tissue of the pericardium obtained post‐mortem from an individual with systemic AL amyloidosis. An apparently identical 12 kD VL fragment with the same N‐terminal sequence constituted the BJ protein present in the urine. This urinary protein exhibited strikingly cathodic electrophoretic mobility on agarose gels and lacked retention by anionic exchange chromatography matrices, indicative of a highly basic pI (>10). When it was subjected to in vitro fibril‐formation experiments, the BJ protein adopted a fibrillar conformation only at acidic pHs, remaining aggregated but not fibrillar at physiological pH. The data indicate that a specific tissue deposition pattern involves not only structural properties of the protein but rather more complex mechanisms in which acidic micro‐environments may contribute to the stabilization of amyloidogenic conformations.