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R Kisilevsky

Publications and source records attributed to R Kisilevsky.

At least 73 records · Page 4Linked to original sources
PubMed1991

Transcription of cRNA for in situ hybridization from polymerase chain reaction-amplified DNA.

The synthesis of cRNA probes for in situ hybridization is usually accomplished with transcription systems using cloning vectors that contain bacteriophage RNA polymerase promoters. In this report we describe an alternative technique for generating cRNA probes that obviates the need for cDNA cloning by using the polymerase chain reaction. The segment of DNA corresponding to the desired RNA sequence was amplified from genomic DNA by the polymerase chain reaction. The bacteriophage SP6 and T7 RNA polymerase promoters were incorporated into the amplified DNA by including the promoter sequences in the 5' termini of the oligonucleotides used to prime the polymerase chain reaction. The promoters permitted the subsequent transcription of sense and antisense cRNA from the amplified DNA. The antisense cRNA was radiolabeled to high specific activity for use as a probe in Northern and mRNA in situ hybridization analyses. This technique offers the advantages of speed and simplicity over conventional transcription systems, which require cDNA cloning. Bypassing the need for cloning in the synthesis of cRNA probes may facilitate the use of these probes in research and diagnostic applications of mRNA in situ hybridization.

Animals↗
PubMed1991

Amyloid and amyloidoses: differences, common themes, and practical considerations.

Amyloidosis is not one entity but a grouping of disorders in all of which there is a tissue protein deposit with similar morphological, structural, and staining properties despite involving fundamentally different proteins or peptides. Each protein is involved in its own specific pathogenetic mechanism. These mechanisms determine, in a manner as yet unknown, which protein will be present in an amyloid deposit. The role of proteolysis in the pathogenesis is questioned. Proteolysis may well be a postfibrillogenic event. Local tissue factors, such as the basement membrane form of heparan sulfate proteoglycan, in addition to perhaps other basement membrane components, may play an important role in the genesis of amyloid. Differences, common themes, and various unanswered questions related to the pathogenesis of the amyloids are presented as are practical diagnostic considerations.

Amyloid↗
PubMed1991

Co-deposition of basement membrane components during the induction of murine splenic AA amyloid.

Past studies have demonstrated that during murine AA amyloid induction there is co-deposition of the AA amyloid peptide and the basement membrane form of heparan sulfate proteoglycan. The synthesis and accumulation of heparan sulfate proteoglycan does not usually occur in the absence of other basement membrane components, such as type IV collagen, laminin, and fibronectin. Using immunohistochemical techniques, the present experiments have demonstrated that in addition to the heparan sulfate proteoglycan, there are other basement membrane components present in splenic AA amyloid deposits and these are present as soon as AA amyloid deposits are detectable. The results indicate that within the time constraints imposed by the experiments, the basement membrane components, fibronectin, laminin, type IV collagen, and heparan sulfate proteoglycan are co-deposited 36 to 48 hours after the AgNO3 and amyloid enhancing factor induction of amyloid, the period when amyloid is first detected. These observations raise the possibility that an abnormality in basement membrane metabolism is a very early event, and potentially plays an integral part in the process of AA amyloidogenesis.

Animals↗
PubMed1990

Inhibition of the initiation of hepatic protein synthesis during ethionine mediated ATP depletion in vivo: modification to ribosomal subunits, evidence of impaired ternary complex formation and a subcellular redistribution of eIF-2 alpha.

Acute ethionine intoxication is known to induce a reversible hepatic injury in female rats by reducing the level of hepatic ATP. The injury indirectly impairs the initiation of hepatic protein synthesis, with resultant polysome disaggregation. Administration of adenine rapidly restores the ATP levels and protein synthesis. Analysis of liver polysome and ribosomal subunits reveals that polysome disaggregation occurs following 3 h of the intoxication, and reaggregation occurs following the administration of adenine. Inactive hepatic ribosomes accumulate as monomers and disomes when analysed by sucrose gradient sedimentation in low-salt buffers. High-salt buffers dissociate the inactive ribosomes into the component 40 S and 60 S subunits. The level of higher density, 1.48 g/cc, 40 S subunit increases during the inhibition of protein synthesis, while the lower density, 1.41 g/cc, 40 S subunit species does not change significantly. Hepatic microsomal and cytosolic extracts examined for their ability to support the formation of the ternary complex of eIF-2-GTP and [35S]Met-tRNAi demonstrate that during acute ethionine intoxication, ternary complex formation in the two extracts decrease 65% and 85%, respectively. These changes are coincident with polysome disaggregation. Administration of adenine to reverse the intoxication restores the ternary complex forming ability of the cytosolic extract, but does not affect the activity of the microsomal salt wash extracts. Mixing experiments indicate the accumulation of an inhibitor of ternary complex formation in the microsomal salt wash fraction. The application of quantitative western blotting demonstrates that the level of antigenic eIF-2 alpha in the microsomal salt wash extract increases 31% during the inhibition. These observations are consistent with the idea that the inhibition of the initiation of hepatic protein synthesis induced by ethionine is mediated by eIF-2 alpha phosphorylation. The latter results in an inhibition of ternary complex formation, redistribution of eIF-2 to the microsome fraction, polysomal disaggregation, and accumulation of inactive ribosomal subunits.

Adenine↗
PubMed1990

Apolipoprotein A-I and apolipoprotein SAA half-lives during acute inflammation and amyloidogenesis.

The half-lives of two apolipoproteins of mouse high-density lipoproteins, apo A-I and apo SAA, were determined in normal animals and compared with those having an inflammatory condition, or inflammation leading to AA amyloid deposition. The apo A-I half-life was considerably shorter in animals with inflammation and in those that were preamyloidotic, than in controls (t1/2 of 3-3.5 h vs. 10-12 h). The average loss of apo A-I in controls over the first 10 h was 31.1 micrograms/ml per h, while that in inflamed animals was 58.7 micrograms/ml per h a 2-fold increase in apo A-I clearance. The apo SAA half-life was similar in all groups of animals and was of the order of 1.5 h. The concentration of apo SAA during inflammation is however considerably higher (500-1000-fold) than in controls, which implies a much greater clearance rate during inflammation and involving a process which is apparently not saturable. In addition to hypertriglyceridemia and Tangier's disease, ordinary acute inflammation can now be added to those pathological conditions which lead to a significant decrease in apo A-I half-life.

Amyloid↗
PubMed1990

Influence of sulphate ions on the structure of AA amyloid fibrils.

To explore the possible interaction of sulphated GAG with AA amyloid peptides, human AA amyloid fibrils were exposed to buffers containing various salts, and the accessibility of free amino groups on the peptides to reductive methylation was examined. Sodium chloride had little effect except at concentrations of 1 M, where it reduced the accessibility of AA peptides to labelling. In contrast 70 mM Na2SO4 led to a significant increase in peptide accessibility to labelling. The results suggest that, at least in part, GAG interact with AA peptides through their sulphate moieties.

Chemical Phenomena↗
PubMed1990

Amyloid enhancing factor-loaded macrophages in amyloid fibril formation.

Amyloid enhancing factor (AEF) is believed to be a key agent that triggers the second (deposition) phase of amyloidogenesis. However, the target cells of AEF activation and their function after the activation have not yet been clearly identified. We found that peritoneal resident cells from amyloidotic mice contained very high AEF activity. With a simultaneous subcutaneous injection of 1.0 ml of the casein-adjuvant emulsion, an intravenous injection of 10,000 cells was consistently capable of inducing amyloidosis in a recipient mouse in 72 hours. After 2-hour cultures, the major AEF activity was found in the adherent cells (macrophages). An intravenous injection of 5 to 10 million of the live macrophages with the casein-adjuvant injection caused amyloid deposits in the recipient not only in the spleen and the liver but also in the lung (an extremely rare site of AA amyloid deposition). We have interpreted this finding to indicate that the injected AEF-loaded macrophages, while still residing in the lung and exposed to the blood stream, processed SAA to form amyloid. We further tested this postulate in an in vitro system. In a 4-day culture of the AEF-loaded macrophages in a medium containing SAA-rich mouse serum, small masses (less than 15 microns in diameter) of Congo red positive substance were observed scattered adjacent to or surrounded by the macrophages. The present observations lend strong credence to the conclusion that AEF-loaded macrophages are fully capable of processing SAA to AA and further to amyloid fibrils, and that they indeed play a role in the second phase of amyloidogenesis in vivo.

Amyloid↗
PubMed1989

Differential induction of the serum amyloid A gene family in response to an inflammatory agent and to amyloid-enhancing factor.

The murine serum amyloid A1 (SAA1), SAA2, and SAA3 genes are expressed in various tissues in response to acute inflammation. Prolonged expression may be accompanied by amyloid deposition in liver, spleen, and kidney. Shortly before and during deposition, an amyloid-enhancing factor (AEF) can be extracted from these tissues which accelerates amyloid formation when administered with an inflammatory agent. We have investigated the ability of liver AEF to alter expression of the three SAA genes in liver, spleen, and kidney when administered to normal mice or to mice in which inflammation was created with the injection of silver nitrate. In liver, both AEF and silver nitrate induce SAA1 and SAA2 mRNA accumulation. However, AEF elicits a more rapid response and also acts as a potent inducer of hepatic SAA3 mRNA. Silver nitrate does not induce any SAA mRNA species in kidney, whereas AEF induces all three species. In contrast, AEF induces only SAA3 mRNA in the spleen. We also show that the elevation in hepatic SAA mRNA levels induced by either AEF or silver nitrate is associated with a transient increase in the length of the poly(A) tail.

Animals↗
PubMed1989

Sulfated glycosaminoglycans in amyloid plaques of prion diseases.

Brain sections from cases of human Creutzfeldt-Jakob disease, Gerstmann-Sträussler syndrome, kuru, and hamster scrapie containing amyloid were examined for the presence of sulfated glycosaminoglycans (GAGs), the anionic component of proteoglycans, using the sulfated Alcian blue method and Alcian blue technique with 0.3 M and 0.7 M magnesium chloride. These studies suggest that sulfated glycosaminoglycans are part of the CNS amyloid plaques in each of the above human prion disorders as well as in experimental scrapie. All the amyloid plaques stained positively with Alcian blue at 0.3 M, and less so at 0.7 M magnesium chloride indicating the presence of sulfated GAGs. Therefore, the amyloid plaques of prion diseases possess similar histochemical features to those found in Alzheimer's disease.

Alzheimer Disease↗
PubMed1989

The ultrastructural localization of sulfated proteoglycans is identical in the amyloids of Alzheimer's disease and AA, AL, senile cardiac and medullary carcinoma-associated amyloidosis.

The cationic dyes cuprolinic blue and ruthenium red were used to ultrastructurally localize proteoglycans (PGs) within the neuritic plaque and neurofibrillary tangle of Alzheimer's disease. Highly sulfated PGs were specifically localized to the amyloid fibril of the neuritic plaque and the paired filaments of the neurofibrillary tangle. This demonstrates that highly sulfated PGs either comprise part of the Alzheimer's amyloid fibril and paired filament or are intimately associated with them. Four unrelated types of amyloid--AA (inflammation-associated), AL (immunoglobulin light chain), senile cardiac (prealbumin) and medullary carcinoma-associated amyloid (procalcitonin)--showed an identical pattern of localization of highly sulfated PG to the different amyloid fibrils. This constant close spatial relationship between PGs and diverse amyloid proteins suggests that PGs may play a role in amyloidogenesis.

Alzheimer Disease↗
PubMed1988

Circular-dichroism studies on two murine serum amyloid A proteins.

C.d. studies have shown that mouse SAA2 (serum amyloid A2) protein has about one-half of the alpha-helix content of the SAA1 (serum amyloid A1) analogue (15 as against 32%), although secondary-structure prediction analyses based on sequence data do not suggest such a large difference between the forms. The decreased helical content may be a reflection or indication of a stronger propensity to aggregation of the SAA2 form compared with SAA1. The main elements of secondary structure in both proteins are beta-sheets/turns. Interactions with Ca2+ are accompanied by small losses in alpha-helix content, whereas binding to chondroitin-6-sulphate in the presence of millimolar Ca2+ also decreases the amount of secondary structure. However, SAA2 binding to heparan sulphate increases its beta-sheet structure, whereas with SAA1 secondary structure is not apparently altered by its interaction with heparan sulphate. Computer-generated surface profiles show slight differences in accessibility, hydrophilicity and flexibility between the proteins. Understanding these differences may help to explain why SAA2 is found in amyloid fibrils whereas SAA1 is not. In particular, a stronger tendency to aggregation might be the reason why SAA2 is deposited exclusively in these fibrils.

Animals↗
PubMed1988

A reproducible model for the study of factor X kinetics in AA amyloidosis.

Factor X clearance was examined in a model of rapid AA amyloid deposition. Accelerated equilibration with extravascular compartments and accelerated removal postequilibration mimic features seen in patients with AL amyloidosis. The handling of Factor X was different from that of two other proteins, mouse albumin and IgG. Each protein had its own specific characteristic clearance properties, although in amyloidotic animals all proteins were cleared more rapidly in the postequilibration phase. The liver was by far the major site of Factor X clearance but this was true in all control groups as well. No significant difference was seen in tissue clearance site in any of the treatment groups, perhaps because the amount of AA amyloid in each tissue 3 days into the protocol was not yet large. Nevertheless, a reproducible model that possesses accelerated Factor X clearance is now available to study the mechanism of coagulation factor abnormalities in amyloidosis.

Albumins↗
PubMed1988

The potential significance of sulphated glycosaminoglycans as a common constituent of all amyloids: or, perhaps amyloid is not a misnomer.

Amyloid is a generic term referring to a group of diseases in which proteinaceous tissue deposits all have in common specific stain affinities, a common appearance in polarized light, common ultrastructure fibrillary characteristics, and uniform x-ray diffraction and infrared spectral properties. Where groups of diseases have a common underlying pathogenetic process the polypeptide responsible for the protein fibril is the same regardless of the specific disease. Where diseases have a different underlying pathogenesis the polypeptide is unique for each disease. The different amyloidogenic polypeptides are clearly not related in terms of amino acid sequence or function, yet they all tend to fold in such a way as to present the same staining, structural or spectral properties. It is proposed that amyloid fibrils are not only composed of the specific amyloidogenic polypeptide but also highly sulphated glycosaminoglycans or proteoglycans which have a profound influence on the manner in which the peptides fold and interact with each other. It is this highly charged carbohydrate which may be common to all amyloids and which plays a determining role in the final appearance of the deposit. Amyloid should therefore be considered as more than simply a protein entity but, as its name originally implied, one related to carbohydrate deposition as well.

Amyloid↗
PubMed1988

Direct evidence for circulating apoSAA as the precursor of tissue AA amyloid deposits.

A precursor product study was carried out using tritiated, undenatured murine high-density lipoprotein (HDL)-apoSAA, to assess whether circulating HDL-apoSAA is the precursor of tissue AA amyloid deposits. The quantity of label accumulating in AA over a 24-h period was determined per unit weight of spleen. In addition a mathematical assessment of the quantity of label that should have accumulated in splenic AA within 24 h was made, based on the half-life of circulating apoSAA, the rate of change of the specific activity of circulating apoSAA, and rate of deposition of splenic AA. The observed result was approximately 47% of the theoretically predicted value. The latter did not include extraction efficiencies of AA peptide from spleen nor losses which might have occurred during column fractionation of splenic extracts. The observed and predicted results are therefore in remarkably good agreement and indicate that circulating apoSAA is the major source of protein for splenic AA amyloid deposition.

Amyloidosis↗
PubMed1988

Effects of culture substrates and normal hepatic sinusoidal cells on in vitro hepatocyte synthesis of Apo-SAA.

Primary hepatocyte cultures synthesize apo-SAA upon stimulation with supernatant from lipopolysaccharide (LPS)-treated macrophages. The matrices on which the hepatocytes were grown influence their basal apo-SAA synthetic capability. Fibronectin was superior. Coculturing hepatocytes with hepatic sinusoidal cells did not adversely affect the ability of hepatocytes to synthesize and secrete apo-SAA into the culture medium. In 72 h, clear islands of endothelial cells nestled in layers of hepatocytes. Both apo-SAA and apo-SAA were made in considerable quantities but no evidence could be obtained that the apo-SAA were free of apo-A-1. The coculturing of hepatocytes with liver sinusoidal cells, the site of ultimate AA deposition, is a first step in establishing an in vitro system for AA amyloidogenesis.

Animals↗
PubMed1987

A comparison of accessibility of ribosomal proteins on free and membrane-bound ribosomes: the ribosomal proteins potentially involved in ribosome-membrane binding.

The relative accessibility of rat liver ribosomal proteins to reductive methylation was examined using membrane-bound and free ribosomes. Comparisons indicated that 12-13 large ribosomal proteins are masked by ribosomal association with membranes. These consisted of L8, L10, L17, L26-28, L31 and L36, and probably also include L4, L5, L7 and L29. These proteins seem to surround a region centered about L3 and may partly define a ribosomal channel through which the nascent peptide emerges. Approx. 10-20% of the large ribosomal subunit surface area is shielded by the membrane.

Animals↗