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Biomedical subjects

K Iqbal

Publications and source records attributed to K Iqbal.

At least 163 records · Page 9Linked to original sources
PubMed1986

Paired helical filaments associated with Alzheimer disease are readily soluble structures.

Considerable controversy exists concerning the origin and composition of Alzheimer neurofibrillary tangles (ANT) and of paired helical filaments (PHF), the abnormal cytoplasmic fibers which ultrastructurally are the major components of ANT. Thus far, the unusual solubility properties of PHF have hindered the analysis of ANT. A new procedure is presented for isolating purified PHF which are soluble in the presence of sodium dodecyl sulfate. The purification protocol involves differential and rate zonal centrifugation, treatment with the detergents sarcosyl and sulfobetain 3-14, and sonication. The isolated PHF from Alzheimer disease/senile dementia of the Alzheimer type (7 cases) and Down's syndrome (one case) have been characterized structurally by negative-stain electron microscopy, biochemically by PAGE, and immunologically by both the ELISA technique and Western blot analysis using a monoclonal antibody prepared against ANT. Distinct polypeptides were shown to be associated with this structure and not seen in preparations from young and age-matched normal brains.

Adult↗
PubMed1986

Occurrence of neuropil threads in the senile human brain and in Alzheimer's disease: a third location of paired helical filaments outside of neurofibrillary tangles and neuritic plaques.

Paired helical filaments, pathologically changed components of the cytoskeleton of human nerve cells, are demonstrated by a specific silver staining technique, immunostaining and electron microscopy. Accumulations of these filaments are found in the telencephalic cortex of old-aged individuals and patients suffering from Alzheimer's disease. Apart from neurofibrillary tangles and neuritic plaques, paired helical filaments are encountered in neuropil threads. At this third location, pathological filaments occur in small and inconspicuous profiles scattered throughout both allocortical and isocortical areas. The pattern of distribution and packing density of neuropil threads varies between different cortical areas and layers.

Aged↗
PubMed1986

Abnormal phosphorylation of the microtubule-associated protein tau (tau) in Alzheimer cytoskeletal pathology.

A monoclonal antibody to the microtubule-associated protein tau (tau) labeled some neurofibrillary tangles and plaque neurites, the two major locations of paired-helical filaments (PHF), in Alzheimer disease brain. The antibody also labeled isolated PHF that had been repeatedly washed with NaDodSO4. Dephosphorylation of the tissue sections with alkaline phosphatase prior to immunolabeling dramatically increased the number of tangles and plaques recognized by the antibody. The plaque core amyloid was not stained in either dephosphorylated or nondephosphorylated tissue sections. On immunoblots PHF polypeptides were labeled readily only when dephosphorylated. In contrast, a commercially available monoclonal antibody to a phosphorylated epitope of neurofilaments that labeled the tangles and the plaque neurites in tissue did not label any PHF polypeptides on immunoblots. The PHF polypeptides, labeled with the monoclonal antibody to tau, electrophoresed with those polypeptides recognized by antibodies to isolated PHF. The antibody to tau-labeled microtubules from normal human brains assembled in vitro but identically treated Alzheimer brain preparations had to be dephosphorylated to be completely recognized by this antibody. These findings suggest that tau in Alzheimer brain is an abnormally phosphorylated protein component of PHF.

Alzheimer Disease↗
PubMed1985

Alzheimer paired helical filaments: identification of polypeptides with monoclonal antibodies.

Paired helical filaments (PHF) were isolated from autopsied brain of cases of Alzheimer dementia, and their polypeptides were identified with monoclonal antibodies to PHF by Western blots. The PHF polypeptide profile consisted of several bands with a size difference of less than 5 kilodalton (kDa) between adjacent bands; the most prominent bands were in the 45-62 kDa region. These PHF polypeptides were also labeled with tangles reactive antisera to microtubules from normal brain but had electrophoretic patterns different from those of microtubules and neurofilaments. These studies define the molecular weight profile of PHF polypeptides and suggest that they might originate from normal brain proteins.

Aged↗
PubMed1985

Alzheimer paired helical filaments: cross-reacting polypeptide/s normally present in brain.

Antisera to microtubule-enriched fraction from normal human brain (anti-MT sera) label neurofibrillary tangles and neurites of neuritic (senile) plaques in brain sections of cases with Alzheimer disease/senile dementia of the Alzheimer type (AD/SDAT); the plaque core amyloid is not labeled. These anti-MT sera label both tangles in tissue sections and smears of isolated tangles which had been extracted with sodium dodecyl sulfate (SDS) to remove impurities trapped in between the paired helical filaments (PHF). The tangle labeling of anti-MT sera is eliminated on their absorption both with microtubule-enriched fractions from human and animal brain and with the isolated PHF. Neurofilament triplet, actin, myosin, keratin, or fibroblasts do not absorb the tangles staining antibodies. Furthermore, antisera containing antibodies to tubulin, microtubule-associated high mol. wt. polypeptides (MAPS), neurofilament triplet, and the 50,000 mol. wt. contaminant of CNS neurofilament preparations do not label tangles. On immunoblots of SDS-polyacrylamide gels of isolated PHF anti-MT sera label some of the same polypeptides identified with antisera to PHF; affinity-purified antibodies to tubulin used as a control do not label any PHF polypeptide on the immunoblots. The anti-MT sera, when preabsorbed with the PHF polypeptides eluted from SDS-polyacrylamide gels, do not label tangles. These studies demonstrate that a polypeptide/s cross-reactive with Alzheimer PHF is indeed normally present in brain and that it is different from tubulin, neurofilament triplet, actin, myosin, vimentin, and keratin.

Actins↗
PubMed1985

Proteolytic activity in brains of rabbits treated with aluminum.

Aluminum injection in rabbits leads to neurofibrillary changes which are at light microscopic level similar to those found in Alzheimer's disease. We used this animal model to see whether changes in proteolytic activity occur that may affect protein degradation in the altered neurofibrillary structure. Rabbits were injected via the cisterna magna with aluminum chloride, and after ten days tissue was excised from the spinal cord, hippocampus, occipital lobe, and cerebellum. Sections from the hippocampus and spinal cord were examined for neurofibrillary changes; enzyme activity was measured in all four areas. The enzymes studied were cathepsins A, B, and D, and the angiotensin-converting enzyme. No significant differences could be established in enzymatic activity in aluminum-injected animals compared to controls. However, a significant decrease in Triton-soluble proteins was observed in the treated animals, which correlated with changes in neurofibrillary structure. This decrease was most noticeable in the spinal cord (from 16.6 to 12.5 mg/g).

Aluminum↗
PubMed1985

Brain glutamate decarboxylase and cholinergic enzyme activities in scrapie.

C57BL/6J mice, age 6-8 weeks were inoculated intracerebrally with brain homogenate from mice previously infected with the 139A strain of scrapie; control mice were identically treated with brain homogenate from non-infected normal mice. The activities of choline acetyltransferase (CAT), acetyl cholinesterase (AChE), and glutamic acid decarboxylase (GAD) were determined in the forebrain and hindbrain of these animals after 67, 126 and 151 days post-inoculation. There were no significant differences in the activities of CAT and GAD between scrapie and control mice at early, middle or late stages of the disease in the scrapie-infected animals; there was an about 20% decline in AChE activity in the scrapie brain.

Acetylcholinesterase↗
PubMed1984

Alzheimer paired helical filaments: immunochemical identification of polypeptides.

Antisera to isolated Alzheimer neurofibrillary tangles (ANT) of paired helical filaments ( PHF ) were raised in rabbits. These anti- PHF sera immunolabeled both ANT in sections of Alzheimer hippocampus and ANT which were isolated and extracted with sodium dodecyl sulfate (SDS). The immunostaining of ANT in tissue sections was removed by absorption of the anti- PHF serum with small amounts of PHF and also with 40-fold the amount of a fraction prepared identically from normal brain; neurofilament and brain microtubule preparations used at the same concentration as the normal brain control fraction did not eliminate the tangle staining. Furthermore, the tangle staining was also not removed with glial filaments or actin and myosin filaments. No labeling of the neurofilaments of axons and cerebellar basket fibers by anti- PHF sera was observed in tissue sections from non-neurologic brain. On paper blots of SDS-polyacrylamide gels anti- PHF serum reacted with neither polypeptides of the normal brain control fraction nor major microtubule and neurofilament polypeptides. However, the immunoblots of PHF preparations with the anti- PHF serum revealed staining of several polypeptide bands in the 45,000-70,000 molecular weight (MW) region, material on top of the gel and diffuse staining of the high MW region. The tangles staining in tissue sections by the anti- PHF serum was abolished by its absorption with PHF polypeptides extracted from high and low molecular weight areas of SDS polyacrylamide gels but not with identically prepared neurofilament polypeptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1984

Alzheimer neurofibrillary tangles: monoclonal antibodies to inherent antigen(s).

Using isolated Alzheimer neurofibrillary tangles as the immunogen, nine mouse hybridomas were generated which produced antibodies to the tangles as tested in both tissue sections and isolated neurons from Alzheimer brain. Extraction of isolated neurofibrillary tangles with 2% SDS could not remove the antigen(s) with which these monoclonal antibodies reacted. Immunocytochemical study revealed that each of the monoclonal antibodies reacted with one or more of other tissue antigens in addition to the Alzheimer tangles. However, no reaction with either neurofilaments or microtubules was observed with any one of these antibodies. This is the first demonstration of monoclonal antibodies which have been generated against isolated Alzheimer neurofibrillary tangles; these antibodies react with antigen(s) inherent to the tangles.

Alzheimer Disease↗
PubMed1984

Alzheimer paired helical filaments: bulk isolation, solubility, and protein composition.

A method has been developed for the bulk isolation of Alzheimer neurofibrillary tangles (ANT) of paired helical filaments (PHF) from histopathologically confirmed cases of Alzheimer disease/senile dementia of the Alzheimer type (AD/SDAT). The fresh or frozen autopsied cerebral cortex affected with Alzheimer neurofibrillary changes is dissociated by homogenization and sieving through nylon bolting cloth and the ANT are separated by a combination of sucrose discontinuous density gradient centrifugation, glass bead column chromatography, and sodium dodecyl sulfate (SDS) treatment. The isolated ANT produce red-green birefringence when viewed through polarized light after staining with Congo red. Ultrastructurally, the isolated PHF are well preserved and have the dimensions of the PHF seen in situ. Two major Populations of ANT which exist in different proportions in AD/SDAT brains are identified on the basis of their solubility in SDS. The ANT I and the ANT II are soluble and insoluble respectively on treatment with 2% SDS at room temperature for 5 min. Solubilization of the ANT II requires several repeated extractions with a solution containing 10% each of SDS and beta-mercaptoethanol (BME) at 100 degrees C for 10 min. Sonication of the ANT II greatly facilitates their solubilization. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated ANT reveals the presence of two major polypeptides with molecular weights (MW) of 62,000 and 57,000, several minor polypeptides with MW below 57,000, and a significant amount of material not entering the stacking and the resolving gels. Re-electrophoresis of polypeptides extracted from various areas of the resolving gel or of the material which does not enter the gel generates the same polypeptide profile as on the first gel, suggesting that the PHF material which does not enter the gel may result from the reaggregation of the polypeptides that enter the resolving gel. None of the polypeptides that enter the resolving gel. None of the polpeptides observed in the isolated PHF comigrate in the SDS-PAGE with any of the neurofilament polypeptides, tubulin, actin, or myosin.

Actins↗
PubMed1984

Ultracytochemical distribution of myelin basic protein after injection into the cerebrospinal fluid. Evidence for transport through the blood-brain barrier and binding to the luminal surface of cerebral veins.

Distribution of myelin basic protein (MBP) in the central nervous system (CNS) following injection into the cerebrospinal fluid (CSF) was studied by different qualitative and quantitative immunelectron -microscopic techniques. Endogenous MBP was present in myelin sheaths in injected as well as in control animals. After injection of exogenous MBP into CSF this protein was present in the subarachnoid space, on the surface of meningeal cells, on the surface of collagen fibers, in the basement membrane of the glia limitans, in vessel walls, and in the extracellular space of spinal roots. In meningeal veins, endothelial vesicles filled with peroxidase reaction product were found on the abluminal side of endothelial cells, in the endothelial cytoplasm and sometimes opening into the vascularllumen . In addition patchy staining of the luminal surface of endothelial cells was noted, indicating binding of antigen at this location. Quantitative immunelectron microscopy (an indirect technique with rabbit anti-MBP serum as primary layer and gold-labeled anti-rabbit IgG as secondary layer) revealed highly significant MBP binding on the luminal surface of endothelial cells after injection of this antigen into the CSF. The present results indicate that MBP, when liberated in CNS is transported through the blood-brain barrier and presented on the luminal surface of endothelial cells of the cerebral and meningeal veins. This observation may be important in interpretation of pathogenesis of initial inflammatory infiltrates in experimental allergic encephalitis (EAE).

Animals↗
PubMed1984

Ultrastructure of paired helical filaments of Alzheimer's neurofibrillary tangle.

Isolated paired helical filaments (PHF) of Alzheimer's disease and senile dementia of the Alzheimer type (AD/SDAT) were studied by negative stain techniques and electron microscopy (EM). The helical structure of the PHF was confirmed by tilt analysis of negatively stained isolated PHF. A substructure of four protofilaments, each 3-5 nm in diameter, was also observed. A comparison of PHF substructure, neurofilaments and microtubules, revealed no similarity among the three structures.

Aged↗
PubMed1983

Purification and characterization of two forms of Ca2+-activated neutral protease from calf brain.

Two forms (CANP1 and CANP2) of a calcium-activated neutral protease (CANP) have been purified, 1,950- and 1,250-fold, respectively, to near homogeneity from calf brain. The purification procedure involves ammonium sulfate fractionation of the brain cytosol followed by chromatography on DEAE-Sephacel, hydroxylapatite, and alpha-casein-CH-Sepharose 4B affinity gel. A protein with apparent Mr = 17,000 copurifies with each of the proteases. This protein was separated by chromatography on a reactive red-120 agarose. Preliminary experiments indicate that, in the absence of this protein, the activity of each of the proteases was reduced. These observations raise the possibility that the 17,000-Da protein may regulate the activity of these proteases. Each of the proteases have similar apparent Mr = 78,000 as judged on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Except for casein, hemocyanin, and hemoglobin, no other exogenous proteins tested were significantly hydrolyzed by either of the proteases. [ methyl-14C ]alpha-Casein or methemoglobin was routinely used as a substrate for both of the enzymes. The endogenous proteins, neurotubules (microtubule-associated proteins and tubulin), neurofilament triplet proteins and desmin from smooth muscle were extensively hydrolyzed by both of the proteases. A marked difference was found in their requirement for CaCl2. CANP1 was maximally active at 700 microM while CANP2 exhibited highest activity at 2 microM CaCl2. Both displayed maximum activity at pH 7.5, although the overall pH profiles were slightly different. Among the actinomycete protease inhibitors, antipain, leupeptin, and pepstatin, leupeptin was highly effective in inhibiting the activities of both enzymes. Both of the proteases were also inhibited by sulfhydryl modifying agents. Metal ions other than CaCl2 were poor activators of the activity of either protease.

Aluminum↗
PubMed1983

Ultrastructural morphology of amyloid fibrils from neuritic and amyloid plaques.

The structure of partially purified, CNS amyloid fibrils from three different sources have been compared by negative stain EM. The fibrils isolated from brains with senile dementia of Alzheimer type were 4-8 nm in diameter, narrowing every 30-40 nm and apparently composed of two 2-4 nm filaments. The fibrils from a Gerstmann-Sträussler syndrome brain were 7-9 nm in diameter, narrowing every 70-80 nm and with a suggestion that they are composed of two 3-5 nm filaments. The fibrils isolated from 87V scrapie-affected mouse brains were 4-8 nm in diameter with a twist every 15-25 nm presumably composed of two 2-4 nm filaments. The fibrils from the scrapie brains were usually observed in pairs. The shape of the clusters of the isolated amyloid fibrils observed in each disease was similar in negative stain and thin section EM preparations and was related to the characteristic morphology of the amyloid fibrils in the neuritic and amyloid plaques in situ. The structural differences between the CNS amyloid fibrils from the various diseases studied by us may reflect differences in the polypeptides which comprise the fibril and/or a different pathogenesis in the formation of the amyloid fibrils.

Alzheimer Disease↗