Biomedical subjects

K Iqbal

Publications and source records attributed to K Iqbal.

At least 19 recordsLinked to original sources

PubMed1995

Alzheimer paired helical filaments, untreated and pronase digested, studied by vertical platinum-carbon replication and high resolution transmission electron microscopy.

Untreated paired helical filaments (PHF) and pronase treated PHF filaments have been stereoscopically imaged with a freeze-drying vertical platinum-carbon replication preparation method for TEM. The untreated PHF have an average wide region, W = 22.8 +/- 2.4 nm, a narrow region width, T = 10.6 +/- 1.7 nm, and a helical turn period, L = 78.6 +/- 13.4. The widths of the pronase treated PHF were significantly reduced and had average measurements of W = 14.8 +/- 1.2 nm, T = 5.7 +/- 1.0 nm, with the helical period unchanged, L = 75.4 +/- 17 nm. The surfaces of the untreated PHF contained approximately 1.0 and approximately 0.4 nm strands, the size of normal and denatured tau monomer. The pronase treated PHF contained approximately 1.0 and approximately 0.4 nm strands as well as approximately 2.0 nm strands. The stereoscopic images of the untreated and the pronase digested PHF do not support a double helical morphology for the PHF. The PHF appear to be long helical ribbons. The approximately 1.0 and approximately 2 nm substructure has been organized both parallel and orthogonal to the PHF-core axis for distances less than 80 nm. The most frequent structural appearance is of a disorganized PHF core. The surfaces of the untreated PHF also have a similar disorganized appearance.

Alzheimer Disease↗

PubMed1995

Dephosphorylation of Alzheimer paired helical filaments by protein phosphatase-2A and -2B.

Microtubule-associated protein tau is abnormally hyperphosphorylated in the brain of patients with Alzheimer disease and in this form is the major protein subunit of the paired helical filaments (PHF), the most prominent lesion of the disease. In this study the dephosphorylation of sparingly soluble PHF, PHF II-tau by brain protein phosphatase (PP)-2A1 and PP-2B, and the resulting biochemical, biological, and structural alterations were investigated. Both of the phosphatases dephosphorylated PHF II-tau at the sites of Ser-199/Ser-202 and partially dephosphorylated it at Ser-396/Ser-404; in addition, PHF II-tau was dephosphorylated at Ser-46 by PP-2A1 and Ser-235 by PP-2B. The relative electrophoretic mobility of PHF II-tau increased after dephosphorylation by either enzyme. Divalent cations, manganese, and magnesium increased the activities of PP-2A1 and PP-2B toward PHF II-tau. Dephosphorylation both by PP-2B and PP-2A1 decreased the resistance of PHF II-tau to proteolysis by the brain calcium-activated neutral proteases (CANP). The ability of PHF II-tau to promote the in vitro microtubule assembly was restored after dephosphorylation by PP-2A1 and PP-2B. Microtubules assembled by the dephosphorylated PHF II-tau were structurally identical to those assembled by bovine tau used as a control. The dephosphorylation both by PP-2A1 and PP-2B caused dissociation of the tangles and the PHF; some of the PHF dissociated into straight protofilaments/subfilaments. Approximately 25% of the total tau was released from PHF on dephosphorylation by PP-2A1. These observations demonstrate that PHF II-tau is accessible to dephosphorylation by PP-2A1 and PP-2B, and dephosphorylation makes PHF dissociate, accessible to proteolysis by CANP, and biologically active in promoting the assembly of tubulin into microtubules.

Alzheimer Disease↗

PubMed1995

Phosphorylation and accumulation of tau without any concomitant increase in tubulin levels in Chinese hamster ovary cells stably transfected with human tau441.

Eucaryotic expression vectors bearing a 1.4 kb cDNA encoding the 4 repeat isoform of human tau, tau441, in either the sense or anti-sense orientation with respect to a cytomegalovirus (CMV) promoter were constructed. The resulting constructs were used to transiently express tau in Chinese Hamster Ovary cells and to generate non-neuronal stable cell lines. Immunocytochemical studies of these cells show that tau is expressed in the sense but not the anti-sense or vector containing lines. Some of the cells expressing tau showed fine elongated processes which were stained by tau antibodies. The general tau immunostaining pattern appeared diffuse and punctuate. The expressed tau was seen both unbound and bound to microtubules. In some cells labeling with antibodies that specifically recognize hyperphosphorylation of tau was observed. The size of this population increased with increasing numbers of cell passages. However, no increase in steady-state tubulin level was observed following tau441 expression. These studies show that tau can accumulate in the cells without a concomitant increase in tubulin.

Animals↗

PubMed1995

Abnormally phosphorylated tau in SY5Y human neuroblastoma cells.

In Alzheimer disease (AD) the microtubule associated protein (MAP) tau is hyperphosphorylated at several sites. In the present study, like AD tau, tau in the human neuroblastoma SH-SY5Y was found to be hyperphosphorylated, at Ser-199/202, Thr-231, Ser-396 and Ser-404. However, in contrast to AD, the tau in SY5Y cells was not hyperphosphorylated at Ser-235 and there was only one tau isoform. Quantitative analysis revealed that approximately 80% of the SY5Y-tau was phosphorylated at Ser-199/202. The phosphorylated tau was deposited in perikarya and processes of the cells whereas most of the unphosphorylated (at Ser-199/202) tau was localized in the nucleus. Tau from the cell lysates did not bind to taxol-stabilized microtubules. In contrast, MAP1b and MAP2 from cell lysates bound to stabilized microtubules in vitro and were associated to the microtubule network in situ. Phosphorylation of tau at high levels, its inactivity with microtubules and its accumulation in SY5Y cells provide for the first time a cell model of cytoskeletal changes seen in AD.

Cytoplasm↗

PubMed1995

Rapid Alzheimer-like phosphorylation of tau by the synergistic actions of non-proline-dependent protein kinases and GSK-3.

Tau protein from Alzheimer disease (AD) brain is phosphorylated at eleven Ser/Thr-Pro and nine Ser/Thr-X sites. The former sites are phosphorylated by proline-dependent protein kinases (PDPKs), the latter by non-PDPKs. The identities of both the PDPKs and non-PDPKs involved in AD tau hyperphosphorylation are still to be established. In this study we have analyzed the interactions between a PDPK (GSK-3) and several non-PDPKs (A-kinase, C-kinase, CK-1, CaM kinase II) in the phosphorylation of one isoform (tau 39) of human tau. We found that the rate of phosphorylation of tau 39 by GSK-3 was increased several-fold if tau were first prephosphorylated by the non-PDPKs. Further, several Alzheimer-like epitopes in tau can be induced only slowly after phosphorylation of tau by GSK-3 alone. After a prephosphorylation of tau by the non-PDPKs, however, the rate of induction of these epitopes by GSK-3 is increased several-fold. These results suggest that one role of non-PDPK-catalyzed phosphorylation is the modulation of PDPK-catalyzed phosphorylation of tau in AD brain.

Alzheimer Disease↗

PubMed1995

Modulation of GSK-3-catalyzed phosphorylation of microtubule-associated protein tau by non-proline-dependent protein kinases.

The phosphorylation of bovine tau, either by GSK-3 alone or by a combination of GSK-3 and several non-proline-dependent protein kinases (non-PDPKs), was studied. GSK-3 alone catalyzed the incorporation of approximately 3 mol 32P/mol tau at a relatively slow rate. Prephosphorylation of tau by A-kinase, C-kinase, or CK-2 (but not by CK-1, CaM kinase II or Gr kinase) increased both the rate and extent of a subsequent phosphorylation catalyzed by GSK-3 by several-fold. These results suggest that the phosphorylation of tau by PDPKs such as GSK-3 (and possibly MAP kinase, cdk5) may be positively modulated at the substrate level by non-PDPK-catalyzed phosphorylations.

Animals↗

PubMed1995

Bovine and human tau, highly homologous but less crossreactive: implications for Alzheimer disease.

Tau is a highly conserved protein during evolution. However, polymerization of tau into paired helical filaments, a characteristic of Alzheimer disease is unique to humans and has not been seen in animals. The cause of this phenomenon is not known. The cDNA-derived amino acid sequences of bovine and human tau are highly homologous. In this study we show that despite the high homology there are marked differences between the immunoreactivities of human and bovine tau. The four antibodies employed in this study were the monoclonal Tau-1 to the bovine protein, two polyclonal anti-bovine tau 92e and 111e and a polyclonal anti-human tau 113e. The monoclonal antibody Tau-1, the epitope of which lies in the amino acid residues 196-215 of bovine tau and residues 189-207 of human tau differing by a single amino acid, a glycine at position 196 in bovine protein with a proline at position 189 in human protein, reacts 2.3 +/- 0.5 times better with bovine tau than with human tau. Both polyclonal antibodies against bovine tau, 92e and 111e react 25 +/- 7 and 45 +/- 17 folds, respectively, better with bovine tau than human tau, whereas the polyclonal antibody 113e to a human tau peptide does not cross-react with bovine tau. The differences in cross-reactivity that are probably due to differences in conformation of tau from the two species were found using both cytosolic extracts and tau isolated from human and bovine brains.(ABSTRACT TRUNCATED AT 250 WORDS)

Alzheimer Disease↗

PubMed1995

Cranial fasciitis: presentation as a post-auricular mass.

Cranial fasciitis is a rare variant of nodular fasciitis. It is a benign condition with features resembling sarcoma, seen principally in young males. It involves the skull bones and grows at a rapid pace. Accurate diagnosis and surgical excision is the key to management. Prognosis is good with recurrence rare. By 1992, 17 cases had been reported in the literature. The present case is the first reported in Pakistan. It presented as a post-auricular mass.

Child, Preschool↗

PubMed1995

Guanosine triphosphate binding to beta-subunit of tubulin in Alzheimer's disease brain: role of microtubule-associated protein tau.

In Alzheimer's disease, paired helical filaments composed mainly of abnormally phosphorylated tau accumulate in certain selected neurons of the brain, and microtubules are rarely seen in the affected cells. In the present study, the binding of 32P-labeled 8-azidoguanosine triphosphate ([gamma-32P]8N3GTP), the photoaffinity analogue of GTP to the beta-subunit of tubulin in brain homogenates was found to be markedly lower in patients with Alzheimer's disease than in aged control human cases. No significant differences were observed in the levels of the alpha- and beta-subunits of tubulin between Alzheimer's disease and control brains obtained 2-7 h postmortem. In nine of 19 Alzheimer's disease and 11 of 12 control autopsied brains (2-7 h postmortem and stored at -75 degrees C) tubulin was isolated successfully from brain cytosol by in vitro polymerization induced with DEAE-dextran. The GTP binding was observed in the two cycled assembled microtubule preparations from all the normal control, and in eight of nine Alzheimer's disease cases. Alzheimer's disease microtubule preparations contained varying amounts of abnormally phosphorylated tau, whereas no abnormal tau was detected in the control brain preparations. Addition of bovine tau to bovine, normal human, and Alzheimer's disease brain tubulin preparations markedly increased GTP binding to the beta-subunit. An alkaline phosphatase-treated paired helical filament-enriched preparation increased by approximately twofold the GTP binding to bovine brain tubulin. GTP binding to tubulin prepared by phosphocellulose chromatography of two cycled microtubules from three Alzheimer's disease and three normal control brains, revealed insignificant differences between the two groups. These findings have suggested that (1) tau protein promotes the GTP binding to the beta-subunit of tubulin, and (2) the breakdown of the microtubule system in brains of patients with Alzheimer's disease might in part be due to the abnormal phosphorylation of tau which depresses the GTP binding.

Affinity Labels↗

PubMed1995

Phosphorylation of tau protein by casein kinase-1 converts it to an abnormal Alzheimer-like state.

The microtubule-associated protein tau is abnormally hyperphosphorylated in Alzheimer's disease. Both proline-dependent protein kinases (PDPKs) and non-PDPKs are involved in this hyperphosphorylation of tau. Several PDPKs can phosphorylate tau in vitro and induce Alzheimer-like epitopes to many phosphorylation-dependent antibodies. A similar induction has not been reported with non-PDPKs. In this study we have evaluated six non-PDPKs [cyclic AMP-dependent (A-kinase), calcium/phospholipid-dependent (C-kinase), casein kinase-1 (CK-1), casein kinase-2 (CK-2), calcium/calmodulin-dependent protein kinase II, and calcium/calmodulin-dependent protein kinase from rat cerebellum] for their abilities to induce Alzheimer-like epitopes on tau. Such epitopes were induced by A-kinase, C-kinase, CK-1, and CK-2, but the degree of induction achieved by CK-1 was much greater than with the other kinases. These results suggest that CK-1 may play an important role in the conversion of tau from the normal to the abnormal phosphorylation state in Alzheimer's disease.

Alzheimer Disease↗

PubMed1995

Phosphatase activity toward abnormally phosphorylated tau: decrease in Alzheimer disease brain.

Microtubule-associated protein tau is abnormally hyperphosphorylated and aggregated in affected neurons of Alzheimer disease brain. This hyperphosphorylated tau can be dephosphorylated at some of the abnormal phosphorylated sites by purified protein phosphatase-1, 2A, and 2B in vitro. In the present study, we have developed an assay to measure protein phosphatase activity toward tau-1 sites (Ser199/Ser202) using the hyperphosphorylated tau isolated from Alzheimer disease brain as substrate. Using this assay, we have identified that in normal brain, protein phosphatase-2A and 2B and, to a lesser extent, 1 are involved in the dephosphorylation of tau. The Km values of dephosphorylation of the hyperphosphorylated tau by protein phosphatase-2A and 2B are similar. The tau phosphatase activity is decreased by approximately 30% in brain of Alzheimer disease patients compared with those of age-matched controls. These findings suggest that a defect of protein phosphatase could be the cause of the abnormal hyperphosphorylation of tau in Alzheimer disease.

Aged↗

PubMed1995

Pharyngocutaneous fistula following laryngectomy.

Sixty-eight cases of stage III and IV laryngeal cancer were analysed to determine the frequency and management of post-operative pharyngocutaneous fistula (PCF). The overall incidence in our series is 8.9% (6 cases). In five patients PCF healed spontaneously without any surgical intervention while one patient expired due to concomitant other medical illness. The longest time taken by the fistula to heal by conservative treatment was 7 weeks. Retaining the NG tube for a longer period (14-16 days) and adequate and appropriate antibiotic cover reduces the incidence of fistula formation.

Adult↗

PubMed1994

Expression of protein phosphatases (PP-1, PP-2A, PP-2B and PTP-1B) and protein kinases (MAP kinase and P34cdc2) in the hippocampus of patients with Alzheimer disease and normal aged individuals.

Microtubule-associated protein tau is abnormally hyperphosphorylated in the brain of patients with Alzheimer disease (AD). Previous studies have shown (i) that in vitro tau can be phosphorylated to an Alzheimer abnormally phosphorylated state-like protein by proline-directed protein kinases MAP kinase and p34cdc2, and (ii) that the AD abnormally phosphorylated tau can be in vitro dephosphorylated by protein phosphatases PP-2B, PP-2A and PP-1 and not by PP-2C. However, to have a direct effect on the regulation of phosphorylation of tau, these enzymes should be present in the affected neurons. In the present study immunocytochemical localization of protein phosphatases PP-1, PP-2A, PP-2B and PTP, and protein kinases MAP kinase and p34cdc2 were studied in the hippocampal formation of AD and as a control in non-demented elderly patients. All the protein phosphatases and protein kinases studied were localized to both granular and pyramidal neurons. In the pyramidal neurons, the enzymes staining was observed in neuronal soma and neurites. PTP-1B, PP-1 and PP-2A were also highly expressed in microglia. The topographical distributions of all the enzymes studied were similar, i.e. the intensity of immunostaining in hippocampus in end-plate (CA3 and CA4) > prosubiculum, subiculum > entorhinal cortex > dentate gyrus > CA2 > CA1. Furthermore, the expression of all the enzymes was also observed in the tangle-bearing neurons. The PP-2B staining of the tangle-bearing neurons was weaker than the unaffected neurons in the same tissue section field in AD cases.

Adult↗

PubMed1994

Levels of normal and abnormally phosphorylated tau in different cellular and regional compartments of Alzheimer disease and control brains.

Microtubule associated protein tau is abnormally phosphorylated in Alzheimer disease (AD) brain. In the present study we investigated (i) whether tau is axonal or both axonal and somatodendritic, (ii) whether tau is a marker of Alzheimer neurofibrillary pathology, and (iii) whether the levels of tau in the cytosol (100,000 x g supernate) from AD brain are altered. Frozen autopsied tissue from 20 AD, 17 normal aged and 15 neurological control cases obtained 3-8 h postmortem were analyzed. Levels of normal, total, and abnormally phosphorylated tau were determined by a radioimmunoslot-blot assay using mAb Tau-1 as the primary antibody. Both frontal gray matter homogenate and cytosol from normal brains had 30-45% higher levels of normal tau than the corresponding fractions from the white matter. In AD frontal and temporal cortices, the total tau levels were 6- to 7-fold higher than in cerebellar cortex (P < 0.01 and P < 0.02). Furthermore, tau levels of cerebellar cortex, an area of the brain unaffected with Alzheimer neurofibrillary changes, were indistinguishable between AD and control groups. The levels of normal tau in cytosol from both frontal gray and white matters in AD were reduced by approximately 40% (P < 0.05). The levels of total tau in AD frontal and temporal cortex were 4- to 5-fold higher than in the corresponding tissue from control cases (P < 0.01) and this increase was in the form of abnormally phosphorylated tau. These studies suggest (i) that there is probably at least as much tau in the somatodendritic compartment as in the axonal compartment, (ii) that the abnormally phosphorylated tau is a biochemical marker of the neurofibrillary pathology in AD, and (iii) that the levels of normal tau are significantly reduced in the 100,000 x g brain supernate from AD cases.

Alzheimer Disease↗

PubMed1994

Alzheimer paired helical filaments. Restoration of the biological activity by dephosphorylation.

In a normal mature neuron, microtubule associated protein tau promotes the assembly of tubulin into microtubules and maintains the structure of microtubules. In Alzheimer disease brain, tau is abnormally hyperphosphorylated and is the major protein subunit of paired helical filaments (PHF). In the present study, the biological activity of tau in PHF and the effect of dephosphorylation on this activity were examined. PHF were isolated from Alzheimer disease brains and tau from the untreated or alkaline phosphatase-treated PHF was extracted by ultrasonication in microtubule assembly buffer. Tubulin was isolated by phosphocellulose chromatography of three cycled microtubules from bovine brain. PHF-tau did not promote assembly of bovine tubulin into microtubules whereas tau from the dephosphorylated PHF produced a robust microtubule assembly. These studies suggest (i) that in Alzheimer disease tau in PHF is functionally inactive because of abnormal phosphorylation and (ii) that the abnormally phosphorylated site(s) in PHF that inactivates PHF-tau is accessible to enzymatic dephosphorylation in vitro.

Alkaline Phosphatase↗

PubMed1994

Ubiquitin in cerebrospinal fluid: a rapid competitive enzyme-linked immunoflow assay.

The aims of this study were to develop a rapid immunoassay to determine the levels of ubiquitin in cerebrospinal fluid and to establish the ubiquitin levels in the spinal fluid of normal aged individuals. A competitive enzyme-linked immunoflow assay was developed. In this assay, ubiquitin is bound to nitrocellulose membrane, after which the primary antibody-test sample mixture and the enzyme-labeled secondary antibody under vacuum are applied sequentially. The final reaction product is collected in a microtiter plate by suction. This competitive assay requires only approximately 4 h and is potentially useful for determining in biological fluids the levels of any antigen or its antibodies that might be present. Employing this immunoassay, cerebrospinal fluid ubiquitin levels were found to be 147.5 +/- 5.2 ng ml-1 in non-neurological aged cases.

Aged↗

PubMed1994

Role of abnormally phosphorylated tau in the breakdown of microtubules in Alzheimer disease.

The microtubule assembly-promoting activity of different pools of tau protein isolated from Alzheimer disease (AD) and control brains and the effect of dephosphorylation on this activity were studied. Tau isolated from a 2.5% perchloric extract of AD brain had almost the same activity as that obtained from control brain, and this activity did not change significantly on dephosphorylation. Abnormally phosphorylated tau (AD P-tau) isolated from brain homogenate of AD patients had little activity, and upon dephosphorylation with alkaline phosphatase, its activity increased to approximately the same level as the acid-soluble tau. Addition of AD P-tau to a mixture of normal tau and tubulin inhibited microtubule assembly. AD P-tau bound to normal tau but not to tubulin. These studies suggest that the abnormal phosphorylation of tau might be responsible for the breakdown of microtubules in affected neurons in AD not only because the altered protein has little microtubule-promoting activity but also because it interacts with normal tau, making the latter unavailable for promoting the assembly of tubulin into microtubules.

Alzheimer Disease↗

PubMed1994

Dephosphorylation of microtubule-associated protein tau by protein phosphatase-1 and -2C and its implication in Alzheimer disease.

Microtubule-associated protein tau is abnormally hyperphosphorylated and forms the major protein subunit of paired helical filaments (PHF) in Alzheimer disease brains. The abnormally phosphorylated sites Ser-199, Ser-202, Ser-396 and Ser-404 but not Ser-46 and Ser-235 of Alzheimer tau were found to be dephosphorylated by protein phosphatase-1 and this dephosphorylation was activated by Mn2+. In contrast, protein phosphatase-2C did not dephosphorylate any of these sites. Both protein phosphatase-1 and -2C had high activities towards [32P]tau phosphorylated by cAMP-dependent protein kinase. These results suggest that both protein phosphatase-1 and -2C might be associated with normal phosphorylation state of tau, but only the former and not the latter phosphatase is involved in its abnormal phosphorylation in Alzheimer disease.

Aged↗