The microtubule-associated protein is a family of six isoforms that becomes abnormally hyperphosphorylated and accumulates in the form of paired helical filaments (PHF) in the brains of patients with Alzheimer's disease (AD) and patients with several other tauopathies. Here, we show that the abnormally hyperphosphorylated from AD brain cytosol (AD P-) self-aggregates into PHF-like structures on incubation at pH 6.9 under reducing conditions at 35°C during 90 min. In vitro dephosphorylation, but not deglycosylation, of AD P-inhibits its self-association into PHF. Furthermore, hyperphosphorylation induces self-assembly of each of the six isoforms into tangles of PHF and straight filaments, and the microtubule binding domains͞repeats region in the absence of the rest of the molecule can also self-assemble into PHF. Thus, it appears that self-assembles by association of the microtubule binding domains͞repeats and that the abnormal hyperphosphorylation promotes the self-assembly of into tangles of PHF and straight filaments by neutralizing the inhibitory basic charges of the flanking regions.

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 fitly on dephosphorylation. Abnormally phosphorylated tau (AD P-tau) isolated from brain homogenate of AD patients had little activity, and upon dephosphorylation with alkaline phospbatase, 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.In the brains of patients with Alzheimer disease (AD), the cytoskeleton is progressively disrupted and displaced by the appearance of bundles of paired helical filaments (PHF), which are composed mainly ofhyperphosphorylated forms of tau protein (1, 2). Unlike normal tau, which contains two or three phosphate groups, the soluble hyperphosphorylated tau from AD brain (AD P-tau) contains 5-9 mol of phosphate per mol of the protein (3). Levels of tau are severalfold higher in AD than in the age-matched control brains, and this increase is in the form of the abnormally phosphorylated tau (4). Neurons with neurofibrillary tangles of PHF lack microtubules, and microtubule assembly from brain cytosol in the absence of an added polycation, DEAE-dextran, is not observed (5). The reason for this disruption might therefore be some alteration in either tau or other microtubuleassociated proteins. In AD brain, tau can be isolated from different pools: (i) a cytosolic fraction, (ii) abnormally phosphorylated tau that is not polymerized into PHF and sediments at 200,000 x g, and (iii) as a component of PHF. To understand the role of the abnormal phosphorylation of tau in microtubule disruption in AD brain, we studied the ability of the normal cytosolic tau and the AD P-tau to bind to tubulin and to promote microtubule assembly and investigated the effect of alkaline phosphatase treatment of tau on microtubule assembly. The studies described in this paper suggest that the abnormal phosphorylation of tau is a likely cause of the breakdown of the microtubule system in AD because the AD P-tau does not bind to tubulin and inhibits the in vitro assembly ofnormal tau and tubulin into microtubules. The altered tau inhibits microtubule assembly, probably through its binding to normal tau, making the latter unavailable for interaction with tubulin.

Cystic fibrosis (CF) patients are hypersusceptible to chronic Pseudomonas aeruginosa lung infections. Cultured human airway epithelial cells expressing the ΔF508 allele of the cystic fibrosis transmembrane conductance regulator (CFTR) were defective in uptake of P. aeruginosa compared with cells expressing the wild-type allele. Pseudomonas aeruginosa lipopolysaccharide (LPS)-core oligosaccharide was identified as the bacterial ligand for epithelial cell ingestion; exogenous oligosaccharide inhibited bacterial ingestion in a neonatal mouse model, resulting in increased amounts of bacteria in the lungs. CFTR may contribute to a host-defense mechanism that is important for clearance of P. aeruginosa from the respiratory tract.Among the most serious manifestations of CF are chronic pulmonary infections with the bacterium P. aeruginosa. The basis for hypersusceptibility of CF patients to this bacterium is not well understood, and the role of mutant CFTR, if any, is not clear. Binding and internalization of respiratory pathogens by epithelial cells followed by desquamation could be an important mechanism for clearing bacteria from the lung. This mechanism has been shown to be important in protecting against bladder infections (1).To investigate whether the most common and severe CFTR mutation (ΔF508) affected uptake of P. aeruginosa, we performed bacterial invasion assays (2) with four cell lines: CFT1, an airway epithelial cell line derived from a CF patient homozygous for ΔF508 CFTR and that is transformed with human papilloma virus 18 E6/E7; CFT1-ΔF508, which expresses a third copy of ΔF508 CFTR introduced by a retrovirus; CFT1-LC3, which expresses a control gene (β-galactosidase) introduced by the same retrovirus; and CFT1-LCFSN, which expresses a functional wild-type human CFTR gene (3). We tested a standard laboratory strain of P. aeruginosa, designated PAOI, and two nonmucoid, LPSsmooth clinical isolates from CF patients (4). Compared with CFT1-LCFSN cells, the three lines expressing ΔF508 CFTR internalized significantly fewer bacterial cells (Fig. 1A). The ΔF508 mutation causes inefficient processing of CFTR, a defect that is partially corrected if the cells are grown at 26°C (5). When epithelial cells were cultured for 72 hours at 26°C there were no longer significant differences in uptake of the P. aeruginosa strains by the cells expressing wild-type or mutant CFTR (Fig. 1B). Because the overall uptake of bacteria at 26°C was low, we performed additional experiments with cells grown for 72 hours at 26°C in which the invasion assay was performed at 37°C for 3 hours, conditions under which surface expression of mutant ΔF508 CFTR is maintained (5). No significant difference in bacterial cell uptake was measured (Fig. 1C), and overall amounts of internalization approached those of the CFT1-LCFSN cells at 37°C. Returning cells expressing ΔF508 CFTR to 37°C for 24 hours after growth for 72 hours at 26°C removes CFTR from the cell surface (5); under these conditions internalization of the bacterial strains ...

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The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel, but its relationship to the primary clinical manifestation of CF, chronic Pseudomonas aeruginosa pulmonary infection, is unclear. We report that CFTR is a cellular receptor for binding, endocytosing, and clearing P. aeruginosa from the normal lung. Murine cells expressing recombinant human wild-type CFTR ingested 30-100 times as many P. aeruginosa as cells lacking CFTR or expressing mutant ⌬F508 CFTR protein.Purified CFTR inhibited ingestion of P. aeruginosa by human airway epithelial cells. The first extracellular domain of CFTR specifically bound to P. aeruginosa and a synthetic peptide of this region inhibited P. aeruginosa internalization in vivo, leading to increased bacterial lung burdens. CFTR clears P. aeruginosa from the lung, indicating a direct connection between mutations in CFTR and the clinical consequences of CF.

Microtubule-associated protein T is abnormally hyperphosphorylated and aggregated in affected neurons of Alzheimer disease brain . This hyperphosphorylated -r can be dephosphorylated at some of the abnormal phosphorylated sites by purified protein phosphatase-1, 2A, and 213 in vitro . In the present study, we have developed an assay to measure protein phosphatase activity toward -r-1 sites (Ser' 9'/Ser 212) using the hyperphosphorylated T isolated from Alzheimer disease brain as substrate . Using this assay, we have identified that in normal brain, protein phosphatase-2A and 213 and, to a lesser extent, 1 are involved in the dephosphorylation of r. The Km values of dephosphorylation of the hyperphosphorylated T by protein phosphatase-2A and 2B are similar. The T phosphatase activity is decreased by -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 r in Alzheimer disease . Key Words : Microtubule-associated protein T-Protein phosphatase-Protein dephosphorylation-Alzheimer disease .

Homozygous mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis (CF). In the heterozygous state, increased resistance to infectious diseases may maintain mutant CFTR alleles at high levels in selected populations. Here we investigate whether typhoid fever could be one such disease. The disease is initiated when Salmonella typhi enters gastrointestinal epithelial cells for submucosal translocation. We found that S. typhi, but not the related murine pathogen S. typhimurium, uses CFTR for entry into epithelial cells. Cells expressing wild-type CFTR internalized more S. typhi than isogenic cells expressing the most common CFTR mutation, a phenylalanine deleted at residue 508 (delta508). Monoclonal antibodies and synthetic peptides containing a sequence corresponding to the first predicted extracellular domain of CFTR inhibited uptake of S. typhi. Heterozygous deltaF508 Cftr mice translocated 86% fewer S. typhi into the gastrointestinal submucosa than wild-type Cftr mice; no translocation occurred in deltaF508 Cftr homozygous mice. The Cftr genotype had no effect on the translocation of S. typhimurium. Immunoelectron microscopy revealed that more CFTR bound to S. typhi in the submucosa of Cftr wild-type mice than in deltaF508 heterozygous mice. We conclude that diminished levels of CFTR in heterozygotes may decrease susceptibility to typhoid fever.

Microbial capsular antigens are effective vaccines but are chemically and immunologically diverse, resulting in a major barrier to their use against multiple pathogens. A β-(1→6)-linked poly-N-acetyl-D-glucosamine (PNAG) surface capsule is synthesized by four proteins encoded in genetic loci designated intercellular adhesion in Staphylococcus aureus or polyglucosamine in selected Gram-negative bacterial pathogens. We report that many microbial pathogens lacking an identifiable intercellular adhesion or polyglucosamine locus produce PNAG, including Gram-positive, Gram-negative, and fungal pathogens, as well as protozoa, e.g., Trichomonas vaginalis, Plasmodium berghei, and sporozoites and blood-stage forms of Plasmodium falciparum. Natural antibody to PNAG is common in humans and animals and binds primarily to the highly acetylated glycoform of PNAG but is not protective against infection due to lack of deposition of complement opsonins. Polyclonal animal antibody raised to deacetylated glycoforms of PNAG and a fully human IgG1 monoclonal antibody that both bind to native and deacetylated glycoforms of PNAG mediated complement-dependent opsonic or bactericidal killing and protected mice against local and/or systemic infections by Streptococcus pyogenes, Streptococcus pneumoniae, Listeria monocytogenes, Neisseria meningitidis serogroup B, Candida albicans, and P. berghei ANKA, and against colonic pathology in a model of infectious colitis. PNAG is also a capsular polysaccharide for Neisseria gonorrhoeae and nontypable Hemophilus influenzae, and protects cells from environmental stress. Vaccination targeting PNAG could contribute to immunity against serious and diverse prokaryotic and eukaryotic pathogens, and the conserved production of PNAG suggests that it is a critical factor in microbial biology.immunotherapy | infectious diseases | malaria | carbohydrates | animal models