Tau Released from Paired Helical Filaments with Formic Acid or Guanidine Is Susceptible to Calpain-Mediated Proteolysis

Abstract: Paired helical filaments (PHFs), a characteristic neuropathologic finding in Alzheimer's disease brain, are abnormal fibrillary forms of hyperphosphorylated tau (PHF-tau), which have been shown to be highly resistant to calpain digestion. Either excessive phosphorylation or fibrillary arrangement of tau proteins in PHFs may play a role in proteolytic resistance by limiting access to calpain recognition/digestion sites. To determine the contribution of the fibrillary conformation, isolated PHFs were subjected to treatment with either formic acid or guanidine. Both procedures effectively abolished the fibrillary structure of PHF but preserved PHF-tau immunoreactivity using a panel of antibodies that recognize nonphosphorylated and phosphorylated epitopes. These treatments also significantly increased the sensitivity of PHF-tau polypeptides to calpain proteolysis as shown by significant decreases in the half-life ( t _1/2) from the infinite with native PHF to 44 min and 4.4 min in formic acid- or guanidine-treated samples, respectively. In contrast, the sensitivity of normal fetal tau (3.4 min) was either decreased (5.9 min) or unaffected (3.6 min) by similar treatment. Our results indicate that after guanidine treatment, the sensitivity of PHF to calpain resembles that of fetal tau. These results strongly suggest that the fibrillary structure of PHF-tau, rather than hyperphosphorylation, is the major factor responsible for the resistance of abnormal filaments to calpain-mediated proteolysis.     

Protamines in plant sperm

Sperm protamines have been isolated from representatives of three major plant groups: algae (Chara corallina ), bryophytes ( Marchantia polymorpha), and ferns ( Marsilea vestitia ). We previously reported the complete displacement of histones by protamines in Marchantia (Reynolds W F & Wolfe, S L, Exp cell res 116 (1978) 269 [8] ). Marchantia protamines appear as four components on acid-urea gels, whereas Chara and Marsilea protamines comigrate as a single band with a mobility comparable to salmon protamine. The amino acid compositions of the plant protamines show these to be arginine-rich, highly basic (35-42%) proteins which display overall similarity in amino acid composition (84-91%). The molecular weights of Chara and Marsilea protamines are approx. 4700-5300 D.     

Ammonium-limited growth and uptake by Oscillatoria agardhii in chemostat cultures

Growth of Oscillatoria agardhil was studied in ammonium-limited chemostat cultures, at various dilution rates (=growth rates, μ). The uptake kinetics for ammonium of nitrogen (ammonium or nitrate)-limited chemostat cultures also was investigated. The kinetics of ammonium-limited growth could be adequately described by both the Monod and Droop equations, and were closely similar to the nitrate-limited growth kinetics of this species. The uptake kinetics for ammonium showed similarities as well as differences with the uptake kinetics for nitrate. The similarities were apparent in the uptake capacity values for ammonium and nitrate , which were identical, high and independent of μ. The differences were to be found in the half-saturation constants for ammonium uptake and nitrate uptake , the former being hardly influenced by μ. A consitutive, high affinity, system is likely to operate in the uptake and assimilation of ammonium by nitrogen-limited O. agardhii. The use of ammonium uptake parameters in studies of growth-limiting factors in nature can provide information as to whether a nitrogen-limitation prevails in natural habitats of this species.     

Molecular cloning of rat intestinal mucin. Lack of conservation between mammalian species

We have prepared antisera to deglycosylated rat intestinal mucin and used it to obtain immunoreactive clones from a rat jejunum cDNA library. Four of these clones were sequenced, and all were found to be partial cDNAs that contained 18-base pair tandem repeats characteristic of a mucin. These cDNAs encoded a repetitive peptide with a consensus sequence of TTTPDV. Thus, they bear little resemblance to either of the two human intestinal mucin cDNAs isolated previously (Gum, J. R., Byrd, J. C., Hicks, J. W., Toribara, N. W., Lamport, D. T. A., and Kim, Y. S. (1989) J. Biol. Chem. 264, 6480-6487 and Gum, J. R., Hicks, J. W., Swallow, D. M., Lagace, R. E., Byrd, J. C., Lamport, D. T. A., Siddiki, B., and Kim, Y. S. (1990) Biochem. Biophys. Res. Commun. 171, 407-415). One of these rat mucin clones, designated RMUC 176, was chosen for further analysis. This clone recognized a band of approximately 9 kilobases when used to probe RNA blots. A strong hybridization band was present using rat small intestine and colon RNA but was not detectable when RNA isolated from heart, liver, or kidney was tested. The RMUC 176 clone and the two previously isolated human intestinal mucin cDNA clones were used to probe blots prepared from BamHI-digested DNA of various species. Here, the human probes detected fragments present only in human and chimpanzee DNA, whereas the RMUC 176 clone recognized fragments only in rat and mouse DNA. Thus, the repetitive portions of intestinal mucin genes are apparently not well conserved between phylogenetically distant species.     

Dosage of the smallest chromosome affects both the yeast-hyphal transition and the white-opaque transition of Candida albicans WO-1.

The WO-1 strain of Candida albicans is capable of alternating between two highly distinct yeast cell types termed white and opaque (E. H. A. Rikkerrink, B. B. Magee, and P. T. Magee, J. Bacteriol. 170:895-899, 1988; B. Slutsky, M. Staebell, J. Anderson, L. Risen, M. Pfaller, and D. R. Soll, J. Bacteriol. 169:189-197, 1987). We have isolated WO-1 mutants that show a marked deficiency at being able to switch from the white form to the opaque form under conditions normally favorable for this transition. Pulsed-field electrophoresis demonstrated that one of the initial two spontaneous nonswitching mutants lacked the smallest chromosome that is normally present in WO-1. The availability of a WO-1 derivative whose only functional ADE2 gene is located on this small chromosome made possible, through the induction of chromosome nondisjunction, the isolation of numerous new mutants missing this chromosome as well as mutants containing two copies of the chromosome. Mutants missing the smallest chromosome showed a greatly diminished ability to produce opaque sectors and to produce germ tubes in the presence of human serum. Mutants containing two copies of the small chromosome showed an increased ability to produce germ tubes. These results indicate that this small chromosome carries one or more genes involved in both the white-opaque switch and the yeast-hyphal switch.     

Differential localizations of and requirements for the two Drosophila ninaC kinase/myosins in photoreceptor cells

The ninaC gene encodes two retinal specific proteins (p132 and p174) consisting of a protein kinase domain joined to a domain homologous to the head region of the myosin heavy chain. The putative myosin domain of p174 is linked at the COOH-terminus to a tail which has some similarities to myosin-I tails. In the current report, we demonstrate that the ninaC mutation results in light- and age-dependent retinal degeneration. We also show that ninaC flies display an electrophysiological phenotype before any discernible retinal degeneration indicating that the electrophysiological defect is the primary effect of the mutation. This suggests that ninaC has a role in phototransduction and that the retinal degeneration is a secondary effect resulting from the defect in phototransduction. To examine the requirements for the individual ninaC isoforms, mutant alleles were generated which express only p132 or p174. Elimination of p174 resulted in a ninaC phenotype as strong as the null allele; however, elimination of p132 had little if any effect. As a first step in investigating the basis for the difference in requirements for p174 and p132 we performed immuno-localization at the electron microscopic level and found that the two isoforms display different subcellular distributions in the photoreceptor cells. The p132 protein is restricted primarily to the cytoplasm and p174 to the rhabdomeres, the microvillar structure which is the site of action of many of the steps in phototransduction. This suggests that the p174 myosin-I type tail is the domain responsible for association with the rhabdomeres and that the substrate for the p174 putative kinase may be a rhabdomeric protein important in photo-transduction.     

The human MUC2 intestinal mucin has cysteine-rich subdomains located both upstream and downstream of its central repetitive region.

The human MUC2 mucin is a large secretory glycoconjugate that coats the epithelia of the intestines, airways, and other mucus membrane-containing organs. Previous work has shown that this mucin contains an extended tandem repeat-containing domain rich in Thr and Pro. In the present work we describe two additional regions of this mucin located both upstream and downstream of the tandem repeat array. The carboxyl-terminal domain contains 984 residues and can be divided into mucin-like (139 residues) and cysteine-rich (845 residues) subdomains. This latter subdomain exhibits varying degrees of sequence similarity to a wide range of mucins and mucin-like proteins including those isolated from rats, pigs, cows, and frogs. We also report here the sequence of 1270 residues lying immediately upstream of the tandem repeats. This region contains a repetitive, mucin-like subdomain and a second cysteine-rich stretch of more than 700 residues. Both cysteine-rich subdomains of this mucin have sequence similarity with von Willebrand factor, a serum protein that exists as a disulfide-linked polymer. This suggests that these cysteine-rich subdomains are important in the catenation of mucin monomers into oligomers, the structures that confer viscoelasticity upon mucus.     

Interactions between Pseudomonas aeruginosa and iodophor germicides in milking parlor udder wash water systems.

In a field study of 29 dairy farms, Pseudomonas aeruginosa was isolated more frequently (P = 0.05) from milking parlor udder wash water systems containing iodophor germicides than from those with no germicide. Most available iodine (AI2) concentrations were below the recommended level of 25 ppm (25 microgram/ml). Rubber and polyvinyl chloride hoses caused rapid decreases in the AI2 concentrations of 25 ppm iodophor solutions. AI2 dropped from 25 ppm to 6 ppm or less in 240 min for solutions contained in either polyvinyl chloride or rubber, compared with solutions in glass, which were unchanged in 240 min. Addition of inactivated iodophor solution to aqueous cultures resulted in significantly higher (P less than 0.05) numbers of P. aeruginosa at 10 and 24 h postinoculation. P. aeruginosa was grown in polyvinyl chloride tubing and exposed twice daily to 0, 10, or 25 ppm of AI2. None of the exposure concentrations eliminated the bacteria from the hoses, and bacterial numbers were not significantly different in hoses exposed to 0 and 10 ppm by the eighth treatment day. Bacteria taken from the water in these hoses were exposed to different concentrations of iodophor solution. Iodophor concentrations which will kill 50% of P. aeruginosa cultures previously exposed to 0, 10, and 25 ppm of AI2 were predicted to be 3.0, 11.8, and 20.8 ppm, respectively.     

Chromosomal rearrangements associated with morphological mutants provide a means for genetic variation of Candida albicans.

At frequencies as high as 1.4%, the pathogenic yeast Candida albicans spontaneously gave rise to morphological mutants exhibiting more than 20 different types of abnormal colonies; approximately two-thirds of the mutants were stable, while the other one-third were unstable and produced mixtures of different colonial forms at very high rates. Abnormal electrophoretic karyotypes were observed for all of the 14 mutants that were examined, indicating that they were associated with different types of single and multiple gross chromosomal rearrangements. Because C. albicans is asexual and does not go through a meiotic cycle, we suggest that the high frequency of chromosomal rearrangements provides a means for genetic variation in this organism.