Arginine deiminases: therapeutic tools in the etiology and pathogenesis of Alzheimer's disease

There is, at present, no definitive pre-mortem diagnostic tool for Alzheimer's disease, (AD) which relates to a poor understanding of its etiology. Brains of AD patients contain large amounts of the toxic plaque-forming beta-amyloid1-42 fragment in addition to elevated concentrations of the amino acid L-arginine. This work proposes that lowering levels of arginine in the astrocytes surrounding amyloid plaques may serve as a therapeutic tool in this neurodegenerative disorder. Arginine deiminase (ADI), from Pseudomonas aeruginosa, and peptidylarginine deiminase [PAD II], from bovine brain, are inhibited by amyloid peptides that contain arginine (amyloid1-42) and those that have no arginine (amyloid12-28/22-35). Enhanced activity of PAD II is noted with free L-arginine.     

Abcg5/Abcg8-independent pathways contribute to hepatobiliary cholesterol secretion in mice

The ATP-binding cassette (ABC) half-transporters ABCG5 and ABCG8 heterodimerize into a functional complex that mediates the secretion of plant sterols and cholesterol by hepatocytes into bile and their apical efflux from enterocytes. We addressed the putative rate-controlling role of Abcg5/Abcg8 in hepatobiliary cholesterol excretion in mice during (maximal) stimulation of this process. Despite similar bile salt (BS) excretion rates, basal total sterol and phospholipid (PL) output rates were reduced by 82% and 35%, respectively, in chow-fed Abcg5(-/-) mice compared with wild-type mice. When mice were infused with the hydrophilic BS tauroursodeoxycholate, similar relative increases in bile flow, BS output, PL output, and total sterol output were observed in wild-type, Abcg5(+/-), and Abcg5(-/-) mice. Maximal cholesterol and PL output rates in Abcg5(-/-) mice were only 15% and 69%, respectively, of wild-type values. An infusion of increasing amounts of the hydrophobic BS taurodeoxycholate increased cholesterol excretion by 3.0- and 2.4-fold in wild-type and Abcg5(-/-) mice but rapidly induced cholestasis in Abcg5(-/-) mice. Treatment with the liver X receptor (LXR) agonist T0901317 increased the maximal sterol excretion capacity in wild-type mice (fourfold), concomitant with the induction of Abcg5/Abcg8 expression, but not in Abcg5(-/-) mice. In a separate study, mice were fed chow containing 1% (wt/wt) cholesterol. As expected, hepatic expression of Abcg5 and Abcg8 was strongly induced (fivefold and fourfold) in wild-type but not LXR-alpha-deficient (Lxra(-/-)) mice. Surprisingly, hepatobiliary cholesterol excretion was increased to the same extent, i.e., 2.2-fold in wild-type mice and 2.0-fold in Lxra(-/-) mice, upon cholesterol feeding. Our data confirm that Abcg5, as part of the Abcg5/Abcg8 heterodimer, strongly controls hepatobiliary cholesterol secretion in mice. However, our data demonstrate that Abcg5/Abcg8 heterodimer-independent, inducible routes exist that can significantly contribute to total hepatobiliary cholesterol output.     

In vivo heteromer formation. Expression of soluble betaA4-crystallin requires coexpression of a heteromeric partner

The beta-crystallins are a family of long-lived, abundant structural proteins that are coexpressed in the vertebrate lens. As beta-crystallins form heteromers, a process that involves transient exposure of hydrophobic interfaces, we have examined whether in vivobeta-crystallin assembly is enhanced by protein chaperones, either small heat shock proteins, Hsp27 or alphaB-crystallin, or Hsp70. We show here that betaA4-crystallin is abundantly expressed in HeLa cells, but rapidly degraded, irrespective of the presence of Hsp27, alphaB-crystallin or Hsp70. Degradation is even enhanced by Hsp70. Coexpression of betaA4-crystallin with betaB2-crystallin yielded abundant soluble betaA4-betaB2-crystallin heteromers; betaB1-crystallin was much less effective in solubilizing betaA4-crystallin. As betaB2-crystallin competed for betaA4-crystallin with Hsp70 and the proteasomal degradation pathway, betaB2-crystallin probably captures an unstable betaA4-crystallin intermediate. We suggest that the proper folding of betaA4-crystallin is not mediated by general chaperones but requires a heteromeric partner, which then also acts as a dedicated chaperone towards betaA4-crystallin.     

Crystal structure of truncated human betaB1-crystallin

Crystallins are long-lived proteins packed inside eye lens fiber cells that are essential in maintaining the transparency and refractive power of the eye lens. Members of the two-domain betagamma-crystallin family assemble into an array of oligomer sizes, forming intricate higher-order networks in the lens cell. Here we describe the 1.4 angstroms resolution crystal structure of a truncated version of human betaB1 that resembles an in vivo age-related truncation. The structure shows that unlike its close homolog, betaB2-crystallin, the homodimer is not domain swapped, but its domains are paired intramolecularly, as in more distantly related monomeric gamma-crystallins. However, the four-domain dimer resembles one half of the crystallographic bovine betaB2 tetramer and is similar to the engineered circular permuted rat betaB2. The crystal structure shows that the truncated betaB1 dimer is extremely well suited to form higher-order lattice interactions using its hydrophobic surface patches, linker regions, and sequence extensions.     

Periods of intermittent hypoxic apnea can alter chemoreflex control of sympathetic nerve activity in humans

Obstructive sleep apnea is associated with sustained elevation of muscle sympathetic nerve activity (MSNA) and altered chemoreflex control of MSNA, both of which likely play an important role in the development of hypertension in these patients. Additionally, short-term exposure to intermittent hypoxic apneas can produce a sustained elevation of MSNA. Therefore, we tested the hypothesis that 20 min of intermittent hypoxic apneas can alter chemoreflex control of MSNA. Twenty-one subjects were randomly assigned to one of three groups (hypoxic apnea, hypercapnic hypoxia, and isocapnic hypoxia). Subjects were exposed to 30 s of the perturbation every minute for 20 min. Chemoreflex control of MSNA was assessed during baseline, 1 min posttreatment, and every 15 min throughout 180 min of recovery by the MSNA response to a single hypoxic apnea. Recovery hypoxic apneas were matched to a baseline hypoxic apnea with a similar nadir oxygen saturation. A significant main effect for chemoreflex control of MSNA was observed after 20 min of intermittent hypoxic apneas (P <0.001). The MSNA response to a single hypoxic apnea was attenuated 1 min postexposure compared with baseline (P <0.001), became augmented within 30 min of recovery, and remained augmented through 165 min of recovery (P <0.05). Comparison of treatment groups revealed no differences in the chemoreflex control of MSNA during recovery (P=0.69). These data support the hypothesis that 20 min of intermittent hypoxic apneas can alter chemoreflex control of MSNA. Furthermore, this response appears to be mediated by hypoxia.     

Subunit exchange demonstrates a differential chaperone activity of calf alpha-crystallin toward beta LOW- and individual gamma-crystallins

The chaperone activity of native alpha-crystallins toward beta(LOW)- and various gamma-crystallins at the onset of their denaturation, 60 and 66 degrees C, respectively, was studied at high and low crystallin concentrations using small angle x-ray scattering (SAXS) and fluorescence energy transfer (FRET). The crystallins were from calf lenses except for one recombinant human gamma S. SAXS data demonstrated an irreversible doubling in molecular weight and a corresponding increase in size of alpha-crystallins at temperatures above 60 degrees C. Further increase is observed at 66 degrees C. More subtle conformational changes accompanied the increase in size as shown by changes in environments around tryptophan and cysteine residues. These alpha-crystallin temperature-induced modifications were found necessary to allow for the association with beta(LOW)- and gamma-crystallins to occur. FRET experiments using IAEDANS (iodoacetylaminoethylaminonaphthalene sulfonic acid)- and IAF (iodoacetamidofluorescein)-labeled subunits showed that the heat-modified alpha-crystallins retained their ability to exchange subunits and that, at 37 degrees C, the rate of exchange was increased depending upon the temperature of incubation, 60 or 66 degrees C. Association with beta(LOW)- (60 degrees C) or various gamma-crystallins (66 degrees C) resulted at 37 degrees C in decreased subunit exchange in proportion to bound ligands. Therefore, beta(LOW)- and gamma-crystallins were compared for their capacity to associate with alpha-crystallins and inhibit subunit exchange. Quite unexpectedly for a highly conserved protein family, differences were observed between the individual gamma-crystallin family members. The strongest effect was observed for gamma S, followed by h gamma Srec, gamma E, gamma A-F, gamma D, gamma B. Moreover, fluorescence properties of alpha-crystallins in the presence of bound beta(LOW)-and gamma-crystallins indicated that the formation of beta(LOW)/alpha- or gamma/alpha-crystallin complexes involved various binding sites. The changes in subunit exchange associated with the chaperone properties of alpha-crystallins toward the other lens crystallins demonstrate the dynamic character of the heat-activated alpha-crystallin structure.     

Tubulin Polyglycylation: Differential Posttranslational Modification of Dynamic Cytoplasmic and Stable Axonemal Microtubules in Paramecium

Polyglycylation, a posttranslational modification of tubulin, was discovered in the highly stable axonemal microtubules of Paramecium cilia where it involves the lateral linkage of up to 34 glycine units per tubulin subunit. The observation of this type of posttranslational modification mainly in axonemes raises the question as to its relationship with axonemal organization and with microtubule stability. This led us to investigate the glycylation status of cytoplasmic microtubules that correspond to the dynamic microtubules in Paramecium. Two anti-glycylated tubulin monoclonal antibodies (mAbs), TAP 952 and AXO 49, are shown here to exhibit different affinities toward mono- and polyglycylated synthetic tubulin peptides. Using immunoblotting and mass spectrometry, we show that cytoplasmic tubulin is glycylated. In contrast to the highly glycylated axonemal tubulin, which is recognized by the two mAbs, cytoplasmic tubulin reacts exclusively with TAP 952, and the α- and β- tubulin subunits are modified by only 1–5 and 2–9 glycine units, respectively. Our analyses suggest that most of the cytoplasmic tubulin contains side chain lengths of 1 or 2 glycine units distributed on several glycylation sites. The subcellular partition of distinct polyglycylated tubulin isoforms between cytoplasmic and axonemal compartments implies the existence of regulatory mechanisms for glycylation. By following axonemal tubulin immunoreactivity with anti-glycylated tubulin mAbs upon incubation with a Paramecium cellular extract, the presence of a deglycylation enzyme is revealed in the cytoplasm of this organism. These observations establish that polyglycylation is reversible and indicate that, in vivo, an equilibrium between glycylating and deglycylating enzymes might be responsible for the length of the oligoglycine side chains of tubulin.     

Germinal centres and the B-cell system. V

Summary Thoracic duct lymphocytes (TDL) were studied with respect to their capacity to give rise to germinal centres (GC) and to form primary antibody in an adoptive transfer system of the rat. Challenge with sheep erythrocytes (SRBC) 24h after lethal irradiation (900 rads) and syngeneic TDL reconstitution (10⁸) lead to conspicuous GC activity already 7 days after transfer. In contrast, using syngeneic bone marrow (BM) in the adoptive transfer system, no GC formation was observed over the period studied (14 days after reconstitution). Reconstitution experiments using in vivo-separated T-TDL (1–5 % s-Ig⁺) and B-TDL (>90% s-Ig⁺) subpopulations, either separately or in combination, indicated that GC originate from B-TDL but require T-TDL for induction.Abbreviations BM Bone Marrow - TDL Thoracic Duct Lymphocytes - GC Germinal Centre - SRBC Sheep Red Blood Cells - GCDC Germinal Centre-Derived Cells - GCPC Germinal Centre-Precursor Cells - DAB Mineral Salt Solution Dulbecco A + B - FCS Fetal Calf Serum - PALS Peri-Arteriolar Lymphocyte Sheath - AFCP Antibody-Forming Cell Precursors