Differences in the abilities of human tau isoforms to promote microtubule assembly.

Three isoforms of human tau protein were compared for their abilities to induce microtubule assembly. The three isoforms, tau 3 (tau containing three microtubule-binding domains), tau 4 (tau containing four microtubule-binding domains) and tau 4L (tau containing four microtubule binding domains plus a 58-amino-acid insert near the N-terminus) were expressed in E. coli and purified using ammonium sulfate precipitation, ion exchange, and size exclusion chromatography. All three isoforms induced microtubule assembly at micromolar concentrations and showed similar critical concentrations for assembly of 0.4-0.45 microM. However, tau 4 induced microtubule formation at a rate five- to tenfold faster than either tau 3 or tau 4L. The rate of microtubule elongation seen with tau 4 was twofold greater than with tau 3 or tau 4L, suggesting that the faster rate of microtubule assembly seen with tau 4 was due, at least in part, to faster elongation. Tau 4 induced a greater number of microtubules to form at steady state than did tau 3 or tau 4L. The microtubules generated with each tau isoform had similar steady-state length distributions and were equally susceptible to cold-induced disassembly. These results indicate that the additional microtubule-binding domain in tau 4 enhances microtubule assembly, while the 58-amino-acid insert negates the stimulatory effect of the fourth microtubule-binding domain.     

Priming with a very low dose of DNA complexed with cationic block copolymers followed by protein boost elicits broad and long-lasting antigen-specific humoral and cellular responses in mice

Cationic block copolymers spontaneously assemble via electrostatic interactions with DNA molecules in aqueous solution giving rise to micellar structures that protect the DNA from enzymatic degradation both in vitro and in vivo. In addition, we have previously shown that they are safe, not immunogenic and greatly increased antigen-specific CTL responses following six intramuscular inoculations of a very low dose (1 μg) of the vaccine DNA as compared to naked DNA. Nevertheless, they failed to elicit detectable humoral responses against the antigen. To gain further insight in the potential application of this technology, here we show that a shorter immunization protocol based on two DNA intramuscular inoculations of 1 μg of DNA delivered by these copolymers and a protein boost elicits in mice broad (both humoral and cellular) and long-lasting responses and increases the antigen-specific Th1-type T cell responses and CTLs as compared to priming with naked DNA. These results indicate that cationic block copolymers represent a promising adjuvant and delivery technology for DNA vaccination strategies aimed at combating intracellular pathogens.     

Why do Italian people rate their health worse than French people do? An exploration of cross-country differentials of self-rated health

The prevalence of bad self-rated health (SRH) varies considerably across countries. Here we present the results of a cross-national comparative study based on the data of National Health Surveys conducted in France and Italy. According to these data, 11% of the Italian and 6% of the French adult population aged between 45 and 74 rate their health as bad or very bad. This gap may result from differences in population structure regarding the individual characteristics (sociodemographic characteristics, diseases and disabilities, lifestyle, and others) that impact on SRH i.e., a structural effect. It may also be that the link between these characteristics and SRH is “country-specific” i.e., a contextual effect. We use logistic regression models to assess the contribution of both explanations. We find that the structural effect plays a prominent role in the higher prevalence of bad SRH in Italy compared to France.     

Preoperative Chemotherapy Alone and Combined with Preoperative Radiotherapy in Small-Size Breast Cancer

Abstract: Breast conservation surgery is an effective and safe treatment for many breast carcinomas. It may be possible to further limit the extent of resection (or expand the indication for breast conservation) by the application of preoperative chemotherapy and radiotherapy. We explored the feasibility of this in a pilot study.
Seventy-three patients (mean age 48, 63% premenopausal) with confirmed breast cancer, less than 2.5 cm, received chemotherapy (Group A) or chemotherapy plus radiotherapy (Group B) prior to limited resection (tumorectomy). Axillary dissection was always performed. Results: In 6/31 (19%) Group A and 17/42 (40%) Group B patients the tumor was not palpable after preoperative treatment, with complete pathological remission in 1 and 3 cases respectively. Histologic grading, mitosis, cellular alteration, and cellularity evaluations indicated a consistently greater therapeutic effect with chemoradiotherapy than with chemotherapy alone.
In conclusion, radiotherapy appears useful in the preoperative treatment of breast cancer and its use in association with various drug combinations should be further explored.     

Tau protein induces bundling of microtubules in vitro: comparison of different tau isoforms and a tau protein fragment.

Expression of tau protein in non-neuronal cells can result in a redistribution of the microtubule cytoskeleton into thick bundles of tau-containing microtubules (Lewis et al.: Nature 342:498-505, 1989; Kanai et al.: J Cell Biol 109:1173-1184, 1989). We reconstituted microtubule bundles using purified tubulin and tau in order to study the assembly of these structures. Taxol-stabilized tubulin polymers were incubated with various concentrations of recombinant human tau and examined by electron microscopy. With increasing concentrations of tau 3 (tau isoform containing three microtubule binding domains) or tau 4 (isoform containing four microtubule binding domains) the microtubules changed orientation from a random distribution to loosely and tightly packed parallel arrays and then to thick cables. In contrast, tau 4L, the tau isoform containing four microtubule binding domains plus a 58-amino acid insert near the N-terminus, showed minimal bundling activity. tau 4-induced bundling could be inhibited by the addition of 0.5 M NaCl or 0.4 mM estramustine phosphate, conditions which are known to inhibit tau binding to microtubules. A tau construct that contained only the microtubule binding domains plus 19 amino acids to the C-terminus was fully capable of bundling microtubules. Phosphorylation of tau 3 with cAMP-dependent protein kinase had no effect on its ability to induce microtubule bundling. These results indicate that tau protein is directly capable of bundling microtubules in vitro, and suggests that different tau isoforms differ in their ability to bundle microtubule filaments.     

Pyridoxol L,2-pyrrolidon-5 carboxylate prevents active fibroplasia in CCl4-treated rats.

In the present study we evaluated the protective activity of pyridoxol L,2-pyrrolidon-5 carboxylate (metadoxine) against CCl4 intoxication in rats, especially in relation to liver fibrosis. After 6 consecutive weeks of CCl4 treatment, the animals developed liver fibrosis and inflammation as revealed by histological analysis which also included semiquantitative scoring of these features. In addition the serum levels of the immunoreactive prolyl hydroxylase (SIRPH), an enzyme involved in the hydroxylation of the procollagen molecule, were significantly higher (44.2 +/- 16.3 micrograms/ml; P less than 0.005) in this group of animals than in controls (26.1 +/- 8.06). On the contrary, animals treated with CCl4 + metadoxine (200 mg/kg i.p.) had less severe liver fibrosis and normal SIRPH levels (21.5 +/- 14.6). These data suggest that metadoxine may be an effective pharmacological tool for preventing the progression of liver disease in rats exposed to CCl4 to cirrhosis.     

Metabolism of 7-(1,3-thiazolidin-2-ylmethyl)theophylline by rat liver microsomes. Evidence for a monooxygenase-dependent step in 1,3-thiazolidine ring cleavage.

Metabolic transformation of the mucoregulator and bronchodilator 7-(1,3-thiazolidin-2-ylmethyl)theophylline was studied in vitro with a rat liver microsomal preparation containing a NADPH-generating system. The only metabolite observed was 7-theophyllinacetaldehyde. In contrast to previous literature pointing out the chemical nature of 2-substituted thiazolidine ring cleavage, the formation of 7-theophyllinacetaldehyde was mediated by monooxygenase-dependent oxidation. Possibly an unstable sulfoxide was the first metabolic product, rapidly converted to 7-theophyllinacetaldehyde by hydrolysis. The sulfoxidation was apparently catalyzed mainly by flavin-containing monooxygenases, as selective thermal inactivation and methymazole significantly reduced the rate of formation of the metabolite. No N7-dealkylation pathway producing theophylline was detected, indicating a high regioselectivity in in vitro metabolism, due to the nucleophilicity of the sulfur atom.     

Immunoreactivity of an antibody to a beta/A4 sequence with Alzheimer paired helical filaments and tau protein.

beta/A4, a peptide that forms the extracellular amyloid fibrils of Alzheimer senile plaques, has also been proposed to be a component of Alzheimer paired helical filaments (PHFs). We compared BR88, an antiserum to amino acids 1-12 of beta/A4, with BR126, an antiserum to the sequence SEKLDFKDRVQS in tau protein, since tau protein is the only confirmed component of PHFs. In enzyme-linked immunosorbent assays (ELISAs), both antibodies reacted with pronase-treated PHFs better after PHFs were treated with guanidine. tau protein shares no sequence homology with beta/A4. Nevertheless, BR88 cross-reacted with human recombinant tau isoforms by ELISA and Western blot analysis with potencies comparable to those for anti-tau antibodies. BR88 reacted with a beta/A4 peptide as well on a molar basis as with tau protein and showed some reactivity to the tubulin-binding region of tau protein. In conclusion, the beta/A4 antiserum BR88 cross-reacts with tau protein, possibly explaining its reactivity with PHFs.