Tau assembly in inducible transfectants expressing wild-type or FTDP-17 tau

Conditional expression systems for 4-repeat wild-type (WT) tau or the corresponding mutants V337M and R406W were established in human neuroglioma H4 cells to study the effect of tau mutations on the physicochemical properties of tau, and to develop a cellular model for the formation of filamentous tau characteristic of frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) and Alzheimer's disease. Upon induction tau expression increased, reaching maximal levels at 5 to 7 days. WT tau was phosphorylated at amino acids T181, S202/T205, T231, and S396/S404. The R406W mutation decreased tau phosphorylation at each of these sites as did the V337M mutation except for S396/S404 sites that increased. Most tau in postnuclear cell lysates was recovered in the supernatant fraction after centrifugation at 200,000 x g. The amount of tau in the pellet fraction increased more in mutant transfectants compared to WT when the induction was extended beyond 5 days. This particulate tau could be partially extracted with salt, Triton X-100, or sarkosyl. Of the transfectants, R406W had the highest proportion of sarkosyl-insoluble tau by day 7. This insoluble fraction was thioflavin S-positive and contained 15- to 5-nm-wide filaments with tau immunoreactivities. The R406W filaments were more abundant than those detected in similar preparations from WT or V337M transfectants. At the light microscopy level, most tau was found with microtubules, or diffusely distributed in the cytoplasm, but none of this appeared thioflavin S-positive. The results suggest that conditional tau transfectants are in a pretangle stage making them an attractive model system for studying intracellular tangle accumulation and for testing potential therapeutic agents as inhibitors for tau aggregation.     

Nutrient signalling in the regulation of human muscle protein synthesis

The mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) are important nutrient- and energy-sensing and signalling proteins in skeletal muscle. AMPK activation decreases muscle protein synthesis by inhibiting mTOR signalling to regulatory proteins associated with translation initiation and elongation. On the other hand, essential amino acids (leucine in particular) and insulin stimulate mTOR signalling and protein synthesis. We hypothesized that anabolic nutrients would be sensed by both AMPK and mTOR, resulting in an acute and potent stimulation of human skeletal muscle protein synthesis via enhanced translation initiation and elongation.
We measured muscle protein synthesis and mTOR-associated upstream and downstream signalling proteins in young male subjects ( n = 14) using stable isotopic and immunoblotting techniques. Following a first muscle biopsy, subjects in the 'Nutrition' group ingested a leucine-enriched essential amino acid–carbohydrate mixture (EAC). Subjects in the Control group did not consume nutrients. A second biopsy was obtained 1 h later. Ingestion of EAC significantly increased muscle protein synthesis, modestly reduced AMPK phosphorylation, and increased Akt/PKB (protein kinase B) and mTOR phosphorylation ( P < 0.05). mTOR signalling to its downstream effectors (S6 kinase 1 (S6K1) and 4E-binding protein 1 (4E-BP1) phosphorylation status) was also increased ( P < 0.05). In addition, eukaryotic elongation factor 2 (eEF2) phosphorylation was significantly reduced ( P < 0.05). Protein synthesis and cell signalling (phosphorylation status) was unchanged in the control group ( P > 0.05).
We conclude that anabolic nutrients alter the phosphorylation status of both AMPK- and mTOR-associated signalling proteins in human muscle, in association with an increase in protein synthesis not only via enhanced translation initiation but also through signalling promoting translation elongation.     

Prion domain interaction responsible for species discrimination in yeast [PSI+] transmission

BACKGROUND: The yeast [PSI+] factor is transmitted by a prion mechanism involving self-propagating Sup35 aggregates. As with mammalian prions, a species barrier prevents prion transmission between yeast species. The N-terminal of Sup35 of Saccharomyces cerevisiae, necessary for [PSI+], contains two species-signature elements-a Gln/Asn-rich region (residues 1-41; designated NQ) that is followed by oligopeptide repeats (designated NR). RESULTS: In this study, we show that S. cerevisiae[PSI+] is transmissible through plasmid shuffling and cytoplasmic transfer to heterotypic Sup35s whose NQ is replaced with the S. cerevisiae NQ. In addition to homology, the N-terminal location is essential for NQ mediated susceptibility to [PSI+] transmission amongst heterotypic Sup35s. In vitro, a swap of NQ of S. cerevisiae Sup35 led to cross seeding of amyloid formation. CONCLUSIONS: These findings suggest that NQ discriminates self from non-self, and is sufficient to initiate [PSI+] transmission irrespective of whether NR is heterotypic. NR as well as NQ alone coalesces into existing [PSI+] aggregates, showing their independent potentials to interact with the identical sequence in the [PSI+] conformer. The role of NQ and NR in [PSI+] prion formation is discussed.     

Factors involved in the sensitivity of different hematopoietic cell lines to infection by subgroup C adenovirus: implication for gene therapy of human lymphocytic malignancies

Gene transfer approaches using viruses such human adenovirus (HAdV) may provide an alternative treatment for diseases involving hematopoietic cells. Better understanding of the cellular mechanisms by which the HAdV introduces DNA into these cells should help in vector design. We examined HAdV intracellular delivery in several cell lines including B and T lymphocytes. We demonstrated that HAdV resistance in most B lymphocytes is the result of moderate HAdV uptake. In contrast, high levels of coxsackie and HAdV receptor (hCAR) are expressed on the surface of HSB2 (T cells), allowing efficient binding and uptake but no transgene expression, probably because of deficient endosomolysis and subsequent exocytose. This work demonstrates the existence of hCAR-dependent and -independent endocytic route in hematopoietic cells. Moreover, it precises the intracellular barriers to be overcome by HAdV in such cells to be infectious and gives previous information's to design new vectors for gene transfer.     

Interaction of adenosine and its phosphorylated derivatives with putative purinergic receptors in the gill vascular bed of rainbow trout

1. The haemodynamic responses of trout gill to pulses of adenosine and related nucleotides were recorded in isolated trout head preparations. 2. Pulses of adenosine and related nucleotides induced a vasoconstriction of arterial gill vessels. Theophylline antagonized the resonse to adenosine but had not influence on its metabolism. 3. Dipyridamole and two adenosine deaminase inhibitors [deoxycoformycin and erythro-9(2-hydroxy-3-nonyl) adenine] had no effect on either the haemodynamic response of adenosine or its deamination and its uptake by gill tissues. 4. The adenosine response was neither mediated by cholinergic nor adrenergic receptors. 5. These results suggest the existence of extracellular "purinergic receptors" in the gills of trout.     

High risk pregnancies in hypopituitary women

BACKGROUND: Various short papers have suggested that pregnancies in women with hypopituitarism are high risk but no formal assessment of pregnancy outcome has yet been reported. METHODS: An audit was carried out concerning the outcome of 18 pregnancies in nine women who underwent ovulation induction in a single centre over 20 years. RESULTS: The live birth rate was 61%, miscarriage rate 28% and mid-trimester uterine death rate 11% with no survivors from four sets of twins. The Caesarian section rate was 100% and half of the live births were on or below the 10th centile for weight. One woman successfully breast-fed. CONCLUSIONS: Women with hypopituitarism have high-risk pregnancies, perhaps because of a uterine defect secondary to endocrine deficiency. Fertility treatment must strive for singleton pregnancies with application of particularly strict criteria to avoid twin pregnancies. Early elective Caesarian section is probably warranted in this group.     

Basophils express a type 2 cytokine profile on exposure to proteases from helminths and house dust mites

The proteolytic activities frequently associated with sources of allergens and parasite secretions have been suggested as important immunomodulators. We have investigated whether the protease activity of the house dust mite allergen Der p1 and the secreted proteases of the hookworm Necator americanus are able to directly induce type 2 cytokine production by basophils. Der p1 and the secretions of N. americanus induced interleukin (IL)-4, IL-5, and IL-13 but not interferon-gamma mRNA in KU812 basophils. Enzyme-linked immunosorbent assay confirmed that IL-4 and IL-13 were secreted. A nonproteolytic antigen failed to induce cytokine expression, and preincubation of Der p1 or N. americanus secretions with protease inhibitors inhibited cytokine expression. Data were confirmed using basophils purified from human peripheral blood. We speculate that this innate mechanism may contribute to the development of a cytokine milieu that could promote immunoglobulin E synthesis, eosinophil recruitment, and the development of type 2 T cells.     

Two novel presenilin-1 mutations (Y256S and Q222H) are associated with early-onset Alzheimer's disease

Mutations in the gene encoding presenilin 1 (PS-1) account for 50% of early-onset familial Alzheimer's disease (EOFAD) cases. In this study, we identified two missense mutations in the coding sequence of the presenilin (PS-1) gene in two EOFAD pedigrees. AD was confirmed in one pedigree by autopsy. Mutation analysis of PCR products amplified from genomic DNA templates showed two novel PS-1 mutations resulting in Gln222His and Tyr256Ser. The two novel mutations are located within predicted transmembrane domains five (TM-5) and six (TM-6), respectively, and are associated with very early ages of onset. The Tyr256Ser is associated with one of the youngest age of AD onset, 25 years, which is consistent with a drastic change in function of the altered PS-1 protein. A morphometric analysis of the cortical degenerative changes of the Tyr256Ser case, showed severe involvement of the primary motor cortex, which correlated well with the pyramidal changes, including tetraspasticity. Immunoblot analysis showed the Tyr256Ser case had the greatest expression of Abeta(1-40) and Abeta(1-42), which was confirmed by ELISA, compared to other PS-1 mutant FAD cases and age-matched controls and, thus, contributes to the severity of the disease pathology.     

A murine TSPY

Sequences homologous to human and bovine TSPY were isolated from M. musculus testicular cDNA, and a nearly full-length gene was polymerase chain reaction (PCR) amplified from mouse genomic DNA. This gene is apparently non-functional. Contrary to the situation encountered in species along the primate and artiodactyl lineages, in which TSPY is moderately repetitive, murine Tspy appears to be single copy. Murine Tspy is located on Yp, i.e. in the same syntenic group as in man. Sequence comparisons of murine, human and bovine TSPY exons suggest that TSPY became non-functional during rodent evolution.