Oligopeptide-mediated acceleration of amyloid fibril formation of amyloid beta(Abeta) and alpha-synuclein fragment peptide (NAC).

The effects of oligopeptides on the secondary structures of Abeta and NAC, a fragment of alpha-synuclein protein, were studied by circular dichroism (CD) spectra. The effects of oligopeptides on the amyloid fibril formation were also studied by fluorescence spectra due to thioflavine-T. The oligopeptides were composed of a fragment of Abeta or NAC and were interposed by acidic or basic amino acid residues. The peptide, Ac-ELVFFAKK-NH2, which involved a fragment Leu-Val-Phe-Phe-Ala at Abeta(17-21), had no effect on the secondary structures of Abeta(1-28) in 60% or 90% trifluoroethanol (TFE) solutions at both pH 3.2 and pH 7.2. However, it showed pronounced effects on the secondary structure of Abeta(1-28) at pH 5.4. The Ac-ELVFFAKK-NH2 reduced the alpha-helical content, while it increased the beta-sheet content of Abeta(1-28). In phosphate buffer solutions at pH 7.0, Ac-ELVFFAKK-NH2 had little effect on the secondary structures of Abeta(1-28). However, it accelerated amyloid fibril formation when monitored by fluorescence spectra due to thioflavine-T. On the other hand, LPFFD, a peptide known as a beta-sheet breaker, caused neither an appreciable extent of change in the secondary structure nor amyloid fibril formation in the same buffer solution. The peptide, Ac-ETVK-NH2, which involved a fragment Thr-Val at NAC(21-22), had no effect on the secondary structure of NAC in 90% TFE and in isotonic phosphate buffer. However, Ac-ETVK-NH2 in water with small amounts of NaN3 and hexafluoroisopropanol greatly increased the beta-sheet content of NAC after standing the solution for more than 1 week. Interestingly, in this solution. Ac-ETVK-NH2, accelerated the fibril formation of NAC. It was concluded that an oligopeptide that involves a fragment of amyloidogenic proteins could be a trigger for the formation of amyloid plaques of the proteins even when it had little effect on the secondary structures of the proteins as monitored by CD spectra for a short incubation time.     

Characterization of Oseltamivir-Resistant 2009 H1N1 Pandemic Influenza A Viruses

Influenza viruses resistant to antiviral drugs emerge frequently. Not surprisingly, the widespread treatment in many countries of patients infected with 2009 pandemic influenza A (H1N1) viruses with the neuraminidase (NA) inhibitors oseltamivir and zanamivir has led to the emergence of pandemic strains resistant to these drugs. Sporadic cases of pandemic influenza have been associated with mutant viruses possessing a histidine-to-tyrosine substitution at position 274 (H274Y) in the NA, a mutation known to be responsible for oseltamivir resistance. Here, we characterized in vitro and in vivo properties of two pairs of oseltaimivir-sensitive and -resistant (possessing the NA H274Y substitution) 2009 H1N1 pandemic viruses isolated in different parts of the world. An in vitro NA inhibition assay confirmed that the NA H274Y substitution confers oseltamivir resistance to 2009 H1N1 pandemic viruses. In mouse lungs, we found no significant difference in replication between oseltamivir-sensitive and -resistant viruses. In the lungs of mice treated with oseltamivir or even zanamivir, 2009 H1N1 pandemic viruses with the NA H274Y substitution replicated efficiently. Pathological analysis revealed that the pathogenicities of the oseltamivir-resistant viruses were comparable to those of their oseltamivir-sensitive counterparts in ferrets. Further, the oseltamivir-resistant viruses transmitted between ferrets as efficiently as their oseltamivir-sensitive counterparts. Collectively, these data indicate that oseltamivir-resistant 2009 H1N1 pandemic viruses with the NA H274Y substitution were comparable to their oseltamivir-sensitive counterparts in their pathogenicity and transmissibility in animal models. Our findings highlight the possibility that NA H274Y-possessing oseltamivir-resistant 2009 H1N1 pandemic viruses could supersede oseltamivir-sensitive viruses, as occurred with seasonal H1N1 viruses.     

The structure of S100A11 fragment explains a local structural change induced by phosphorylation

S100A11 protein is a member of the S100 family containing two EF‐hand motifs. It undergoes phophorylation on residue T10 after cell stimulation such as an increase in Ca²⁺ concentration. Phosphorylated S100A11 can be recognized by its target protein, nucleolin. Although S100A11 is initially expressed in the cytoplasm, it is transported to the nucleus by the action of nucleolin. In the nucleus, S100A11 suppresses the growth of keratinocytes through p21^CIP1/WAF1 activation and induces cell differentiation. Interestingly, the N‐terminal fragment of S100A11 has the same activity as the full‐length protein; i.e. it is phosphorylated in vivo and binds to nucleolin. In addition, this fragment leads to the arrest of cultured keratinocyte growth. We examined the solution structure of this fragment peptide and explored its structural properties before and after phosphorylation. In a trifluoroethanol solution, the peptide adopts the α‐helical structure just as the corresponding region of the full‐length S100A11. Phosphorylation induces a disruption of the N‐capping conformation of the α‐helix, and has a tendency to perturb its surrounding structure. Therefore, the phosphorylated threonine lies in the N‐terminal edge of the α‐helix. This local structural change can reasonably explain why the phosphorylation of a residue that is initially buried in the interior of protein allows it to be recognized by the binding partner. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

A WT1 protein‐derived,naturally processed 16‐mer peptide,WT1332, is a promiscuous helper peptide for induction of WT1‐specific Th1‐type CD4+ T cells

The Wilms' tumor gene WT1 is overexpressed in various tumors, and the WT1 protein has been demonstrated to be an attractive target antigen for cancer immunotherapy. A WT1 protein‐derived 16‐mer peptide, WT1₃₃₂ (KRYFKLSHLQMHSRKH), which was naturally generated through processing in cells and could elicit Th1‐type CD4⁺ helper T cell responses with an HLA‐DRB1*0405‐restriction has previously been identified by us. In the present study, it has been demonstrated that WT1₃₃₂ can induce WT1₃₃₂‐specific CD4⁺ T cell responses with the restriction of not only HLA‐DRB1*0405 but also HLA‐DRB1*1501, ‐DRB1*1502, or ‐DPB1*0901. These HLA class II‐restricted WT1₃₃₂‐specific CD4⁺ T cell lines produced IFN‐γ but neither IL‐4 nor IL‐10 with WT1₃₃₂ stimulation, thus showing a Th1‐type cytokine profile. Furthermore, HLA‐DRB1*1501 or ‐DRB1*1502‐restricted WT1₃₃₂‐specific CD4⁺ T cell lines responded to WT1‐expressing transformed cells in an HLA‐DRB1‐restricted manner, which is consistent with our previous finding that WT1₃₃₂ is a naturally processed peptide. These results indicate that the natural peptide, WT1₃₃₂, is a promiscuous WT1‐specific helper epitope. WT1₃₃₂ is expected to apply to cancer patients with various types of HLA class II as a WT1‐specific helper peptide in combination with HLA class I‐restricted WT1 peptides.     

Occurrence of agmatine pathway for putrescine synthesis in Selenomonas ruminatium

Selenomonas ruminantium synthesizes cadaverine and putrescine from L-lysine and L-ornithine as the essential constituents of its peptidoglycan by a constitutive lysine/ornithine decarboxylase (LDC/ODC). S. ruminantium grew normally in the presence of the specific inhibitor for LDC/ODC, DL-alpha-difluoromethylornithine, when arginine was supplied in the medium. In this study, we discovered the presence of arginine decarboxylase (ADC), the key enzyme in agmatine pathway for putrescine synthesis, in S. ruminantium. We purified and characterized ADC and cloned its gene (adc) from S. ruminantium chromosomal DNA. ADC showed more than 60% identity with those of LDC/ODC/ADCs from Gram-positive bacteria, but no similarity to that from Gram-negative bacteria. In this study, we also cloned the aguA and aguB genes, encoding agmatine deiminase (AguA) and N-carbamoyl-putrescine amidohydrolase (AguB), both of which are involved in conversion from agmatine into putrescine. AguA and AguB were expressed in S. ruminantium. Hence, we concluded that S. ruminantium has both ornithine and agmatine pathways for the synthesis of putrescine.     

Phosphorylation of plasma membrane aquaporin regulates temperature-dependent opening of tulip petals

The opening and closing of tulip petals was reproduced in the dark by changing the temperature from 5 degrees C to 20 degrees C for opening and 20 degrees C to 5 degrees C for closing. The opening process was accompanied by (3)H(2)O transport through the stem from the incubation medium to the petals. A Ca(2+)-channel blocker and a Ca(2+)-chelator inhibited petal opening and (3)H(2)O transport. Several proteins in the isolated plasma membrane fraction were phosphorylated in the presence of 25 micro M Ca(2+) at 20 degrees C. The 31-kDa protein that was phosphorylated, was suggested immunologically as the putative plasma membrane aquaporin (PM-AQP). This phosphorylated PM-AQP clearly reacted with the anti-phospho-Ser. In-gel assay revealed the presence of a 45-kDa Ca(2+)-dependent protein kinase in the isolated plasma membrane. Phosphorylation of the putative PM-AQP was thought to activate the water channel composed of PM-AQP. Dephosphorylation of the phosphorylated PM-AQP was also observed during petal closing at 5 degrees C, suggesting the inactivation of the water channel.     

Anti-AIDS agents. Part 57: Actein, an anti-HIV principle from the rhizome of Cimicifuga racemosa (black cohosh), and the anti-HIV activity of related saponins

Actein (1), a tetracyclic triterpenoid from the rhizome of Cimicifuga racemosa (black cohosh), and 82 related triterpenoid and steroidal saponins isolated from higher plants were evaluated for anti-HIV activity as a continuing study to discover potential anti-AIDS agents from natural products. Actein showed potent activity and another twelve saponins showed moderate activity. The active compounds included two steroidal, seven tetracyclic triterpenoid, and four pentacyclic triterpenoid compounds.     

Occupancy dynamics in small-stream habitats: niches define the responses to floods by two neotropical fishes

We modeled the occupancy dynamics of the benthic chum-chum (Pimelodella cf. gracilis) and the midwater threespot leporinus (Leporinus friderici) to determine how these fish species respond to disturbances caused by stream flooding. Fieldwork was conducted in two neotropical streams in central Brazil. Both species occupied fewer sites during floods than during periods with stable hydrology. The percentage of sites occupied by threespot leporinus increased from 57 to 75 %, and the percentage occupied by chum-chum increased from 27 to 87 %. Threespot leporinus moved upstream after floods, first recolonizing the sites farthest downstream and then recolonizing those farthest upstream. In contrast, chum-chum remained in several scattered sites along the stream during floods and recolonized nearby sites from these sources (i.e., colonization was not spatially concentrated). During wet hydroperiods, chum-chum used sites with water velocities ranging from 0.35 to 0.75 m/s. During the dry period, this range increased to 0.20–0.90 m/s (in sites that were colonized after the floods ended). Our results also showed that both species were more easily detected by snorkeling during the dry hydroperiod and that characteristics such as the substrate type and water velocity had different effects on the detectability of the two species. Overall, our study indicated that the annual persistence of threespot leporinus depends largely on seasonal upstream-biased movement, whereas the chum-chum population depends on the seasonal scattered colonization of temporary sites.