The novel presenilin-1-associated protein is a proapoptotic mitochondrial protein

Recent studies have suggested a possible role for presenilin proteins in apoptotic cell death observed in Alzheimer's disease. The mechanism by which presenilin proteins regulate apoptotic cell death is not well understood. Using the yeast two-hybrid system, we previously isolated a novel protein, presenilin-associated protein (PSAP) that specifically interacts with the C terminus of presenilin 1 (PS1), but not presenilin 2 (PS2). Here we report that PSAP is a mitochondrial resident protein sharing homology with mitochondrial carrier protein. PSAP was detected in a mitochondria-enriched fraction, and PSAP immunofluorescence was present in a punctate pattern that colocalized with a mitochondrial marker. More interestingly, overexpression of PSAP caused apoptotic death. PSAP-induced apoptosis was documented using multiple independent approaches, including membrane blebbing, chromosome condensation and fragmentation, DNA laddering, cleavage of the death substrate poly(ADP-ribose) polymerase, and flow cytometry. PSAP-induced cell death was accompanied by cytochrome c release from mitochondria and caspase-3 activation. Moreover, the general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, which blocked cell death, did not block the release of cytochrome c from mitochondria caused by overexpression of PSAP, indicating that PSAP-induced cytochrome c release was independent of caspase activity. The mitochondrial localization and proapoptotic activity of PSAP suggest that it is an important regulator of apoptosis.     

Cloning and sequencing of complete tau-crystallin cDNA from embryonic lens of Crocodylus palustris

τ-Crystallin is a taxon-specific structural protein found in eye lenses. We present here the cloning and sequencing of complete τ-crystallin cDNA from the embryonic lens ofCrocodylus palustris and establish it to be identical to the α-enolase gene from non-lenticular tissues. Quantitatively, the τ-crystallin was found to be the least abundant crystallin of the crocodilian embryonic lenses. Crocodile τ-crystallin cDNA was isolated by RT-PCR using primers designed from the only other reported sequence from duck and completed by 5′- and 3′-rapid amplification of cDNA ends (RACE) using crocodile gene specific primers designed in the study. The complete τ-crystallin cDNA of crocodile comprises 1305 bp long ORF and 92 and 409 bp long untranslated 5′- and 3′-ends respectively. Further, it was found to be identical to its putative counterpart enzyme α-enolase, from brain, heart and gonad, suggesting both to be the product of the same gene. The study thus provides the first report on cDNA sequence of τ-crystallin from a reptilian species and also re-confirms it to be an example of the phenomenon of gene sharing as was demonstrated earlier in the case of peking duck. Moreover, the gene lineage reconstruction analysis helps our understanding of the evolution of crocodilians and avian species.     

Airway hyperresponsiveness, late allergic response, and eosinophilia are reversed with mycobacterial antigens in ovalbumin-presensitized mice

Pretreatment with mycobacterial Ags has been shown to be effective in preventing allergic airway inflammation from occurring in a mouse model. Because most asthmatics are treated after the development of asthma, it is crucial to determine whether mycobacterial Ags can reverse established allergic airway inflammation in the presensitized state. Our hypothesis, based upon our previous findings, is that mycobacteria treatment in presensitized mice will suppress the allergic airway inflammation with associated clinical correlates of established asthma, with the noted exception of factors associated with the early allergic response (EAR). BALB/c mice sensitized and challenged with OVA were evaluated for pulmonary functions during both the EAR and late allergic response, and airway hyperresponsiveness to methacholine. Following this, sensitized mice were randomized and treated with placebo or a single dose (1 x 10(5) CFUs) of bacillus Calmette-Guérin (BCG) or Mycobacterium vaccae via nasal or peritoneal injection. One week later, the mice were rechallenged with OVA and methacholine, followed by bronchoalveolar lavage (BAL) and tissue collection. Mice treated with intranasal BCG were most significantly protected from the late allergic response (p < 0.02), airway hypersensitivity (p < 0.001) and hyperreactivity (p < 0.05) to methacholine, BAL (p < 0.05) and peribronchial (p < 0.01) eosinophilia, and BAL fluid IL-5 levels (p < 0.01) as compared with vehicle-treated, sensitized controls. Intranasal M. vaccae treatment was less effective, suppressing airway hypersensitivity (p < 0.01) and BAL eosinophilia (p < 0.05). No changes were observed in the EAR, BAL fluid IL-4 levels, or serum total and Ag-specific IgE. These data suggest that mycobacterial Ags (BCG>M. vaccae) are effective in attenuating allergic airway inflammation and associated changes in pulmonary functions in an allergen-presensitized state.     

A C-reactive protein mutant that does not bind to phosphocholine and pneumococcal C-polysaccharide

C-reactive protein (CRP), the major human acute-phase plasma protein, binds to phosphocholine (PCh) residues present in pneumococcal C-polysaccharide (PnC) of Streptococcus pneumoniae and to PCh exposed on damaged and apoptotic cells. CRP also binds, in a PCh-inhibitable manner, to ligands that do not contain PCh, such as fibronectin (Fn). Crystallographic data on CRP-PCh complexes indicate that Phe(66) and Glu(81) contribute to the formation of the PCh binding site of CRP. We used site-directed mutagenesis to analyze the contribution of Phe(66) and Glu(81) to the binding of CRP to PCh, and to generate a CRP mutant that does not bind to PCh-containing ligands. Five CRP mutants, F66A, F66Y, E81A, E81K, and F66A/E81A, were constructed, expressed in COS cells, purified, and characterized for their binding to PnC, PCh-BSA, and Fn. Wild-type and F66Y CRP bound to PnC with similar avidities, while binding of E81A and E81K mutants to PnC was substantially reduced. The F66A and F66A/E81A mutants did not bind to PnC. Identical results were obtained with PCh-BSA. In contrast, all five CRP mutants bound to Fn as well as did wild-type CRP. We conclude that Phe(66) is the major determinant of CRP-PCh interaction and is critical for binding of CRP to PnC. The data also suggest that the binding sites for PCh and Fn on CRP are distinct. A CRP mutant incapable of binding to PCh provides a tool to assess PCh-inhibitable interactions of CRP with its other biologically significant ligands, and to further investigate the functions of CRP in host defense and inflammation.     

Induction of preference and performance after acclimation to novel hosts in a phytophagous spider mite: adaptive plasticity?

We examined induction of preference and performance on novel host plants for two laboratory populations of the polyphagous spider mite Tetranychus urticae, with one population adapted to bean and the other population adapted to tomato. We bred four isofemale lines of the bean population only and used them in all the assays. The bean population had a 30% lower fecundity on tomato than on bean, while the tomato population had equal fecundity on both host plants. Acclimation of adult females to the novel host plant for both populations increased acceptability of that novel host but did not increase rejection of the original host. The bean population experienced a 60% benefit and a 30% cost in terms of egg production for acclimating to tomato, thus exemplifying adaptive plasticity. The tomato population showed a 23% benefit for acclimating to bean but no cost. Mites from the bean population that were acclimated to tomato fed more on tomato than did mites that were not acclimated to tomato. When these mites were fed inhibitors of cytochrome P-450 detoxification enzymes, their performance was severely depressed (84%) on tomato but not on bean. However, mites that were fed inhibitors of P-450 enzymes did not reduce their acceptance of tomato as a host. Thus, performance on novel hosts (but not preference) in this species is likely correlated with the induction of detoxifying enzymes. Spider mites are known to form host races rapidly on novel hosts. Induction of preference and physiological acclimation via detoxification enzymes may enhance performance and, thus, strongly contribute to initial stages of host race formation.     

Proteome analysis of differentially displayed proteins as a tool for investigating ozone stress in rice (Oryza sativa L.) seedlings

Employing classical two-dimensional electrophoresis (2-DE), amino acid sequencing and immunoblot analysis, we examine for the first time the effect of ozone, a highly notorious environmental pollutant, on rice seedling proteins. Drastic visible necrotic damage to leaf by ozone and consequent increase in ascorbate peroxidase protein(s) was accompanied by rapid changes in the 2-DE protein profiles, over controls. Out of a total of 56 proteins investigated, which were reproducible in repeated experiments, 52 protein spots were visually identified as differentially expressed over controls. Six proteins were N-terminally blocked, and the sequence of 14 proteins could not be determined, whereas 36 proteins were N-terminally and one was internally sequenced. Ozone caused drastic reductions in the major leaf photosynthetic proteins, including the abundantly present ribulose-1, 5-bisphosphate carboxylase/oxygenase, and induction of various defense/stress related proteins. Most prominent change in leaves, within 24 h post-treatment with ozone, was the induced accumulation of a pathogenesis related (PR) class 5 protein, three PR 10 class proteins, ascorbate peroxidase(s), superoxide dismutase, calcium-binding protein, calreticulin, a novel ATP-dependent CLP protease, and an unknown protein. Present results demonstrate the highly damaging effect of ozone on rice seedlings at the level of the proteome.     

On the topological evidences for modelling lipophilicity

Topological evidences for modelling lipophilicity of a large series of diversed compounds have been provided on the basis of distance-based topological indices. A pool of topological indices along with indicator parameters related to the type of the compounds present in the set of 140 compounds were used for this purpose. The results have shown that topology as well as the type of compounds are the responsible parameters for modelling lipophilicity.