Nested capillary anti-resonant silica fiber with mid-infrared transmission and low bending sensitivity at 4000 nm.

We report a silica glass nested capillary anti-resonant nodeless fiber with transmission and low bending sensitivity in the mid-infrared around 4000 nm. The fiber is characterized in terms of transmission over 1700-4200 nm wavelengths, revealing a mid-infrared 3500-4200 nm transmission window, clearly observable for a 12 m long fiber. Bending loss around 4000 nm is 0.5 dB/m measured over three full turns with 40 mm radius, going up to 5 dB/m for full turns with 15 mm radius. Our results provide experimental evidence of hollow-core silica fibers in which nested, anti-resonant capillaries provide high bend resistance in the mid-infrared. This is obtained for a fiber with a large core diameter of over 60 µm relative to around 30 µm capillaries in the cladding, which motivates its application in gas fiber lasers or fiber-based mid-infrared spectroscopy of COx or NxO analytes.

Hydrogen peroxide induces apoptosis-like death in Entamoeba histolytica trophozoites.

Programmed cell death (PCD) is an essential process in the growth and development of multicellular organisms. However, accumulating evidence indicates that unicellular eukaryotes can also undergo PCD with apoptosis-like features. This study demonstrates that after exposure to 0.8 mM H(2)O(2) for 9 h Entamoeba histolytica presents morphological and biochemical evidence of apoptosis-like death. Morphological characteristics of apoptosis-like death including DNA fragmentation, increased vacuolization, nuclear condensation and cell rounding were observed for H(2)O(2)-exposed trophozoites with preservation of membrane integrity. Biochemical alteration in ion fluxes is also a key feature in PCD, and H(2)O(2)-exposed trophozoites showed overproduction of reactive oxygen species, increased cytosolic Ca(2+) and decreased intracellular pH. Phosphatidylserine was also found to be expressed in the outer leaflet of the plasma membrane of the H(2)O(2)-treated trophozoites. Pretreatment with the cysteine protease inhibitor E-64d, the extracellular and intracellular Ca(2+) chelators EGTA and BAPTA/AM, and the Ca(2+) influx inhibitor verapamil prior to H(2)O(2) exposure abolished DNA fragmentation. The oxidatively stressed trophozoites also showed an increased calpain activity, indicating involvement of Ca(2+)-dependent calpain-like cysteine proteases in PCD of E. histolytica. A homogeneous caspase assay showed no significant caspase activity, and administration of caspase 1 inhibitor also did not prevent the death phenotype for the oxidatively stressed trophozoites, indicating a caspase-independent apoptosis-like death. Our observations clearly demonstrate that there is a distinct calpain-dependent but caspase-independent pathway for apoptosis-like death in oxidatively stressed E. histolytica trophozoites.

Tau protein- induced sequestration of the eukaryotic ribosome: Implications in neurodegenerative disease.

The human tau is a microtubule-associated intrinsically unstructured protein that forms intraneuronal cytotoxic deposits in neurodegenerative diseases, like tauopathies. Recent studies indicate that in Alzheimer's disease, ribosomal dysfunction might be a crucial event in the disease pathology. Our earlier studies had demonstrated that amorphous protein aggregation in the presence of ribosome can lead to sequestration of the ribosomal components. The present study aims at determining the effect of incubation of the full-length tau protein (Ht40) and its microtubule binding 4-repeat domain (K18) on the eukaryotic ribosome. Our in vitro studies show that incubation of Ht40 and the K18 tau variants with isolated non-translating yeast ribosome can induce a loss of ribosome physical integrity resulting in formation of tau-rRNA-ribosomal protein aggregates. Incubation with the tau protein variants also led to a disappearance of the peak indicating the ribosome profile of the HeLa cell lysate and suppression of translation in the human in vitro translation system. The incubation of tau protein with the ribosomal RNA leads to the formation of tau-rRNA aggregates. The effect of K18 on the yeast ribosome can be mitigated in the presence of cellular polyanions like heparin and tRNA, thereby indicating the electrostatic nature of the aggregation process.

Sequestration of Ribosome during Protein Aggregate Formation: Contribution of ribosomal RNA.

An understanding of the mechanisms underlying protein aggregation and cytotoxicity of the protein aggregates is crucial in the prevention of several diseases in humans. Ribosome, the cellular protein synthesis machine is capable of acting as a protein folding modulator. The peptidyltransferase center residing in the domain V of large ribosomal subunit 23S rRNA is the centre for the protein folding ability of the ribosome and is also the cellular target of several antiprion compounds. Our in vitro studies unexpectedly reveal that the partial unfolding or aggregation of lysozyme under reducing conditions in presence of the ribosome can induce aggregation of ribosomal components. Electrostatic interactions complemented by specific rRNA-protein interaction drive the ribosome-protein aggregation process. Under similar conditions the rRNA, especially the large subunit rRNA and in vitro transcribed RNA corresponding to domain V of 23S rRNA (bDV RNA) stimulates lysozyme aggregation leading to RNA-protein aggregate formation. Protein aggregation during the refolding of non-disulfide containing protein BCAII at high concentrations also induces ribosome aggregation. BCAII aggregation was also stimulated in presence of the large subunit rRNA. Our observations imply that the specific sequestration of the translation machine by aggregating proteins might contribute to their cytotoxicity.

Withaferin A induced impaired autophagy and unfolded protein response in human breast cancer cell-lines MCF-7 and MDA-MB-231.

The autophagy-lysosome pathway and the ubiquitin-proteasome systems are the two major routes for eukaryotic intracellular protein clearance. Cancerous cells often display elevated protein synthesis and byproduct disposal, thus, inhibition of the protein degradation pathways became an emerging approach for cancer therapy. The present study revealed that withaferin-A (WA), the biologically active withanolide derived from Withania somnifera, initially induced formation of autophagosomes in human breast cancer cell-lines, MCF-7 and MDA-MB-231. WA treatment elevated the levels of autophagic substrate p62/SQSTM1 (p62) and both LC3-II and LC3-I (microtubule-associated protein 2 light chain 3) and simultaneously reduced the upstream autophagy markers like beclin-1 and ATG5-ATG12 complex, which indicate accumulation of autophagosomes in the cells. WA induced disruption of microtubular network through inhibition of tubulin polymerization and its hyper-acetylation, thus prevent the formation of autolysosome (by merging of autophagosomes with lysosomes) and its recycling process, leading to incomplete autophagy. Further, WA caused ER (Endoplasmic Reticulum) stress, which is evident from the activation of ER-related caspase-4 and increased levels of ER stress marker proteins. Thus, these findings altogether indicate that WA mediated inhibition of proteasomal degradation system and perturbation of autophagy, i.e. suppression of both the intracellular degradation systems caused accumulation of ubiquitinated proteins, which in turn led to unfolded protein response and ER stress mediated proteotoxicity in human breast cancer cell-lines, MCF-7 and MDA-MB-231.

The ribosome can prevent aggregation of partially folded protein intermediates: studies using the Escherichia coli ribosome.

BACKGROUND: Molecular chaperones that support de novo folding of proteins under non stress condition are classified as chaperone 'foldases' that are distinct from chaperone' holdases' that provide high affinity binding platform for unfolded proteins and prevent their aggregation specifically under stress conditions. Ribosome, the cellular protein synthesis machine can act as a foldase chaperone that can bind unfolded proteins and release them in folding competent state. The peptidyl transferase center (PTC) located in the domain V of the 23S rRNA of Escherichia coli ribosome (bDV RNA) is the chaperoning center of the ribosome. It has been proposed that via specific interactions between the RNA and refolding proteins, the chaperone provides information for the correct folding of unfolded polypeptide chains. RESULTS: We demonstrate using Escherichia coli ribosome and variants of its domain V RNA that the ribosome can bind to partially folded intermediates of bovine carbonic anhydrase II (BCAII) and lysozyme and suppress aggregation during their refolding. Using mutants of domain V RNA we demonstrate that the time for which the chaperone retains the bound protein is an important factor in determining its ability to suppress aggregation and/or support reactivation of protein. CONCLUSION: The ribosome can behave like a 'holdase' chaperone and has the ability to bind and hold back partially folded intermediate states of proteins from participating in the aggregation process. Since the ribosome is an essential organelle that is present in large numbers in all living cells, this ability of the ribosome provides an energetically inexpensive way to suppress cellular aggregation. Further, this ability of the ribosome might also be crucial in the context that the ribosome is one of the first chaperones to be encountered by a large nascent polypeptide chains that have a tendency to form partially folded intermediates immediately following their synthesis.

Oxidative stress-induced cell cycle blockage and a protease-independent programmed cell death in microaerophilic Giardia lamblia.

Giardia lamblia is a microaerophilic human gastrointestinal parasite and considered as an early-diverged eukaryote. In vitro oxidative stress generation plays a significant role in cell cycle progression and cell death of this parasite. In the present study hydrogen peroxide, metronidazole, and a modified growth medium without cysteine and ascorbic acid have been chosen as oxidative stress-inducing agents. Cell cycle progression has been found to be regulated by different types of oxidative stresses. Apoptosis is not an established pathway in Giardia, which is devoid of ideal mitochondria, but in the present investigation, apoptosis-like programmed cell death has been found by the experiments like AnnexinV-FITC assay, DNA fragmentation pattern, etc. On the contrary, Caspase-9 assay, which confirms the caspase-mediated apoptotic pathway, has been found to be negative in all the stress conditions. Protease inhibitor assay confirmed that, even in absence of any proteases, programmed cell death does occur in this primitive eukaryote. All these results signify a novel pathway of programmed suicidal death in Giardia lamblia under oxidative stress. This is the first demonstration of protease-independent programmed cell death regulation in Giardia exclusive for its own specialties.

Alprazolam intercalates into DNA.

In vitro interaction of a benzodiazepine group of drugs Alprazolam (Alp), a hypnotic and tranquilizer, with DNA was studied by various methods. Absorption spectrophotometric study showed that Alp binds strongly with supercoiled pUC 19 DNA and the calculated binding constant is 8.245x10(3) M(-1) in 10 mM Tris-Cl buffer, pH 7.4. Spectrofluorometric study showed that ethidium bromide induced DNA fluorescence intensity was reduced completely after addition of Alp. But Alp did not interfere with the interaction of Hoechst 33258, a DNA minor groove binder, with plasmid DNA. Circular dichroic spectroscopic study showed that with the gradual increase in Alp concentrations, both the positive and the negative peaks of DNA were gradually decreased and at higher concentrations of Alp (60 microM and 80 microM), the negative peaks became positive indicating the intercalation and the conformational change in the DNA. Binding of Alp with DNA increased the thermal stability of DNA by 6 degrees C with respect to the mock treated sample. Gel electrophoresis study of supercoiled pUC 19 DNA showed more compact structure as a result of Alp binding. Transmission electron microscopic observations also confirmed this compactness. Thus, our observations suggest the strong interaction of Alp with DNA, which may raise serious questions about the random uses of Alprazolam.

Uptake of Pb(2+ )by a cyanobacterium belonging to the genus Synechocystis, isolated from East Kolkata Wetlands.

East Kolkata Wetlands is a conserved wetland utilizing sewage and garbage, generated by Kolkata Municipal Corporation area for cultivation purpose. Cyanobacteria are the photosynthetic prokaryotes having bioremedial capacity. We have isolated a cyanobacterium from the sewage recycling fish-pond of East Kolkata Wetlands. Partial sequence of 16S rDNA gene of the isolated strain showed 100% similarity with that of genus Synechocystis. Isolated strain and Synechocystis sp. PCC6803 survived up to 300 mug ml(-1) Pb(2+ )and growth was completely inhibited at 400 mug ml(-1) Pb(2+). All experiments were carried out with 100 mug ml(-1) Pb(2+) in which growth was the maximum. 91.67% of the total Pb(2+) got adsorbed to the outer surface of the cell and 1% of the total Pb(2+) entered the cell of the isolated strain as estimated by atomic absorption spectrometry, but in Synechocystis sp. PCC6803 72.72% adsorbed and 0.96% penetrated. Intracellular and periplasmic depositions of Pb(2+) were observed in both the strain. A filamentous structure developed outside the cell wall of the isolated cyanobacterium, but very little change was observed in Synechocystis sp. PCC6803. ZiaR-SmtB like regulator gene was expressed in both the strains after Pb(2+) induction. The cDNA sequence of ZiaR of the isolated cyanobacterium shows 100% homology with that of Synechocystis sp. PCC6803. Upon Pb(2+) induction, expression of SOD gene increased. cDNA sequence of the SOD gene from the isolated strain showed 98% homology with that of Synechocystis sp. PCC6803. Enzymatic activity of catalase and SOD was also increased. No DNA damage was monitored upon induction with Pb(2+).

Role of MMP-2 in inhibiting Na+ dependent Ca2+ uptake by H2O2 in microsomes isolated from pulmonary smooth muscle.

Treatment of microsomes (preferentially enriched with endoplasmic reticulum) isolated from bovine pulmonary artery smooth muscle tissue with H2O2 (1 mM) markedly stimulated matrix metalloproteinase activity and also inhibited Na+ dependent Ca2+ uptake. Electron micrograph revealed that H2O2 (1 mM) does not cause any damage to the microsomes. MMP-2 and TIMP-2 were determined to be the ambient protease and corresponding antiprotease of the microsomes. Pretreatment with vitamin E (1 mM) and TIMP-2 (50 microg/ml) reversed the effect produced by H2O2 (1 mM) on Na+ dependent Ca2+ uptake in the microsomes. However, H2O2 (1 mM) caused changes in MMP-2 activity and Na+ dependent Ca2+ uptake were not reversed upon pretreatment of the microsomes with a low concentration of 5 microg/ml of TIMP-2 which otherwise reversed MMP-2 (1 microg/ml) mediated increase in 14C-gelatin degradation and inhibition of Na+ dependent Ca2+ uptake. Combined treatment of the microsomes with a low dose of MMP-2 (0.5 microg/ml) and H2O2 (0.5 mM) inhibited Na+ dependent Ca2+ uptake in the microsomes compared to the respective low dose of either of them. Direct treatment of TIMP-2 (5 microg/ml) with H2O2 (1 mM) abolished the inhibitory effect of the inhibitor on 14C-gelatinolytic activity elicited by 1 microg/ml of MMP-2. Thus, one of the mechanisms by which H2O2 activates MMP-2 could be due to inactivation of TIMP-2 by the oxidant. The resulting activation of MMP-2 subsequently inhibits Na+ dependent Ca2+ uptake in the microsomes.