Destabilization of ß-amyloid aggregates by thrombin derived peptide: plausible role of thrombin in neuroprotection.

ß-amyloid (Aß) aggregates involved in Alzheimer's disease (AD) are resistant to proteases but could be destabilized by small peptides designed to target specific hydrophobic regions of Aß that take part in aggregate assembly. Since thrombin and AD are intricately connected, and elastase modulates thrombin activity, elastase-digested thrombin peptides were verified for intervention in the Aß-aggregation pathway. Intact or elastase-digested thrombin destabilized Aß fibril, as demonstrated by thioflavin T assay. Peptides were synthesized employing thrombin as a template, of which, a hexapeptide (T3) showed maximum destabilization at 1 µm. ExPASy peptide cutter software coupled with mass spectrometric analysis confirmed the generation of T3 peptide from elastase-digested thrombin. TEM micrographs revealed that 30-day incubation of preformed Aß fibrils or monomers with T3 resulted in destabilization or inhibition, respectively, leading mostly to particles of 1.74 ± 0.17 nm, which roughly corresponded to Aß monomer. Surface plasmon resonance employing CM5 chip coupled with Aß40 mouse monoclonal antibody showed a drop in response when T3 was incubated with Aß fibrils between 2 and 8 h. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide and confocal microscopy demonstrated the ability of T3 to rescue neuroblastoma cells from Aß oligomer-induced cytotoxic damage. Although no [Aß-T3] adduct could be detected by mass spectrometry, an initial interaction appeared to facilitate the process of destabilization/inhibition of aggregation. T3 was comparable to standard ß-sheet breaker peptides, LPFFD and KLVFF in terms of Aß aggregate destabilization. High hydrophobicity values coupled with recognition and breaking elements make T3 a potential candidate for future therapeutic applications.

Degradation of fibrin-ß amyloid co-aggregate: A novel function attributed to ubiquitin.

Human placental extract contains numerous bioactive components that are effective wound healing, antimicrobial and anti-inflammatory agents. During our investigation on the therapeutic potency of human placental extract, we have purified ubiquitin-like molecules that showed strong fibrino(geno)lytic activity. Further investigation confirmed similar potency of ubiquitin purified from adult human erythrocyte. Additionally, ubiquitin efficiently degraded disordered amyloid ß 42 peptide (Aß42) aggregate and fibrin-Aß42 co-aggregate in vitro and reduced co-aggregate induced cytotoxicity in SH-SY5Y human neuroblastoma cells as compared to plasmin. Ubiquitin also degraded abnormal co-aggregates of fibrin with other plasma proteins such as fibronectin, albumin, lysozyme, tranthyretin and α-synuclein. To elucidate the mechanism of degradation, synthetic peptides (ADG, GKT, DQQ, QRL, LIF, AGK, HLVL) derived from ubiquitin template as well as synthetic ubiquitin (8565.32 Da) were employed. Synthetic ubiquitin completely degraded preformed Aß 42 aggregate and fibrin-Aß42 co-aggregate, whereas, the smaller synthetic peptides showed varying degrees of degradation. These observations attribute a novel function of ubiquitin that may be used for degrading abnormal fibrin clots in human body. Thorough investigation might unfold a novel molecular mechanism of ubiquitin in protein homeostasis.

Waste electrical and electronic equipment management and Basel Convention compliance in Brazil, Russia, India, China and South Africa (BRICS) nations.

Brazil, Russia, India, China and South Africa (BRICS) nations account for one-quarter of the world's land area, having more than 40% of the world's population, and only one-quarter of the world gross national income. Hence the study and review of waste electrical and electronic equipment management systems in BRICS nations is of relevance. It has been observed from the literature that there are studies available comparing two or three country's waste electrical and electronic equipment status, while the study encompassing the BRICS nations considering in a single framework is scant. The purpose of this study is to analyse the existing waste electrical and electronic equipment management systems and status of compliance to Basel convention in the BRICS nations, noting possible lessons from matured systems, such as those in the European Union EU) and USA. The study introduced a novel framework for a waste electrical and electronic equipment management system that may be adopted in BRICS nations and revealed that BRICS countries have many similar types of challenges. The study also identified some significant gaps with respect to the management systems and trans-boundary movement of waste electrical and electronic equipment, which may attract researchers for further research.

Human Amniotic Membrane-Derived Mesenchymal and Epithelial Cells Exert Different Effects on Monocyte-Derived Dendritic Cell Differentiation and Function.

We previously demonstrated that mesenchymal cells from human amniotic membrane (hAMTCs) inhibit the generation and maturation of monocyte-derived dendritic cells (DCs) in vitro. Considering the crucial role of DCs in the immune response and that epithelial cells of the human amniotic membrane (hAECs) share some of the immunoregulatory properties of hAMTCs, we investigated whether hAECs also modulate monocyte-derived DCs. We compared hAECs with hAMTCs in a cell-to-cell contact setting and their secreted factors in modulating DC differentiation and function. First, we demonstrated that primary and expanded hAMTCs strongly inhibited the differentiation of DCs and induced a shift toward M2-like macrophages. This was observed when hAMTCs were cultured in contact (hAMTC-DC(cont)) or in Transwells (hAMTC-DC(tw)) with monocytes and even when medium conditioned by hAMTCs was used instead of hAMTCs. hAECs also prevented DC development, but to a lesser extent than hAMTCs. hAECs were more effective when cultured in contact with monocytes (hAEC-DC(cont)) rather than in Transwells (hAEC-DC(tw)). The modulatory capacity of hAECs changed during passaging unlike the hAMSCs. The ability to stimulate CD4(+) and CD8(+) T-cell proliferation was almost completely abolished by hAMTC-DC(cont), whereas hAMTC-DC(tw) and hAEC-DC(cont) displayed only a reduced ability to stimulate CD8(+) T cells. Furthermore, monocytes cocultured with hAMTCs and hAECs showed some similarities, but also differences in cytokine/chemokine secretion. Similarities were observed in the inhibition of IL-12p70 and TNF-α and the increase in IL-10 in supernatants taken from monocyte-DCs cocultured with hAMTCs and hAECs in contact and Transwell settings. The inflammatory factors IL-8, CXCL9, and MIP-1α were significantly lower in hAMTC-DC(cont), hAMTC-DC(tw), and hAEC-DC(cont) conditions. In contrast, only hAMTCs (in both contact and Transwell conditions) were able to significantly increase IL-1ß and CCL2. Altogether, we demonstrated that hAMTCs and hAECs affect DC differentiation, but that hAMTCs exerted a stronger inhibitory effect, abolished T-cell proliferation, and also induced more changes in cytokine/chemokine production.

Human placental extract mediated inhibition of proteinase K: implications of heparin and glycoproteins in wound physiology.

Efficient debridement of the wound bed following the removal of microbial load prevents its progression into a chronic wound. Bacterial infection and excessive proteolysis characterize impaired healing and therefore, their inhibition might restore the disturbed equilibrium in the healing process. Human placental extract exhibits reversible, non-competitive inhibition towards Proteinase K, a microbial protease, by stabilizing it against auto-digestion. Scattering and fluorescence studies followed by biochemical analysis indicated the involvement of a glycan moiety. Surface plasmon resonance demonstrated specific interaction of heparin with Proteinase K having Kd in µM range. Further, Proteinase K contains sequence motifs similar to other heparin-binding proteins. Molecular docking revealed presence of clefts suitable for binding of heparin-derived oligosaccharides. Comprehensive analysis of this inhibitory property of placental extract partly explains its efficacy in curing wounds with common bacterial infections.

Ubiquitin-like protein from human placental extract exhibits collagenase activity.

An aqueous extract of human placenta exhibits strong gelatinase/collagenase activity in zymography. 2-D gel electrophoresis of the extract with gelatin zymography in the second dimension displayed a single spot, identified as ubiquitin-like component upon MALDI/TOF MS/MS analysis. Immunoblot indicated presence of ubiquitin and absence of collagenase in the extract. Collagenase activity of the ubiquitin-like component was confirmed from the change in solubility of collagen in aqueous buffer, degradation of collagen by size-exclusion HPLC and atomic force microscopy. Quantification with DQ-gelatin showed that the extract contains 0.04 U/ml of collagenase activity that was inhibited up to 95% by ubiquitin antibody. Ubiquitin from bovine erythrocytes demonstrated mild collagenase activity. Bioinformatics studies suggest that placental ubiquitin and collagenase follow structurally divergent evolution. This thermostable intrinsic collagenase activity of placental extract might have wide physiological relevance in degrading and remodeling collagen as it is used as a drug for wound healing and pelvic inflammatory diseases.

Regulation of trypsin activity by peptide fraction of an aqueous extract of human placenta used as wound healer.

An aqueous extract of human placenta, used as wound healer, shows stabilization of trypsin against autodigestion as one of the peptides of the extract binds very strongly with the protease. Trypsin retains 40% of activity at constant level between 20 and 26 days in presence of the extract against complete inactivation in its absence. Inhibition of esterolytic activity and inability to react with p-nitrophenyl-p'-guanidinobenzoate, HCl, an active site directed reagent, by trypsin in presence of a peptide fraction of the extract indicated blocking of the catalytic site of the enzyme. Rayleigh scattering, size-exclusion HPLC, fluorescence resonance energy transfer, and surface plasmon resonance show that fibronectin type III-like peptide present in the extract interacts with trypsin. The peptide-trypsin complex is dissociated in presence of high concentration of substrates. Thus, regulation of trypsin activity by the placental extract is evident.

Analysis of free and bound NADPH in aqueous extract of human placenta used as wound healer.

NADPH is an important biomolecule involved in cellular regeneration. The distribution of free and bound NADPH in aqueous extract of human placenta used as a potent wound healer has been analyzed. Quantification from fluorescence and immuno-affinity chromatography indicates that 75.1+/-2.2% of NADPH present in the extract exists as free nucleotide or bound to very small peptides or amino acids whereas the rest remains bound to large peptides. Inability to dissociate the bound form of the nucleotide from the large peptides using urea or guanidium hydrochloride indicates that the binding is covalent. Identification of a fragmented mass of m/z 382.94 (nicotinamide+sugar+phosphate) from the NADPH-peptide conjugates supported the intactness of the nicotinamide moiety. Glutathione reductase assay indicated that 95.2+/-3.5% of the total NADPH pool of the extract can act as cosubstrate of the enzyme. This indicates that while a major fraction of free NADPH of the extract is easily available for cellular processes, the rest can also function locally where the conjugated peptides are deposited.