Hydrolysis of amelogenin by matrix metalloprotease-20 accelerates mineralization in vitro

In the following respects, tooth enamel is a unique tissue in the mammalian body: (a) it is the most mineralized and hardest tissue in it comprising up to 95 wt% of apatite; (b) its microstructure is dominated by parallel rods composed of bundles of 40–60 nm wide apatite crystals with aspect ratios reaching up to 1:10,000 and (c) not only does the protein matrix that gives rise to enamel guides the crystal growth, but it also conducts its own degradation and removal in parallel. Hence, when mimicking the process of amelogenesis in vitro, crystal growth has to be coupled to proteolytic digestion of the amelogenin assemblies that are known to play a pivotal role in conducting the proper crystal growth. Experimental settings based on controlled and programmable titration of amelogenin sols digested by means of MMP-20 with buffered calcium and phosphate solutions were employed to imitate the formation of elongated, plate-shaped crystals. Whilst amelogenin can act as a promoter of nucleation and crystal growth alone, in this study we show that proteolysis exerts an additional nucleation- and growth-promoting effect. Hydrolysis of full-length amelogenin by MMP-20 decreases the critical time needed for the protein and peptides to adhere and to cover the substrate. The formation and immobilization of a protein layer subsequently reduces the time for calcium phosphate crystallization. Coupling the proteolytic reaction to titration in the presence of 0.4 mg/ml rH174 has been shown to have the same effect on the crystal growth promotion as quadrupling the concentration of rH174 to 1.6 mg/ml. Controlling the rate and the extent of the proteolytic cleavage can thus be used to control the nucleation and growth rates in a protein-guided crystallization system.     

Implementation of a mass measles campaign in central Afghanistan,December 2001 to May 2002

In Afghanistan health services have been disrupted by 23 years of conflict and 1 of 4 children die before age 5 years. Measles accounts for an estimated 35,000 deaths annually. Surveillance data show a high proportion of measles cases (38%) among those >/=5 years old. In areas with complex emergencies, measles vaccination is recommended for those aged 6 months to 12-15 years. From December 2001 to May 2002, Afghan authorities and national and international organizations targeted 1,748,829 children aged 6 months to 12 years in five provinces in central Afghanistan for measles vaccinations. Two provinces reported coverage of >90% and two >80%. Coverage in Kabul city was 62%. A subsequent cluster survey in the city found 91% coverage (95% confidence interval [CI], 0.85-0.91) among children 6-59 months and 88% (95% CI, 0.87-0.95) among those 5-12 years old. Thus, this campaign achieved acceptable coverage despite considerable obstacles.     

Role of intra-arterial steroid administration in the management of steroid-refractory acute gastrointestinal graft-versus-host disease

We report here a case of severe steroid-refractory gastrointestinal graft-versus-host disease treated with intra-arterial administration of corticosteroids. A 53-year-old female with non-Hodgkin's lymphoma received peripheral blood hematopoietic stem cell transplant from her HLA-matched sibling. She developed grade II skin and grade IV gastrointestinal graft-versus-host disease with no hepatic involvement. Therapy with oral prednisone easily controlled her skin rash but she had profuse diarrhea that did not respond to high dose intravenous corticosteroids and denileukin diftitox. Infusion of methyl-prednisolone into superior and inferior mesenteric arteries produced dramatic improvement of diarrhea, with complete resolution of gastrointestinal graft-versus-host disease.     

Effect of Pluchea lanceolata bioactives in LPS-induced neuroinflammation in C6 rat glial cells

Neuroinflammation plays a significant role in various chronic and acute pathological conditions of the central nervous system. In the Indian system of medicine, Pluchea lanceolata is used to treat the neurological disorders. We investigated the effect of major pentacyclic triterpene and its naturally occurring acetate derivative isolated from P. lanceolata on lipopolysaccharide (LPS)-stimulated neuroinflammatory condition associated to inflammatory cytokine production in rat astrocytoma cell line (C6). The log concentration dependence of Pluchea bioactive taraxasterol (Tx) significantly (p?<?0.05) attenuates the release of pro-inflammatory cytokines, such as TNF-α, IFN-γ, and IL-6, while its in situ produced acetyl derivative, i.e., taraxasterol acetate (TxAc), did not inhibit the LPS-induced IL-6 production at lower concentration (p?>?0.05). Surflex-Dock molecular modeling study was performed to simulate the binding capacity of compounds into the active site of the TNF-α (2AZ5), tumor protein P53 (2VUK), and NF-kappa-B (1RAM). The differential inhibition of cytokines by Tx and TxAc was further confirmed by high docking scores showing the high affinity to target proteins. Findings of the study demonstrated the comparatively greater role of Pluchea triterpene than its in situ produced acetate derivate in neuroinflammation-associated disorders.     

4‐Hydroxy‐α‐Tetralone and its Derivative as Drug Resistance Reversal Agents in Multi Drug Resistant Escherichia coli

The purpose of present investigation was to understand the drug resistance reversal mechanism of 4‐hydroxy‐α‐tetralone ( 1 ) isolated from Ammannia spp. along with its semi‐synthetic derivatives ( 1a – 1e ) using multidrug resistant Escherichia coli (MDREC). The test compounds did not show significant antibacterial activity of their own, but in combination, they reduced the minimum inhibitory concentration (MIC) of tetracycline (TET). In time kill assay, compound 1 and its derivative 1e in combination with TET reduced the cell viability in concentration dependent manner. Compounds 1 and 1e were also able to reduce the mutation prevention concentration of TET. Both compounds showed inhibition of ATP dependent efflux pumps. In real time polymerase chain reaction (RT‐PCR) study, compounds 1 and 1e alone and in combination with TET showed significant down expression of efflux pump gene (yojI) encoding multidrug ATP binding cassettes (ABC) transporter protein. Molecular mechanism was also supported by the in silico docking studies, which revealed significant binding affinity of compounds 1 and 1e with YojI. This study confirms that compound 1 and its derivative 1e are ABC efflux pump inhibitors which may be the basis for development of antibacterial combinations for the management of MDR infections from inexpensive natural product.     

On the mechanism of sensing unfolded protein in the endoplasmic reticulum

Unfolded proteins in the endoplasmic reticulum (ER) activate the ER transmembrane sensor Ire1 to trigger the unfolded protein response (UPR), a homeostatic signaling pathway that adjusts ER protein folding capacity according to need. Ire1 is a bifunctional enzyme, containing cytoplasmic kinase and RNase domains whose roles in signal transduction downstream of Ire1 are understood in some detail. By contrast, the question of how its ER-luminal domain (LD) senses unfolded proteins has remained an enigma. The 3.0-A crystal structure and consequent structure-guided functional analyses of the conserved core region of the LD (cLD) leads us to a proposal for the mechanism of response. cLD exhibits a unique protein fold and is sufficient to control Ire1 activation by unfolded proteins. Dimerization of cLD monomers across a large interface creates a shared central groove formed by alpha-helices that are situated on a beta-sheet floor. This groove is reminiscent of the peptide binding domains of major histocompatibility complexes (MHCs) in its gross architecture. Conserved amino acid side chains in Ire1 that face into the groove are shown to be important for UPR activation in that their mutation reduces the response. Mutational analyses suggest that further interaction between cLD dimers is required to form higher-order oligomers necessary for UPR activation. We propose that cLD directly binds unfolded proteins, which changes the quaternary association of the monomers in the membrane plane. The changes in the ER lumen in turn position Ire1 kinase domains in the cytoplasm optimally for autophosphorylation to initiate the UPR.     

TRARESA: a tissue microarray-based hospital system for biomarker validation and discovery

Formalin fixed and paraffin embedded tissue (FFPE) collections in pathology departments are the largest resource for retrospective biomedical research studies. Based on the literature analysis of FFPE related research, as well as our own technical validation, we present the Translational Research Arrays (TRARESA), a tissue microarray centred, hospital based, translational research conceptual framework for both validation and/or discovery of novel biomarkers. TRARESA incorporates the analysis of protein, DNA and RNA in the same samples, correlating with clinical and pathological parameters from each case, and allowing (a) the confirmation of new biomarkers, disease hypotheses and drug targets, and (b) the postulation of novel hypotheses on disease mechanisms and drug targets based on known biomarkers. While presenting TRARESA, we illustrate the use of such a comprehensive approach. The conceptualisation of the role of FFPE-based studies in translational research allows the utilisation of this commodity, and adds to the hypothesis-generating armamentarium of existing high-throughput technologies.