The chromophore structures of the Pr States in plant and bacterial phytochromes

The resonance Raman spectra of the Pr state of the N-terminal 65-kDa fragment of plant phytochrome phyA have been measured and analyzed in terms of the configuration and conformation of the tetrapyrroles methine bridges. Spectra were obtained from phyA adducts reconstituted with the natural chromophore phytochromobilin as well as phycocyanobilin and its isotopomers labeled at the terminal methine bridges through (13)C/(12)C and D/H substitution. Upon comparing the resonance Raman spectra of the various phyA adducts, it was possible to identify the bands that originate from normal modes dominated by the stretching coordinates of the terminal methine bridges A-B and C-D. Quantum chemical calculations of the isolated tetrapyrroles reveal that these modes are sensitive indicators for the methine bridge configuration and conformation. For all phyA adducts, the experimental spectra of Pr including this marker band region are well reproduced by the calculated spectra obtained for the ZZZasa configuration. In contrast, there are substantial discrepancies between the experimental spectra and the spectra calculated for the ZZZssa configuration, which has been previously shown to be the chromophore geometry in the Pr state of the bacterial, biliverdin-binding phytochrome from Deinococcus radiodurans (Wagner, J. R., J. S. Brunzelle, K. T. Forest, R. D. Vierstra. 2005. Nature. 438:325-331). The results of this work, therefore, suggest that plant and bacterial (biliverdin-binding) phytochromes exhibit different structures in the parent state although the mechanism of the photoinduced reaction cycle may be quite similar.     

Quercetin-3-rutinoside Inhibits Protein Disulfide Isomerase by Binding to Its b′x Domain

Quercetin-3-rutinoside inhibits thrombus formation in a mouse model by inhibiting extracellular protein disulfide isomerase (PDI), an enzyme required for platelet thrombus formation and fibrin generation. Prior studies have identified PDI as a potential target for novel antithrombotic agents. Using a fluorescence enhancement-based assay and isothermal calorimetry, we show that quercetin-3-rutinoside directly binds to the b′ domain of PDI with a 1:1 stoichiometry. The binding of quercetin-3-rutinoside to PDI induces a more compact conformation and restricts the conformational flexibility of PDI, as revealed by small angle x-ray scattering. The binding sites of quercetin-3-rutinoside to PDI were determined by studying its interaction with isolated fragments of PDI. Quercetin-3-rutinoside binds to the b′x domain of PDI. The infusion of the b′x fragment of PDI rescued thrombus formation that was inhibited by quercetin-3-rutinoside in a mouse thrombosis model. This b′x fragment does not possess reductase activity and, in the absence of quercetin-3-rutinoside, does not affect thrombus formation in vivo. The isolated b′ domain of PDI has potential as an antidote to reverse the antithrombotic effect of quercetin-3-rutinoside by binding and neutralizing quercetin-3-rutinoside.     

Molecular Pathology of Rare Bleeding Disorders (RBDs) in India: A Systematic Review

Background

Though rare in occurrence, patients with rare bleeding disorders (RBDs) are highly heterogeneous and may manifest with severe bleeding diathesis. Due to the high rate of consanguinity in many caste groups, these autosomal recessive bleeding disorders which are of rare occurrence in populations across the world, may not be as rare in India.

Objectives

To comprehensively analyze the frequency and nature of mutations in Indian patients with RBDs.

Methods

Pubmed search was used (www.pubmed.com) to explore the published literature from India on RBDs using the key words “rare bleeding disorders”, “mutations”, “India”, “fibrinogen”, “afibrinogenemia”, “factor II deficiency”, “prothrombin” “factor VII deficiency”, “factor V deficiency”, “factor X deficiency”, “factor XI deficiency”, “combined factor V and VIII deficiency”, “factor XIII deficiency”, “Bernard Soulier syndrome” and “Glanzmanns thrombasthenia” in different combinations. A total of 60 relevant articles could be retrieved. The distribution of mutations from India was compared with that of the world literature by referring to the Human Gene Mutation Database (HGMD) (www.hgmd.org).

Results

Taken together, 181 mutations in 270 patients with different RBDs have been reported from India. Though the types of mutations reported from India and their percentage distribution with respect to the world data are largely similar, yet much higher percentage of small deletions, duplication mutations, insertions, indels were observed in this analysis. Besides the identification of novel mutations and polymorphisms, several common mutations have also been reported, which will allow to develop a strategy for mutation screening in Indian patients with RBDs.

Conclusion

There is a need for a consortium of Institutions working on the molecular pathology of RBDs in India. This will facilitate a quicker and cheaper diagnosis of RBDs besides its utility in first trimester prenatal diagnosis of the affected families.     

Tools for high efficiency genetic manipulation of the human pathogen Penicillium marneffei

Penicillium marneffei is an opportunistic pathogen of humans and displays a temperature dependent dimorphic transition. Like many fungi, exogenous DNA introduced by DNA mediated transformation is integrated randomly into the genome resulting in inefficient gene deletion and position-specific effects. To enhance successful gene targeting, the consequences of perturbing components of the non-homologous end joining recombination pathway have been examined. The deletion of the KU70 and LIG4 orthologs, pkuA and ligD, respectively, dramatically enhanced the observed homologous recombination frequency leading to efficient gene deletion. While ΔpkuA was associated with reduced genetic stability over-time, ΔligD represents a suitable recipient strain for downstream applications and combined with a modified Gateway? system for the rapid generation of gene deletion constructs, this represents an efficient pipeline for characterizing gene function in P. marneffei.     

Characterization of Mce4A protein of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>: role in invasion and survival

Background  

The mce4 operon is one of the four homologues of mammalian cell entry (mce) operons of Mycobacterium tuberculosis. The mce4A (Rv3499c) gene within this operon is homologous to mce1A (Rv0169), that has a role in host cell invasion by M. tuberculosis. Our earlier reports show that mce4 operon is expressed during the stationary phase of growth of the bacillus in culture and during the course of infection in mammalian hosts. M. tuberculosis carrying mutation in mce4 operon shows growth defect and reduced survival in infected mice. However, the intracellular localization of Mce4A protein and its direct role in cell entry or survival of the bacillus has not been demonstrated so far.     

Luminescence,circular dichroism and in silico studies of binding interaction of synthesized naphthylchalcone derivatives with bovine serum albumin

Chalcones possess various biological properties, for example, antimicrobial, anti‐inflammatory, analgesic, antimalarial, anticancer, antiprotozoal and antitubercular activity. In this study, naphthylchalcone derivatives were synthesized and characterized using ¹H NMR ¹³C NMR, Fourier transform infrared and mass techniques. Yields for all derivatives were found to be >90%. Protein–drug interactions influence the absorption, distribution, metabolism and excretion (ADME) properties of a drug. Therefore, to establish whether the synthesized naphthylchalcone derivatives can be used as drugs, their binding interaction toward a serum protein (bovine serum albumin) was investigated using fluorescence, circular dichroism and molecular docking techniques under physiological conditions. Fluorescence quenching of the protein in the presence of naphthylchalcone derivatives, and other derived parameters such as association constants, number of binding sites and static quenching involving confirmed non‐covalent binding interactions in the protein–ligand complex were observed. Circular dichroism clearly showed changes in the secondary structure of the protein in the presence of naphthylchalcones, indicating binding between the derivatives and the serum protein. Molecular modelling further confirmed the binding mode of naphthylchalcone derivatives in bovine serum albumin. A site‐specific molecular docking study of naphthylchalcone derivatives with serum albumin showed that binding took place primarily in the aromatic low helix and then in subdomain II. The dominance of hydrophobic, hydrophilic and hydrogen bonding was clearly visible and was responsible for stabilization of the complex.     

Fully integrated downstream process to enable next-generation manufacturing

Next-generation manufacturing (NGM) has evolved over the past decade to a point where large biopharmaceutical organizations are making large investments in the technology and considering implementation in clinical and commercial processes. There are many well-considered reasons to implement NGM. For the most part, organizations will not fund NGM unless the implementation benefits the funding organization by providing reduced costs, reduced time, or additional needed capabilities. Productivity improvements gained from continuous purification are shown in this work, which used a new system that fully integrates and automates several downstream unit operations of a biopharmaceutical process to provide flexibility and easy implementation of NGM. The equipment and automation needed to support NGM can be complicated and expensive. Biopharmaceutical Process Development considered two options as follows: (1) design its own NGM system or (2) buy a prebuilt system. PAK BioSolutions offers a turn-key automated and integrated system that can operate up to four continuous purification stages simultaneously, while maintaining a small footprint in the manufacturing plant. The system provides significant cost benefits (~10× lower) compared with the alternative—integration of many different pieces of equipment through a Distributed Control System that would require significant engineering time for design, automation, and integration. Integrated and Continuous Biomanufacturing can lead to significant reductions in facility size, reduced manufacturing costs, and enhanced product quality when compared with the traditional batch mode of operation. The system uses new automation strategies that robustly link unit operations. We present the optimized process fit, sterility and bioburden control strategy, and automation features (such as pH feedback control and in-line detergent addition), which enabled continuous operation of a 14-day end-to-end monoclonal antibody purification process at the clinical manufacturing scale.     

Prostaglandins E1 and E2 stimulate the proliferation of pulmonary artery smooth muscle cells.

Prostaglandins E1 and E2 are thought to be inhibitors of the growth of systemic vascular smooth muscle cells (SMC). However, their effect on the proliferation of SMC from the pulmonary artery (PA) has not been described and was the subject of this investigation. Cultures of bovine PA SMC were exposed to PGE1 and PGE2 under various conditions and their growth was assessed. PGE1 and PGE2 did not inhibit the growth of PA SMC in 10% fetal calf serum (FCS), but instead caused a dose dependent (10 ng - 1 microgram/ml) increase in [3H]-thymidine incorporation when added to cultures containing 0.5% FCS; the highest doses resulted in 95% and 75% increases in [3H]-thymidine uptake at 24 hours with PGE1 and PGE2 respectively. This was accompanied by a modest increase in actual cell numbers (e.g., 20% with 1 microgram/ml PGE1). Furthermore, PGE1 could mimic insulin-like growth factor (IGF-1) by potentiating the stimulation of SMC growth by fibroblast growth factor, suggesting that PGE1 may act as a progression factor in the growth cycle of these cells. There was, however, no effect of PGE1 on the proliferation of bovine aortic SMC. We conclude that, contrary to most reported effects on systemic SMC, PGE1 and PGE2 do not inhibit the proliferation of PA SMC but rather stimulate it.