pp 1-1 March 2014
pp 3-11 March 2014 Clipboard
Secretion is widespread in all eukaryotic cells: all of us experience this in the course of daily life – saliva, mucus, sweat, tears, bile juice, adrenalin, etc. – the list is extremely long. How does a cell manage to repeatedly spit out some stuff without losing the rest? The answer is: through regulated vesicle trafficking within the cell. The Nobel Prize in Physiology or Medicine 2013 was awarded to Drs Randy Schekman, James E Rothman and Thomas C Südhof for their ‘discoveries of machinery regulating vesicle traffic, a major transport system in our cells’. Dr Randy Schekman and his colleagues discovered a number of genes required for vesicle trafficking from the endoplasmic reticulum (ER) and Golgi; the James E Rothman group unravelled the protein machinery that allows vesicles to bud off from the membrane and fuse to their targets; and Dr Thomas C Südhof along with his colleagues revealed how calcium ions could instruct vesicles to fuse and discharge their contents with precision. These enabled the biotechnology industry to produce a variety of pharmaceutical and industrial products like insulin and hepatitis B vaccines, in a cost-efficient manner, using yeast and tissue cultured cells.
pp 13-27 March 2014 Commentary
Biochemical information has been crucial for the development of evolutionary biology. On the one hand, the sequence information now appearing is producing a huge increase in the amount of data available for phylogenetic analysis; on the other hand, and perhaps more fundamentally, it allows understanding of the mechanisms that make evolution possible. Less well recognized, but just as important, understanding evolutionary biology is essential for understanding many details of biochemistry that would otherwise be mysterious, such as why the structures of NAD and other coenzymes are far more complicated than their functions would seem to require. Courses of biochemistry should thus pay attention to the essential role of evolution in selecting the molecules of life.
pp 29-32 March 2014 Series
pp 33-41 March 2014 Brief communication
Chloroplasts offer high-level transgene expression and transgene containment due to maternal inheritance, and are ideal hosts for biopharmaceutical biosynthesis via multigene engineering. To exploit these advantages, we have expressed 12 enzymes in chloroplasts for the biosynthesis of artemisinic acid (precursor of artemisinin, antimalarial drug) in an alternative plant system. Integration of transgenes into the tobacco chloroplast genome via homologous recombination was confirmed by molecular analysis, and biosynthesis of artemisinic acid in plant leaf tissues was detected with the help of 13C NMR and ESI-mass spectrometry. The excess metabolic flux of isopentenyl pyrophosphate generated by an engineered mevalonate pathway was diverted for the biosynthesis of artemisinic acid. However, expression of megatransgenes impacted the growth of the transplastomic plantlets. By combining two exogenous pathways, artemisinic acid was produced in transplastomic plants, which can be improved further using better metabolic engineering strategies for commercially viable yield of desirable isoprenoid products.
pp 43-52 March 2014 Articles
Protein factors involved in lipofection pathways remain elusive. Using avidin-biotin affinity chromatography and mass finger printing analysis technique, herein we report the identification of a 70 kDa size protein (bovine serum albumin precursor, BSAP) which binds strongly with lipoplexes and may play role in lipofection pathway. Using multiple cultured animal cells and three structurally different cationic transfection lipids, we show that the efficiencies of liposomal transfection vectors get significantly enhanced (by ∼2.5- to 5.0-fold) in cells pre-transfected with lipoplexes of reporter plasmid construct encoding BSAP. Findings in the cellular uptake experiments in A549 cells cultured in DMEM supplemented with 10% (w/w) BODIPY-labelled BSAP are consistent with the supposition that BSAP enters cell cytoplasm from the cell culture medium (DMEM supplemented with 10% FBS) used in lipofection. Cellular uptake studies by confocal microscopy using BODIPY-labelled BSAP and FITC-labelled plasmid DNA revealed co-localization of plasmid DNA and BSAP within the cell cytoplasm and nucleus. In summary, the present findings hint at the possible involvement of BSAP in lipofection pathway.
pp 53-61 March 2014 Articles
Many horizontally acquired genes (xenogenes) in the bacterium Escherichia coli are maintained in a silent transcriptional state by the nucleoid-associated transcription regulatory protein H-NS. Recent evidence has shown that antibiotic-mediated inhibition of the transcription terminator protein Rho leads to de-repression of horizontally acquired genes, akin to a deletion of hns. The mechanism behind this similarity in outcomes between the perturbations of two distinct processes remains unclear. Using ChIP-seq of H-NS in wild-type cells, in addition to that in cells treated with bicyclomycin – a specific inhibitor of Rho, we show that bicyclomycin treatment leads to a decrease in binding signal for H-NS to the E. coli chromosome. Rho inhibition leads to RNA polymerase readthrough, which in principle could displace H-NS from the DNA, thus leading to transcriptional derepression of H-NS-silenced genes. Other possible mediators of the effect of Rho on H-NS are discussed. A possible positive feedback between Rho and H-NS might help reinforce xenogene silencing.
pp 63-74 March 2014 Articles
Classical swine fever virus (CSFV), the pathogen of classical swine fever (CSF), causes severe hemorrhagic fever and vascular necrosis in domestic pigs and wild boar. A large number of evidence has proven that non-structural 5A (NS5A) is not only a very important part of viral replication complex, but also can regulate host cell’s function; however, the underlying mechanisms remain poorly understood. In the current study, aiming to find more clues in understanding the molecular mechanisms of CSFV NS5A’s function, the yeast two-hybrid (Y2H) system was adopted to screen for CSFV NS5A interactive proteins in the cDNA library of the swine umbilical vein endothelial cell (SUVEC). Alignment with the NCBI database revealed 16 interactive proteins: DDX5, PSMC3, NAV1, PHF5A, GNB2L1, CSDE1, HSPA8, BRMS1, PPP2R3C, AIP, TMED10, POLR1C, TMEM70, METAP2, CHORDC1 and COPS6. These proteins are mostly related to gene transcription, protein folding, protein degradation and metabolism. The interactions detected by the Y2H system should be considered as preliminary results. Since identifying novel pathways and host targets, which play essential roles during infection, may provide potential targets for therapeutic development. The finding of proteins obtained from the SUVEC cDNA library that interact with the CSFV NS5A protein provide valuable information for better understanding the interactions between this viral protein and the host target proteins.
pp 75-84 March 2014 Articles
Dictyostelium discoideum encodes a single Rheb protein showing sequence similarity to human homologues of Rheb. The DdRheb protein shares 52% identity and 100% similarity with the human Rheb1 protein. Fluorescence of Rheb yellow fluorescent protein fusion was detected in the D. discoideum cytoplasm. Reverse transcription-polymerase chain reaction and whole-mount in situ hybridization analyses showed that rheb is expressed at all stages of development and in prestalk cells in the multicellular structures developed. When the expression of rheb as a fusion with lacZ was driven under its own promoter, the 𝛽-galactosidase activity was seen in the prestalk cells. D. discoideum overexpressing Rheb shows an increase in the size of the cell. Treatment of the overexpressing Rheb cells with rapamycin confirms its involvement in the TOR signalling pathway.
pp 85-95 March 2014 Articles
One of the pathological hallmarks of Alzheimer’s disease is the presence of insoluble extracellular amyloid plaques. These plaques are mainly constituted of amyloid beta peptide (A𝛽), a proteolytic product of amyloid precursor protein (APP). APP processing also generates the APP intracellular domain (AICD). We have previously demonstrated that AICD interacts with FKBP12, a peptidyl-prolyl cis-trans isomerase (PPIase) ubiquitous in nerve systems. This interaction was interfered by FK506, a clinically used immunosuppressant that has recently been reported to be neuroprotective. To elucidate the roles of FKBP12 in the pathogenesis of Alzheimer’s disease, the effect of FKBP12 overexpression on APP processing was evaluated. Our results revealed that APP processing was shifted towards the amyloidogenic pathway, accompanied by a change in the subcellular localization of APP, upon FKBP12 overexpression. This FKBP12-overexpression-induced effect was reverted by FK506. These findings support our hypothesis that FKBP12 may participate in the regulation of APP processing. FKBP12 overexpression may lead to the stabilization of a certain isomer (presumably the cis form) of the Thr668-Pro669 peptide bond in AICD, therefore change its affinity to flotillin-1 or other raft-associated proteins, and eventually change the localization pattern and cause a shift in the proteolytic processing of APP.
pp 97-105 March 2014 Articles
Alzheimer’s disease (AD) is a progressive neurodegenerative disorder. The deterioration of subcellular organelles, including the mitochondria, is another major ultrastructural characteristic of AD pathogenesis, in addition to amyloid plaque deposition. However, the three-dimensional (3-D) study of mitochondrial structural alteration in AD remains poorly understood. Therefore, ultrastructural analysis, 3-D electron tomography, and immunogold electron microscopy were performed in the present study to clarify the abnormal structural alterations in mitochondria caused by the progression of AD in APP/PSEN1 transgenic mice, expressing human amyloid precursor protein, as a model for AD. Amyloid 𝛽 (A𝛽) plaques accumulated and dystrophic neurites (DN) developed in the hippocampus of transgenic AD mouse brains. We also identified the loss of peroxiredoxin 3, an endogenous cytoprotective antioxidant enzyme and the accumulation of A𝛽 in the hippocampal mitochondria of transgenic mice, which differs from those in age-matched wild-type mice. The mitochondria in A𝛽 plaque-detected regions were severely disrupted, and the patterns of ultrastructural abnormalities were classified into three groups: disappearance of cristae, swelling of cristae, and bulging of the outer membrane. These results demonstrated that morpho-functional alterations of mitochondria and AD progression are closely associated and may be beneficial in investigating the function of mitochondria in AD pathogenesis.
pp 107-117 March 2014 Articles
Rhythmic sound or music is known to improve cognition in animals and humans. We wanted to evaluate the effects of prenatal repetitive music stimulation on the remodelling of the auditory cortex and visual Wulst in chicks. Fertilized eggs (0 day) of white leghorn chicken (Gallus domesticus) during incubation were exposed either to music or no sound from embryonic day 10 until hatching. Auditory and visual perceptual learning and synaptic plasticity, as evident by synaptophysin and PSD-95 expression, were done at posthatch days (PH) 1, 2 and 3. The number of responders was significantly higher in the music stimulated group as compared to controls at PH1 in both auditory and visual preference tests. The stimulated chicks took significantly lesser time to enter and spent more time in the maternal area in both preference tests. A significantly higher expression of synaptophysin and PSD-95 was observed in the stimulated group in comparison to control at PH1-3 both in the auditory cortex and visual Wulst. A significant inter-hemispheric and gender-based difference in expression was also found in all groups. These results suggest facilitation of postnatal perceptual behaviour and synaptic plasticity in both auditory and visual systems following prenatal stimulation with complex rhythmic music.
pp 119-126 March 2014 Articles
A plant that is in part infected by a pathogen is more resistant throughout its whole body to subsequent infections – a phenomenon known as systemic acquired resistance (SAR). Mobile signals are synthesized at the site of infection and distributed throughout the plant through vascular tissues. Mechanism of SAR development subsequent to reaching the mobile signal in the distal tissue is largely unknown. Recently we showed that FLOWERING LOCUS D (FLD) gene of Arabidopsis thaliana is required in the distal tissue to activate SAR. FLD codes for a homologue of human-lysine-specific histone demethylase. Here we show that FLD function is required for priming (SAR induced elevated expression during challenge inoculation) of WRKY29 and WRKY6 genes. FLD also differentially influences basal and SAR-induced expression of WRKY38, WRKY65 and WRKY53 genes. In addition, we also show that FLD partly localizes in nucleus and influences histone modifications at the promoters of WRKY29 and WRKY6 genes. The results altogether indicate to the possibility of FLD’s involvement in epigenetic regulation of SAR.
pp 127-137 March 2014 Articles
Hpa1 is a harpin protein produced by Xanthomonas oryzae, an important bacterial pathogen of rice, and has the growth-promoting activity in plants. To understand the molecular basis for the function of Hpa1, we generated an inactive variant protein, Hpa1𝛥NT, by deleting the nitroxyl-terminal region of the Hpa1 sequence and compared Hpa1𝛥NT with the full-length protein in terms of the effects on vegetative growth and related physiological responses in Arabidopsis. When Hpa1 was applied to plants, it acted to enhance the vegetative growth but did not affect the floral development. Enhanced plant growth was accompanied by induced expression of growth-promoting genes in plant leaves. The growth-promoting activity of Hpa1 was further correlated with a physiological consequence shown as promoted leaf photosynthesis as a result of facilitated CO2 conduction through leaf stomata and mesophyll cells. On the contrary, plant growth, growth-promoting gene expression, and the physiological consequence changed little in response to the Hpa1𝛥NT treatment. These analyses suggest that Hpa1 requires the nitroxyl-terminus to facilitate CO2 transport inside leaf cells and promote leaf photosynthesis and vegetative growth of the plant.
pp 139-144 March 2014 Reviews
Etoposide, a semi-synthetic derivative of podophyllotoxin, is one of the most active and useful antineoplastic agent used routinely in firstline combination chemotherapy of testicular cancer, small-cell lung cancer and non-Hodgkin’s lymphoma. Etoposide displays narrow therapeutic index, erratic pharmacokinetics and dose individualization that needs to be achieved for overcoming inter- and intra-patient variability (25–80%), so as to maintain proper drug exposure within a therapeutic range. Etoposide posses high plasma protein binding (97%) and is degraded via complex metabolic pathways. The main pharmacokinetic determinants of etoposide are still not completely defined in order to optimize the pharmaco-therapeutic parameters including dose, therapeutic schedule and route of administration. Much research has been done to determine drug–drug and herb–drug interactions for improving the bioavailability of etoposide. The present article gives insight on pharmaceutical and pharmacological attempts made from time to time to overcome the erratic inter- and intra-patient variability for improving the bioavailability of etoposide.
pp 145-155 March 2014 Reviews
Apico-basal polarity is a cardinal molecular feature of adult eukaryotic epithelial cells and appears to be involved in several key cellular processes including polarized cell migration and maintenance of tissue architecture. Epithelial cell polarity is maintained by three well-conserved polarity complexes, namely, PAR, Crumbs and SCRIB. The location and interaction between the components of these complexes defines distinct structural domains of epithelial cells. Establishment and maintenance of apico-basal polarity is regulated through various conserved cell signalling pathways including TGF𝛽, Integrin and WNT signalling. Loss of cell polarity is a hallmark for carcinoma, and its underlying molecular mechanism is beginning to emerge from studies on model organisms and cancer cell lines. Moreover, deregulated expression of apico-basal polarity complex components has been reported in human tumours. In this review, we provide an overview of the apico-basal polarity complexes and their regulation, their role in cell migration, and finally their involvement in carcinogenesis.
pp 157-169 March 2014 Reviews
Regenerative medicine is an evolving interdisciplinary topic of research involving numerous technological methods that utilize stem cells to repair damaged tissues. Particularly, mesenchymal stem cells (MSCs) are a great tool in regenerative medicine because of their lack of tumorogenicity, immunogenicity and ability to perform immunomodulatory as well as anti-inflammatory functions. Numerous studies have investigated the role of MSCs in tissue repair and modulation of allogeneic immune responses. MSCs derived from different sources hold unique regenerative potential as they are self-renewing and can differentiate into chondrocytes, osteoblasts, adipocytes, cardiomyocytes, hepatocytes, endothelial and neuronal cells, among which neuronal-like cells have gained special interest. MSCs also have the ability to secrete multiple bioactive molecules capable of stimulating recovery of injured cells and inhibiting inflammation. In this review we focus on neural differentiation potential ofMSCs isolated from different sources and how certain growth factors/small molecules can be used to derive neuronal phenotypes from MSCs. We also discuss the efficacy of MSCs when transplanted in vivo and how they can generate certain neurons and lead to relief or recovery of the diseased condition. Furthermore, we have tried to evaluate the appropriatemerits of different sources of MSCs with respect to their propensity towards neurological differentiation as well as their effectiveness in preclinical studies.