Life Sciences

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Shiv Nadar University

The TIM23 (translocase of the mitochondrial inner membrane) complex mediates translocation of preproteins across and their insertion into the mitochondrial inner membrane. How the translocase mediates sorting of preproteins into the two different subcompartments is poorly understood. In particular, it is not clear whether association of two operationally defined parts of the translocase, the membrane-integrated part and the import motor, depends on the activity state of the translocase. We established conditions to in vivo trap the TIM23 complex in different translocation modes. Membrane-integrated part of the complex and import motor were always found in one complex irrespective of whether an arrested preprotein was present or not. Instead, we detected different conformations of the complex in response to the presence and, importantly, the type of preprotein being translocated. Two non-essential subunits of the complex, Tim21 and Pam17, modulate its activity in an antagonistic manner. Our data demonstrate that the TIM23 complex acts as a single structural and functional entity that is actively remodelled to sort preproteins into different mitochondrial subcompartments. © 2008 European Molecular Biology Organization | All Rights Reserved.

World University root node

(SoNS) School of Natural Sciences

Fulltext

EMBO Journal

Active remodelling of the TIM23 complex during translocation of preproteins into mitochondria

Microbiological Research

Erratum to "In silico mining in expressed sequences of Neurospora crassa for identification and abundance of microsatellites". [Microbiol. Res. 162 (2007) 250-256] (DOI:10.1016/j.micres.2006.05.012)

The mechanism of DNA replication initiation and progression is poorly understood in the parasites, including human malaria parasite Plasmodium falciparum. Using bioinformatics tools and yeast complementation assay, we identified a putative homologue of Saccharomyces cerevisiae origin recognition complex subunit 5 in P. falciparum (PfORC5). PfORC5 forms distinct nuclear foci colocalized with the replication foci marker proliferating cell nuclear antigen (PfPCNA) and co-immunoprecipitates with PCNA during early-to-mid trophozoite stage replicating parasites. Interestingly, these proteins separate from each other at the non-replicating late schizont stage, citing the evidence of the presence of both PCNA and ORC components in replication foci during eukaryotic DNA replication. PfORC1, another ORC subunit, colocalizes with PfPCNA and PfORC5 at the beginning of DNA replication, but gets degraded at the late schizont stage, ensuring the regulation of DNA replication in the parasites. Further, we have identified putative PCNA-interacting protein box in PfORC1 that may explain in part the colocalization of PfORC and PfPCNA. Additionally, use of specific DNA replication inhibitor hydroxyurea affects ORC5/PCNA foci formation and parasitic growth. These results strongly favour replication factory model in the parasites and confer great potential to understand the co-ordination between ORC and PCNA during eukaryotic DNA replication in general. © 2008 The Authors.

Molecular Microbiology

Plasmodium falciparum origin recognition complex subunit 5: Functional characterization and role in DNA replication foci formation

Rap1 GTPase activation by its cAMP responsive nucleotide exchange factor Epac present in endothelial cells increases endothelial cell barrier function with an associated increase in cortical actin. Here, Epac1 was shown to be responsible for these actin changes and to colocalize with microtubules in human umbilical vein endothelial cells. Importantly, Epac activation with a cAMP analogue, 8-pCPT-2′O-Me-cAMP resulted in a net increase in the length of microtubules. This did not require cell- cell interactions or Rap GTPase activation, and it was attributed to microtubule growth as assessed by time-lapse microscopy of human umbilical vein endothelial cell expressing fluorophore-linked microtubule plus-end marker end-binding protein 3. An intact microtubule network was required for Epac-mediated changes in cortical actin and barrier enhancement, but it was not required for Rap activation. Finally, Epac activation reversed microtubuledependent increases in vascular permeability induced by tumor necrosis factor-alpha; and transforming growth factor-β. Thus, Epac can directly promote microtubule growth in endothelial cells. This, together with Rap activation leads to an increase in cortical actin, which has functional significance for vascular permeability. © 2008 by The American Society for Cell Biology.

Molecular Biology of the Cell

Role of Epac1, an exchange factor for Rap GTPases, in endothelial microtubule dynamics and barrier function

The late Neogene section of ODP Hole 763A, Exmouth Plateau, southeast Indian Ocean, has been biostratigraphically subdivided into eight planktic foraminiferal zones. The zones are similar to those of the tropical northern Indian and tropical western Pacific Oceans. The sequential order of planktic foraminiferal events has been determined, and 32 late Neogene planktic foraminiferal events have been identified. A major faunal turnover (22 events) occurs between 80 and 45 meters below seafloor (mbsf), spanning 1.6 million years between 3.6 Ma and 2 Ma and may be related to the stresses transmitted to the upper ocean due to major cooling associated with Northern Hemisphere glaciation. Based on integration with magnetostratigraphy, numerical age estimates of planktic foraminiferal events have been determined for the first time from the southeast Indian Ocean. A comparison with the published dates for these events from other parts of the world suggests that 14 events are reliable for correlation over a wide geographic range. The useful synchronous events are the Globorotafta tosaensis FO (2.84 Ma), the Globorotalia tosaensis LO (0.59 Ma), the Globigerinoides extremus LO (1.87 Ma), the Globigerinoides fistulosus FO (3.26 Ma), the Globigerinoides fistulosus LO (1.73 Ma), the Dentoglobigerina altispira LO (3.05 Ma), the Dentoglobigerina globosa LO (3.18), the Neogloboquadrina dutertrei FO (2.77 Ma), the Sphaeroidinellopsis LO (3.35), the Globorotalia margaritae LO (3.38 Ma), the Globorotalia crassaformis FO (4.41 Ma), the Globoturborotalita nepenthes LO (3.44 Ma), the Globorotalia tumida tumida FO (5.10 Ma) and the Pulleniatina primalis FO (5.70 Ma). In contrast, the other events are diachronous and considered unsuitable for inter-oceanic correlation. The Pliocene-Pleistocene boundary is marked at ODP Hole 763A by the LO of Globigerinoides fistulosus, which is a synchronous event over a wide latitudinal range and occurs close to the top of the Olduvai Event (Chron C2n; 1.77 Ma). The Miocene-Pliocene boundary is approximated by the first evolutionary appearance of Globorotalia tumida tumida from its ancestor Globorotalia plesiotumida and occurs close to base of Thvera magnetic event (C3n.4n; 5.23 Ma). Recognition of both of these epoch boundaries is in accord with the International Commission on Stratigraphy. The detailed planktic foraminiferal biochronology developed for Hole 763A will aid correlation and dating of various paleoceanographic events over the last 6 million years in the southeast Indian Ocean.

Journal of Foraminiferal Research

Late neogene planktic foraminiferal biochronology of the ODP site 763A, Exmouth Plateau, southeast Indian Ocean

Plasmodium falciparum sirtuin, PfSir2, contains histone deacetylase (HDAC) activity that may be central to the regulation of virulence gene expression in the parasites. Although a few reports have been published recently regarding in vitro and in vivo function of PfSir2, expression of the endogenous protein (c. 30 kDa) has not been shown yet. Here we report the presence of PfSir2 in the parasite at the protein level by specific antibodies. HDAC activity of PfSir2 can be inhibited by nicotinamide, a product of sirtuin reaction. Surprisingly, we find that nicotinamide also delays parasite growth significantly in culture. These findings further our knowledge on PfSir2 and raise the possibility of using an inexpensive agent like nicotinamide as an antimalarial in combination with other antiparasitic drugs. © 2008 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

FEMS Microbiology Letters

Nicotinamide inhibits Plasmodium falciparum Sir2 activity in vitro and parasite growth

Aspartate semialdehyde dehydrogenase from Mycobacterium tuberculosis (Asd, ASADH, Rv3708c), which is the second enzyme in the lysine/homoserine- biosynthetic pathways, has been expressed heterologously in Escherichia coli. The enzyme was purified using affinity and gel-filtration chromatographic techniques and crystallized in two different crystal forms. Preliminary diffraction data analysis suggested the presence of up to four monomers in the asymmetric unit of the orthorhombic crystal form A and of one or two monomers in the cubic crystal form B. {\textcopyright} International Union of Crystallography All rights reserved.

Acta Crystallographica Section F: Structural Biology and Crystallization Communications

Purification, crystallization and preliminary X-ray diffraction analysis of aspartate semialdehyde dehydrogenase (Rv3708c) from Mycobacterium tuberculosis

Vascular endothelial-cadherin (VE-cad) is localized to adherens junctions at endothelial cell borders and forms a complex with α-, β-, γ-, and p120-catenins (p120). We previously showed that the VE-cad complex disassociates to form shortlived "gaps" during leukocyte transendothelial migration (TEM); however, whether these gaps are required for leukocyte TEM is not clear. Recently p120 has been shown to control VE-cad surface expression through endocytosis. We hypothesized that p120 regulates VE-cad surface expression, which would in turn have functional consequences for leukocyte transmigration. Here we show that endothelial cells transduced with an adenovirus expressing p120GFP fusion protein significantly increase VE-cad expression. Moreover, endothelial junctions with high p120GFP expression largely prevent VE-cad gap formation and neutrophil leukocyte TEM; if TEM occurs, the length of time required is prolonged. We find no evidence that VE-cad endocytosis plays a role in VE-cad gap formation and instead show that this process is regulated by changes in VE-cad phosphorylation. In fact, a nonphosphorylatable VE-cad mutant prevented TEM. In summary, our studies provide compelling evidence that VE-cad gap formation is required for leukocyte transmigration and identify p120 as a critical intracellular mediator of this process through its regulation of VE-cad expression at junctions. © 2008 by The American Society of Hematology.

Blood

p120-catenin regulates leukocyte transmigration through an effect on VE-cadherin phosphorylation

In rod-shaped bacteria, septal peptidoglycan synthesis involves the late recruitment of the ftsI gene product (PBP3 in Escherichia coli) to the FtsZ ring. We show that in Caulobacter crescentus, PBP3 accumulates at the new pole at the beginning of the cell cycle. Fluorescence recovery after photobleaching experiments reveal that polar PBP3 molecules are, constantly and independently of FtsZ, replaced by those present in the cellular pool, implying that polar PBP3 is not a remnant of the previous division. By the time cell constriction is initiated, all PBP3 polar accumulation has disappeared in favour of an FtsZ-dependent localization near midcell, consistent with PBP3 function in cell division. Kymograph analysis of time-lapse experiments shows that the recruitment of PBP3 to the FtsZ ring is progressive and initiated very early on, shortly after FtsZ ring formation and well before cell constriction starts. Accumulation of PBP3 near midcell is also highly dynamic with a rapid exchange of PBP3 molecules between midcell and cellular pools. Localization of PBP3 at both midcell and pole appears multifactorial, primarily requiring the catalytic site of PBP3. Collectively, our results suggest a role for PBP3 in pole morphogenesis and provide new insights into the process of peptidoglycan assembly during division. © 2008 Blackwell Publishing Ltd.

Localization of PBP3 in Caulobacter crescentus is highly dynamic and largely relies on its functional transpeptidase domain

The paper describes the major circulation changes in the eastern Indian Ocean during last 5 million years based on variation in planktic foraminiferal assemblages in various ODP and DSDP cores examined by various workers. The Late Miocene/Pliocene transition is characterized by weakening of the Leeuwin Current and migration of the polar front towards north. The early Pliocene is marked by closing of the Indonesian Seaway for surface waters and strengthening of the Leeuwin Current, a general warming of the surface waters and thickening of the mixed layer in the eastern Indian Ocean. The Late Pliocene and Pleistocene witnessed severe climatic fluctuations and episodic weakening and strengthening of the Leeuwin Current in response to either ENSO induced changes in the Western Pacific Warm Pool or lowering of sea level and reduction in Indonesian throughflow due to ice sheet expansion.