Effect of long and short photoperiod on vasotocin neurons of paraventricular nuclei and adrenal function of water deprived Japanese quail

The responses of magnocellular neurons of paraventricular nuclei (PVN) and changes to adrenal activity to water deprivation in Japanese quail maintained under gonado-inhibitory and stimulatory photoperiods were examined. Water deprivation of 4 days resulted in a 12% decrease in body weight of sexually regressed short day (SD, 6L:18D) quail, while the decrease was more (18%) in sexually stimulated long day (LD, 16L:8D) quail. The increase in plasma osmolality following water deprivation was also more (47%) in LD than to SD quail (36%). Under the LD condition, quail had increased numbers, sizes and immunostaining of ir-AVT neurons of PVN compared to SD condition. A significant increase in the number of ir-AVT neurons was observed following 4 days of water deprivation in both SD and LD quail compared to their respective fully hydrated controls. However, the degree of response was more under the LD compared to the SD condition suggesting that gonado-stimulatory long days increase the activity/response of the AVT system. Increased adrenal ascorbic acid content (i.e., activity) was also observed to quail of LD when compared to SD treatment. However, osmotic stress led to adrenal hypertrophy and hyperactivity of quail of both of the photoperiodic regimes. Our findings indicate that not only osmotic stress but also photo-gonadal stimulation upregulates the expression of hypothalamic AVT genes and increases the localization of ir-AVT in many neurons of PVN. The above results support the existence of a parallel adrenal-gonad relationship and increase in adrenal function during osmotic stress, which also leads to simultaneous increase in AVT system. We conclude that photo-sexual conditions alter hypothalamic vasotocinergic and adrenal activity in Japanese quail and the degree of stimulation of the two systems following osmotic stress is higher under gonado-stimulatory LD conditions.     

Kinase activation of ClC-3 accelerates cytoplasmic condensation during mitotic cell rounding

"Mitotic cell rounding" describes the rounding of mammalian cells before dividing into two daughter cells. This shape change requires coordinated cytoskeletal contraction and changes in osmotic pressure. While considerable research has been devoted to understanding mechanisms underlying cytoskeletal contraction, little is known about how osmotic gradients are involved in cell division. Here we describe cytoplasmic condensation preceding cell division, termed "premitotic condensation" (PMC), which involves cells extruding osmotically active Cl(-) via ClC-3, a voltage-gated channel/transporter. This leads to a decrease in cytoplasmic volume during mitotic cell rounding and cell division. Using a combination of time-lapse microscopy and biophysical measurements, we demonstrate that PMC involves the activation of ClC-3 by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in human glioma cells. Knockdown of endogenous ClC-3 protein expression eliminated CaMKII-dependent Cl(-) currents in dividing cells and impeded PMC. Thus, kinase-dependent changes in Cl(-) conductance contribute to an outward osmotic pressure in dividing cells, which facilitates cytoplasmic condensation preceding cell division.     

Investigate the chronic neurotoxic effects of diquat

Chronic exposure to agricultural chemicals (pesticides/herbicides) has been shown to induce neurotoxic effects or results in accumulation of various toxic metabolic by-products. These substances have the relevant ability to cause or increase the risk for neurodegeneration. Diquat is an herbicide that has been extensively used in the United States of America and other parts of the world. Diquat is constantly released into the environment during its use as a contact herbicide. Diquat structurally resembles 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) and paraquat. Rotenone, paraquat, maneb and MPTP reproduce features of movement disorders in experimental animal models. Based on the structural similarity to other neurotoxins, chronic exposure of diquat can induce behavioral and neurochemical alterations associated with dopaminergic neurotoxicity. However, in the present study, diquat unlike other neurotoxins (rotenone, 6-hydroxydopamine, MPTP, paraquat and maneb) did not induce dopamine depletion in the mouse striatum. Although, notable exacerbation in motor impairment (swimming score, akinesia and open field) were evident that may be due to the decreased dopamine turnover and mild nigrostriatal neurodegeneration. These data indicate that, despite the apparent structural similarity to other dopaminergic neurotoxins, diquat did not exert severe deleterious effects on dopamine neurons in a manner that is unique to rotenone and MPTP.     

Novel 16S rRNA based PCR method targeting Deinococcus spp. and its application to assess the diversity of deinococcal populations in environmental samples

The members of the genus Deinococcus are extensively studied because of their exemplary radiation resistance. Both ionizing and non-ionizing rays are routinely employed to select upon the radiation resistant deinococcal population and isolate them from the majority of radiation sensitive population. There are no studies on the development of molecular tools for the rapid detection and identification of deinococci from a mixed population without causing the bias of radiation enrichment. Here we present a Deinococcus specific two-step hemi-nested PCR for the rapid detection of deinococci from environmental samples. The method is sensitive and specific to detect deinococci without radiation exposure of the sample. The new protocol was successfully employed to detect deinococci from several soil samples from different geographical regions of India. The PCR method could be adapted to a three-step protocol to study the diversity of the environmental deinococcal population by denaturing gradient gel electrophoresis (DGGE). Sequence analysis of the DGGE bands revealed that the samples harbor diverse populations of deinococci, many of which were not recovered by culturing and may represent novel clades. We demonstrate that the genus specific primers are also suitable for the rapid identification of the bacterial isolates that are obtained from a typical radiation enrichment isolation technique. Therefore the primers and the protocols described in this study can be used to study deinococcal diversity from environmental samples and can be employed for the rapid detection of deinococci in samples or identifying pure culture isolates as Deinococcus species.     

Neuroprotection from Tissue Inhibitor of Metalloproteinase-1 and its nanoparticles

Matrix metalloproteinases (MMPs) are family of zinc dependent endopeptidases, which cleave extracellular matrix proteins, and play an important role in tissue remodelling in physiological and pathological processes. There is enhanced expression of MMPs, in particular MMP-9, during numerous pathological conditions, including epilepsy and ischemic stroke. Therefore, inhibition of MMP-9 is considered as a potential therapeutic target. Tissue Inhibitor of Matrix Metalloproteinase-1 (TIMP-1) is a 28 kDa endogenous inhibitor of MMP-9. In this study we examined recombinant mouse TIMP-1 for its in-vitro neuroprotective effects, against Kainic Acid (KA) induced excitotoxicity in organotypic hippocampal slice culture (OHC) model. We also studied, sustained release effects of TIMP-1 in OHC by using poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs). TIMP-1 and TIMP-1 PLGA NPs were added to the slice cultures at different time points, i.e., 30 min before treatment with KA and 6 h after KA treatment. Propidium iodide staining was used to reveal cell toxicity in the cultures. In addition, neurotoxicity was assessed using standard lactate dehydrogenase (LDH) release assay. Gelatinolytic activity in conditioned cultured medium of OHC was accessed by a fluorescent substrate assay. Briefly, our result show that TIMP-1 provided significant level of neuroprotection, especially when given before 30 min of KA and released from the NPs. Since gelatinolytic activity assay showed a decrease in MMP-9 activity, it can be suggested that this neuroprotection might be mediated by the gelatinase inhibition.     

Protein-protein-interaction Network Organization of the Hypusine Modification System

Hypusine modification of eukaryotic initiation factor 5A (eIF-5A) represents a unique and highly specific post-translational modification with regulatory functions in cancer, diabetes, and infectious diseases. However, the specific cellular pathways that are influenced by the hypusine modification remain largely unknown. To globally characterize eIF-5A and hypusine-dependent pathways, we used an approach that combines large-scale bioreactor cell culture with tandem affinity purification and mass spectrometry: “bioreactor-TAP-MS/MS.” By applying this approach systematically to all four components of the hypusine modification system (eIF-5A1, eIF-5A2, DHS, and DOHH), we identified 248 interacting proteins as components of the cellular hypusine network, with diverse functions including regulation of translation, mRNA processing, DNA replication, and cell cycle regulation. Network analysis of this data set enabled us to provide a comprehensive overview of the protein-protein interaction landscape of the hypusine modification system. In addition, we validated the interaction of eIF-5A with some of the newly identified associated proteins in more detail. Our analysis has revealed numerous novel interactions, and thus provides a valuable resource for understanding how this crucial homeostatic signaling pathway affects different cellular functions.Cellular homeostasis is controlled by signaling networks that communicate through post-translational modifications (PTM)¹ of proteins, including phosphorylation, acetylation and methylation (1–3). These modifications are typically attached to various types of proteins by multiple independent enzymes, and thereby simultaneously regulate a wide range of protein functions. Consequently, most signaling pathways are highly redundant, enabling maintenance of cellular integrity even if the modification of a single signaling molecule is disrupted (4). A striking exception is hypusine. This essential PTM is limited to a single protein: the eukaryotic initiation factor 5A (eIF-5A) (5). Disruption of this PTM leads to growth arrest in proliferating eukaryotic cells and is fatal for the developing mammalian embryo (6, 7). During hypusine biosynthesis, the lysine residue at position 50 (Lys₅₀) in eIF-5A is converted into the unusual amino acid hypusine (N^ε-(4-amino-2-hydroxybutyl)lysine; depicted in Fig. 1A) (5). This process activates eIF-5A and is mediated by two enzymatic reactions: first, deoxyhypusine synthase (DHS) catalyzes the transfer of the 4-aminobutyl moiety of spermidine to the ε-amino group of Lys₅₀ to form an intermediate residue, deoxyhypusine (Dhp₅₀) (8). Subsequently, deoxyhypusine hydroxylase (DOHH) mediates the formation of hypusine (Hyp₅₀) by addition of a hydroxyl group to the deoxyhypusine residue (9). eIF-5A, DHS and DOHH are all essential for proliferation of higher eukaryotic cells (10, 11), and eIF-5A is strictly conserved throughout eukaryotic evolution (12).Open in a separate windowFig. 1.The hypusine modification and TAP fusion proteins employed in this study. A, The hypusine modification pathway and major proposed eIF-5A functions. B, Structure of the plasmid inserts coding for SG-tagged bait proteins. The amino acid positions of eIF-5A mutants are indicated in italic. SBP, streptavidin binding peptide. C, Metabolic incorporation of ³H-labeled spermidine into eIF-5A. Arrowheads indicate bands of SG-tagged and endogenous eIF-5A proteins. D, Anti-Myc-tag Western blot of cell lysates from retrovirally transduced Ba/F3 p210 cell lines for the quantification of constitutively expressed SG-tagged bait proteins. E, Representative TAP outputs for MS/MS analysis, after 1D PAGE separation and Coomassie staining. Separation distance varies from ∼2 to 4 cm.The eIF-5A protein has been proposed to promote various different cellular processes that potentially regulate proliferation, including translation initiation (13) and elongation (14) as well as nucleocytoplasmic transport of RNA or other cargoes (15, 16). Using inhibitors of DHS and DOHH or eIF-5A mutants deficient for hypusine modification, it has also been shown that this modification is a prerequisite of at least a subset of known eIF-5A functions (10, 11, 17, 18). The eIF-5A protein has also been implicated in numerous pathologic conditions including various types of cancer (19–23), β-cell inflammation (and therefore diabetes) (24) and HIV-1 infection (25). Human and rodent cells carry two highly homologous eIF-5A genes coding for distinct isoforms. Although eIF-5A1 is expressed at high levels throughout all tissues, eIF-5A2 is detectable only in a few embryonic tissues as well as adult testis, central nervous system (26), and cancer tissue (21, 22, 27–29).Although there have been ample reports suggesting eIF-5A is involved in translational control, the molecular mechanisms through which it ultimately influences cellular physiology and leads to disease remain unclear. Moreover, it remains equally possible that at least some of eIF-5A''s effects on cellular functions might not involve direct effects on translation. Also, there is no information available on whether the two isoforms of mammalian eIF-5A are functionally congruent.To address these fundamental questions systematically and comprehensively, we employed a bioreactor-based tandem affinity purification (TAP) approach followed by MS identification of purified protein complexes (“bioreactor-TAP-MS/MS”). To obtain a complete interaction map of the proteins involved in hypusine modification, we used this approach to identify interaction partners of both isoforms of eIF-5A, as well as the hypusine modification enzymes DHS and DOHH. In total, we identified 248 proteins that either directly interact with these bait proteins or are components of higher complexes containing the aforementioned proteins. Furthermore, we validated a subset of putative interaction partners of both eIF-5A isoforms, using Western blots of reciprocal TAP experiments, as well as a live-cell protein-fragment complementation assay (PCA). Our analysis provides a molecular framework for a detailed understanding on how this signal transduction pathway affects different crucial cellular functions.     

Stretch-induced actin remodeling requires targeting of zyxin to stress fibers and recruitment of actin regulators

Reinforcement of actin stress fibers in response to mechanical stimulation depends on a posttranslational mechanism that requires the LIM protein zyxin. The C-terminal LIM region of zyxin directs the force-sensitive accumulation of zyxin on actin stress fibers. The N-terminal region of zyxin promotes actin reinforcement even when Rho kinase is inhibited. The mechanosensitive integrin effector p130Cas binds zyxin but is not required for mitogen-activated protein kinase-dependent zyxin phosphorylation or stress fiber remodeling in cells exposed to uniaxial cyclic stretch. α-Actinin and Ena/VASP proteins bind to the stress fiber reinforcement domain of zyxin. Mutation of their docking sites reveals that zyxin is required for recruitment of both groups of proteins to regions of stress fiber remodeling. Zyxin-null cells reconstituted with zyxin variants that lack either α-actinin or Ena/VASP-binding capacity display compromised response to mechanical stimulation. Our findings define a bipartite mechanism for stretch-induced actin remodeling that involves mechanosensitive targeting of zyxin to actin stress fibers and localized recruitment of actin regulatory machinery.