ACUTE EFFECTS OF MOVEMENT VELOCITY ON BLOOD LACTATE AND GROWTH HORMONE RESPONSES AFTER ECCENTRIC BENCH PRESS EXERCISE IN RESISTANCE-TRAINED MEN

This study aimed to compare the effects of different velocities of eccentric muscle actions on acute blood lactate and serum growth hormone (GH) concentrations following free weight bench press exercises performed by resistance-trained men. Sixteen healthy men were divided into two groups: slow eccentric velocity (SEV; n = 8) and fast eccentric velocity (FEV; n = 8). Both groups performed four sets of eight eccentric repetitions at an intensity of 70% of their one repetition maximum eccentric (1RMecc) test, with 2-minute rest intervals between sets. The eccentric velocity was controlled to 3 seconds per range of motion for SEV and 0.5 seconds for the FEV group. There was a significant difference (P < 0.001) in the kinetics of blood lactate removal (at 3, 6, 9, 15, and 20 min) and higher mean values for peak blood lactate (P = 0.001) for the SEV group (9.1 ± 0.5 mM) compared to the FEV group (6.1 ± 0.4 mM). Additionally, serum GH concentrations were significantly higher (P < 0.001) at 15 minutes after bench press exercise in the SEV group (1.7 ± 0.6 ng · mL⁻¹) relative to the FEV group (0.1 ± 0.0 ng · mL⁻¹). In conclusion, the velocity of eccentric muscle action influences acute responses following bench press exercises performed by resistance-trained men using a slow velocity resulting in a greater metabolic stress and hormone response.     

Molecular Architecture and Function of the SEA Complex,a Modulator of the TORC1 Pathway

The TORC1 signaling pathway plays a major role in the control of cell growth and response to stress. Here we demonstrate that the SEA complex physically interacts with TORC1 and is an important regulator of its activity. During nitrogen starvation, deletions of SEA complex components lead to Tor1 kinase delocalization, defects in autophagy, and vacuolar fragmentation. TORC1 inactivation, via nitrogen deprivation or rapamycin treatment, changes cellular levels of SEA complex members. We used affinity purification and chemical cross-linking to generate the data for an integrative structure modeling approach, which produced a well-defined molecular architecture of the SEA complex and showed that the SEA complex comprises two regions that are structurally and functionally distinct. The SEA complex emerges as a platform that can coordinate both structural and enzymatic activities necessary for the effective functioning of the TORC1 pathway.The highly conserved Target of Rapamycin Complex 1 (TORC1)¹ controls eukaryotic cell growth and cellular responses to a variety of signals, including nutrients, hormones, and stresses (1, 2). In a nutrient-rich environment, TORC1 promotes anabolic processes including ribosome biogenesis and translation. Nutrient limitation or treatment with rapamycin inhibits the Tor1 kinase and initiates autophagy, a catabolic process that mediates the degradation and recycling of cytoplasmic components. However, the nutrient-sensing function of TORC1 is not fully understood, and the mechanisms of TORC1 modulation by amino acid and nitrogen availability are not yet clear.In the yeast Saccharomyces cerevisiae, the TOR1 complex is composed of four subunits (Tor1, Kog1, Tco89, and Lst8) and is localized to the vacuole membrane. Amino acid levels are signaled to TORC1 (at least partially) via the EGO complex (Ragulator-Rag in mammals), which consists of Ego1, Ego3, Gtr1 (RagA/RagB), and Gtr2 (RagC/RagD) (3–6). The small GTPases Gtr1 and Gtr2 function as heterodimers and in their active form exist as the Gtr1-GTP/Gtr2-GDP complex. Amino acid sensing via the EGO complex involves the conserved vacuolar membrane protein Vam6, a member of the HOPS tethering complex. Vam6 is a GDP exchange factor that regulates the nucleotide-binding status of Gtr1 (6). At the same time, the GTP-bound state of Gtr1 is controlled by a leucyl t-RNA synthetase (7). In mammals, amino acids promote interaction of Ragulator-Rag with mTORC1 and its translocation to the lysosomal membrane (3, 4). Ragulator interacts with the v-ATPase complex at the lysosomal membrane (8), and leucyl t-RNA synthetase binds to RagD to activate mTORC1 (9).A genome-wide screen for TORC1 regulators in yeast identified two proteins, Npr2 and Npr3, as proteins that mediate amino acid starvation signal to TORC1 (10). Npr2 and Npr3 are both members of the SEA complex that we discovered recently (11–13). Besides Npr2 and Npr3, the SEA complex also contains four previously uncharacterized proteins (Sea1–Sea4) and two proteins also found in the nuclear pore complex, Seh1 and Sec13, the latter of which is additionally a component of the endoplasmic-reticulum-associated COPII coated vesicle. However, the SEA complex localizes to the vacuole membrane, and not to the nuclear pore complex or endoplasmic reticulum.The Sea proteins contain numerous structural elements present in intracellular structural trafficking complexes (11). For example, proteins Sea2–Sea4 are predicted to possess β-propeller/α-solenoid folds and contain RING domains, architectural combinations characteristic to protein complexes that form coats around membranes (e.g. coated vesicles, nuclear pore complexes) or participate in membrane tethering (e.g. HOPS, CORVET complexes). Npr2 and Npr3 possess a longin domain, found in many guanine nucleotide exchange factors (GEFs) (14–16), and Sea1/Iml1 is a GTPase activating protein (GAP) for Gtr1 (17). These structural characteristics, taken together with functional data, indicate a role for the SEA complex in intracellular trafficking, amino acid biogenesis, regulation of the TORC1 pathway, and autophagy (11–13, 17–20). A mammalian analog of the SEA complex, termed GATOR1/GATOR2, has recently been identified (21). GATORS are localized at the lysosome membrane and serve as upstream regulators of mammalian TORC1 via GATOR1 GAP activity toward RagA and RagB (21).In this study, we characterized the structural and functional organization of the yeast SEA complex. We present here a well-defined molecular architecture of the SEA complex obtained via an integrative modeling approach based on a variety of biochemical data. The structure reveals the relative positions and orientations of two SEA subcomplexes, Sea1/Npr2/Npr3 (or SEACIT (19)) and Sea2/Sea3/Sea4/Sec13/Seh1 (or SEACAT (19)), and identifies the Sea3/Sec13 dimer as a major interacting hub within the complex. We describe how the SEA complex interacts physically with TORC1 and the vacuole and is required for the relocalization of Tor1, and how every member of the Sea1/Npr2/Npr3 subcomplex is required for general autophagy.     

Plant regeneration from leaf explants of Aloe barbadensis Mill. and genetic fidelity assessment through DNA markers

An efficient plant regeneration protocol was developed from leaf explants of Aloe barbadensis Mill on Murashige and Skoog’s (MS) medium supplemented with 2.0 mg/l 6-benzyladenine (BA) or Kinetin (Kn), 0.25–0.5 mg/l NAA (1-napthalene acetic acid) and 3 % (w/v) sucrose within 4 weeks of culture. The maximum number of shoot buds were obtained on MS medium supplemented with 2.0 mg/l BA, 0.5 mg/l NAA, 40 mg/l Ads (adenine sulphate) within 4–6 weeks of subculture. Inclusion of 0.25–0.50 mg/l gibberellic acid into the medium, the shoot buds became elongated. Repeated subculture on regeneration medium induces higher rate of shoot regeneration. The root induction from excised microshoots was achieved on half-strength MS medium supplemented with 0.25–1.0 mg/l NAA or indole-3-butyric acid (IBA) and 2 % (w/v) sucrose. Maximum percentage of rooting was achieved on medium having 0.5 mg/l NAA with 3 % (w/v) sucrose. About 80 % of in vitro raised plantlets were hardened in the greenhouse and successfully established in the soil. Both Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) markers were used to detect the variability among the regenerated plants developed in vitro. The results showed that there was no polymorphism among the regenerated plantlets. This study will help for propagation of quality planting material of Aloe barbadensis for commercialization.     

Enriching the Pore: Splendid Complexity from Humble Origins

The nucleus is the defining intracellular organelle of eukaryotic cells and represents a major structural innovation that differentiates the eukaryotic and prokaryotic cellular form. The presence of a nuclear envelope (NE) encapsulating the nucleus necessitates a mechanism for interchange between the contents of the nuclear interior and the cytoplasm, which is mediated via the nuclear pore complex (NPC), a large protein assembly residing in nuclear pores in the NE. Recent advances have begun to map the structure and functions of the NPC in multiple organisms, and to allow reconstruction of some of the evolutionary events that underpin the modern NPC form, highlighting common and differential NPC features across the eukaryotes. Here we discuss some of these advances and the questions being pursued, consider how the evolution of the NPC has been constrained, and finally propose a model for how the NPC evolved.      

Tissue culture of ornamental pot plant: a critical review on present scenario and future prospects

Recent modern techniques of propagation have been developed which could help growers to meet the demand of the horticultural industry in the next century. An overview on the in vitro propagation via thin cell layer, meristem culture, regeneration via organogenesis and somatic embryogenesis is presented. Available methods for the transfer of genes could significantly simplify the breeding procedures and overcome some of the agronomic and environmental problems, which other wise would not be achievable through conventional propagation methods. The development and remarkable achievements with biotechnology in ornamental pot plants made during the three decades have been reviewed. The usefulness of the pot plants in commercial industry as well as propagation techniques, screening for various useful characteristics and selection of somaclonal variation is also discussed.     

Enhanced CellClassifier: a multi-class classification tool for microscopy images

Background  

Light microscopy is of central importance in cell biology. The recent introduction of automated high content screening has expanded this technology towards automation of experiments and performing large scale perturbation assays. Nevertheless, evaluation of microscopy data continues to be a bottleneck in many projects. Currently, among open source software, CellProfiler and its extension Analyst are widely used in automated image processing. Even though revolutionizing image analysis in current biology, some routine and many advanced tasks are either not supported or require programming skills of the researcher. This represents a significant obstacle in many biology laboratories.     

A new family of yeast nuclear pore complex proteins

We have identified a novel family of yeast nuclear pore complex proteins. Three individual members of this family, NUP49, NUP100, and NUP116, have been isolated and then characterized by a combination of molecular genetics and immunolocalization. Employing immunoelectron and immunofluorescence microscopy on yeast cells, we found that the binding of a polyspecific monoclonal antibody recognizing this family was predominantly at the nuclear pore complexes. Furthermore, the tagging of NUP49 with a unique epitope enabled the immunolocalization of this protein to the nuclear pore complex by both fluorescence and electron microscopy. DNA sequence analysis has shown that the amino-terminal regions of NUP49, NUP100, and NUP116 share repeated "GLFG" motifs separated from each other by glutamine, asparagine, serine and threonine rich spacers. All three proteins lack a repetitive domain found in the two precisely described yeast nuclear pore complex proteins. Only NUP49 is essential for cell viability. NUP116-deficient cells grow very slowly and are temperature sensitive, whereas the lack of NUP100 has no detectable phenotype. NUP100 and NUP116 are homologous over their entire lengths. Interestingly, NUP100 and NUP116 are both flanked by a histidine tRNA gene and a transposon element suggesting that they may have arisen by gene duplication. We propose that subfamilies of pore complex proteins can be defined by their characteristic combinations of different modular domains.     

Proteomic elucidation of the targets and primary functions of the picornavirus 2A protease

Picornaviruses are small RNA viruses that hijack host cell machinery to promote their replication. During infection, these viruses express two proteases, 2A^pro and 3C^pro, which process viral proteins. They also subvert a number of host functions, including innate immune responses, host protein synthesis, and intracellular transport, by utilizing poorly understood mechanisms for rapidly and specifically targeting critical host proteins. Here, we used proteomic tools to characterize 2A^pro interacting partners, functions, and targeting mechanisms. Our data indicate that, initially, 2A^pro primarily targets just two cellular proteins: eukaryotic translation initiation factor eIF4G (a critical component of the protein synthesis machinery) and Nup98 (an essential component of the nuclear pore complex, responsible for nucleocytoplasmic transport). The protease appears to employ two different cleavage mechanisms; it likely interacts with eIF3L, utilizing the eIF3 complex to proteolytically access the eIF4G protein but also directly binds and degrades Nup98. This Nup98 cleavage results in only a marginal effect on nuclear import of proteins, while nuclear export of proteins and mRNAs were more strongly affected. Collectively, our data indicate that 2A^pro selectively inhibits protein translation, key nuclear export pathways, and cellular mRNA localization early in infection to benefit viral replication at the expense of particular cell functions.     

Immunohistochemical demonstration of mycobacterial antigens in intracranial tuberculoma.

Mycobacterial antigens have been demonstrated immunohistochemically in the paraffin sections of 10 intracranial tuberculous granulomas and the results were compared with the detection of acid fast bacilli by conventional Ziehl-Neelsen method. In none of the 10 specimens, acid fast bacilli were demonstrated while mycobacterial antigens were characterised as diffusely staining granular brownish-pink material within the cytoplasm of giant cells and macrophages. In 14 specimens of granulomatous lesions due to non-tuberculous aetiology, immunohistochemical stains were negative for mycobacterial antigen. Thus demonstration of mycobacterial antigen will be not only useful in establishing mycobacterial aetiology of a caseating intracranial granuloma but also can be used as an alternative method to the conventional Ziehl-Neelsen method.