Function Of Cellular Activity Research Articles

Domain collapse and active site ablation generate a widespread animal mitochondrial seryl-tRNA synthetase.

Through their aminoacylation reactions, aminoacyl tRNA-synthetases (aaRS) establish the rules of the genetic code throughout all of nature. During their long evolution in eukaryotes, additional domains and splice variants were added to what is commonly a homodimeric or monomeric structure. These changes confer orthogonal functions in cellular activities that have recently been uncovered. An unusual exception to the familiar architecture of aaRSs is the heterodimeric metazoan mitochondrial SerRS. In contrast to domain additions or alternative splicing, here we show that heterodimeric metazoan mitochondrial SerRS arose from its homodimeric ancestor not by domain additions, but rather by collapse of an entire domain (in one subunit) and an active site ablation (in the other). The collapse/ablation retains aminoacylation activity while creating a new surface, which is necessary for its orthogonal function. The results highlight a new paradigm for repurposing a member of the ancient tRNA synthetase family.

Glutaraldehyde and uranyl acetate dual fixation combined sputtering/unroofing enables intracellular fatty acids TOF-SIMS imaging with organelle-corresponding subcellular distribution.

Fatty acids (FAs) have diverse functions in cellular activities. The intracellular distribution of FAs is critical for their functions. Imaging of FAs by time-of-flight secondary ion mass spectrometry (TOF-SIMS) has been achieved. However, TOF-SIMS images of FAs so far do not have subcellular distribution due to inadequate sample preparation methods. In this study, we developed a chemical fixation method using glutaraldehyde (GA) with uranyl acetate (UA), which preserved cellular structure and intracellular FA distribution well. Combining GA+UA fixation with sputtering-based methods and unroofing-based methods, respectively, we successfully imaged intracellular lipids with the subcellular distribution.

Expression of p53 isoforms in response to two levels of hypoxia and effects of p53 knock‐down in glyceraldehyde‐3‐phosphate dehydrogenase in the white shrimp Litopenaeus vannamei

In response to hypoxia, aquatic organisms rely on anaerobic glycolysis. In the white shrimp Litopenaeus vannamei, hypoxia induces glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression, a key enzyme in glycolysis. In mammals, GAPDH has additional functions in cellular activities besides glucose metabolism, such as interaction with the transcription factor p53 to form a complex that participates in apoptosis. We investigated the effect of two levels of hypoxia (1.5 and 1.0 mg dissolved oxygen (DO) L−1) on the expression of the two shrimp p53 isoforms known, FLp53 and ∆Np53, and the effect of p53 silencing during hypoxia on GAPDH expression and activity in hepatopancreas. The FLp53 transcripts were down-regulated after 24 and 48 h exposure to both hypoxic conditions, but not the ∆Np53. p53 silencing using RNAi, induced expression of GAPDH at 48 h in the 1.5 mg DO L−1 hypoxic condition compared with the unsilenced shrimp, contrary to the 1.0 mg DO L−1 where expression was induced in the silenced animals in normoxia after 48 h, but no significant changes were detected in enzyme activity in any case. These results indicate differential expression of p53 isoforms under two hypoxic conditions and maintenance of GAPDH transcripts and activity in p53-silenced shrimp.

Deciphering the Inhibition Mechanism of under Trial Hsp90 Inhibitors and Their Analogues: A Comparative Molecular Dynamics Simulation.

Heat shock protein 90 (Hsp90) performs functions in cellular activities together with other signaling pathways. Hsp90 is evolutionarily conserved and universally articulated as a human cancer-causing agent involved in lung cancer and breast cancer followed by colon and rectum cancers. It has emerged as an effective drug candidate, and inhibition may affect several signaling pathways associated with cancer spread. Therefore, in-silico approaches, molecular docking, molecular dynamics simulation, and binding free energy calculations were applied to create insights into the inhibition mechanism against Hsp90 to identify new cancer therapeutic drugs. Top-docked Hsp90-inhibitor complexes with their analogues were selected as the best complexes based on the GOLD fitness score and orientation. The significant interaction of Hsp90 inhibitors and their analogues were observed to be bound with active site residues as well as residing within the same cavity region. System stability factors RMSD, RMSF, beta-factor, and radius of gyration were analyzed for top-docked complexes and ensure strong binding interaction between inhibitors and the Hsp90 cavity. Cavity bound inhibitors were found to retain consistent hydrogen bonding during the simulation. The radial distribution function (RDF) illustrated that interacting active site residues drive the binding and stability of the inhibitors. Similarly, the axial frequency distribution, which is an indigenously developed analytical tool, produced noteworthy knowledge of the hydrogen-bonding pattern. Results yielded new insights into the design of cancer therapeutic drugs against Hsp90. This finding suggests that under trial Hsp90 inhibitors MPC-3100 could be a potential starting point into the development of potential anticancer agents with the possibility of future directions for the improvement of early existing Hsp90 inhibitors CNF-2024 and SNX-5422 as an anticancer agent.

Membrane Heterogeneity and its Role in Immune Signaling Elucidated by Spectral Imaging

Nature of the cellular membrane heterogeneity has been a long-standing question in cell biology. One crucial measure for the membrane heterogeneity is the lipid packing. Measuring changes of lipid packing in the plasma membrane during cellular events has been an important part of elucidating the biological function of membrane physical chemistry. Here, we apply conventional and spectral imaging to figure out the nature of membrane heterogeneity and its role in immune signalling events.We observe bio-membranes sampling different lipid packing states that form coexisting domains (i.e., relatively ordered and disordered domains) with a variety of distinct compositions and functional characteristics. We first investigate the connection between membrane composition and lipid packing in synthetic liposomes and show that both the relatively ordered and disordered phase, and the difference between them, are tuned by lipid composition. We then give an example of live cells regulating the lipid packing phenotypes of their plasma membranes, accessing a variety of both ordered and disordered domains as a function of cellular activity. Finally, we find that in both synthetic and natural membranes, the tunable interdomain lipid packing disparity (i.e. the difference between coexisting domains) regulates component partitioning between domains and the functionality of membrane embedded lipidic receptors. By applying advanced image processing with spectral imaging, we show that even the marginal modulation of membrane heterogeneity is crucial for immune signalling events such as FCR and B-cell signalling.Membrane heterogeneity is an organizing principle for the membrane bio-activity by either concentrating certain molecules in transient domains or by physically adjusting the conformity of the molecules to favour or hinder their interactions in the membrane.

Recent advances in understanding plant nuclear envelope proteins involved in nuclear morphology.

The nuclear envelope (NE) is a fundamental structure of the nucleus and plays an important role in nuclear morphology through the strict regulation of NE protein function. Beyond its physical barrier function between nucleoplasm and cytoplasm, recent studies of the plant NE have provided novel insights into basic aspects of nuclear morphology as well as cellular organization. In this review, we focus on plant NE proteins that have emerged from recent studies in nuclear morphology, and we discuss their physiological functions in cellular activities. A better understanding of the NE protein functions should provide key insights into the physiological significance of proper nuclear structure in plants.

Long-time course of protease-activated receptor-1 expression after intracerebral hemorrhage in rats

Thrombin plays an important role in brain injuries associated with intracerebral hemorrhage (ICH). The protease-activated receptor (PAR)-1 is responsible for the vast majority of the thrombin's cellular activation functions. We tested the hypothesis that thrombin-induced brain damage after ICH, at least in part, is mediated by PAR-1. We report that there are significant differences between PAR-1 positive cell number and PAR-1 mRNA absorbance ratio between ICH model group (at 6 h, 24 h, 3 d, 7 d and 14 d) and normal group ( P < 0.05). These results suggest that the long-time course of PAR-1 expression may be partly involved in the mechanism of thrombin-induced brain damage after ICH.

Angiopoietin: A TIE(d) Balance in Tumor Angiogenesis

Angiopoietins (ANG-1 and ANG-2) and their TIE-2 receptor tyrosine kinase have wide-ranging effects on tumor malignancy that includes angiogenesis, inflammation, and vascular extravasation. These multifaceted pathways present a valuable opportunity in developing novel inhibition strategies for cancer treatment. However, the regulatory role of ANG-1 and ANG-2 in tumor angiogenesis remains controversial. There is a complex interplay between complementary yet conflicting roles of both the ANGs in shaping the outcome of angiogenesis. Embryonic vascular development suggests that ANG-1 is crucial in engaging interaction between endothelial and perivascular cells. However, recruitment of perivascular cells by ANG-1 has recently been implicated in its antiangiogenic effect on tumor growth. It is becoming clear that TIE-2 signaling may function in a paracrine and autocrine manner directly on tumor cells because the receptor has been increasingly found in tumor cells. In addition, alpha(5)beta(1) and alpha(v)beta(5) integrins were recently recognized as functional receptors for ANG-1 and ANG-2. Therefore, both the ligands may have wide-ranging functions in cellular activities that affect overall tumor development. Collectively, these TIE-2-dependent and TIE-2-independent activities may account for the conflicting findings of ANG-1 and ANG-2 in tumor angiogenesis. These uncertainties have impeded development of a clear strategy to target this important angiogenic pathway. A better understanding of the molecular basis of ANG-1 and ANG-2 activity in the pathophysiologic regulation of angiogenesis may set the stage for novel therapy targeting this pathway.

Essential Role for Nuclear PTEN in Maintaining Chromosomal Integrity

A broad spectrum of mutations in PTEN, encoding a lipid phosphatase that inactivates the P13-K/AKT pathway, is found associated with primary tumors. Some of these mutations occur outside the phosphatase domain, suggesting that additional activities of PTEN function in tumor suppression. We report a nuclear function for PTEN in controlling chromosomal integrity. Disruption of Pten leads to extensive centromere breakage and chromosomal translocations. PTEN was found localized at centromeres and physically associated with CENP-C, an integral component of the kinetochore. C-terminal PTEN mutants disrupt the association of PTEN with centromeres and cause centromeric instability. Furthermore, Pten null cells exhibit spontaneous DNA double-strand breaks (DSBs). We show that PTEN acts on chromatin and regulates expression of Rad51, which reduces the incidence of spontaneous DSBs. Our results demonstrate that PTEN plays a fundamental role in the maintenance of chromosomal stability through the physical interaction with centromeres and control of DNA repair. We propose that PTEN acts as a guardian of genome integrity.

Identification of thrombin receptors in rat brain capillary endothelial cells.

Both thrombin and plasmin induce contraction of brain endothelial cells, which may increase capillary permeability thereby leading to disruption of the blood-brain barrier. Identification of thrombin receptors, as well as the influence of plasmin on their activation, in capillary endothelial cells and astrocytes are therefore essential for understanding injury-related actions of thrombin in the brain. Using the reverse transcriptase-polymerase chain reaction method, the present study shows that primary cultures of rat brain capillary endothelial (RBCE) cells and astrocytes derived from rat brain express two different thrombin receptors. The first is proteolytically activated receptor (PAR)-1, the receptor responsible for the vast majority of the thrombin's cellular activation functions; the second is PAR-3, a receptor described to be essential for normal responsiveness to thrombin in mouse platelets. In addition to these thrombin receptors, the mRNA (messenger RNA) for PAR-2, a possible trypsin receptor, was also identified. Functional significance of thrombin receptors was indicated by changes in [Ca2+]i in response to thrombin, as measured by FURA-2 fluorescence in RBCE cells. Thrombin as low as 4 nmol/L induced an abrupt increase in [Ca2+]i whereas, upon addition of active site-blocked thrombin or plasmin, [Ca2+]i remained unchanged. The [Ca2+]i signal attributable to thrombin was smaller in a low Ca2+-containing medium, indicating that an influx of Ca2+ from the extracellular medium makes a contribution to the overall [Ca2+]i rise. The amplitude of the transient [Ca2+]i signal was dependent on the concentration of thrombin, and repeated application of the enzyme caused an essentially complete and long-term desensitization of the receptor. The PAR-1 agonist peptide SFLLRN also elicited a transient increase in [Ca2+]i. After activation by SFLLRN, cells showed a diminished response to thrombin, but the response was not absent, indicating that PAR-3 might contribute to the generation of the [Ca2+]i signal. Pretreatment of RBCE cells with 100 nmol/L plasmin completely prevented [Ca2+]i rise attributable to thrombin. These data show that RBCE cells and astrocytes express at least two receptors for thrombin, PAR-1 and PAR-3, and probably both receptors are involved in thrombin-induced [Ca2+]i signals. Plasmin itself does not elevate [Ca2+]i but prevents the activation of receptors by thrombin.

Variations in signal-mediated transport through the nuclear pores as a function of cellular activity

Previously, we utilized colloidal gold particles coated with nucleoplasmin, which contains well characterized nuclear localization signals (NLS), to study signal-mediated nuclear transport in proliferating and quiescent BALB/c 3T3 cells. The particles were injected into the cytoplasm, and nuclear uptake (N/C gold ratios) was later determined by TEM. The functional size of the transport channels was also analyzed by measuring the particles that entered the nucleoplasm. When large gold particles (110 to 270 Åin diameter) were injected, nuclear uptake was significantly greater in proliferating as compared to quiescent cells. The average fonctional pore diameter was approximately 230 Å during proliferation, and 140 Åor less in quiescent populations.