Expression of semaphorin 3A in the rat corneal epithelium during wound healing

The neural guidance protein semaphorin 3A (Sema3A) is expressed in corneal epithelial cells of the adult rat. We have now further investigated the localization of Sema3A in the normal rat corneal epithelium as well as changes in its expression pattern during wound healing after central corneal epithelial debridement. The expression pattern of Sema3A was compared with that of the tight-junction protein zonula occludens-1 (ZO-1), the gap-junction protein connexin43 (Cx43), or the cell proliferation marker Ki67. Immunofluorescence analysis revealed that Sema3A was present predominantly in the membrane of basal and wing cells of the intact corneal epithelium. The expression of Sema3A at the basal side of basal cells was increased in the peripheral epithelium compared with that in the central region. Sema3A was detected in all layers at the leading edge of the migrating corneal epithelium at 6 h after central epithelial debridement. The expression of Sema3A was markedly up-regulated in the basal and lateral membranes of columnar basal cells apparent in the thickened, newly healed epithelium at 1 day after debridement, but it had largely returned to the normal pattern at 3 days after debridement. The expression of ZO-1 was restricted to superficial epithelial cells and remained mostly unchanged during the wound healing process. The expression of Cx43 in basal cells was down-regulated at the leading edge of the migrating epithelium but was stable in the remaining portion of the epithelium. Ki67 was not detected in basal cells of the central epithelium at 1 day after epithelial debridement, when Sema3A was prominently expressed. Immunoblot analysis showed that the abundance of Sema3A in the central cornea was increased 1 day after epithelial debridement, whereas that of ZO-1 or Cx43 remained largely unchanged. This increase in Sema3A expression was accompanied by up-regulation of the Sema3A coreceptor neuropilin-1. Our observations have thus shown that the expression of Sema3A is increased markedly in basal cells of the newly healed corneal epithelium, and that this up-regulation of Sema3A is not associated with cell proliferation. They further suggest that Sema3A might play a role in the regulation of corneal epithelial wound healing.     

CpG island hypermethylation of multiple tumor suppressor genes associated with loss of their protein expression during rat lung carcinogenesis induced by 3-methylcholanthrene and diethylnitrosamine

The epigenetic mechanisms underlying the tumorigenesis caused by polycyclic aromatic hydrocarbons and nitrosamine compounds such as 3-methylcholanthrene (MCA) and diethylnitrosamine (DEN) are currently unknown. We reported previously that dynamic changes in DNA methylation occurred during MCA/DEN-induced rat lung carcinogenesis. Here, we used the same animal model to further study the evolution of methylation alterations in tumor suppressor genes (TSGs) DAPK1, FHIT, RASSF1A, and SOCS-3. We found that none of these genes were methylated in either normal or hyperplasia tissue. However, as the severity of the cancer progressed through squamous metaplasia and dysplasia to carcinoma in situ (CIS) and infiltrating carcinoma, so methylation became more prevalent. Particularly dramatic increases in the level of methylation, the average number of methylated genes, and the incidence of concurrent methylation in three genes were observed in CIS and infiltrating carcinoma. Similar but less profound changes were seen in squamous metaplasia and dysplasia. Furthermore, methylation status was closely correlated to loss of protein expression for these genes, with protein levels markedly declining along the continuum of carcinogenesis. These results suggest that progressive CpG island hypermethylation leading to inactivation of TSGs might be a vital molecular mechanism in the pathogenesis of MCA/DEN-induced multistep rat lung carcinogenesis.     

Silencing of the PP2A catalytic subunit causes HER-2/neu positive breast cancer cells to undergo apoptosis

Protein phosphatase 2A (PP2A), in its activated form as a phosphatase, is a tumour suppressor. However, when PP2A is phosphorylated at the tyrosine residue (pY307), it loses its phosphatase activity and becomes inactivated. In our previous study, we found a higher expression of pY307-PP2A in HER-2/neu positive breast tumour samples and significantly correlated to tumour progression, and in this context, it could function as a proto-oncogene. The above and subsequent findings led us to postulate that the critical role of PP2A in maintaining the balance between cell survival and cell death may be linked to its phosphorylation status at its Y307 residue. Hence, we further investigated the effects of knocking down the PP2A catalytic subunit which contains the Y307 amino acid residue in two HER-2/neu positive breast cancer cell lines, BT474 and SKBR3. We showed that this causes the silenced HER-2/neu breast cancer cells to undergo apoptosis and furthermore, that such apoptosis is mediated by p38 MAPK-caspase 3/PARP activation. Understanding the role of PP2A in HER2/neu positive cells might thus provide insight into new targets for breast cancer therapy.     

Microtubule destruction induces tau liberation and its subsequent phosphorylation

Neurofibrillary tangle-bearing neurons, a pathological hallmark of Alzheimer’s disease, are mostly devoid of normal microtubule (MT) structure and instead have paired helical filaments that are composed of abnormal hyperphosphorylated tau. However, a causal relationship between tau phosphorylation and MT disruption has not been clarified. To examine whether MT disruption induces tau phosphorylation, stathmin, an MT-disrupting protein, was co-expressed with tau in COS-7 cells. Stathmin expression induced apparent MT catastrophe and tau hyperphosphorylation at Thr-181, Ser-202, Thr-205, and Thr-231 sites. In contrast, c-Jun N-terminal kinase activation, or phosphatase inhibition, led to significant tau phosphorylation without affecting MT structure. These findings suggest that MT disruption induces subsequent tau phosphorylation.     

Effects of decomposition and storage conditions on the δ13C and δ15N isotope values of killer whale (Orcinus orca) skin and blubber tissues

Several different factors in the collection and preservation of whale skin and blubber samples were examined to determine their effect on the results obtained by stable nitrogen and carbon isotope (δ¹⁵N and δ¹³C) analysis. Samples of wet killer whale skin retained their original stable isotope values for up to 14 d at 4°C or lower. However, decomposition significantly changed the δ¹⁵N value within 3 d at 20°C. Storage at ?20°C was as effective as ?80°C for the preservation of skin and blubber samples for stable isotope analysis for at least a year. By contrast, once a skin sample had been freeze‐dried and lipid extracted, the stable isotope values did not change significantly when it was stored dry at room temperature for at least 12 mo. Preservation of whale skin samples for a month in DMSO‐salt solution, frozen or at room temperature, did not significantly change the δ¹⁵N and δ¹³C values of lipid extracted tissues, although the slight changes seen could influence results of a study if only small changes are expected.     

Computational modelling and analysis of mechanical conditions on cell locomotion and cell–cell interaction

Between other parameters, cell migration is partially guided by the mechanical properties of its substrate. Although many experimental works have been developed to understand the effect of substrate mechanical properties on cell migration, accurate 3D cell locomotion models have not been presented yet. In this paper, we present a novel 3D model for cells migration. In the presented model, we assume that a cell follows two main processes: in the first process, it senses its interface with the substrate to determine the migration direction and in the second process, it exerts subsequent forces to move. In the presented model, cell traction forces are considered to depend on cell internal deformation during the sensing step. A random protrusion force is also considered which may change cell migration direction and/or speed. The presented model was applied for many cases of migration of the cells. The obtained results show high agreement with the available experimental and numerical data.     

HIV-1 Tat protein induces glial cell autophagy through enhancement of BAG3 protein levels

BAG3 protein has been described as an anti-apoptotic and pro-autophagic factor in several neoplastic and normal cells. We previously demonstrated that BAG3 expression is elevated upon HIV-1 infection of glial and T lymphocyte cells. Among HIV-1 proteins, Tat is highly involved in regulating host cell response to viral infection. Therefore, we investigated the possible role of Tat protein in modulating BAG3 protein levels and the autophagic process itself. In this report, we show that transfection with Tat raises BAG3 levels in glioblastoma cells. Moreover, BAG3 silencing results in highly reducing Tat- induced levels of LC3-II and increasing the appearance of sub G0/G1 apoptotic cells, in keeping with the reported role of BAG3 in modulating the autophagy/apoptosis balance. These results demonstrate for the first time that Tat protein is able to stimulate autophagy through increasing BAG3 levels in human glial cells.     

The TRAF3 adaptor protein drives proliferation of anaplastic large cell lymphoma cells by regulating multiple signaling pathways

T cells devoid of tumor necrosis factor receptor associated factor-3 (Traf3) exhibit decreased proliferation, sensitivity to apoptosis, and an improper response to antigen challenge. We therefore hypothesized that TRAF3 is critical to the growth of malignant T cells. By suppressing TRAF3 protein in different cancerous T cells, we found that anaplastic large cell lymphoma (ALCL) cells require TRAF3 for proliferation. Since reducing TRAF3 results in aberrant activation of the noncanonical nuclear factor-κB (NF-κB) pathway, we prevented noncanonical NF-κB signaling by suppressing RelB together with TRAF3. This revealed that TRAF3 regulates proliferation independent of the noncanonical NF-κB pathway. However, suppression of NF-κB-inducing kinase (NIK) along with TRAF3 showed that high levels of NIK have a partial role in blocking cell cycle progression. Further investigation into the mechanism by which TRAF3 regulates cell division demonstrated that TRAF3 is essential for continued PI3K/AKT and JAK/STAT signaling. In addition, we found that while NIK is dispensable for controlling JAK/STAT activity, NIK is critical to regulating the PI3K/AKT pathway. Analysis of the phosphatase and tensin homolog (PTEN) showed that NIK modulates PI3K/AKT signaling by altering the localization of PTEN. Together our findings implicate TRAF3 as a positive regulator of the PI3K/AKT and JAK/STAT pathways and reveal a novel function for NIK in controlling PI3K/AKT activity. These results provide further insight into the role of TRAF3 and NIK in T cell malignancies and indicate that TRAF3 differentially governs the growth of B and T cell cancers.     

The Intrasectional Chemotaxonomic Placement of Hypericum elegans Stephan ex Willd. Inferred from the Essential‐Oil Chemical Composition

Here we report, for the first time, the results of detailed GC and GC/MS analyses of the essential oil of a rare taxon in Serbia, Hypericum elegans Stephan ex Willd . One hundred and sixty two constituents identified accounted for 98.6% of the oil. The major components of the oil were undecane (31.9%), α‐pinene (16.7%), nonane (6.1%), bicyclogermacrene (5.8%), 2‐methyloctane (3.7%), and germacrene D (3.6%). Non‐terpenoids as chemotaxonomic markers constituted the main fraction of H. elegans oil, whereby n‐alkanes were the most abundant contributors of this fraction. Based on these results and previously published ones, we performed an intrasectional multivariate statistical comparison of corresponding essential‐oil chemical compositions. Principal component analysis (PCA) and agglomerative hierarchical clustering (AHC) of the data on the volatile profiles of section Hypericum taxa revealed that H. elegans either represents an oil chemotype of its own (AHC) or could be considered related to H. perforatum (PCA).     

Extensive and Modular Intrinsically Disordered Segments in C. elegans TTN-1 and Implications in Filament Binding, Elasticity and Oblique Striation

TTN-1, a titin like protein in Caenorhabditis elegans, is encoded by a single gene and consists of multiple Ig and fibronectin 3 domains, a protein kinase domain and several regions containing tandem short repeat sequences. We have characterized TTN-1's sarcomere distribution, protein interaction with key myofibrillar proteins as well as the conformation malleability of representative motifs of five classes of short repeats. We report that two antibodies developed to portions of TTN-1 detect an ∼ 2-MDa polypeptide on Western blots. In addition, by immunofluorescence staining, both of these antibodies localize to the I-band and may extend into the outer edge of the A-band in the obliquely striated muscle of the nematode. Six different 300-residue segments of TTN-1 were shown to variously interact with actin and/or myosin in vitro. Conformations of synthetic peptides of representative copies of each of the five classes of repeats—39-mer PEVT, 51-mer CEEEI, 42-mer AAPLE, 32-mer BLUE and 30-mer DispRep—were investigated by circular dichroism at different temperatures, ionic strengths and solvent polarities. The PEVT, CEEEI, DispRep and AAPLE peptides display a combination of a polyproline II helix and an unordered structure in aqueous solution and convert in trifluoroethanol to α-helix (PEVT, CEEEI, DispRep) and β-turn (AAPLE) structures, respectively. The octads in BLUE motifs form unstable α-helix-like structures coils in aqueous solution and negligible heptad-based, α-helical coiled-coils. The α-helical structure, as modeled by threading and molecular dynamics simulations, tends to form helical bundles and crosses based on its 8-4-2-2 hydrophobic helical patterns and charge arrays on its surface. Our finding indicates that APPLE, PEVT, CEEEI and DispRep regions are all intrinsically disordered and highly reminiscent of the conformational malleability and elasticity of vertebrate titin PEVK segments. The proposed presence of long, modular and unstable α-helical oligomerization domains in the BLUE region of TTN-1 could bundle TTN-1 and stabilize oblique striation of the sarcomere.