Tubulohelical membrane arrays: From the initial observation to the elucidation of nanophysical properties and cellular function

Lipids undergo self-assembly to form ordered nonlamellar, nanoperiodic arrays both in vitro and in vivo. While engineering of such membrane arrays for technical devices is envisaged, we know little about their cellular function. Do they represent building blocks of an inherent cellular nanotechnology? Prospects for answering this question could be improved if the nanophysical properties of the membrane arrays could be studied in the context of specific cellular functions. Therefore, we draw attention to exceptional complex membrane arrays found in the renal epithelial cell line PtK2 that could provide perfect conditions for both biophysical and cell functional studies. The so-called tubulohelical membrane arrays (TUHMAs) combine nanoperiodicity of lipid membranes with that of helix-like proteinaceous core structures. Strikingly, they show several characteristics of dynamic, microtubule-associated single organelles. Our initial data indicate that TUHMA formation occurs in the depth of the cytoplasm under participation of cytoplasmic nucleoporins. Once matured, they may fuse with the nuclear membrane in polarized positions, either perpendicularly or in parallel to the nucleus. As a starting point for the initiation of functional studies we found a connection between TUHMAs and primary cilia, indicated by immunolabeling patterns of detyrosynated tubulin and cytoplasmic nucleoporins. We discuss these observations in the context of the ciliary cycle and of the specific requirement of ciliated renal epithelial cells for oriented cell division. Finally, we raise the question of whether putative nanooptical properties of TUHMAs could serve for communicating orientation between dividing cells

Minocycline Synergizes with N-Acetylcysteine and Improves Cognition and Memory Following Traumatic Brain Injury in Rats

Background: There are no drugs presently available to treat traumatic brain injury (TBI). A variety of single drugs have failed clinical trials suggesting a role for drug combinations. Drug combinations acting synergistically often provide the greatest combination of potency and safety. The drugs examined (minocycline (MINO), N-acetylcysteine (NAC), simvastatin, cyclosporine A, and progesterone) had FDA-approval for uses other than TBI and limited brain injury in experimental TBI models. Methodology/Principal Findings: Drugs were dosed one hour after injury using the controlled cortical impact (CCI) TBI model in adult rats. One week later, drugs were tested for efficacy and drug combinations tested for synergy on a hierarchy of behavioral tests that included active place avoidance testing. As monotherapy, only MINO improved acquisition of the massed version of active place avoidance that required memory lasting less than two hours. MINO-treated animals, however, were impaired during the spaced version of the same avoidance task that required 24-hour memory retention. Coadministration of NAC with MINO synergistically improved spaced learning. Examination of brain histology 2 weeks after injury suggested that MINO plus NAC preserved white, but not grey matter, since lesion volume was unaffected, yet myelin loss was attenuated. When dosed 3 hours before injury, MINO plus NAC as single drugs had no effect on interleukin-1 formation; together they synergistically lowered interleukin-1 levels. This effect on interleukin-1 was not observed when th

In Vitro Antibacterial Activity of Cysteine Protease Inhibitor from Kiwifruit (Actinidia deliciosa)

The need for replacing traditional pesticides with alternative agents for the management of agricultural pathogens is rising worldwide. In this study, a cysteine proteinase inhibitor (CPI), 11 kDa in size, was purified from green kiwifruit to homogeneity. We examined the growth inhibition of three plant pathogenic Gram-negative bacterial strains by kiwi CPI and attempted to elucidate the potential mechanism of the growth inhibition. CPI influenced the growth of phytopathogenic bacteria Agrobacterium tumefaciens (76.2 % growth inhibition using 15 mu M CPI), Burkholderia cepacia (75.6 % growth inhibition) and, to a lesser extent, Erwinia carotovora (44.4 % growth inhibition) by inhibiting proteinases that are excreted by these bacteria. Identification and characterization of natural plant defense molecules is the first step toward creation of improved methods for pest control based on naturally occurring molecules

CLIMP-63 is a gentamicin-binding protein that is involved in drug-induced cytotoxicity

Aminoglycoside-induced nephrotoxicity and ototoxicity is a major clinical problem. To understand how aminoglycosides, including gentamicin, induce cytotoxicity in the kidney proximal tubule and the inner ear, we identified gentamicin-binding proteins (GBPs) from mouse kidney cells by pulling down GBPs with gentamicin–agarose conjugates and mass spectrometric analysis. Among several GBPs specific to kidney proximal tubule cells, cytoskeleton-linking membrane protein of 63 kDa (CLIMP-63) was the only protein localized in the endoplasmic reticulum, and was co-localized with gentamicin-Texas Red (GTTR) conjugate after cells were treated with GTTR for 1 h. In western blots, kidney proximal tubule cells and cochlear cells, but not kidney distal tubule cells, exhibited a dithiothreitol (DTT)-resistant dimer band of CLIMP-63. Gentamicin treatment increased the presence of DTT-resistant CLIMP-63 dimers in both kidney proximal (KPT11) and distal (KDT3) tubule cells. Transfection of wild-type and mutant CLIMP-63 into 293T cells showed that the gentamicin-dependent dimerization requires CLIMP-63 palmitoylation. CLIMP-63 siRNA transfection enhanced cellular resistance to gentamicin-induced toxicity, which involves apoptosis, in KPT11 cells. Thus, the dimerization of CLIMP-63 is likely an early step in aminoglycoside-induced cytotoxicity in the kidney and cochlea. Gentamicin also enhanced the binding between CLIMP-63 and 14-3-3 proteins, and we also identified that 14-3-3 proteins are involved in gentamicin-induced cytotoxicity, likely by binding to CLIMP-63

IGF-I activates caspases 3/7, 8 and 9 but does not induce cell death in colorectal cancer cells

Background: Colorectal cancer is the third most common cancer in the western world. Chemotherapy is often ineffective to treat the advanced colorectal cancers due to the chemoresistance. A major contributor to chemo-resistance is tumour-derived inhibition or avoidance of apoptosis. Insulin-like growth factor I (IGF-I) has been known to play a prominent role in colorectal cancer development and progression. The role of IGF-I in cancer cell apoptosis is not completely understood.Methods: Using three colorectal cancer cell lines and one muscle cell line, associations between IGF-I and activities of caspase 3/7, 8 and 9 have been examined; the role of insulin-like growth factor I receptor (IGF-IR) in the caspase activation has been investigated.Results: The results show that exogenous IGF-I significantly increases activity of caspases 3/7, 8 and 9 in all cell lines used; blocking IGF-I receptor reduce IGF-I-induced caspase activation. Further studies demonstrate that IGF-I induced caspase activation does not result in cell death. This is the first report to show that while IGF-I activates caspases 3/7, 8 and 9 it does not cause colorectal cancer cell death.Conclusion: The study suggests that caspase activation is not synonymous with apoptosis and that activation of caspases may not necessarily induce cell death

CLIMP-63 is a gentamicin-binding protein that is involved in drug-induced cytotoxicity

Aminoglycoside-induced nephrotoxicity and ototoxicity is a major clinical problem. To understand how aminoglycosides, including gentamicin, induce cytotoxicity in the kidney proximal tubule and the inner ear, we identified gentamicin-binding proteins (GBPs) from mouse kidney cells by pulling down GBPs with gentamicin–agarose conjugates and mass spectrometric analysis. Among several GBPs specific to kidney proximal tubule cells, cytoskeleton-linking membrane protein of 63 kDa (CLIMP-63) was the only protein localized in the endoplasmic reticulum, and was co-localized with gentamicin-Texas Red (GTTR) conjugate after cells were treated with GTTR for 1 h. In western blots, kidney proximal tubule cells and cochlear cells, but not kidney distal tubule cells, exhibited a dithiothreitol (DTT)-resistant dimer band of CLIMP-63. Gentamicin treatment increased the presence of DTT-resistant CLIMP-63 dimers in both kidney proximal (KPT11) and distal (KDT3) tubule cells. Transfection of wild-type and mutant CLIMP-63 into 293T cells showed that the gentamicin-dependent dimerization requires CLIMP-63 palmitoylation. CLIMP-63 siRNA transfection enhanced cellular resistance to gentamicin-induced toxicity, which involves apoptosis, in KPT11 cells. Thus, the dimerization of CLIMP-63 is likely an early step in aminoglycoside-induced cytotoxicity in the kidney and cochlea. Gentamicin also enhanced the binding between CLIMP-63 and 14-3-3 proteins, and we also identified that 14-3-3 proteins are involved in gentamicin-induced cytotoxicity, likely by binding to CLIMP-63

Cyclin-Dependent Kinase Activity Controls the Onset of the HCMV Lytic Cycle

The onset of human cytomegalovirus (HCMV) lytic infection is strictly synchronized with the host cell cycle. Infected G0/G1 cells support viral immediate early (IE) gene expression and proceed to the G1/S boundary where they finally arrest. In contrast, S/G2 cells can be infected but effectively block IE gene expression and this inhibition is not relieved until host cells have divided and reentered G1. During latent infection IE gene expression is also inhibited, and for reactivation to occur this block to IE gene expression must be overcome. It is only poorly understood which viral and/or cellular activities maintain the block to cell cycle or latency-associated viral IE gene repression and whether the two mechanisms may be linked. Here, we show that the block to IE gene expression during S and G2 phase can be overcome by both genotoxic stress and chemical inhibitors of cellular DNA replication, pointing to the involvement of checkpoint-dependent signaling pathways in controlling IE gene repression. Checkpoint-dependent rescue of IE expression strictly requires p53 and in the absence of checkpoint activation is mimicked by proteasomal inhibition in a p53 dependent manner. Requirement for the cyclin dependent kinase (CDK) inhibitor p21 downstream of p53 suggests a pivotal role for CDKs in controlling IE gene repression in S/G2 and treatment of S/G2 cells with the CDK inhibitor roscovitine alleviates IE repression independently of p53. Importantly, CDK inhibiton also overcomes the block to IE expression during quiescent infection of NTera2 (NT2) cells. Thus, a timely block to CDK activity not only secures phase specificity of the cell cycle dependent HCMV IE gene expression program, but in addition plays a hitherto unrecognized role in preventing the establishment of a latent-like state

Targeting Hypoxia in Cancer Cells by Restoring Homeodomain Interacting Protein-Kinase 2 and p53 Activity and Suppressing HIF-1α

BACKGROUND:The tumor suppressor homeodomain-interacting protein kinase-2 (HIPK2) by phosphorylating serine 46 (Ser46) is a crucial regulator of p53 apoptotic function. HIPK2 is also a transcriptional co-repressor of hypoxia-inducible factor-1alpha (HIF-1alpha) restraining tumor angiogenesis and chemoresistance. HIPK2 can be deregulated in tumors by several mechanisms including hypoxia. Here, we sought to target hypoxia by restoring HIPK2 function and suppressing HIF-1alpha, in order to provide evidence for the involvement of both HIPK2 and p53 in counteracting hypoxia-induced chemoresistance. METHODOLOGY/PRINCIPAL FINDINGS:Upon exposure of colon and lung cancer cells to hypoxia, by either low oxygen or cobalt, HIPK2 function was impaired allowing for increased HIF-1alpha expression and inhibiting the p53-apoptotic response to drug. Cobalt suppressed HIPK2 recruitment onto HIF-1alpha promoter. Hypoxia induced expression of the p53 target MDM2 that downregulates HIPK2, thus MDM2 inhibition by siRNA restored the HIPK2/p53Ser46 response to drug. Zinc supplementation to hypoxia-treated cells increased HIPK2 protein stability and nuclear accumulation, leading to restoration of HIPK2 binding to HIF-1alpha promoter, repression of MDR1, Bcl2, and VEGF genes, and activation of the p53 apoptotic response to drug. Combination of zinc and ADR strongly suppressed tumor growth in vivo by inhibiting HIF-1 pathway and upregulating p53 apoptotic target genes. CONCLUSIONS/SIGNIFICANCE:We show here for the first time that hypoxia-induced HIPK2 deregulation was counteracted by zinc that restored HIPK2 suppression of HIF-1 pathway and reactivated p53 apoptotic response to drug, underscoring the potential use of zinc supplementation in combination with chemotherapy to address hypoxia and improve tumor treatment

Cyclic AMP induces IPC leukemia cell apoptosis via CRE-and CDK-dependent Bim transcription

The IPC-81 cell line is derived from the transplantable BNML model of acute myelogenic leukemia (AML), known to be a reliable predictor of the clinical efficiency of antileukemic agents, like the first-line AML anthracycline drug daunorubicin (DNR). We show here that cAMP acted synergistically with DNR to induce IPC cell death. The DNR-induced death differed from that induced by cAMP by (1) not involving Bim induction, (2) being abrogated by GSK3β inhibitors, (3) by being promoted by the HSP90/p23 antagonist geldanamycin and truncated p23 and (4) by being insensitive to the CRE binding protein (CREB) antagonist ICER and to cyclin-dependent protein kinase (CDK) inhibitors. In contrast, the apoptosis induced by cAMP correlated tightly with Bim protein expression. It was abrogated by Bim (BCL2L11) downregulation, whether achieved by the CREB antagonist ICER, by CDK inhibitors, by Bim-directed RNAi, or by protein synthesis inhibitor. The forced expression of BimL killed IPC-81WT cells rapidly, Bcl2-overexpressing cells being partially resistant. The pivotal role of CREB and CDK activity for Bim transcription is unprecedented. It is also noteworthy that newly developed cAMP analogs specifically activating PKA isozyme I (PKA-I) were able to induce IPC cell apoptosis. Our findings support the notion that AML cells may possess targetable death pathways not exploited by common anti-cancer agents