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Background Although adjacent segmental intervertebral disc degeneration (ASDD) is one of the most common complications after lumbar fusion, its exact mechanism remains unclear. As an antibody to RANKL, denosumab (Dmab) effectively reduces bone resorption and stimulates bone formation, which can increase bone mineral density (BMD) and improve osteoporosis. However, it has not been confirmed whether Dmab has a reversing or retarding effect on ASDD. Methods Three-month-old female Sprague-Dawley rats that underwent L4–L5 posterolateral lumbar fusion (PLF) with spinous-process wire fixation 4 weeks after bilateral ovariectomy (OVX) surgery were given Dmab 4 weeks after PLF surgery (OVX+PLF+Dmab group). In addition, the following control groups were defined: Sham, OVX, PLF, and OVX+PLF (n=12 each). Next, manual palpation and X-ray were used to evaluate the state of lumbar fusion. The bone microstructure in the lumbar vertebra and endplate as well as the disc height index (DHI) of L5/6 was evaluated by microcomputed tomography (μCT). The characteristic alterations of ASDD were identified via Safranin-O green staining. Osteoclasts were detected using tartrate-resistant acid phosphatase (TRAP) staining, and the biomechanical properties of vertebrae were evaluated. Aggrecan (Agg), metalloproteinase-13 (MMP-13), and a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS-4) expression in the intervertebral disc were detected by immunohistochemistry and real-time polymerase chain reaction (RT-PCR) analysis. In addition, the expression of CD24 and Sox-9 was assessed by immunohistochemistry. Results Manual palpation showed clear evidence of the fused segment’s immobility. Compared to the OVX+PLF group, more new bone formation was observed by X-ray examination in the OVX+PLF+Dmab group. Dmab significantly alleviated ASDD by retaining disc height index (DHI), decreasing endplate porosity, and increasing vertebral biomechanical properties and BMD. TRAP staining results showed a significantly decreased number of active osteoclasts after Dmab treatment, especially in subchondral bone and cartilaginous endplates. Moreover, the protein and mRNA expression results in discs (IVDs) showed that Dmab not only inhibited matrix degradation by decreasing MMP-13 and ADAMTS-4 but also promoted matrix synthesis by increasing Agg. Dmab maintained the number of notochord cells by increasing CD24 but reducing Sox-9. Conclusions These results suggest that Dmab may be a novel therapeutic target for ASDD treatment.

Aims The prevalent human hepatocellular carcinoma (HCC) is a leading cause of global cancer-related mortality. The small molecular weight triepoxide derivative, 1,3,5-triazine-2,4,6(1H,3H,5H)-tri-one-1,3,5-tri-(oxiranylmethyl) (teroxirone), has been proved effective against the proliferation of lung cancer cells. The purpose is to further examine if teroxirone regulate growth and metastatic potential of HCC cells with aims at disclosing more of the reaction mechanisms. Main methods Measurements of cell viability and flow cytometry were conducted to test sensitivities of teroxirone against HCC cells. The signaling pathway leading to apoptotic death was unraveled by Western blotting analysis. The metastatic progression was evaluated by cell-based phenotype assay that included migration, invasion, gelatin zymography and wound assay. The in vivo drug efficiency was done in immune-deficient mice with the established xenograft tumors. Key findings Teroxirone inhibited growth of HCC cells, but not hepatic cells. The drug induced apoptosis in HCC cells bearing mutant p53. Pretreatment of caspase-3 inhibitor restored cell viabilities by suppressing extrinsic pathway-mediated cell death. More experiments suggested that sub-apoptotic concentrations of teroxirone mitigated migration, invasion and wound healing of HCC cells. The drug reduced growth of the xenograft tumors as established in animal models by activating apoptotic death. Significance The findings asserted that teroxirone is an eligible addition to the existing options as an anticancer agent to eliminate HCC.

To fight cancer at its roots by targeting cancer stem cells is a promising approach for therapy. Previously, an indolylquinoline derivative, 3-((7-ethyl-1H-indol-3-yl)-methyl)-2-methylquinoline (EMMQ), was reported effectively inhibiting the growth of lung cancer cells through impairment of cellular mitochondria functions. To address more on drug efficiency, the study further exploited if EMMQ can impede the propagation of tumorspheres stemmed from non-small cell lung cancer cells. EMMQ inhibited proliferation of spheroids in culture. In animal models, administration of the drug attenuated the spheroid tumorigenicity. The activated apoptosis alleviated growth of xenograft tumors in immune-deficient mice as established by the enriched tumorspheres. More evidence suggested that the reduced stemness of the spheroid tumors is attributed to apoptotic death. The findings supported that EMMQ is an eligible approach to eradicate the minor but tumorigenic lung cancer tumorspheres.

The neuroprotective evaluation of ligustrazine derivatives has become a research focus all over the world. A novel ligustrazine derivative, (3,5,6-Trimethylpyrazin-2-yl)methyl(E)-3-(4-((3,5,6-trimethylpyrazin-2-l)methoxy)phenyl)acrylate (T-CA), has shown protective effects against CoCl2-induced neurotoxicity in a differentiated PC12 cell model and middle cerebral artery occlusion (MCAO) model in our previous studies. However, nearly none of the parent drugs existed after rapid metabolism due to uncertain reasons. Thus, the fragmentation regularities of mass spectra, and metabolites, of T-CA in rats were examined using liquid chromatography-electrospray ionizationion trap mass spectrometry (LC/LTQ-Orbitrap MS) in this research. The main fragment ion, mass spectrum characteristics, and the structural information were elucidated. When compared with a blank sample, we identified five kinds of T-CA metabolites, including three phase I metabolites and two phase II metabolites. The results showed that the metabolic pathways of T-CA in rats via oral administration were hydrolysis (ether bond rupture, ester bond rupture), oxidation, reduction, glucose aldehyde acidification, etc. In addition, three main metabolites were synthesized and their structures were identified by superconducting high-resolution NMR and high-resolution mass spectroscopy (HR-MS). The neuroprotective activity of these metabolites was validated in a PC12 cell model. One of the metabolites (M2) showed significant activity (EC50 = 9.67 μM), which was comparable to the prototype drug T-CA (EC50 = 7.97 μM). The current study provides important information for ligustrazine derivatives, pertaining to the biological conversion process in vivo.

Numerous approaches suggested that compounds with conjugated triazole moieties or benzoxazone pharmacores are effective to antagonize proliferation of human tumors. The current study reported that a synthetic triazole-conjugated benzoxazone, 4-((5-benzyl-1H-1,2,3-triazol-3-yl)-methyl)-7-methoxy-2H-benzo[b][1,4]-oxazin-3(4H)-one (BTO), inhibited growth rates of human non-small cell lung cancer cells. The cytotoxicity can be enhanced with increasing drug concentrations. More evidence supported that the induced reactive oxygen species lead to ultimate apoptotic cell death by recruiting autophagy. The mechanistic pathway as elucidated involved tumor suppressor p53 activation and LC3-1 conversion followed by PARP and procaspase-3 cleavage. Autophagy inhibition reverted apoptotic death and restored cell viabilities. BTO suppressed the development of A549 cell xenograft tumors by activating autophagy and apoptosis simultaneously. As an efficient tumor growth inhibitor with relatively small molecular weight, BTO is a viable addition to the existing list of lung cancer treatment.

Glycyrrhetinic Acid (GA), a triterpenoid aglycone component of the natural product glycyrrhizinic acid, was found to possess remarkable anti-proliferative and apoptosis-inducing activity in various cancer cell lines. Though GA was not as active as other triterpenes, such as betulinic acid and oleanolic acid, it could trigger apoptosis in tumor cells and it can be obtained easily and cheaply, which has stimulated scientific interest in using GA as a scaffold to synthesize new antitumor agents. The structural modifications of GA reported in recent decades can be divided into four groups, which include structural modifications on ring-A, ring-C, ring-E and multiple ring modifications. The lack of a comprehensive and recent review on this topic prompted us to gather more new information. This overview is dedicated to summarizing and updating the structural modification of GA to improve its antitumor activity published between 2005 and 2016. We reviewed a total of 210 GA derivatives that we encountered and compiled the most active GA derivatives along with their activity profile in different series. Furthermore, the structure activity relationships of these derivatives are briefly discussed. The included information is expected to be of benefit to further studies of structural modifications of GA to enhance its antitumor activity.

In this work, we demonstrated that the growth of human non-small-cell-lung-cancer cells H460 and A549 cells can be inhibited by low concentrations of an epoxide derivative, teroxirone, in both in vitro and in vivo models. The cytotoxicity was mediated by apoptotic cell death through DNA damage. The onset of ultimate apoptosis is dependent on the status of p53. Teroxirone caused transient elevation of p53 that activates downstream p21 and procaspase-3 cleavage. The presence of caspase-3 inhibitor reverted apoptotic phenotype. Furthermore, we showed the cytotoxicity of teroxirone in H1299 cells with stable ectopic expression of p53, but not those of mutant p53. A siRNA-mediated knockdown of p53 expression attenuated drug sensitivity. The in vivo experiments demonstrated that teroxirone suppressed growth of xenograft tumors in nude mice. Being a potential therapeutic agent by restraining cell growth through apoptotic death at low concentrations, teroxirone provides a feasible perspective in reversing tumorigenic phenotype of human lung cancer cells.

As a practical and safe herbal medicine, the seeds of Brucea javanica (L.) Merr., were used to cure patients suffering from infectious diseases such as malaria. Recent advances revealed that the herb could also be a useful cancer therapy agent. The study demonstrated that aqueous B. javanica (BJ) extract attenuated the growth of human non-small-lung cancer cells bearing mutant L858R/T790M epidermal growth factor receptor (EGFR). The reduced cell viability in H1975 cells was attributed to apoptosis. Transfection of EGFR small hairpin RNA reverted the sensitivities. When nude mice were fed BJ extract, the growth of xenograft tumors, as established by H1975 cells, was suppressed. Additional histological examination and fluorescence analysis of the resected tissues proved that the induced apoptosis mitigated tumor growth. The work proved that the BJ extract exerted its effectiveness by targeting lung cancer cells carrying mutated EGFR while alleviating tumorigenesis. Aqueous BJ extract is a good candidate to overcome drug resistance in patients undergoing target therapy.

Activated hepatic stellate cells (HSCs) are the main extracellular matrix (ECM)-producing cells in the injured liver and the key mediators of liver fibrosis; they also promote the progression of hepatocellular carcinoma (HCC). In the acidic extracellular microenvironment of HCC, HSCs are activated to promote the migration of HCC cells. It is worth attempting to alter the weak acidic microenvironment to promote activated HSC apoptosis to treat liver fibrosis and liver cancer. In the present study, a series of novel OA-amino acids analogues were designed and synthesized to introduce different amino acids in the 3-hydroxyl of OA using the ester condensation reaction to enhance hydrophilicity, alkalinity, and biological activity. We found that OA-lysine derivative (3g) could improve the hydrophilic of OA and induce HSCs apoptosis via inducing MMP depolarization and increasing intracellular Ca2+ levels. Additionally, 3g displayed a better hepatoprotective effect than OA (20 mg/kg, intragastric administration) against the acute liver injury induced by carbon tetrachloride (CCl4) in mice. The results suggested that basic amino acids (lysine) could effectively enhance OA’s hydrophilicity, alkalinity, and hepatoprotective activity in vitro and in vivo, which might be likely associated with increasing bioavailability and altering an extracellular weak acidic microenvironment with further verification. Therefore, the OA-lysine derivative (3g) has the potential to be developed as an agent with hepatoprotective activity.

Bimodal targeting of the endoplasmic reticular protein, cytochrome P4501A1 (CYP1A1), to mitochondria involves activation of a cryptic mitochondrial targeting signal through endoprotease processing of the protein. Here, we characterized the endoprotease that regulates mitochondrial targeting of CYP1A1. The endoprotease, which was induced by beta-naphthoflavone, was a dimer of 90 kDa and 40 kDa subunits, each containing Ser protease domains. The purified protease processed CYP1A1 in a sequence-specific manner, leading to its mitochondrial import. The glucocorticoid receptor, retinoid X receptor, and p53 underwent similar processing-coupled mitochondrial transport. The inducible 90 kDa subunit was a limiting factor in many cells and some tissues and, thus, regulates the mitochondrial levels of these proteins. A number of other mitochondria-associated proteins with noncanonical targeting signals may also be substrates of this endoprotease. Our results describe a new mechanism of mitochondrial protein import that requires an inducible cytoplasmic endoprotease for activation of cryptic mitochondrial targeting signals.

We have mapped the sites of ischemia/reperfusion-induced phosphorylation of cytochrome c oxidase (CcO) subunits in rabbit hearts by using a combination of Blue Native gel/Tricine gel electrophoresis and nano-LC–MS/MS approaches. We used precursor ion scanning combined with neutral loss scanning and found that mature CcO subunit I was phosphorylated at tandem Ser115/Ser116 positions, subunit IVi1 at Thr52 and subunit Vb at Ser40. These sites are highly conserved in mammalian species. Molecular modeling suggests that phosphorylation sites of subunit I face the inter membrane space while those of subunits IVi1 and Vb face the matrix side.

The concerted actions of molecular networks determine how cells undergo proliferation, death or aging. Here we show that the highly invasive, tumorigenic human non-small-cell-lung cancer (NSCLC) cells carrying mutated p53 alleles were transfected with herpes simplex virus- thymidine kinase (HSV- tk ) cDNA and the selected clone was susceptible to exogenous ganciclovir (GCV). The work further indicated that, in the stable HSV- tk transfectants, GCV suppressed cell proliferation by inducing G 2 /M cell cycle arrest and premature senescence and the potency can be amplified through bystander effect. The growth suppression of the established tumor xenografts in nude mice can be successfully targeted by GCV. These data showed that the GCV-suppressed tumor cell proliferation can be coordinated by cell cycle arrest and cellular senescence in HSV- tk transfectant lacking wild-type p53.

Human non-small cell lung cancer (NSCLC) cells were transfected with recombinant prodrug herpes simplex virus type I thymidine kinase (HSV-tk) cDNA, and the selected clones underwent apoptosis in response to induction by antiviral ganciclovir (GCV). The efficiency of GCV-induced growth inhibition and the extent of the bystander effect were associated with the expression level of HSV-TK in stable transfectants. Development in the HSV-tk/GCV system toward cell death was initiated with cell-cycle accumulation at S and G(2)/M phases, immediately followed by the appearance of sub-G(0)/G(1) cells after drug exposure. To investigate the regulation of cell-cycle modulators during drug treatment, we analyzed release of the apoptosis initiator cytochrome c and activation of the downstream effectors caspase-9, caspase-3 and poly(ADP-ribose)polymerase 16 hr after GCV sensitization, followed by transient escalation of tumor-suppressor p53 and cell-cycle modulators cyclin A and B(1) before committing to programmed cell death. Furthermore, tumor regression was proportional to the degree of ectopic expression of the transferred HSV-tk gene. Our results demonstrate that the HSV-tk/GCV system effectively inhibits the proliferation of NSCLC cells in vitro and in vivo through potent induction of apoptosis, thus providing a rationale for further development.

Mouse monoclonal antibody (MAb-18) against yellow grouper nervous necrosis virus (YGNNV) coat protein was developed and assayed for its neutralization ability. In the present study, we cloned and sequenced the cDNAs encoding heavy (gamma) and light (kappa) chains by constructing a cDNA library of the MAb-18 hybridoma. Three expression vectors, pCMV-NNV-18H (gamma), pCMV-NNV-18L (kappa), and pCMV-NNV-18HL (both gamma and kappa chains) were constructed and successfully expressed in grouper brain (GB) cells. Western blotting results indicated the secretion of antibody in to the medium with successful folding. Extracellular antibodies secreted by the pCMV-NNV-18HL transfected GB cells, neutralized well with YGNNV and showed the highest neutralization index (log(10) of NI) value 4. A significant reduction of titre (99.9%) was observed, when the intracellularly immunized GB cells were propagated with the YGNNV. These preliminary demonstrations suggest the immunoprophylactic use of plasmid constructs encoding the genes of mouse MAbs and the possibility of producing transgenic pathogen-free spawners and larvae, which contain freely available MAbs against pathogen in the circulation.

Bone marrow mesenchymal stem cells (BMSCs) are a kind of pluripotent stem cells derived from bone marrows, which can not only support hematopoiesis, but also have capabilities of multidifferentiation, high-proliferation and self-renewing. They have become one of hotspots in stem cell studies. Studies on in vitro intervention with BMSCs with TCMs have made remarkable progress in recent years. According to the findings, some traditional Chinese medicines can promote proliferation of BMSCs, some can inhibit the apoptosis of BMSCs, while others can induce BMSCs to differentiate into multiple cell types, such as osteoblast. Furthermore, some studies also involved relevant action mechanisms. The authors summarized the advance in relevant studies by reference to relevant literatures of this field.

Hepatic mitochondria contain an inducible cytochrome P450, referred to as P450 MT5, which cross-reacts with antibodies to microsomal cytochrome P450 2E1. In the present study, we purified, partially sequenced, and determined enzymatic properties of the rat liver mitochondrial form. The mitochondrial cytochrome P450 2E1 was purified from pyrazole-induced rat livers using a combination of hydrophobic and ion-exchange chromatography. Mass spectrometry analysis of tryptic fragments of the purified protein further ascertained its identity. N-terminal sequencing of the purified protein showed that its N terminus is identical to that of the microsomal cytochrome P450 2E1. In reconstitution experiments, the mitochondrial cytochrome P450 2E1 displayed the same catalytic activity as the microsomal counterpart, although the activity of the mitochondrial enzyme was supported exclusively by adrenodoxin and adrenodoxin reductase. Mass spectrometry analysis of tryptic fragments and also immunoblot analysis of proteins with anti-serine phosphate antibody demonstrated that the mitochondrial cytochrome P450 2E1 is phosphorylated at a higher level compared with the microsomal counterpart. A different conformational state of the mitochondrial targeted cytochrome P450 2E1 (P450 MT5) is likely to be responsible for its observed preference for adrenodoxin and adrenodoxin reductase electron transfer proteins.

Hepatic mitochondria from different mammalian species contain varying levels of glutathione S-transferase (GST) activities. More than 70% of the activity detectable in the mouse liver mitochondria is associated with the soluble matrix. The mouse mitochondrial matrix GST was purified using a combination of (NH4)2SO4 fractionation, Sephadex gel filtration and affinity chromatography on glutathione (GSH) conjugated Sepharose. The purified GST comigrates with the mouse cytosolic MI (or alpha form), and exhibits an apparent molecular mass of 25 kD on sodium dodecyl sulfate-polyacrylamide gels. Polyclonal antibody to the purified mitochondrial GST cross-reacted with the similarly migrating cytosolic MI GST, suggesting extensive immunochemical relatedness between these two forms. As previously demonstrated for the cytosolic alpha form, the mitochondrial GST catalyzes aflatoxin B1-GSH conjugation (6.3 nmol/mg protein/min) and exhibits peroxidase activity (6.7 mumol/mg protein/min). The putative mitochondrial GST only in intact mitochondria, but not in sonic disrupted mitochondria, is resistant to proteolytic digestion with trypsin, demonstrating its intramitochondrial location. Isoelectric focusing on the flat bed polyacrylamide gel system resolves the mitochondrial GST into two distinct components with apparent pI of 9.9 and 9.7, both of which cross-react with polyclonal antibody to the mitochondrial GST. Under the identical conditions, the most cationic form of cytosolic GST cross-reacting intensely with the antibody resolves as a single component with an apparent pI of 9.4. Thus the mitochondrial GST resembles the alpha family of isoenzymes, though it appears to represent independent molecular species different from the cytosolic forms.

Mitochondrial dysfunction and altered transmembrane potential initiate a mitochondrial respiratory stress response, also known as mitochondrial retrograde response, in a wide spectrum of cells. The mitochondrial stress response activates calcineurin, which regulates transcription factors, including a new nuclear factor-κB (NF-κB) pathway, different from the canonical and noncanonical pathways. In this study using a combination of small interfering RNA-mediated mRNA knock down, transcriptional analysis, and chromatin immunoprecipitation, we report a common mechanism for the regulation of previously established stress response genes Cathepsin L, RyR1, and Glut4. Stress-regulated transcription involves the cooperative interplay between NF-κB (cRel: p50), C/EBPδ, cAMP response element-binding protein, and nuclear factor of activated T cells. We show that the functional synergy of these factors requires the stress-activated heterogeneous nuclear ribonucleoprotein (hnRNP) A2 as a coactivator. HnRNP A2 associates with the enhanceosome, mostly through protein–protein interactions with DNA-bound factors. Silencing of hnRNP A2 as well as other DNA binding signature factors prevents stress-induced transcriptional activation and reverses the invasiveness of mitochondrial DNA-depleted C2C12 cells. Induction of mitochondrial stress signaling by electron transfer chain inhibitors also involved hnRNPA2 activation. We describe a common mechanism of mitochondrial respiratory stress-induced activation of nuclear target genes that involves hnRNP A2 as a transcription coactivator.

Euonymus alatus (E. alatus) has been used as a folk medicine for diabetes in China for more than one thousand years. In order to identify major active components, effects of different fractions of E. alatus on the plasma glucose levels were investigated in normal mice and alloxan-induced diabetic mice. Our results show that ethyl acetate fraction (EtOAc Fr.) displayed significant effects on reducing plasma glucose. In oral glucose tolerance, EtOAc Fr. at 17.2 mg/kg could significantly decrease the blood glucose of both normal mice and diabetic mice. After 4 weeks administration of the EtOAc Fr, when compared with the diabetic control, there were significant difference in biochemical parameters, such as glycosylated serum protein, superoxide dismutase and malondial dehyde, triglyceride, and total cholesterol, between alloxan-induced diabetic mice and the control group. Additional histopathological studies of pancreatic islets also showed EtOAc Fr. has beneficial effects on diabetic mice. Chemical analysis with three-dimensional HPLC demonstrated that the major components from EtOAc Fr were flavonoids and phenolic acids, which had anti-oxidative effects on scavenging DPPH-radical in vitro. All these experimental results suggest that EtOAc Fr. is an active fraction of E. alatus and can prevent the progress of diabetes. The mechanism of E. alatus for glucose control may be by stimulating insulin release, improving glucose uptake and improving oxidative-stress.

DNA damage is lethal and capable of inducing cellular aging or apoptosis. In this work, the highly tumorigenic and cisplatin-resistant human non-small cell lung cancer (NSCLC) cells were transfected with construct encoding the complete sequence of p16INK4a (p16). The stable clones with elevated p16 exhibited enhanced sensitivities to low concentration cisplatin treatment. Further study indicated that cisplatin arrested cells at G2/M phase and the effectiveness is proportional to the level of p16 expressed. The growth of the xenograft tumors established by p16 transfectants in nude mice was also suppressed by cisplatin by inducing senescence-like phenotype. The data altogether indicated that, in cisplatin-resistant tumor cells with basal endogenous p16, the growth suppression by drugs can be greatly improved by ectopic gene transfer.

Euonymus alatus as a folk medicine in China has been clinically used to treat type 2 diabetes for many years, and also exerts beneficial effects on hyperglycemia of diabetic animals. Our previous studies have isolated kaempferol and quercetin from the extract of E. alatus. In the present study, we investigated the possible mechanism of antidiabetic activity of these compounds. Kaempferol and quercetin could significantly improve insulin-stimulated glucose uptake in mature 3T3-L1 adipocytes. In addition, further experiments showed that kaempferol and quercetin served as weak partial agonists in the peroxisome proliferator-agonist receptor gamma (PPARgamma) reporter gene assay. Kaempferol and quercetin could not induce differentiation of 3T3-L1 preadipocytes as traditional PPARgamma agonist. When added together with the PPARgamma agonist rosiglitazone to 3T3-L1 preadipocytes, they could inhibit 3T3-L1 differentiation in a dose-dependent manner. Competitive ligand-binding assay confirmed that kaempferol and quercetin could compete with rosiglitazone at the same binding pocket site as PPARgamma. Kaempferol and quercetin showed significant inhibitory effects on NO production in response to lipopolysaccharide treatment in macrophage cells in which the PPARgamma was overexpressed; rosiglitazone was less potent than kaempferol and quercetin. These observations suggest that kaempferol and quercetin potentially act at multiple targets to ameliorate hyperglycemia, including by acting as partial agonists of PPARgamma.

A large number of mitochondrial proteins lack canonical mitochondrial-targeting signals. The bimodal transport of cytochromes P450 (CYPs) to endoplasmic reticulum and mitochondria (MT), reported previously by us, likely represents one mode of non-canonical protein targeting to MT. Herein, we have studied the mechanism of mouse MT-CYP1A1 targeting to gain insight into the regulatory features and evolutionary conservation of bimodal targeting mechanism. Mouse MT-CYP1A1 consists of two NH2-terminal-truncated molecular species, +91A1 and +331A1. Mutations Pro-2 --> Leu and Tyr-5 --> Leu, which increase the signal recognition particle (SRP) binding, diminished MT targeting of the protein in intact cells. By contrast, mutations Leu-7 --> Asn and Leu-17 --> Asn, which decreased SRP-binding affinity, enhanced MT targeting, thus suggesting that SRP binding is an important regulatory step that modulates bimodal targeting. Protein kinase C (PKC)-mediated phosphorylation of nascent chains at Thr-35 vastly decreased affinity for SRP binding suggesting an important regulatory step. In support of these results, COS cell transfection experiments show that phosphomimetic mutation Thr-35 --> Asp or induced cellular PKC caused increased CYP1A1 targeting to MT and correspondingly lower levels to the endoplasmic reticulum. Results suggest evolutionary conservation of chimeric signals and bimodal targeting of CYP1A1 in different species. The mouse MT-CYP1A1 is an extrinsic membrane protein, which exhibited high FDX1 plus FDXR-mediated N-demethylation of a number of tricyclic antidepressants, pain killers, anti-psychotics, and narcotics that are poor substrates for microsomal CYP1A1.

Four tropical marine fish cell lines have been established from the eye, fin, heart and swim bladder of grouper, Epinephelus awoara (Temminck & Schlegel). Optimum media and temperature conditions for maximum growth were standardized. The eye and swim bladder cells were mostly epithelial, but the fin and heart cells were mostly fibroblastic. The viability of cells was 95% after 1 year of storage in liquid nitrogen (-196 degrees C). Besides these four cell lines, previously established grouper brain, kidney and liver cell lines were also used for a viral susceptibility study which showed that all the cell lines were sensitive to grouper iridovirus, whereas only brain, fin and liver cell lines were susceptible to the yellow grouper nervous necrosis virus (a nodavirus). Electron microscopy studies of the grouper irido- and nodaviruses in ultrathin sections of infected cells showed an abundance of viral particles in the cytoplasm of the virus-infected cells indicating the effective replication of these two viruses. It is suggested that these cell lines can be used for the isolation of putative fish specific viruses and provide a valuable tool to study the mechanisms of host-pathogen interactions. Furthermore, these cell lines upon transfection, using pEGFP-C1 and pEGFP-aMT2.5 (ayu metallothionein promoter), produced significant fluorescent signals indicating their utility for exogenous studies.

Termination of mitochondrial (mt) H-strand transcription in mammalian cells occurs at two distinct sites on the genome. The first site of termination, referred to as mt-TERM occurs beyond the 16 S rRNA gene. However, the second and final site of termination beyond the tRNAThr gene remains unclear. In this study we have characterized the site of termination of the polycistronic distal gene transcript beyond the D-loop region, immediately upstream of the tRNAPhe gene. This region, termed D-TERM, maps to nucleotides 16274–16295 of the mouse genome and includes a conserved A/T rich sequence motif AATAAA as a part of the terminator. Gel-shift analysis showed that the 22 bp D-TERM DNA forms two major complexes with mouse liver mt extract in a sequence-specific manner. Protein purification by DNA-affinity chromatography yielded two major proteins of 45 kDa and 70 kDa. Finally, the D-TERM DNA can mediate transcription termination in a unidirectional manner in a HeLa mt transcription system, only in the presence of purified mouse liver mt D-TERM DNA binding proteins. We have therefore characterized a novel mt transcription termination system, similar in some properties to that of sea urchin, as well as the nuclear RNA Pol I and Pol II transcription termination systems.

The mitochondrial respiratory chain, which consumes approx. 85–90% of the oxygen utilized by cells, is a major source of reactive oxygen species (ROS). Mitochondrial genetic and biosynthetic systems are highly susceptible to ROS toxicity. Intramitochondrial glutathione (GSH) is a major defence against ROS. In the present study, we have investigated the nature of the glutathione S-transferase (GST) pool in mouse liver mitochondria, and have purified three distinct forms of GST: GSTA1-1 and GSTA4-4 of the Alpha family, and GSTM1-1 belonging to the Mu family. The mitochondrial localization of these multiple GSTs was confirmed using a combination of immunoblot analysis, protease protection assay, enzyme activity, N-terminal amino acid sequencing, peptide mapping and confocal immunofluorescence analysis. Additionally, exogenously added 4-hydroxynonenal (HNE), a reactive byproduct of lipid peroxidation, to COS cells differentially affected the cytosolic and mitochondrial GSH pools in a dose- and time-dependent manner. Our results show that HNE-mediated mitochondrial oxidative stress caused a decrease in the GSH pool, increased membrane lipid peroxidation, and increased levels of GSTs, glutathione peroxidase and Hsp70 (heat-shock protein 70). The HNE-induced oxidative stress persisted for longer in the mitochondrial compartment, where the recovery of GSH pool was slower than in the cytosolic compartment. Our study, for the first time, demonstrates the presence in mitochondria of multiple forms of GSTs that show molecular properties similar to those of their cytosolic counterparts. Our results suggest that mitochondrial GSTs may play an important role in defence against chemical and oxidative stress.

Transforming growth factor-alpha (TGF-alpha)-mediated autocrine regulation in human non-small-cell lung cancer (NSCLC) cells NCI-H226 and its brain metastatic variant H226Br were compared. An enhanced TGF-alpha-induced dose-dependent mitogenic responsiveness in H226Br cells was observed. Neutralising antibody that binds TGF-alpha inhibits H226Br cell growth more effectively than NCI-H226 cell growth. Binding assay with 125I-labelled epidermal growth factor (EGF) revealed that H226Br has two types of EGF receptors (EGFRs), whereas the parental cell line, NCI-H226, has only one. H226Br cells contain twice as many EGFRs as H226 cells, as proved by Scatchard analysis and immune kinase assay. Northern analysis indicated that there is more EGFR transcript in H226Br than in NCI-H226, indicating a transcriptional EGFR gene elevation during metastasis progression. The level of accumulated immunoactive TGF-alpha is lower in the conditioned medium of H226Br than in that of NCI-H226. demonstrating down-regulation of TGF-alpha transcript. The accumulated data suggest an elevated and sensitive autocrine modulation by TGF-alpha and EGFR in immortalising the brain metastatic variant cells that were derived from a human NSCLC squamous cell line. Images Figure 4 Figure 5 Figure 6 Figure 7

Insulin-like growth factor (IGF-I) is associated with autocrine and paracrine stimulation for cell growth and development of brain tumor cells. The function of IGF-I in the brain metastatic variant of human lung cancer cells is investigated. The cells used here were derived in vivo with intracarotid injection of human non-small cell lung carcinoma NCI-H226. The tumor was developed as a cultured cell line, H226Br. Unlike the parental cells, H226Br was tumorigenic in nu/nu nude mice. Reverse transcriptase-polymerase chain reaction showed that IGF-I transcript of H226Br is increased compared to that of parental cells. The amount of IGF-I secreted in cultured medium of H226Br is higher than that of cultured parental cells. The IGF-I receptor-specific antibody, alpha IR3, inhibits H226Br growth in serum-free culture. The results established that IGF-I is an autocrine growth regulator for human non-small cell lung cancer cells that progressed to brain.

We studied the biological response to and production of transforming growth factor-alpha (TGF-alpha) by the non-small cell lung carcinoma (NSCLC) clonal cell lines H226b, H322a, H460a, H596b. Each of these cell lines expressed epidermal growth factor receptor (EGFR) as determined by [125I]EGF competitive binding and Scatchard analysis and by phosphorylation. The receptors were functionally active as determined in immune complex kinase assays. H322a, H226b, H460a, and H596b cells showed stimulated [3H]thymidine (Thd) uptake in response to TGF-alpha. Exogenously added TGF-alpha increased colony formation in soft agar for three of the cell lines in media containing serum. All cell lines expressed TGF-alpha detected by immunohistochemistry and TGF-alpha mRNA, although to differing degrees. Cell lysates and spent media competed for EGFR binding with EGF, thus demonstrating production of TGF-alpha-like activity. The anti-TGF-alpha monoclonal antibody AB-3 inhibited the uptake of [3H]Thd by proliferating H322a and H226b cells but not H460a and H596b cells. No inhibition occurred with MOPC21 antibody and inhibition was completely reversed by addition of TGF-alpha to the culture. Suramin inhibited cell proliferation and [3H]Thd uptake by all cell lines. Inhibition of H460a and H596b cells was reversed with exogenous TGF-alpha but not PDGF. Our data suggests that TGF-alpha is a mediator of autocrine growth stimulation for NSCLC cells, and that for some NSCLC cells cytoplasmic binding of receptor and ligand is the primary mechanism for autocrine growth stimulation.

The amino acid sequence of bovine heart mitochondrial coupling factor 6 (F6) has been determined by automated Edman degradation of the whole protein and derived peptides. Preparations based on heat precipitation and ethanol extraction showed allotypic variation at three positions while material further purified by HPLC yielded only one sequence that also differed by a Phe-Thr replacement at residue 62. The mature protein contains 76 amino acids with a calculated molecular weight of 9006 and a pI of approximately equal to 5, in good agreement with experimentally measured values. The charged amino acids are mainly clustered at the termini and in one section in the middle; these three polar segments are separated by two segments relatively rich in nonpolar residues. Chou-Fasman analysis suggests three stretches of alpha-helix coinciding (or within) the high-charge-density sequences with a single beta-turn at the first polar-nonpolar junction. Comparison of the F6 sequence with those of other proteins did not reveal any homologous structures.

Telomeres protect against chromosomal damage. Accelerated telomere loss has been associated with premature aging syndromes such as Werner's syndrome and Dyskeratosis Congenita, while, progressive telomere loss activates a DNA damage response leading to chromosomal instability, typically observed in cancer cells and senescent cells. Therefore, identifying mechanisms of telomere length maintenance is critical for understanding human pathologies. In this paper we demonstrate that mitochondrial dysfunction plays a causal role in telomere shortening. Furthermore, hnRNPA2, a mitochondrial stress responsive lysine acetyltransferase (KAT) acetylates telomere histone H4at lysine 8 of (H4K8) and this acetylation is associated with telomere attrition. Cells containing dysfunctional mitochondria have higher telomere H4K8 acetylation and shorter telomeres independent of cell proliferation rates. Ectopic expression of KAT mutant hnRNPA2 rescued telomere length possibly due to impaired H4K8 acetylation coupled with inability to activate telomerase expression. The phenotypic outcome of telomere shortening in immortalized cells included chromosomal instability (end-fusions) and telomerase activation, typical of an oncogenic transformation; while in non-telomerase expressing fibroblasts, mitochondrial dysfunction induced-telomere attrition resulted in senescence. Our findings provide a mechanistic association between dysfunctional mitochondria and telomere loss and therefore describe a novel epigenetic signal for telomere length maintenance.