Hydrostatic pressure (HP) has a profound effect on cartilage metabolism in normal and pathological conditions, especially in weight-bearing areas of the skeletal system. As an important component of overall load, HP has been shown to affect the synthetic capacity and well-being of chondrocytes, depending on the mode, duration and magnitude of pressure. In this study we examined the effect of continuous HP on the gene expression profile of a chondrocytic cell line (HCS-2/8) using a cDNA array containing 588 well-characterized human genes under tight transcriptional control. A total of 51 affected genes were identified, many of them not previously associated with mechanical stimuli. Among the significantly up-regulated genes were immediate-early genes, and genes involved in heat-shock response (hsp70, hsp40, hsp27), and in growth arrest (GADD45, GADD153, p21(Cip1/Waf1), tob). Markedly down-regulated genes included members of the Id family genes (dominant negative regulators of basic helix-loop-helix transcription factors), and cytoplasmic dynein light chain and apoptosis-related gene NIP3. These alterations in the expression profile induce a transient heat-shock gene response and activation of genes involved in growth arrest and cellular adaptation and/or differentiation.

A small family of necrosis-inducing peptides has been identified as virulence factors of Rhynchosporium secalis, a fungal pathogen of barley (Hordeum vulgare L.) Two members of this family, NIP1 and NIP3, were found to stimulate the phosphohydrolyzing activity of the Mg2+-dependent, K+-stimulated H+-ATPase of plasma membrane vesicles isolated from barley leaves by partitioning in an aqueous two-phase system. Stimulation of enzyme activity was saturated by 10 to 15 [mu]M fungal protein. Another member of the peptide family, NIP2, did not affect the enzyme, indicating that it has a different mode of action.

The adenovirus E1B19K protein inhibits apoptosis induced by E1A and other divergent signals. The cellular proteins that interact with E1B19K have been analyzed by isolating cDNA clones by the yeast two hybrid system. One of these clones encodes B5 which consists of 219 amino acid residues and contains the putative BH3 and transmembrane regions. B5 binds strongly to Nip3 and itself, weakly to E1B19K, but not to Bcl-2 and localizes in nuclear envelope, endoplasmic reticulum and mitochondria. B5 has sequence homology with Nip3 in the middle and C-terminal regions, but not in the N-terminal region. Unlike other E1B19K binding BH3 proteins so far characterized, B5 does not induce apoptosis, but inhibits apoptosis induced by Nip3. However the deletion mutant B5Delta1-31 lacking the N-terminus does induce apoptosis, although weaker than does Nip3, suggesting that the N-terminal region is masking the apoptosis-inducing capacity of B5.

Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) plays a critical role in EBV-induced transformation. An earlier report (Y. Kawaguchi et al., J. Virol. 74: 10104-10111, 2000) showed that EBNA-LP interacts with a cellular protein HS1-associated protein X-1 (HAX-1). The predicted amino acid sequence of HAX-1 exhibits similarity to that of another cellular protein Nip3 which has been shown to interact with cellular and viral anti-apoptotic proteins such as Bcl-2 and BHRF1, an EBV homolog of Bcl-2. Here we investigated whether HAX-1, like Nip3, interacts with Bcl-2 proteins and report the following. (i) A purified chimeric protein consisting of gluthathione S-transferase (GST) fused to BHRF1 (GST-BHRF1) or Bcl-2 (GST-Bcl-2) specifically pulled down HAX-1 transiently expressed in COS-7 cells. (ii) GST-BHRF1 or GST-Bcl-2 was not able to pull down EBNA-LP transiently expressed in COS-7 cells, whereas each of the GST fusion proteins formed complexes with EBNA-LP in the presence of RAX-1. These results indicated that EBNA-LP interacts with the viral and cellular Bcl-2 proteins through HAX-1, suggesting that EBNA-LP possesses a potential function in the regulation of apoptosis in EBV-infected cells.

We have examined the recognition of the variable (V) domain of the heavy (VH) and light (V lambda 2) chains of mouse myeloma protein 315 by helper T cells. Mice were primed with the isolated V domain in complete Freund's adjuvant, and carrier (V domain)-primed spleen cells were transferred together with hapten (NIP)-primed spleen cells to recipient mice that were boosted with NIP3-Fab-315. The helper cell response to both domains was governed by H-2-linked immune response (Ir) genes, and VH-315 and V lambda 2 displayed different Ir phenotypes. H-2k conferred high responsiveness to VH on three different genetic backgrounds, BALB/c, C3H, and B10; mice of the d and b haplotypes were low responders. Conversely, H-2d conferred high responsiveness to V lambda 2 on two backgrounds, BALB/c and C3H, whereas mice of the k haplotype were low responders to this domain. Non-H-2 genes of the B10 background extinguished the helper cell response to V lambda 2 in animals with the high responder d haplotype. The VH Ir gene mapped to the K-A interval of the H-2 complex. Unfolded (completely reduced and alkylated) V domains primed helper cells as efficiently as folded domains for responses to NIP3-Fab-315, indicating that the helper cells recognized an antigenic determinant that was not conformation-dependent. The data indicate that there exists helper T cells which recognize each member of the M315 pair of V domains independent of the other, and that these V domains are recognized like conventional extrinsic protein antigens.

Interleukin-21 (IL-21) has been recently shown to modulate the growth of specific types of B-cell neoplasm. Here, we studied the biological effects of IL-21 in mantle cell lymphoma (MCL). All MCL cell lines and tumors examined expressed the IL-21 receptor. Addition of recombinant IL-21 (rIL-21) in vitro effectively induced STAT1 activation and apoptosis in MCL cells. As STAT1 is known to have tumor-suppressor functions, we hypothesized that STAT1 is important in mediating IL-21-induced apoptosis in MCL cells. In support of this hypothesis, inhibition of STAT1 expression using siRNA significantly decreased the apoptotic responses induced by IL-21. To further investigate the mechanism of IL-21-mediated apoptosis, we employed oligonucleotide arrays to evaluate changes in the expression of apoptosis-related genes induced by rIL-21; rIL-21 significantly upregulated three proapoptotic proteins (BIK, NIP3 and HARAKIRI) and downregulated two antiapoptotic proteins (BCL-2 and BCL-XL/S) as well as tumor necrosis factor-alpha. Using an ELISA-based assay, we demonstrated that rIL-21 significantly decreased the DNA binding of nuclear factor-kappaB, a transcriptional factor known to be a survival signal for MCL cells. To conclude, IL-21 can effectively induce apoptosis in MCL via a STAT1-dependent pathway. Further understanding of IL-21-mediated apoptosis in MCL may be useful in designing novel therapeutic approaches for this disease.

Hypoxia is a key phenomenon in tumor behavior, selecting for resistance to apoptosis, conferring resistance to radiotherapy and chemotherapy, and also inducing angiogenic factors such as vascular endothelial growth factor (VEGF). Exochelins are naturally evolved iron chelators produced by Mycobacterium tuberculosis. Because iron chelation has been reported to activate the hypoxia-inducible factor (HIF), we investigated the effects of an exochelin [desferri-exochelin (DFE) 772SM] on this hypoxia-inducible pathway and downstream target genes. DFE induced HIF-1alpha and HIF-2alpha transcription factors regulating the hypoxic response in the breast tumor cell line MDA468. DFE was 10 times more potent and more rapid in onset of effect than the clinically used iron chelator deferoxamine. The expression of downstream hypoxia-responsive target genes VEGF and the proapoptotic protein NIP3 was activated by transcription. MDA468 proliferation was inhibited via HIF-independent pathways, related to other effects of iron chelation. DFE inhibited effects of VEGF on endothelial cell proliferation. DFE potentially could be useful in cancer therapy by inducing apoptosis via NIP3 in conjunction with other non-HIF-related growth inhibitory pathways and blocking endothelial proliferation despite the presence of VEGF.

Normal spleen cells were separated in dishes coated with thin layers of DNP-gelatin or NIP-gelatin into binding and nonbinding cells and stimulated in vitro with DNP- and/or NIP-conjugated polymerized flagellin (POL). Hapten-specific unresponsiveness was induced in the binding cell population by melting the gel at 37 degrees C or in unfractionated cells by pretreatment with soluble hapten-gelatin and could be reversed by treatment with collagenase. A specific enrichment of anti-DNP and anti-NIP antibody-forming cell precursors (AFCP) could be demonstrated in the binding cell populations after treatment with collagenase in cultures with or without "feeder" cells. However, the response of small numbers of unfractionated and purified hapten-specific spleen cells was suboptimal even in the presence of mitomycin-treated or irradiated feeder cells. Optimal numbers of anti-DNP (anti-NIP) antibody-forming cells were generated by small numbers of normal or purified spleen cells in the presence of spleen cells depleted of anti-DNP (anti-NIP) AFCP. In this system the response of only 2 times 10-4 purified hapten-specific cells was higher than the response of 10-6 unfractionated cells. Purified DNP-specific cells responded only to DNP-POL but not to NIP-POL and purified NIP-specific cells responded only to NIP-POL but not to DNP-POL. The degree of enrichment of anti-DNP AFCP decreased with increasing numbers of binding cells. NIP3-gelatin layers bound four to five times less spleen cells than DNP2-gelatin layers and the enrichment of anti-NIP AFCP (about 300-fold) was three times greater than the enrichment of anti-DNP AFCP (about 100-fold). The immunological significance of hapten-gelatin binding cells which apparently failed to respond to antigen is discussed.

Studies examining the effects of hypoxia upon osteoclast biology have consistently revealed a stimulatory effect; both osteoclast differentiation and resorption activity have been shown to be enhanced in the presence of hypoxia. In the present study we examined the effects of the hypoxia mimetics dimethyloxallyl glycine (DMOG) and desferrioxamine (DFO) upon osteoclastogenesis. In contrast to hypoxia, our studies revealed a dose-dependent inhibition of osteoclast formation from macrophages treated with DMOG and DFO. Moreover, expression of a constitutively active form of hypoxia-inducible factor 1alpha (HIF-1alpha) did not enhance osteoclastogenesis and actually attenuated the differentiation process. DMOG did not affect cell viability or receptor activator of nuclear factor kappaB ligand (RANKL)-dependent phosphorylation of mitogen-activated protein (MAP) kinases. However, RANKL-dependent transcription of tartrate-resistant acid phosphatase (TRAP) was reduced in the presence of DMOG. Additionally, DMOG promoted transcription of the pro-apoptotic mediator B-Nip3. These studies suggest that a hypoxia-responsive factor other than HIF-1alpha is necessary for enhancing the formation of osteoclasts in hypoxic settings.

The aryl hydrocarbon receptor nuclear translocator (Arnt) is a basic helix-loop-helix (bHLH) protein that also contains a Per-Arnt-Sim (PAS) domain. In addition to forming heterodimers with many other bHLH-PAS proteins, including the aryl hydrocarbon receptor (AhR) and hypoxia-inducible factors 1alpha, 2alpha and 3alpha, Arnt can also form homodimers when expressed from its cDNA in vitro or in vivo. However, target genes of the Arnt/Arnt homodimer remain to be identified. In this study, we have elucidated the profile of genes responsive to the reintroduction of Arnt expression in an Arnt-deficient mouse hepatoma cell line (c4), using DNA microarray analysis. The expression of 27 genes was upregulated by 1.5-fold or more in c4 cells infected with a retroviral vector expressing mouse Arnt, while no genes were found to be downregulated. Among the upregulated genes, BCL2/adenovirus E1B 19 kDa-interacting protein 1 (NIP3), serine (or cysteine) proteinase inhibitor, clade E, member 1 (PAI1), and N-myc downstream regulated-like (NDR1), were confirmed to be induced by Arnt using real-time PCR. We also found that the 5' promoter region of 15 out of 20 upregulated genes contain the type 2 E-box 5'-CACGTG-3' Arnt/Arnt binding sequence, consistent with the notion that they represent target genes for Arnt.

The hypoxia-inducible factor (HIF)-mediated signaling pathway is an adaptive and protective mechanism that is triggered by hypoxia, ischemia, and other pathophysiological conditions. The expression of HIF-1α and downstream genes, some of which are pro-apoptotic whereas others are pro-survival, is up-regulated in ischemic stroke. Interestingly, however, the effects of HIF-1α activation are different in the early and late stages of acute cerebral ischemia, and these differences may depend on the duration and severity of hypoxia. Therefore, in the present study, we investigated the effect of HIF-1α activation in chronic cerebral hypoperfusion, which plays an important role in the development of dementia. Permanent bilateral common carotid artery occlusion (2VO) was used to induce chronic global cerebral hypoperfusion in rats. The expression of HIF-1α protein and the transcription of downstream genes were measured at different time points, including 0 h, 12 h, 24 h, 3 days, 7 days, 14 days, 28 days, 42 days, and 56 days after 2VO. HIF-1α increased as early as 12 h after the occlusion and remained high for at least 56 days. Interestingly, mRNA levels of both pro-apoptotic (Bcl-2/adenovirus EIB 19 kDa-interacting protein 3, NADPH oxidase activator 1, and NIP3-like protein X) and pro-survival (vascular endothelial growth factor, glucose transporter-1) genes were up-regulated at the early stage after 2VO, followed by a gradual decline to baseline/control levels. Thus, HIF-1α increased consistently during chronic cerebral hypoperfusion, whereas both pro-apoptotic and pro-survival downstream genes were up-regulated only early after 2VO. This mismatch in gene expression may contribute to the lack of a protective effect of highly expressed HIF-1α during the chronic stage of cerebral hypoperfusion.

Cortical dysplasia (CD) is a well-recognized cause of intractable epilepsy, especially in children and is characterized histologically by derangements in cortical development and organization. The objective of this study was to expand the current knowledge of altered gene expression in CD as a first step towards in the identification of additional genes operative in the evolution of CD. Surgical specimens were obtained from eight patients (4 males and 4 females; age range 2-38 years; mean 15 years) with a pathologic diagnosis of CD. Nondysplastic temporal neocortex was obtained from a 2-year-old boy with intractable epilepsy and medial temporal lobe ganglioglioma. After total RNA isolation from frozen brain tissues, we carried out gene expression profiling using a cDNA expression array. Differences in gene expressions between CD and the nondysplastic neocortex were confirmed by semi-quantitative conventional reverse transcription-PCR. Three genes (recombination activating gene 1 (RAG1), heat shock 60 kDa protein 1 (HSP-60), and transforming growth factor beta1 (TGF beta1)) were found to be up-regulated more than two-fold in CD, whereas four genes (phosphoinositide-3-kinase regulatory subunit polypeptide 1 [p85 alpha] (PI3K), frizzled homolog 2 [Drosophila], Bcl-2/adenovirus E1B 19 kDa interacting protein (NIP3), and glia maturation factor beta (GMF beta)) were down-regulated to less than 50% of their normal levels. Interestingly, the majority of genes showing altered expression were associated with apoptosis. Our study demonstrates diverse changes in gene expression in CD. However, it remains to be shown which of these are causally related to the evolution of CD.

OBJECTIVE

To study the long-term transcriptomic effects of dihydrotestosterone (DHT) in adipose tissue. Fat distribution is regulated by sexual hormones. It is still unclear if androgens are promoting or reducing intra-abdominal fat accumulation.

METHODS

Retroperitoneal adipose tissue were isolated from each group of gonadectomized (GDX) C57BL6 male mice treated with vehicle or DHT for 21 days. Serial analysis of gene expression (SAGE) was performed to generate approximately 150,000 SAGE tags from each sample.

RESULTS

Among the numerous genes regulated by DHT, transcripts involved in glycolysis, such as aldolase 1 A isoform and pyruvate kinase muscle as well as lipogenic transcripts, such as malic enzyme supernatant and ELOVL family member 6 elongation of long chain fatty acids were down-regulated by androgen supplementation. In contrast, transcripts involved in lipolysis and fatty acid oxidation, such as carboxylesterase 3, acetyl-coenzyme A acyltransferase 1, 3-ketoacyl-CoA thiolase B and enoyl-coenzyme A hydratase/3-hydroxyacyl coenzyme A dehydrogenase were up-regulated by DHT. Pro-apoptotic transcripts such as cell death-inducing DFFA-like effector c, BCL2/adenovirus E1B 19 kDa-interacting protein 1 NIP3 and -interacting protein 3-like were up-regulated by DHT, whereas transcripts involved in promotion of cell cycle such as cyclin D2 were down-regulated by DHT.

CONCLUSIONS

These results suggest that chronic androgen treatment may help to improve metabolic profile by regulating various critical pathways involved in adipose tissue physiology. In addition, several genes associated with a healthier metabolic profile, such as adiponectin and CD36 antigen, were up-regulated by 21 days of DHT treatment.

The barley pathogen Rhynchosporium commune secretes necrosis-inducing proteins NIP1, NIP2, and NIP3. Expression analysis revealed that NIP1 transcripts appear to be present in fungal spores already, whereas NIP2 and NIP3 are synthesized after inoculation of host plants. To assess the contribution of the three effector proteins to disease development, deletion mutants were generated. The development of these fungal mutants on four barley cultivars was quantified in comparison with that of the parent wild-type strain and with two fungal strains failing to secrete an "active" NIP1 avirulence protein, using quantitative polymerase chain reaction as well as microscopic imaging after fungal green fluorescent protein tagging. The impact of the three deletions varied quantitatively depending on the host genotype, suggesting that the activities of the fungal effectors add up to produce stronger growth patterns and symptom development. Alternatively, recognition events of differing intensities may be converted into defense gene expression in a quantitative manner.

The present study was performed to determine the effect of waterborne CdCl2 exposure influencing lipid deposition and metabolism, oxidative stress and mitochondrial dysfunction, and explore the underlying molecular mechanism of cadmium (Cd)-induced disorder of hepatic lipid metabolism in fish. To this end, adult zebrafish were exposed to three waterborne CdCl2 concentrations (0(control), 5 and 25 μg Cd/l, respectively) for 30 days. Lipid accumulation, the activities of enzymes related to lipid metabolism and oxidative stress, as well as the expression level of genes involved in lipid metabolism and mitophagy were determined in the liver of zebrafish. Waterborne CdCl2 exposure increased hepatic triglyceride (TG) and Cd accumulation, the activities of fatty acid synthase (FAS), 6-phosphogluconate dehydrogenase (6PGD), glucose 6-phosphate dehydrogenase (G6PD) and malic enzyme (ME), and the mRNA level of fatty acid synthase (fas), acetyl-CoA carboxylase alpha (acaca), glucose 6-phosphate dehydrogenase (g6pd) and malic enzyme (me), but reduced the mRNA level of carnitine palmitoyl transferase 1 (cpt1), hormone-sensitive lipase alpha (hsla), and adipose triacylglyceride lipase (atgl). The activities of superoxide dismutase (SOD), glutathoinine peroxidase (GPx) and cytochrome c oxidase (COX) and the ATP level were significantly reduced after CdCl2 exposure. CdCl2 exposure significantly increased the mRNA level of genes (microtubule-associated protein light chain 3 alpha (lc3a), PTEN-induced putative kinase 1 (pink1), NIP3-like protein X (nix) and PARKIN (parkin)) related to mitophagy. To elucidate the mechanism, reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) and the mitochondrial permeability transition (MPT) inhibitor cyclosporine A (CsA) were used to verify the role of ROS and mitochondrial dysfunction in Cd-induced disorder of lipid metabolism. NAC pretreatment reversed the Cd-induced up-regulation of TG accumulation and activities of lipogenic enzymes, and the Cd-induced down-regulation of mRNA levels of lipolytic genes. Meanwhile, NAC pretreatment also blocked the mitochondrial membrane potential (MMP) collapse and decreased the ATP level, suggesting that ROS played a crucial role in regulating the Cd-induced mitochondrial dysfunction. Taken together, our findings, for the first time, highlight the importance of the oxidative stress and mitochondrial dysfunction in Cd-induced disorder of hepatic lipid metabolism, which proposed a novel mechanism for elucidating metal element exposure inducing the disorder of lipid metabolism in vertebrates.

Dietary soy and soy isoflavones are neuroprotective in experimental cerebral ischemia. Because these isoflavones have estrogenic properties, we hypothesized that, like estrogens, they would inhibit acute vascular injury and the detrimental acute increase in hypoxia-induced vascular endothelial growth factor (VEGF) that leads to cerebral edema after stroke. Mature ovariectomized female Sprague Dawley rats were fed soy-free or soy-containing diets for 4 weeks followed by 90 minutes of transient middle cerebral artery occlusion. Similar to estrogens, dietary soy significantly reduced cerebral edema and vascular apoptosis 24 hours after stroke. Soy also inhibited the ischemia-induced increase in cortical VEGF and VEGF receptor (VEGFR)-2 protein expression observed 4 and 24 hours after stroke, although mRNA levels increased. The reduction in VEGF/VEGFR-2 was associated both with decreases in receptor phosphorylation and signaling to AKT and endothelial nitric oxide synthase. Furthermore degradation of the VEGFR-2 was increased with dietary soy. The primary ischemic stimulus for VEGF, hypoxia-inducible factor 1α (HIF1α), was similarly reduced by dietary soy 4 hours after transient middle cerebral artery occlusion in both the cortex and striatum. The inhibition of HIF1α activity was further confirmed by a significant decrease in the HIF1α-activated apoptotic mediator BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (Nip3-like protein X). These data suggest that soy isoflavones target events early in the ischemic cascade as part of their neuroprotective actions and counterbalance some of the detrimental effects of the endogenous response to cerebral injury.

Mitophagy is an autophagic process through which damaged or dysfunctional mitochondria are specifically degraded to maintain cellular homeostasis. It is highly regulated by various signaling pathways such as the PTEN-induced putative kinase 1 (PINK1)/Parkin and NIP3-like protein X (NIX)/BNIP3 pathways. Additionally, it plays a crucial role in inducing some pathological processes. Notably, some evidence suggesting the association of mitophagy with the occurrence of chronic diseases such as Parkinson's disease (PD), cancer, diabetes, atherosclerosis (AS), and myocardial ischemia reperfusion (MIR) injury is available. Particularly, it has been reported that mitophagy could hinder the development of PD by activating the PINK1/Parkin pathway and acting as a defense mechanism against the induction of diabetes. Conversely, the induction of mitophagy plays dual roles in driving the process of cancer, AS, and MIR injury. In this review, we have explained the role and regulatory mechanisms through which mitophagy plays a role in these chronic pathologies. Importantly, the pharmacological targeting of mitophagy might prove to be a potential alternative for the treatment of these chronic diseases.

Ni2P/Ni12P5 nanoparticles were obtained by thermal decomposition of nickel organometallic salt at low temperature. The use of different characterization techniques allowed us to determine that this process produced a mixture of two nickel phosphide phases: Ni2P and Ni12P5. These nickel phosphides nanoparticles, supported on mesoporous silica, showed activity and high selectivity for producing the hydrogenation of the acetophenone carbonyl group to obtain 1-phenylethanol. This is a first report that demonstrates the ability of supported Ni2P/Ni12P5 nanoparticles to produce the chemoselective hydrogenation of acetophenone. We attribute these special catalytic properties to the particular geometry of the Ni-P sites on the surface of the nanoparticles. This is an interesting result because the nickel phosphides have a wide composition range (from Ni3P to NiP3), with different crystallographic structures, therefore we think that different phases could be active and selective to hydrogenate many important molecules with more than one functional group.

Iterative screening of a phage display cDNA expression library of the third-stage larvae (L3) of Brugia malayi with sera from putatively immune individuals (endemic normal, EN) identified a novel clone with insert showing significant homology to Onchocerca volvulus novel immunogenic protein-3 (Ov-NIP3) gene and Caenorhabditis elegans NIP3-like protein and hence the gene was designated Brugia malayi NIP3-like protein (BmNIP3). EST database analysis showed that ESTs from several gastrointestinal nematodes such as Ancylostoma caninum, Teladorsagia circumcincta, Haemonchus contortus and Strongyloides stercoralis has BmNIP3 homologues, but the gene has not been described from these parasites. Sequence analyses showed that BmNIP3 has three potential mucin-type O-glycosylation sites and several serine/threonine phosphorylation sites. As expected, BmNIP3 protein isolated from the parasite was serine/threonine phosphorylated. Further analyses showed that BmNIP3 is differentially transcribed, with highest level of expression present in the larval (L3 and L4) stages. Mice immunized with rBmNIP3 developed strong antibody responses predominantly of the IgG1 and IgG2a subtype. A similar analyses of the sera samples from EN individuals showed that they also carry high levels of IgG1 and IgG2 antibodies against BmNIP3, whereas, chronically infected patients carry largely IgG3 antibodies and MF individuals carry high levels of IgG1 antibodies against BmNIP3. This study thus describes a novel protein from B. malayi that appears to be highly immunogenic in both humans and mice.

Apoptosis is regulated by interaction of antiapoptotic Bcl-2 family proteins with various proapoptotic proteins, several of which are also members of the Bcl-2 family. BNIP3 (formerly NIP3) is a proapoptotic mitochondrial protein classified in the Bcl-2 family based on limited sequence homology-3 (BH3) domain and COOH-terminal transmembrane domain. Sequence comparison of BNIP3 has indicated that there are several BNIP3 human homologs of this protein, like BNIP3L, Nix and BNIP3. We have cloned a new member of BNIP3 family from the cDNA library prepared from human dermal papilla cells and designated as BNIP3h. BNIP3h shows substantial homology with other BNIP3 family proteins. BNIP3h induced apoptosis from 24 hours after transfection in MCF7 cell lines and its apoptosis inducing activity is extended until 72 hours after transfection.

Bronchopulmonary dysplasia (BPD), also known as chronic lung disease, is one of the most common respiratory diseases in premature new‑born humans. Mitochondria are not only the main source of reactive oxygen species but are also critical for the maintenance of homeostasis and a wide range of biological activities, such as producing energy, buffering cytosolic calcium and regulating signal transduction. However, as a critical quality control method for mitochondria, little is known about the role of mitophagy in BPD. The present study assessed mitochondrial function in hyperoxia‑exposed alveolar type II (AT‑II) cells of rats during lung development. New‑born Sprague‑Dawley rats were divided into hyperoxia (85% oxygen) and control (21% oxygen) groups. Histopathological and morphological properties of the lung tissues were assessed at postnatal days 1, 3, 7 and 14. Ultrastructural mitochondrial alteration was observed using transmission electron microscopy and the expression of the mitophagy proteins putative kinase (PINK)1, Parkin and Nip3‑like protein X (NIX) in the lung tissues was evaluated using western blotting. Immunofluorescence staining was used to determine the co‑localisation of PINK1 and Parkin. Real‑time analyses of extracellular acidification rate and oxygen consumption rate were performed using primary AT‑II cells to evaluate metabolic changes. Mitochondria in hyperoxia‑exposed rat AT‑II cells began to show abnormal morphological and physiological features. These changes were accompanied by decreased mitochondrial membrane potential and increased expression levels of PINK1‑Parkin and NIX. Increased binding between a mitochondria marker (cytochrome C oxidase subunit IV isoform I) and an autophagy marker (microtubule‑associated protein‑1 light chain‑3B) was observed in primary AT‑II cells and was accompanied by decreased mitochondrial metabolic capacity in model rats. Thus, mitophagy mediated by PINK1, Parkin and NIX in AT‑II cells occurred in hyperoxia‑exposed new‑born rats. These findings suggested that the accumulation of dysfunctional mitochondria may be a key factor in the pathogenesis of BPD and result in attenuated alveolar development.

BACKGROUND

Chronic intermittent hypoxia (CIH) is the most important pathophysiologic feature of sleep apnea syndrome (SAS). To explore the relationship between SAS and dementia, the effects of CIH on the expression of Nip3, neuron apoptosis and beta-amyloid protein deposit in the brain cortex of the frontal lobe of mice were evaluated in this study.

METHODS

Thirty male ICR mice were divided into four groups: control group (A, n = 10, sham hypoxia/reoxygenation), 2 weeks CIH group (B, n = 5), 4 weeks CIH group (C, n = 5), and 8 weeks CIH group (D, n = 10). The ICR mice were placed in a chamber and exposed to intermittent hypoxia (oxygen concentration changed periodically from (21.72 +/- 0.55)% to (6.84 +/- 0.47)% every two minutes, eight hours per day). Neuron apoptosis of the cortex of the frontal lobe was detected by means of terminal deoxy-nucleotidyl transferase-mediated in situ end labeling (TUNEL). Immunohistochemical staining was performed for measuring expression of Nip3 and beta-amyloid protein. The ultrastructure of neurons was observed under a transmission electron microscope.

RESULTS

TUNEL positive neurons in each square millimeter in the cortex of the frontal lobe were categorized by median or Ri into group A (1, 5.5), group B (133, 13), group C (252, 21), and group D (318, 24). There were significant differences among the above four groups (P = 0.000). The significance test was performed between the control group and each CIH group respectively: group A and B (P>> 0.05); group A and C (P < 0.01); and group A and D (P < 0.005). The number of apoptotic neurons kept increasing in the ICR mice under CIH condition, and reached the peak in the group D, but there was no significant difference between groups B and C, between groups B and D, and between groups C and D. Nip3 positive neurons in each square millimeter in the cortex of the frontal lobe in each group were calculated by median or Ri as follows: group A (2, 5.5), group B (117, 13), group C (227, 26.2), and group D (479, 21.4). There were significant differences among the four groups (P = 0.000). The statistical test was performed between the control group and each CIH group respectively: groups A and B (P>> 0.05); groups A and C (P < 0.005); and groups A and D (P < 0.005). There was no significant difference between groups B and C, groups B and D, and groups C and D. The expression of Nip3 was closely correlated with neuron apoptosis in the brain (P < 0.05). The expression of beta-amyloid protein in the brain of mice was negative in all CIH groups and the control group. Ultrastructure observation showed karyopyknosis of nucleus, swelling of chondriosomes, deposit of lipofuscins and degeneration of neural sheath in all CIH groups but not in the control group.

CONCLUSIONS

The results of this study indicate that CIH could up-regulate the expression of Nip3, and result in neuron apoptosis and ultrastructural changes in neurons of the frontal cortex.

The sophisticated uptake and translocation regulation of the essential element boron (B) in plants is ensured by two transmembrane transporter families: the Nodulin26-like Intrinsic Protein (NIP) and BOR transporter family. Though the agriculturally important crop Brassica napus is highly sensitive to B deficiency, and NIPs and BORs have been suggested to be responsible for B efficiency in this species, functional information of these transporter subfamilies is extremely rare. Here, we molecularly characterized the NIP and BOR1 transporter family in the European winter-type cv. Darmor-PBY018. Our transport assays in the heterologous oocyte and yeast expression systems as well as in growth complementation assays in planta demonstrated B transport activity of NIP5, NIP6, NIP7 and BOR1 isoforms. Moreover, we provided functional and quantitative evidence that also members of the NIP2, NIP3 and NIP4 groups facilitate the transport of B. A detailed B- and tissue-dependent B-transporter expression map was generated by quantitative polymerase chain reaction. We showed that NIP5 isoforms are highly upregulated under B-deficient conditions in roots, but also in shoot tissues. Moreover, we detected transcripts of several B-permeable NIPs from various groups in floral tissues that contribute to the B distribution within the highly B deficiency-sensitive flowers.

The sensitivities of apoptosis induced by E1A, c-Myc, Bax, and Nip3 to wild-type (wt) and mutated p53 and Id proteins were analyzed by transient transfection followed by flow cytometry with p53 null mouse cerebellum cell lines W7 and M13 that express wt and mutated p53 in response to dexamethasone, respectively. Apoptosis induced by c-Myc was stimulated weakly by wt p53, strongly by Ids, but suppressed completely by mutated p53 irrespective of coexpression with Ids, while apoptosis induced by E1A was suppressed by mutated p53 but stimulated when coexpressed with Ids. Apoptosis induced by Bax was little affected by wt and mutated p53, but inhibited by Ids, while apoptosis induced by Nip3 was inhibited by both wt and mutated p53 and inhibition was stimulated by Ids. Caspase-1 was activated only by Bax significantly when coexpressed with mutated p53 but not with wt p53. These results indicate that the apoptotic processes elicited by these inducers are different and differently affected by wt and mutated p53 and by Ids.