Genotoxic stress/p53-induced DNAJB9 inhibits the pro-apoptotic function of p53.

DNAJB9 is a recently isolated member of the molecular chaperone gene family, whose precise function is largely unknown. In the present study, we have identified DNAJB9 as an inducible gene of the tumor suppressor p53. DNAJB9 expression was induced by p53 or genotoxic stress in a p53-dependent manner, which was mediated by the Ras/Raf/ERK pathway. In addition, depletion of DNAJB9 by using siRNAs greatly increased genotoxic stress/p53-induced apoptosis, suggesting that DNAJB9 inhibits the pro-apoptotic function of p53. We also found that DNAJB9 physically interacts with p53 through its J domain, through which it inhibits the pro-apoptotic function of p53. Moreover, DNAJB9 colocalized with p53 in both cytoplasm and nucleus under genotoxic conditions. Together, these results demonstrate that DNAJB9 is a downstream target of p53 that belongs to the group of negative feedback regulators of p53.

Sds22/PP1 links epithelial integrity and tumor suppression via regulation of myosin II and JNK signaling.

Loss of epithelial integrity often correlates with the progression of malignant tumors. Sds22, a regulatory subunit of protein phosphatase 1 (PP1), has recently been linked to regulation of epithelial polarity in Drosophila. However, its role in tumorigenesis remains obscure. In this study, using Drosophila imaginal tissue as an in vivo model system, we show that sds22 is a new potential tumor suppressor gene in Drosophila. Without sds22, cells lose epithelial architecture, and become invasive and tumorigenic when combined with Ras overexpression; conversely, sds22 overexpression can largely suppress tumorigenic growth of Ras(V12)scrib(-/-) mutant cells. Mechanistically, we show that sds22 prevents cell invasion and metastasis by inhibiting myosin II and Jun N-terminal kinase (JNK) activity downstream of PP1. Loss of this inhibition causes cells to lose epithelial organization and promotes cell invasion. Finally, human Sds22 is focally deleted and downregulated in multiple carcinomas, and this downregulation correlates with tumor progression, suggesting that sds22 inactivation may contribute to tumorigenesis and metastatic potential in human cancers via a similar mechanism.

Podocyte biology in diabetic nephropathy.

Glomerular visceral epithelial cells, namely podocytes, are highly specialized cells and give rise to primary processes, secondary processes, and finally foot processes. The foot processes of neighboring podocytes interdigitate, leaving between them filtration slits. These are bridged by an extracellular substance, known as the slit diaphragm, which plays a major role in establishing size-selective barrier to protein loss. Furthermore, podocytes are known to synthesize matrix molecules to the glomerular basement membrane (GBM), including type IV collagen, laminin, entactin, and agrin. Because diabetic nephropathy is clinically characterized by proteinuria and pathologically by glomerular hypertrophy and GBM thickening with foot process effacement, podocytes have been the focus in the field of research on diabetic nephropathy. As a result, many investigations have demonstrated that the diabetic milieu per se, hemodynamic changes, and local growth factors such as transforming growth factor-beta and angiotensin II, which are considered mediators in the pathogenesis of diabetic nephropathy, induce directly and/or indirectly hypertrophy, apoptosis, and structural changes, and increase type IV collagen synthesis in podocytes. This review explores some of the structural and functional changes of podocytes under diabetic conditions and their role in the development and progression of diabetic nephropathy.

Activation of the renin-angiotensin system within podocytes in diabetes.

The autocrine and paracrine activation of the renin-angiotensin system (RAS) within cells of the kidney plays a role in the overall pathophysiology of the renal disease due to diabetes. In this study, we focus on components of the RAS in the podocyte as these cells are important in the pathogenesis of glomerulosclerosis and proteinuria. Immortalized mouse podocytes were exposed to media containing normal glucose (NG) or high glucose (HG) for in vitro studies. In vivo studies utilized kidney tissue obtained from rats treated for 3 months with streptozotocin to induce diabetes. Angiotensinogen (AGT) and the angiotensin II (AII) type 1 receptor mRNA and protein were significantly increased in the podocytes cultured under the high glucose conditions. Both angiotensins I and II levels were significantly higher in cell lysates and the conditioned media of cells grown in high glucose. There were no differences in renin activity, angiotensin-converting enzyme level, or AII type 2 receptor level. Glomerular AGT and AII type 1 receptor assessed by means of immunohistochemistry were increased in diabetic rats compared with the control rats. Other measured components of the RAS within the glomeruli were not different. We suggest that increased AGT, an attendant increase in AII and increased AII type 1 receptor in podocytes experiencing diabetic conditions play an important role in the pathogenesis of diabetic nephropathy.

shl, a New set of Arabidopsis mutants with exaggerated developmental responses to available red, far-red, and blue light.

The interaction of light perception with development is the subject of intensive genetic analysis in the model plant Arabidopsis. We performed genetic screens in low white light-a threshold condition in which photomorphogenetic signaling pathways are only partially active-for ethyl methane sulfonate-generated mutants with altered developmental phenotypes. Recessive mutants with exaggerated developmental responses were obtained in eight complementation groups designated shl for seedlings hyperresponsive to light. shl1, shl2, shl5, and shl3 shl4 (double mutant) seedlings showed limited or no phenotypic effects in darkness, but showed significantly enhanced inhibition of hypocotyl elongation in low-white, red, far-red, blue, and green light across a range of fluences. These results reflect developmental hyper-responsiveness to signals generated by both phytochrome and cryptochrome photoreceptors. The shl11 mutant retained significant phenotypic effects on hypocotyl length in both the phyA mutant and phyB mutant backgrounds but may be dependent on CRY1 for phenotypic expression in blue light. The shl2 phenotype was partially dependent on PHYB, PHYA, and CRY1 in red, far-red, and blue light, respectively. shl2 and, in particular, shl1 were partially dependent on HY5 activity for their light-hyperresponsive phenotypes. The SHL genes act (genetically) as light-dependent negative regulators of photomorphogenesis, possibly in a downstream signaling or developmental pathway that is shared by CRY1, PHYA, and PHYB and other photoreceptors (CRY2, PHYC, PHYD, and PHYE).

Production and characterization of immortalized rat hepatocytes secreting hepatocyte growth factor/scatter factor.

BACKGROUND/AIMS: Hepatocyte transplantation is a recently attractive field in the treatment of liver failure and enzyme-deficient diseases. However, procurement of sufficient quantities of hepatocytes is almost impossible. We attempted to create a hepatocyte cell line that could be used for hepatocyte transplantation. METHODOLOGY: L2A2 is a conditionally immortalized rat hepatocyte cell line produced by transfection of temperature-sensitive simian virus T antigen to the hepatocytes in the Lewis rat. Hepatocyte Growth Factor/Scatter Factor (HGF/SF)-secreting L2A2 cells, designated as SF-21, was produced by transfecting human HGF/SF cDNA into L2A2 cells. RESULTS: This cell line was able to produce HGF/SF at the rate of 5-10 ng/10(6) cells/24 hrs, and the recombinant HGF/SF was of the expected size and was functionally active in that it could scatter Madin-Darby canine kidney cells. The SF-21 cells grew faster than its parental cell clone, and survived and proliferated at 37 degrees C in vitro. Also, the SF-21 cells were able to survive and proliferate when transplanted into the spleen of syngeneic rat, and expressed glucose-6-phosphatase. CONCLUSIONS: These HGF/SF-secreting hepatocytes can be used as a model system to test a feasibility of using genetically engineered hepatocyte cell line for hepatocyte transplantation in the rat.

Stereochemical requirement at 4-position of 4-phenyl-1-arylsulfonylimidazolidinones for their cytotoxicities.

In order to investigate the stereochemical requirements of planar structure at 4-position of 4-phenyl-1-arylsulfonylimidazolidinones (1) for their cytoxicities against human cancer cell lines, the size, the distance from imidazolidinone ring, and the conformation of this moiety were variegated. Replacement of phenyl moiety with naphthyl in compounds 2 and 3 or benzyl moiety in compound 4 sharply reduced activity of 1. Conformational restriction on phenyl ring in compound 5 also resulted in the loss of activity of 1. Therefore, phenyl moiety without any substituents directly attached to imidazolidinone ring of 1 should be considered as an essential pharmacophore for this analog.

Expression of SET is modulated as a function of cell proliferation.

We explored a biological role of SET as it relates to cell proliferation and differentiation. Immunohistochemical staining demonstrated that the expression of SET was ubiquitous and diffuse over the whole embryo on gestational day 15. At a later stage of development, SET was expressed at relatively lower levels and localized to specific tissues and cells. On embryonic day 19, specific SET immunoreactivity was found in the epithelium of skin, respiratory tract, intestine, and retina as well as in muscle and cartilage. In these cells SET was stained mostly in the nucleus, which was supported indirectly by nuclear transport of enhanced green fluorescence protein-SET fusion proteins in ECV304 endothelial cells. Set mRNA expression was further confirmed in various cultured cells, including NIH 3T3 cells, L6 myoblast cells, human umbilical vein endothelial cells, and ECV304 cells. Using F9 teratocarcinoma cell lines, which were stimulated to differentiate into the two different cell lineages of parietal and visceral endoderm, we have further examined the role of SET. The expression of set mRNA and SET protein was diminished about three-fold in both differentiated endoderm cells compared to the undifferentiated F9 cells. However, when F9 cells were subjected to serum starvation, reduction of set mRNA abundance also took place at a similar level to that observed in response to differentiation. Consistent with this, quiescent L6 myoblast showed a marked downregulation of set mRNA compared to proliferating cells. These results suggest that SET is involved mainly in the regulation of cell proliferation rather than differentiation during embryonic development.

Aging process is accompanied by increase of transglutaminase C.

Crosslinking has been suggested as one of the mechanisms involved in the aging process. Among the various random or enzyme-mediated crosslinking reactions, transglutaminase (TGase)-catalyzed crosslinking activity has been proposed for its possible involvement in cell proliferation, differentiation, carcinogenesis, programmed death, and aging. Moreover, recent findings of TGase C as a putative signal transducer and cell cycle regulator has renewed interest in the study of TGase C in relation to aging phenomena. The ubiquitous presence of TGase C compared to the organ-specific localization of other types of TGases has attracted special attention as a cellular aging device. In the present investigation for in vitro studies, we have compared the pattern of TGase C in young and old human red blood cells, separated by density differentiation, and in early and late-passage or hydrogen peroxide-treated human primary fibroblasts. For in vivo study, we monitored the age-dependent changes of TGase C in the liver and brain tissues of 4, 12, 18, and 24-month-old Sprague-Dawley rats. We obtained evidence that both the activity and protein levels of TGase C were high in old RBC and late-passage or hydrogen peroxide-treated fibroblasts. Similar findings were seen in liver and brain tissue such as age-dependent increases in TGase activity and protein level in an organ-specific pattern. These data suggest that TGase C might play an active role in the cellular process with age.

Transcriptional control of the glnD gene is not dependent on nitrogen availability in Escherichia coli.

Glutamine synthetase (GS) is one of the most important enzymes in the assimilation of nitrogenous compounds in Escherichia coli and related bacteria. For the control of its activity and biosynthesis, tricyclic cascades of uridylylation/deuridylylation of PII protein, adenylylation/deadenylylation of glutamine synthetase, and phosphorylation/dephosphorylation of Ntr1 are operating, where the regulation of uridylylation/deuridylylation by uridylyl transferase-uridylyl removing enzyme (UT-UR) (the product of the glnD gene) would play the ultimate nitrogen sensing role. However, the possible nitrogen-regulatable element in the upstream of the glnD gene has been debated. In the present experiment, we have cloned and sequenced the four minute regions of the Escherichia coli chromosome, where rpsB, map, glnD, and dapD genes have been identified in sequence. We could localize the transcriptional start site at seven nucleotides upstream of the translation initiation codon by primer extension analysis. The nitrogen dependency of the glnD gene has been analyzed by Northern blot, RNase protection, and promoter-luciferase activity assays. These data suggested a constitutive expression of the glnD gene independent of nitrogen availability. From these results, it could be concluded that the ultimate nitrogen sensing device for the bacteria should be the UT-UR itself, through modulation of its activity in response to the nitrogen status rather than its biosynthetic mechanism.

Metabolic modulation of cellular redox potential can improve cardiac recovery from ischemia-reperfusion injury.

OBJECTIVE: Ischemia-reperfusion heart injury is an important pathologic condition against which many strategies, mainly involving antioxidants or radical scavengers, have been developed, but without satisfactory results. In the present experiment, modulation of the cytosolic NADH/NAD ratio by pyruvate and aspartate was tested in order to protect the heart from ischemia-reperfusion injury. METHODS: Effects of pyruvate and aspartate on cardiac function recovery and redox potential were analyzed in the isolated heart of male Sprague-Dawley rats. Hearts were made globally ischemic for 20 min and then reperfused for 30 min. RESULTS: Pyruvate and aspartate protected against tissue injury and improved contractile function after reperfusion of ischemic hearts, and these substances effectively decreased the tissue and cytosolic NADH/NAD ratio of the myocardium in a dose-dependent manner. Postischemic cardiac functions were negatively related to tissue and cytosolic NADH/NAD ratios. Increased NADH selectively inhibited myocardial xanthine dehydrogenase in vitro. It was thus expected that a decrease of NADH might limit the production of reactive oxygen species through the recovery of xanthine dehydrogenase activity. CONCLUSION: These results indicate that a decrease of NADH is related to pyruvate and aspartate-induced protection of ischemic myocardium.

Isolation and characterization of brain-specific transglutaminases from rat.

The relevance of transglutaminases with neural function and several disorders has been emphasized recently. Especially, many polypeptides associated with neurodegenerative diseases are suggested to be putative transglutaminase substrates such as beta amyloid protein of Alzheimer's disease, microtubule-associated proteins and neurofilaments, etc. In addition, the CAG repeated gene products with probable polyglutamine tract, putative transglutaminase substrates, were identified in several neurodegenerative disorders. However, the identity of the brain transglutaminase has not been confirmed, because of enzymic stability and low activity. In the present experiment, we have isolated brain-specific transglutaminases, designated as TGase NI and TGase NII, which are different from other types of transglutaminases in respects of molecular weights (mw. 45 kDa, 29 kDa respectively), substrate affinity, elution profile on ion-exchange chromatography, sensitivity to proteases and ethanol, and immunological properties. The enzymes were localized specifically in the brain tissues but not in the liver tissue. And neural cells such as pheochromocytoma cell, glioma cell, primary neuronal and glial cells were shown to be enriched with TGase NI and TGase NII. The possible biological roles of the enzymes were discussed not only on the aspect of crosslinking activity but also of signal transducing capacity of the enzyme in the brain.

Characterization of the WW domain of human yes-associated protein and its polyproline-containing ligands.

We had previously identified the WW domain as a novel globular domain that is composed of 38-40 semiconserved amino acids and is involved in mediating protein-protein interaction. The WW domain is shared by proteins of diverse functions including structural, regulatory, and signaling proteins in yeast, nematode, and mammals. Functionally it is similar to the Src homology 3 domain in that it binds polyproline ligands. By screening a 16-day mouse embryo expression library, we identified two putative ligands of the WW domain of Yes kinase-associated protein which we named WW domain-binding proteins 1 and 2. These proteins interacted with the WW domain via a short proline-rich motif with the consensus sequence of four consecutive prolines followed by a tyrosine. Herein, we report the cDNA cloning and characterization of the human orthologs of WW domain-binding proteins 1 and 2. The products encoded by these cDNA clones represent novel proteins with no known function. Furthermore, these proteins show no homology to each other except for a proline-rich motif. By fluorescence in situ hybridization on human metaphase chromosomes, we mapped the human genes for WW domain-binding proteins 1 and 2 to chromosomes 2p12 and 17q25, respectively. In addition, using site-directed mutagenesis, we determined which residues in the WW domain of Yes kinase-associated protein are critical for binding. Finally, by synthesizing peptides in which the various positions of the four consecutive proline-tyrosine motif and the five surrounding residues were replaced by all possible amino acid residues, we further elucidated the binding requirements of this motif.

Planar structural requirement at 4-position of 1-arylsulfonyl-4-phenyl-4,5-dihydro-2-imidazolones for their cytotoxicity.

In order to identify the structural requirement at 4-position of 1-arylsulfonyl-4-phenyl-4,5-dihydro-2-limidazolones3 for their cytotoxicity, the corresponding 1-arylsulfonyl-4-cyclohexyl-4,5-dihydro-2-imidazolones4 were synthesized and theirin vitro cytotoxicity against human solid tumor cell lines were measured. Unlike compounds3a-c, cyclohexyl analogues4a-c do not show the cytotoxicity. This dramatic loss of activity of these analogues on the volume change with the bulkier cyclohexyl group indicates that the planar structure at 4-position of 1-arylsulfonyl-4-phenyl-4,5-dihydro-2-limidazolones3 is required for their activity as an important pharmacophoric moiety.