Adenovirus-mediated REIC/Dkk-3 gene transfer inhibits tumor growth and metastasis in an orthotopic prostate cancer model.

We had previously reported that REIC/Dkk-3, a member of the Dickkopf (Dkk) gene family, works as a tumor suppressor. In this study, we evaluated the therapeutic effects of an intratumoral injection with adenoviral vector encoding REIC/Dkk-3 gene (Ad-REIC) using an orthotopic mouse prostate cancer model of RM-9 cells. We also investigated the in vivo anti-metastatic effect and in vitro anti-invasion effect of Ad-REIC gene delivery. We demonstrated that the Ad-REIC treatment inhibited prostate cancer growth and lymph node metastasis, and prolonged mice survival in the model. These therapeutic responses were consistent with the intratumoral apoptosis induction and in vitro suppression of cell invasion/migration with reduced matrix metalloprotease-2 activity. We thus concluded that in situ Ad-REIC/Dkk-3 gene transfer may be a promising therapeutic intervention modality for the treatment of prostate cancer.

Hornerin, a novel profilaggrin-like protein and differentiation-specific marker isolated from mouse skin.

A novel mouse cDNA named hornerin was isolated by RNA differential display applied to developing mouse skin. Hornerin, which has 2,496 amino acids, comprises EF-hand domains at the N terminus followed by a spacer sequence and a large repetitive domain, indicating that hornerin is a novel member of the "fused gene"-type cornified envelope precursor protein family. The repetitive domain of hornerin was found to be rich in glycine, serine, and glutamine. Hornerin was expressed in the tongue, esophagus, forestomach, and skin among the adult mouse tissues examined, all of them cornifying stratified epithelium. In the embryonic mouse skin, hornerin mRNA was first detected on gestational day 15.5 in the epidermis coincidentally with the formation of a granular layer. In accordance with this, hornerin was detected in the granular and cornified layers of the mature epidermis. In the granular cells of the epidermis, the hornerin protein was detected in keratohyalin granules together with profilaggrin. Furthermore, Western blot analysis of the mouse skin showed that the hornerin protein was cleaved during the process of epidermal differentiation, indicating possible posttranslational proteolytic processing as is observed in profilaggrin. Differentiation of primary mouse epidermal keratinocytes with 0.12 mm Ca(2+) resulted in the induction of hornerin. These results indicate that hornerin is structurally as well as functionally most similar to profilaggrin among the family members and possibly plays pleiotropic roles, including a role in cornification.

PKCtheta II, a new isoform of protein kinase C specifically expressed in the seminiferous tubules of mouse testis.

Protein kinase C (PKC) theta, a Ca(2+)-independent isoform of PKC, has been known to be expressed in skeletal muscle and T cells. In the present study, we isolated and characterized a smaller transcript expressed in the mouse testis, the cDNA of which is referred hereafter as PKCthetaII and the original PKCtheta as PKCthetaI. The cDNA clone of PKCthetaII has 2184 base pairs and 464 amino acids in the possible open reading frame, consisting of the 5' unique sequence of 20 amino acids and the PKCthetaI sequence of 444 amino acids. Genomic DNA analysis revealed that transcription of PKCthetaII is initiated from the PKCthetaII-specific exon, which is located between exons 7 and 8 of the PKCtheta gene, indicating that alternative splicing is the mechanism by which PKCthetaII is generated. PKCthetaII is expressed exclusively in the testis in an age-dependent manner with sexual maturation. In situ hybridization and reverse transcription-polymerase chain reaction of microdissected tissues clearly demonstrated that PKCthetaII is expressed in the seminiferous tubules of the mouse testis. Consistent with its molecular structure lacking the C1 regulatory domain, PKCthetaII is constitutively active as determined by an in vitro kinase assay, being independent of PKC activators, e.g. phosphatidylserine and phorbol ester. PKCthetaII may play a crucial role in spermatogenesis or some related function of the testis.

Molecular cloning and sequencing of myosin light chains in human megakaryoblastic leukemia cells.

Myosin light chain genes of hematopoietic cells have yet to be characterized. We cloned the full-length cDNAs of 20 kDa regulatory myosin light chain (MLC-2) and 17 kDa essential myosin light chain (MLC-3) from Meg-01, a human megakaryoblastic leukemia cell line. Both MLC-2 and MLC-3 gene are transcribed ubiquitously in various hematopoietic cells. The MLC-2 open reading frame of 516 nucleotides encoding a protein of 172 residues was detected in cloned cDNA of 967 nucleotides. The Ca2+-binding domain and five phosphorylation sites were highly conserved. The deduced amino acid sequence has a 99.4% and 100% homology with that of human fetus brain and human lymphocyte, respectively. The MLC-3 open reading frame of 453 nucleotides encoding a protein of 151 residues was detected in cloned cDNA of 742 nucleotide. The MLC-3 protein is 99.3% identical to that of human fibroblasts. These results suggest that hematopoietic myosin light chain proteins are similar to those of other nonmuscle cells and smooth muscle, thus differing from skeletal and cardiac muscles.

Calmin, a protein with calponin homology and transmembrane domains expressed in maturing spermatogenic cells.

A cDNA named calmin of approximately 3.2 kb was isolated by RNA differential display applied to developing mouse skin. Calmin cDNA encodes 1021 amino acids with two calponin homology (CH) domains in tandem on the N-terminal side and a transmembrane domain on the C-terminal side. The region covering the CH domains showed a high level of homology with beta-spectrin, alpha-actinin, and dystrophin. Among the proteins with the tandem CH domains, calmin is unique in having a transmembrane domain. Three alternative splicing sites were identified at the 3'-side of calmin, giving rise to polymorphic protein products with or without the transmembrane domain. The calmin transcript was detected in adult testis, liver, kidney, and large intestine; the expression in testis was far stronger than that in the other tissues. In situ hybridization and immunostaining revealed that calmin was expressed in maturing spermatogenic cells at later stages. Human calmin cDNA was also isolated, and its exon/intron organization was determined.

Long-term prognosis of BSSO mandibular relapse and its relation to different facial types.

The bilateral sagittal split osteotomy (BSSO) has evolved into an effective and preferred surgical procedure for mandibular setbacks. As with all surgical procedures designed to setback the mandible, relapse occasionally occurs after BSSO procedures. Several factors have been suggested to play a contributory role in this relapse. The present study was performed to determine the stability of the mandibular position over the course of long-term observation. Different facial patterns that could potentially be used as predictors of relapse were examined. The study included cases of skeletal mandibular prognathism, with the patient in each case having undergone surgical correction involving a BSSO at least 5 years prior to the study. Lateral cephalograms were analyzed in order to classify facial patterns. Angular and linear cephalometric measurements, consisting of SN-Pogonion angle, SN-occlusal plane angle, and Pogonion depth and height, were compared at 1 year postoperatively and at the long-term follow-up. A significant correlation between facial type and relapse pattern was confirmed at the long-term assessment of prognosis.

A tetratricopeptide repeat-containing protein gene, tpis, whose expression is induced with differentiation of spermatogenic cells.

The tetratricopeptide repeat (TPR) is a degenerate 34-amino-acid sequence which forms scaffolds to mediate protein-protein interactions. We have isolated a cDNA named tpis from mouse embryonic skin and found that the deduced 529-amino-acid sequence contained 5 TPRs. In addition to skin, the transcript of tpis was detected in tissues with stratified squamous epithelium, e.g., tongue, esophagus, and forestomach. tpis was most strongly expressed in testis among adult tissues examined. The transcript of tpis from testis was longer, encoding 372 additional amino acid residues at the 5'-side with 3 more TPRs. In situ hybridization revealed specific expression of tpis at a distinct differentiation stage of spermatogenic cells, indicating involvement of tpis in spermatogenesis. Chromosomal localization of the tpis gene was determined as 18.10 cM of chromosome 15.

A longitudinal study of the physical growth of Japanese infants.

In order to clarify the characteristics of the physical growth of Japanese infants up to 1 year of age, the data of 3183 infants from 53 hospitals in Japan were collected. Percentile values were calculated from the measurement of body weight, recumbent length, and head circumference, with some additional data interpolated to fill the gaps in the measurements. Compared to the National Center for Health Statistics (NCHS) standard, the recumbent length and head circumference of these infants were smaller throughout the observation period, while body weight was larger at 0-6 months of age, and smaller at 8-12 months of age. The cases were divided into five groups according to birth weight, to examine the influence of foetal growth. The 50th percentile curves of each group were parallel and the intervals between the 3rd and 97th percentile curves in each group became wider in the first 6 months of life, up to the interval of 2.20-3.18 Kg. There was no significant difference in body weight among three groups according to feeding methods, in any age period.

Isolation and characterization of a putative keratin-associated protein gene expressed in embryonic skin of mice.

Embryonic mouse skin undergoes substantial morphologic changes from 13 days post-coitus (dpc) to 16 dpc, i.e., from simple layers of epithelial cells and periderm at 13.5 dpc to almost fully differentiated stratified epithelium with the rudiments of hair follicles at 16.5 dpc. Using RNA differential display, we isolated a gene involved in the development of mouse epidermis. This gene, tentatively designated as 4C32, encodes 197 amino acids containing six direct repeats of 10 amino acids with the CQ motif. The repetitive structure with the CQ motif is seen in most keratin-associated protein families, which are known to be specifically expressed in hair follicles. 4C32 is expressed in the outermost layer of the embryonic epidermis at 15.5 and 16.5 dpc, and abruptly disappears at 17.5 dpc, suggesting that 4C32 is expressed in the periderm. The periderm is a superficial layer of embryonic epidermis, and is known to disappear at 17 dpc in mouse embryos. The 4C32 transcripts were also detected in the developing and matured tongue tissues and in the tail scale, but not at any stage in hair follicles.

Oxidative stress on mitochondria and cell membrane of cultured rat hepatocytes and perfused liver exposed to ethanol.

BACKGROUND & AIMS: The precise pathogenic significance of oxidative injury in the evolution of alcohol-induced liver disease is still obscure. The present report was designed to investigate whether ethanol alters the production of active oxidants and biological activities of hepatocytes. METHODS: The following parameters in rat hepatocytes were investigated by using fluorescence probes in vitro and ex vivo: (1) mitochondrial membrane potential and membrane permeability transition, (2) oxygen radicals generation, (3) membrane barrier function, and (4) glutathione level. RESULTS: Ethanol (50 mmol/L) increased oxidative stress in hepatocytes and subsequently induced an increased mitochondrial permeability transition and a decreased membrane potential. These ethanol-induced alterations were attenuated by an inhibitor of alcohol dehydrogenase and an intracellular oxidant scavenger, whereas they were enhanced by diethyl maleic acid, a glutathione depletor. Ethanol plus diethyl maleic acid but not ethanol alone increased the number of hepatocytes with membrane barrier dysfunction. A continuous infusion of ethanol (50 mmol/L) increased oxidative stress and decreased mitochondrial membrane potential in the pericentral area of isolated perfused rat liver. CONCLUSIONS: Active oxidants generated during ethanol metabolism increase mitochondrial permeability transition and modulate mitochondrial energy synthesis in hepatocytes. Reduction of glutathione level enhances mitochondrial dysfunction and impairs membrane barrier function of hepatocytes.

CD18/ICAM-1-dependent oxidative NF-kappaB activation leading to nitric oxide production in rat Kupffer cells cocultured with syngeneic hepatoma cells.

Previous studies have indicated that nitric oxide (NO) released from Kupffer cells modulates biological viability of cocultured hepatoma cells. This study was designed to evaluate the mechanisms by which Kupffer cells synthesize and release NO in reponse to cocultured hepatoma cells. Kupffer cells isolated from male Wistar rats were cocultured with rat hepatoma cell line, AH70 cells. The sum of nitrite and nitrate levels increased in the culture medium of Kupffer cells with AH70 cells as compared with those of Kupffer cells or AH70 cells alone. Increased expressions of iNOS and iNOS mRNA in Kupffer cells cocultured with AH70 cells were detected by an immunofluorescence staining and a fluorescence in situ hybridization study, respectively. A fluorescence in situ DNA-protein binding assay revealed that NF-kappaB activation occurs in Kupffer cells and activated NF-kappaB moved into the nuclei preceding to an increased production of NO. Oxidative stress indicated by dichlorofluorescein fluorescence was observed in Kupffer cells cocultured with AH70 cells. An increased calcium mobilization indicated as increased fluo-3-associated fluorescence was also induced in Kupffer cells after coculture with AH70 cells. Monoclonal antibodies directed against rat CD18 and ICAM-1, as well as TMB-8, a calcium inhibitor, prevented the calcium mobilization, active oxygen production, and NF-kappaB activation in addition to the increased production of NO. Pyrrolidine dithiocarbamate, an inhibitor of oxidative NF-kappaB activation, diphenylene iodonium, an NADPH oxidase inhibitor, and quinacrine, a phospholipase A2 inhibitor, significantly attenuated the increase in dichlorofluorescein fluorescence, NF-kappaB activation, and NO production. Therefore, this study suggests that CD18/ICAM-1-dependent cell-to-cell interaction with hepatoma cells causes calcium mobilization and oxidative activation of NF-kappaB, which may lead to the increased production of NO in Kupffer cells.

Oxidative stress-mediated apoptosis of hepatocytes exposed to acute ethanol intoxication.

The present study was designed to investigate whether acute ethanol intoxication increases the production of active oxidants, and subsequently promotes apoptosis of hepatocytes. Hepatocytes were isolated from male Wistar rats, and cultured in the presence or absence of ethanol. The fluorescence in situ nick end labeling method and an enzyme-linked immunosorbent assay (ELISA) system to quantify fragmented DNA were used to estimate apoptotic change in hepatocytes. Nuclear morphological alterations and membrane barrier dysfunction of hepatocytes were assessed by staining with Hoechst 33342 and propidium iodide (PI). Intracellular glutathione level was determined as the fluorescence of monochlorobimane (MCLB), which forms conjugate with glutathione to become fluorescent. Ethanol (100 mmol/L) increased the amount of fragmented DNA and the number of apoptotic hepatocytes in vivo as well as in vitro. These ethanol-induced alterations in hepatocytes were attenuated by simultaneous incubation with either 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, or dimethylthiourea, an intracellular oxidant scavenger. Diethyl maleic acid (DMA), a glutathione depletor, enhanced the induction of apoptotic change, and decreased membrane barrier function in ethanol-treated hepatocytes, whereas ethanol per se did not increase the number of PI-positive hepatocytes. Furthermore, combination of ethanol and DMA but not ethanol alone decreased the hepatocyte MCLB fluorescence. Taken together, the present study suggests that active oxidants produced during ethanol metabolism mediate fragmentation of DNA in hepatocytes, and that intracellular antioxidants such as glutathione play a critical role in the cytoprotective mechanisms of hepatocyte against lethal cell death, ie, apoptosis, induced by ethanol.

CD18/ICAM-1-dependent nitric oxide production of Kupffer cells as a cause of mitochondrial dysfunction in hepatoma cells: influence of chronic alcohol feeding.

The present study was designed to monitor the process for hepatoma cell injury induced by Kupffer cells. The non-activated Kupffer cells isolated from male Wistar rats reduced the mitochondrial membrane potential in the cocultured AH70 cells, which was indicated by the decreased rhodamine 123 (Rh123) fluorescence. Increased level of nitrite and nitrate in the medium and induction of iNOS in Kupffer cells were observed after coculture with AH70 cells. Incubation with either NG-monomethyl-L-arginine or aminoguanidine attenuated the increased nitric oxide (NO) production of Kupffer cells and the decreased Rh123 fluorescence of AH70 cells. Fluo-3, a calcium-sensitive probe, fluorescence in Kupffer cells increased after coculture with AH70 cells. Addition of TMB-8, a calcium inhibitor, or monoclonal antibody directed against ICAM-1 or CD18 prevented the increases in fluo-3 fluorescence and NO production of Kupffer cells and Kupffer cell-induced mitochondrial dysfunction in AH70 cells, suggesting the involvement of calcium mobilization and CD18/ICAM-1. It is therefore suggested that the Kupffer cell-mediated mitochondrial dysfunction of hepatoma cells largely depends on NO production by iNOS, and that the NO production by Kupffer cells is triggered by CD18/ICAM-1-dependent interaction with hepatoma cells and subsequent calcium mobilization. In other series of experiments, male Wistar rats fed ethanol for 4 weeks were used. The NO production and calcium mobilization of Kupffer cells and reduction of the mitochondrial membrane potential in cocultured hepatoma cells were diminished in the case of Kupffer cells isolated from chronically ethanol-fed rats, while CD18 and ICAM-1 expression was still observed. Thus, the present study further suggests that NO-dependent anti-hepatoma cell activity of Kupffer cells is suppressed in chronically ethanol-fed animals.

Ethanol-induced leukocyte adherence and albumin leakage in rat mesenteric venules: role of CD18/intercellular adhesion molecule-1.

The adherence and emigration of leukocytes have been implicated as a rate-limiting step in the microvascular disturbance in a variety of pathogenic events. The objective of the present study was to investigate leukocyte-endothelial cell adhesion and endothelial barrier function in rat mesenteric microvessels exposed to ethanol, which is known to cause inflammation and injury in various organs. Mesentery of male Wistar rats was used for intravital microscopic observations. Leukocyte adherence and albumin leakage were monitored in single postcapillary venules using the intravital fluorescence microscope. Superfusion of 50 mM ethanol elicited the leukocyte adherence and albumin leakage within 60 min. Pretreatment with a monoclonal antibody directed against either CD18 or intercellular adhesion molecule-1 (ICAM-1) significantly prevented the ethanol-induced increase in leukocyte adherence and decrease in barrier function of endothelium. These results suggest that ethanol-induced leukocyte adherence is mediated by CD18 on leukocytes and ICAM-1 on endothelial cells. The present study further supports that CD18/ ICAM-1-dependent leukocyte-endothelial adhesive interactions lead to macromolecular leakage in the postcapillary venules exposed to ethanol.

Increased nitric oxide synthase activity as a cause of mitochondrial dysfunction in rat hepatocytes: roles for tumor necrosis factor alpha.

Kupffer cells have been implicated in playing an important role in the pathogenesis of endotoxemia-associated liver injury. The present study was designed to investigate whether Kupffer cell-derived mediators alter the mitochondrial oxidative phosphorylation of hepatocytes in the endotoxemic condition. Liver cells were isolated from male Wistar rats. Oxidative phosphorylation was monitored as the fluorescence of rhodamine 123 (Rh123), which is the fluorescent cationic dye used to indicate mitochondrial energy synthesis. Two hours after coculture of hepatocytes with lipopolysaccharide (LPS)-pretreated Kupffer cells, a marked decrease in hepatocyte rhodamine 123 fluorescence was observed. The hepatocyte mitochondrial dysfunction was attenuated by the addition of either N(G)-monomethyl-L-arginine (L-NMMA), an inhibitor of nitric oxide (NO) synthesis, or aminoguanidine, an inducible-type of NO synthase inhibitor, to the culture medium of cocultures, to the pretreatment of LPS-activated Kupffer cells with antisense oligodeoxynucleotides against iNOS messenger RNA (mRNA), or to tumor necrosis factor alpha (TNF-alpha) mRNA. Four hours after the coculture, hepatocyte Rh123 fluorescence further decreased, and an iNOS induction as well as an increased NO production were observed in hepatocytes that were cocultured with LPS-pretreated Kupffer cells. The membrane barrier dysfunction of hepatocytes, indicated by propidium iodide staining, was also induced by a 4-hour coculture with LPS-pretreated Kupffer cells. These late-phase changes were inhibited either by the pretreatment of hepatocytes with antisense oligodeoxynucleotides against iNOS mRNA or by treatments that are effective in the early phase (within 2 hours). Incubation with recombinant rat TNF-alpha decreased hepatocyte Rh123 fluorescence within 2 hours. Thus, the present study suggests that NO and TNF-alpha released from LPS-pretreated Kupffer cells directly inhibit the hepatocyte mitochondrial function in the early phase, and then NO synthesized by TNF-alpha-induced hepatocyte iNOS causes lethal hepatocyte injury, characterized by diminished mitochondrial energization and membrane barrier function in the late phase.

Rat Kupffer cell-derived nitric oxide suppresses proliferation and induces apoptosis of syngeneic hepatoma cells.

BACKGROUND & AIMS: Evidence increasingly indicates that nitric oxide plays an important role in antitumor mechanisms. The aim of this study was to investigate the role of NO in the mechanisms regulating the proliferation and death of hepatoma cells cocultured with Kupffer cells. METHODS: Kupffer cells were isolated from male Wistar rats and cocultured with rat hepatoma AH70 cells. Proliferation was determined by calculating the number of total and 5-bromodeoxyuridine-positive AH70 cells. Apoptosis was assessed by electron-microscopic and fluorescence-microscopic observations and in situ nick end labeling method. Immunofluorescence and in situ hybridization studies were performed to investigate the induction of inducible NO synthase (iNOS). RESULTS: Kupffer cells reduced proliferation and induced apoptosis of AH70 cells, which were attenuated by the NO synthesis inhibitors NG-monomethyl-L-arginine and aminoguanidine. Increased inductions of iNOS messenger RNA and iNOS were observed in Kupffer cells cocultured with AH70 cells. Addition of monoclonal antibody directed against either rat CD18 or intercellular adhesion molecule 1 also attenuated the increased NO production of Kupffer cells and the alterations of AH70 cells. CONCLUSIONS: Kupffer cell-derived NO suppresses proliferation and induces apoptosis of hepatoma cells. The CD18 intercellular adhesion molecule 1-dependent adhesive interaction with hepatoma cells triggers NO production by Kupffer cells.

Clinical usefulness of continuous administration of Nocardia rubra cell wall skeleton (N-CWS) in diffuse panbronchiolitis (DPB).

Eight patients with diffuse panbronchiolitis (DPB) who had repeated intractable airway infections were continuously treated with Nocardia rubra cell wall skeleton (N-CWS), a biological response modifier. As a result, subjective symptoms were reduced in 6 patients. Antibiotics therapy could be discontinued completely in two patients and the dose of antibiotics could be reduced considerably in two other patients. No adverse reactions in relation to N-CWS were observed. These results suggest that N-CWS is effective in treating erythromycin-resistant DPB.

Prediction of smoking behavior in Japanese young adults.

Eighty-eight second grade students of a senior high school in Saitama prefecture in Japan participated in a prospective study to predict cigarette smoking behavior 3.5 years later. Predictor variables include sex, knowledge, beliefs and attitudes toward smoking, previous smoking behavior, and smoking behavior of their families. Stepwise discriminant analyses revealed that 90% of the smokers and 65% of the non-smokers were correctly classified. In this model, previous smoking behavior proved to be the best predictor. Attitude toward adult male's smoking, sex and smoking behavior of subjects' families were also related. These four variables explained 35% of the variance in smoking behavior. As for stepwise discriminant analyses among those who had not smoked at baseline, 78% of the smokers and 76% of the non-smokers were correctly classified. Attitude toward adult male's smoking, sex, knowledge about long-term effects of cigarette smoking and smoking behavior of their families entered the model in this order. These four variables explained 37% of the variance. Implications of this study for smoking prevention programs in Japan are discussed.