Expression of pulmonary VEGF family declines with age and is further down-regulated in lipopolysaccharide (LPS)-induced lung injury

Vascular endothelial growth factor (VEGF) is a survival factor in endothelial cells and a promoter of angiogenesis that reportedly plays a pivotal role protecting against injury. In aged humans and animals, lung injuries are generally more serious and cause higher mortality. We thus hypothesized that the expression of VEGF and its related molecules in the lung declines with age. In this study, we first examined the expression of VEGF family (VEGF-A, -B, -C and -D), VEGF-A isoforms (VEGF120, 164, 188), and VEGF-specific receptors (VEGFR-1: Flt-1; VEGFR-2: Flk-1 and VEGFR-3: Flt-4) by quantitative RT-PCR in lungs from young and old mice. Expression of all these except for VEGF-D was significantly lower in old mice than in young mice. We then subjected young and old mice to lipopolysaccaride (LPS)-induced lung injury. Old animals demonstrated poor survival and prolonged lung inflammation when compared with young counterparts. At 24 and 72 h after intratracheal LPS administration, expression of the examined factors was down-regulated in the lungs irrespective of age. In conclusion, pulmonary expression of the VEGF family and their receptors declines with age, and is further down-regulated in LPS-induced lung injury, although the mechanism of age- and/or injury-related down-regulation of VEGF remains unknown.     

Nonmammalian gonadotropin-releasing hormone molecules in the brain of promoter transgenic rats

Mammalian gonadotropin-releasing hormone (GnRH1) and nonmammalian immunoreactive GnRH subtypes were examined in transgenic rats carrying an enhanced GFP (EGFP) reporter gene driven by a rat GnRH1 promoter. Double-label immunocytochemistry was performed on EGFP(+)/GnRH1 brain sections by using antisera against GnRH1, GnRH2 (chicken II), GnRH3 (salmon), or seabream GnRH. EGFP(+)/GnRH1 neurons were in the septal-preoptic hypothalamus but not in the midbrain, consistent with GnRH1-immunopositive neurons in WT rats. Apparent coexpression of EGFP(+)/GnRH1 with other GnRH subtypes was observed. All EGFP(+) neurons in the septal-preoptic hypothalamus were GnRH1-immunopositive. However, only approximately 80% of GnRH1-immunopositive neurons were EGFP(+), which awaits further elucidation. GnRH subtypes-immunopositive fibers and EGFP(+)/GnRH1 fibers were conspicuous in the organum vasculosum of the lamina terminalis, median eminence, and surrounding the ependymal walls of the third ventricle and the aqueduct in the midbrain. These results demonstrate that the expression of the EGFP-GnRH1 transgene is restricted to the bona fide GnRH1 population and provide clear morphological evidence supporting the existence of GnRH1 neuronal subpopulations in the septal-preoptic hypothalamus, which might be driven by different segments of the GnRH promoter. This genetic construct permits analyses of promoter usage in GnRH neurons, and our histochemical approaches open questions about functional relations among isoforms of this peptide, which regulates reproductive physiology in its behavioral and endocrine aspects.     

Comparative genomic hybridization analysis for pancreatic cancer specimens obtained by endoscopic ultrasonography-guided fine-needle aspiration

Background Comparative genomic hybridization (CGH) analysis of pancreatic cancer has been done exclusively for surgical and autopsy specimens, because of the difficulty of tissue sampling without surgery. To overcome this difficulty, we applied CGH technology to cells obtained by endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA).Methods In the present study, we performed EUS-FNA for 17 patients with pancreatic cancer before surgery. Tumor cells were selected by microdissection. DNA was extracted from the cells and amplified by degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR). Then CGH was carried out.Results In the 15 patients with tubular adenocarcinoma, the most common loci of gains (including amplification) were 5p, 8q, and 20q (60% of the patients); and 1q, 7p, and 12p (27%). The most frequent losses were 17p (73%); 9p, 18q, and 19p (47%); and 8p (33%). These findings were similar to our previously reported data. Both of the patients with acinar cell carcinoma showed gains of 2q and 5p, and losses of 1p, 9p, 9q, 11p, 11q, 14q, 17p, 17q, and 18q.Conclusions The results of this study suggest that comprehensive genetic analysis is possible for EUS-FNA biopsy specimens, with a combination of microdissection and DOP-PCR. This analytical strategy will enable us to evaluate the biological characteristics of pancreatic cancer before treatment.     

Antibacterial Effect of Amino Acid–Silver Complex Loaded Montmorillonite Incorporated in Dental Acrylic Resin

Several dental materials contain silver for antibacterial effect, however the effect is relatively low. The reason for the lower antibacterial efficacy of silver is considered to be the fact that silver ions bind to chloride ions in saliva. To develop new effective silver antibacterial agents that can be useful in the mouth, we synthesized two novel amino acid (methionine or histidine)–silver complexes (Met or His–Ag) loaded with montmorillonite (Mont) and analyzed their antibacterial efficacy. At first the complexes were characterized using nuclear magnetic resonance (NMR), and amino acid–Ag complex-loaded Mont (amino acid–Ag–Mont) were characterized using X-ray diffraction (XRD) and scanning electron microscopy (SEM). The antibacterial efficacy of these materials in dental acrylic resin was then investigated by bacterial growth measurement using a spectrophotometer. As controls, commercially available silver-loaded zeolite and silver-zirconium phosphate were also tested. Dental acrylic resin incorporating His–Ag–Mont strongly inhibited Streptococcus mutans growth. This was explained by the fact that His-Ag complex revealed the highest amounts of silver ions in the presence of chloride. The structure of the amino acid–Ag complexes affected the silver ion presence in chloride and the antibacterial efficacy. His–Ag–Mont might be used as antibacterial agents for dental materials.     

Maintenance of serum immunoglobulin G antibodies to Epstein-Barr virus (EBV) nuclear antigen 2 in healthy individuals from different age groups in a Japanese population with a high childhood incidence of asymptomatic primary EBV infection

Immunoglobulin G (IgG) antibodies to Epstein-Barr virus (EBV) nuclear antigens 2 and 1 (EBNA-2 and EBNA-1, respectively) were studied using sera from healthy individuals of a population with a high incidence of asymptomatic primary EBV infections during infancy or childhood in Japan. Two CHO-K1 cell lines expressing EBNA-2 and EBNA-1 were used for anticomplement and indirect immunofluorescence assays. The positivity rate for EBNA-2 IgG rose in the 1- to 2-year age group, increased and remained at a plateau ( approximately 45%) between 3 and 29 years of age (3- to 4-, 5- to 9-, 10- to 14-, and 15- to 29-year age groups), and then reached 98% by age 40 (>/== 40-year age group). Both seropositivity for EBNA-1 and seropositivity for EBNAs in Raji cells (EBNA/Raji) were detected in the 1- to 2-year age group, remained high, and finally reached 100% by age 40. The geometric mean titer (GMT) of EBNA-2 IgG reached a plateau in the 5- to 9- and 10- to 14-year-old groups and remained elevated in the older age groups (15 to 29 and >/== 40 years). The GMT of EBNA-1 IgGs increased to a plateau in the 1- to 2-year-old group and remained unchanged in the older age groups. The GMT of EBNA/Raji IgGs also reached a plateau in the 1- to 2-year-old group, remained level throughout the 3- to 14-year age groups, and decreased in the 15- to 29-year-olds. EBNA-2 IgGs emerged earlier than EBNA-1 IgGs in 8 of 10 patients with infectious mononucleosis, who were between 1 and 27 years old, and declined with time in three of eight cases. These results suggest that EBNA-2 IgG antibodies evoked in young children by asymptomatic primary EBV infections remain elevated throughout life, probably because of reactivation of latent and/or exogenous EBV superinfection.     

Molecular mechanism underlying activation of superoxide-producing NADPH oxidases: roles for their regulatory proteins

The phagocyte NADPH oxidase is dormant in resting cells but becomes activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants, thereby playing a crucial role in host defence. The catalytic core of this enzyme comprises the two membranous subunits gp91phox/Nox2 and p22phox. The oxidase activation requires the small GTPase Rac and the SH3 domain-containing proteins p47phox and p67phox; they normally exist in the cytoplasm and translocate upon cell stimulation to the membrane. The translocation depends on a stimulus-induced conformational change of p47phox, which leads to the SH3 domain-mediated interaction with p22phox, a binding required for the gp91phox/Nox2-dependent superoxide production. Activation of Nox1, an oxidase that is likely involved in host defence at the colon, requires novel proteins homologous to p47phox and p67phox, designated Noxo1 and Noxa1, respectively. Noxo1 and Noxa1, both expressed abundantly in the colon, are capable of constitutively activating Nox1. The constitutive activation may be due to the property of Noxo1: in contrast with p47phox, Noxo1 seems to normally associate with p22phox, which is required for the Nox1 activation. We will also describe the mechanism underlying regulation of the third oxidase Nox3, which exits in fetal kidney and inner ears.     

Immunosuppressive dose of azathioprine inhibits replication of human cytomegalovirus in vitro

Summary Azathioprine (Aza) was found to have anti-human cytomegalovirus (HCMV) activity in vitro at concentrations used for immunosuppression therapy. The dose of Aza for 50% plaque reduction was 0.592µg/ml for HCMV in human embryonic lung (HEL) cells, but those of Aza for 50% plaque reduction for herpes simplex virus (HSV) and varicella-zoster virus were more than 20µg/ml. The dose of Aza for 50% reduction of the HCMV yield in infected cells was 0.25µg/ml, while that for 50% reduction of the HSV yield in infected cells was more than 50µg/ml. The dose of Aza for 50% growth inhibition of HEL cells was 30µg/ml, and 50.7 and 120 times greater than the doses for 50% reduction of the plaque formation and the yield of HCMV, respectively. Thus Aza was found to have a strong anti-HCMV activity at concentrations used for immunosuppression. When HCMV infected cells were treated with cyclosporine (CsA: 0.2µg/ml) and prednisolone (Pred: 0.3µg/ml) simultaneously with Aza, the doses of Aza for 50% reduction of plaque formation and the yield of HCMV were 0.73 and 0.32µg/ml, respectively. Thus an inhibitory effect of Aza was also observed in HCMV-infected cells treated with CsA and Pred at their concentrations used for immunosuppression. Maintenance of an anti-HCMV dose of Aza in combination with CsA and Pred might establish not only satisfactory immunosuppression but also suppression of HCMV infection in transplant recipients.