Patency of the preterm fetal ductus arteriosus is regulated by endothelial nitric oxide synthase and is independent of vasa vasorum in the mouse

Patency of the fetal ductus arteriosus (DA) is maintained in an environment of low relative oxygen tension and a preponderance of vasodilating forces. In addition to prostaglandins, nitric oxide (NO), a potent vasodilator in the pulmonary and systemic vasculatures, has been implicated in regulation of the fetal DA. To further define the contribution of NO to DA patency, the expression and function of NO synthase (NOS) isoforms were examined in the mouse DA on days 17-19 of pregnancy and after birth. Our results show that endothelial NOS (eNOS) is the predominant isoform expressed in the mouse DA and is localized in the DA endothelium by in situ hybridization. Despite rapid constriction of the DA after birth, eNOS expression levels were unchanged throughout the fetal and postnatal period. Pharmacological inhibition of prostaglandin vs. NO synthesis in vivo showed that the preterm fetal DA on day 16 is more sensitive to NOS inhibition than the mature fetal DA on day 19, whereas prostaglandin inhibition results in marked DA constriction on day 19 but minimal effects on the day 16 DA. Combined prostaglandin and NO inhibition caused additional DA constriction on day 16. The contribution of vasa vasorum to DA regulation was also examined. Immunoreactive platelet endothelial cell adhesion molecule and lacZ tagged FLK1 localized to DA endothelial cells but revealed the absence of vasa vasorum within the DA wall. Similarly, there was no evidence of vasa vasorum by vascular casting. These studies indicate that eNOS is the primary source of NO in the mouse DA and that vasomotor tone of the preterm fetal mouse DA is regulated by eNOS-derived NO and is potentiated by prostaglandins. In contrast to other species, mechanisms for DA patency and closure appear to be independent of any contribution of the vasa vasorum.     

Identification of stathmin as a novel marker of cell proliferation in the recovery phase of acute ischemic renal failure

Ischemic renal injury can be classified into the initiation and extension phase followed by the recovery phase. The recovery phase is characterized by increased dedifferentiated and mitotic cells in the damaged tubules. Suppression subtractive hybridization was performed by using RNA from normal and ischemic kidneys to identify the genes involved in the physiological response to ischemia-reperfusion injury (IRI). The expression of stathmin mRNA increased by fourfold at 24 h of reperfusion. The stathmin mRNA did not increase in sodium-depleted animals or in animals with active, persistent injury secondary to cis-platinum. Immunofluorescent labeling demonstrated that the expression of stathmin increased dramatically at 48 h of reperfusion. Labeling with antibodies to stathmin and proliferating cell nuclear antigen (PCNA) indicates that the expression of stathmin was induced before the upregulation of PCNA and that all PCNA-positive cells expressed stathmin. Double immunofluorescent labeling demonstrated the colocalization of stathmin with vimentin, a marker of dedifferentiated cells. Stathmin expression was also significantly enhanced in acute tubular necrosis in humans. On the basis of its induction profile in IRI, the data indicating its enhanced expression in proliferating cells and regenerating organs, we propose that stathmin is a marker of dedifferentiated, mitotically active epithelial cells that may contribute to tubular regeneration and could prove useful in distinguishing the injury phase from recovery phase in IRI.     

Comparison of real-time and nested PCR assays for detection of herpes simplex virus DNA

We performed a real-time PCR assay to detect herpes simplex virus (HSV) DNA, and compared it prospectively with a nested PCR assay in 164 clinical samples (109 cerebrospinal fluid and 55 sera) from patients suspected of having neonatal HSV infection or HSV encephalitis. In 25 of 164 samples, HSV DNA was detected by the nested PCR assay. All samples positive for HSV DNA in the nested PCR assay were also positive in the real-time PCR assay, and all but two samples negative for HSV DNA in the nested assay were negative in the real-time assay. The real-time PCR assay thus had a sensitivity of 100% and a specificity of 99%, when compared with the nested assay. Sequential assays in a case of disseminated HSV showed that a decrease in HSV DNA paralleled clinical improvement. Quantification of HSV DNA by real-time PCR was useful for diagnosing and monitoring patients with HSV encephalitis and neonatal HSV infection.     

Genetic and geographical differentiation of <Emphasis Type="Italic">Pandaka</Emphasis> gobies in Japan

The mitochondrial DNA (mtDNA) phylogeny of Japanese Pandaka species (Perciformes: Gobiidae) was inferred from partial nucleotide sequences of the mitochondrial 12S and 16S rRNA genes (1083bp). The resultant mtDNA tree showed two major clades (clade I and clade II), which were inconsistent with the present taxonomic classification. One of the major clades was further divided into two geographical groups, distributed on the Japanese Major Islands (clade I-A) and from Amami-oshima Island to Iriomote Island (clade I-B). The mtDNA haplotypes in clade II were found only on Iriomote Island. The mtDNA divergences in clade I indicated that the Japanese Major Island (clade I-A) and Ryukyu (clade I-B) groups have been geographically isolated from each other for millions of years, based on the putative molecular divergence rate. The geographical distributions of mtDNA haplotypes in clade I-A and clade I-B also suggested that Pandaka gobies had not dispersed to distant offshore islands, indicating that their geographical differentiation may be closely associated with the geological history of the Japanese and Ryukyu Archipelagos.This revised version was published online in January 2005 with corrections to the repetition of the 1st authors name.     

Mitochondrial genomes and phylogeny of the ocean sunfishes (Tetraodontiformes: Molidae)

We determined the complete nucleotide sequences of the mitochondrial genomes for the three currently recognized species of ocean sunfish: Mola mola, Masturus lanceolatus, and Ranzania laevis (Tetraodontiformes: Molidae). Each genome contained the 37 genes as found in teleosts, with the typical gene order in teleosts. Bayesian, maximum-likelihood, and maximum-parsimony analyses were conducted with the data set comprising concatenated nucleotide sequences from 36 genes (excluding the ND6 gene) of three molids and four outgroups (three tetraodontiforms plus a caproid). The resultant trees supported monophyly of the Molidae and its intrarelationships ((Mola, Masturus), Ranzania), which were congruent with previous morphology-based hypotheses.     

Stereo- and biochemical profiles of the 5-6- and 6-6-junction isomers of alpha-D-mannopyranosyl [60]fullerenes

The 5-6- and 6-6-junction isomers of alpha-D-mannopyranosyl [60]fullerene were studied by means of circular dichroism (CD), deuterium labeling, 1H-NMR, molecular-dynamics (MD) calculations, and a lectin-binding assay. The CD spectra of the O-acetylated derivatives allowed clear discrimination of the isomers, while the 1H-NMR spectra, with assistance from deuterium labeling and MD calculations, served to disclose the unique conformation and molecular geometry of each acetylated isomer in chloroform solution. The deprotected 5-6- and 6-6-isomers, which gave colloidal suspensions in aqueous mixtures, displayed marked activity in blocking lectin-induced hemagglutination by concanavalin A.     

PCR-Based Approach for Sequencing Mitochondrial Genomes of Decapod Crustaceans,with a Practical Example from Kuruma Prawn (<Emphasis Type="Italic">Marsupenaeus japonicus</Emphasis>)

An approach for sequencing the entire mitochondrial genomes (mitogenomes) of decapod crustaceans using 79 newly designed and 7 published polymerase chain reaction (PCR) primers is described. The approach comprises the following steps: (1) the entire mitogenome is amplified in 2 or 3 long PCRs; (2) the 86 primers are used in different combinations to amplify contiguous, overlapping short segments of the entire mitogenome with the diluted long PCR products as templates; (3) direct cycle sequencing is conducted using the short PCR products. This strategy allows a more rapid determination of decapod mitogenomic sequences than a traditional method using cloned mitochondrial DNA and primer walking strategy. As a practical example, the mitogenomic sequence for a kuruma prawn Marsupenaeus japonicus (Crustacea: Decapoda), was determined using the PCR-based approach.     

Liposomes with differential lipid components exert differential adjuvanticity in antigen-liposome conjugates via differential recognition by macrophages

We previously reported that liposomes having differential lipid components displayed differential adjuvant effects when antigen was coupled with liposomes via glutaraldehyde. In the present study, antigen-liposome conjugates prepared using liposomes having differential lipid components were added to the macrophage culture, and phagocytosis and the antigen digest of liposome-coupled antigen by macrophages were then investigated. Antigen presentation by macrophages to an antigen-specific T-cell clone was further investigated using the same conjugates. Antigen-liposome conjugates which induced higher levels of antibody production in vivo were recognized more often, and the liposome-coupled antigen was digested to a greater degree by macrophages than antigen-liposome conjugates which induced lower levels of antibody production. These results correlated closely with those regarding antigen presentation by macrophages; when antigen was coupled to liposomes showing higher adjuvant effect, macrophages cocultured with antigen-liposome conjugates activated antigen-specific T-cells at a higher degree. The concentration of OVA in the macrophage culture added as antigen-liposome conjugates was approximately 32 microg/mL. However, the extent of T-cell activation was almost equal to that when 800 microg/mL of soluble OVA was added to the culture. The results of the present study demonstrated that the adjuvant activity of liposomes observed primary in vivo correlated closely with the recognition of antigen-liposome conjugates and antigen presentation of liposome-coupled antigen by macrophages, suggesting that the adjuvant effects of liposomes are exerted at the beginning of the immune response, i.e., recognition of antigen by antigen-presenting cells.     

BIG2, a guanine nucleotide exchange factor for ADP-ribosylation factors: its localization to recycling endosomes and implication in the endosome integrity

Small GTPases of the ADP-ribosylation factor (ARF) family play a key role in membrane trafficking by regulating coated vesicle formation, and guanine nucleotide exchange is essential for the ARF function. Brefeldin A blocks the ARF-triggered coat assembly by inhibiting the guanine nucleotide exchange on ARFs and causes disintegration of the Golgi complex and tubulation of endosomal membranes. BIG2 is one of brefeldin A-inhibited guanine nucleotide exchange factors for the ARF GTPases and is associated mainly with the trans-Golgi network. In the present study, we have revealed that another population of BIG2 is associated with the recycling endosome and found that expression of a catalytically inactive BIG2 mutant, E738K, selectively induces membrane tubules from this compartment. We also have shown that BIG2 has an exchange activity toward class I ARFs (ARF1 and ARF3) in vivo and inactivation of either ARF exaggerates the BIG2(E738K)-induced tubulation of endosomal membranes. These observations together indicate that BIG2 is implicated in the structural integrity of the recycling endosome through activating class I ARFs.