The PTEN/PI3K pathway governs normal vascular development and tumor angiogenesis

PTEN is an important tumor suppressor gene. Hereditary mutation of PTEN causes tumor-susceptibility diseases such as Cowden disease. We used the Cre-loxP system to generate an endothelial cell-specific mutation of Pten (Tie2CrePten) in mice. Tie2CrePten(flox/+) mice displayed enhanced tumorigenesis due to an increase in angiogenesis driven by vascular growth factors. This effect was partially dependent on the PI3K subunits p85alpha and p110gamma. In vitro, Tie2CrePten(flox/+) endothelial cells showed enhanced proliferation/migration. Tie2CrePten(flox/flox) mice died before embryonic day 11.5 (E11.5) due to bleeding and cardiac failure caused by impaired recruitment of pericytes and vascular smooth muscle cells to blood vessels, and of cardiomyocytes to the endocardium. These phenotypes depend strongly on p110gamma rather than on p85alpha and were associated with decreased expression of Ang-1, VCAM-1, connexin 40, and ephrinB2 but increased expression of Ang-2, VEGF-A, VEGFR1, and VEGFR2. Pten is thus indispensable for normal cardiovascular morphogenesis and post-natal angiogenesis, including tumor angiogenesis.     

Biological properties and gene expression associated with metastatic potential of human osteosarcoma

Lung metastasis has a great influence on the prognosis of patients with osteosarcoma. We previously established two high-metastatic sublines, M112 and M132, from the HuO9 human osteosarcoma cell line by in vivo selection. In this study, we newly isolated a high-metastatic subline, H3, and three low-metastatic sublines, L6, L12 and L13, from HuO9 by the dilution plating method. Three high-metastatic sublines produced more than 200 metastatic nodules in the lung, while three low-metastatic sublines produced no or few nodules after injection of 2 × 10⁶ cells into the tail vein of nude mice. There were significant differences in the motility and invasiveness between high- and low-metastatic sublines, whereas the growth rates in vitro and the tumorigenicity in vivo showed no correlation with their metastatic abilities. Early adherence to culture plates was significantly lower in two of three low-metastatic sublines, which occupied smaller surface areas on the culture plates than other sublines did. Comparison of the expression of 637 cancer-related genes by cDNA microarray revealed that seven genes were differentially expressed between high- and low-metastatic sublines. Among them, five genes (AXL, TGFA, COLL7A1, WNT5A, and MKK6) were associated with adherence, motility, and/or invasiveness. These results suggest that the differences in motility/invasiveness and adhesive abilities are key determinants of lung metastasis in osteosarcoma. This revised version was published online in July 2006 with corrections to the Cover Date.     

Development of a PCR method for rapid identification of new Streptococcus mutans serotype k strains

In a previous study, we isolated and characterized a new serotype k of Streptococcus mutans from human blood and oral cavities. Analysis of the genes involved in biosynthesis of the serotype-specific polysaccharide of serotype k strains revealed that the serotype k-specific nucleotide alignment was commonly present in the 5' region of the rgpF gene (350 bp from the initial sequence) compared to the reference strains, and then a method for rapid identification of serotype k strains was developed by use of PCR with primers designed on the basis of the sequence of the variable region. PCR assays with primers specific for amplification of serotype k strains showed a negative reaction with serotype c, e, and f strains and a positive reaction with serotype k strains, with the sensitivity for identification of the serotype k strains shown to range from 5 to 50 cells. Next, the frequency of positive reactions for serotype k-specific primers was surveyed with DNA taken from saliva samples from 200 subjects (2 to 18 years of age), and 10 of those showed a positive reaction, which was higher than the frequency in our previous survey with a serological method. In addition, all saliva samples from subjects with serotype k strains in our previous study were shown to be positive with the serotype k-specific primers. These results indicate that this new PCR method is effective for identification of subjects with S. mutans serotype k.     

Genotyping of Mycobacterium leprae on the basis of the polymorphism of TTC repeats for analysis of leprosy transmission

The polymorphism of TTC repeats in Mycobacterium leprae was examined using the bacilli obtained from residents in villages at North Maluku where M. leprae infections are highly endemic (as well as from patients at North Sulawesi of Indonesia) to elucidate the possible mode of leprosy transmission. TTC genotypes are stable for several generations of passages in nude mice footpads and, hence, are feasible for the genotyping of isolates and epidemiological analysis of leprosy transmission. It was found that bacilli with different TTC genotypes were distributed among residents at the same dwelling in villages in which leprosy is endemic and that some household contacts harbored bacilli with a different genotype from that harbored by the patient. Investigations of a father-and-son pair of patients indicated that infections of bacilli with 10 and 18 copies, respectively, had occurred. Genotypes of TTC repeats were found to differ between a son under treatment and two brothers. These results reveal the possibility that in addition to exposure via the presence of a leprosy patient with a multibacillary infection who was living with family members, there might have been some infectious sources to which the residents had been commonly exposed outside the dwellings. A limited discriminative capacity of the TTC polymorphism in the epidemiological analysis implies the need of searching other useful polymorphic loci for detailed subdivision of clinical isolates.     

Use of conventional or replicating nucleic acid-based vaccines and recombinant Semliki forest virus-derived particles for the induction of immune responses against hepatitis C virus core and E2 antigens

Replicating and nonreplicating nucleic acid-based vaccines as well as Semliki Forest-recombinant Viruses (rSFVs) were evaluated for the development of a vaccine against hepatitis C virus (HCV). Replicating SFV-DNA vaccines (pSFV) and rSFVs expressing HCV core or E2 antigens were compared with classical CMV-driven plasmids (pCMV) in single or bimodal vaccine protocols. In vitro experiments indicated that all vaccine vectors produced the HCV antigens but to different levels depending on the antigen expressed. Both replicating and nonreplicating core-expressing plasmids induced, upon injection in mice, specific comparable CTL responses ranging from 10 to 50% lysis (E:T ratio 100:1). Comparison of different injection modes (intramuscular versus intraepidermal) and the use of descalating doses of DNA (1-100 microgram) did not show an increased efficacy of the core-SFV plasmid compared with the CMV-driven one. Surprisingly, rSFVs yielded either no detectable anticore CTL or very low anti-E2 antibody titers following either single or bimodal administration together with CMV-expressing counterparts. Prime-boost experiments revealed, in all cases, the superiority of DNA-based only vaccines. The anti-E2 antibody response was evaluated using three different assays which indicated that all generated anti-E2 antibodies were targeted at similar determinants. This study emphasizes the potential of DNA-based vaccines for induction of anti-HCV immune responses and reveals an unexpected and limited benefit of SFV-based vaccinal approaches in the case of HCV core and E2.     

Activities of a soluble extract from lymphoid cells of MRL mice. Effect on B cell differentiation in vitro

Soluble extract (sEx) was prepared from lymphoid cells of MRL/Mp-lpr/lpr(MRL/l) mice with early lupus nephritis and also of MRL/Mp-+/+ (MRL/n) mice. sEx from lymph node and spleen T cells of MRL/l mice had an activity for B cells to differentiate into immunoglobulin-producing cells but that of MRL/n mice did not show such an activity. sEx of MRL/l mice also enhanced the in vitro response of B cells to a suboptimal dose of lipopolysaccharide. Implication of these phenomena in the development of lupus nephritis is discussed.     

The effect of vitamin A depletion on antigen-stimulated trapping of peripheral lymphocytes in local lymph nodes of rats

The effect of vitamin A depletion on antigen-stimulated trapping of peripheral lymphocytes in lymphatic organs was studied in rats. Distribution of [3H]-uridine-labelled syngenic peripheral lymphocytes was quantified by assaying radioactive content of brachial and axillary lymph nodes, spleen and liver of normal and vitamin A-depleted F344/Ducrj rats immunized with sheep red blood cells. Localization of labelled cells in the ipsilateral brachial lymph nodes of the normal rats was stimulated by three times upon immunization with sheep erythrocytes as compared with the contralateral nodes. Recruitment of cells in axillary lymph nodes, spleen and liver was not significantly different from non-immunized values. The vitamin A-depleted rats exhibited marked deterioration in antigen-stimulated trapping of labelled cells in the draining brachial lymph nodes. These results suggest that this effect of vitamin A depletion is due to derangement of integrity of lymphocyte-trapping mechanism in the draining lymph nodes and not to any change in nature of lymphocytes per se.     

Rapid screening of point mutations of the Neisseria gonorrhoeae parC gene associated with resistance to quinolones.

To detect quinolone resistance-associated mutations within the Asp-86, Ser-87, Ser-88, and Glu-91 codons of the Neisseria gonorrhoeae parC gene, we developed a rapid and simple assay based on amplification of the regions of the parC gene containing the mutations sites by PCR and digestion of the PCR products with restriction enzymes. By using the method of primer-specified restriction site modification, artificial SalI, PstI, EcoRI, and HinfI restriction sites were created in the regions containing the Asp-86, Ser-87, Ser-88, and Glu-91 codons, respectively. The mutations generating alterations at Asp-86, Ser-87, Ser-88, and Glu-91 were detected as failures of SalI, PstI, EcoRI, and HinfI to digest the respective PCR products. Fifty-five clinical strains of N. gonorrhoeae were examined for mutations in the parC gene by this assay. Appropriate mutations at either the Asp-86, Ser-87, Ser-88, or Glu-91 codon were detected in each of 11 strains in which a mutation had previously been observed by DNA sequencing. This rapid and simple assay could be a useful device for screening genetic alterations in the parC gene associated with resistance to quinolones in N. gonorrhoeae.     

Vesicles in the subacrosomal space and partial diaphragms in the subacrosomal nuclear envelope of round spermatids of a rat injected intravenously with gold labeled-testosterone-bovine serum albumin conjugate: vesicular trafficking from acrosome to nucleus

Colloidal gold labeled-testosterone-bovine serum albumin conjugate (testosterone-BSA-gold) injected into the vascular system of rats is taken up by endocytosis into round spermatids. Based on observation of silver deposits indicating testosterone-BSA-gold with silver enhancement, we have suggested that testosterone-BSA-gold enters the nuclei through not only the postacrosomal nuclear envelope but also the subacrosomal nuclear envelope (SNE) via the acrosome (Nishimura and Nakano, 1997). However, it was unclear how testosterone-BSA-gold in the acrosome entered the nucleoplasm. Spermatids showing silver deposits on the subacrosomal space were observed under electron microscope without silver enhancement, to clarify the courses of translocation. In the spermatids, vesicles with the gold particles were seen in the subacrosomal space. Some of the vesicles were in contact with the SNE. A part of the outer nuclear membrane projected into the space. Furthermore, local single-bilayer nuclear membranes, which seemed to partially lack nuclear lamina, were present in the SNE. These results indicate the possibility that the vesicles mediate the transport of testosterone-BSA-gold from acrosome to nucleus, and that the vesicle membrane fuses with not only the outer nuclear membrane but also a shared bilayer in the SNE.