Collagenofibrotic glomerulopathy with a widespread expression of type-V collagen

Collagenofibrotic glomerulopathy is considered as a form of glomerulopathy in which organized collagen type III progressively deposits. We report a case of this disease with widespread expression of collagen type V. A 65-year-old woman was admitted to our hospital for further evaluation of nephrotic-range proteinuria. The patient had had anemia and hypertension for 9 years, and proteinuria for 3 years. A renal biopsy specimen showed a remarkable mesangial expansion with Congo red-negative and periodic acid-Schiff-positive deposits. At the ultrastructural level, two forms of bundling fibers were found in the mesangium and subendothelial side of the glomerular basement membranes (GBM). The GBM itself appeared normal. Immunohistochemical investigation showed that the glomerular lesions were strongly reactive with both anti-collagen type-III and -V antibodies. Immunoelectron microscopy demonstrated collagen type V in both forms of bundling fibers. Despite therapy, her renal function declined. The clinical course and renal pathology of this case were in accordance with collagenofibrotic glomerulopathy except for the widespread expression of collagen type V. Collagen type V is a fibrillar collagen capable of forming banding fibrils. This report poses the question whether collagen type V accumulates only in this particular case or whether it is a normal component in collagenofibrotic glomerulopathy.     

Real-time imaging of individual fluorescent-protein color-coded metastatic colonies in vivo

We have established stable, bright green fluorescent protein (GFP)- or red fluorescent protein (RFP)-expressing HT-1080 human fibrosarcoma clones. These cell lines showed similar cell proliferation rates and high-frequency experimental lung metastasis. The HT-1080-GFP and -RFP clones enable simultaneous real-time dual-color imaging in the live animal. HT-1080 cells were transduced with retroviral vectors containing GFP or RFP and the neomycin resistance gene. Stable transformants were selected stepwise with G418 up to 800 μl/ml. Subsequently, high GFP- or RFP-expressing clones, HT-1080-GFP or HT-1080-RFP, respectively, were selected. 3×10⁶ cells from each clone were mixed and injected into the tail vein of SCID mice. The cells seeded the lung at high frequency with subsequent formation of pure green and pure red colonies as well as mixed yellow colonies with different patterns visualized directly on excised lungs. The lung metastases were also visualized by external fluorescence imaging in live animals through skin-flap windows over the chest wall. Lung metastases were observed on the lung surface of all mice. SCID mice well tolerated multiple surgical procedures for direct-view imaging via skin-flap windows. Real-time metastatic growth of the two different colored clones in the same lung was externally imaged with resolution and quantification of green, red, or yellow colonies in live animals. The color coding enabled determination of whether the colonies grew clonally or were seeded as a mixture with one cell type eventually dominating, or whether the colonies grew as a mixture. The simultaneous real-time dual-color imaging of metastatic colonies described in this report gives rise to the possibility of color-coded imaging of clones of cancer cells carrying various forms of gene of interest. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Dominant-negative hypoxia-inducible factor-1 alpha reduces tumorigenicity of pancreatic cancer cells through the suppression of glucose metabolism

In the tumor cells exposed to hypoxia, hypoxia-inducible factor-1 (HIF-1)-mediated adaptation responses such as angiogenesis and anaerobic metabolism are induced for their survival. We have recently reported that the constitutive expression of HIF-1 alpha renders pancreatic cancer cells resistant to apoptosis induced by hypoxia and glucose deprivation. We then established dominant-negative HIF-1 alpha (dnHIF-1 alpha) transfectants and examined their susceptibility to apoptosis and growth inhibition induced by hypoxia and glucose deprivation in vitro and their tumorigenicity in SCID mice. We further examined the expressions of aldolase A and Glut-1 in vitro and Glut-1 expression and glucose uptake in the tumor tissues and microvessel counts in the tumor tissues. As a result, dnHIF-1 alpha rendered the pancreatic cancer cells sensitive to apoptosis and growth inhibition induced by hypoxia and glucose deprivation. Also it abrogated the enhanced expression of Glut-1 and aldolase A mRNAs under hypoxia and reduced the expression of Glut-1 and the glucose uptake in the tumor tissues and consequently in vivo tumorigenicity. We found no significant difference in the microvessel counts among the tumor tissues. From these results, we suggest that the disruption of the HIF-1 pathway might be effective in the treatment of pancreatic cancers.     

Impaired liver regeneration after partial hepatectomy in db/db mice

Fatty liver is the most common hepatic disorder in humans and supposed to be a cause of poor prognosis after liver transplantation and hepatic resection which could be resulted from impaired liver regeneration. This study was carried out to analyze the process of liver regeneration in db/db mice which show severe steatosis because of abnormal leptin receptor. We performed 70% partial hepatectomy (PH) on db/db mice and normal +m/+m mice, and then sacrificed the animals 1, 2, 3, 5, 7 and 10 days later. The liver samples were weighed and examined histologically or immunohistochemically. As a result, the liver mass restitution was significantly inhibited in db/db mice compared with +m/+m mice. The BrdU labelling index peaked at 2 days after PH in both strains, although the value was lower in db/db mice. After that, interestingly, it decreased to the control level at 5 days in +m/+m mice while the recovery was delayed in db/db mice. Similar sequence was also observed in the PCNA labelling index. In addition, the peak time of the mitosis index was 2 days and 5 days after PH in +m/+m mice and in db/db mice, respectively. Thus, although not significant, the proliferative response of hepatocytes to PH occurred somewhat more transient and sharply in +m/+m mice while it lasted somewhat longer in db/db mice. This suggests that db/db mice may be valuable as one of the animal models for the investigation of the effects of steatosis on the liver regeneration.     

A novel serine protease of the mammalian HtrA family is up-regulated in mouse uterus coinciding with placentation

This paper characterizes a novel gene, previously identified as uniquely regulated at implantation in mouse uterus. We cloned its full mRNA sequence encoding a serine protease possessing an IGF-binding domain and named it pregnancy-related serine protease (PRSP). PRSP is structurally similar to mammalian HtrA1 (56% amino acid similarity). Northern analysis revealed that the expression of PRSP mRNA was low before pregnancy, but it was increased at implantation and markedly up-regulated post-implantation. In-situ hybridization localized low levels of mRNA expression to the epithelium and stroma during very early pregnancy, but high expression to the decidual cells on day 8.5, primarily at the mesometrial pole where the placenta was forming. By day 10.5, PRSP mRNA was detected in the placenta. We also cloned an alternatively spliced PRSP mRNA that is expressed at a very low level. We located PRSP gene on chromosome 5 and established its intron/exon structure, which unambiguously explains how the two mRNA variants are produced through alternative splicing. Based on PRSP protein domain structure and its unique expression during pregnancy, we propose that PRSP plays an important role in the formation/function of the placenta.     

Pulmonary metastases from a low-grade endometrial stromal sarcoma confirmed by chromosome aberration and fluorescence in-situ hybridization approaches: a case of recurrence 13 years after hysterectomy

Pulmonary metastasis from low-grade endometrial stromal sarcomas (ESSs) occasionally are found after long, disease-free periods, mostly as incidental histological or radiological discoveries. We describe a case of low-grade ESS presenting as nodular pulmonary metastases finally diagnosed by estrogen-receptor staining, cytogenetic and fluorescence in situ hybridization (FISH) analyses, and perusal of the histology of hysterectomy material. An abnormal nodule in the lung field was discovered by means of chest X-ray of a 47-year-old woman. She had been disease free for 13 years after hysterectomy for an alleged leiomyoma. A computed tomographic scan revealed nodules, with fluctuation in size over the 2-year period, in both lungs. Finally the lesion in the left lung was resected, and pulmonary endometriosis was suspected because of the lack of stromal cell nuclear atypia and positive immunohistochemical reactions for estrogen and progesterone receptors. However, a characteristic karyotype was identified cytogenetically: 46, XX, t(7;17)(p15;q11), the translocation of which, specific to ESS, was confirmed by FISH analysis. A final diagnosis of pulmonary metastases from an ESS could be made by reviewing the histology of the previous uterine tumor. In this case, metastatic lesions from an ESS showed a decrease as well as an increase in size, despite the malignant potential. Immunostaining for estrogen and progesterone receptors and cytogenetic and FISH analyses, together with clinical information on the past gynecological history, are valuable diagnostic keys.     

Prediction of clinically insignificant prostate cancer by detection of allelic imbalance at 6q, 8p and 13q

The criterion tumor volume (TV) for clinically insignificant prostate cancer has been reported, but it differs from study to study: some have reported TV < 200 mm³; others, < 500 mm³. The aim of the present study was to distinguish clinically insignificant cancers from significant ones using molecular biological methods. A total of 184 microscopic cancers (MC) defined as limited within a 3 mm circle and 82 main tumor (MT) nodules were selected. Thirteen microsatellite loci at 6q22, 8p23.2–23, 13q14 and 13q33 were evaluated for loss of heterozygosity (LOH). MT were subgrouped as TV ≥ 500 mm³ or <500 mm³; TV ≥ 200 mm³ or < 200 mm³; and TV < 200 mm³, 200 mm³ ≤ TV < 500 mm³ or TV ≥ 500 mm³; and frequencies of LOH were compared between these three groups. Frequencies of LOH at 6q16–21, 6q22, 8p23.1, 8p23.2, 13q14 were significantly lower in MC (1.0%, 2.7%, 1.9%, 1.1% and 5.4%) than in MT (30.9%, 40.4%, 12%, 8.7% and 20.6%), but no significant differences in LOH frequency were found within each of the three TV groups, between each cut-off. When insignificant tumor is defined as TV < 200 mm³ or < 500 mm³, it should include tumors with malignant potential equivalent to larger tumors. It is suggested that in order to identify insignificant tumor within a strict safety range, TV should be set lower.     

Stabilization of tRNA (mG37) methyltransferase [TrmD] from Aquifex aeolicus by an intersubunit disulfide bond formation

Recombinant Aquifex aeolicus TrmD protein has a Cys20–Cys20 disulfide bond between its two subunits. This was demonstrated by SDS–polyacrylamide gel analysis of wild-type enzyme and C20S mutant protein (in which the Cys20 residue is substituted by serine), in the absence or presence of various concentrations of dithiothreitol. Analytical gel-filtration chromatography revealed that the C20S mutant protein forms a dimer structure even though it is missing the disulfide bond. Western blotting analysis suggests that the Cys20–Cys20 disulfide bond is formed in native TrmD protein in living A. aeolicus cells. Incubation at 85 °C for 20 min caused the precipitation of more than half of the C20S protein, while more than 70% of the wild-type enzyme was soluble at that temperature. This assay clearly demonstrates that the disulfide bond enhances the protein stability at 85 °C. A kinetic assay showed that the methyl-transfer activity of the C20S mutant protein was slightly less than that of the wild-type enzyme at 70 °C. Comparison of the CD-spectra of wild-type and C20S proteins reveals that some of the α-helices in the C20S mutant protein are less tightly packed than those of the wild-type enzyme at 70 °C.     

Soluble G protein of respiratory syncytial virus inhibits Toll-like receptor 3/4-mediated IFN-beta induction

Monocyte-derived dendritic cells (mDCs) recognize viral RNA extrinsically by Toll-like receptor (TLR) 3 on the membrane and intrinsically retinoic acid-inducible gene I (RIG-I)/melanoma differentiation-associated gene 5 (MDA5) in the cytoplasm to induce type I IFNs and mDC maturation. When mDCs were treated with live or UV-irradiated respiratory syncytial virus (RSV), early ( approximately 4 h) induction of IFN-beta usually occurs in other virus infections was barely observed. Live RSV subsequently replicated to activate the cytoplasmic IFN-inducing pathway leading to robust type I IFN induction. We found that RSV initial attachment to cells blocked polyI:C-mediated IFN-beta induction, and this early IFN-beta-modulating event was abrogated by antibodies against envelope proteins of RSV, demonstrating the presence of a IFN-regulatory mode by early RSV attachment to host cells. By IFN-stimulated response element (ISRE) reporter analysis in HEK293 cells, polyI:C- or LPS-mediated ISRE activation was dose dependently inhibited by live and inactive RSV to a similar extent. Of the RSV envelope proteins, simultaneously expressed or exogenously added RSV G or soluble G (sG) proteins inhibited TLR3/4-mediated ISRE activation in HEK293 cells. sG proteins expressed in cells did not affect the RIG-I/MDA5 pathway but inhibited the TLR adaptor TRIF/TICAM-1 pathway for ISRE activation. Finally, extrinsically added sG protein suppressed the production of IFN-beta in mDCs. Although the molecular mechanism of this extrinsic functional mode of the RSV G glycoprotein (G protein) remains undetermined, G proteins may neutralize the fusion glycoprotein function that promotes IFN-mediated mDC modulation via TLR4 and may cause insufficient raising cell-mediated immunity against RSV.