Availability of subfertile transgenic rats expressing the c-myc gene as recipients for spermatogonial transplantation

The spermatogonial transplantation system was applied to evaluate stem cell kinetics and niche quality and to produce gene-modified animals using the stem cells after homologous recombination-based selection. This study was designed to determine whether the transplanted spermatogonia were able to proliferate and differentiate in male rats expressing the c-myc transgene under control of the human metallothionein IIA promoter (MT-myc Tg rats). Donor testicular cells were prepared from heterozygous chicken beta actin (CAG)/enhanced green fluorescent protein (EGFP)-transgenic rats (EGFP Tg rats) during the second week after birth and injected into the seminiferous tubules of the MT-myc Tg rats (line-A and -B; both subfertile) or rats pretreated with busulfan to remove endogenous spermatogonia. Three to four months after transplantation, cell colonies with EGFP fluorescence were detected in 36% (4/11), 40% (8/20), and 71% (5/7) of the transplanted testes in line-A MT-myc Tg rats, line-B MT-myc Tg rats, and busulfan-treated rats, respectively. No EGFP-positive colonies were detected when wild-type male rats were used as recipients (0/7; testis-basis). The histopathological and immunofluorescent examination of the serial sections from the transplanted testes showed normal spermatogenesis of the donor spermatogonia, but atrophy of the recipient seminiferous tubules. Microinsemination with round spermatids and mature spermatozoa derived from EGFP-positive testes in line-A rats resulted 26% (10/39 transferred) and 23% (11/48 transferred) full-term offspring, respectively. Thus, the MT-myc Tg male rats were suitable as potent recipients for spermatogonial transplantation without any chemical pretreatment to remove the endogenous spermatogonia.     

High-performance liquid chromatographic method to measure protein L-isoaspartyl/D-aspartyl o-methyltransferase activity in cell lysates

Protein l-isoaspartyl/d-aspartyl o-methyltransferase (PIMT) is a widely expressed protein repair enzyme that restores isomerized aspartyl residues to their normal configuration. Current methods for measuring PIMT activity have limited sensitivity or require radioactivity. We have developed a highly sensitive new assay method to measure PIMT activity in cell lysates. As a substrate, we used a fluorescently labeled delta sleep-inducing peptide (DSIP) that contains an isoaspartyl residue: 7-nitro-2,1,3-benzoxadiazole (NBD)-DSIP(isoAsp). The PIMT-catalyzed transfer of a methyl group onto this substrate can be detected with a simple high-performance liquid chromatography (HPLC) procedure. After the enzyme reaction, the methylated form of the peptide is stable and can be reproducibly separated from the unmethylated form in an acidic solvent and fluorometrically detected by HPLC. The limit of detection was estimated to be approximately 1 pmol of NBD-DSIP(isoAsp) (signal/noise ratio [S/N] = 3), and the quantitation limit of the activity was approximately 18 μg of total cell lysate from HEK293 cells (10.7 pmol/min/mg protein). This assay method is sensitive enough to detect PIMT activity in biological samples without the use of radioisotopes, offering significant advantages over previously reported methods.     

Amyloid‐β up‐regulates complement factor B in retinal pigment epithelial cells through cytokines released from recruited macrophages/microglia: Another mechanism of complement activation in age‐related macular degeneration

One of the earliest signs of age‐related macular degeneration (AMD) is the formation of drusen which are extracellular deposits beneath the retinal pigmented epithelium (RPE). To investigate the relationship between drusen and AMD, we focused on amyloid β (Aβ), a major component of drusen and also of senile plaques in the brain of Alzheimer's patients. We previously reported that Aβ was accumulated in drusen‐like structure in senescent neprilysin gene‐disrupted mice. The purpose of this study was to investigate the influence of Aβ on factor B, the main activator of the complement alternative pathway. The results showed that Aβ did not directly modulate factor B expression in RPE cells, but increased the production of monocyte chemoattractant protein‐1 (MCP‐1). Aβ also increased the production of IL‐1β and TNF‐α in macrophages/microglia, and exposure of RPE cells to IL‐1β and TNF‐α significantly up‐regulated factor B. Co‐cultures of RPE cells and macrophages/microglia in the presence of Aβ significantly increased the expression of factor B in RPE. These findings indicate that cytokines produced by macrophages/microglia that were recruited by MCP‐1 produced in RPE cells stimulated by Aβ up‐regulate factor B in RPE cells. Thus, a combined mechanism exists for Aβ‐induced for the activation of the complement alternative pathway in the subretinal space; cytokine‐induced up‐regulation of activator factor B and dysfunction of the inhibitor factor I by direct binding to Aβ as suggested in our earlier study. J. Cell. Physiol. 220: 119–128, 2009. © 2009 Wiley‐Liss, Inc.     

Lysosomal-Associated Protein Multispanning Transmembrane 5 Gene (LAPTM5) Is Associated with Spontaneous Regression of Neuroblastomas

Background

Neuroblastoma (NB) is the most frequently occurring solid tumor in children, and shows heterogeneous clinical behavior. Favorable tumors, which are usually detected by mass screening based on increased levels of catecholamines in urine, regress spontaneously via programmed cell death (PCD) or mature through differentiation into benign ganglioneuroma (GN). In contrast, advanced-type NB tumors often grow aggressively, despite intensive chemotherapy. Understanding the molecular mechanisms of PCD during spontaneous regression in favorable NB tumors, as well as identifying genes with a pro-death role, is a matter of urgency for developing novel approaches to the treatment of advanced-type NB tumors.

Principal Findings

We found that the expression of lysosomal associated protein multispanning transmembrane 5 (LAPTM5) was usually down-regulated due to DNA methylation in an NB cell-specific manner, but up-regulated in degenerating NB cells within locally regressing areas of favorable tumors detected by mass-screening. Experiments in vitro showed that not only a restoration of its expression but also the accumulation of LAPTM5 protein, was required to induce non-apoptotic cell death with autophagic vacuoles and lysosomal destabilization with lysosomal-membrane permeabilization (LMP) in a caspase-independent manner. While autophagy is a membrane-trafficking pathway to degrade the proteins in lysosomes, the LAPTM5-mediated lysosomal destabilization with LMP leads to an interruption of autophagic flux, resulting in the accumulation of immature autophagic vacuoles, p62/SQSTM1, and ubiqitinated proteins as substrates of autophagic degradation. In addition, ubiquitin-positive inclusion bodies appeared in degenerating NB cells.

Conclusions

We propose a novel molecular mechanism for PCD with the accumulation of autophagic vacuoles due to LAPTM5-mediated lysosomal destabilization. LAPTM5-induced cell death is lysosomal cell death with impaired autophagy, not cell death by autophagy, so-called autophagic cell death. Thus LAPTM5-mediated PCD is closely associated with the spontaneous regression of NBs and opens new avenues for exploring innovative clinical interventions for this tumor.     

Influences of forested watershed conditions on fluctuations in stream water temperature with special reference to watershed area and forest type

In order to gain insight into the effect of watershed conditions on fluctuations in stream water temperature, we statistically analyzed water temperature data for 1 year, using root mean square (Rms) and harmonic (A Amplitude, φ delay time) methods. The average values of delay time (days) between air and water temperatures (T _a and T _w) of small (< 0.5 ha), medium (0.5–100 ha) and large (> 100 ha) watersheds were 4.53 ± 0.82 days, 11.83 ± 3.88 days and 4.45 ± 1.52 days, respectively. Fluctuations in stream water temperature expressed by Rms (Rms T _w/Rms T _a) and harmonic methods (A −T _w/A −T _a) in the medium-sized watersheds with moderate slope gradients were 0.37 ± 0.09 and 0.56 ± 0.14, respectively. These values increased in the larger watersheds with low slope gradients, including five large rivers covered by various landscapes, with their averages of 0.53 ± 0.09 and 0.78 ± 0.09, respectively, indicating the influences of solar radiation and heat transfer processes. In the smaller watersheds with high slope gradients, these values were 0.73 ± 0.02 and 0.87 ± 0.03, respectively, suggesting that shorter passage time affected water temperatures. With respect to forest type, these values at badly managed hinoki forest watersheds (0.45 ± 0.04 and 0.73 ± 0.07) were larger than those at broadleaf forest (0.34 ± 0.04 and 0.51 ± 0.12) and well-managed hinoki forest (0.33 ± 0.04 and 0.51 ± 0.07) watersheds, indicating different proportions of flow paths.     

Improvement of the transformation efficiency of Flammulina velutipes Fv-1 using the glyceraldehyde-3-phosphate dehydrogenase gene promoter

To improve the expression level of heterologous genes in Flammulina velutipes Fv-1, we constructed new vectors having glyceraldehydes-3-phosphate dehydrogenase (gpd) gene promoter to control the expression of target genes. When the hygromycin B phosphotransferase (hph) gene from Escherichia coli was controlled by the gpd promoter, transformation efficiency was 3-fold higher than the case of that controlled by the tryptophan synthetase gene (trp1) promoter.     

Geographical distribution of Metagonimus yokogawai and M. miyatai in Shizuoka Prefecture, Japan, and their site preferences in the sweetfish, Plecoglossus altivelis, and hamsters

Sweetfish, Plecoglossus altivelis, obtained from 18 rivers in Shizuoka Prefecture were examined for metacercarial infection of 2 flukes, Metagonimus yokogawai and Metagonimus miyatai. The infection rate and density of metacercariae in the fish were higher in eastern and western regions than in central region of the prefecture. After infection of hamsters with metacercariae derived from the scale, 98.7% of the adult worms obtained from the intestine was found to be M. miyatai. Conversely, from infection with metacercariae from the flesh, 90.0% of the worms was M. yokogawai. Since the worms had no exclusivity in the tissues, we conclude that the flukes have location preference with the former primarily preferring the scale, and the latter the flesh. Fish from two rivers located in adjacent areas in the western region had relatively a higher ratio of M. yokogawai in the scale relative to other rivers, suggesting an intraspecific genetic variation due to geographical isolation. On examination of adult worms in the hamster's intestine, M. yokogawai was mainly located towards the anterior part of the intestine, unlike M. miyatai, suggesting that in mammalian host too, the parasites have site preference.     

UV-irradiation induces oxidative damage to mitochondrial DNA primarily through hydrogen peroxide: analysis of 8-oxodGuo by HPLC

Roles of reactive oxygen species (ROS) in damage to mitochondrial DNA (mtDNA) following ultraviolet (UV)-irradiation were investigated in the human hepatoma cell line SK-HEP-1. We altered the intracellular status of ROS by the overexpression of manganese superoxide dismutase (MnSOD) and/or catalase. Using HPLC, we analyzed 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), known as a marker of damage to DNA molecules. UV-irradiation resulted in the accumulation of 8-oxodGuo in these cells. The overexpression of MnSOD enhanced the accumulation of 8-oxodGuo by UV. The co-overexpression of catalase inhibited the accumulation of 8-oxodGuo by UV in MnSOD-transfectants. The overexpression of MnSOD reduced the colony forming capacity in SK-HEP-1 cells and the co-overexpression of catalase with MnSOD stimulated the capacity compared to control. UV-irradiation inhibited the colony forming capacity in these cells; no difference was observed among the capacities of control, MnSOD- and catalase-transfectants. However, the overexpression of MnSOD/catalase significantly rescued the reduction of colony forming capacity by UV-irradiation. Our results suggest that the accumulation of hydrogen peroxide plays a key role in the oxidative damage to mtDNA of UV-irradiated cells, and also that the overexpression of both MnSOD and catalase reduces the mtDNA damage and blocks the growth inhibition by UV. Our results also indicate that the increased activity of MnSOD may lead to a toxic effect on mtDNA by UV-irradiation.