Protective effects of mangafodipir against chemotherapy-induced ovarian damage in mice

Background

Given the seriousness of chemotherapy-induced ovarian injury in female cancer patients, the preservation of fertility, including through the use of cryopreservation technology and pharmaceuticals, requires investigation. Previous studies have shown that damage to the ovaries is related to oxidative stress caused by anticancer drugs. Therefore, superoxide dismutase (SOD) may represent a key factor in the pharmacological protection of the ovaries. The aim of our study was to identify the effects of mangafodipir, a manganese chelate and SOD-mimetic, on suppression of apoptosis in granulosa cells and primordial follicle activation induced by anticancer drugs.

Methods

Cell viability assays using methyltrichlorosilane solutions and immunoblotting for cleaved caspase-3 were performed in in vitro experiments with the simultaneous addition of mangafodipir to human non-luteinized granulosa cell line (HGrC) cultures treated with hydrogen peroxide (H₂O₂), cisplatin, or paclitaxel. Count and morphological analyses of follicles at each developing stage in the ovaries and immunohistochemistry for cleaved caspase-3, Ki67 and 4-hydroxynonenal, a marker for oxidative stress, were also performed using mangafodipir-injected 6-week-old female ICR mice treated with cisplatin or paclitaxel. Further, mangafodipir was injected into 6-week-old female BALB/c mice inoculated with ES-2 to analyze whether mangafodipir inhibits the anti-tumor effects of cisplatin or paclitaxel treatment.

Results

Mangafodipir attenuated apoptosis induced by H₂O₂ and anticancer drugs in vitro. Mangafodipir also decreased the expression of 4-hydroxynonenal and reduced cisplatin- and paclitaxel-induced apoptosis in granulosa cells in vivo. In addition, mangafodipir inhibited the loss of primordial follicles. Tumor xenograft studies in mice showed that mangafodipir did not affect anticancer drug antitumor effects.

Conclusions

Oxidative stress might be one of the mechanisms of cisplatin- and paclitaxel-induced the loss of primordial follicles. Mangafodipir can reduce cisplatin- and paclitaxel-induced apoptosis in granulosa cells and primordial follicle activation partially via its SOD activity. At the same time, mangafodipir might have other potential mechanisms to inhibit the activation of primordial follicles. Further, mangafodipir attenuated the ovarian damage caused by cisplatin and paclitaxel without affecting their antitumor activities. Mangafodipir, therefore, though its efficacy might be limited, may be a new option for the preservation of fertility during anticancer treatment.

     

Genetic characteristics and possible introduced origin of the paradise fish <Emphasis Type="Italic">Macropodus opercularis</Emphasis> in the Ryukyu Archipelago,Japan

It has hitherto been unknown whether the paradise fish Macropodus opercularis that inhabits the Ryukyu Archipelago, Japan, is native to the region or was introduced. This study examined the genetic identity of fish from five islands in the Ryukyu Archipelago (Okinoerabu, Okinawa, Yagaji, Kume, and Minamidaito islands) and compared it with those from Taiwan Island, mainland China, and Hainan Island. Analyses of the mtDNA control (760 bp) and cytb (660 bp) regions showed that haplotypes of specimens from the Ryukyu Archipelago were the same as or were very similar (with a 1- or 2-bp difference) to those from Taiwan. In addition, haplotypes from the Ryukyu Archipelago also showed lower genetic diversity than those from Taiwan Island, mainland China, and Hainan Island. These results suggest a high likelihood that the fish in the Ryukyu Archipelago were artificially introduced from Taiwan. However, the possibility that the fish is indigenous to the Ryukyu Archipelago cannot be completely ruled out, because some haplotypes and a clade from the Ryukyu Archipelago have not been found in the other areas. Regardless of its origin, we emphasize the importance of the conservation of the paradise fish in the Ryukyu Archipelago as an indicator of the threatened wetland environment as well as for its cultural value.     

Variation in growth performance of Ryukyu-ayu, <Emphasis Type="Italic">Plecoglossus altivelis ryukyuensis</Emphasis>, inferred from otolith analysis

Ryukyu-ayu (Plecoglossus altivelis ryukyuensis) is an amphidromous fish species that migrates between the sea and rivers over its one-year life span. Although growth performance during the early marine stage may affect growth in the later riverine stage of this species’ life cycle, no studies have specifically examined this relationship in P. a. ryukyuensis. In the present study, we reconstructed the growth trajectories of P. a. ryukyuensis individuals collected from the Yakugachi River, Amami-Oshima Island, Japan in 2016 (n?=?47) throughout their growth period in both the sea and river by using otolith analysis. Using this, we determined the age and body size of individuals at the time of their upstream migration, as well as their growth rates during the marine and riverine stages. Results showed that body size at upstream migration significantly affected body size at the riverine stage, indicating that juveniles with larger body size in the sea had better growth performance in the river. Individuals with higher growth rates during the marine stage tended to enter the river younger and at larger body sizes than those with lower marine growth rates. Our results demonstrated the close linkage between the growth performance in the sea and in rivers of P. a. ryukyuensis. This information will contribute to better understanding variations in growth patterns of this endangered species and potentially aid in its conservation.     

Monitoring of Ralstonia eutropha KT1 in Groundwater in an Experimental Bioaugmentation Field by In Situ PCR

Ralstonia eutropha KT1, which degrades trichloroethylene, was injected into the aquifer after activation with toluene, and then the number of bacteria was monitored by in situ PCR targeting the phenol hydroxylase gene and by fluorescent in situ hybridization (FISH) targeting 16S rRNA. Before injection of the bacterial suspension, the total concentration of bacteria in the groundwater was approximately 3 × 10⁵ cells/ml and the amount of Ralstonia and bacteria carrying the phenol hydroxylase gene as a percentage of total bacterial cells was less than 0.1%. The concentration of bacteria carrying the phenol hydroxylase gene detected by in situ PCR was approximately 3 × 10⁷ cells/ml 1 h after injection, and the concentration of Ralstonia detected by FISH was similar. The number of bacteria detected by in situ PCR was similar to that detected by FISH 4 days after the start of the extraction of groundwater. On and after day 7, however, the number of bacterial cells detected by FISH was less than that detected by in situ PCR.     

Variant of Penicillinase Mediated by an R Factor in Escherichia coli

The penicillinase from an Escherichia coli strain harboring an R factor R(GN823) was purified and its properties were compared with those of a known type I penicillinase mediated by R factors. The molecular weight and S(20,w) of the enzyme were 22,600 and 2.42S, respectively. The isoelectric point of the enzyme was 6.9. These values are clearly different from those of type I penicillinase. The specific activity of the enzyme was 84,700 units per mg of the purified enzyme protein, which is about 20 times higher than that of the type I penicillinase. However, similarities were observed between the enzyme and the type I-penicillinase at optimal pH (6.5 to 7.0), optimal temperature (40 to 45C), substrate specificity, Michaelis constants for penicillins and cephaloridine, and effect of inhibitors. Furthermore, antiserum against type I penicillinase showed cross-reaction against this enzyme. The enzyme was named type Ib penicillinase, and the original type I penicillinase was renamed type Ia-penicillinase.     

Glucose 1-phosphate increases active transport of calcium in intestine

Active calcium transport in intestine is essential for serum calcium homeostasis as well as for bone formation. It is well recognized that vitamin D is a major, if not sole, stimulator of intestinal calcium transport activity in mammals. Besides vitamin D, endogenous glucose 1-phosphate (G1P) affects calcium transport activity in some microorganisms. In this study, we investigated whether G1P affects intestinal calcium transport activity in mammals as well. Of several glycolytic intermediates, G1P was the sole sugar compound in stimulating intestinal calcium uptake in Caco-2 cells. G1P stimulated net calcium influx and expression of calbindin D9K protein in rat intestine, through an active transport mechanism. Calcium uptake in G1P-supplemented rats was greater than that in the control rats fed a diet containing adequate vitamin D3. Bone mineral density (BMD) of aged rat femoral metaphysis and diaphysis was also increased by feeding the G1P diet. G1P did not affect serum levels of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] at all. These results suggest that exogenously applied G1P stimulates active transport of calcium in intestine, independent of vitamin D, leading to an increase of BMD.     

Kinetic analysis on nitric oxide binding of recombinant Prolixin-S, a nitric oxide transport protein from the bloodsucking bug, Rhodnius prolixus.

Kinetics of the NO binding and removal reaction of recombinant Prolixin-S (rProlixin-S) were analyzed using stopped-flow spectrophotometry. The reaction was observed as a biphasic process. The rate constant of the fast phase increased linearly as NO concentration increased. The rate constant at the slow phase increased as NO concentrations increased at low NO concentration, then reached a plateau at high NO concentration. These NO dependencies of the reaction are characteristic of a bimolecular two-step consecutive reaction. The reaction consisted of the fast NO binding reaction of rProlixin-S and the following slow structural change of NO-protein complex. Kinetic studies revealed that the NO binding rate constant was independent of pH, but the rate constant of the NO removal reaction increased as pH increased. The apparent NO dissociation constant (Kd) of rProlixin-S was also calculated from the values of the kinetic parameters obtained in this work. The Kd value increased as pH and temperature increased. The Kd value of rProlixin-S and NO was 10-300 nM in regular physiological condition, which is 103 higher and 103 lower than those of the other ferric and ferrous hemoproteins and NO, respectively. These results indicate that Prolixin-S is one of NO transport proteins regulating blood pressure.     

Disappearance of a Novel Protein Component of the 26S Proteasome duringXenopusOocyte Maturation

We have prepared polyclonal antibodies againstXenopus20S proteasomes. The antibodies cross-react with several proteins that are common to 20S and 26S proteasomes and with at least two proteins that are unique to 26S proteasomes. The antibodies were used to analyze changes in the components of proteasomes during oocyte maturation and early development ofXenopus laevis.A novel protein with a molecular weight of 48 kDa, p48, was clearly detected in immature oocytes, but was found at very low levels in mature oocytes and ovulated eggs. p48 was reduced to low levels during oocyte maturation, after maturation-promoting factor was activated. The amount of p48 in eggs remained low during early embryonic development, but increased again after the midblastula transition. These results show that at least one component of 26S proteasomes changes during oocyte maturation and early development and suggest that alterations in proteasome function may be important for the regulation of developmental events, such as the rapid cell cycles, of the early embryo.