Biomedical subjects

Y Kaneda

Publications and source records attributed to Y Kaneda.

At least 271 records · Page 15Linked to original sources

PubMed1997

Chest wall implantation of lung cancer at the drainage tube site: report of a case.

We report herein the case of a 70-year-old man in whom a chest wall implantation of adenocarcinoma of the lung at the drainage tube site was found 4 months after a right lower lobectomy with mediastinal lymph node dissection had been performed for adenocarcinoma of the right lower lobe. The lesion was successfully treated by tumor extirpation. We believe that tumor seeding to the chest wall occurred at the time of thoracotomy. To prevent such tumor seeding, the pleural cavity should be washed out routinely with a massive volume of physiological saline solution prior to closure of the chest wall.

Adenocarcinoma↗

PubMed1997

Experimental bronchiolitis obliterans induced by in vivo HVJ-liposome-mediated endothelin-1 gene transfer.

BACKGROUND: Bronchiolitis obliterans (OB) is a lesion that results when injury to small conducting airways is repaired by a proliferation of fibrous granulation tissue. Bronchiolitis obliterans has emerged as a main cause of morbidity and mortality in the setting of lung and heart-lung transplantation. Endothelin-1 (ET-1), initially discovered as a vasoconstrictive peptide, has a mitogenic activity on vascular smooth cells and airway epithelial cells. Overproduction of endothelin has been reported in patients with OB or chronic rejection after lung transplantation. It is still undetermined whether locally overexpressed ET-1 has a potential impact in the pathogenesis of OB. METHODS: We locally overexpressed ET-1 using ultraviolet irradiation-inactivated hemagglutinating virus of Japan (HVJ)-liposome-mediated in vivo gene transfer. Plasmid DNA of prepro-ET-1 and high mobility group 1 protein were coencapsulated in liposomes, and were introduced into airway epithelial cells by HVJ-mediated membrane fusion. Control animals received instillation of HVJ-liposome with an empty expression cassette. To confirm the efficiency of transfection, HVJ liposome with beta-galactosidase gene was introduced. The expression of ET-1 and beta-galactosidase was assessed by immunohistochemistry. RESULTS: Bronchial epithelium alveolar cells and alveolar macrophage were stained blue (X-Gal) 1 week after in vivo gene transfer of beta-galactosidase gene, indicating beta-gal activity. In animals 1 to 2 weeks after in vivo transfection of prepro-ET-1 gene, hyperplastic connective tissue plaque was seen in the alveolar duct and small conducting airway, indicating histologically distinctive bronchiolitis obliterans. Strong ET-1-like immunoactivities were seen in the airway epithelial, hyperplastic connective tissue, and alveolar cells. No histopathologic changes were seen in the control animals. CONCLUSIONS: These results suggested that ET-1 may play an important role in the pathogenesis of OB. The effective pharmacologic antagonist or inhibitor may possibly control the progression of disease in patients of OB.

Animals↗

PubMed1997

Isolated lung perfusion with doxorubicin prolongs survival in a rodent model of pulmonary metastases.

BACKGROUND: We developed a rodent model of unilateral pulmonary metastases to evaluate long-term survival after isolated lung perfusion with doxorubicin. METHODS: In the model development study, on day 0, two groups of F344 rats (n = 15) underwent transient right pulmonary artery occlusion for either 5 or 10 minutes at the time of intravenous injection of methylcholantrene-induced sarcoma cells. On day 14, all animals were sacrificed and lung nodules counted. In the survival study, on day 0, 21 rats received intravenous injection of sarcoma cells with concomitant 10-minute right pulmonary artery occlusion. On day 7, eight rats underwent left isolated lung perfusion with doxorubicin (6.4 mg/kg); five rats underwent perfusion with buffered Hespan; six untreated rats were studied as controls. RESULTS: Ten of fifteen animals (67%) in the model study with 5-minute pulmonary artery occlusion had right-sided tumor nodules. Ten-minute occlusion resulted in a tumor-free right lung in all animals. In the survival study, all animals in the Hespan and control groups died of massive tumor replacement of the left lung, with median survival times of 20 and 18 days, respectively. The median survival time of 36 days for the animals undergoing isolated lung perfusion with doxorubicin was significantly longer (p < 0.00001). The left lung of two of the doxorubicin perfused rats was tumor-free at 6 weeks. CONCLUSIONS: Isolated lung perfusion with doxorubicin results in a durable response and prolongs survival in the treatment of experimental sarcoma pulmonary metastases.

Animals↗

PubMed1997

Efficient gene transfer method into the whole heart through the coronary artery with hemagglutinating virus of Japan liposome.

OBJECTIVE: To confirm gene transfer techniques especially into the whole heart, we tried out a gene transfer method involving liposome with the viral envelope hemagglutinating virus of Japan liposome as an alternative to existing techniques such as cationic lipofection or other viral vectors. METHOD: For this study, hemagglutinating virus of Japan liposome (H group) or cationic liposome (L group) was used to compare the efficacy of gene transfection of oligonucleotide labeled with fluorescein isothiocyanate and cDNA of beta-galactosidase and human manganese-superoxide dismutase. Fluorescein-labeled oligonucleotide, cDNA of beta-galactosidase, or manganese-superoxide dismutase was complexed with liposomes, DNA-binding nuclear protein, and the viral protein coat of hemagglutinating virus of Japan. After donor rat hearts arrested by cardioplegia had been harvested, the coronary artery during cardioplegic arrest was infused via an aortic cannula with the liposome-gene complex. Next, the hearts were transplanted into the abdomen of recipient rats of the same strain, and all recipients were put to death after 3 days of transfection. RESULTS: Fluorescein isothiocyanate was detected in the nuclei of more than 70% of the myocytes (75% +/- 14%, n = 5) in the H group compared with fewer than 10% in the L group (7% +/- 5%, n = 5). The intensity of fluorescein isothiocyanate was significantly higher in the H group (979 +/- 112 FI) than in the L group (116 +/- 68 FI). beta-Galactosidase was expressed in the cytosol of more than 50% of the myocytes in the H group (61% +/- 7%, n = 5) compared with none in the L group (0%, n = 5). After 3 days of gene transfection, and when exposed to ischemia (30 minutes, 37 degrees C) and reperfusion (30 minutes, 37 degrees C) with Langendorff apparatus, the hearts transfected with manganese-superoxide dismutase (S group, n = 5) showed a significantly higher percentage of recovery of left ventricular end-diastolic pressure (S vs C, 86% +/- 3% vs 54% +/- 12%) and coronary flow (98% +/- 2% vs 66% +/- 12%) than did the control hearts (C group, n = 5). Western blotting analysis showed an apparent increased expression of manganese-superoxide dismutase in the hearts transfected with manganese-superoxide dismutase compared with the control hearts. These results clearly demonstrated that the donor hearts were transfected with fluorescein-labeled oligonucleotide and the beta-galactosidase gene as a result of coronary infusion of the hemagglutinating virus of Japan liposome during cardioplegic arrest at the time of harvest. Furthermore, the hearts transfected with manganese-superoxide dismutase showed significant improvement in tolerance against ischemia reperfusion injury. CONCLUSION: We believe that this method represents a novel in vivo gene transfer technique for the heart and thus may provide a new tool for research and therapy of heart transplantation.

Animals↗

PubMed1997

Pathogenesis of murine toxoplasmic hydrocephalus.

The prevailing hypothesis concerning the pathogenesis of toxoplasmic hydrocephalus alleges that (a) parasites invade and destroy the ependymal lining of the lateral ventricles, followed by (b) the sloughing of masses of degenerating ependymal and inflammatory cells leading to obstruction of the ventricular foramina and aqueduct of Sylvius, thereby initiating the hydrocephalus. Our observations in chronically infected mice indicate otherwise. Parasite invasion of the ependyma was not detected; the intraventricular masses of cellular 'debris' contained neither ependymal nor inflammatory cells; and obstruction of the ventricular foramina and/or aqueduct was not seen. As an alternative hypothesis, we suggest the development of hydrocephalus in the infected mice was consequent to severe leptomeningeal inflammation blocking the subarachnoid space and impeding the resorption of cerebrospinal fluid by the arachnoid villi. Narrowing of the aqueduct of Sylvius, when present, was adjudged the result, not the cause of the hydrocephalus, due to compression of the midbrain by the enlarging lateral ventricles.

Animals↗

PubMed1997

In vivo transfection of cis element "decoy" against nuclear factor-kappaB binding site prevents myocardial infarction.

The transcriptional factor nuclear factor-kappaB (NFkappaB) plays a pivotal role in the coordinated transactivation of cytokine and adhesion molecule genes that might be involved in myocardial damage after ischemia and reperfusion. Therefore, we hypothesized that synthetic double-stranded DNA with high affinity for NFkappaB could be introduced in vivo as "decoy" cis elements to bind the transcriptional factor and to block the activation of genes mediating myocardial infarction, thus providing effective therapy for myocardial infarction. Treatment before and after infarction by transfection of NFkappaB decoy, but not scrambled decoy, oligodeoxynucleotides before coronary artery occlusion or immediately after reperfusion had a significant inhibitory effect on the area of infarction. Here, we report the first successful in vivo transfer of NFkappaB decoy oligodeoxynucleotides to reduce the extent of myocardial infarction following reperfusion, providing a new therapeutic strategy for myocardial infarction.

Animals↗

PubMed1997

Prevention of graft coronary arteriosclerosis by antisense cdk2 kinase oligonucleotide.

Graft coronary arteriosclerosis, which limits the long-term survival of allograft recipients, is characterized by diffuse intimal thickening composed of proliferative smooth muscle cells. We observed that messenger RNA of the cell cycle regulatory enzyme cyclin-dependent kinase (cdk) 2 kinase, which mediates smooth muscle cell proliferation, was elevated in the thickened intima of coronary arteries of murine heterotopic cardiac allografts. We studied the effects of antisense phosphorothioate oligodeoxynucleotide (ODN) against this enzyme using gene transfer mediated by a hemagglutinating virus of Japan (HVJ)-liposome complex intraluminally delivered to inhibit the intimal hyperplasia. At 30 days after transplantation, antisense cdk2 kinase ODN treatment had dramatically inhibited neointimal formation in the allografts. Expression of vascular cell adhesion molecule-1 was also suppressed by antisense cdk2 kinase. However, these effects were not observed in the sense or scrambled ODN-treated allografts. Thus, an intraluminal administration of antisense ODN directed to a specific cell cycle regulatory gene can inhibit neointimal formation after cardiac transplantation.

Animals↗

PubMed1997

Survival of grafts of genetically modified cardiac myocytes transfected with FITC-labeled oligodeoxynucleotides and the beta-galactosidase gene in the noninfarcted area, but not the myocardial infarcted area.

Since one of the attractions of gene therapy in the heart is the implantation of genetically modified cultured cells, we employed genetically modified myocytes transfected with FITC-labeled oligodeoxynucleotides (ODN) and beta-galactosidase gene in this study, to investigate the cellular localization and fate of grafted myocytes in the heart. In addition, we examined the feasibility of myocytes grafting into a myocardial infarction model. Delivery of FITC-labeled ODN with the HVJ-liposome method resulted in sustained fluorescence as compared to direct transfer of 'naked' ODN. Interestingly, implantation of cardiac myocytes transfected with FITC-labeled ODN ex vivo by the HVJ-liposome method resulted in sustained fluorescence for at least 1 week in the noninfarcted area, whereas little fluorescence was detected in the area of infarction. This observation was confirmed by measurement of fluorescence, which showed significantly higher levels in the noninfarcted area than the infarcted area. Positive staining for beta-galactosidase protein was also clearly observed 7 days after grafting of transfection of the beta-galactosidase gene, while no staining was detected in grafted myocytes in control rats. Survival of implanted genetically modified cardiac myocytes in the noninfarcted, but not infarcted area, provides new information for the local delivery of recombinant molecules to the heart using gene therapy.

Animals↗

PubMed1997

HVJ (Sendai virus)-cationic liposomes: a novel and potentially effective liposome-mediated technique for gene transfer to the airway epithelium.

We designed a novel technique for targeted gene transfer into the airway epithelium. This was constructed using multilamellar cationic liposomes, containing N-(alpha-trimethylammonioacetyl)-didodecyl-D-glutamate chloride, phosphatidylcholine and cholesterol and fused with haemagglutinating virus of Japan (HVJ), namely HVJ cationic liposomes. Single aerosol delivery of this novel vector to the airway of rats led to a highly efficient and widespread transduction of fluorescein isothiocyanate-labeled oligonucleotides or lacZ gene into the bronchial epithelium and alveolar macrophages, but not into the alveolar epithelium. The efficiency of gene transfer to the airway epithelium with a single administration of the lacZ gene was about 47.6% in the trachea, 39.0% in the bronchi and proximal bronchioli, and 2.9% in the terminal bronchioli, respectively (mean value, n = 6). Expression level of the luciferase gene delivered with this novel system was much higher than that without HVJ, in both the trachea and lung tissue. Two pretreatment HVJ-cationic liposome vehicles every other week resulted in minimal inflammatory infiltration in the sub-epithelial layer with no significant reduction in efficiency of the following gene transfer. We propose that this novel HVJ cationic liposome-mediated gene transfer system may be suitable for clinical gene therapy to treat subjects with lethal lung diseases such as cystic fibrosis.

Animals↗

PubMed1997

Gene delivery by HVJ-liposome in the experimental gene therapy of murine glioma.

We investigated the delivery of foreign genes into mouse glioma cells in vivo using hemagglutinating virus of Japan (HVJ)-liposomes, which are coated by Sendai virus envelope protein. HVJ-liposomes, containing lacZ gene or herpes simplex virus thymidine kinase (HSVtk) gene-bearing plasmid DNA were applied in the meningeal gliomatosis (MG) mouse model system. Highly efficient delivery was observed in disseminated glioma cells, and 80% of MG mice expressing the HSVtk gene were cured by treatment with ganciclovir. These results suggest that this novel gene delivery system may be applicable for the in vivo gene therapy of human malignant glioma.

Animals↗

PubMed1997

Induction of antibody response to human tumor antigens by gene therapy using a fusigenic viral liposome vaccine.

Development of effective cancer vaccines would help prevent and control tumor progression. A novel approach of immunizing against tumor antigens is in vivo gene vaccination. We have developed a fusigenic viral liposome vector using HVJ (hemagglutinating virus of Japan) and liposome to deliver human tumor antigen genes effectively to cells in vivo. Plasmids containing the human tumor antigen genes MAGE-1 and MAGE-3 were encapsulated in fusigenic viral liposomes and injected into mice intramuscularly. MAGE-1 and -3 recombinant proteins were used in Western blotting and affinity ELISA for assessment of antibody responses. Mice immunized with MAGE-1 and -3 gene vaccine individually were shown to produce anti-MAGE-1 and -3 IgG antibody responses respectively. Animals immunized with plasmid alone did not induce anti-MAGE-1 or -3 IgG responses. Antibody responses could be enhanced on reimmunization with the gene vaccines. Muscle biopsies taken after vaccine injection were verified to express gene-specific mRNA transcripts. Mice immunized with MAGE-1 or -3 gene vaccines were shown to induce antibodies that could cross-react with the respective recombinant proteins. This study demonstrates that in vivo immunization using HVJ-liposome containing human tumor antigen genes can effectively deliver and induce immune responses to the respective whole proteins.

Animals↗

PubMed1997

Antimetastatic effect of synthetic Glu-Ile-Leu-Asp-Val peptide derivatives containing D-amino acids.

The aim of this study was to increase the antimetastatic potency of the fibronectin-related peptide, Glu-Ile-Leu-Asp-Val (EILDV), and to determine the minimal core sequence of EILDV required to inhibit tumor metastasis in vivo. The EILDV subpeptide analog, ILDV, markedly inhibited the adhesion of B16-BL6 melanoma cells to fibronectin. EILD and ILD were only slightly inhibitory, and the smaller overlapping tripeptide, LDV, was inactive. The inhibitory activities of ILDV and LDV on the migration of B16-BL6 melanoma cells were as potent as those of EILDV, whereas ILD did not inhibit cell migration. These results suggested that the minimal sequences essential for cell adhesion and migration are ILD and LDV, respectively. However, the antimetastatic effects of all subpeptide analogs were lower than that of EILDV. In order to improve the stability in vivo, we synthesized various EILDV-related peptides substituted with a D-amino acid. EILDV containing D-Glu or D-Ile inhibited cell adhesion and migration as potent as EILDV, whereas replacing Leu, Asp or Val with the corresponding D-isomer reduced the antiadhesive activities. The inhibitory effect of EILDV-related peptides containing D-Leu, D-Asp or D-Val on migration was also lower than that of EILDV. All synthetic EILDV-related peptides containing D-amino acids inhibited metastasis by B16-BL6 melanoma cells to the same extent as EILDV, whereas the specific activity of EILDV was decreased by the D-amino acid substitution. These results indicated that the balance of stability in vivo and biological activity in vitro is important in inhibiting tumor metastasis.

Amino Acid Sequence↗

PubMed1997

In vivo transfer of a foreign gene to keratinocytes using the hemagglutinating virus of Japan-liposome method.

The hemagglutinating virus of Japan (HVJ)-liposome method involves the entrapment of DNA and nuclear protein within liposomes and the use of HVJ to enhance liposome fusion with cell membranes. This method has been used successfully for in vivo gene transfer to various types of tissue. In this study, we investigated whether this method transfers genes effectively to normal and malignantly transformed keratinocytes in vivo. We applied HVJ-liposome complex (HLC) containing the beta-galactosidase gene to the tape-stripped skin of hairless rats and detected the enzyme activity in the keratinocytes of the treated skin. Comparison of this method with the naked DNA injection method, which was shown recently to be useful for in vivo gene transfer to keratinocytes, demonstrated that the transfer efficiency of the latter was about 5 times higher than that of the former. We assessed the efficacy of the HVJ-liposome method for gene transfer to transformed keratinocytes by examining the effect of HLC containing the herpes simplex virus thymidine kinase gene on the growth of mouse squamous cell carcinomas. Local injection of HLC into the tumors followed by administration of ganciclovir to mice resulted in tumor growth inhibition. These results indicate that the HVJ-liposome method is suitable for in vivo gene transfer to keratinocytes; also that this method may prove a good tool for basic research into keratinocyte biology and future keratinocyte gene therapy.

Animals↗

PubMed1997

Preparation of a monoclonal antibody specific for Entamoeba dispar and its ability to distinguish E. dispar from E. histolytica.

A monoclonal antibody (MAb), MAb ED17 (immunoglobulin G2a [IgG2a]), prepared against trophozoites of Entamoeba dispar SAW1734RclAR cultured monoxenically with Crithidia fasciculata, reacted with 25 of 26 isolates of E. dispar by an indirect fluorescent-antibody test. In contrast, the MAb failed to react with any of 20 isolates of E. histolytica or other enteric protozoan parasites. Western blot (immunoblot) analysis showed that the molecular mass of the E. dispar antigen recognized by the MAb was 160 kDa under reduced conditions. Immunoelectron microscopy revealed that the antigen was mainly located on digested C. fasciculata, but not on undigested organisms. Double staining with a mixture of MAb ED17 and MAb 4G6 (an IgG1 MAb which reacts exclusively with E. histolytica), followed by incubation with a mixture of fluorescein isothiocyanate-labeled anti-mouse IgG2a and tetramethylrhodamine isothiocyanate-labeled anti-mouse IgG1 antibodies, simultaneously identified mixed populations of E. dispar and E. histolytica. This method may prove to be useful for the accurate identification of E. dispar and E. histolytica, even in mixed infections.

Animals↗

PubMed1997

Abnormal structure of the Y chromosome detected in bovine gonadal hypoplasia (XY female) by FISH.

The structure of the Y chromosome was investigated by FISH and G banding in 3 cases of bovine XY females shown by PCR to lack the SRY gene. Although 2 different repeat-DNA sequences (BC1.2 and btDYZ-1) hybridized to the short arm of the Y chromosome in a normal bull, they hybridized to both arms of the Y chromosome in the XY females. In the Y chromosome from XY females, only the centromeric region was darkly stained by G banding, in comparison with dark staining on the long arm of a normal Y chromosome. From the results obtained, it seemed that the Y chromosome from the XY females was structurally abnormal, namely an isochromosome with an identical short arm. Our results also confirm that SRY is located on the long arm of the Y chromosome in bovine.

Animals↗

PubMed1997

Molecular Delivery System for Antisense Oligonucleotides: Enhanced Effectiveness of Antisense Oligonucleotides by HVJ-liposome Mediated Transfer.

BACKGROUND: The effectiveness of antisense oligodeoxynucleotides for in vitro and in vivo studies is limited by a low efficiency of cellular uptake and instability due to degradation by nucleases. To overcome some of these problems, we recently developed a transfer method that utilizes inactivated Sendai virus (hemagglutinating virus of Japan [HVJ]) complexed with liposomes to deliver antisense oligodeoxynucleotides. In this study, we compared the effectiveness of the HVJ-liposome method versus a cationic liposome method versus passive uptake to deliver antisense oligodeoxynucleotides against basic fibroblast growth factor on angiotensin (Ang) II-induced rat vascular smooth muscle cell growth. METHODS AND RESULTS: Twenty to twenty-eight hours after transfection, antisense fibroblast growth factor oligodeoxynucleotides introduced by passive uptake and HVJ-liposome method decreased basal DNA synthesis significantly as compared to the sense, control, and scrambled oligodeoxynucleotides groups; however, 60-68 hours after transfection, only antisense fibroblast growth factor oligodeoxynucleotides transduced by the HVJ-liposome method resulted in a significant inhibition of DNA synthesis under basal and Ang II-(10(-;6)M) stimulated conditions. The IC(25) of oligodeoxynucleotides assessed by the inhibition of thymidine incorporation was significantly lower using the HVJ-liposome method than those using the other transfer methods. To clarify the mechanisms of cellular uptake of oligodeoxynucleotides with the HVJ-liposome method, we studied the cellular fate of fluorescein isothiocyanate (FITC)-labeled oligodeoxynucleotides FITC-oligodeoxynucleotides was localized in nuclei at 5 minutes after transfection with the HVJ-liposome method. In contrast, FITC-oligodeoxynucleotides introduced by passive uptake was detected in nonnuclear cellular compartments, possibly endosomes, but not the nuclei. Cellular fluorescence of oligodeoxynucleotides introduced by passive uptake disappeared within 24 hours, while that introduced by the HVJ-liposome method could be observed up to 72 hours. CONCLUSION: These results demonstrate that the HVJ-liposome transfer enhanced the effectiveness of AS-fibroblast growth factor via its specific molecular mechanisms of transfer.

Journal Article↗

PubMed1997

Amino acids and peptides. XXX. Preparation of Arg-Gly-Asp (RGD) hybrids with poly(ethylene glycol) analogs and their antimetastatic effect.

Hybrids of a fibronectin-related peptide[Arg-Gly-Asp (RGD)] with poly(ethylene glycol) (PEG) analogs were prepared by a simple and easy procedure. Two amino-PEG analogs were used as carriers for hybrid formation of the RGD. One was poly(oxyethylene)dipropylamine and the other was Jeffamine ED type, which has branched chains. RGD peptides were formed stepwise on PEG analogs by the diisopropylcarbodiimide method. The synthetic intermediates were easily purified by molecular-sieve gel chromatography and the final products were purified by molecular-sieve gel chromatography, followed by HPLC. This simple and easy preparation procedure using molecular-sieve gel chromatography for purification of synthetic intermediates is advantageous for the preparation of peptide-polymer hybrids. We found that PEG is stable to HF treatment at 0 degree C for 1 h. The inhibitory effect of the RGD hybrids on experimental metastasis of B16-BL6 was examined in mice. The Jeffamine type hybrid showed no inhibitory effect at the dose of 1 mg/mouse, but poly(oxyethylene)dipropylamine type hybrid was inhibitory at the same dose. The effect of the latter hybrid was about the same as that of 1 mg of RGD. One mg of the hybrid contains 0.18 mumol of RGD and 1 mg of RGD is 2.38 mumol. Thus it can be said that the inhibitory effect of RGD was potentiated by hybrid formation with poly(oxyethylene)diisopropylamine.

Animals↗

PubMed1997

The possibility of early estimation for fertility in bovine heterosexual twin females.

To diagnose the possibility of early estimation for fertility in bovine heterosexual twin females, we designed a new diagnostic program. The 9 freemartins (FM) and 5 normal females (Normal) were used in this study. All 14 cases, at 4 months of age, were given Pregnant Mare Serum Gonadotrophin (PMSG) and human Chorionic Gonadotrophin (hCG) 1.5-2 days later. Thereafter, the concentration of estradiol-17 beta (E2) was determined by RIA, and that of progesterone (P) was done by RIA and EIA (Ovcheck EIA Kit). The concentration of E2 in the Group of Normal rapidly increased after administration of PMSG, but in the Group of FM, the concentration of E2 changed in very low levels over 14 days. The concentration of P in the Group of Normal rapidly increased after administration of PMSG, but in the Group of FM, the concentration of P changed in very low levels over 14 days.

Aging↗