Aggregated chromosomes/chromatin transfer: a novel approach for mitochondrial replacement with minimal mitochondrial carryover: the implications of mouse experiments for human aggregated chromosome transfer.

Nuclear transfer techniques, including spindle chromosome complex (SC) transfer and pronuclear transfer, have been employed to mitigate mitochondrial diseases. Nevertheless, the challenge of mitochondrial DNA (mtDNA) carryover remains unresolved. Previously, we introduced a method for aggregated chromosome (AC) transfer in human subjects, offering a potential solution. However, the subsequent rates of embryonic development have remained unexplored owing to legal limitations in Japan, and animal studies have been hindered by a lack of AC formation in other species. Building upon our success in generating ACs within mouse oocytes via utilization of the phosphodiesterase inhibitor 3-isobutyl 1-methylxanthine (IBMX), this study has established a mouse model for AC transfer. Subsequently, a comparative analysis of embryo development rates and mtDNA carryover between AC transfer and SC transfer was conducted. Additionally, the mitochondrial distribution around SC and AC structures was investigated, revealing that in oocytes at the metaphase II stage, the mitochondria exhibited a relatively concentrated arrangement around the spindle apparatus, while the distribution of mitochondria in AC-formed oocytes appeared to be independent of the AC position. The AC transfer approach produced a marked augmentation in rates of fertilization, embryo cleavage, and blastocyst formation, especially as compared to scenarios without AC transfer in IBMX-treated AC-formed oocytes. No significant disparities in fertilization and embryo development rates were observed between AC and SC transfers. However, relative real-time PCR analyses revealed that the mtDNA carryover for AC transfers was one-tenth and therefore significantly lower than that of SC transfers. This study successfully accomplished nuclear transfers with ACs in mouse oocytes, offering an insight into the potential of AC transfers as a solution to heteroplasmy-related challenges. These findings are promising in terms of future investigation with human oocytes, thus advancing AC transfer as an innovative approach in the field of human nuclear transfer methodology.

Research Note: Expression of IGF-1 and IGF-1 receptor proteins in skeletal muscle fiber types in chickens with hepatic fibrosis.

We investigated the expression of insulin-like growth factor 1 (IGF-1) and IGF-1 type 1 receptor (IGF-1R) in skeletal muscle fiber types in chickens with hepatic fibrosis induced by bile duct ligation (BDL). Eleven hens, approximately 104 weeks old, were randomly assigned to BDL (n = 4) and sham surgery (SHAM; n = 7) groups. In BDL hens, histopathology revealed marked bile duct proliferation and liver fibrosis. The cross-sectional area (CSA) of myofibers from both the pectoralis (PCT) muscles significantly decreased in the BDL group compared with the SHAM group (P < 0.01). In contrast, the CSA of myofibers from the femorotibialis lateralis (FTL) muscle did not decrease in the BDL group. Type I fibers were large, round, and hypertrophic. Elongated type IIA and IIB fibers were also present. For IGF-1 immunostaining, the immunoreaction intensity was higher in the PCT in the BDL group than the SHAM group. Within the BDL group, type I fibers from FTL had a stronger immunoreaction intensity than the type II fibers. For IGF-1R immunostaining, the intensity of the immunoreactions was similar within the PCT in the BDL group compared with the SHAM group. For FTL, type I fibers had stronger reactions to IGF-1R than type II fibers in the BDL group. These results suggest that type I fibers express both IGF-1 and IGF-1R and become hypertrophic in chickens with hepatic fibrosis.

Morphological characterization of small hepatocytes after bile duct ligation in chicken.

Small hepatocytes have hepatocyte-like characteristics and high proliferation activity. Most small hepatocyte studies report on in vitro rat or human hepatocytes. Only a few studies of small hepatocytes after bile duct ligation have been reported, and none of these have focused on these cells in birds. In this study, small hepatocytes appearing in bile duct ligation chicken liver were examined using the morphological method with histological, immunohistochemical, and ultrastructural studies. Nine Boris Brown hens (over 744-d old) were used. In all chickens, both the common hepatoenteric duct and hepatocystic duct were ligated, and the livers were examined 1, 4, 6, 9, and 13 weeks after bile duct ligation. Histologically, the small cells were half the size of normal liver cells, and mitotic figures were often observed. The nuclei showed two forms: large and small. Many small cells were negative for periodic acid-Schiff stain, but positive cells were rarely observed. The cells existed in colonies on the side of the sinusoid of the hepatic lamina. Immunohistochemically, the small cells with large nuclei were strongly positive for CD44, albumin and proliferation cell nuclear antigen, and the cells with small nuclei were weakly positive. In the CD44-positive cell colony, negative cells were often observed to have mature hepatocyte-like morphology. Moreover, many of the cells were PAN cytokeratin negative. Ultrastructurally, the small cells had more nuclei with rich heterochromatin, poor cytoplasmic organelles, and narrow cytoplasm with a high electron density than mature hepatocytes. Moreover, cells having a middle ultrastructural characteristic existed between the small cells and mature hepatocytes. The small hepatocellular colony of the chicken appeared as a regeneration-related change in the liver after bile duct ligation. The cell had high cell proliferation activity and morphological, immunohistochemical, and ultrastructural characteristics similar to those of the mammalian small hepatocyte, as well as a similar progenitor cell.

Lower thigh muscle mass is associated with all-cause and cardiovascular mortality in elderly hemodialysis patients.

BACKGROUND/OBJECTIVES: Higher body mass index appears protective in hemodialysis patients, although it remains to be determined which component of muscle or fat mass is primarily associated with this survival advantage. SUBJECTS/METHODS: Eighty-one hemodialysis patients in our institution were prospectively followed from July 2011 to August 2015. Muscle and fat mass were evaluated by measuring the cross-sectional areas of the thigh and abdomen using computed tomography. The relationship between muscle and fat mass, and all-cause and cardiovascular mortality was studied using the Kaplan-Meier analyses and multivariate Cox proportional hazard models. RESULTS: During more than 4 years of follow-up, 26 patients (32%) died. In the Kaplan-Meier curve analyses, lower thigh muscle mass was significantly associated with all-cause and cardiovascular mortality (log-rank test, P<0.01 and P<0.001, respectively), but there was no such association with thigh fat, abdominal muscle and fat mass levels. In multivariate Cox proportional hazard models, each 0.1 cm2/kg increase in the thigh muscle area adjusted by dry weight was associated with an estimated 22% lower risk of all-cause mortality (95% confidence interval (95% CI), 0.64-0.95, P<0.05) and a 30% lower risk of cardiovascular mortality (95% CI, 0.54-0.90, P<0.01). CONCLUSIONS: Lower thigh muscle mass is significantly associated with all-cause and cardiovascular mortality in hemodialysis patients. Our findings indicate the importance of focusing on the muscle mass of lower extremities to predict the clinical outcomes of hemodialysis patients.

Abundant FUS-immunoreactive pathology in the skin of sporadic amyotrophic lateral sclerosis.

OBJECTIVES: The fused in sarcoma (FUS) protein is a 526 amino acid and its expression is ubiquitous. Recently, mutations in a gene coding FUS have been identified in familial amyotrophic lateral sclerosis (ALS). Also, FUS has been found in neuronal cytoplasmic inclusions in sporadic forms of ALS, suggesting that FUS has an important role in the neurodegeneration occurring in sporadic disease. However, there has been no study of FUS in ALS skin. MATERIAL AND METHODS: We made a quantitative immunohistochemical study of the expression of FUS in the skin from patients with sporadic ALS and controls. RESULTS: The proportion of FUS-immunoreactive (ir) cells in the epidermis in ALS patients was significantly higher (P < 0.001) than in controls. There was a significant positive relationship (r = 0.78, P < 0.001) between the proportion and duration of illness in ALS patients. The optical density of FUS-ir cells in the epidermis in ALS patients is markedly stronger (P < 0.001) than in controls. There was a significant positive relation (r = 0.49, P < 0.05) between the immunoreactivity and duration of illness in ALS patients. CONCLUSIONS: These data suggest that changes of FUS in ALS skin are related to the disease process, and that metabolic alterations of FUS may take place in the skin of patients with ALS.

Protein degradation by the ubiquitin-proteasome pathway and organ fibrosis.

Abnormal and exaggerated deposition of extracellular matrix proteins is the common feature of fibrotic diseases. The resulting fibrosis disrupts the normal architecture of the affected organs and finally leads to their dysfunction and failure. At present, there are no effective therapies for fibrotic diseases. Protein degradation via the ubiquitin-proteasome system is the major pathway for non-lysosomal proteolysis and controls many critical cellular functions including cell-cycle progression, deoxyribonucleic acid repair, growth and differentiation. Therefore, aberration of the system leads to dysregulation of cellular homeostasis and development of many diseases such as cancers, degenerative diseases and fibrotic diseases. Although the ubiquitin-proteasome system has mainly been investigated in the field of cancers so far and several anti-cancer drugs that modulate the activity of the system have been used clinically, the recent findings regarding the system and fibrosis can provide a rational basis for the discovery of novel therapy for fibrotic diseases. In this article, we discuss (i) the basic mechanism of the ubiquitin-proteasome system and (ii) the recent findings regarding the association between the system and pathological organ fibrosis. These examples indicate that the ubiquitin-proteasome system plays diverse roles in the progression of fibrotic diseases, and further studies of the system are expected to reveal new strategies for overcoming pathological fibrosis.

Towards retinoid therapy for Alzheimer's disease.

Alzheimer's disease(AD) is associated with a variety of pathophysiological features, including amyloid plaques, inflammation, immunological changes, cell death and regeneration processes, altered neurotransmission, and age-related changes. Retinoic acid receptors (RARs) and retinoids are relevant to all of these. Here we review the pathology, pharmacology, and biochemistry of AD in relation to RARs and retinoids, and we suggest that retinoids are candidate drugs for treatment of AD.

Ubiquitin-dependent degradation of SnoN and Ski is increased in renal fibrosis induced by obstructive injury.

Transforming growth factor-beta (TGF-beta) plays a critical role in the progression of renal fibrosis. The activity of TGF-beta is tightly controlled by various mechanisms, among which antagonizing Smad-mediated gene transcription by co-repressors represents one of the important components. We investigated the expression, degradation, and ubiquitination of Smad transcriptional co-repressors SnoN (ski-related novel gene N) and Ski (Sloan-Kettering Institute proto-oncogene) in renal fibrogenesis. We also studied the involvement of Smad-ubiquitination regulatory factor 2 (Smurf2) in ubiquitination of SnoN protein. The kidneys of mice with unilateral ureteral obstruction (UUO) and those of sham-operated mice were used. Renal lesions and the expression of TGF-beta1, type I collagen, SnoN, Ski, and Smurf2 were examined by immunohistochemistry, Western blot, and/or real-time reverse transcriptase-polymerase chain reaction. Degradation and ubiquitination of SnoN/Ski proteins were also investigated. The obstructed kidneys of UUO mice showed progressive tubulointerstitial fibrosis, high expression levels of TGF-beta1, type I collagen, SnoN and Ski mRNAs, and low levels of SnoN and Ski proteins. Both degradation and ubiquitination of SnoN/Ski proteins were markedly increased in the obstructed kidneys, in which Smurf2 expression was increased. Smurf2 immunodepletion in extracts of obstructed kidneys resulted in reduced ubiquitination of SnoN. Our results suggest that the reduction of SnoN/Ski proteins resulting from increased ubiquitin-dependent degradation is involved in the progression of tubulointerstitial fibrosis.

HER-2/neu expression in ovarian clear cell carcinomas.

HER-2 /neu is a 185-kDa glycoprotein and a transmembrane receptor with tyrosine kinase activity. Its overexpression is observed in 25-30% of primary breast carcinomas and is associated with a poor clinical prognosis. Recently, the U.S. Food and Drug Administration and the Japanese Ministry of Health, Welfare, and Labor approved the use of trastuzumab (Herceptin, Genentech, South San Francisco, CA) for the treatment of patients with metastatic breast carcinomas overexpressing HER-2 /neu. Results of clinical trials with Herceptin suggest that it may prolong the survival of patients with advanced metastatic breast carcinoma. Relatively little is known concerning the relationship between HER-2 /neu status and ovarian clear cell carcinoma. If HER-2 /neu overexpression status were demonstrable in ovarian clear cell carcinoma and a clinical correlation between overexpression and prognosis could be established, a rationale for clinical use of Herceptin for this tumor could be established. Our aim was to evaluate HER-2 /neu status in ovarian clear cell carcinomas. Fifteen ovarian clear cell carcinoma cases were immunostained for HER-2 /neu using HercepTest (DAKO, Glostrup, Denmark). Overexpression of HER-2 /neu was detected in only one case. Unlike in breast carcinoma, HER-2 /neu overexpression appeared to be uncommon in ovarian clear cell carcinomas. Herceptin may thus target only a small proportion of ovarian clear cell carcinomas and be of limited clinical value for treatment of this carcinoma.

Focal segmental glomerulosclerosis in a case of panhypopituitarism: a possible role of growth hormone treatment.

Panhypopituitarism manifests various symptoms including growth failure, hypothyroidism, adrenal insufficiency and hypogonadism. Dwarfism is an important problem in children with this condition, and long-term treatment with recombinant human growth hormone (GH) is usually required. We report a 24-year-old man with panhypopituitarism complicated by focal segmental glomerulosclerosis (FSGS). The patient had been treated with GH for hypopituitary dwarfism from 3 years of age. Proteinuria was initially noticed at 15 years of age and persisted despite cessation of GH supplementation at 18 years of age. A renal biopsy specimen showed glomerular hypertrophy and limited glomerulosclerosis, compatible with FSGS. To our knowledge, this is the first reported case of panhypopituitarism complicated by FSGS. Our case suggests that GH treatment for dwarfism may induce irreversible glomerular disease.

Renin-producing serous cystoadenocarcinoma of the ovary: a case report.

BACKGROUND: Only a few renin-producing ovarian tumors have been reported, and most such ovarian tumors have been sex cord/stromal tumors. Renin-producing ovarian epithelial tumors are quite rare. CASE: A 46-year-old woman presented with hypertension and hypokalemia. Examinations of the patient revealed elevated plasma renin activity, hyperaldosteronism and a pelvic mass. Subsequently, a right ovarian tumor mass was resected. Microscopic observation of the tumor revealed a well-differentiated serous cystadenocarcinoma. Immediately after surgery, blood pressure, serum potassium, plasma renin activity and plasma aldosterone levels returned to normal ranges. RT-PCR analysis and immunohistochemical staining of this tumor indicated that it was producing renin. CONCLUSION: This is the first report of a renin-producing ovarian serous cystadenocarcinoma.

Pseudohyperkalemia occurring in a patient with chronic renal failure and polycythemia vera without severe leukocytosis or thrombocytosis.

Pseudohyperkalemia is defined as a serum potassium concentration 0.4 mEq/l greater than the plasma concentration. The basis of this phenomenon is the release of intracellular potassium from platelets, leukocytes, or erythrocytes, commonly in the setting of extreme leukocytosis (> 10 x 10(4)/microl) or thrombocytosis (> 60 x 10(4)/microl). We report a case of pseudohyperkalemia in a patient with chronic renal failure and polycythemia vera without the finding of severe leukocytosis or thrombocytosis (white blood cell count 1.88 x 10(4)/microl and platelet count 37.9 x 10(4)/microl, respectively). The serum potassium concentration was 8.2 mEq/l, while the plasma potassium level was 6.4 mEq/l in a sample obtained simultaneously. The concentrations of platelet factor IV and beta-thromboglobulin, known to be markers of platelet activation, were greater than 100 ng/ml and 200 ng/ml, respectively, indicating that platelet activation may have been related to the development of pseudohyperkalemia in this patient. These findings suggest that pseudohyperkalemia should be considered when hyperkalemia is seen in a patient with chronic renal failure and myeloproliferative disorders.

[A case of ovarian cancer with metastatic tumor in the liver with paclitaxel and carboplatin systemic chemotherapy was very effective].

An 82-year-old woman was admitted to the Dept. of Obstetrics and Gynecology, Yamanashi Medical University to try to identify the origin of a liver metastatic tumor. CT examination revealed a small tumor located adjacent to the uterine cervix in a cul-de-sac. With biopsy using MR, it was clearly shown histologically that the origin of the tumor was the ovary. Systemic chemotherapy with paclitaxel and carboplatin was selected as the most reasonable treatment for this case because of the patient's age. After 6 courses of this chemotherapy, the tumor in the cul-de-sac disappeared and the tumor in the liver decreased markedly. Furthermore, no severe side effects were seen during this treatment. This result indicated that systemic chemotherapy with paclitaxel and carboplatin is effective and safe in cases of advanced ovarian cancer.

Hypercalcemia in a patient with B-cell acute lymphoblastic leukemia: a role of proinflammatory cytokine.

The complication of hypercalcemia is reported to occur only in 2.5-4.8% of patients with acute lymphoblastic leukemia (ALL). We herein report a 53-year-old female patient with early B-cell ALL, complicated with extreme hypercalcemia (15.2 mg/dL). Bone X-ray revealed osteolytic changes in many locations. Serum 1,25(OH)2vitaminD3 and parathyroid hormone (PTH) levels were suppressed below normal ranges on admission. The circulating parathyroid hormone-related protein (PTHrP) value was within a normal range (< 1.1 pmol/L). Serum concentrations of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and soluble IL-2 receptor were increased to 72 pg/ml, 25.3 pg/ml, and 1469 U/ml, respectively. Following the induction chemotherapy, the serum calcium level was promptly normalized accompanied with decreases in serum TNF-alpha, IL-6 and soluble IL-2 receptor values to 34 pg/ml, 6.35 pg/ml, and 737 U/ml, respectively. Serum PTHrP values remained within detectable levels. To our knowledge, this is the first case of B-cell ALL in a patient who developed hypercalcemia with elevated concentrations of TNF-alpha, IL-6, and soluble IL-2 receptor, but not related to PTHrP. High circulating proinflammatory cytokines may have contributed to development of ALL-induced osteolysis and hypercalcemia in the present case.

Potent estrogen agonists based on carborane as a hydrophobic skeletal structure. A new medicinal application of boron clusters.

BACKGROUND: Carboranes (dicarba-closo-dodecaboranes) are a class of carbon-containing polyhedral boron-cluster compounds having remarkable thermal stability and exceptional hydrophobicity. Applications of the unique structural and chemical properties offered by icosahedral carboranes in boron neutron capture therapy have received increasing attention over the past 30 years. However, these features of carboranes may allow another application as a hydrophobic pharmacophore in biologically active molecules that interact hydrophobically with receptors. RESULTS: We have designed candidate estrogen-receptor-binding compounds having carborane as a hydrophobic skeletal structure and synthesized them. The most potent compound bearing a carborane cage exhibited activity at least 10-fold greater than that of 17beta-estradiol in the luciferase reporter gene assay. Estrogen receptor-alpha-binding data for the compound were consistent with the results of the luciferase reporter gene assay. The compound also showed potent in vivo effects on the recovery of uterine weight and bone loss in ovariectomized mice. CONCLUSION: Further development of the potent carborane-containing estrogenic agonists described here, having a new skeletal structure and unique characteristics, should yield novel therapeutic agents, especially selective estrogen receptor modulators. Furthermore, the suitability of the spherical carborane cage for binding to the cavity of the estrogen receptor-alpha ligand-binding domain should provide a basis for a similar approach to developing novel ligands for other steroid receptors.

Novel retinoidal tropolone derivatives. Bioisosteric relationship of tropolone ring with benzoic acid moiety in retinoid structure.

Several tropolone derivatives (4-7) were designed as novel retinoids on the assumption that the tropolone ring may mimic the benzoic acid moiety in retinoid structures, such as Am80 (2). Among the synthesized compounds, 5-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)ethynyl]tropolone (7a) showed moderate potency as a differentiation-inducer of HL-60 cells. The activities of the tropolones were greatly enhanced in the presence of HX630, an RXR agonist (retinoid synergist).

Tuberculous arthritis mimicking neoplasm in a hemodialysis patient.

Hemodialysis patients are known to develop the complication of extrapulmonary tuberculosis more frequently than the general population. Tuberculous arthritis is a rare form of extrapulmonary tuberculosis and is reported to occur in approximately 1% of cases in nonuremic patients. Only 3 cases in dialysis patients, who were not proven by a bacterial culture or had died before treatment, have been reported. We report herein a culture-proven case of tuberculous arthritis developing at the sternoclavicular joint, which initially mimicked an apparent neoplasm in a hemodialysis patient. A favorable outcome was obtained after antituberculous therapy. Tuberculosis must be considered one of the most significant diagnoses in hemodialysis patients who present with a tumor-like lesion.

Retinoidal pyrimidinecarboxylic acids. Unexpected diaza-substituent effects in retinobenzoic acids.

Several pyridine- and pyrimidine-carboxylic acids were synthesized as ligand candidates for retinoid nuclear receptors, retinoic acid receptors (RARs) and retinoic X receptors (RXRs). Although the pyridine derivatives, 6-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carbamoyl]pyri dine-3-carboxylic acid (2b) and 6-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]py ridine-3-carboxylic acid (5b) are more potent than the corresponding benzoic acid-type retinoids, Am80 (2a) and Am580 (5a), the replacement of the benzene ring of Am580 (5a), Am555 (6a), or Am55 (7a) with a pyrimidine ring caused loss of the retinoidal activity both in HL-60 cell differentiation assay and in RAR transactivation assay using COS-1 cells. On the other hand, pyrimidine analogs (PA series, 10 and 11) of potent RXR agonists (retinoid synergists) with a diphenylamine skeleton (DA series, 8 and 9) exhibited potent retinoid synergistic activity in HL-60 cell differentiation assay and activated RXRs. Among the synthesized compounds, 2-[N-n-propyl-N-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)a mino]pyrimidine-5-carboxylic acid (PA013, 10e) is most active retinoid synergist in HL-60 assay.

Enhancing effect of tumor promoters, phorbol esters and teleocidins on nuclear receptor-mediated transcription.

Interaction between a tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA), and ligands of nuclear receptors has been interpreted as the result of crosstalk between the nuclear receptors and oncogenic transcription factor AP-1. We examined the effects of various tumor promoters on transcription mediated by several nuclear receptors (RAR, TR, and ROR) by using thymidine kinase promoter-based reporter systems. TPA-type and other types of tumor promoters (okadaic acid, thapsigargin) enhanced reporter gene transcription independently of the cognate ligands for the receptors. Various kinds of TPA-type tumor promoters, teleocidine and its synthetic derivatives (indolactam, benzolactams) enhanced reporter gene transcription in proportion to their differentiation-inducing activities. Although TPA is known to activate protein kinase C (PKC), some PKC inhibitors did not inhibit the effect of TPA on reporter gene transcription. Interestingly, staurosporin, a strong PKC inhibitor and also a tumor promoter, enhanced the effect of TPA and weakly enhanced the reporter transcription itself. These results suggest this reporter system is useful for the evaluation of effects on the gene expression of various tumor promoters, including non-TPA type.

Electric field assisted thermal desorption ionization using an infrared laser.

A new technique has been developed for the formation of gas-phase electrons and ions by electric field assisted thermal desorption ionization at atmospheric pressure. Experiments were carried out using a sharp tungsten wire tip coated with a thin solid sample film which was irradiated by a 10.6 µm infrared laser. By applying a strong electric field on the laser-irradiated tungsten wire tip, abundant ions such as alkali ions, halide ions, and also multiply charged negative ions S(2)O(6)(2-) and S(2)O(8)(2-), were formed. Copyright 1999 John Wiley & Sons, Ltd.