Safer Vitrification of Mouse and Human Embryos Using the Novel Cryoroom Vitrification System for Assisted Reproductive Technology.

BACKGROUND: Vitrification is widely used for assisted reproductive technology (ART). Most vitrification devices require the skillful placement of embryos into the carrier and aspiration of excessive vitrification solution. OBJECTIVE: To evaluate the efficacy and safety of the Cryoroom as a vitrification device. MATERIALS AND METHODS: Mouse and human embryos were vitrified with Cryoroom or Cryotop, and the developmental potency was assessed in vitro. Mouse monozygotic twin blastocysts were vitrified with Cryoroom or Cryotop for microarray analysis. RESULTS AND DISCUSSION: In mouse and human embryos, there were no differences between the survival and developmental progress in each device. In silico, the Cryoroom device showed no changes, particularly in DNA methylation after vitrification compared with the Cryotop. These results showed that the form and function of the device may affect the gene expression levels in vitrified embryos. CONCLUSION: The Cryoroom represents a safe and potentially revolutionary vitrification device for ART.

Comparison of mouse and human ankles and establishment of mouse ankle osteoarthritis models by surgically-induced instability.

OBJECTIVE: Prevalence of ankle osteoarthritis (OA) is lower than that of knee OA, however, the molecular mechanisms underlying the difference remain unrevealed. In the present study, we developed mouse ankle OA models for use as tools to investigate pathophysiology of ankle OA and molecular characteristics of ankle cartilage. DESIGN: We anatomically and histologically examined ankle and knee joints of C57BL/6 mice, and compared them with human samples. We examined joints of 8-week-old and 25-month-old mice. For experimental models, we developed three different ankle OA models: a medial model, a lateral model, and a bilateral model, by resection of respective structures. OA severity was evaluated 8 weeks after the surgery by safranin O staining, and cartilage degradation in the medial model was sequentially examined. RESULTS: Anatomical and histological features of human and mouse ankle joints were comparable. Additionally, the mouse ankle joint was more resistant to cartilage degeneration with aging than the mouse knee joint. In the medial model, the tibiotalar joint was markedly affected while the subtalar joint was less degenerated. In the lateral model, the subtalar joint was mainly affected while the tibiotalar joint was less altered. In the bilateral model, both joints were markedly degenerated. In the time course of the medial model, TdT-mediated dUTP nick end labeling (TUNEL) staining and Adamts5 expression were enhanced at early and middle stages, while Mmp13 expression was gradually increased during the OA development. CONCLUSION: Since human and mouse ankles are comparable, the present models will contribute to ankle OA pathophysiology and general cartilage research in future.

Pigmented spots as a sign of mammary Paget's disease.

Pigmented mammary Paget's disease is a rare variant of mammary Paget's disease. The clinical appearance mimics malignant melanoma. This paper describes a case of asymptomatic, slightly pigmented spots on the right mammary nipple. The pigmented nipple was histopathologically diagnosed as mammary Paget's disease with an underlying intraductal carcinoma. This case suggests the importance of conducting skin biopsies of developing pigmented spots on the nipples in elderly people.

Glenoid osteotomy for atraumatic posteroinferior shoulder instability associated with glenoid dysplasia.

Aims: We report the clinical results of glenoid osteotomy in patients with atraumatic posteroinferior instability associated with glenoid dysplasia. Patients and Methods: The study reports results in 211 patients (249 shoulders) with atraumatic posteroinferior instability. The patients comprised 63 men and 148 women with a mean age of 20 years. The posteroinferior glenoid surface was elevated by osteotomy at the scapular neck. A body spica was applied to maintain the arm perpendicular to the glenoid for two weeks postoperatively. Clinical results were evaluated using the Rowe score and Japan Shoulder Society Shoulder Instability Score (JSS-SIS); bone union, osteoarthrosis, and articular congruity were examined on plain radiographs. Results: The Rowe score improved from 36 to 88 points, and the JSS-SIS improved from 47 to 81 points. All shoulders exhibited union without progression of osteoarthritis except one shoulder, which showed osteoarthritic change due to a previous surgery before the glenoid osteotomy. All but three shoulders showed improvement in joint congruency. Eight patients developed disordered scapulohumeral rhythm during arm elevation, and 12 patients required additional open stabilization for anterior instability. Conclusion: Good results can be expected from glenoid osteotomy in patients with atraumatic posteroinferior instability associated with glenoid dysplasia. Cite this article: Bone Joint J 2018;100-B:331-7.

Molecular structure and transcriptional regulation of the gene for the platelet-derived growth factor alpha receptor in cultured vascular smooth muscle cells.

PDGF has been shown to contribute to hypertrophy in vascular smooth muscle cells (VSMC). PDGF-AA differentially promotes protein synthesis in VSMC from spontaneously hypertensive rats (SHR) but not in those from Wistar-Kyoto rats (WKY). This observation has led us to postulate a role for PDGF alpha receptor (PDGFR-alpha) in the hypertensive hypertrophy of blood vessels. Western and Northern blot analyses demonstrated a high and specific expression of the PDGFR-alpha protein and mRNA in SHR cells but not in WKY cells. To clarify the mechanism of the differential expression of the PDGFR-alpha gene, we isolated the promoter region of the gene. Studies on the promoter functions indicated that this promoter is active in SHR cells but not in WKY cells. The regulatory domain responsible for this difference was narrowed to the sequence between -246 and -139, which enhanced the promoter activity of SHR fivefold over the basal activity. DNase I footprinting and gel-shift assay indicated that this sequence specifically interact with nuclear proteins from VSMC through the binding site for CCAAT/enhancer-binding proteins, and members of the C/enhancer-binding protein family play a significant role in the strain-specific transcription of the PDGFR-alpha gene.

Alterations of the double-strand break repair gene MRE11 in cancer.

MRE11 plays a role in DNA double-strand break repair. Hypomorphic mutations of MRE11 have been demonstrated in ataxia-telangiectasia (AT)-like disorder. ATM mutations play a causal role in AT and have been demonstrated in lymphoid malignancies in patients without AT histories. By analogy with the relationship of ATM to lymphoid malignancies, it is probable that alterations of MRE11 are associated with tumor formation. We performed a mutation analysis of MRE11 in 159 unselected primary tumors. Three missense mutations at conserved positions were found in breast and lymphoid tumors. Additionally, an aberrant transcript without genomic mutation was found in a breast tumor. These findings suggest an occasional role for MRE11 alterations in the development of primary tumors.

Mutations in the RAD54 recombination gene in primary cancers.

Association of a recombinational repair protein RAD51 with tumor suppressors BRCA1 and BRCA2 suggests that defects in homologous recombination are responsible for tumor formation. Also recent findings that a protein associated with the MRE11/RAD50 repair complex is mutated in Nijmegen breakage syndrome characterized by increased cancer incidence and ionizing radiation sensitivity strongly support this idea. However, the direct roles of BRCA proteins and the protein responsible for NBS in recombinational repair are not clear though they are associated with the recombinational repair complexes. Since RAD51 forms a complex with other members of the RAD52 epistasis group and with BRCA proteins, it is reasonable to ask if alterations of members of the RAD52 epistasis group lead to tumor development. Here we describe missense mutations at functional regions of RAD54 and the absence of the wild-type RAD54 expression resulting from aberrant splicing in primary cancers. Since RAD54 is a recombinational protein associated with RAD51, this is the first genetic evidence that cancer arises from a defect in repair processes involving homologous recombination.

Mutations of a novel human RAD54 homologue, RAD54B, in primary cancer.

Association of breast tumor susceptibility gene products BRCA1 and BRCA2 with the RAD51 recombination protein suggested that cancer could arise through defects in recombination. The identification of NBS1, responsible for Nijmegen breakage syndrome, from the MRE11/RAD50 recombination protein complex also supports this hypothesis. However, our mutation analysis revealed that known members of the RAD52 epistasis group are rarely mutated in human primary cancer. Here we describe the isolation of a novel member of the SNF2 superfamily, characterized with sequence motifs similar to those in DNA and RNA helicases. The gene, designated RAD54B, is significantly homologous to the RAD54 recombination gene. The expression of RAD54B was high in testis and spleen, which are active in meiotic and mitotic recombination. These findings suggest that RAD54B may play an active role in recombination processes in concert with other members of the RAD52 epistasis group. RAD54B maps to human chromosome 8q21.3-q22 in a region associated with cancer-related chromosomal abnormalities. Homozygous mutations at highly conserved positions of RAD54B were observed in human primary lymphoma and colon cancer. These findings suggest that some cancers arise through alterations of the RAD54B function.

The intron 5-inserted form of rat erythropoietin receptor is expressed as a membrane-bound form.

The cDNA encoding an intron 5-inserted form of the erythropoietin receptor (I5Epo-R) has been cloned from rat. DNA sequence analysis reveals that the insertion of intron 5, which consists of 79 bp, causes a shift in reading frame and results in termination in the region of exon 7. The deduced amino acid sequence is composed of 316 amino acid residues, which is a molecular weight of 34220. To study the function of rat I5Epo-R, we established a Chinese hamster ovary cell line expressing rat I5Epo-R. Western blot analysis and binding studies with 125I-recombinant human erythropoietin showed that the transfected cells expressed rat I5Epo-R with a molecular size of 36 kDa as a membrane-bound form, but not as a soluble form, and had a single class of binding sites with a Kd of 700 pM.

Disordered expression of the sucrase-isomaltase complex in the small intestine in Otsuka Long-Evans tokushima fatty rats, a model of non-insulin-dependent diabetes mellitus with insulin resistance.

To clarify the relationship between diabetes mellitus and carbohydrate digestion, the activities of sucrase and isomaltase, which form a complex enzyme (SI complex) on the brush border membranes, were compared in the progression of diabetes mellitus in Otsuka Long-Evans Tokushima fatty (OLETF) rats, a model of human non-insulin-dependent diabetes mellitus with insulin resistance, and Long-Evans Tokushima Otsuka (LETO) rats as non-diabetic controls. Until 40 weeks of age, OLETF rats were obese and had a high plasma glucose level, compared to age-matched LETO rats, but the sucrase and isomaltase activities showed no significant differences between the two strains. Oral glucose tolerance test revealed that during 40-48 weeks of age, NIDDM became very severe with advancing insulin resistance in OLETF rats. In OLETF rats, in contrast to LETO rats, at 48 weeks of age, abnormal increases in the sucrase and isomaltase activities occurred, along with a remarkable decrease in body weight and a further great increase in the plasma glucose level in the non-fasting state. Hyperinsulinemia occurred in 20-week-old OLETF rats; however, at 40 and 48 weeks of age, the plasma insulin level in the non-fasting state in OLETF rats was not significantly different from that in LETO rats. The level of mRNA encoding the SI complex increased abnormally in 48-week-old OLETF rats. These results suggest that the advance of insulin resistance leads to an increase in the expression of the SI complex on the transcriptional level.

Cobalamin deficiency results in severe metabolic disorder of serine and threonine in rats.

Dietary cobalamin (vitamin B12; Cbl) deficiency caused significant increases in plasma serine, threonine, glycine, alanine, tyrosine, lysine and histidine levels in rats. In particular, the serine and threonine levels were over five and eight times, respectively, higher in the Cbl-deficient rats than those in the sufficient controls. In addition, some amino acids, including serine and threonine, were excreted into urine at significantly higher levels in the deficient rats. When Cbl was supplemented into the deficient rats for 2 weeks, in coincidence with the disappearance of the urinary excretion of methylmalonic acid (an index of Cbl deficiency), the plasma serine and threonine levels were normalized. These results indicate that Cbl deficiency results in metabolic disorder of certain amino acids, including serine and threonine. The expression level of hepatic serine dehydratase (SDH), which catalyzes the conversion of serine and threonine to pyruvate and 2-oxobutyrate, respectively, was significantly lowered by Cbl deficiency, even though Cbl does not participate directly in the enzyme reaction. The SDH activity in the deficient rats was less than 20% of that in the sufficient controls, and was normalized 2 weeks after the Cbl supplementation. It is thus suggested that the decrease of the SDH expression relates closely with the abnormalities in the plasma and urinary levels of serine and threonine in the Cbl-deficient rats.

Regulation of vascular angiotensin release.

To investigate the regulatory mechanism of the vascular renin-angiotensin system, we perfused isolated rat hind legs with plasma-free buffer and quantified angiotensin peptides in the perfusate. Angiotensin release from hind legs was increased in rats pretreated with losartan (DuP 753) and rats fed a low sodium diet with subsequent furosemide and was decreased in nephrectomized rats and rats given dexamethasone, ethynylestradiol, and triiodothyronine. Using these models, we have attempted to identify which step or component of angiotensin metabolism determines angiotensin release level. Changes caused by these manipulations in plasma renin concentration and basal angiotensin release from hind legs were almost parallel, whereas plasma angiotensinogen concentration and the angiotensin release changed in opposite directions. Infusion of renin in hind legs caused a marked increase in angiotensin release and continued even 1 hour after cessation of renin infusion. Infusion of angiotensinogen did not alter the angiotensin release. Angiotensin clearance and angiotensin I conversion were not affected by either nephrectomy or losartan pretreatment. Aortic renin messenger RNA level was extremely low and not increased by nephrectomy or losartan pretreatment, although kidney renin messenger RNA level was increased by losartan pretreatment. These results provide evidence that plasma renin of kidney origin is the major source of vascular functional renin and plays the determining role in the regulation of vascular angiotensin release. Plasma-derived or locally produced angiotensinogen, locally produced renin, converting enzyme, and angiotensin clearance are not considered to be the primary determinant in the regulation of vascular angiotensin release in these acute and subacute experimental models.

Oscillation of ADP-ribosyl cyclase activity during the cell cycle and function of cyclic ADP-ribose in a unicellular organism, Euglena gracilis.

In Euglena gracilis, the activity of ADP-ribosyl cyclase, which produces cyclic ADP-ribose, oscillated during the cell cycle in a synchronous culture induced by a light-dark cycle, and a marked increase in the activity was observed in the G2 phase. Similarly, the ADP-ribosyl cyclase activity rose extremely immediately before cell division started, when synchronous cell division was induced by adding cobalamin (which is an essential growth factor and participates in DNA synthesis in this organism) to its deficient culture. Further, cADPR in these cells showed a maximum level immediately before cell division started. A dose-dependent Ca2+ release was observed when microsomes were incubated with cADPR.

Induction chemoradiotherapy for advanced stage III non-small cell lung cancer: long-term follow-up in 42 patients.

This multi-institutional phase II study was designed to assess the feasibility, efficacy, toxicity, and long-term survival of induction chemoradiotherapy followed by surgery in previously untreated patients with advanced stage III non-small cell lung cancer. Chemotherapy regimen included cisplatin 20 mg/m2 on days 1-5 and 29-33, and VP-16 40 mg/m2 on days 1-5 and 29-33. Radiotherapy (50 Gy in 25 fractions) began on day 1. Clinically downstaged patients underwent thoracotomy 3-5 weeks after the completion of radiotherapy. Forty-two eligible patients (ten stage IIIA and 32 IIIB) were followed for a median period of 64 months. The response rate was 81%, and 20 patients had a clinically good response. Twenty-one patients underwent thoracotomy. Nineteen patients had complete resections and there were seven pathologic complete responses. There were four treatment related deaths (all stage IIIBs). There were significant survival differences between stage IIIA versus IIIB patients (P = 0.028; median survivals, 24.9 vs. 11.1 months; 5-year survival rates, 20% vs. 8.3%), and patients that achieved pathologic complete response (CR) versus those that did not (P = 0.045; median survivals 30.1 vs. 11.1 months; 5-year survival rates, 28.6% vs. 8.3%). Although the induction chemoradiotherapy employed in this study was not appropriate for stage IIIB patients, it proved feasible in stage IIIA patients in whom it resulted in good 5-year survival rates. It also provided good survival rates in patients achieving pathologic CR.

Purification and some properties of short chain-length specific trans-2-enoyl-CoA reductase in mitochondria of Euglena gracilis.

Short chain-length specific trans-2-enoyl-CoA reductase (reductase I), which contributed to mitochondrial fatty acid synthesis, was purified about 200-fold from crude extract of mitochondria in Euglena gracilis. It had a molecular weight of 39,000, and consisted of two dissimilar subunits with molecular weights of 15,000 and 25,000. The enzyme utilized crotonyl-CoA as the most active substrate and showed negative cooperativity in the reaction with the substrate. NADH was the sole electron donor. Some divalent cations were inhibitory to the enzyme when incubated with the enzyme prior to the start of the reaction. The reductase apparently contained loosely bound FAD.

Occurrence of oxygen-sensitive, NADP+-dependent pyruvate dehydrogenase in mitochondria of Euglena gracilis.

Pyruvate dehydrogenase found in mitochondria of Euglena gracilis was active on NADP+ but not NAD+, and FAD and methyl viologen also served as electron acceptors. For 2-oxoglutarate dehydrogenase both NAD+ and NADP+ were utilized and the ratio of its activity on NAD+ and NADP+ was about 1:5. The activity of pyruvate dehydrogenase was inhibited by pyruvate in aerobiosis, while not in anaerobiosis.

Purification and characterization of ADP-ribosyl cyclase from Euglena gracilis.

ADP-ribosyl cyclase, which catalyzes the conversion from NAD+ to cyclic adenosine diphosphoribose (cADPR), is proposed to participate in cell cycle regulation in Euglena gracilis. This enzyme, which was found as a membrane-bound protein, was purified almost the homogeneity after solubilization with deoxycholate, and found to be a monomeric protein with a molecular mass of 40 kDa. Its Km value for NAD+ was estimated to be 0.4 mM, and cADPR, a product of the enzyme, inhibited the enzyme competitively with respect to NAD+ whereas another product, nicotinamide, showed noncompetitive (mixed-type) inhibition. In contrast to mammalian CD38 and BST-1, Euglena ADP-ribosyl cyclase lacked cADPR hydrolase activity.

Mitochondrial alcohol dehydrogenase from ethanol-grown Euglena gracilis.

The inducing effects of ethanol on alcohol dehydrogenase and the key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, in Euglena cells were investigated. Ethanol as the sole carbon source resulted in increases in alcohol dehydrogenase and the two glyoxylate cycle enzymes. The experimental results indicated that ethanol is assimilated by alcohol dehydrogenase and the glyoxylate cycle in Euglena. Mitochondria from aerobically grown Euglena contain a unique type of alcohol dehydrogenase that accounts for their ability to respire with ethanol as a substrate. This alcohol dehydrogenase was purified to homogeneity from ethanol-grown Euglena gracilis. The mitochondrial alcohol dehydrogenase was NAD(+)-specific but not NADP(+)-specific. Ethanol was the most active substrate, but the enzyme was also active towards 1-butanol, 1-heptanol, cinnamyl alcohol, and myristyl alcohol. These results indicated that mitochondrial alcohol dehydrogenase participated in alcohol metabolism in Euglena gracilis.

Alcohol induced B-A transition of DNAs with different base compositions studied by circular dichroism.

The circular dichroic (CD) spectra of natural DNAs (from Cl. perfringens, T2 phage, calf thymus, E. coli, and M. lysodeikticus) as well as duplexes of synthetic DNAs (poly(dA) X poly(dT), poly(dA-dT), and poly(dG-dC] were measured in water-ethanol mixtures with 0.3 mM NaCl. A conformational change from the B to the A form was observed for the natural DNAs on adding ethanol. The ethanol concentration that induces the transition and the extent of the change in the CD spectrum are different for the five natural DNAs depending on their GC contents. The higher the GC content is, the more easily the transition to the A form takes place. The results indicate that the GC content of a DNA is an important factor for induction of the B-A transition. The results for the synthetic DNAs show that their properties cannot be inferred by simple extrapolation of those of natural DNAs. Coexisting ions and the molecular weight of a DNA were also found to affect the induction of the B-A transition.

Enhancement of metabolizing herbicides in young tubers of transgenic potato plants with the rat CYP1A1 gene.

A rat P450 monooxygenase gene ( CYP1A1) was introduced into potato plants to enhance the metabolism of the environmental contaminants in subterranean organs. The CYP1A1 gene was kept under the control of the potato patatin promoter to enhance tuber-specific expression. A total of 106 transgenic plants (PAT1A1 plants) were obtained following selection by a resistance test to kanamycin and PCR analysis. PAT1A1 plants treated with 10% exogenous sucrose showed a higher activity of monooxgenase in the leaves than the non-transgenic plants. This indicated that the activity enhanced by 10% sucrose was due to the patatin promoter containing the sucrose-inducted elements. One representative transgenic plant, Ag2197, was selected on the basis of monooxgenase activity in the leaves and Western blot analysis. Ag2197 was found to accumulate a large amount of CYP1A1 mRNA and protein in the developing tuber but not in the mature tuber. The residual herbicides, atrazine and chlortoluron, were analyzed in the micro-tubers of Ag2197 and non-transgenic plants. The amount of residual herbicides in Ag2197 was much lower than that in the non-transgenic plant, indicating that the transgenic plant metabolized the herbicides to a detoxified form. The transgenic plants produced in this study might be useful for the phytoremediation of chemical pollution in the soil.