Diagnostic and Prognostic Value of 11C-Methionine PET for Nonenhancing Gliomas.

BACKGROUND AND PURPOSE: Noninvasive radiologic evaluation of glioma can facilitate correct diagnosis and detection of malignant transformation. Although positron-emission tomography is considered valuable in the care of patients with gliomas, (18)F-fluorodeoxyglucose and (11)C-methionine have reportedly shown ambiguous results in terms of grading and prognostication. The present study compared the diagnostic and prognostic capabilities of diffusion tensor imaging, FDG, and (11)C-methionine PET in nonenhancing gliomas. MATERIALS AND METHODS: Thirty-five consecutive newly diagnosed, histologically confirmed nonenhancing gliomas that underwent both FDG and (11)C-methionine PET were retrospectively investigated (23 grade II and 12 grade III gliomas). Apparent diffusion coefficient, fractional anisotropy, and tumor-to-normal tissue ratios of both FDG and (11)C-methionine PET were compared between grade II and III gliomas. Prognostic values of these parameters were also tested by using progression-free survival. RESULTS: Grade III gliomas showed significantly higher average tumor-to-normal tissue and maximum tumor2-to-normal tissue than grade II gliomas in (11)C-methionine (P = .013, P = .0017, respectively), but not in FDG-PET imaging. There was no significant difference in average ADC, minimum ADC, average fractional anisotropy, and maximum fractional anisotropy. (11)C-methionine PET maximum tumor-to-normal tissue ratio of 2.0 was most suitable for detecting grade III gliomas among nonenhancing gliomas (sensitivity, 83.3%; specificity, 73.9%). Among patients not receiving any adjuvant therapy, median progression-free survival was 64.2 ± 7.2 months in patients with maximum tumor-to-normal tissue ratio of <2.0 for (11)C-methionine PET and 18.6 ± 6.9 months in patients with maximum tumor-to-normal tissue ratio of >2.0 (P = .0044). CONCLUSIONS: (11)C-methionine PET holds promise for World Health Organization grading and could offer a prognostic imaging biomarker for nonenhancing gliomas.

Microtubule assembly and in vitro development of bovine oocytes with increased intracellular glutathione level prior to vitrification and in vitro fertilization.

Although vitrification is a useful technique for preservation of bovine oocytes, the yield of blastocysts derived from the vitrified oocytes is still low. We have recently reported a new type of cryoinjury, multiple aster formation, by which pronuclear migration and development of vitrified-warmed and in vitro-fertilized bovine oocytes are impaired. The aim of the present study was to investigate the effect of glutathione (GSH) content of vitrified bovine oocytes on multiple aster formation and subsequent in vitro development. Treatment of bovine cumulus-oocyte complexes with ß-mercaptoethanol (ßME) and L-cysteine (Cys) during in vitro maturation resulted in 2.5-fold higher GSH content not only in fresh control but also in vitrified-warmed oocytes. The percentage of normally fertilized zygotes exhibiting sperm aster(s) was >95% in all four groups (with or without ßME/Cys × fresh control or vitrified). The frequency of multiple aster formation in vitrified oocytes (three-fold higher than that in fresh control oocytes) was not affected by the increased level of intracellular GSH with ßME/Cys. Consequently, the migration and development of pronuclei as well as the yield of blastocysts from vitrified-warmed oocytes (17 versus 41%) were not improved. In addition, there was no effect of increased GSH level on the yield of blastocysts in fresh control groups.

Social rank-dependent expression of arginine vasotocin in distinct preoptic regions in male Oryzias latipes.

This study examined arginine vasotocin (AVT) expression in the brains of dominant and subordinate male medaka Oryzias latipes after short- and long-term competition. High AVT expression in distinct preoptic regions was found in dominants and subordinates within minutes of encountering each other. During long-term competition, AVT expression remained high in dominants but not in subordinates.

Radiation-induced anaplastic ependymoma with a remarkable clinical response to temozolomide: a case report.

Radiation-induced gliomas are uncommon and therapeutic options are limited due to prior exposure to radiotherapy. Meanwhile, the chemotherapeutic response of anaplastic ependymoma, another rare entity in adults, is often disappointing. We report on the first recorded case of radiation-induced anaplastic ependymoma, in which an excellent clinical response to temozolomide was demonstrated.

High incidence of multiple aster formation in vitrified-warmed bovine oocytes after in vitro fertilization.

In vitro-matured bovine oocytes do not tolerate vitrification as well as mature murine or human oocytes. Delayed first cleavage in vitrified and in vitro-fertilized bovine oocytes may be responsible for the decreased yield of blastocysts in vitro. Because formation of sperm-aster and the subsequent assembly of microtubule network play an important role for migration and fusion of both pronuclei, aster formation in vitrified-warmed oocytes was analyzed by confocal laser-scanning microscopy. At 10 h post-insemination (hpi), proportions of oocytes fertilized normally were comparable between the vitrified and fresh control groups (67 and 70%, respectively). Proportions of oocytes that exhibited microtubule assembly were similar between the two groups (95% each), but the proportion of oocytes with multiple asters was higher in the vitrified group when compared with the fresh control group (68 vs 29%, P < 0.05). Both migration and development of two pronuclei were adversely affected by multiple aster formation. In the next experiment, multiple asters observed in 5.5 vs 8 hpi pronuclear zygotes were located near the male pronucleus, suggesting that those multiple asters were not the cytoplasmic asters of maternal origin. In conclusion, multiple aster formation frequently observed in vitrified-warmed bovine oocytes may be related to loss of ooplasmic function responsible for normal microtubule assembly from the sperm-aster.

In vivo survival of domestic cat oocytes after vitrification, intracytoplasmic sperm injection and embryo transfer.

We evaluated: (1) cleavage rate after IVF or intracytoplasmic sperm injection (ICSI) of in vivo- and in vitro-matured oocytes after vitrification (experiment 1); and (2) fetal development after transfer of resultant ICSI-derived embryos into recipients (experiment 2). In vivo-matured cumulus-oocyte complexes (COCs) were recovered from gonadotropin-treated donors at 24 h after LH treatment. In vitro-matured oocytes were obtained by mincing ovaries (from local veterinary clinics) and placing COCs into maturation medium for 24 h. Mature oocytes were denuded and cryopreserved in a vitrification solution of 15% DMSO, 15% ethylene glycol, and 18% sucrose. In experiment 1, for both in vivo- and in vitro-matured oocytes, cleavage frequencies after IVF of control and vitrified oocytes and after ICSI of vitrified oocytes were not different (P > 0.05). After vitrification, blastocyst development occurred only in IVF-derived, in vitro-matured oocytes. In experiment 2, 18 presumptive zygotes and four two-cell embryos derived by ICSI of vitrified in vitro-matured oocytes and 19 presumptive zygotes produced from seven in vivo- and 12 in vitro-matured oocytes were transferred by laparoscopy into the oviducts of two recipients, respectively. On Day 21, there were three fetuses in one recipient and one fetus in the other. On Days 63 and 66 of gestation, four live kittens were born. In vivo viability of zygotes and/or embryos produced via ICSI of vitrified oocytes was established by birth of live kittens after transfer to recipients.

Tetrad-FISH analysis reveals recombination suppression by interstitial heterochromatin sequences in rye (Secale cereale).

Tetrad analysis is a genetic method that can locate genes and centromeres on a linkage map with a high degree of precision. Despite its effectiveness and accuracy, application of this method is generally limited to fungi, algae and mosses. Here we demonstrate a new method of tetrad analysis that is applicable to other organisms. This combines tetrad analysis with fluorescence in situ hybridization (FISH), and is thus referred to as tetrad-FISH analysis. We demonstrate the effectiveness of this method using tetrads of rye, Secale cereale. The rye strain JNK contains interstitial heterochromatin in a region of Chromosome 2R. We have previously cloned the tandemly repeated sequence forming this heterochromatin in the plasmid pScJNK. We performed FISH using pScJNK as the probe on tetrads obtained from heterozygotes for the heterochromatin region. The frequency of tetrads demonstrating positive signals in two cells that are diagonally opposite one another must correspond to the frequency of recombination in the interval between the heterochromatin and the centromere. Comparison between the results of tetrad-FISH analysis and linkage maps based on RFLP markers clearly indicated that heterochromatin strongly suppresses recombination of whole chromosomal regions. We discuss the effectiveness of tetrad-FISH analysis, particularly for the localization of functional centromeres in linkage maps.

Osmotic stress induced by carbohydrates enhances expression of foreign proteins in Escherichia coli.

Arabinose has been serendipitously observed to enhance the expression of P450s in Escherichia coli. To understand the mechanism of this arabinose-dependent enhancement, the effects of various carbohydrates were investigated. Surprisingly, a series of sugars, including pentoses and hexoses, enhanced the foreign gene expression in a manner similar to arabinose. Furthermore, glycerol, a poor carbon source, also enhanced P450 expression. These results indicate that the enhancement is independent of the specific efficiency of the carbon source and also suggest the involvement of osmotic stress. Therefore, the effect of the sigma(s) (also termed sigma(38)) factor, a sigma subunit of RNA polymerase that plays a central role in regulating the expression of osmotic stress response genes, has been examined. We found that the glycerol-dependent increase in P450 expression was not observed in sigma(s)-deficient E. coli, indicating that carbohydrates enhance the foreign gene expression in E. coli via the induction of the osmotic stress response. The results suggest the important role of the osmotic stress response in posttranscriptional processes required for producing functional proteins.

Intraoperative ultrasound arteriography with the "Coded Harmonic Angio" technique. Report of three cases.

The result of combining the ultrasound Coded Excitation method and an ultrasound contrast agent (UCA), the Coded Harmonic Angio (CHA) technique provides arterial images with exceptional spatial, temporal, and contrast resolution that are comparable to those produced by conventional digital subtraction angiography. The authors report on their experience with intraoperative ultrasound arteriography performed using the transdural CHA technique in three patients: one harboring a meningioma, another with a middle cerebral artery aneurysm, and a third with an arteriovenous malformation. The present study demonstrates how intraoperative cerebral ultrasound arteriography can be applied to assess the adequacy of neurovascular procedures without the presence of an experienced operator.

Microsomal p450s use specific proline-rich sequences for efficient folding, but not for maintenance of the folded structure.

The amino-terminal region of microsomal P450s contains three distinct sequence motifs, the signal-anchor sequence (SA), the basic sequence (BS), and the proline-rich sequence (PR). Studies with two P450s of the CYP2C subfamily, P4502C11 (CYP2C11) and P4502C2 (CYP2C2), have indicated that upon expression in eukaryotic cells (yeast, COS cells, and insect cells), specific proline residues in PR are important for proper folding. In the present study, we have established that the PR region in a very different CYP gene family, P450c17 (CYP17), is also important for efficient folding. These studies have been carried out using expression in Escherichia coli. Using P4502C11, we have established that the folding requirements for P450s in bacteria are very similar to those in eukaryotic cells. Interestingly, when the PR from P450c17 is swapped for that of P4502C11 and visa versa, complete misfolding is observed. However, both the BS and SA can be swapped between these P450s without affecting folding. After proper folding of P450c17, removal of the PR by factor Xa protease has no effect on the maintenance of the P450 structure. Inspection of the sequences of many different CYP gene families indicates that the PR sequence is conserved within a gene family but varies considerably between families. We conclude that PR is important for directing the folding pathway leading to the functional P450, but not for maintaining the functional form.

Importance of a proline-rich sequence in the amino-terminal region for correct folding of mitochondrial and soluble microbial p450s.

All microsomal P450s have a proline-rich sequence (PR) in the amino-terminal region that is needed for proper folding [Kusano, K., Sakaguchi, M., Kagawa, N., Waterman, M.R. and Omura, T. (2001) J. Biochem., 129, 259-269]. There are also multiple proline residues near the amino-termini of the mature forms of all mitochondrial P450s and the amino-termini of soluble microbial P450s. To examine the functional significance of the PR in mitochondrial P450s, we expressed human P450c27 (CYP27) and bovine P450scc (CYP11A1) in an Escherichia coli heterologous expression system, and found that in each one specific proline residue is important for correct folding. Deletions from the amino-terminus further indicated the importance of the PR for the expression of a spectrally normal P450c27. Essentially the same results were obtained with two soluble microbial P450s, P450cam (CYP101) and P450nor, in each of which a PR is important for proper folding. We conclude that in all P450s (mitochondrial, microbial and microsomal P450s), a proline-rich sequence located in the amino-terminal region is important for proper folding. Furthermore, we predict that the importance of the PR in P450 folding is to reduce the tendency of the polypeptide to misfold prior to heme binding.

Aluminium and cadmium inhibit vitellogenin and its mRNA induction by estradiol-17 beta in the primary culture of hepatocytes in the rainbow trout Oncorhynchus mykiss.

Effects of Al and Cd on vitellogenin (VTG) and VTG mRNA induction by estradiol-17 beta (E(2)) were examined in primary hepatocyte cultures of rainbow trout. Hepatocytes were precultured for 2 days and then E(2) (2 x 10(-6) M) and Al (10(-6)-10(-4) M) or Cd (10(-9)-10(-6) M) were simultaneously added to the incubation medium. Hepatocytes were cultured for 5 more days. Media and hepatocytes were then analyzed by SDS-PAGE and Northern blotting for VTG and VTG mRNA, respectively. These metals had no appreciable effect on the viability of hepatocytes in culture. However, Al and Cd interfered with VTG production and VTG mRNA expression. Al reduced VTG production in a concentration-dependent way and a significant reduction occurred at Al concentrations greater than 5 x 10(-5) M. VTG mRNA expression also decreased with a negative correlation with Al concentrations (r = -0.98). The inhibition of VTG production by Cd was not concentration-dependent. This metal markedly inhibited VTG production and VTG mRNA expression at 10(-6) M. The Al-induced inhibition of VTG production was restored 7 days after Al removal, but the Cd-induced inhibition was not restored. These results suggest that Al and Cd inhibit VTG production at the transcriptional level to reduce VTG mRNA expression by different mechanisms.

Three zinc finger nuclear proteins, Sp1, Sp3, and a ZBP-89 homologue, bind to the cyclic adenosine monophosphate-responsive sequence of the bovine adrenodoxin gene and regulate transcription.

Adrenocorticotropin acting through cyclic adenosine monophosphate (cAMP) regulates transcription of the bovine adrenodoxin (Adx) gene in the adrenal cortex. The bovine Adx cAMP-responsive transcription sequence (CRS) has previously been found to contain two consensus GC boxes. By use of nuclear extracts from adrenocortical cells, Sp1 and Sp3 are shown here to bind to CRS. Mutations designed to enhance the identification of additional CRS binding proteins by reducing Sp protein binding showed the presence of an additional DNA-binding protein (Adx factor). Adx factor binding is inhibited by the zinc-chelating agent, 1,10-o-phenanthroline, suggesting it might be a zinc finger protein. By a fractionation/renaturation technique the Adx factor in mouse Y1 adrenocortical cells was found to be in the size range of 106-115 kDa by gel mobility shift assay. On the basis of size, the CRS sequence to which it binds, and its tentative identification as a zinc finger protein, Adx factor has been identified as a Krüppel-like zinc finger protein (a mouse ZBP-89 homologue). Further mutagenesis of CRS demonstrates that it can further be divided into two similar cAMP-responsive elements, and elimination of ZBP-89 binding does not affect cAMP responsiveness of either. Expression of these three nuclear proteins in Drosophila SL2 cells has been used to decipher the role of Adx CRS binding proteins in regulating transcription. Sp1 and Sp3 confer basal transcriptional activities, yet only Sp1 confers cAMP-responsive activity. ZBP-89 represses basal transcriptional activity.

Exposure of Goldfish (Carassius auratus) to Bluegills (Lepomis macrochirus) Enhances Expression of Stress Protein 70 mRNA in the Brains and Increases Plasma Cortisol Levels.

The effect of psychological stress on HSP70 mRNA in the brains and plasma cortisol levels in goldfish was examined. Stress was induced by exposure to a predator (bluegills). HSP70 mRNA and cortisol were determined by Northern blotting and ELISA, respectively. Goldfish exposed to four predators in the same tank without a partition showed marked increases in HSP70 mRNA and cortisol levels 6 hr and 12 hr after commencement of exposure. When goldfish were separated from bluegills with a net partition, HSP70 mRNA expression was enhanced after 6 hr, and returned to the control level after 12 hr. Plasma cortisol levels increased after 2 hr, and returned to the control level after 6 hr. When goldfish were placed in a transparent tank around which bluegills were swimming, HSP70 mRNA expression and cortisol levels increased after 6 hr and 12 hr. Goldfish exposed to water circulating through a tank with bluegills showed no sign of changes in HSP70 mRNA expression or cortisol levels. These results suggest that psychological stress enhanced HSP70 mRNA expression in the brains and increased plasma cortisol levels via visual perception.

Protein synthesis inhibitors and ethanol selectively enhance heterologous expression of P450s and related proteins in Escherichia coli.

The antibiotics chloramphenicol (Cm), tetracycline, and erythromycin, which inhibit bacterial protein synthesis and are known to induce the cold shock response, unexpectedly enhance the heterologous expression of P450s and related proteins in Escherichia coli. In contrast, antibiotics that mimic heat shock in E. coli such as puromycin, streptomycin, and kanamycin decrease the expression of the same proteins. A sublethal dose of Cm (1 microgram/ml) effectively enhances the expression of both membrane-bound proteins (microsomal and mitochondrial P450s) and a soluble mitochondrial protein (adrenodoxin) over the range of two- to eightfold. The expression level of N-terminal truncated P450c17 (1600 nmol/liter culture without Cm), for instance, reached 3500 nmol/liter culture by the addition of Cm, approximately 8.4% of the total cellular protein. Cm also enabled expression at useful levels of active P450s previously difficult to express in E. coli. In contrast, the expression of P450scc, a mitochondrial protein, is decreased by Cm but enhanced by ethanol, a powerful elicitor of heat shock response in E. coli. These results show that both the cold shock response induced by some antibiotics and the heat shock response induced by ethanol may lead to enhanced expression of certain heterologous proteins in E. coli. This study also indicates that protein synthesis inhibitors associated with the cold shock response may act as protein synthesis enhancers under certain conditions.

[The regulation of steroidogenesis by 17 alpha-hydroxylase/17,20-lyase (P450c17)].

Cytochrome P450c17 (P450c17) catalyzes 17 alpha-hydroxylation and 17, 20-lyase reactions. This enzyme plays a key role in determination of the balance between glucocorticoids and steroid sex hormones. In this review we discuss recent progress in the studies of both transcriptional regulation of CYP17 encoding P450c17 and enzymatic regulation of P450c17. Several transcription factors involved in cAMP-dependent transcription of the gene have been isolated and identified to be members of the atypical homeodomain "TALE" superfamily containing Pbx, Meis, Pknox and TGIF families. The studies of enzymatic regulation of P450c17 suggest that cytochrome b5 (b5), a heme protein, may switch the reaction of P450c17 by enhancing the 17, 20-lyase activity to increase the level of plasma C19 steroids. The importance of b5 in the synthesis of C19 steroids has also been shown in a clinical study reporting that the external genitalia was abnormal in a patient having a defect in b5. Therefore, this enhancement by b5 on the lyase activity of P450c17 may be essential to normal sexual differentiation in humans and also important in control of an optimal balance between sex steroid hormones and glucocorticoids. In addition, the age-dependent expression of P450c17 in immature rat livers is also discussed.

Evidence that immature rat liver is capable of participating in steroidogenesis by expressing 17alpha-hydroxylase/17,20-lyase P450c17.

Steroid hydroxylase cytochrome P450c17 has been previously purified from microsomal fractions of immature rat livers. In this study, we investigated the expression of P450c17 in rat livers to understand a role of steroidogenesis in the extrasteroidogenic tissue. Upon immunoblot analysis utilizing liver microsomes from rats, P450c17 was detected in 1 and 3 week old rats but not in adult rats. Data from immunohistochemical studies also showed a similar age-dependent expression of P450c17 and indicated that P450c17 detected in immature rat livers is localized in cells surrounding interlobular veins. This age-dependent expression of P450c17 in rat livers was observed in both sexes. Upon enzymatic analysis utilizing microsomal fractions from livers, levels of 17alpha-hydroxylase and 17,20-lyase activity for pregnenolone and progesterone increased by 3 weeks and dramatically reduced at 7 weeks, which is consistent with the expression level of P450c17. These data clearly indicate that P450c17 is expressed in immature rat liver to produce 17alpha-hydroxysteroids and C19-steroids. Based upon immunoblot analysis, the expression level of P450c17 in immature rat livers was approximately one third of that in testis. Compared expression level of P450c17 and total volume of organs between liver and testis, the total amount of steroid metabolites produced by liver P450c17 could be greater than that produced by gonadal P450c17. Because of the absence of P450c17 in rat adrenal glands, rat liver could be the major site for producing 17alpha-hydroxysteroids and C19-steroids in this particular period of life. Although physiological products formed by P450c17 in liver and their roles remain to be elucidated, this study suggests a large capacity of prepubertal rat liver for participating the production of steroid hormones and a putative importance of 17alpha-hydroxysteroids and C19-steroids, such as cortisol and androstendione, which are generally believed to be minor components of steroid hormones in rodents.

Members of the meis1 and pbx homeodomain protein families cooperatively bind a cAMP-responsive sequence (CRS1) from bovine CYP17.

The mammalian Pbx homeodomain proteins provide specificity and increased DNA binding affinity to other homeodomain proteins. A cAMP-responsive sequence (CRS1) from bovine CYP17 has previously been shown to be a binding site for Pbx1. A member of a second mammalian homeodomain family, Meis1, is now also demonstrated to be a CRS1-binding protein upon purification using CRS1 affinity chromatography. CRS1 binding complexes from Y1 adrenal cell nuclear extract contain both Pbx1 and Meis1. This is the first transcriptional regulatory element reported as a binding site for members of the Meis1 homeodomain family. Pbx1 and Meis1 bind cooperatively to CRS1, whereas neither protein can bind this element alone. Mutagenesis of the CRS1 element indicates a binding site for Meis1 adjacent to the Pbx site. All previously identified Pbx binding partners have Pbx interacting motifs that contain a tryptophan residue amino-terminal to the homeodomain that is required for cooperative binding to DNA with Pbx. Members of the Meis1 family contain one tryptophan residue amino-terminal to the homeodomain, but site-directed mutagenesis indicates that this residue is not required for cooperative CRS1 binding with Pbx. Thus, the Pbx-Meis1 interaction is unique among Pbx complexes. Meis1 also cooperatively binds CRS1 with the Pbx homologs extradenticle from Drosophila melanogaster and ceh-20 from Caenorhabditis elegans, indicating that this interaction is evolutionarily conserved. Thus, CYP17 CRS1 is a transcriptional regulatory element containing both Pbx and Meis1 binding sites, which permit these two homeodomain proteins to bind and potentially regulate cAMP-dependent transcription through this sequence.

[A case of progressive right coronary ostial stenosis after Carrel patch method using gelatin-resorcin-formalin glue].

A 31-year-old man with type A chronic aortic dissection associated with annuloaortic ectasia underwent the concomitant graft replacement of the total aortic root and the transverse aortic arch. The two coronary arteries were reconstructed using the Carrel patch method. The false lumen of right coronary artery was closed by injection of GRF glue into the dissected space and compressing the dissected layers. Postoperative course was uneventful, and the patient has returned to normal daily life 2 months after surgery. Remarkable progression of the right coronary artery ostial stenosis was observed by coronary angiography 6 months after surgery. The remarkable progression of stenosis may occur in association with injection of GRF glue into the dissected space, although the exact etiology of the progressive stenosis remains obscure.