Cerebral Blood Volume Analysis in Glioblastomas Using Dynamic Susceptibility Contrast-Enhanced Perfusion MRI: A Comparison of Manual and Semiautomatic Segmentation Methods

Purpose

To compare the reproducibilities of manual and semiautomatic segmentation method for the measurement of normalized cerebral blood volume (nCBV) using dynamic susceptibility contrast-enhanced (DSC) perfusion MR imaging in glioblastomas.

Materials and Methods

Twenty-two patients (11 male, 11 female; 27 tumors) with histologically confirmed glioblastoma (WHO grade IV) were examined with conventional MR imaging and DSC imaging at 3T before surgery or biopsy. Then nCBV (means and standard deviations) in each mass was measured using two DSC MR perfusion analysis methods including manual and semiautomatic segmentation method, in which contrast-enhanced (CE)-T1WI and T2WI were used as structural imaging. Intraobserver and interobserver reproducibility were assessed according to each perfusion analysis method or each structural imaging. Interclass correlation coefficient (ICC), Bland-Altman plot, and coefficient of variation (CV) were used to evaluate reproducibility.

Results

Intraobserver reproducibilities on CE-T1WI and T2WI were ICC of 0.74–0.89 and CV of 20.39–36.83% in manual segmentation method, and ICC of 0.95–0.99 and CV of 8.53–16.19% in semiautomatic segmentation method, repectively. Interobserver reproducibilites on CE-T1WI and T2WI were ICC of 0.86–0.94 and CV of 19.67–35.15% in manual segmentation method, and ICC of 0.74–1.0 and CV of 5.48–49.38% in semiautomatic segmentation method, respectively. Bland-Altman plots showed a good correlation with ICC or CV in each method. The semiautomatic segmentation method showed higher intraobserver and interobserver reproducibilities at CE-T1WI-based study than other methods.

Conclusion

The best reproducibility was found using the semiautomatic segmentation method based on CE-T1WI for structural imaging in the measurement of the nCBV of glioblastomas.     

MR Imaging Evaluation of Intracerebral Hemorrhages and T2 Hyperintense White Matter Lesions Appearing after Radiation Therapy in Adult Patients with Primary Brain Tumors

The purpose of our study was to determine the frequency and severity of intracerebral hemorrhages and T2 hyperintense white matter lesions (WMLs) following radiation therapy for brain tumors in adult patients. Of 648 adult brain tumor patients who received radiation therapy at our institute, magnetic resonance (MR) image data consisting of a gradient echo (GRE) and FLAIR T2-weighted image were available three and five years after radiation therapy in 81 patients. Intracerebral hemorrhage was defined as a hypointense dot lesion appearing on GRE images after radiation therapy. The number and size of the lesions were evaluated. The T2 hyperintense WMLs observed on the FLAIR sequences were graded according to the extent of the lesion. Intracerebral hemorrhage was detected in 21 (25.9%) and 35 (43.2) patients in the three- and five-year follow-up images, respectively. The number of intracerebral hemorrhages per patient tended to increase as the follow-up period increased, whereas the size of the intracerebral hemorrhages exhibited little variation over the course of follow-up. T2 hyperintense WMLs were observed in 27 (33.3%) and 32 (39.5) patients in the three and five year follow-up images, respectively. The age at the time of radiation therapy was significantly higher (p < 0.001) in the patients with T2 hyperintense WMLs than in those without lesions. Intracerebral hemorrhages are not uncommon in adult brain tumor patients undergoing radiation therapy. The incidence and number of intracerebral hemorrhages increased over the course of follow-up. T2 hyperintense WMLs were observed in more than one-third of the study population.     

Insulin Directly Regulates Steroidogenesis via Induction of the Orphan Nuclear Receptor DAX-1 in Testicular Leydig Cells

Testosterone level is low in insulin-resistant type 2 diabetes. Whether this is due to negative effects of high level of insulin on the testes caused by insulin resistance has not been studied in detail. In this study, we found that insulin directly binds to insulin receptors in Leydig cell membranes and activates phospho-insulin receptor-β (phospho-IR-β), phospho-IRS1, and phospho-AKT, leading to up-regulation of DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1) gene expression in the MA-10 mouse Leydig cell line. Insulin also inhibits cAMP-induced and liver receptor homolog-1 (LRH-1)-induced steroidogenic enzyme gene expression and steroidogenesis. In contrast, knockdown of DAX-1 reversed insulin-mediated inhibition of steroidogenesis. Whether insulin directly represses steroidogenesis through regulation of steroidogenic enzyme gene expression was assessed in insulin-injected mouse models and high fat diet-induced obesity. In insulin-injected mouse models, insulin receptor signal pathway was activated and subsequently inhibited steroidogenesis via induction of DAX-1 without significant change of luteinizing hormone or FSH levels. Likewise, the levels of steroidogenic enzyme gene expression and steroidogenesis were low, but interestingly, the level of DAX-1 was high in the testes of high fat diet-fed mice. These results represent a novel regulatory mechanism of steroidogenesis in Leydig cells. Insulin-mediated induction of DAX-1 in Leydig cells of testis may be a key regulatory step of serum sex hormone level in insulin-resistant states.     

Novel Protective Mechanism against Irreversible Hyperoxidation of Peroxiredoxin: N��-TERMINAL ACETYLATION OF HUMAN PEROXIREDOXIN II*

Peroxiredoxins (Prxs) are a group of peroxidases containing a cysteine thiol at their catalytic site. During peroxidase catalysis, the catalytic cysteine, referred to as the peroxidatic cysteine (C_P), cycles between thiol (C_P-SH) and disulfide (–S–S–) states via a sulfenic (C_P-SOH) intermediate. Hyperoxidation of the C_P thiol to its sulfinic (C_P-SO₂H) derivative has been shown to be reversible, but its sulfonic (C_P-SO₃H) derivative is irreversible. Our comparative study of hyperoxidation and regeneration of Prx I and Prx II in HeLa cells revealed that Prx II is more susceptible than Prx I to hyperoxidation and that the majority of the hyperoxidized Prx II formation is reversible. However, the hyperoxidized Prx I showed much less reversibility because of the formation of its irreversible sulfonic derivative, as verified with C_P-SO₃H-specific antiserum. In an attempt to identify the multiple hyperoxidized spots of the Prx I on two-dimensional PAGE analysis, an N-acetylated Prx I was identified as part of the total Prx I using anti-acetylated Lys antibody. Using peptidyl-Asp metalloendopeptidase (EC 3.4.24.33) peptide fingerprints, we found that N^α-terminal acetylation (N^α-Ac) occurred exclusively on Prx II after demethionylation. N^α-Ac of Prx II blocks Prx II from irreversible hyperoxidation without altering its affinity for hydrogen peroxide. A comparative study of non-N^α-acetylated and N^α-terminal acetylated Prx II revealed that N^α-Ac of Prx II induces a significant shift in the circular dichroism spectrum and elevation of T_m from 59.6 to 70.9 °C. These findings suggest that the structural maintenance of Prx II by N^α-Ac may be responsible for preventing its hyperoxidation to form C_P-SO₃H.Peroxiredoxins (Prxs)⁴ are a family of peroxidases that possess a conserved cysteine residue at the catalytic site for the reduction of peroxide/peroxynitrite. Using thiol-based reducing equivalents, like thioredoxin, Prxs catalyze the reduction of hydrogen peroxide, alkylhydroperoxides, and peroxynitrite to water, corresponding alcohols, and nitrite, respectively (1–8). Based on the number and location of conserved cysteine residue(s) directly involved in peroxide reduction, the six isotypes of mammalian Prx can be grouped into three distinct subgroups as follows: 2-Cys Prx, atypical 2-Cys Prx, and 1-Cys Prx, (1–2, 5). Human Prx I (hPrx I) and Prx II (hPrx II) are members of the 2-Cys Prx subgroup and thus contain two conserved cysteine residues that are directly involved in peroxidase activity. Cys⁵² for hPrx I and Cys⁵¹ for hPrx II are designated the peroxidatic cysteines (C_P). These residues attack the O–O bond of the peroxide (ROOH) substrate to form the product (ROH) and the sulfenic derivative C_P-SOH. This sulfenic derivative then forms a disulfide bond with the other conserved cysteine residue, which is referred to as the resolving cysteine (C_R; Cys¹⁷³ in hPrx I and Cys¹⁷² in hPrx II). In the case of 2-Cys Prxs, the disulfide partners, C_P and C_R, reside within different subunits; therefore, the disulfide bond established between C_P and C_R (C_P-S–S-C_R) is intermolecular. The reduced thioredoxin molecule is responsible for reducing the C_P-S–S-C_R disulfide bond to generate sulfhydryls (1–3, 5, 9).The C_P of eukaryotic 2-Cys Prxs is vulnerable to hyperoxidation, which results in the loss of its peroxidase activity. This feature is referred to as the “floodgate” mechanism, by which Prxs function as a redox sensor for the regulation of cell signaling (10–11). Hyperoxidation of C_P does not occur when the disulfide bond (C_P-S–S-C_R) is formed. However, the thiol (C_P-SH) can be hyperoxidized via the sulfenic (C_P-SOH) derivative intermediate in the absence of C_P-S-S-C_R formation during catalysis (12). Two different hyperoxidation products of C_P, the reversible sulfinic (C_P-SO₂H) derivative and the irreversible sulfonic (C_P-SO₃H) derivative, have been identified. The irreversible C_P-SO₃H was reported in Tsa1p, a yeast 2-Cys Prx, based on in vivo and in vitro regeneration assay results, and a stronger reactivity to an anti-Tsa1p-SO₃H antibody, which exhibits high specificity toward Tsa1p-C_P-SO₃H relative to Tsa1p-C_P-SO₂H (13). Both forms of hyperoxidized Prxs, C_P-SO₂H and C_P-SO₃H, are superimposed on the acidic migrated spot instead of the Prx-SH spot on a two-dimensional polyacrylamide gel because of the introduction of one negative charge by hyperoxidation (12–16). The protein sulfinic acid reductase, sulfiredoxin, is responsible for reversing 2-Cys Prx-SO₂H to Prx-SH in the presence of ATP and thiol-reducing equivalents like thioredoxin or glutathione (17–24). Until now, an intracellular enzymatic regeneration system for Prx-SO₃H has not been reported.Because mammalian Prx I and Prx II have been studied independently in a number of different organisms and cultured cells, the comparative biochemical data supporting their distinctive functional identities is still very limited. Recombinant Prx I (rPrx I) showed a 2.6-fold higher specific activity as a peroxidase than the recombinant Prx II (rPrx II) without any obvious catalytic or mechanistic differences (25, 26). Recent competition kinetics studies of hPrx II revealed a rate constant of 1.3 × 10⁷ m^–1 s^–1, which is fast enough to favor an intracellular hydrogen peroxide target even in competition with catalase or glutathione peroxidase (27, 28). The kinetic parameters of the competition assay for hPrx I are still not available. Mammalian Prx I interacts with and regulates a broad spectrum of proteins, such as the Src homology domain 3 of c-Abl (29), the Myc box II (MBII) domain of c-Myc (30), the macrophage migration inhibitory factor (MIF, 31), the androgen receptor (32), and the apoptosis signal-regulating kinase-1 (ASK-1) (33). The suggested roles of Prx I in interactions with these molecules are those of a tumor repressor, a survival enhancer, and a growth regulator. Although these suggested functions are controversial (34), all of them can be attributed to the peroxide-scavenging capacity of Prx I (at least in part), except for the enhancement of androgen receptor transactivation (32). Prx II interacts with platelet-derived growth factor receptor and functions as a negative regulator for platelet-derived growth factor signaling (35). Prx II also binds to phospholipase D1 (PLD1) and functions as a hydrogen peroxide-stimulated PLD1 signal terminator (36). Both of these suggested Prx II roles are attributable to the peroxidase activity of Prx II. The major phenotypes of Prx I knock-out mice involve the development of a variety of age-related cancers, hemolytic anemia (37), and dramatic shifts in subcellular reactive oxygen species localization (38). Prx II knock-out mice exhibit splenomegaly and a lack of tumor development in any cell type or tissue (39). Until now, the protein molecule that interacts with Prx I and Prx II has not been characterized, and there is no indication of a heterodimer between Prx I and Prx II. Despite their similar peroxide-scavenging capacities, it is reasonable to conclude that the Prx I and Prx II are unable to compensate for each other in terms of physiological roles. There are several examples of tissue- or cell type-specific expression patterns, such as exclusive Prx I expression in astrocytes and Leydig cells and Prx II expression in neurons and Sertoli cells (40, 41); however, Prx I and Prx II are coexpressed in the majority of mammalian cells and tissues, suggesting distinguished biochemical characteristics of their cellular regulatory mechanisms. Recently, the unique presence of Cys⁸³ in hPrx I, which contributes to the stability of the dimer-dimer interface and suppresses local unfolding, has been claimed to be prone to overoxidation of Prx I (42). The contribution of the dimer-decamer interconversion to the regulation of Prx I activity has also been reported (43).In this study, we confirmed that hPrx II was more susceptible to hyperoxidation as well as more prone to regeneration than hPrx I in HeLa cells. We also found that the difficulty in regenerating hPrx I was caused by irreversible sulfonic (C_P-SO₃H) hyperoxidation. Using AspN (EC 3.4.24.33) peptide fingerprints, we identified the N^α-terminal acetylation exclusively on hPrx II. In addition, we provide evidence for the structural maintenance offered by N^α-terminal acetylation of hPrx II, which possibly contributes to preventing irreversible overoxidation of C_P-SO₃H.     

Enhanced production of ethanol from glycerol by engineered Hansenula polymorpha expressing pyruvate decarboxylase and aldehyde dehydrogenase genes from Zymomonas mobilis

To improve production of ethanol from glycerol, the methylotrophic yeast Hansenula polymorpha was engineered to express the pdc and adhB genes encoding pyruvate decarboxylase and aldehyde dehydrogenase II from Zymomonas mobilis, respectively, under the control of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter. The ethanol yield was 3.3-fold higher (2.74 g l^?1) in the engineered yeast compared with the parent strain (0.83 g l^?1). Further engineering to stimulate glycerol utilization in the recombinant strain via expression of dhaD and dhaKLM genes from Klebsiella pneumoniae encoding glycerol dehydrogenase and dehydroxyacetone kinase, respectively, resulted in a 3.7-fold increase (3.1 g l^?1) in ethanol yield.     

A continuous spectrophotometric assay for NADPH-cytochrome P450 reductase activity using 1,1-diphenyl-2-picrylhydrazyl

NADPH-cytochrome P450 reductase (CPR) transfers electrons from NADPH to cytochrome P450, and catalyzes the one-electron reduction of many drugs and foreign compounds. Various forms of spectrophotometric titration have been performed to investigate the electron-accepting properties of CPR, particularly, to examine its ability to reduce cytochrome c and ferricyanide. In this study, the reduction of 1,1-diphenyl-2-picrylhydrazyl (DPPH) by CPR was assessed as a means of monitoring CPR activity. The principle advantage of DPPH is that its reduction can be assayed directly in the reaction medium by a continuous spectrophotometry. Thus, electrons released from NADPH by CPR were transferred to DPPH, and DPPH reduction was then followed spectrophotometrically by measuring A(520) reduction. Optimal assay concentrations of DPPH, CPR, potassium phosphate buffer, and NADPH were first established. DPPH reduction activity was found to depend upon the strength of the buffer used, which was optimal at 100 mM potassium phosphate and pH 7.6. The extinction coefficient of DPPH was 4.09mM(-1) cm(-1). DPPH reduction followed classical Michaelis-Menten kinetics (K(m) = 28 microM, k(cat) = 1690 min(-1)). This method uses readily available materials, and has the additional advantages of being rapid and inexpensive.