Metabolic biomarkers for non-alcoholic fatty liver disease induced by high-fat diet: In vivo magnetic resonance spectroscopy of hyperpolarized [1-13C] pyruvate.

Hyperpolarized 13C magnetic resonance spectroscopy (MRS) to assess hepatic metabolism in non-alcoholic fatty liver disease (NAFLD) has not been reported. This study searched for cellular metabolism-based biomarkers for NAFLD induced by a high-fat diet (HFD) in rats. Also, correlations of the biomarkers with enzyme levels and histopathology were identified during a 6-week follow-up. Six rats were fed a control diet (CD) and seven rats were fed the HFD for 6 weeks. Hyperpolarized 13C dynamic MRS was performed on rat liver following an injection of hyperpolarized [1-13C] pyruvate. Compared with CD-fed rats, HFD-fed rats showed significant increases in the levels of serum alanine aminotransferase and low-density lipoprotein cholesterol at weeks 4 and 6 of follow-up. After the 6-week HFD, the ratios of [1-13C] alanine/pyruvate and [1-13C] lactate/pyruvate were significantly increased, as were the levels of alanine aminotransferase and lactate dehydrogenase, which are potentially associated with hepatosteatosis. The results implicate [1-13C] alanine and [1-13C] lactate as potentially useful noninvasive biomarkers of hepatosteatosis occurring in NAFLD.

Time-course metabolic changes in high-fat diet-induced obesity rats: A pilot study using hyperpolarized (13)C dynamic MRS.

The purpose of this study was to investigate the time-course metabolic changes based on hyperpolarized (13)C magnetic resonance spectroscopy (MRS) in high-fat diet (HFD)-induced obesity rats and the correlation between metabolic and serum enzyme levels. Sprague-Dawley rats were fed either HFD (60% fat) or normal diet (10% fat) for 6weeks. A HyperSense DNP was used to hyperpolarize [1-(13)C] pyruvic acid and the hyperpolarized (13)C MRS was examined every 2weeks in the course of 6weeks using a 3T GE MR750 scanner. The body weight of HFD-induced obese rats was significantly increased compared to normal rats at the 6th week after the onset of feeding (p=0.05). Simultaneously, the HFD-induced obese rats showed significantly increased levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and low-density lipoprotein (LDL)-cholesterol compared to normal rats (p≤0.05). In the dynamic (13)C MR spectra acquired at the 6th week, the obese rats showed significantly increased ratios of [1-(13)C] lactate/[1-(13)C] pyruvate and [1-(13)C] alanine/[1-(13)C] pyruvate (p=0.05). The (13)C spectral outcomes are positively correlated with the enzyme levels of ALT and LDH in the HFD-induced obesity. The [1-(13)C] lactate and [1-(13)C] alanine are potentially considered as noninvasive biomarkers for the HFD-induced obesity.

Intravoxel incoherent motion diffusion-weighted imaging for monitoring chemotherapeutic efficacy in gastric cancer.

AIM: To assess intravoxel incoherent motion diffusion-weighted imaging (IVIM-DWI) for monitoring early efficacy of chemotherapy in a human gastric cancer mouse model. METHODS: IVIM-DWI was performed with 12 b-values (0-800 s/mm(2)) in 25 human gastric cancer-bearing nude mice at baseline (day 0), and then they were randomly divided into control and 1-, 3-, 5- and 7-d treatment groups (n = 5 per group). The control group underwent longitudinal MRI scans at days 1, 3, 5 and 7, and the treatment groups underwent subsequent MRI scans after a specified 5-fluorouracil/calcium folinate treatment. Together with tumor volumes (TV), the apparent diffusion coefficient (ADC) and IVIM parameters [true water molecular diffusion coefficient (D), perfusion fraction (f) and pseudo-related diffusion coefficient (D(*))] were measured. The differences in those parameters from baseline to each measurement (ΔTV%, ΔADC%, ΔD%, Δf% and ΔD(*)%) were calculated. After image acquisition, tumor necrosis, microvessel density (MVD) and cellular apoptosis were evaluated by hematoxylin-eosin (HE), CD31 and terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) staining respectively, to confirm the imaging findings. Mann-Whitney test and Spearman's correlation coefficient analysis were performed. RESULTS: The observed relative volume increase (ΔTV%) in the treatment group were significantly smaller than those in the control group at day 5 (ΔTVtreatment% = 19.63% ± 3.01% and ΔTVcontrol% = 83.60% ± 14.87%, P = 0.008) and day 7 (ΔTVtreatment% = 29.07% ± 10.01% and ΔTVcontrol% = 177.06% ± 63.00%, P = 0.008). The difference in ΔTV% between the treatment and the control groups was not significant at days 1 and 3 after a short duration of treatment. Increases in ADC in the treatment group (ΔADC%treatment, median, 30.10% ± 18.32%, 36.11% ± 21.82%, 45.22% ± 24.36%) were significantly higher compared with the control group (ΔADC%control, median, 4.98% ± 3.39%, 6.26% ± 3.08%, 9.24% ± 6.33%) at days 3, 5 and 7 (P = 0.008, P = 0.016, P = 0.008, respectively). Increases in D in the treatment group (ΔD%treatment, median 17.12% ± 8.20%, 24.16% ± 16.87%, 38.54% ± 19.36%) were higher than those in the control group (ΔD%control, median -0.13% ± 4.23%, 5.89% ± 4.56%, 5.54% ± 4.44%) at days 1, 3, and 5 (P = 0.032, P = 0.008, P = 0.016, respectively). Relative changes in f were significantly lower in the treatment group compared with the control group at days 1, 3, 5 and 7 follow-up (median, -34.13% ± 16.61% vs 1.68% ± 3.40%, P = 0.016; -50.64% ± 6.82% vs 3.01% ± 6.50%, P = 0.008; -49.93% ± 6.05% vs 0.97% ± 4.38%, P = 0.008, and -46.22% ± 7.75% vs 8.14% ± 6.75%, P = 0.008, respectively). D* in the treatment group decreased significantly compared to those in the control group at all time points (median, -32.10% ± 12.22% vs 1.85% ± 5.54%, P = 0.008; -44.14% ± 14.83% vs 2.29% ± 10.38%, P = 0.008; -59.06% ± 19.10% vs 3.86% ± 5.10%, P = 0.008 and -47.20% ± 20.48% vs 7.13% ± 9.88%, P = 0.016, respectively). Furthermore, histopathologic findings showed positive correlations with ADC and D and tumor necrosis (r s = 0.720, P < 0.001; r s = 0.522, P = 0.007, respectively). The cellular apoptosis of the tumor also showed positive correlations with ADC and D (r s = 0.626, P = 0.001; r s = 0.542, P = 0.005, respectively). Perfusion-related parameters (f and D(*)) were positively correlated to MVD (r s = 0.618, P = 0.001; r s = 0.538, P = 0.006, respectively), and negatively correlated to cellular apoptosis of the tumor (r s = -0.550, P = 0.004; r s = -0.692, P < 0.001, respectively). CONCLUSION: IVIM-DWI is potentially useful for predicting the early efficacy of chemotherapy in a human gastric cancer mouse model.

Elevated Coexpression of KITENIN and the ErbB4 CYT-2 Isoform Promotes the Transition from Colon Adenoma to Carcinoma Following APC loss.

PURPOSE AND EXPERIMENTAL DESIGN: The molecular events in the malignant progression of colon adenoma after loss of adenomatous polyposis coli (APC) are not fully understood. KITENIN (KAI1 C-terminal interacting tetraspanin) increases the invasiveness of colorectal cancer cells, and we identified a novel EGFR-independent oncogenic signal of EGF that works under coexpressed KITENIN and ErbB4. Here we tested whether elevated KITENIN and ErbB4 contribute to further progression of intestinal adenoma following APC loss. RESULTS: The intestinal tissues of villin-KITENIN transgenic mice in which villin-driven KITENIN expression induces increased c-Jun expression exhibit mild epithelial cell proliferation but no epithelial lineage changes compared with those of nontransgenic mice. Among the four ErbB4 isoforms, JM-a/CYT-2 and JM-b/CYT-2 exhibited the highest AP-1 activity when cells coexpressing KITENIN and each isoform were stimulated by EGF. Interestingly, predominant overexpression of the ErB4-CYT-2 mRNA as well as increased EGFR expression were observed in intestinal adenoma of APC(min/+) mice, which makes the microenvironment of activated EGF signaling. When we crossed villin-KITENIN mice with APC(min/+) mice, intestinal tumor tissues in the crossed mice showed the characteristics of early-stage invading adenocarcinoma. In patients with colorectal cancer, ErbB4-CYT-2 mRNA expression was significantly greater in tumor tissues than in normal adjacent tissues, but no significant differences in tumor tissue expression were found between different colorectal cancer stages. Furthermore, the mRNA expression of KITENIN and that of ErbB4-CYT-2 were positively correlated in human colorectal cancer tissue. CONCLUSIONS: Elevated coexpression of KITENIN and ErbB4-CYT-2 promotes the transition of colon adenoma to adenocarcinoma within an APC loss-associated tumor microenvironment.

Efficacy of the mineral oil and hyaluronic acid mixture eye drops in murine dry eye.

PURPOSE: To investigate the therapeutic effects of mineral oil (MO) and hyaluronic acid (HA) mixture eye drops on the tear film and ocular surface in a mouse model of experimental dry eye (EDE). METHODS: Eye drops consisting of 0.1% HA alone or mixed with 0.1%, 0.5%, or 5.0% MO were applied to desiccating stress-induced murine dry eyes. Tear volume, corneal irregularity score, tear film break-up time (TBUT), and corneal fluorescein staining scores were measured at 5 and 10 days after treatment. Ten days after treatment, goblet cells in the conjunctiva were counted after Periodic acid-Schiff staining. RESULTS: There was no significant difference in the tear volume between desiccating stress-induced groups. The corneal irregularity score was lower in the 0.5% MO group compared with the EDE and HA groups. The 0.5% and 5.0% MO groups showed a significant improvement in TBUT compared with the EDE group. Mice treated with 0.1% and 0.5% MO mixture eye drops showed a significant improvement in fluorescein staining scores compared with the EDE group and the HA group. The conjunctival goblet cell count was higher in the 0.5% MO group compared with the EDE group and HA group. CONCLUSIONS: The MO and HA mixture eye drops had a beneficial effect on the tear films and ocular surface of murine dry eye. The application of 0.5% MO and 0.1% HA mixture eye drops could improve corneal irregularity, the corneal fluorescein staining score, and conjunctival goblet cell count compared with 0.1% HA eye drops in the treatment of EDE.

Effect of detrusor overactivity on the expression of aquaporins and nitric oxide synthase in rat urinary bladder following bladder outlet obstruction.

BACKGROUND: Aquaporins (AQPs) have recently been reported to be expressed in rat and human urothelium. Nitric oxide (NO) is thought to play a role in the bladder overactivity related to bladder outlet obstruction (BOO). The purpose of this study is to investigate the effect of BOO on the expression of AQP2-3 and nitric oxide synthase (NOS) isoforms in rat urothelium. METHODS: Female Sprague-Dawley rats (230-240 g, n = 60) were divided into 2 groups. The control group (n = 30) and the partial bladder outlet obstruction (BOO) group (n = 30). After 4 weeks, we performed a urodynamic study to measure the contraction interval and contraction pressure. The expression and cellular localization of AQP2-3, endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) were determined by Western blot and immunohistochemistry. RESULTS: On the cystometrogram, the estimated contraction interval time (minutes, mean ± SE) was significantly lower in the BOO group (3.0 ± 0.9) than in the control group (6.3 ± 0.4; p < 0.05). AQP2 was localized in the cytoplasm of the epithelium, whereas AQP3 was found only in the cell membrane of the epithelium. The protein expression of AQP2-3, eNOS and nNOS was significantly increased in the BOO group. CONCLUSION: Detrusor overactivity induced by BOO causes a significant increase in the expression of AQP2-3, eNOS, and nNOS in rat urinary bladder. This may imply that the AQPs and NOS isoforms have a functional role in the bladder dysfunction that occurs in association with BOO.

Nrf2 and Sp family synergistically enhance the expression of ion transporters in potassium-depleted conditions.

INTRODUCTION: During potassium (K) depletion, many adaptive responses are likely mediated through a complex network that involves expression of a variety of genes. We identified that the Nrf2 gene was differentially expressed between normal and K-depleted rat kidney. METHODS: To investigate the effect of Nrf2 on colonic H/K-ATPase and kNBC1, overexpression of Nrf2 was carried out in 293T and CV1 cell lines, and experiments were conducted in low-K media. Sp family was cotransfected with Nrf2 to examine the relationship between the 2 molecules and their effect on ion transporters. RESULTS: Ion transporters were activated by overexpression of Nrf2 and cotransfection of Nrf2 with Sp family genes showed additional enhancement of colonic H/K-ATPase and kNBC1 expression and their promoter activities. Pretreatment with low-K media increased the transcriptional activity of Nrf2, colonic H/K-ATPase and kNBC1. Furthermore, transfection of dominant-negative Nrf2 completely abolished low-K-mediated expression of the ion transporters. CONCLUSION: These results suggest that Nrf2 mediates transcriptional activation of colonic H/K-ATPase and kNBC1 in response to K-depleted stress and augments Sp family-mediated expression of these ion transporters.

Bilateral asymmetric supernumerary heads of biceps brachii.

Anatomical variations of the biceps brachii have been described by various authors, but the occurrence of bilateral asymmetric supernumerary heads is rare and has not been reported. We found three accessory heads of the biceps brachii muscle on right arm and an anomalous third head of biceps brachii on left arm. The third, fourth, and fifth heads of right arm originated from the body of humerus at the insertion site of coracobrachialis and inserted into the distal part of biceps brachii short head in order. The third head of left arm originated from humerus at the insertion site of coracobrachialis and combined with the distal part of biceps brachii and continued to the proximal part of common biceps tendon. Understanding the existence of bilateral asymmetric supernumerary heads of biceps brachii may influence preoperative diagnosis and surgery on the upper limbs.

Tear production and ocular surface changes in experimental dry eye after elimination of desiccating stress.

PURPOSE: To investigate the severity and duration of desiccating stress-induced dry eye disease between mice with and without a genetic predisposition to spontaneous autoimmunity. METHODS: Experimental dry eye was induced in 12- to 16-week-old wild-type C57BL/6 and autoimmune NOD.B10.H2(b) mice by subcutaneous injection of scopolamine with exposure to an air draft for 10 days. Tear volume and corneal smoothness were measured at baseline, 5 and 10 days after desiccating stress, and 3, 7, 14, and 28 days after the removal of desiccating stress. Periodic acid-Schiff staining and immunohistochemistry were performed to evaluate the densities of conjunctival goblet cells and CD4(+) T cells in each group. Interleukin (IL)-1ß and IL-6 concentrations in conjunctival tissues were measured by multiplex immunobead assay. RESULTS: Signs of experimental dry eye were noted at 5 and 10 days after desiccating stress in both strains. After the removal of desiccating stress, in C57BL/6 mice, tear production and corneal smoothness improved at 3 and 7 days, respectively, and conjunctival goblet cells and CD4(+) T-cell densities and cytokine levels returned to baseline levels at 14 days. In contrast, in NOD.B10.H2(b) mice, none of the parameters recovered to baseline levels during a period of 28 days after the removal of desiccating stress. CONCLUSIONS: After the removal of desiccating stress in experimental dry eye, tear volume and ocular surface parameters recovered within 2 weeks in C57BL/6 mice, whereas they remained unchanged in NOD mice. In contrast to autoimmune mice, experimental dry eye can be reversed after the elimination of desiccating stress in nonsusceptible mice.

Gene expression profiling of mouse aborted uterus induced by lipopolysac charide.

To identify genes that participate in the abortion process, normal pregnant uteri were compared to lipopolysaccharide (LPS)-induced abortion uteri. At day 6 of pregnancy, mice were treated with LPS at various time points to induce an abortion. Total RNAs were applied to a cDNA microarray to analyze genes with altered expression. At the early stage (2 hours) of LPS-induced abortion, upregulated genes were mainly composed of immune responsive genes, including Ccl4, Ccl2, Cxcl13, Gbp3, Gbp2, Mx2, H2-Eb1, Irf1 and Ifi203. Genes related to toll-like receptor signaling were also overexpressed. At late stages of abortion (12-24 hours), many genes were suppressed rather than activated, and these were mainly related to the extracellular matrix, cytoskeleton, and anti-apoptosis. Altered expression of several selected genes was confirmed by real time reverse transcription-polymerase chain reaction. The results demonstrated that many known genes were altered in the LPS-treated pregnant uterus, implying that the molecular mechanisms of the genes involved in LPS-induced abortion are complicated. Further analysis of this expression profile will help our understanding of the pathophysiological basis for abortion.

Effects of androgen on the expression of vascular endothelial growth factor in the penile corpus cavernosum.

OBJECTIVES: To investigate the effect of orchiectomy and androgen replacement on the expression of vascular endothelial growth factor (VEGF) in rat penile tissue. MATERIAL AND METHODS: Adult male Sprague-Dawley rats (12 weeks old) were left intact (control) or surgically castrated. Orchiectomized rats were left untreated or received testosterone propionate (TP) for 7 days, beginning 1 or 2 weeks after castration. Erectile function was assessed by measuring intracavernosal pressure in response to cavernous nerve stimulation, and the expression of VEGF protein and mRNA was determined by immunohistochemistry, Western blot analysis, and reverse transcriptase-polymerase chain reaction. Serum testosterone values were measured in each animal by radioimmunoassay. RESULTS: Serum androgen levels decreased significantly in castrated animals, whereas TP injection normalized the serum levels of testosterone. Intracavernosal pressure was significantly decreased in untreated castrated rats (31.3 ± 15.7% at 2 weeks postcastration; 18.6 ± 4.6% at 3 weeks postcastration) compared with intact controls (58.0 ± 11.4% and 58.9 ± 8.2%, respectively). Erectile function was normalized in androgen-replaced rats, irrespective of treatment was initiation 1 or 2 weeks after orchiectomy. The expression of VEGF protein and mRNA was decreased in the corpus cavernosum of castrated animals compared with controls, whereas androgen replacement normalized the expression of VEGF. These results were consistently observed by all 3 methods of assessment. CONCLUSIONS: These data suggest that androgen regulates the expression of VEGF in rat penile corpus cavernosum and confirms the importance of androgens in the maintenance of erectile function.

Distribution of interstitial cells of cajal in menopausal rat urinary bladder showing detrusor overactivity.

PURPOSE: Recent studies have showed that interstitial cells of Cajal (ICCs) are widely distributed in the genitourinary tract and have suggested their involvement in spontaneous electrical activity and muscle contraction. The purposes of this study were to investigate the effect of estrogen on ICCs in rat urinary bladder from the detrusor overactivity induced by ovariectomy. MATERIALS AND METHODS: Female Sprague-Dawley rats (230-240 g, N=60) were divided into three groups: control (N=20), bilateral ovariectomy (Ovx, N=20), and bilateral ovariectomy followed by subcutaneous injections of 17 ß-estradiol (50 mg/kg/day, Ovx + Est, N=20). After 4 weeks, urodynamic studies measuring contraction interval and contraction pressure were done. The cellular localization of ICCs was determined by immunohistochemistry in the rat urinary bladder. RESULTS: Filling cystometry studies demonstrated a reduced interval between voiding contractions and an increased voiding pressure in Ovx group. The approximate the contraction interval (min) was (3.9±0.25) significantly decreased in the Ovx group compared to the control group (6.7±0.15), which was increased after estrogen treatment (9.7±0.22) (p<0.05). Conversely, the average contraction pressures (mmHg) were increased in the Ovx group (28.9±2.1) compared to the control group (21.2±1.45), and decreased after estrogen treatment (24.8±2.21) (p<0.05). The population of c-Kit immunoreactive ICCs was decreased in both the urothelial and muscle layers in Ovx bladders, which increased to the control value after estrogen treatment. CONCLUSIONS: These results demonstrated an decreased immunoreactivity of ICCs in the menopausal rat model and suggest that thedecreased population of ICCs expression may contribute to the modulation of bladder overactivity induced by menopause.

The Expression of AQP1 and eNOS in Menopausal Rat Urinary Bladder.

PURPOSE: Aquaporins (AQPs) have been reported to be expressed in rat and human urothelium. Nitric oxide (NO) is thought to play an important role in the bladder overactivity related to menopause. The purpose of this study was to investigate the effect of hormonal alteration on the expression of AQP1 and eNOS in menopausal rat urinary bladder. MATERIALS AND METHODS: Female Sprague-Dawley rats (230-240 g, N=30) were divided into three groups: control (N=10), bilateral ovariectomy (Ovx, N=10), and bilateral ovariectomy followed by subcutaneous injections of 17ß-estradiol (50 mg/kg/day, Ovx+Est, N=10). After 4 weeks, urodynamic studies measuring the contraction interval and contraction pressure were done. The expression and cellular localization of AQP1 and eNOS were determined by performing Western blotting and immunohistochemistry on the rat urinary bladder. RESULTS: The approximate contraction interval (min) was significantly decreased in the Ovx group (3.9±0.25) compared to the control group (6.7±0.15), and was increased after estrogen treatment (9.7±0.22) (p<0.05). The AQP1 and eNOS immunoreactivities were localized in the same areas: capillaries, arterioles, and venules of the lamina propria. The protein expression of AQP1 was not changed significantly, whereas eNOS expression was significantly decreased in the Ovx group and restored to the control value in the Ovx+Est group. CONCLUSIONS: This study showed that ovariectomy causes a significant change in e-NOS expression without a change in AQP1 in menopausal rat urinary bladder. This may imply that e-NOS has a functional role in the bladder overactivity that occurs in association with menopause.

Glycoprotein 90K, downregulated in advanced colorectal cancer tissues, interacts with CD9/CD82 and suppresses the Wnt/beta-catenin signal via ISGylation of beta-catenin.

BACKGROUND AND AIMS: 90K, a tumour-associated glycoprotein, interacts with galectins and has roles in host defence by augmenting the immune response, but the serum 90K level was suggested to indicate poor prognosis in several cancers. The cellular mechanisms of 90K action on colorectal cancer (CRC) cell motility and its effect on CRC progression were investigated. METHODS: The impact of 90K was analysed by combining cell cultures, in vitro assays, and immunohistochemistry. RESULTS: Secreted 90K suppresses CRC cell invasion, but this action of 90K is masked through binding with extracellular galectins. A novel pathway is identified comprising a secretory 90K and a CD9/CD82 tetraspanin web; in this pathway, 90K interacts with CD9/CD82, suppresses the Wnt/beta-catenin signal via a novel proteasomal-ubiquitination mechanism of beta-catenin that is dependent on ISG15 (interferon-stimulated gene-15) modification (ISGylation) but not on glycogen synthase kinase 3beta (GSK-3beta) and Siah/Adenomatous polyposis coli (APC). In a syngeneic mouse colon tumour model, tumour growth and lung metastasis were increased with 90K knockdown. In colon tissues from stage IV human CRC and invading cancer cells of corresponding metastatic liver tissues, in which beta-catenin and galectin expression was higher, immunostained 90K and CD9/CD82 were lower than in adjacent hepatic tissues or colon tissues from stage I. CONCLUSIONS: 90K itself has antitumour activity in CRC cells via suppression of Wnt signalling with a novel mechanism of ISGylation-dependent ubiquitination of beta-catenin when it interacts with CD9/CD82, but is downregulated in advanced CRC tissues. The data suggest a strategy of strengthening this novel pathway with concomitant knockdown of galectins as a potential therapeutic approach to CRC progression.

HOXB13 promotes androgen independent growth of LNCaP prostate cancer cells by the activation of E2F signaling.

BACKGROUND: Androgen signaling plays a critical role in the development of prostate cancer and its progression. However, androgen-independent prostate cancer cells emerge after hormone ablation therapy, resulting in significant clinical problems. We have previously demonstrated that the HOXB13 homeodomain protein functions as a prostate cancer cell growth suppressor by inhibiting androgen-mediated signals. However, the role of the HOXB13 in androgen-independent growth of prostate cancer cells remains unexplained. RESULTS: In this report, we first demonstrated that HOXB13 was highly overexpressed in hormone-refractory tumors compared to tumors without prostate-specific antigen after initial treatment. Functionally, in an androgen-free environment minimal induction of HOXB13 in LNCaP prostate cancer cells, to the level of the normal prostate, markedly promoted cell proliferation while suppression inhibited cell proliferation. The HOXB13-mediated cell growth promotion in the absence of androgen, appears to be mainly accomplished through the activation of RB-E2F signaling by inhibiting the expression of the p21waf tumor suppressor. Indeed, forced expression of HOXB13 dramatically decreased expression of p21waf; this inhibition largely affected HOXB13-mediated promotion of E2F signaling. CONCLUSIONS: Taken together, the results of this study demonstrated the presence of a novel pathway that helps understand androgen-independent survival of prostate cancer cells. These findings suggest that upregulation of HOXB13 is associated with an additive growth advantage of prostate cancer cells in the absence of or low androgen concentrations, by the regulation of p21-mediated E2F signaling.

Testicular steroidogenesis is decreased by hyperthermia in old rats.

PURPOSE: To evaluate the effects of hyperthermia on testicular steroidogenesis in a rat model. MATERIALS AND METHODS: Three-month-old and 20-month-old male Sprague-Dawley rats were randomly divided into 4 groups of 10 rats each, a control group and a hot-bath group for each age. The rats in the hot-bath groups received multiple 10-min treatments in a hot bath (41-43 degrees C) over a period of 4 weeks. Testicular testosterone, serum testosterone and serum luteinizing hormone levels were measured. The protein levels of 2 steroidogenic enzymes, StAR and P450c17, were measured by Western blot. The testes were examined histologically by light microscopy. RESULTS: Testicular testosterone levels of the 20-month-old, but not the 3-month-old, rats in the hot-bath group were significantly lower than those in the control group (p < 0.05). Serum testosterone levels of both the old and the young hot-bath groups tended to decrease compared with their corresponding controls, although the differences were not statistically significant. Serum luteinizing hormone levels changed insignificantly after the hot baths in both age groups. The hot-bath treatment had no significant effect on P450c17 protein levels, whereas the protein level of StAR was significantly lower in the old hot-bath group than in the same-age control group (p < 0.05). CONCLUSIONS: Hyperthermia significantly decreased the testicular testosterone level in old male rats and significantly lowered the StAR protein level. These data imply that frequent hot baths might impair testicular steroidogenesis, especially in old men.

HOXB13 is co-localized with androgen receptor to suppress androgen-stimulated prostate-specific antigen expression.

During the prostate cancer (PCa) development and its progression into hormone independency, androgen receptor (AR) signals play a central role by triggering the regulation of target genes, including prostate-specific antigen. However, the regulation of these AR-mediated target genes is not fully understood. We have previously demonstrated a unique role of HOXB13 homeodomain protein as an AR repressor. Expression of HOXB13 was highly restricted to the prostate and its suppression dramatically increased hormone-activated AR transactivation, suggesting that prostate-specific HOXB13 was a highly potent transcriptional regulator. In this report, we demonstrated the action mechanism of HOXB13 as an AR repressor. HOXB13 suppressed androgen-stimulated AR activity by interacting with AR. HOXB13 did neither bind to AR responsive elements nor disturb nuclear translocation of AR in response to androgen. In PCa specimen, we also observed mutual expression pattern of HOXB13 and AR. These results suggest that HOXB13 not only serve as a DNA-bound transcription factor but play an important role as an AR-interacting repressor to modulate hormone-activated androgen receptor signals. Further extensive studies will uncover a novel mechanism for regulating AR-signaling pathway to lead to expose new role of HOXB13 as a non-DNA-binding transcriptional repressor.

Distribution and three-dimensional appearance of the interstitial cells of Cajal in the rat stomach and duodenum.

The relationship between the interstitial cells of Cajal (ICC) and enteric nerves or smooth muscles cells is not fully defined. Presently, distribution and appearance of ICC in the rat stomach and duodenum was studied by immunohistochemistry, electron microscopy, and three-dimensional reconstruction. c-kit expressing ICC were regularly observed in the Auerbach's myenteric plexus (AP) of the stomach and duodenum. ICC in stomach and duodenum muscle layers was dissimilarly distributed. c-kit immunoreactive cells were sparsely distributed in the stomach circular muscle layer but were abundant in the duodenum deep muscular plexus (DMP). Electron microscopy revealed that stomach ICC-AP were irregular ovals with few cytoplasmic processes, and possessed an electron-dense cytoplasm, numerous mitochondria, intermediate filaments, and caveolae. Duodenum and stomach ICC-AP were similar in appearance. Ultrastructure observations and three-dimensional reconstructions revealed ICC-AP processes wrapping the nerve fibers and projecting into the space between smooth muscle cells. While ICC-AP was occasionally close to enteric nerves or smooth muscle cells, no connections were observed. ICC-DMP in duodenum was elongated and adopted the same cell axis orientation as the circular muscle cells. Unlike ICC-AP, ICC-DMP formed gap junctions with smooth muscle cells and had close contact with nerves. These results indicate that ICC-AP is regularly distributed in stomach and duodenum, while ICC-DMP is exclusively located in the duodenum. ICC-DMP, which possess gap junctions and closely contacts nerves, may participate in neuromuscular transmission.

Differential expression of osteocalcin during the metastatic progression of prostate cancer.

Osteocalcin expression is restricted to osteoblasts and serum osteocalcin level is elevated in metastatic bone tumors including prostate tumors, which predominantly metastasizes to the bone and causes typical osteoblastic lesions. Previously, we have reported that osteocalcin RNA is widely expressed but incompletely spliced in the prostate including prostate tumors. Considering that many studies using osteocalcin-driven gene therapy have been conducted to treat hormone refractory metastatic tumors, detailed mechanisms controlling osteocalcin expression needs to be clarified. We aim to learn how osteocalcin expression is regulated during the metastatic process of prostate cancer. We applied assays of immunohistochemistry and RNA in situ hybridization in prostate tumors acquired from prostate (15) and metastatic sites, 13 from lymph node and 14 from bone. RT-PCR analysis in various cultured prostate cells was also performed. As predicted, osteocalcin RNA was highly expressed in most prostate epithelial cells of tumors, regardless of metastatic status of the tumor. However, osteocalcin protein was undetectable in tumors acquired from the primary site or lymph nodes whereas protein was highly expressed in the majority of bone-metastasized prostate tumors. RT-PCR analysis demonstrated that there was more completely spliced form of osteocalcin RNA present in bone-derived prostate cancer cells. Our data suggest that osteocalcin RNA was expressed but not completely spliced in non-bone environment, ultimately resulting in improper production of osteocalcin protein. This study explains why serum osteocalcin level is increased in patients with bone-metastasized prostate cancers. Yet, it remains to be clarified what regulates bone-specific osteocalcin RNA splicing in prostate tumors.

Photodynamic therapy with verteporfin for corneal neovascularization.

PURPOSE: To investigate the efficacy of photodynamic therapy with verteporfin for the treatment of patients with corneal neovascularization. DESIGN: Prospective interventional case series. METHODS: Eighteen eyes of 18 patients with stable corneal neovascularization who were refractory to conventional treatment were treated with photodynamic therapy with verteporfin (6 mg/m(2)). Five patients were treated following penetrating keratoplasty (PK), and two patients were treated before PK. Anterior segment photography was performed before and after treatment. Best-corrected visual acuity (BCVA) and area of corneal neovascularization were measured. RESULTS: At the one-year follow-up, 14 eyes (77.8%) showed a decrease in corneal neovascularization, and nine eyes (50.0%) showed complete vascular occlusion. In five patients who had corneal allograft, complete or partial occlusion was achieved in all eyes. Two patients who underwent subsequent keratoplasty did not manifest allograft rejection or revascularization. Seventeen eyes (94.4%) had stable or improved vision. The mean area of corneal neovascularization significantly decreased from 25.5 +/- 14.2 mm(2) to 14.9 +/- 14.6 mm(2) (P < .01), respectively. No significant complications associated with photodynamic therapy were observed except mild stromal haze in one eye. CONCLUSION: Photodynamic therapy with verteporfin may be effective for the treatment of corneal neovascularization.