Reduced translation efficiency due to novel splicing variants in 5' untranslated region and identification of novel cis-regulatory elements in canine and human BRCA2.

BACKGROUND: Breast cancer 2, early onset (BRCA2) is a tumor suppressor gene. The protein encoded by this gene plays an important role in homologous recombination (HR)-mediated DNA repair. Deleterious mutations in BRCA2 and downregulation of its expression have been associated with tumorigenesis in dogs and humans. Thus, regulation of BRCA2 expression level is important for maintaining homeostasis in homologous recombination. RESULTS: In this study, the mechanisms that regulate the expression of BRCA2 were proposed. Novel splicing variants were identified in the 5' untranslated region (UTR) of canine and human BRCA2 in canine testis, canine ovary, and canine and human cultured cell lines. In cultured cells, the ratio of BRCA2 splicing variants at the 5' UTR was altered by serum starvation. These novel splicing variants, excluding one of the canine splicing variants, were found to reduce the translational efficiency. Additionally, the DNA sequence in human BRCA2 intron 1 harbored novel cis-regulatory elements. Three silencer and two enhancer cis-regulatory elements were identified in human BRCA2 intron 1. CONCLUSIONS: This study demonstrates that BRCA2 expression level is regulated via 5' UTR splicing variants and that the BRCA2 intron 1 region harbors cis-regulatory elements.

High glucose stimulates expression of aldosterone synthase (CYP11B2) and secretion of aldosterone in human adrenal cells.

Aldosterone synthase is the key rate-limiting enzyme in adrenal aldosterone production, and induction of its gene (CYP11B2) results in the progression of hypertension. As hypertension is a frequent complication among patients with diabetes, we set out to elucidate the link between diabetes mellitus and hypertension. We examined the effects of high glucose on CYP11B2 expression and aldosterone production using human adrenal H295R cells and a stable H295R cell line expressing a CYP11B2 5'-flanking region/luciferase cDNA chimeric construct. d-glucose (d-glu), but not its enantiomer l-glucose, dose dependently induced CYP11B2 transcription and mRNA expression. A high concentration (450 mg·dL-1) of d-glu time dependently induced CYP11B2 transcription and mRNA expression. Moreover, high glucose stimulated secretion of aldosterone into the media. Transient transfection studies using deletion mutants/nerve growth factor-induced clone B (NGFIB) response element 1 (NBRE-1) point mutant of CYP11B2 5'-flanking region revealed that the NBRE-1 element, known to be activated by transcription factors NGFIB and NURR1, was responsible for the high glucose-mediated effect. High glucose also induced the mRNA expression of these transcription factors, especially that of NURR1, but NURR1 knockdown using its siRNA did not affect high glucose-induced CYP11B2 mRNA expression. Taken together, it is speculated that high glucose may induce CYP11B2 transcription via the NBRE-1 element in its 5'-flanking region, resulting in the increase in aldosterone production although high glucose-induced NURR1 is not directly involved in the effect. Additionally, glucose metabolism and calcium channels were found to be involved in the high glucose effect. Our observations suggest one possible explanation for the high incidence of hypertension in patients with diabetes.

Foot Pressure Pattern During Walking in Individuals with Anterior Cruciate Ligament Injury.

BACKGROUND: We evaluated foot pressure distribution during walking in individuals with anterior cruciate ligament (ACL) injury. METHODS: Our study included 24 ACL-deficient patients and 22 healthy young adults as controls. The former group was divided into the dominant-side ACL-deficient group (n = 17) and the nondominant-side ACL-deficient group (n = 7). The following parameters were calculated from the path of the center of pressure (COP) on a foot pressure distribution system: percentage of COP (%COP; the ratio of anteroposterior COP path length to foot length), percentage of COP locus area (%CLA; the ratio of the area encompassed by the COP path and a line between the start and end points of the COP path to foot area), and the value of maximum amplitude of COP (MACOP; the maximum perpendicular length from the COP path to a line between the start and end points of the COP). RESULTS: In the nondominant-side ACL-deficient group, %COP (P = .026), %CLA (P = .001), and MACOP (P =.012) on the injured side were significantly lower than those in the nondominant leg side of the control group. No significant differences were found between the dominant-side ACL-deficient group and the control group. CONCLUSIONS: Shortening of the COP trace in the nondominant-side ACL-deficient group may be associated with knee flexion during walking at heel contact. Because the parameters used herein can be obtained easily, repetitively, and quantitatively, they should be used in evaluating the gait of patients with ACL-deficient knees.

Proton beam therapy for locally advanced and unresectable (T4bN0M0) squamous cell carcinoma of the ethmoid sinus: A report of seven cases and a literature review.

The present study reports treatment outcomes of locally advanced and unresectable squamous cell carcinoma of the ethmoid sinus (SCC-ES) following proton beam therapy (PBT). Between January 1997 and December 2012, 7 patients (median age, 63 years) with SCC-ES underwent definitive PBT. All tumors were categorized as T4bN0M0 (2009 UICC tumor-node-metastasis classification) and were treated using conventional fractionation at a median total dose of 72 Gy equivalents (GyE; range, 70.4-76 GyE). Imaging diagnosis for the initial treatment effect within 3 months of PBT revealed that a complete response (CR) was achieved in 2 patients and a partial response (PR) in 5 patients. The overall median survival time of the patients was 43 months (range, 12-62 months), and 4 patients survived for ≥3 years. No recurrence was observed in the 2 patients who exhibited an initial CR treatment effect; however, locoregional recurrences occurred in 4/5 patients who exhibited a PR. No grade 3 or severe acute toxicities were observed, but the late toxicities of grade 3 contralateral optic nerve damage and cataracts developed in 1 and 2 patients, respectively. Based on the findings of the present study, intensification of the local treatment effect may be important for yielding favorable treatment outcomes, since no distant metastasis was observed. PBT is therefore a potentially useful treatment tool for unresectable SCC-ES.

Proton beam therapy for a patient with a giant thymic carcinoid tumor and severe superior vena cava syndrome.

Surgical resection is the first choice for treatment of a thymic carcinoid tumor and radiotherapy is often performed as adjuvant therapy. Here, we report a case of an unresectable and chemoresistant thymic carcinoid tumor that was treated successfully using standalone proton beam therapy (PBT). The patient was a 66-year-old woman in whom surgical resection of the tumor was impossible because of cardiac invasion. Therefore, chemotherapy was administered. However, the tumor grew to 15 cm in diameter and she developed severe superior vena cava (SVC) syndrome. She was referred to our hospital and received PBT at a dose of 74 GyE in 37 fractions. PBT was conducted without severe early toxicities. After PBT, the tumor mildly shrunk to 13 cm in diameter and SVC syndrome almost disappeared. Subsequently, the tumor has continued to decrease in size slowly over the last 2 years and late toxicities have not been observed. Our experience with this case suggests that PBT may be effective for an unresectable thymic carcinoid tumor.

Angiotensin II receptor blockers differentially affect CYP11B2 expression in human adrenal H295R cells.

We generated a stable H295R cell line expressing aldosterone synthase gene (CYP11B2) promoter/luciferase chimeric reporter construct that is highly sensitive to angiotensin II (AII) and potassium, and defined AII receptor blocker (ARB) effects. In the presence of AII, all ARBs suppressed AII-induced CYP11B2 transcription. However, telmisartan alone increased CYP11B2 transcription in the absence of AII. Telmisartan dose-dependently increased CYP11B2 transcription/mRNA expression and aldosterone secretion. Experiments using CYP11B2 promoter mutants indicated that the Ad5 element was responsible. Among transcription factors involved in the element, telmisartan significantly induced NGFIB/NURR1 expression. KN-93, a CaMK inhibitor, abrogated the telmisartan-mediated increase of CYP11B2 transcription/mRNA expression and NURR1 mRNA expression, but not NGFIB mRNA expression. NURR1 over-expression significantly augmented the telmisartan-mediated CYP11B2 transcription, while high-dose olmesartan did not affect it. Taken together, telmisartan may stimulate CYP11B2 transcription via NGFIB and the CaMK-mediated induction of NURR1 that activates the Ad5 element, independent of AII type 1 receptor.

[A case of calcineurin-inhibitor induced pain syndrome associated with tacrolimus therapy for ulcerative colitis].

A 23-year-old woman was admitted with a relapse of ulcerative colitis. Tacrolimus therapy was initiated following inadequate response to corticosteroid therapy. Although the symptoms partially improved, she suddenly developed severe pain localized to the lower limbs on day 16 of tacrolimus therapy. By day 17, she was unable to move. Magnetic resonance imaging revealed born marrow edema in the lower limbs. We suspected calcineurin-inhibitor induced pain syndrome (CIPS) due to tacrolimus therapy. The pain improved within approximately four weeks of tacrolimus cessation. CIPS that is not associated with organ transplantation is a rare occurrence. Here we report a rare case of CIPS that was caused by tacrolimus therapy in a patient with ulcerative colitis.

Foot pressure pattern and its correlation with knee range of motion limitations for individuals with medial knee osteoarthritis.

OBJECTIVE: To determine the foot pressure pattern of individuals with medial knee osteoarthritis (OA) and to analyze its relation with knee flexion/extension range of motion. DESIGN: Descriptive. SETTING: Rehabilitation center. PARTICIPANTS: Individuals with unilateral, painful medial knee OA (n=50; mean age, 75y; OA group) were enrolled as subjects, and young, healthy persons (n=50; mean age, 28y; young group) and elderly persons (n=44; mean age, 74y; elderly group) without any pain, deformity, or apparent OA changes in either knee were enrolled as controls. INTERVENTION: Walking 10m at a comfortable speed. MAIN OUTCOME MEASURES: Partial foot pressures as percentages of body weight (%PFP), anteroposterior length of the center of pressure (COP) path as a percentage of foot length (%Long), transverse width of the COP path as a percentage of foot width (%Trans), knee flexion/extension range of motion (in the OA group). RESULTS: The %PFP values for the heel and hallux, %Long, and %Trans were all significantly lower in the OA group than in controls (P<.001, all). Within the OA group, a limited range of knee extension was significantly associated with a short %Long (P<.001) but not with %Trans. CONCLUSIONS: Individuals with medial knee OA exhibited low pressure on the heel and hallux and short %Long of the COP path, and limitation of knee extension was associated with shortness of %Long. The shortness of %Long in the OA group likely resulted from insufficient knee extension during the heel-contact phase associated with low pressure on the heel. %Long is a useful parameter in gait analysis by using a foot pressure measurement system.

Flare up of ulcerative colitis during pregnancy treated by adsorptive granulocyte and monocyte apheresis: therapeutic outcomes in three pregnant patients.

PURPOSE: Treatment of ulcerative colitis with drugs during pregnancy potentially may harm the mother and the unborn child. Granulocytapheresis depletes elevated/activated myeloid lineage leucocytes as sources of inflammatory cytokines. We were interested in the safety and efficacy of granulocytapheresis in patients who had ulcerative colitis flare up during pregnancy. METHODS: Three pregnant cases with active ulcerative colitis received Adacolumn granulocytapheresis, up to 10 sessions within 3-6 weeks. Case 1: a 33-year-old woman with left-sided colitis and bloody diarrhoea 7-9 times/day showed loss of mucosal vascular patterns, and contact bleeding from the rectum to the sigmoid colon. Case 2: a 36-year-old woman with pancolitis and bloody diarrhoea 6-8 times/day had loss of mucosal vascular patterns and pus from the rectum to the sigmoid colon. Case 3: a 36-year-old woman with pancolitis and diarrhoea 4-5 times/day (first episode) had erosions and pus in the mucosa from the rectum to the transverse colon. RESULTS: Colitis flare was in weeks 5, 13 and 22 of pregnancy in cases 1, 2, 3, respectively. The corresponding granulocytapheresis sessions were 5, 7, and 10, reflecting an increasing trend with the pregnancy week. Patients 1 and 2 achieved complete remission, patient 3 achieved clinical remission. CONCLUSION: In these three cases with active ulcerative colitis during pregnancy, granulocytapheresis as a non-pharmacologic treatment was effective and safe. In case 3 that did not respond well to the initial granulocytapheresis sessions, a moderate dose of prednisolone enhanced the efficacy of granulocytapheresis and tapering of prednisolone shortly after administration was not associated with relapse.

[Successful treatment of a case of advanced sigmoid colon cancer with occlusion of the common celiacomesenteric trunk].

A 74-year-old man was admitted to our hospital with abdominal pain and bloody stool. The patients' history showed that he had had occlusion of the proximal common trunk of the celiac artery (CA) and the superior mesenteric artery (SMA). The inferior mesenteric artery (IMA), and the marginal artery of the colon had developed well. It was assumed that almost the entire visceral blood might be supplied by the IMA to the CA and the SMA. Our investigation revealed that the patient had advanced cancer of the sigmoid colon, which had caused intestinal obstruction. Sigmoidectomy was performed with care to avoid injuring the IMA and the marginal arcade artery. Normal hemodynamics were successfully established followed by sigmoidectomy, and cure was obtained in this patient.

Analysis of nucleotides by pressure-assisted capillary electrophoresis-mass spectrometry using silanol mask technique.

A method for the determination of nucleotides based on pressure-assisted capillary electrophoresis-electrospray ionization mass spectrometry (PACE-MS) is described. To prevent multi-phosphorylated species from adsorbing onto the fused-silica capillary, silanol groups were masked with phosphate ions by preconditioning the capillary with the background electrolyte containing phosphate. During preconditioning, nebulizer gas was turned off to avoid contamination of MS detector with phosphate ions. To detect nucleotides using the CE positive mode at a pH 7.5, it was necessary to apply air pressure to the inlet capillary during electrophoresis to supplement the electroosmotic flow (EOF) toward the cathode. Moreover, we exchanged the running electrolyte every analysis using the buffer replenishment system to obtain the required reproducibility. Under the optimized conditions, 14 phosphorylated species such as nucleotides, nicotinamide-adenine dinucleotides and coenzyme A (CoA) compounds were well determined in less than 20 min. The relative standard deviations (n=6) of the method were better than 0.9% for migration times and between 1.7% and 8.1% for peak areas. The detection limits for these species were between 0.5 and 1.7 micromol/L with pressure injection of 50 mbar for 30 s (30 nL) at a signal-to-noise ratio of 3. This approach is robust and quantitative compared to the previous method, and its utility is demonstrated by the analysis of intracellular nucleotides and CoA compounds extracted from Escherichia coli wild type, pfkA and pfkB knockout mutants. The methodology was used to suggest that pfkA is the main functional enzyme.

Direct measurement of isotopomer of intracellular metabolites using capillary electrophoresis time-of-flight mass spectrometry for efficient metabolic flux analysis.

We have developed a metabolic flux analysis method that is based on (13)C-labeling patterns of the intracellular metabolites directly measured by capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). The flux distribution of the central carbon metabolism in Escherichia coli was determined by this new approach and the results were compared with findings obtained by conventional GC-MS analysis based on isotopomer of the proteinogenic amino acids. There were some differences in estimation results between new approach using CE-TOFMS and conventional approach using GC-MS. These were thought to be attributable to variations in measured mass distributions between amino acids and the corresponding precursors and to differences in the sensitivity of the exchange coefficients to mass distributions. However, our CE-TOFMS method facilitates high-throughput flux analysis without requiring complicated sample preparation such as hydrolysis of proteins and derivatization of amino acids.

Multiple high-throughput analyses monitor the response of E. coli to perturbations.

Analysis of cellular components at multiple levels of biological information can provide valuable functional insights. We performed multiple high-throughput measurements to study the response of Escherichia coli cells to genetic and environmental perturbations. Analysis of metabolic enzyme gene disruptants revealed unexpectedly small changes in messenger RNA and proteins for most disruptants. Overall, metabolite levels were also stable, reflecting the rerouting of fluxes in the metabolic network. In contrast, E. coli actively regulated enzyme levels to maintain a stable metabolic state in response to changes in growth rate. E. coli thus seems to use complementary strategies that result in a metabolic network robust against perturbations.