Overexpression of O-GlcNAc by prostate cancer cells is significantly associated with poor prognosis of patients.

BACKGROUND: O-linked ß-N-acetylglucosamine (O-GlcNAc) is a glycan essential for fundamental cellular processes such as transcription/translation, nuclear transport, protein stability and protein-protein interactions. However, the role of O-GlcNAc in prostate cancer progression of patients remains poorly unknown. Here we investigated the clinicopathological significance of O-GlcNAc expression level in prostate cancer. METHODS: O-GlcNAc expression level in prostate cancer cells was determined by immunohistochemistry of prostate biopsy specimens obtained from 56 patients later treated with hormone deprivation therapy comparing with adjacent normal prostate glands in the same sections. Overall survival was determined by the Kaplan-Meier and Cox proportional hazards methods with univariate and multivariate models. The effects of reduced O-GlcNAc expression level on proliferation and invasion of prostate cancer LNCaP cells were examined using small interfering RNA (siRNA) targeting O-GlcNAc transferase (OGT), the enzyme responsible for O-GlcNAc biosynthesis. RESULTS: Defining cancer cells showing stronger cytoplasmic staining than normal prostate glands as overexpression of O-GlcNAc, 39% of prostate cancer patients were categorized as overexpression. The Kaplan-Meier and Cox proportional hazards methods with univariate model analysis revealed that O-GlcNAc overexpression was associated with overall survival (P=0.0012 for the Kaplan-Meier and P=0.0021 for Cox univariate hazard model analysis). Furthermore, O-GlcNAc was the only item in which a significant difference was observed at overall survival by multivariate analysis (P=0.0475). Finally, siRNA-mediated OGT knockdown in LNCaP cells resulted in decreased expression of O-GlcNAc and promoted decreased proliferation and tumor cell invasion compared with control siRNA-transfected LNCaP cells. CONCLUSIONS: These results indicate that O-GlcNAc expression level in prostate cancer cells is associated with poor prognosis of prostate cancer patients and likely enhances tumor cell proliferation and invasion.

Clinical features of a new disease concept, IgG4-related thyroiditis.

OBJECTIVES: Immunoglobulin (Ig)G4-related disease is a recently proposed systemic disorder that includes autoimmune pancreatitis (AIP), Mikulicz's disease, and various other organ lesions. In the present retrospective study, we examined whether thyroid lesions should also be included in IgG4-related disease (Ig4-RD) under the new term IgG4-related thyroiditis. METHOD: We enrolled 114 patients with Ig4-RD, including 92 patients with AIP, 15 patients with Mikulicz's disease, and seven patients with IgG4-related cholangitis, and analysed clinical findings, function, serum values of activity markers, computed tomography (CT) images, and histology of the thyroid gland. RESULTS: Among the 22 patients (19%) in our cohort who were found to have hypothyroidism [thyroid stimulating hormone (TSH) > 4 mIU/L], 11 patients had clinical hypothyroidism [free thyroxine (FT4) < 1 ng/dL] and 11 patients had subclinical hypothyroidism (FT4 ≥ 1 ng/dL). Serum concentrations of IgG, IgG4, circulating immune complex (CIC), and ß2-microglobulin (ß2-MG) were significantly higher in the hypothyroidism group compared with the remaining 92 euthyroid patients, and serum C3 concentration was significantly lower. After prednisolone treatment, TSH values had decreased significantly (p = 0.005) in this group and FT4 values had increased significantly (p = 0.047). CT images showed that the thyroid glands of patients with clinical hypothyroidism had a significantly greater volume than those of the euthyroid and other groups. Pathological analysis of one resected thyroid gland disclosed a focused lesion with infiltration of lymphocytes and IgG4-bearing plasma cells and loss of thyroid follicles. CONCLUSIONS: Thyroid lesions associated with hypothyroidism can be considered as a new disease termed IgG4-related thyroiditis. Awareness of this condition should lead to appropriate corticosteroid treatment that may prevent progression to a fibrous state.

The effects of various therapeutic measures on shoulder range of motion and cross-sectional areas of rotator cuff muscles after baseball pitching.

AIM: The purpose of this study was to investigate the effects of various therapeutic measures on the shoulder range of motion (ROM) and muscle cross-sectional area (mCSA) of rotator cuff muscles after baseball pitching. EXPERIMENTAL DESIGN: a mode of therapeutic measures was classified in 4 groups; the control (CON), ice treatment (IT), light shoulder exercise (LSE) and ice treatment with LSE (ILSE) groups. Each therapeutic measure was performed after pitching. PARTICIPANTS: 7 healthy, skilled baseball pitchers. MEASURES: ROM and mCSA were measured before pitching, immediately after pitching, at the time of the therapeutic measure, and 24 hours after pitching. Shoulder ROM at 90 inverted exclamation mark of abduction included internal rotation (IROM), maximum internal rotation (IMROM), external rotation (EROM) and maximum external rotation (EMROM). RESULTS: In all groups, both IROM and IMROM were significantly decreased after pitching compared with the pre-exercise values and conversely both EROM and EMROM were significantly increased. The mCSA of all rotator cuff muscles were increased significantly after pitching. For IMROM, ILSE showed a significant recovery at the post-therapeutic measure compared with the others and at 24 hours after pitching compared with IT, respectively. For IROM, both LSE and ILSE showed significant recovery compared with CON at the post-therapeutic measure. For the mCSA of external muscles, ILSE showed a greater decrease at the post-therapeutic measure than the others, and at 24 hours after pitching than CON. CONCLUSION: This study suggested the possibility that ILSE was more effective to recover ROM and decrease mCSA than the other methods.

The effects of various therapeutic measures on shoulder strength and muscle soreness after baseball pitching.

AIM: This study was intended as an investigation of the effects of various therapeutic measures on the shoulder strength and muscle soreness after baseball pitching. EXPERIMENTAL DESIGN: participants threw 98 pitches in a simulated single game. The mode of the therapeutic measures after pitching were classified into 4 groups; the control group (CON), the ice treatment group (IT), the light shoulder exercise group (LSE) and the ice treatment with LSE group (ILSE). Each therapeutic measure was applied to the dominant shoulder immediately after pitching. PARTICIPANTS: 7 healthy, skilled baseball pitchers. MEASURES: both shoulder strength and muscle soreness were measured before pitching, immediately after pitching (Post-P), at the time of the therapeutic measure (Post-TM), and 24 hours after pitching (Post-24 h). RESULTS: All 4 groups showed shoulder strength losses in shoulder abduction, internal/external rotation with no shoulder abduction or with the shoulder abducted to 90 degrees immediately after pitching. ILSE had greater recovery from Post-P values at Post-TM or Post-24 h than the other methods in all 5 shoulder strengths. On the other hand, the soreness in shoulder internal rotation was increased significantly from Post-P and continued by Post-24 h. Both IT and ILSE had beneficial effects on reducing the shoulder muscle soreness at Post-TM or Post-24 h. CONCLUSION: The findings of this study suggested that ILSE was the optimal therapeutic measure against decreased shoulder strength or increased shoulder muscle soreness resulting from the repetitive baseball pitching.

Endurance training under 2500-m hypoxia does not increase myoglobin content in human skeletal muscle.

The present study was carried out to determine whether myoglobin (Mb) concentration ([Mb]) in human skeletal muscle is influenced by 8 weeks of endurance training under normal conditions, and under hypoxic conditions equivalent to an altitude of 2500 m. Fourteen healthy but sedentary male adults who did not participate in any regular exercise program took part in this study. They were divided into two groups according to the training regime to which they were submitted: the N group, who exercised under normobaric conditions, and the H group, who exercised under hypobaric conditions. All subjects performed an incremental cycling exercise at sea level to evaluate their maximal O2 uptake (VO2max) before and after the 8-week endurance training course period. Muscle tissue samples were obtained by needle biopsy from the vastus lateralis muscle for histochemical and biochemical analysis. Training induced an increase in VO2max in both the N and H groups (P < 0.05), although there was no significant difference in these changes between groups. The 8-week training had no effect on [Mb] in either group. Muscle fiber composition was also unaffected by the training course. In contrast, citrate synthase activity in both groups increased by [mean (SD)] 28.2 (33.3)% (N: P < 0.01) and 32.0 (18.2)% (H: P < 0.05) after training, and the number of capillaries (capillary:fiber ratio) increased by 47.7 (33.8)% (N: P < 0.01) and 32.3 (20.6)% (H: P < 0.05). There were no significant differences in these parameters between the N and H groups. These results suggest that significant improvement of aerobic potential as a result of endurance training are not accompanied by increases in [Mb] in human skeletal muscle. In addition, a lower absolute workload may not be sufficient to stimulate Mb synthesis in humans, even where endurance training is carried out under hypoxia.

Effects of hyperbaric oxygen on procollagen messenger RNA levels and collagen synthesis in the healing of rat tendon laceration.

In the present study, we examined the effect of intermittent exposure to hyperbaric oxygen (HBO) on experimentally induced tendon laceration in the right hind limb of 56 male Wistar rats. We compared animals treated by HBO (2 atmospheres absolute [ATA] 100% O(2) x 60 min x once per day) with control animals (without HBO) at 3, 7, 14, and 28 days after wound creation. Histology, and pro-alpha1 (I) mRNA by Northern hybridization, pro-alpha1 (III) mRNA, transforming growth factor-beta1 (TGF-beta1) mRNA by RT-PCR were studied. Our results indicate the use of HBO influences the pro-a1 (I) mRNA expression. The level of pro-alpha1 (I) mRNA was especially increased at 7 14 days after laceration by HBO. These results suggest that intermittent oxygen exposure may play a significant role in collagen synthesis and be beneficial for producing the extracellular matrices in tissue engineering.

Early phase adaptations of muscle use and strength to isokinetic training.

The purpose of this study was to investigate the effect of short periods of isokinetic resistance training on muscle use and strength. Seven men trained the right quadriceps femoris muscles (QF) 9 d for 2 wk using 10 sets of 5 knee extensions each day. Isometric and isokinetic torques of QF were measured at six angular velocities. Cross-sectional areas (CSA) of QF were determined from axial images using magnetic resonance imaging (MRI). Transverse relaxation time (T2) and activated area of QF, which represented the area greater than the mean resting T2 + ISD in MR[pixels, were calculated at rest and immediately after repetitive isokinetic knee extensions based on T2-weighted MR images. Muscle fiber types, fiber area, and phosphofructokinase (PFK) activities were determined from biopsies of the vastus lateralis muscle. No changes were found in CSA of QF, muscle fiber types, fiber area, and PFK activities after the training. Isometric and isokinetic peak torques at 60-240 degrees x s(-1) and relative area of QF activated by knee extensions increased significantly after the training. These results suggest that muscle strength increases after short periods of isokinetic resistance training without muscle hypertrophy would be due to increased muscle contractile activity.

Maintenance of myoglobin concentration in human skeletal muscle after heavy resistance training.

The aim of this study was to determine the effects of 8 weeks of resistance training (RT) on the myoglobin concentration ([Mb]) in human skeletal muscle, and to compare the change in the [Mb] in two different RT protocols. The two types of protocol used were interval RT (IRT) of moderate to low intensity with a high number of repetitions and a short recovery time, and repetition RT (RRT) of high intensity with a low number of repetitions and a long recovery time. A group of 11 healthy male adults voluntarily participated in this study and were divided into IRT (n = 6) and RRT (n = 5) groups. Both training protocols were carried out twice a week for 8 weeks. At the completion of the training period, the one-repetition maximal force values and isometric force were increased significantly in all the subjects, by about 38.8% and 26.0%, respectively (P < 0.01). The muscle fibre composition was unchanged by the 8 weeks of training. The muscle fibre cross-sectional areas were increased significantly by both types of training in all fibre types (I, IIa and IIb, mean + 16.1 %, P < 0.05). The [Mb] showed no significant changes at the completion of the training [IRT from 4.63 (SD 0.63) to 4.48 (SD 0.72), RRT from 4.47 (SD 0.75) to 4.24 (SD 0.80) mg x g(-1) wet tissue] despite a significant decrease in citrate synthase activity [IRT from 5.27 (SD 1.45) to 4.49 (SD 1.48), RRT from 5.33 (SD 2.09) to 4.85 (SD 1.87) micromol x min(-1) x g(-1) wet tissue; P < 0.05] observed after both protocols. These results suggested that myoglobin and mitochondria enzymes were regulated by different mechanisms in response to either type of RT. Moreover, the maintained [Mb] in hypertrophied muscle should preserve oxygen transport from capillaries to mitochondria even when diffusion distance is increased.

Preexisting membranous nephropathy in allograft kidney.

A case of membranous nephropathy, preexisting in a donor kidney, will be reported. A 41-year-old man underwent a cadaver renal transplantation. An allograft biopsy specimen obtained during the operation showed spike formation on periodic acid-silver methenamine staining and deposition of IgG along the glomerular capillary loop on immunoperoxidase staining. Immunofluorescence staining for IgG remained in the specimens obtained on day 11 and after 4 weeks, but markedly decreased in the specimen obtained 7 weeks after transplantation. Electron-dense deposits also decreased in amount, but irregular thickening of the glomerular basement membrane with spikes, electron-lucent washout lesions, and small amounts of electron-dense deposits remained 20 months after the transplantation. These findings suggest that membranous nephropathy, as well as IgA nephritis and diabetic nephropathy, resolve after renal transplantation and that deposition of IgG markedly decreases within a few months after transplantation, but that complete histological restoration of the basement membrane needs at least a few years.

Adaptation and distortion of podocytes in rat remnant kidney.

A sequence of alterations of podocytes as well as the development of glomerulosclerosis was analyzed in a five-sixths nephrectomy rat model. Morphometric measurements showed a gradual enlargement in glomerular size without any increase in the number of podocytes in the glomerulus. Structural changes of podocytes appeared as early as 4 weeks after operation, and increased their degree and extent with time. In electron microscopic studies, changes of podocytes in the early stage consisted of foot process stretching and thinning, and cell body attenuation accompanied by pseudocyst formation. As weeks passed, these changes became prominent and were occasionally associated with podocyte detachment from the glomerular basement membrane followed by tuft adhesion and the development of segmental sclerosis. In parallel to the structural changes, immunohistochemical staining demonstrated a marked increase of desmin expression in the glomerulus with intense staining in podocytes. Podocyte alterations were identified as an adaptation to the enlargement of the glomerulus and, consequently, the podocytes seemed to fall to adapt to the structural changes of the glomerulus. The present study supports the hypothesis that podocyte defects are responsible for both the initiation and progression of glomerular damage. Furthermore, the findings may provide the hypothesis that the native disposition of podocytes in the glomerulus participates in the development of glomerular lesions in a focal and segmental fashion.

Mature teratoma of the placenta.

A rare case of a mature teratoma of the placenta is reported. The tumor lay between the amnion and the chorion and contained skin and its appendages, bone, cartilage, fat and ganglia, without organization. There was no evidence of a recognizable umbilical cord. We concluded that the tumor was a mature teratoma of the placenta. The distinction between a teratoma and a fetus acardius amorphus, and the possible origin of the tumor in this site, are discussed with a brief review of the literature.

Relationships between fiber composition and NMR measurements in human skeletal muscle.

The purpose of this study was to examine the relationships between the relative contents of phosphocreatine (PCr), inorganic phosphate (Pi), beta-adenosine triphosphate (ATP), and transverse relaxation time (T2) with fiber composition, which determined histochemically in the human skeletal muscle. The vastus lateralis muscles of 28 volunteers were subjected to phosphorus nuclear magnetic resonance (31P NMR) spectroscopy, magnetic resonance imaging (MRI) and muscle biopsy. Muscle fibers were divided into type I and type II fibers using myosin ATPase stain. A wide range of fiber composition levels were observed in the subjects (27.3-74.6% type I fibers). The PCr/ATP, Pi/ATP and (PCr + Pi)/ATP ratios were positively related to the percentage of type II fibers (r = 0.695, p < 0.001, r = 0.429, p < 0.05 and r = 0.773, p < 0.001, respectively). There was no correlation between fiber composition and the PCr/Pi ratio (r 0.127, n.s.) or intracellular pH (r = 0.305, n.s.). Moreover, no correlation was found between T2 and fiber type (r = 0.144, n.s.). These results suggest that 31P NMR can detect the differences in relative content of phosphates between type I and type II fibers, thereby noninvasively evaluating fiber composition in human skeletal muscle.

Tears of cruciate ligaments and menisci: evaluation with cine MR imaging.

A cine magnetic resonance (MR) imaging technique, involving the acquisition of kinematic sagittal images during knee movement, was used to evaluate 52 symptomatic knee joints. Results were compared with those obtained by means of static three-dimensional (3D) MR imaging. Twenty-seven of the 28 anterior cruciate ligament (ACL) tears and 22 of 24 normal ligaments were correctly identified at cine MR imaging for a sensitivity of 96% and a specificity of 92%. Static 3D MR imaging yielded a sensitivity of 71% and a specificity of 88%. All four posterior cruciate ligament tears were identified at cine and 3D MR imaging. For meniscal tears, cine MR imaging yielded a sensitivity of 48% and a specificity of 96%; the sensitivity and specificity for 3D MR imaging were 71% and 96%, respectively. Cine MR imaging proved to be more useful than static MR imaging in assessing the tightness of cruciate ligaments, especially of those that were partially torn, and in assessing the movement of meniscal-free fragments. The increased information obtained with cine MR imaging may warrant continued investigation and clinical application.

Analysis of integrated hepatitis B virus DNA and cellular flanking sequences cloned from a hepatocellular carcinoma.

By digestion with HindIII restriction enzyme, a human hepatocellular carcinoma was shown to contain only 2 hepatitis B virus (HBV) DNA inserts. Both HindIII fragments (8 and 16 kb) were molecularly cloned and the structures of HBV DNA and adjacent host sequences were analyzed. One clone (lambda YH 8) contained part of pre-S(I) through the entire S gene and the other (lambda YH 16) had the middle section of the S to the end of X gene of HBV DNA. No gross rearrangements were observed in either HBV or cellular DNA sequences in lambda YH 16 clone. However, the 8 kb HindIII fragment was considered to be amplified together with flanking cellular sequences. Furthermore, the HBV DNA was integrated into cellular genome at the Alu repeated sequences in lambda YH 8.

Novel RNA family structure of hepatitis B virus expressed in human cells, using a helper-free adenovirus vector.

Ad5-HBL is a type 5 adenovirus bearing the large BglII fragment (2.8 kilobases; 87% of the total genome) of hepatitis B virus (HBV), subtype adr. Eight HBV RNAs expressed in HeLa cells infected with Ad5-HBL were mapped by the nuclease S1 technique. Three major RNAs spanning 2.4, 2.0, and 0.7 kilobases of the HBV sequences cover the coding regions of "presurface" plus surface antigen, surface antigen alone, and "X" protein, respectively. The 5' segment of an RNA which could code for core antigen (HBcAg) was also detected. All major HBV RNAs initiate from mutually exclusive 5' ends, terminate at the unique 3' end within the HBcAg coding region (except readthrough species), and have no spliced deletion, forming a novel RNA family structure. No TATA box-like sequences were found near the 5' end of these RNAs, except in the case of the 2.4-kilobase RNA. About two thirds of total HBV RNA does not terminate at the mapped 3'-end position, suggesting the termination signal is functionally inefficient. Since the potential 5' end of HBcAg mRNA was mapped at the same position as the minus-strand nick of HBV DNA previously reported, we propose a model that requires inefficient poly(A) addition to produce an RNA which serves both as HBcAg mRNA and as the putative RNA template of minus-strand DNA synthesis in the HBV life cycle.

Construction of nondefective adenovirus type 5 bearing a 2.8-kilobase hepatitis B virus DNA near the right end of its genome.

A novel helper-free adenovirus type 5 (Ad5) vector system, which utilizes a cloning site 0.2 kilobase (kb) from the right end of the genome, has been developed. To construct a nondefective Ad5 bearing the 2.8-kb DNA fragment of hepatitis B virus (HBV) at this site, we deleted the 2.1-kb nonessential E3 fragment from cloned DNA covering the right one-fourth of the Ad5 genome (76 to 100 map units), inserted the HBV DNA into this site, ligated the recombinant DNA to the rest of the Ad5 genome, and transfected the ligated DNA into human embryo kidney cells. Most of the recovered virus clones had only the E3 deletion and no HBV insertion, suggesting that a homologous recombination occurs between transfected DNAs in these cells. The isolated Ad5 virus bearing the HBV DNA (Ad5-HBL) grew without helper virus in HeLa cells as efficiently as wild-type Ad5, although the 1.9-kb major E4 transcript was detected only poorly in the early phase in the Ad5-HBL-infected cells, suggesting that the HBV DNA inserted upstream of the E4 promoter reduces the E4 transcript. HBV mRNAs transcribed from the inserted DNA were at least as abundant as Ad5 early mRNAs in the late phase of Ad5-HBL infection, but the HBV surface antigen was barely detectable in the infected-cell lysate and culture medium. This result suggests that HBV mRNAs can be transcribed from the inserted genes but no protein can be translated from the HBV mRNAs, presumably because of the translational suppression of cellular mRNAs caused by adenovirus in its late phase.

Potency test of hepatitis B vaccines by the parallel line assay method in mice.

Adequate conditions for the potency test of hepatitis B (HB) vaccines in mice were looked for. Preliminary tests showed that BALB/c female mice of 5 weeks of age are adequate for the test. An immunization period of 5 weeks was found satisfactory for the test. Under these conditions, mice were immunized with each of serial dilutions of the test vaccine and a reference vaccine. The use of the parallel line assay method and the expression of the potency relative to that of the reference vaccine gave a reliable estimate of the potency of HB vaccine.