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Distal femoral fractures are commonly treated with osteosynthesis. However, in older patients with osteoarthritis of the knee, acute primary total knee arthroplasty (TKA) may be performed to treat these fractures. Notably, no studies have documented the use of osteosynthesis in combination with distal femoral osteotomy (DFO) for treating distal femoral fractures in patients with knee osteoarthritis. This report presents the case of a 66-year-old woman with lateral compartment osteoarthritis accompanied by severe valgus knee deformity who underwent osteosynthesis for a distal femoral fracture combined with medial closing-wedge distal femoral osteotomy (MCWDFO) to correct the knee valgus deformity. She experienced a distal femoral fracture (AO/OTA 33B1.1) of the right knee because of a fall. Before the injury, she exhibited a limp due to severe knee pain with some limitations in knee flexion. Non-weight-bearing radiographs of the entire lower extremity suggested a percentage mechanical axis (%MA) of 115%, indicating severe valgus deformity. On day nine after the injury, we performed osteosynthesis for the distal femoral fracture and conducted an MCWDFO to correct the right knee valgus deformity. After MCWDFO, the %MA was corrected to 70%. Partial weight-bearing was initiated three weeks postoperatively and progressed to full weight-bearing at six weeks. To facilitate bone healing, low-intensity pulsed ultrasound (LIPUS) was applied for three months after surgery. Bone union was successfully achieved by month five. Some medial knee pain persisted for six months after surgery; nonetheless, the patient could walk without a limp. We considered that the integration of MCWDFO with osteosynthesis could provide a treatment option for patients with distal femoral fractures and lateral compartment osteoarthritis., Competing Interests: Human subjects: Consent was obtained or waived by all participants in this study. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work., (Copyright © 2024, Fujita et al.)

Study investigators encountered a female Becker muscular dystrophy (BMD) carrier with advanced heart failure (HF) and identified a stop-gain variant in procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 ( PLOD3 ) as a potential second-hit variant. Isogenic induced pluripotent stem cells (iPSCs) with dominant expression of WT- DMD , Δ45-48- DMD , or Δ45-48- DMD with corrected PLOD3 variant were established. Microforce testing using 3-dimensional self-organized tissue rings (SOTRs) generated from iPSC-derived cardiomyocytes (iPSC-CMs) demonstrated that correction of the heterozygous PLOD3 variant did not improve the reduced force, but it significantly recovered the reduced stiffness in Δ45-48- DMD SOTRs. Correction of the PLOD3 variant restored collagen synthesis in iPSC-CMs. Our findings revealed the pathogenesis underlying advanced HF in a female BMD carrier., Competing Interests: This work was supported by JSPS KAKENHI grant numbers 19K08489, 19K12801, 20K21602, 21H02915, and 22K19526, grants-in-aid from the Japanese Ministry of Health, Labor, and Welfare, the Japan agency for medical research and development (21bm0804008h0005, 22bm0804035h0001). This work was also supported by the Cell Science Research Foundation and the Grant for Basic Research of the Japanese Circulation Society (2018), SENSHIN Medical Research Foundation, Daiichi Sankyo, Mitsubishi Tanabe Pharma, and the Center of Medical Innovation and Translational Research and the Center for Medical Research and Education of the Graduate School of Medicine, Osaka University. The Department of Medical Therapeutics for Heart Failure was an endowment department supported by Actelion Pharmaceuticals Japan (2015-2020); the Department of Medical Therapeutics for Heart Failure is currently a Joint Research Department with TOA EIYO Pharmaceutical Company. The authors have reported that they have no relationships relevant to the contents of this paper to disclose., (© 2023 The Authors.)

Purpose: To thoroughly compare the outcomes between exposed and buried Kirschner wires (K-wires) in fixation for pediatric supracondylar humerus fractures., Methods: We examined patients who underwent surgery at our institution between January 2007 and June 2021. We investigated their age, sex, fracture pattern, number of K-wires used, whether they were exposed or buried, operative time, postoperative complications, number of outpatient visits, duration from surgery to K-wire removal, total length of hospitalization, and perioperative radiographic parameters. After propensity score matching, intergroup comparisons were performed to assess the differences in postoperative complication rate, number of outpatient visits, duration from surgery to K-wire removal, total length of hospitalization, and loss of reduction., Results: Propensity score matching resulted in 43 pairs in both groups. Although more patients complained of skin irritation in the buried K-wire group, there was more backing out of the K-wire in the exposed K-wire group ( p < 0.01). There were no significant differences in other complications. There were more outpatient visits in the buried K-wire group ( p < 0.01). The duration from surgery to K-wire removal and the total length of hospitalization were significantly longer in the buried K-wire group ( p < 0.01). There was no significant difference in the loss of Baumann's angle ( p = 0.61), tilting angle ( p = 0.48), or the development of rotation ( p > 0.99) between groups., Conclusion: More outpatient visits and longer lengths of hospitalization in the buried K-wire group may lead to increased costs and burden on parents.

Introduction: Research regarding the epidemiology of paediatric trauma is limited. Using our unique classification, we describe paediatric trauma cases in a 10-year single-centre study to improve paediatric care., Materials and Methods: Data regarding all paediatric trauma cases were extracted using a computerised medical record system that detected fracture diagnosis and epiphyseal injury. Registry search identified cases from January 2008 to December 2017. Age, sex, type of fracture, and details of injury mechanism were analysed, and we categorised the 'falls/turnover' mechanism using a new trauma energy classification based on speed and height., Results: A total of 1379 cases (953 boys and 426 girls) were included. The highest number of injuries (553 cases, 40%) was seen in school children (aged 6 to 10 years). Forearm fracture occurred most frequently, followed by humeral fracture. The most frequent injury mechanism in falls/turnover (969 cases, 70%) was sports in 272 cases (28%), playground equipment in 179 cases (18%), furniture in 102 cases (11%), and bicycling in 87 cases (9%). We classified 956 cases of falls/turnover using our trauma energy classification scheme. Most cases (29%) were classified into the L2 category (low height and high speed), followed by the L1 category (low height and low speed) (p < 0.01). Subcategory analysis using the classification revealed that younger children were more likely to be injured by falling from high places because of their physique, whereas older children were more likely to be injured by a turnover from lower places and at higher speed., Conclusion: We describe the epidemiology of fractures in detail and present a new classification system, which may aid in understanding the injury mechanism independent of children's height. The fact that paediatric fractures occur at relatively low energy levels and are trended by age, activity, and sex, could be of potential universal use for their prevention and parent education., Competing Interests: Declaration of Competing Interest None., (Copyright © 2022. Published by Elsevier Ltd.)

Background: We have been using a simplified Sauvé-Kapandji (SK) procedure using a headless compression screw for the treatment of osteoarthritis (OA) of the distal radio-ulnar joint (DRUJ). Unlike a standard SK procedure, the simplified SK procedure does not require exposure of the DRUJ to prepare the opposing surfaces of the sigmoid fossa and the ulnar head or any procedures to stabilise the proximal stump of the ulna. The aim of this study is to report the radiological outcomes of the simplified SK procedure. Methods: We retrospectively reviewed data of all patients who underwent a simplified SK procedure for osteoarthritis of the DRUJ at our hospital between October 2008 and September 2020 with a follow-up of at least 25 weeks. Baseline demographic and clinical characteristics were investigated. We also measured radiographic parameters and performed statistical analyses to evaluate the shelf-forming region, ulnar stump and deviation of the carpal region. Results: Our study included 10 patients with a mean follow-up of 52.3 weeks. There was no instance of complications such as nonunion of DRUJ arthrodesis, callus formation at ulnar pseudoarthrosis and painful instability at the proximal stump of the ulna. According to the Wilcoxon signed rank test, no radiographic parameters significantly changed during the follow-up period. There was a significant negative correlation between radioulnar width and the variation of ulnar translation index. Conclusions: All radiographic parameters were well maintained, at least during the short follow-up period. There was no instance of nonunion of DRUJ arthrodesis. The simplified SK with simple headless compression screw insertion may be a reliable method for treating OA of the DRUJ. Level of Evidence: Level IV (Therapeutic).

Duchenne muscular dystrophy (DMD) is characterized by progressive muscle degeneration accompanied by dilated cardiomyopathy. Recently, abnormality of yes-associated protein (YAP) has been reported as the pathogenesis of muscle degeneration of DMD; however YAP activity remains unclear in dystrophic heart of DMD. Herein, we investigated YAP activity using disease-specific induced pluripotent stem cell (iPSC) derived cardiomyocytes (CMs) in DMD. DMD-iPSCs were generated from DMD patient with exon 48-54 deletion in DMD, and genome-edited (Ed)-DMD-iPSCs with in-frame (Ed-DMD-iPSCs) were created using CRISPR/Cas9. Nuclear translocation of YAP [nuclear (N)/cytoplasmic (C) ratio] was significantly lower in DMD-iPSC-CMs than in Ed-DMD-iPSC-CMs. In addition, Ki67 expression, indicating proliferative ability, was significantly lower in DMD-iPSC-CMs than Ed-DMD-iPSC-CMs. Therefore, immunofluorescent staining showed that actin stress fibers associated with YAP activity by mechanotransduction were disorganized in DMD-iPSC-CMs. Lysophosphatidic acid (LPA), a known lipid mediator on induction of actin polymerization, significantly increased YAP activity and actin dynamics in DMD-iPSC-CMs using live cell imaging. These results suggested that altered YAP activity due to impaired actin dynamics reduced proliferative ability in DMD-iPSC-CMs. Hence, decreased YAP activity in dystrophic heart may contribute to DMD-cardiomyopathy pathogenesis.

Human induced pluripotent stem (hiPS) cells have been used as a cell source for regenerative therapy and disease modeling. The purity of hiPS-cardiomyocytes (hiPS-CMs) has markedly improved with advancements in cell culture and differentiation protocols. However, the morphological features and molecular properties of the relatively immature cells are still unclear, which has hampered their clinical application. The aim of the present study was to investigate the extent to which topographic substrates actively influence hiPS-CMs. hiPS-CMs were seeded on randomized oriented fiber substrate (random), anisotropic aligned fiber substrate (align), and flat non-scaffold substrate (flat). After culturing for one week, the hiPS-CMs on the aligned patterns showed more mature-like properties, including elongated rod shape, shorter duration of action potential, accelerated conduction velocity, and elevated cardiac gene expression. Subsequently, to determine whether this development was irreversible or was altered after withdrawal of the structural support, the hiPS-CMs were harvested from the three different patterns and reseeded on the non-scaffold (flat) pattern. After culturing for one more week, the improvements in morphological and functional properties diminished, although hiPS-CMs pre-cultured on the aligned pattern retained the molecular features of development, which were even more significant as compared to that observed during the pre-culture stage. Our results suggested that the anisotropic fiber substrate can induce the formation of geometrical mimic-oriented heart tissue in a short time. Although the morphological and electrophysiological properties of hiPS-CMs obtained via facilitated maturation somehow rely on the existence of an exterior scaffold, the molecular developmental features were preserved even in the absence of the external support, which might persist throughout hiPS-CM development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Li, Lee, Miwa, Kuramoto, Masuyama, Yasutake, Tomoyama, Nakanishi and Sakata.)

Background: Although it is important to understand the risk factors affecting the reduction of pediatric supracondylar humerus fractures (PSHFs), no inclusive study has been conducted so far. We performed a retrospective cohort study to identify the risk factors affecting the quality and difficulty of reduction for PSHFs., Methods: We reviewed 160 cases with a PSHF that had been surgically treated in our hospital between January 2007 and December 2019. We investigated age, sex, injured side, body mass index (BMI), modified Gartland classification, neurological deficit or an absent radial artery pulse at an initial examination, start time of the operation, waiting time from injury to surgery, operative time, reduction technique, and perioperative radiographic parameters. We calculated independent predictors of unsuccessful closed reduction, prolonged operative time, and malreduction using multivariate analyses., Results: In a logistic regression analysis, modified Gartland type III was a significant predictor of unsuccessful closed reduction (OR 14.50 [95% CI 4.03-51.90]; P < 0.01) and BMI was a significant predictor of malrotation (OR 1.59 [95% CI 1.06-2.39]; P = 0.025). In a multiple linear regression analysis, BMI and open reduction were significant predictors of prolonged operative time (BMI, P = 0.011; open reduction, P < 0.01)., Conclusions: If closed reduction fails, we should not hesitate to immediately switch to other methods. Obesity was an independent predictor of both prolonged operative time and malrotation, a finding that has not been previously reported. Rotational alignment should be carefully checked, especially for obese children, and accurately be reduced. Open reduction also extended operative time.

Background: Pediatric supracondylar humerus fracture (PSHF) is one of the most common fractures of the elbow seen among boys aged between 5 and 7 years. The timing of surgical treatment for this type of fracture is still controversial. Thus, we aimed to investigate whether the timing of surgery for PSHFs affects the incidence of early postoperative complications and reduction of PSHFs. Methods: We retrospectively reviewed the medical records of PSHF patients who underwent surgery at our hospital between January 2007 and March 2019. We classified patients who underwent surgery within 12 h and more than 12 h after the fracture as the early and delayed groups, respectively. The outcome measures compared between the two groups were the incidence of postoperative early complications such as neurological deficits, including iatrogenic ulnar nerve injury, vascular compromise, compartment syndrome, K-wire migration, and unplanned returns to the operating room. We also examined surgical time, reduction procedure, and perioperative radiographic parameters. Results: There was no significant difference in the incidence of early complications between the early and delayed groups for either modified Gartland type II or type III fractures. There was also no significant difference in surgical time, reduction procedure, or perioperative radiographic parameters between the two groups. Conclusions: Delayed surgery was not associated with an increased rate of postoperative early complications in either type II or type III fractures. The timing of surgery does not affect the difficulty or quality of reduction.

Ectopic foci from pulmonary veins (PVs) comprise the main trigger associated with the initiation of atrial fibrillation (AF). An abrupt anatomical narrow-to-wide transition, modeled as in vitro geometrical patterning with similar configuration in the present study, is located at the junction of PVs and the left atrium (LA). Complex cellular composition, i.e., constituent cell heterogeneity, is also observed in PVs and the PVs-LA junction. High frequency triggers accompanied with anatomical irregularity and constituent cell heterogeneity provoke impaired conduction, a prerequisite for AF genesis. However, few experiments investigating the effects of these factors on electrophysiological properties using human-based cardiomyocytes (CMs) with atrial properties have been reported. The aim of the current study was to estimate whether geometrical patterning and constituent cell heterogeneity under high frequency stimuli undergo conduction disturbance utilizing an in vitro two-dimensional (2D) monolayer preparation consisting of atrial-like CMs derived from human induced pluripotent stem cells (hiPSCs) and atrial fibroblasts (Fbs). We induced hiPSCs into atrial-like CMs using a directed cardiac differentiation protocol with the addition of all- trans retinoic acid (ATRA). The atrial-like hiPSC-derived CMs (hiPSC-CMs) and atrial Fbs were transferred in defined ratios (CMs/Fbs: 100%/0% or 70%/30%) on manually fabricated plates with or without geometrical patterning imitating the PVs-LA junction. High frequency field stimulation emulating repetitive ectopic foci originated in PVs were delivered, and the electrical propagation was assessed by optical mapping. We generated high purity CMs with or without the ATRA application. ATRA-treated hiPSC-CMs exhibited significantly higher atrial-specific properties by immunofluorescence staining, gene expression patterns, and optical action potential parameters than those of ATRA-untreated hiPSC-CMs. Electrical stimuli at a higher frequency preferentially induced impaired electrical conduction on atrial-like hiPSC-CMs monolayer preparations with an abrupt geometrical transition than on those with uniform geometry. Additionally, the application of human atrial Fbs to the geometrically patterned atrial-like hiPSC-CMs tended to further deteriorate the integrity of electrical conduction compared with those using the atrial-like hiPSC-CM alone preparations. Thus, geometrical narrow-to-wide patterning under high frequency stimuli preferentially jeopardized electrical conduction within in vitro atrial-like hiPSC-CM monolayers. Constituent cell heterogeneity represented by atrial Fbs also contributed to the further deterioration of conduction stability.

Competing Interests: Conflicts of Interest: None declared.

A 67-year-old man with a history of aortic mechanical valve replacement exhibited an abnormal mass on the sewing ring of the prosthesis on echocardiography. Despite receiving strong anticoagulation, he developed acute cerebral infarction due to the formation of emboli resulting from the thrombus and underwent urgent re-aortic valve replacement. Based on the microscopic findings of the resected mass, he was finally diagnosed as having nonbacterial thrombotic endocarditis (NBTE) of the mechanical prosthetic valve, which was thought to be associated with colorectal cancer. We herein report the first known case of an antemortem diagnosis of NBTE on a mechanical heart valve.

Background: Locking plates are an internal fixation material useful in the treatment of bone fractures, which provides effective stabilization between the plate and locking head screws (LHSs) via the locking mechanism. However, difficulty in removing LHSs is relatively common, and such cases can require long surgical procedures or use of special removal equipment. The purpose of this study was to report the incidence and risk factors for difficult removal of LHSs., Methods: During the 5-year-6-month period from April 2006 to September 2011, 83 locking plates containing a total of 482 LHSs were removed in 80 patients at our institution. Out of 482 LHSs, there were 118 LHSs with a 2.4-2.7 mm diameter, 308 LHSs with a 3.5 mm diameter and 56 LHSs with a 5.0 mm diameter. The incidence of removal difficulty was examined on the basis of screw diameter. In addition, the risk factors were assessed in only LHSs with a 3.5 mm diameter. LHSs with a 3.5 mm diameter were divided into 2 groups, the difficult removal group and the easy removal group, and the data were examined based on age, sex, time between insertion and removal, and screw position. The incidence of removal difficulty in LHSs with a 3.5 mm diameter was examined every 6 months between insertion and removal., Results: Difficulty in removal was encountered in none (0 %) of 118 LHSs with a 2.4-2.7 mm diameter, 15 (4.9 %) of 308 LHSs with a 3.5 mm diameter, and none (0 %) of 56 LHSs with a 5.0 mm diameter. In only LHSs with a 3.5 mm diameter, the mean ages of the patients in the difficult removal group and the easy removal group were 32.1 and 45.6 years, respectively. The average time between insertion and removal in the difficult removal group and the easy removal group was 529.2 and 389.2 days, respectively. There was a statistically significant difference in age and time between insertion and removal. Removal was difficult in 15 (9.1 %) of 165 LHSs with a 3.5 mm diameter in those with >1 year between insertion and removal., Conclusion: This study suggests that (1) the use of LHSs with a 3.5 mm diameter is a necessary condition for difficulty in screw removal, and that (2) longer time from internal fixation to removal, and (3) younger age, are risk factors for it. When removing LHSs with a 3.5 mm diameter, appropriate instruments and sufficient training are necessary.

The accurate and high-resolution measurement of surface pressure is achieved by a pressure/ temperature-sensitive composite paint (bi-PSP), whereas the pressure-sensitive dye photodegraded the temperature sensitive dye in close arrangement of both dyes. In the present study, an attempt was made to synthesize a homogeneous bi-PSP membrane without light-induced degradation of the dye using mesoporous silica. Mesoporous silica as a molecular sieve was the separation of pressure- and temperature-sensitive dyes. Both achievement of control of photodegradation in temperature-sensitive paints with molecule-screening capacity and macroscopically uniform placement of insoluble pigments in the respective solvent, was accomplished using the mesoporous silica nanoparticles in a compound PSP.

Cellular retinol-binding protein type II (CRBPII) is exclusively expressed in the small intestinal absorptive cells. We previously reported that dietary fat induces CRBPII expression within 12 h of fat intake. To examine at which locus of the villus-crypt axis this response to dietary fat occurs, 6-wk-old rats were fed a low-fat diet (7% energy) for 7 d, and then given free access to a high-fat diet (70% energy) for the subsequent 12, 24 or 48 h. Cryostat sectioning of jejunal segments followed by RNA blot hybridization of the transcripts revealed that CRBPII mRNA was expressed maximally in the lower villus, and the immunoreactive protein of CRBPII was expressed maximally in the mid-villus. Feeding the high-fat diet caused a pronounced increase in CRBPII mRNA level from the lower- to middle-villus within 12 h. These results suggest that the CRBPII gene is maximally expressed in the lower villus, and that dietary fat causes an enhancement of CRBPII gene expression in the villus cells.

Cellular retinol-binding protein type II (CRBPII) is involved in the transport of vitamin A and its metabolism in the small intestine. In the present study, we demonstrated diet-related variations in CRBPII expression in rat jejunum. The CRBPII protein and mRNA levels increased in parallel after the start of feeding period regardless of whether the feeding period was restricted to the hours of darkness or of light. In addition, this variation was observed in the rats fed high-fat diet or low-fat diets, but not in those fed a fat-free diet or in fasted rats. A similar diet-induced variation was seen in the mRNA of liver-type fatty acid-binding protein in rat jejunum. In the transient transfection experiment, unsaturated fatty acid increased rat CRBPII gene promoter activity via the PPARalpha/retinoid X receptor-alpha heterodimer. Taken together, these results suggest that the diet-related variation in CRBPII expression in rat jejunum may be brought about by the transcriptional induction of CRBPII gene expression mainly triggered by dietary fatty acids.

Background: It is not known whether cardiac markers and cyclic variations of integrated backscatter can be used to detect cardiac sarcoidosis., Methods: We studied 62 patients with sarcoidosis affecting the lung, eyes, skin, or heart (27 patients with cardiac involvement and 35 patients without). The cyclic variation of integrated backscatter and wall thickening was evaluated in the left ventricular anterior septum and posterior wall. Plasma A-type natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) concentrations and serum cardiac troponin T were also determined., Results: Plasma natriuretic peptide concentrations were higher in the cardiac involvement group (ANP: 15.5 [interquartile range (IQR) 2.5-34.0] vs. 12.0 [10.0-16.5] pg/ml, P=0.25; BNP: 28.6 [5.9-141] vs. 10.1 [4.8-15.4] pg/ml, P=0.049). However, cardiac troponin T concentration was <0.01 ng/ml in all patients. Receiver-operator characteristic (ROC) analysis showed that both ANP and BNP could identify patients with high-degree atrioventricular block, ventricular tachyarrhythmias, or symptomatic heart failure (the areas under the ROC curve were 0.94 and 0.97, respectively). The cardiac involvement group could be distinguished from the noninvolvement group by combining cutoff values for the magnitude of integrated backscatter cyclic variation (5.5 dB) and wall thickening (30%), albeit only for the posterior wall., Conclusion: Both ANP and BNP are useful markers for identifying patients with sarcoidosis and cardiac complication(s). Moreover, evaluation of integrated backscatter cyclic variation combined with wall thickening may be of help in detecting cardiac involvement in the posterior wall.

From 1996 to 2002 we used intramedullary Kirschner wires to treat 19 femoral shaft fractures in 19 children (11 boys, 8 girls). Their ages ranged from 2 to 13 years (mean 5.8 years). The mean follow-up time was 3 years (range 4 months to 4 years 9 months). The technique was intramedullary pinning using a 2.5- to 3.0-mm Kirschner wire introduced through the greater trochanter followed by spica casting. All cases achieved bone union within 3 months. The mean angular deformity was 1.7 degrees (range 0 degrees -5 degrees ), and one patient had an external rotational deformity. The mean overgrowth was 7.5 mm, but no patient had residual problems during activities of daily living. Our method has the following advantages: no need of traction, leaving bed earlier, ease of nursing, fewer radiographic examinations, less angular deformity. It also has several disadvantages: the need for an operation under general anesthesia, the need for metal removal, and an operation scar. This method can be considered as one option for treating femoral shaft fractures in children.

We describe a consecutive series of 26 patients with simple bone cysts who were treated by curettage, multiple drilling and continuous decompression by the insertion of either a cannulated screw or a pin. In the first 15 patients we used titanium cannulated screws (group 1) and in the next 11 a cannulated hydroxyapatite pin (group 2). Satisfactory healing was achieved in 12 patients in group 1 (80%) and in all in group 2. This technique seems to be a promising option for the treatment of simple bone cysts. The cannulated hydroxyapatite pin is recommended because of its higher success rate and the fact that it does not need to be removed.

Lactase-phlorizin hydrolase (LPH) and sucrase-isomaltase (SI) are two disaccharidases specifically expressed in small intestinal absorptive cells. We previously showed that the transcripts of both genes are elevated within 12 h of carbohydrate intake. To examine at which locus of villus-crypt axis this response to dietary carbohydrate occurs, 6-wk-old rats were fed a low-carbohydrate diet (5% energy) for 7 d, and then force-fed either the low-carbohydrate diet or a sucrose (40% energy) diet during the last 6 h. Cryostat sectioning of jejunal segments followed by RNA blot hybridizations of the transcripts revealed that, unlike SI mRNA which was expressed maximally in the lower villus, maximal LPH mRNA level was attained at the upper villus. The distribution of the respective immunoreactive protein and the enzymatic activity was shifted more toward the villus tips for LPH than for SI. Force-feeding the sucrose diet caused an abrupt increase in SI mRNA level in the lower villus within 3 h, while the rise in LPH mRNA level occurred in the mid- and upper-villus. The diet-induced increases in the LPH mRNA and SI mRNA levels were abolished along the entire villus by the administration of actinomycin D. These results suggest that LPH gene is maximally expressed in more apical villus cells than SI gene, and that dietary sucrose elicits enhancement of the gene expressions in the villus cells which are accumulating the respective transcripts.

Six cyclic peptides with 5, 7, 9, 11, 13, and 15 amino acids, with the inhibitory sequence of the alpha-amylase inhibitor tendamistat, were synthesized. The 11-residue peptide inhibited porcine pancreatic alpha-amylase most potently (K1 0.29 +/- 0.09 microM). For the 11-residue peptide, the circular dichroism study suggested a preliminary relationship between its inhibitory activity and structural property.

Knowledge of the elastic properties of actin filaments is crucial for considering its role in muscle contraction, cellular motile events, and formation of cell shape. The stiffness of actin filaments in the directions of stretching and bending has been determined. In this study, we have directly determined the torsional rigidity and breaking force of single actin filaments by measuring the rotational Brownian motion and tensile strength using optical tweezers and microneedles, respectively. Rotational angular fluctuations of filaments supplied the torsional rigidity as (8.0 +/- 1.2) x 10(-26) Nm2. This value is similar to that deduced from the longitudinal rigidity, assuming the actin filament to be a homogeneous rod. The breaking force of the actin-actin bond was measured while twisting a filament through various angles using microneedles. The breaking force decreased greatly under twist, e.g., from 600-320 pN when filaments were turned through 90 degrees, independent of the rotational direction. Our results indicate that an actin filament exhibits comparable flexibility in the rotational and longitudinal directions, but breaks more easily under torsional load.

Endothelium-dependent relaxations are achieved by a combination of endothelium-derived prostacyclin (PGI2), nitric oxide (NO), and endothelium-derived hyperpolarizing factor (EDHF). However, it remains to be fully clarified whether the relative contribution of these three mechanisms to endothelium-dependent relaxations varies as a function of the vessel size. This study was designed to clarify this point. Acetylcholine (ACh)-induced endothelium-dependent relaxations were examined in isolated blood vessels taken from the aorta and the proximal and distal mesenteric arteries of the rat. The contributions of PGI2, NO, and EDHF were evaluated by the inhibitory effects of indomethacin, N omega-nitro-L-arginine methyl ester (L-NAME) in the presence of indomethacin, and KCl in the presence of indomethacin and L-NAME, respectively. The membrane potentials were recorded with microelectrodes. The expression of endothelial No synthase (eNOS) was examined by both immunostaining and immunoblotting. The contribution of PGI2 was negligible in three different-sized blood vessels. The contribution of NO was most prominent in the aorta, whereas that of EDHF was most prominent in the distal mesenteric arteries. The resting membrane potential was significantly deeper and the ACh-induced hyperpolarization was greater in the distal mesenteric arteries than those in the aorta. The expression of eNOS was the highest in the aorta and the lowest in the distal mesenteric arteries. These results indicate that the importance of EDHF increases as the vessel size decreases in endothelium-dependent relaxations in the rat mesenteric circulation.

Background: We previously reported that chronic inhibition of nitric oxide synthesis by administration of N omega-nitro-L-arginine methyl ester (L-NAME) causes microvascular hyperreactivity to 5-hydroxytryptamine (5-HT) and vascular structural changes in pigs in vivo. In the present study, we investigated the relative contributions of 5-HT receptor subtypes to microvascular hyper-reactivity in this animal model., Methods and Results: Coronary vasomotor response was studied in 16 pigs treated with oral L-NAME for 4 weeks (L group) and in 11 control pigs (C group). Intracoronary administration of 5-HT at 30 micrograms/kg decreased coronary blood flow (CBF) in the two groups. The decrease in CBF by 5-HT was greater (P < .01) in the L group than in the C group. The decrease in CBF by 5-HT in the C group was blocked completely by pretreatment with ketanserin, a 5-HT2 antagonist. In contrast, the augmented decrease in CBF by 5-HT in the L group was only partly inhibited by ketanserin alone and was blocked completely by ketanserin and methiothepin, a 5-HT1/5-HT2 antagonist. The decrease in CBF caused by prostaglandin F2 alpha and the increase in CBF caused by nitroglycerin were comparable between the two groups and were not affected by the 5-HT antagonists., Conclusions: These results suggest that the 5-HT-induced microvascular hyperreactivity may be mediated by relative changes in affinity for 5-HT receptors or de novo expression of 5-HT1 receptors in microvascular smooth muscle cells in our animal model.

The effects of sodium orthovanadate, an inhibitor of protein tyrosine phosphatases, on the endothelial nitric oxide (NO) pathway were studied in vitro. Vanadate caused endothelium-dependent relaxations in isolated porcine coronary arteries, which were abolished by N omega-nitro-L-arginine methyl ester. The relaxations were also abolished by pertussis toxin, an inhibitor of certain G proteins. Tyrosine kinase inhibitors, genistein and alpha-cyano-3-ethoxy-4-hydroxy-5-phenyl-methylcinnamamide (ST-638), significantly attenuated the vanadate-induced relaxations. Vanadate also caused pertussis toxin-sensitive, endothelium-dependent relaxations in isolated porcine renal and femoral arteries and jugular veins. Immunoblots, using an antibody to phosphotyrosines and to c-Src in native porcine aortic endothelial cells, respectively, showed that vanadate induced an elevation of phosphotyrosine proteins and a decrease in the amount of the active form of c-Src family kinases; both changes were markedly suppressed by cotreatment with ST-638. These results indicate that in porcine blood vessels, vanadate causes a synthesis of endothelium-derived NO for which endothelial tyrosine kinases and pertussis toxin-sensitive G protein are considered to be closely involved.

To explore the mechanisms by which jejunal lactase activity is modified by carbohydrate and/or fat intake, mRNA levels and the absolute synthesis rate of lactase-phlorizin hydrolase (LPH) were determined in 6-wk-old rats that were fed either low-starch diets containing long-chain triacylglycerol (LCT, 73% energy as corn oil) or medium-chain triacylglycerol (MCT, 66% energy as MCT, 7% energy as corn oil), or a high-starch diet (70% energy as cornstarch) for 7 days. LPH mRNA levels in the jejunum were similar between LCT-fed and MCT-fed rats, but animals fed the high-starch diet exhibited a greater (2x) LPH mRNA level than other groups. The absolute synthesis rate of LPH, estimated by the flooding dose technique using [3H]phenylalanine, was greater (2.4x) in rats fed the high-starch diet than in other groups. A short-term force-feeding experiment revealed that sucrose was able to evoke LPH mRNA levels within 12 h but that a nonmetabolizable sugar (alpha-methylglucoside) was unable to enhance it. By contrast, animals fed the high-LCT diet showed a lower (by 30%) lactase activity than rats fed the low-starch, high-MCT diet, which was accompanied by not only a reduction of immunoreactive LPH in brush-border membranes but also a reduction in lactase activity per unit weight of immunoreactive LPH. These results suggest that both gene expression and posttranslational events of LPH might be influenced by dietary manipulations; carbohydrate intake primarily increases LPH mRNA levels, and LCT accelerates inactivation and/or degradation of lactase.

Human osteosarcoma cells, that were cultured to confluence and maintained under low nutrient conditions, showed potentially lethal damage repair after cisplatin treatment. This repair was inhibited by a non-toxic dose of caffeine. Flow cytometric analysis indicated that cisplatin-treated cells accumulated in the S phase by 24 hr, followed by accumulation in the G2/M phase. Caffeine inhibited the initial accumulation in the S phase and the release of cells from the G2/M block. DNA synthesis was also inhibited by the cisplatin treatment. The addition of caffeine significantly reversed this inhibition. The content of DNA-bound platinum decreased over time after cisplatin treatment. Caffeine did not influence platinum content, nor did it directly affect the DNA excision repair. Cisplatin inhibits DNA synthesis in the S phase, but caffeine reduces this inhibition. Caffeine-treated cells that pass through S phase are incapable of recovery.

L-Arginine analogues have been widely used to examine the role of endothelium-derived nitric oxide (NO) in vascular responses; however, the effects of the agents on coronary vasomotion are not fully understood. In this study, we examined the effects of the analogues on vasomotion of isolated porcine coronary arteries. Strips of the porcine coronary artery were suspended for isometric tension recording in Krebs-Henseleit solution. L-Arginine analogues, N omega-nitro-L-arginine methyl ester (L-NAME, 10(-9)-10(-3) M), NG-monomethyl-L-arginine (L-NMMA, 10(-9)-10(-3) M), and NG-nitro-L-arginine (L-NNA, 10(-9)-10(-3) M), caused dose-dependent contractions, which were greater in strips with than in those without endothelium. Those endothelium-dependent contractions were almost abolished by indomethacin (10(-5) M) and FeCl2 (10(-3) M). The latter reduces prostaglandin H2 to 12-heptadecatrienoic acid, which has no vasoconstrictor effect. These results indicate that the L-arginine analogues cause endothelium-dependent contractions that are mediated by prostaglandin endoperoxides and suggest that they have properties other than simple inhibition of NO synthesis in porcine coronary arteries.

We have previously demonstrated that intake of fat as well as carbohydrate affects the activity and immunoreactive amount of sucrase-isomaltase (S-I) in rat jejunum. To examine whether diet-related changes in sucrase and isomaltase activities are accompanied by the variations of sucrase-isomaltase mRNA levels, 7-week-old rats were fed either a high-long-chain triacylglycerols diet (73 energy% as corn oil), a high-medium-chain triacylglycerols (MCT) diet (66 energy% as MCT, 7 energy% as corn oil) or a high-carbohydrate diet (70 energy% as corn starch) for 7 days. Northern blot analysis revealed that S-I mRNA levels were abundant in the jejunum of rats fed the high-MCT diet; the levels were similar to those in the rats fed the high-carbohydrate diet. Force-feeding a high-sucrose diet (40 energy% as sucrose) brought about a parallel rise in both S-I mRNA and sodium/D-glucose cotransporter (SGLT1) mRNA levels within 12 h. Force-feeding the high-MCT diet also produced an elevation of S-I mRNA and SGLT1 mRNA. However, force-feeding a diet containing alpha-methylglucoside, a non-metabolizable but actively transported sugar, did not increase S-I mRNA or SGLT1 mRNA level; sucrase activity was nevertheless elevated by feeding alpha-methylglucoside diet. These results suggest that not only carbohydrate intake but also MCT intake might influence S-I mRNA and SGLT1 mRNA levels in the jejunum, presumably through common metabolite(s) of carbohydrates and MCT, and that carbohydrate may play another role in enhancement of the sucrase activity through modulation of translation and/or posttranslational modifications of the sucrase-isomaltase complex.

Cellular retinol-binding protein II (CRBP II) is an abundant cytosolic protein of intestinal absorptive cells. In this study, we examined whether dietary fat modulates the expression of CRBP II in the small intestine. In the rats fed a diet rich in long-chain triacylglycerols (LCT), both CRBP II mRNA and CRBP II protein levels in the jejunum were more than two-fold greater than in the rats fed a low fat diet and a diet rich in medium-chain triacylglycerols (MCT). The mRNA abundance of a retinoid X receptor (RXR alpha), which is thought to interact with the cis-element located in the CRBP II promoter, was elevated in the jejunum of rats fed high-LCT and high-MCT diets as compared with that of animals fed a low-fat diet, but the levels of RXR alpha mRNA of the LCT diet group was similar to that of MCT diet group. These results suggest that the expression level of the CRBP II gene is not directly related to the RXR alpha expression, and that it might be modulated by long-chain fatty acids or their metabolites.

We report on the effects of intraarterial cisplatin and caffeine with/without doxorubicin on high-grade spindle cell sarcomas of bone and soft tissue based on the fact that caffeine enhances cytocidal effects of DNA-damaging agents. Intraarterial cisplatin and caffeine with/without doxorubicin was preoperatively given three times to ail patients and two courses of high-dose methotrexate with the citrovorum factor and vincristine were administered to the patients with skeletal spindle cell sarcoma. Tumor response was assessed radiographically and histologically. Seventeen (90%) of 19 patients with bone sarcoma and 7 (70%) of 10 patients with soft-tissue sarcoma showed good response. All patients with osteosarcoma demonstrating good radiological response underwent marginal excision without subsequent local tumor recurrence. Histologically, there were no viable cells in resected specimen of 14 patients with bone sarcoma. Other 8 cases with soft-tissue sarcoma treated by unplanned surgery were included to assess side effects. Twenty-five out of 37 patients are still free of disease. There was local tumor recurrence in 2 patients who did not respond to the chemotherapy. Toxic effects noted in the clinical study included moderate myelosuppression, nausea and vomiting, renal insufficiency and cutaneous injury. No toxic effects were directly attributable to 1.2-1.5 g/m(2) caffeine. The intraarterial infusion of cisplatin and caffeine combined with/without doxorubicin was tolerable in all patients and led to good response in high-grade spindle cell sarcomas. In patients with good response, limb-salvage surgery can be conducted safely without local relapse and good limb function is preserved by chemotherapy and marginal excision.

Nine patients with osteosarcoma were treated by chemotherapy combined with caffeine and surgery. All primary tumors showed complete histological response to preoperative chemotherapy consisting of three intraarterial infusions of cisplatin and caffeine without/with doxorubicin and two systemic high-dose methotrexate combined with vincristine. Limb-salvage surgery was performed in eight patients with marginal procedure, which led to the preservation of good limb function. Below-knee amputation was done in one patient with calcaneal osteosarcoma. There has been neither local recurrence nor lung metastasis in seven patients with conventional osteosarcoma during a median follow-up period of 28 months. Lung metastases leading to death were observed in one patient with small-cell osteosarcoma despite complete destruction of the primary tumor by preoperative chemotherapy. Chemotherapy combined with caffeine administration deserves further extensive and large-scale study in osteosarcoma.