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Objective: Muscle mass is typically assessed by abdominal computed tomography, magnetic resonance imaging, and dual-energy x-ray absorptiometry. However, these tests are not routinely performed in patients with head and neck cancer (HNC), making sarcopenia assessment difficult. The aim of this study was to develop and validate equations for predicting appendicular skeletal muscle mass (ASM) from data obtained in daily medical practice, with bioelectrical impedance analysis (BIA)-measured appendicular skeletal muscle mass (BIA-ASM) as a reference., Methods: This cross-sectional study included 103 men with HNC who were randomly placed into development and validation groups. The prediction equations for BIA-ASM were developed by multiple regression analysis and validated by Bland-Altman analyses. The estimated skeletal muscle mass index (eSMI) was also statistically evaluated to discriminate the cutoff value for BIA-measured SMI according to the Asian Working Groups for Sarcopenia., Results: Two practical equations, which included 24-h urinary creatinine excretion volume (24hUCrV), handgrip strength (HGS), body weight (BW), and body height (BHt), were developed: ASM (kg) = -39.46 + (3.557 × 24hUCrV [g]) + (0.08872 × HGS [kg]) + (0.1263 × BW [kg]) + (0.2661 × BHt [cm]) if available for 24hUCrV (adjusted R 2  = 0.8905), and ASM (kg) = -42.60 + (0.1643 × HGS [kg]) + (0.1589 × BW [kg]) + (0.2807 × BHt [cm]) if not (adjusted R 2  = 0.8589). ASM estimated by these two equations showed a significantly strong correlation with BIA-ASM (R > 0.900). Bland-Altman analyses showed a good agreement, and eSMI accuracy was high (>80%) in both equations., Conclusions: These two equations are a valid option for estimating ASM and diagnosing sarcopenia in patients with HNC in all facilities without special equipment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Objectives: The aims of this study were to investigate the correlation of phase angle (PhA) with other parameters (e.g., muscle mass/quality/strength and physical function), assess the prognostic relevance of prechemoradiotherapy (CRT) PhA, and suggest a reference value of PhA in Asian patients with head and neck cancer (HNC)., Methods: Ninety-six patients with HNC who underwent CRT were divided into two groups- maintained-PhA group and low-PhA group-according to the PhA 25th percentile values by sex. Pretreatment PhA was measured using direct segmental multifrequency bioelectrical impedance analysis, and muscle quality was assessed using echo intensity in ultrasound images. Correlation of PhA with other parameters was investigated, and between-group differences with respect to adverse events, treatment interruption, and 3-y survival were assessed., Results: PhA showed a positive correlation with isometric knee extension force (R = 0.710), handgrip strength (R = 0.649), skeletal muscle mass index (R = 0.620), and maximum gait speed (R = 0.543; P < 0.001). PhA showed a negative correlation with echo intensity (R = -0.439) and five times sit-to-stand test (R = -0.505; P < 0.01). The low-PhA group had a higher incidence of severe anemia (52% in low-PhA versus 17% in maintained-PhA), aspiration (17 versus 1%), radiotherapy interruption (17 versus 3%), and poor 3-y survival (47 versus 81%) than the maintained-PhA group (P < 0.05)., Conclusion: PhA was correlated with muscle mass/quality/strength, and physical function. Low PhA was associated with severe adverse events, treatment interruption, and shorter survival. These findings suggested that 4.6° for men and 4° for women may be useful as prognostic reference values in Asian patients with HNC., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Objectives: Sarcopenia has been reported as a prognostic risk factor in patients with gastrointestinal (GI) and hepatobiliary pancreatic (HBP) cancers. This study aimed to investigate whether the loss of muscle mass or strength is a stronger prognostic factor, and explore the cutoff values of skeletal muscle mass index (SMI) and handgrip strength (HGS) based on the survival outcome in patients with GI and HBP cancers., Methods: A total of 480 elderly patients with primary GI and HBP cancers who underwent their first resection surgery were analyzed retrospectively. Patients were divided into four groups: Appropriate SMI and HGS, low SMI alone, low HGS alone, and low SMI and HGS. Low SMI was derived from a bioelectrical impedance analysis, and low HGS was defined according to the Asian Working Group for Sarcopenia 2019 criteria., Results: The multivariate analysis showed that low SMI was a significant risk factor for mortality in men only, but low HGS was significant in both sexes. From the multivariate analysis of the four groups, low HGS alone and low SMI and HGS showed a significantly higher hazard ratio than appropriate SMI and HGS in both sexes. An SMI of 7.21 kg/m 2 and HGS of 28 kg were obtained as cutoff values based on the 3-y survival outcomes in men., Conclusions: Low muscle strength was a stronger prognostic factor than low muscle mass. Therefore, measuring muscle strength in all patients is essential., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: The WHO-recommended tuberculosis screening and diagnostic algorithm in ambulatory people living with HIV is a four-symptom screen (known as the WHO-recommended four symptom screen [W4SS]) followed by a WHO-recommended molecular rapid diagnostic test (eg Xpert MTB/RIF [hereafter referred to as Xpert]) if W4SS is positive. To inform updated WHO guidelines, we aimed to assess the diagnostic accuracy of alternative screening tests and strategies for tuberculosis in this population., Methods: In this systematic review and individual participant data meta-analysis, we updated a search of PubMed (MEDLINE), Embase, the Cochrane Library, and conference abstracts for publications from Jan 1, 2011, to March 12, 2018, done in a previous systematic review to include the period up to Aug 2, 2019. We screened the reference lists of identified pieces and contacted experts in the field. We included prospective cross-sectional, observational studies and randomised trials among adult and adolescent (age ≥10 years) ambulatory people living with HIV, irrespective of signs and symptoms of tuberculosis. We extracted study-level data using a standardised data extraction form, and we requested individual participant data from study authors. We aimed to compare the W4SS with alternative screening tests and strategies and the WHO-recommended algorithm (ie, W4SS followed by Xpert) with Xpert for all in terms of diagnostic accuracy (sensitivity and specificity), overall and in key subgroups (eg, by antiretroviral therapy [ART] status). The reference standard was culture. This study is registered with PROSPERO, CRD42020155895., Findings: We identified 25 studies, and obtained data from 22 studies (including 15 666 participants; 4347 [27·7%] of 15 663 participants with data were on ART). W4SS sensitivity was 82% (95% CI 72-89) and specificity was 42% (29-57). C-reactive protein (≥10 mg/L) had similar sensitivity to (77% [61-88]), but higher specificity (74% [61-83]; n=3571) than, W4SS. Cough (lasting ≥2 weeks), haemoglobin (<10 g/dL), body-mass index (<18·5 kg/m 2 ), and lymphadenopathy had high specificities (80-90%) but low sensitivities (29-43%). The WHO-recommended algorithm had a sensitivity of 58% (50-66) and a specificity of 99% (98-100); Xpert for all had a sensitivity of 68% (57-76) and a specificity of 99% (98-99). In the one study that assessed both, the sensitivity of sputum Xpert Ultra was higher than sputum Xpert (73% [62-81] vs 57% [47-67]) and specificities were similar (98% [96-98] vs 99% [98-100]). Among outpatients on ART (4309 [99·1%] of 4347 people on ART), W4SS sensitivity was 53% (35-71) and specificity was 71% (51-85). In this population, a parallel strategy (two tests done at the same time) of W4SS with any chest x-ray abnormality had higher sensitivity (89% [70-97]) and lower specificity (33% [17-54]; n=2670) than W4SS alone; at a tuberculosis prevalence of 5%, this strategy would require 379 more rapid diagnostic tests per 1000 people living with HIV than W4SS but detect 18 more tuberculosis cases. Among outpatients not on ART (11 160 [71·8%] of 15 541 outpatients), W4SS sensitivity was 85% (76-91) and specificity was 37% (25-51). C-reactive protein (≥10 mg/L) alone had a similar sensitivity to (83% [79-86]), but higher specificity (67% [60-73]; n=3187) than, W4SS and a sequential strategy (both test positive) of W4SS then C-reactive protein (≥5 mg/L) had a similar sensitivity to (84% [75-90]), but higher specificity than (64% [57-71]; n=3187), W4SS alone; at 10% tuberculosis prevalence, these strategies would require 272 and 244 fewer rapid diagnostic tests per 1000 people living with HIV than W4SS but miss two and one more tuberculosis cases, respectively., Interpretation: C-reactive protein reduces the need for further rapid diagnostic tests without compromising sensitivity and has been included in the updated WHO tuberculosis screening guidelines. However, C-reactive protein data were scarce for outpatients on ART, necessitating future research regarding the utility of C-reactive protein in this group. Chest x-ray can be useful in outpatients on ART when combined with W4SS. The WHO-recommended algorithm has suboptimal sensitivity; Xpert for all offers slight sensitivity gains and would have major resource implications., Funding: World Health Organization., Competing Interests: Declaration of interests AC reports grants from National Institutes of Health (NIH), Global Health Labs, and Stop TB Partnership, and consulting fees from the US Centers for Disease Control and Prevention (CDC). AK reports grants from Sanofi. FAK reports grants from WHO, Canadian Institutes of Health Research, Fonds de Recherche Quebec, and McGill Interdisciplinary Initiative on Infection and Immunity. GT reports receipt of consumables and equipment from Boditech and Cepheid. NM reports grants from Pfizer and Roche. REC reports grants from NIH, CDC, and Unitaid, and consulting fees from Sanofi. SML reports grants from NIH and CDC. SSk reports grants from Swedish Heart-Lung Foundation. TKr reports consulting fees from WHO. All other authors declare no competing interests., (© 2022 World Health Organization; licensee Elsevier. This is an Open Access article published under the CC BY 3·0 IGO license which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In any use of this article, there should be no suggestion that WHO endorses any specific organisation, products or services. The use of the WHO logo is not permitted. This notice should be preserved along with the article's original URL.)

Background: Novel skin-based tests for tuberculosis infection might present suitable alternatives to current tests; however, diagnostic performance of new tests compared with the purified protein derivative-tuberculin skin test (TST) or interferon-γ release assays (IGRA) needs systematic assessment., Methods: In this systematic review and meta-analysis, we searched English (Medline OVID), Chinese (Chinese Biomedical Literature Database and the China National Knowledge Infrastructure), and Russian (e-library) databases from the inception of each database to May 15, 2019, (with updated search of the Russian and English databases on Oct, 20 2020) using terms "ESAT6" OR "CFP10" AND "skin test" AND "Tuberculosis" OR "C-Tb" OR "Diaskintest". We included studies reporting on the performance of index tests alone or compared with a comparator. Inclusion criteria varied according to review objectives and performance outcome, but reporting of test cut-offs for positivity applied to study population was required from all studies. We used a hierarchy of reference standards for tuberculosis infection consistent with the 2020 WHO framework to evaluate diagnostic performance. Two authors independently reviewed the titles and abstracts for English and Chinese (LF and MK) and Russian studies (MK and VN). Study quality was assessed with QUADAS-2. Pooled random-effects estimates are presented when appropriate for total agreement proportion, sensitivity in microbiologically confirmed tuberculosis and specificity in cohorts with low risk of tuberculosis infection. This study is registered with PROSPERO, CRD42019135572., Findings: We identified 1466 original articles, of which 37 (2·5%) studies, including 10 915 individuals (7111 Diaskintest, 2744 C-Tb, 887 EC, 173 DPPD), were included in the qualitative analysis (29 [78%] studies of Diaskintest, five [15%] studies of C-Tb, two [5%] studies of EC-skintest, and one [3%] study of DPPD). 22 (1·5%) studies including 5810 individuals (3143 Diaskintest, 2129 C-Tb, 538 EC-skintest) were included in the quantitative analysis: 15 (68%) of Diaskintest, five (23%) of C-Tb, and two (9%) of EC-skintest. Tested sub-populations included individuals with HIV, children (0-18 years), and individuals exposed to tuberculosis. Studies were heterogeneous with moderate to high risk of bias. Nine head-to-head studies of index test versus TST and IGRA permitted direct comparisons and pooling. In a mixed cohort of people with and without tuberculosis, Diaskintest pooled agreement with IGRA was 87·16% (95% CI 79·47-92·24) and 55·45% (46·08-64·45) with TST-5 mm cut-off (TST 5 mm ). Diaskintest sensitivity was 91·18% (95% CI 81·72-95·98) compared with 88·24% (78·20-94·01) for TST 5 mm , 89·66 (78·83-95·28) for IGRA QuantiFERON, and 90·91% (79·95-96·16) for TSPOT.TB. C-Tb agreement with IGRA in individuals with active tuberculosis was 79·80% (95% CI 76·10-83·07) compared with 78·92% (74·65-82·63) for TST5 mm/15 mm cut-off (TST 5 mm/15 mm ). TST 5/15mm reflects threshold in cohorts that applied stratified cutoffs: 5 mm for HIV-infected, immunocompromised, or BCG-naive individuals, and 15mm for BCG-vaccinated immunocompetent individuals. C-Tb sensitivity was 74·52% (95% CI 70·39-78·25) compared with a sensitivity of 78·18% (67·75-85·94) for TST 5 mm/15 mm , and 71·67% (63·44-78·68) for IGRA. Specificity was 97·85% (95% CI 93·96-99·25) for C-Tb versus 93·31% (90·22-95·48) for TST 15 mm cut-off and 99·15% (79·66-99·97) for IGRA. EC-skintest sensitivity was 86·06% (95% CI 82·39-89·07)., Interpretation: Novel skin-based tests for tuberculosis infection appear to perform similarly to IGRA or TST; however, study quality varied. Evaluation of test performance, patient-important outcomes, and diagnostic use in current clinical algorithms will inform implementation in key populations., Funding: StopTB (New Diagnostics Working Group) and FIND., Translations: For the Chinese and Russian translations of the abstract see Supplementary Materials section., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Objective: Phase angle (PhA), by bioelectrical impedance analysis, has been used in patients with several diseases; however, its prognostic value in patients with gastrointestinal (GI) and hepatobiliary-pancreatic (HBP) cancer is unclear. The aim of the present study was to investigate the effects of PhA on postoperative short- outcomes and long-term survival in these patients., Methods: This retrospective study reviewed data from 501 patients with GI and HBP cancers who underwent first resection surgery. The data were divided into the following groups according to the preoperative PhA quartile values by sex: high-PhA group with the highest quartile (Q4), normal-PhA group with middle quartiles (Q3 and Q2), and low-PhA group with the lowest quartile (Q1). Preoperative nutritional statuses, postoperative short-term outcomes during hospitalization, and 5-y survival between three groups were compared. Cox proportional hazard models were used to evaluate the prognostic effect of PhA., Results: PhA positively correlated with body weight, skeletal muscle mass, and handgrip strength, and negatively correlated with age and levels of C-reactive protein. The low-PhA group showed a higher prevalence of malnutrition (48%) than normal-PhA (25%), and high-PhA groups (9%; P < 0.001). The incidence of postoperative severe complications was 10% for all patients (14% in low-PhA, 12% in normal-PhA, and 4% in high-PhA; P = 0.018). The incidence of prolonged stays in a postoperative high-care or intensive care unit was 8% in all patients (16% in low-PhA, 8% in normal-PhA, and 2% in high-PhA; P < 0.001). The 5-y survival rate was 74% in all patients (68% in low-PhA, 74% in normal-PhA, and 79% in high-PhA; P < 0.001). The multivariate analysis demonstrated that a low-PhA group was an independent risk factor for mortality (hazard ratio, 1.99; 95% confidence interval, 1.05-3.90; P = 0.034)., Conclusion: PhA is a useful short-term and long-term postoperative prognostic marker for patients with GI and HBP cancers., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Pre-meal drink ingestion is an effective method of controlling energy intake in humans. However, no studies have addressed the influence of differences in the volume of drink intake on gastric motility and energy intake. The purpose of the present study was to examine the effects of differences in the volume of drink intake before a meal on subsequent gastric motility and energy intake in healthy young men. Twelve men completed two, one-day trials in a random order. Subjects visited the laboratory after a 10-h overnight fast and consumed the nutrient drink (0.84 MJ) in either 100 mL or 600 mL quantities over a 5-min period. Then, the subjects sat on a chair for over 2 h to measure their cross-sectional gastric antral areas and gastric contractions with an ultrasound imaging system. Thereafter, the subjects consumed a test meal until they felt completely full. Energy intake was calculated from the amount of food consumed. Energy intake in the 600 mL trial was 12% higher than the 100 mL trial (5.1 ± 1.3 vs. 4.6 ± 1.4 MJ, P = 0.046). The antral area (P = 0.046) and the frequency of the gastric contraction (P = 0.001) over 2 h after consuming the nutrient drink were higher in the 600 mL trial than the 100 mL trial. These findings demonstrated that consumption of a 600 mL nutrient drink increased energy intake. The modulation of gastric motility might have some effects on energy intake., (Copyright © 2019. Published by Elsevier Inc.)

The periosteum and mineralized bone are innervated by nerves that sense pain. These include both myelinated and unmyelinated neurons with either free nerve endings or bearing nociceptors. Parasympathetic and sympathetic autonomic nerves also innervate bone. However, little is known about the route sensory nerves take leaving the epiphyses of long bones at the adult knee joint. Here, we used transgenic mice that express fluorescent Venus protein in Schwann cells (Sox10-Venus mice) to visualize myelinated and unmyelinated nerves in the tibial epiphysis. Immunofluorescence to detect a pan-neuronal marker and the sensory neuron markers calcitonin gene-related peptide (CGRP) and tropomyosin receptor kinase A (TrkA) also revealed Schwann cell-associated sensory neurons. Foramina in the intercondylar area of the tibia were conserved between rodents and primates. Venus-labeled fibers were detected within bone marrow of the proximal epiphysis, exited through foramina along with blood vessels in the intercondylar area of the tibia, and joined Venus-labeled fibers of the synovial membrane and meniscus. These data suggest that innervation of the subchondral plate and trabecular bone within the tibial epiphysis carries pain signals from the knee joint to the brain through intercondylar foramina., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Traction forces generated at cellular focal adhesions (FAs) play an essential role in regulating various cellular functions. These forces (1-100 nN) can be measured by observing the local displacement of a flexible substrate upon which cells have been plated. Approaches employing this method include using microfabricated arrays of poly(dimethylsiloxane) (PDMS) micropillars that bend by cellular traction forces. A tool capable of applying a force to FAs independently, by actively moving the micropillars, should become a powerful tool to delineate the cellular mechanotransduction mechanisms. Here, we developed a patterned magnetic micropillar array PDMS substrate that can be used for the mechanical stimulation of cellular FAs and the measurement of associated traction forces. The diameter, length, and center-to-center spacing of the micropillars were 3, 9, and 9 µm, respectively. Iron particles were embedded into the micropillars, enabling the pillars to bend in response to an external magnetic field, which also controlled their location on the substrate. Applying a magnetic field of 0.3 T bent the pillars by ∼4 µm and allowed transfer of external forces to the actin cytoskeleton through FAs formed on the pillar top. Using this approach, we investigated the traction force changes in cultured aortic smooth muscle cells (SMCs) after local compressive stimuli to release cell pretension. The mechanical responses of SMCs were roughly classified into two types: almost a half of the cells showed a little decrease of traction force at each pillar following compressive stimulation, although cell area increased significantly; and the rest showed the opposite, with increased forces and a simultaneous decrease in area. The traction forces of SMCs fluctuated markedly during the local compression. The root mean square of traction forces significantly increased during the compression, and returned to the baseline level after its release. These results suggest that the fluctuation of forces may be caused by active reorganization of the actin cytoskeleton and/or its dynamic interaction with myosin molecules. Thus, our magnetic micropillar substrate would be useful in investigating the mechanotransduction mechanisms of cells., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Excess carbohydrate intake causes obesity in humans. On the other hand, acute administration of fructose, glucose or sucrose in experimental animals has been shown to increase the plasma concentration of anti-obesity hormones such as glucagon-like peptide 1 (GLP-1) and Fibroblast growth factor 21 (FGF21), which contribute to reducing body weight. However, the secretion and action of GLP-1 and FGF21 in mice chronically fed a high-sucrose diet has not been investigated. To address the role of anti-obesity hormones in response to increased sucrose intake, we analyzed mice fed a high-sucrose diet, a high-starch diet or a normal diet for 15 weeks. Mice fed a high-sucrose diet showed resistance to body weight gain, in comparison with mice fed a high-starch diet or control diet, due to increased energy expenditure. Plasma FGF21 levels were highest among the three groups in mice fed a high-sucrose diet, whereas no significant difference in GLP-1 levels was observed. Expression levels of uncoupling protein 1 (UCP-1), FGF receptor 1c (FGFR1c) and β-klotho (KLB) mRNA in brown adipose tissue were significantly increased in high sucrose-fed mice, suggesting increases in FGF21 sensitivity and energy expenditure. Expression of carbohydrate responsive element binding protein (ChREBP) mRNA in liver and brown adipose tissue was also increased in high sucrose-fed mice. These results indicate that FGF21 production in liver and brown adipose tissue is increased in high-sucrose diet and participates in resistance to weight gain., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: Serum diamine oxidase (DAO) activity varies to a greater extent in women than in men. DAO activity during the luteal phase was higher than that during the follicular phase in healthy women. Recent reports have indicated that duodenal lipid infusion increased DAO activity in the intestinal lymph in rats. The aim of this study was to elucidate the effect of dietary nutrient intake on serum DAO activity in healthy women., Methods: Thirty-four healthy Japanese women were recruited. Food surveys were performed using dietary records for 3 d during both the follicular and luteal phases. Nutrient intake was calculated and expressed as the energy intake ratio. The correlation between DAO activity and nutrient intake was analyzed., Results: Serum DAO activity in both phases was positively correlated with intake of long-chain fatty acids, saturated fatty acids, and monounsaturated fatty acids (P < 0.05). Intake of phosphorus, calcium, zinc, magnesium, iron, and vitamin B 12 during the luteal phase was positively correlated with serum DAO activity (P < 0.05)., Conclusion: In healthy women, serum DAO activity was influenced by dietary fatty acid and micronutrient intake., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Interaction between adipocytes and macrophages has been suggested to play a central role in the pathogenesis of obesity. Ceramide, a sphingolipid de novo synthesized from palmitate, is known to stimulate pro-inflammatory cytokine secretion from multiple types of cells. To clarify whether de novo synthesized ceramide contributes to cytokine dysregulation in adipocytes and macrophages, we observed cytokine secretion in mature 3T3-L1 adipocytes (L1) and RAW264.7 macrophages (RAW) cultured alone or co-cultured under the suppression of de novo ceramide synthesis. Palmitate enhanced ceramide accumulation and stimulated the expression and secretion of interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) in L1. The suppression of serine-palmitoyl transferase, a rate-limiting enzyme of de novo ceramide synthesis, by myriocin or siRNA attenuated those palmitate-induced alterations, and a ceramide synthase inhibitor fumonisin B1 showed similar results. In contrast, the inhibitor of sphingosine kinase or a membrane-permeable ceramide analogue augmented the cytokine secretion. Myriocin effects on the palmitate-induced changes were not abrogated by toll-like receptor-4 blockade. Although palmitate stimulated RAW to secrete tumor necrosis factor-α (TNF-α), it did not significantly increase ceramide content, and neither myriocin nor fumonisin B1 attenuated the TNF-α hypersecretion. The co-culture of L1 with RAW markedly augmented IL-6 and MCP-1 levels in media. Myriocin or fumonisin B1 significantly lowered these cytokine levels and suppressed the gene expression of TNF-α and MCP-1 in RAW and of IL-6 and MCP-1 in L1. In conclusion, de novo synthesized ceramide partially mediates the palmitate effects on pro-inflammatory adipokines and is possibly involved in the interaction with macrophages., (Copyright © 2014 Elsevier Inc. All rights reserved.)

We have reported potent peptidic and non-peptidic BACE1 inhibitors with a hydroxymethylcarbonyl (HMC) isostere as a substrate transition-state mimic. However, our potent inhibitors possess a tetrazole ring at the P1' position. It is desirable that central nervous system (CNS) drugs do not possess an acidic moiety. In this study, we synthesized non-acidic BACE1 inhibitors with heterocyclic derivatives at the P1' position. KMI-1764 (27) exhibited potent inhibitory activity (IC50=27nM). Interestingly, these non-acidic inhibitors tended to follow the quantitative structure-activity relationship (QSAR) equation and interacted with BACE1-Arg235 in the binding model., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Objectives: Serum diamine oxidase (DAO) level is employed as a useful marker of intestinal mucosal integrity. As reported previously, the range of serum DAO levels in women is wider than that in men. We hypothesized that the menstrual cycle may affect DAO levels., Design and Methods: Thirty-six women of Japanese descent were recruited. All participants, aged 20-29 years, were healthy. Food surveys utilized in this study were based on questionnaires validated by dietitians. Complete blood counts, biochemical parameters, female hormones, and serum DAO levels during the follicular and luteal phases were measured in each subject., Results: Biochemical parameters, except for DAO levels, were comparable between the two phases. However, serum DAO levels during the luteal phase were significantly higher than those during the follicular phase., Conclusions: Serum DAO levels were influenced by the menstrual cycle. Furthermore, our findings suggest that serum DAO levels should be interpreted cautiously in premenopausal women., (Copyright © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Previously, we reported potent pentapeptidic BACE1 inhibitors with the hydroxymethylcarbonyl isostere as a substrate transition-state mimic. To improve the in vitro potency, we further reported pentapeptidic inhibitors with carboxylic acid bioisosteres at the P(4) and P1' positions. In the current study, we screened new P1' position 1-phenylcycloalkylamine analogs to find non-acidic inhibitors that possess double-digit nanomolar range IC(50) values. An extensive structure-activity relationship study was performed with various amine derivatives at the P1' position. The most potent inhibitor of this pentapeptide series, KMI-1830, possessing 1-phenylcyclopentylamine at the P1' position had an IC(50) value of 11.6 nM against BACE1 in vitro enzymatic assay., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Peripheral neuropathy has been reported to prevail in obese or pre-diabetic individuals, yet its etiology remains unknown. Palmitate, a saturated fatty acid increased in obesity and diabetes, is known to induce apoptosis in multiple types of cells and this effect may be mediated by ceramide, a member of the sphingolipid family. To clarify whether de novo ceramide synthesis from palmitate contributes to apoptosis of Schwann cells, we cultured immortalized mouse Schwann cells (IMS) and rat primary Schwann cells with palmitate, a ceramide analogue C2-ceramide as well as inhibitors of the de novo ceramide synthesis (myriocin and fumonisin B1). Apoptosis of IMS detected by nuclear staining and cell membrane inversion was significantly increased by incubation with palmitate for 48 h in a dose-dependent fashion. This enhanced apoptosis was partially but significantly suppressed by myriocin and fumonisin B1. Western blot analysis and immunostaining revealed that palmitate clearly activated caspase-3 in IMS. Unexpectedly, the ceramide synthesis inhibitors failed to suppress the palmitate-induced caspase-3 activation in spite of complete restoration in ceramide accumulation. The results seemed relevant to the observations that C2-ceramide did not activate caspase-3 while provoking apoptosis with a clear dose-dependency. In agreement, the pro-apoptotic action of C2-ceramide was not attenuated by caspase inhibitors that partially suppressed palmitate-induced apoptosis. These results in IMS were well reproducible in rat primary Schwann cells, indicating that the observed phenomena are not specific to the cell line. Collectively, we have reached a conclusion that palmitate induces apoptosis in Schwann cells via both a ceramide-mediated, caspase-3-independent pathway and ceramide-independent, caspase-3-dependent pathways. Given the fact that palmitate and ceramide are increased in obese or pre-diabetic subjects, these lipids may be implicated in the pathogenesis of peripheral neuropathy observed in these disorders., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

We previously reported potent BACE1 inhibitors KMI-420 and KMI-570 possessing a hydroxymethylcarbonyl isostere as a substrate transition-state mimic. Acidic moieties at the P(1)(') and P(4) positions of KMI inhibitors are thought to be unfavorable in terms of membrane permeability across the blood-brain barrier. Herein, we replaced acidic moieties at the P(4) position with hydrogen bond accepting groups and acidic moieties at the P(1)(') position with less acidic and similar molecular-size moieties (carboxylic acid or tetrazole bioisosteres). These inhibitors exhibited improved BACE1 inhibitory activities and a thorough quantitative structure-activity relationship study was performed., ((c) 2010 Elsevier Ltd. All rights reserved.)

Recently, the binding sequence Ser-Val-Val-Tyr-Gly-Leu-Arg (SVVYGLR) was found adjacent to the RGD sequence in osteopontin, suggesting involvement in osteo-immune cross-talk. The aim of this study was to investigate bioactive functions of a synthetic SVVYGLR peptide in osteoprogenitor cells and osteoclasts, and to examine potential applications in bone regeneration. The SVVYGLR peptide significantly enhanced the adhesion and proliferation of several human mesenchymal cells including bone marrow-derived mesenchymal stem cells, and alphavbeta3 integrin was involved in cell attachment to the peptide. Additionally, the peptide reduced the number of TRAP-positive multinucleated cells during osteoclastogenesis of RAW264.7 cells and normal murine pre-osteoclasts, and also suppressed NFAT activity and expression of osteoclastogenesis-related mRNAs. When standardized bone defects in rat calvariae were filled with a collagen sponge containing the peptide or PBS (control), the number of TRAP-positive osteoclasts in the grafted sites after 3 weeks was significantly lower in the peptide group. By the 5th week, significantly enhanced resorption of the grafted collagen sponge and new bone formation was observed within and surrounding the sponge in the peptide group. These data suggest that SVVYGLR is an effective bioactive peptide for bone tissue regeneration that promotes attachment and proliferation of osteogenic cells while also suppressing osteoclastogenesis.

Diabetes mellitus induces alterations in bone and mineral metabolism. Diabetic bone disorder causes an increase in bone fractures, delays healing of fractures, and affects the quality of life. There are few optimal therapies for these disorders and the mechanisms responsible for their complications have not been clearly identified. Bone histology studies in humans and animals have demonstrated that decreased bone formation is a critical mechanism of bone mass reduction in diabetes. A major hypothesis about the mechanisms of diabetic complications is a diabetes-induced increase in oxidative stress, because reactive oxygen species (ROS) are increased under diabetic conditions and are known to induce cellular dysfunction in a wide variety of cell types. Oxidative stress is induced by a variety of mechanisms including formation of increased advanced glycation end-products (AGEs), increased polyol pathway flux, activation of protein kinase C isoforms, glucose autoxidation, and mitochondrial overproduction of superoxide under diabetic conditions. Other circulating factors that are elevated in diabetes, such as free fatty acids and leptin, also contribute to increased ROS generation. It is now widely accepted that ROS can cause severe damage to DNA, proteins, and lipids. Concerning bone metabolism, in vitro studies have shown that oxidative stress inhibits osteoblastic differentiation and induces osteoblast insults and apoptosis. Moreover, we have demonstrated that both streptozotocin-induced diabetic mice, an animal model of type 1 diabetes, and spontaneously diabetic Torii (SDT) rats, an animal model of type 2 diabetes, have low-turnover osteopenia associated with increased oxidative stress and that markers of oxidative stress are inversely associated with the histomorphometric parameters of bone formation. Growing evidence suggests that the increase in oxidative stress may at least partly contribute to the development of diabetic osteopenia. This review focuses on the impact of diabetes-induced oxidative stress in the development of diabetic bone disorder.

Diabetes mellitus is associated with increased risk of osteopenia and bone fracture. However, the mechanisms accounting for diabetic bone disorder are unclear. We have previously reported that streptozotocin-induced diabetic mice develop low turnover osteopenia associated with increased oxidative stress in the diabetic condition. To determine the role of oxidative stress in the development of diabetic osteopenia, we presently investigated the effect of overexpression of thioredoxin-1 (TRX), a major intracellular antioxidant, on the development of diabetic osteopenia, using TRX transgenic mice (TRX-Tg). TRX-Tg are C57BL/6 mice that carry the human TRX transgene under the control of beta-actin promoter. Eight-week-old male TRX-Tg mice and wild type (WT) littermates were intraperitoneally injected with either streptozotocin or vehicle. Mice were grouped as 1) non-diabetic WT, 2) non-diabetic TRX-Tg, 3) diabetic WT, and 4) diabetic TRX-Tg. After 12 weeks of streptozotocin treatment, oxidative stress on the whole body and bone was evaluated, and the physical properties of the femora, and histomorphometry parameters of the tibiae were assessed. TRX overexpression did not affect either body weight or hemoglobin A1c levels. There were no significant differences in renal function and in serum levels of calcium, phosphate, and intact parathyroid hormone among the four groups. On the other hand, urinary excretion of 8-hydroxydeoxyguanosine (8-OHdG), a marker of oxidative DNA damage, was significantly elevated in diabetic WT and attenuated in diabetic TRX-Tg. Immunohistochemical staining for 8-OHdG revealed marked intensity in the bone tissue of diabetic WT compared with non-diabetic WT, while staining was attenuated in diabetic TRX-Tg. TRX overexpression partially restored reduced bone mineral density and prevented the suppression of bone formation observed in diabetic WT. Increased oxidative stress in diabetic condition contributes to the development of diabetic osteopenia. Suppression of increased oxidative stress by TRX induction could be a potential therapeutic approach for diabetic osteopenia.

It is well known that patients with type 1 diabetes mellitus exhibit bone abnormalities as one of the complications of the disease. Whether this occurs in type 2 diabetes is controversial. This uncertainty could be because type 2 diabetes includes several pathological types such as obese and non-obese. To examine the bone abnormalities in non-obese type 2 diabetes, we used Spontaneously Diabetic Torii (SDT) rats, which is a newly established model of non-obese type 2 diabetes. Sprague-Dawley (SD) rats were used as a control group (n=17). SDT rats were divided into two groups: the diabetic (DM) group (n=18) and the DM+insulin (INS) group (n=18) at 20 weeks of age. The DM+INS group received subcutaneously implanted insulin pellets every 2 weeks. At 36 weeks of age, the rats were killed, and we evaluated bone formation and the effect of insulin on bone formation, blood and urine analyses, bone mineral density (BMD), histomorphometry, and mRNA expression of alkaline phosphatase (ALP) and osteocalcin (OCN). Despite renal function not being impaired, BMD and bone strength were significantly lower in the DM group than in the control group. Osteoid volume per bone volume, osteoblast surface per bone surface, eroded surface per bone surface, osteoclast surface per bone surface, the mineral apposition rate, and the bone formation rate per bone surface were significantly lower in the DM group than in the control and DM+INS groups. The mRNA expression of ALP and OCN was significantly lower in the DM group than in the control group. Furthermore, 8-hydroxydeoxyguanosine, which is an oxidative stress marker, was remarkably elevated in the DM group. These abnormalities were recovered by insulin therapy. Our data support the notion that non-obese type 2 diabetes is associated with a low turnover of bone and that the abnormalities are ameliorated by insulin. The SDT rat may be a useful animal model for examining the mechanisms of bone abnormalities in non-obese type 2 diabetes.

Diabetic osteopenia causes an increase in bone fracture and a delay in healing of fractures, and affects the quality of life. However, the mechanisms responsible for the disease have not been clearly identified. Oxidative stress may be a potential candidate for the pathogenesis, since it is increased under diabetic conditions and is known to induce cellular dysfunction in a wide variety of cell types. Although in vitro studies have shown that oxidative stress inhibits osteoblastic differentiation and induces osteoblast insults and apoptosis, the relationship between diabetic osteopenia and oxidative stress remains unclear. To explore these issues, analysis of a mouse model that represents the diabetic osteopenia as seen in patients with diabetes is necessary. However, there are few reports of such a model. Therefore, we focused on the streptozotocin (STZ)-induced diabetic mouse, one of the most common animal models of type 1 diabetes. Eight-week-old male C57BL/6 mice were randomly assigned to the following three groups: 1) control group, 2) diabetic group, and 3) insulin-treated diabetic group. After 12 weeks of STZ treatment, the physical properties of the femora, and the static and dynamic parameters of bone histomorphometry of the tibiae from STZ-induced diabetic mice (STZ-mice) were assessed, and oxidative stress in the whole body and bone of the mice was evaluated. Renal function was comparable in all three groups at the end of the experimental period. In addition, no significant difference in serum PTH, Ca, and P was found among the three groups. In contrast, radiological analysis demonstrated a significant decrease in trabecular bone volume, and histomorphometric analyses confirmed that parameters for both bone formation (OV/BV, OS/BS, and BFR/BS) and bone resorption (ES/BS and Oc.S/BS) were also significantly lower in STZ-mice. In addition, urinary excretion of 8-hydroxydeoxyguanosine, a marker of oxidative DNA damage, was elevated in STZ-mice. Further immunohistological studies showed intensified immunostaining of an oxidative stress marker in bone tissue including the osteoblasts of diabetic mice. Here, we demonstrated that STZ-mice exhibit low-turnover osteopenia associated with increased oxidative stress.

This study examines the possibility of using hydroxyapatite (HAp) particles as a controlled release carrier of protein. In order to achieve effective protein release from HAp particles, it is necessary to regulate the conjugated amount of protein on HAp and the resorption of HAp. HAp particles were synthesized at different temperatures (40 degrees C, 60 degrees C, 80 degrees C) in wet condition and the physico-chemical properties of synthesized HAp particles were examined. HAp particles synthesized at low temperatures showed low crystallinity, high solubility and large specific surface area. The useful growth factors for bone regeneration, such as BMP, bFGF and TGF-beta, are basic proteins, so cytochrome c (pI=10.2) was used as a model protein and the adsorptive property of protein on HAp particles was investigated. The protein adsorption on HAp particles changed depending on its specific surface area and the chart of protein adsorption on HAp particles showed a typical Langmuir curve. These findings suggest that the adsorbed amount of protein on HAp particles could be regulated by HAp synthesizing temperature and the concentrations of protein solution. The release kinetics of protein from the HAp particles that adsorbed the protein (HAp-pro) was also evaluated in different pH solutions (pH 4.0 and 7.0). The released protein gradually increased time dependently when HAp-pro were immersed in pH 4.0 solution, but the released protein was significantly smaller when HAp-pro were immersed in pH 7.0 solution. Moreover, the release rate of protein from HAp-pro differed in each HAp that was synthesized at different temperatures, suggesting that the release of protein from HAp-pro depended on HAp resorption. These results suggest that HAp particles synthesized at different temperature are useful as a controlled release carrier of protein.

To improve the low water-solubility of HIV protease inhibitors, we synthesized water-soluble prodrugs of KNI-727, a potent small-sized dipeptide-type HIV-1 protease inhibitor consisting of an Apns-Dmt core (Apns; allophenylnorstatine, Dmt; (R)-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid) as inhibitory machinery. These prodrugs contained an O-acyl peptidomimetic structure with an ionized amino group leading to an increase in water-solubility, and were designed to regenerate the corresponding parent drugs based on the O-->N intramolecular acyl migration reaction via a five-membered ring intermediate at the alpha-hydroxy-beta-amino acid residue, that is Apns. The synthetic prodrug 3a improved the water-solubility (13 mg/mL) more than 8000-fold in comparison with the parent compound, which is the practically acceptable value as water-soluble drug. Furthermore, to understand the structural effects of the O-acyl moiety on the migration rate, we evaluated several phenylacetyl-type and benzoyl-type prodrugs. These prodrugs were stable as an HCl salt and in a strongly acidic solution corresponding to gastric juice (pH 2.0), and could be converted to the parent compounds promptly under aqueous conditions from slightly acidic to basic pH at 37 degrees C.

To improve the low water-solubility of HIV protease inhibitors, we synthesized water-soluble prodrugs of KNI-272 and KNI-279 which are potent HIV-1 protease inhibitors consisting of an Apns-Thz core structure (Apns; allophenylnorstatine, Thz; thiazolidine-4-carboxylic acid) as an inhibitory machinery. The prodrugs, which contained an O-acyl peptidomimetic structure with an ionized amino group leading to the increase of water-solubility, were designed to regenerate the corresponding parent drugs based on the O-->N intramolecular acyl migration reaction at the alpha-hydroxy-beta-amino acid residue, that is allophenylnorstatine. The synthetic prodrugs 3, 4, 6, and 7 improved the water-solubility (>300mg/mL) more than 4000-fold in comparison with the parent compounds, which is the practically acceptable value as water-soluble drugs. These prodrugs were stable as an HCl salt and in a strongly acidic solution corresponding to gastric juice (pH 2.0), and could be converted to the parent compounds promptly in the aqueous condition from slightly acidic to basic pH at 37 degrees C, with the suitable migration rate, via a five-membered ring intermediate. Using a similar method, we synthesized a prodrug (12) of ritonavir, a clinically useful HIV-1 protease inhibitor as an anti-AIDS drug. In contrast to the prodrugs 3, 4, 6, and 7, the prodrug 12 was very slowly converted to ritonavir probably through a six-membered ring intermediate, with the t(1/2) value of 32h that may not be suitable for practical use.

Hydroxyapatite (HAp) was synthesized in the presence of a variety of amino acids in order to investigate the effect of amino acid on the crystallinity and the solubility characteristics of HAp in the HAp-synthesizing condition. In the results of X-ray diffraction analysis, HAp synthesized in the presence of glycine (HAp-Gly), serine (HAp-Ser), aspartic acid (HAp-Asp) and glutamic acid (HAp-Glu) showed poor crystallinity compared with HAp synthesized in the absence of amino acid (HAp-con). The results of Fourier transform infrared spectroscopy suggested the adsorption of these amino acids on HAp. Scanning electron microphotographs showed that the size and morphology of HAp adsorbed amino acids changed significantly. Furthermore, the solubility of these HAps increased significantly compared to HAp-con, each differing in four amino acids. However, other amino acids did not affect the crystallinity and morphology of HAp and had no significant change in their solubility. Collectively, this study suggests that the crystallinity and the solubility of synthesized HAp are different owing to the variation of amino acids in the HAp synthesizing condition. It is expected that digestion-regulated HAp materials could be synthesized by using amino acid in their synthesizing condition.

Aminoguanidine is a well-known inhibitor of the formation of advanced glycation end products and is considered to be promising for the treatment of diabetic complications. We recently reported, however, that administration of aminoguanidine caused the formation of a Schiff base adduct between aminoguanidine and pyridoxal phosphate in the liver and kidney of mice and a concomitant decrease in the amount of liver pyridoxal phosphate. Our study led us to hypothesize that the Schiff base adduct and/or another Schiff base adduct formed from aminoguanidine and pyridoxal might be a better compound than aminoguanidine. In the present study, we examined the in vitro inhibitory potency of the latter adduct against advanced glycation end product formation and its effect on the tissue contents of pyridoxal and its phosphate. Aminoguanidine-pyridoxal phosphate adduct was not employed in this study because of its poor solubility in water. Aminoguanidine-pyridoxal adduct was hydrolyzed by only about 15% during 10 days at pH 7.4 and 37 degrees C. The adduct at 1 mM did not inhibit Amadori product formation induced by incubation of albumin with 100 mM mannose for 10 days. The adduct, when tested at 1 and 2 mM, dose-dependently inhibited advanced glycation end product formation induced by incubation of albumin with mannose; and the inhibitory potency of the adduct was similar to or higher than that of aminoguanidine. The presence of an appreciable amount of aminoguanidine-pyridoxal adduct in the kidney of mice given the adduct suggested that at least part of the adduct administered was absorbed from the gastrointestinal duct. The amounts of pyridoxal and its phosphate in tissues were not at all decreased by administration of the aminoguanidine-pyridoxal Schiff base. We conclude that the Schiff base may be a more promising inhibitor of advanced protein glycation than aminoguanidine.

Localization and function of 5-HT4 receptors in the stomach were examined in mucosa-free preparations of antrum, corpus and fundus from guinea pig stomach by determination of acetylcholine release and in vitro receptor autoradiography. Specific [125I]SB207710, (1-n-butyl-4-piperidinyl) methyl-8-amino-7-iodo-1,4-benzodioxane-5-carboxylate, binding sites were detected in 3 regions of the stomach. High densities of binding were observed in the myenteric plexus of antrum and corpus, but not fundus. In mucosa-free preparations treated with 5-HT1, 5-HT2 and 5-HT3 receptor antagonists, 5-HT (10(-8)-10(-6) M) potentiated the electrically stimulated (0.5 Hz, 1 ms) outflow of [3H]acetylcholine from antrum and corpus strips preloaded with [3H]choline, but not from fundus strips, and the potentiation was antagonized by SB204070, (1-n-butyl-4-piperidinyl) methyl-8-amino-7-chloro-1,4-benzodioxane-5-carboxylate. Thus, 5-HT4 receptors are located on myenteric cholinergic neurons in the antrum and corpus of guinea pig stomach and their activation evokes the release of acetylcholine.

Cortical oscillations have been reported in relation to animal behavior and may have important roles in neuronal information processing such as "binding". We considered oscillatory activities in the primary somatosensory cortex of rats as a preparatory state for sensory processing and investigated the temporal relationship between gamma oscillation (25-45 Hz) onset in the somatosensory cortex (the barrel cortex) and rat's whisking as tactile exploratory behavior. The gamma oscillations of local field potentials preceded the whisking in 52/83 cases (63%). The mean lead time of the oscillations, defined as time from the oscillation onset to the whisking, was 268 ms (+/- 123, S.D.). These preceding gamma oscillations lasted to the onset of whisking. Furthermore, we investigated the temporal relationship during the other two behavioral states: a still but alert state and a state with tactile stimulation of the whiskers. In the state with tactile stimulation, gamma oscillations followed the stimulation, and in the still but alert state, significant gamma oscillation was not observed. The gamma oscillations that precede the whisking are thought to reflect anticipatory activities in the barrel cortex for the subsequent somatosensory input from the whiskers, which could represent a mechanism for rapid processing of expected sensory information.

In Swiss 3T3 fibroblasts, changing the culture medium prior to stimulation resulted in an augmentation of bradykinin-induced prostaglandin E2 synthesis. The augmentation depended on the duration of the exposure to the fresh medium, with a maximum effect at 1 h. Fetal calf serum in the fresh medium was essential for augmented prostaglandin E2 synthesis. The medium change slightly augmented the bradykinin-induced increase in intracellular free Ca2+ concentration and phosphoinositide hydrolysis with a different time course from that for prostaglandin E2 synthesis. 4',5,7-Trihydroxyisoflavone (genistein) and 3,4-dihydroxybenzylidene-malononitrile (tyrphostin 23), inhibitors of tyrosine kinases, and 2'-amino-3'-methoxyflavone (PD98059), an inhibitor of mitogen-activated protein kinase (MAPK) kinase, attenuated the increase in prostaglandin E2 synthesis. Bradykinin caused phosphorylation of cytosolic phospholipase A2 and p42/p44 MAPK, which was augmented by the medium change. From the results, it is concluded that activation of MAPK and cytosolic phospholipase A2 is involved in the augmentation of prostaglandin E2 synthesis produced by the medium change.

Aminoguanidine (AG) is considered to be a promising compound for the treatment of diabetic complications. We examined the in vitro and in vivo formation of Schiff bases of AG with pyridoxal 5'-phosphate (PLP) and pyridoxal (PL). AG reacted in vitro far more rapidly with PLP to form a Schiff base (PLP-AG) than with PL to form another Schiff base (PL-AG). Administration of AG at 7 mM in drinking water for 18 weeks caused the formation of PLP-AG in the liver and kidney of mice (12.1 +/- 1.6 and 3.8 +/- 0.64 nmol/g of tissue, respectively, mean +/- SD, N = 6). The amount of PLP in the liver of mice AG administered was significantly lower than that of control mice (4.0 +/- 1.4 vs 17.4 +/- 1.3 nmol/g of wet tissue, mean +/- SD, N = 6). Simultaneous administration of pyridoxine (1 mM in drinking water) with AG (7 mM in drinking water) did not ameliorate the decrease in tissue PLP and caused the excess formation of PLP-AG. The results suggest that attention should be paid to the deficiency of tissue PLP in the clinical use of AG.

The effect of an aldose reductase inhibitor, [5-(3-thienyl) tetrazol-1-yl] acetic acid (TAT), on the electroretinogram was determined in rats with streptozotocin-induced diabetes. Laboratory chow containing 0.05% TAT was given to rats for 2 months, while other diabetic rats were untreated. Groups of TAT-treated and untreated normal rats were also studied. Treatment with TAT produced significant improvement of the electroretinogram. TAT shortened the peak latencies of the b-wave oscillatory potentials, which were significantly prolonged in untreated diabetic rats (P < 0.0001 vs. untreated normal rats). This was accompanied by a significant decrease in the retinal sorbitol and fructose concentrations (by 46.5% and 25.7%, respectively). TAT treatment of diabetic rats also markedly reduced ADP-induced platelet aggregation and significantly increased the red blood cell 2,3-diphosphoglycerate level, accompanied by a marked reduction in sorbitol and fructose concentrations of platelet and red blood cells. There were significant correlations between the summed b-wave peak latencies and platelet aggregation or the 2,3-diphosphoglycerate level in diabetic rats. These findings suggest that an aldose reductase inhibitor, TAT, has therapeutic value for diabetic retinopathy.

4-Hydroxy-2-nonenal (HNE), one of the major products of lipid peroxidation, inactivated the rate-limiting enzymes (from animal sources) of the glycolytic pathway and the pentose phosphate pathway when incubated at 37 degrees C for 1 h in the absence of glutathione (GSH). The HNE concentration for half-maximal inactivation of 6-phosphofructokinase (PFK) and glyceraldehyde-3-phosphate dehydrogenase was 3-10 microM; and that value for pyruvate kinase, glucose-6-phosphate dehydrogenase, and hexokinases I and II was 0.15-0.6 mM. In the presence of 5 mM GSH, however, only PFK, irrespective of the source (muscle, liver, or erythrocyte), was inactivated by 40-50% when incubated with 0.1 mM HNE for 1 h. Even PFK was not inactivated in the presence of both GSH and its substrate, ATP (2 mM). Glycolysis in human erythrocytes was not affected by treatment of cells with 0.1 mM HNE at 37 degrees C for 30 min. The results suggest that HNE, at concentrations observable under physiological and pathological conditions, hardly affects glycolysis in cells.

To investigate the pathogenesis of diabetic neuropathy in non-insulin-dependent diabetes mellitus, Otsuka Long-Evans Tokushima Fatty rats, an animal model of non-insulin-dependent diabetes mellitus, and non-diabetic Long-Evans Tokushima Otsuka rats were fed with or without sucrose and/or cilostazol, an anticoagulant, for 8 weeks. Sucrose-fed diabetic rats showed a delayed motor nerve conduction velocity, decreased R-R interval variability of electrocardiogram, reduced sciatic nerve blood flow, increased platelet aggregability and a decreased erythrocyte 2,3-diphosphoglycerate concentration compared with non-sucrose-fed diabetic rats and non-diabetic rats. These abnormalities were significantly prevented by treatment with cilostazol without changes in the nerve tissue levels of polyols. These findings indicate that sucrose-fed Otsuka Long-Evans Tokushima Fatty rats may be a useful animal model of neuropathy in non-insulin-dependent diabetes mellitus, and that cilostazol may prevent the development of diabetic neuropathy by modifying vascular factors.

The effect of propionyl-L-carnitine, an analogue of L-carnitine, and insulin on the oscillatory potentials of the electroretinogram was determined in rats with streptozotocin-induced diabetes. Propionyl-L-carnitine was administered at a daily dose of 0.5 g/kg by gavage for 4 weeks, while other rats were treated with subcutaneous injections of insulin (8-10 U/day). Both treatments shortened the peak latencies of the oscillatory potentials in the electroretinogram, which were significantly prolonged in untreated diabetic rats (O1, O2 and O3, and sigma (O1 + O2 + O3)) (P < 0.0001 vs. untreated normal rats). A significant decrease in the erythrocyte free carnitine level in diabetic rats was prevented by both treatments. Insulin produced a significant reduction of retinal glucose, sorbitol and fructose levels in diabetic rats, while propionyl-L-carnitine failed to do so. However, both treatments markedly reduced serum lipids levels in the diabetic rats. These findings provide information on the pathogenesis of diabetic retinopathy as well as suggesting the potential therapeutic value of propionyl-L-carnitine for retinopathy.

Nitric oxide (NO) was measured directly after spinal cord injury (SCI) in rats by an ESR spin-trapping technique using Fe2+ and diethyldithiocarbamate (DETC). The levels of NO and lipid peroxides expressed as thiobarbituric acid reactive substances (TBARS) were increased by SCI in the injured region and the adjacent central region. Pretreatment with 30 mg/kg of NG-nitro-L-arginine methylester (L-NAME), an inhibitor of NO synthase, accelerated increases of the TBARS level and myeloperoxidase (MPO) activity in the injured tissue and caused deterioration of hind limb motor function after SCI, suggesting that NO formation by constitutive NO synthase (c-NOS) has a protective effect against cellular damage resulting from ischemia-reperfusion after SCI. Though c-NOS mRNA expression was not altered after SCI, inducible NO synthase (i-NOS) mRNA expression increased to a maximum of 24 h after SCI with progress of motor dysfunction. Intravenous injection of L-NAME (0.1 mg/kg) 6, 24, 48, and 72 h after SCI reduced the motor disturbance. These results indicate that NO induced by i-NOS may be neurotoxic in the subacute phase after SCI.

The relationship between the 2,3-diphosphoglycerate concentration in red blood cells as a biological indicator of tissue hypoxia and diabetic neuropathy, and the effect of a potent aldose reductase inhibitor, (2S,4S)-6-fluoro-2'5'-dioxospiro [chroman-4,4'-imidazolidine]-2-carboxamide (SNK-860), on both were investigated in streptozotocin-induced diabetic rats. Diabetic rats demonstrated significantly delayed motor nerve conduction velocity and reduced sciatic nerve blood flow. Altered biochemical features in the sciatic nerves, including a marked accumulation of sorbitol and fructose, myo-inositol depletion and decreased Na+/K(+)-ATPase activity were also detected in diabetic rats. These defects were accompanied by a decrease in the red blood cell 2,3-diphosphoglycerate concentration. Treatment with SNK-860 partially or completely ameliorated these abnormalities. These observations suggest that a decrease in the red blood cell 2,3-diphosphoglycerate concentration is one of the factors contributing to tissue hypoxia, which results in diabetic neuropathy, and that this decrease is mediated through an aldose reductase inhibitor-sensitive pathway.

The effects of the prostacyclin analog beraprost sodium on motor nerve function and nerve blood-flow were examined in streptozotocin-induced diabetic rats. Oral administration of beraprost sodium 0.1 mg/kg/day for 8 wks significantly (P < 0.001) improved caudal motor nerve conduction velocity and sciatic nerve blood-flow, both of which are impaired in diabetic rats. Beraprost sodium did not affect glucose, sorbitol, or fructose levels in the sciatic nerve. However, a decreased content of cyclic AMP in the sciatic nerve and higher level of thromboxane B2 in the thoracic aorta of diabetic rats, as compared with those in normal rats, were reversed by the treatment with beraprost sodium (P < 0.01). Results suggest that beraprost sodium may have value in treating diabetic neuropathy, mainly by improving endoneurial blood-flow.

This study investigated the mechanism of activation of K+ channels by ritodrine hydrochloride in human myometrial smooth muscle cells. The patch-clamp technique was used for recording single channel currents. Ritodrine (10(-5) M) activated two types of K+ channels in cultured uterine smooth muscle cells from pregnant women: the Ca(2+)-activated K+ (KCa) channel and the ATP-sensitive K+ (KATP) channel. Forskolin (10(-4) M), an activator of adenylate cyclase, and protein kinase A activated the KCa channel. In addition, 10(-4) M GTP activated the KCa channel in inside-out patches using a pipette containing 10(-5) M ritodrine. The KATP channel was activated by protein kinase A, but not by 10(-4) M GTP. The beta-adrenoceptor agonist ritodrine activates two types of K+ channels: the KCa channel via direct gating by GTP-binding proteins and possibly via cAMP-dependent phosphorylation, and the KATP channel possibly via cAMP-dependent phosphorylation. These mechanisms partially explain the relaxing effect of ritodrine hydrochloride.

A new method for pelvic arteriography using a disposable elastic vein needle is presented. The technique is carried out by percutaneous transfemoral artery puncture and contrast medium is injected by a power air-driven injector which makes bilateral pelvic arteriography possible. Neither a radioopaque long catheter nor a guide wire are necessary in the present method. No major complications have been observed in a series of 162 cases. The present method is advantageous due to its simplicity and safety compared with ordinary Seldinger's method which is not always free of complications.

Six mature female rhesus monkeys were treated with HMG-HCG in control cycles at doses adjusted to induce ovulation while avoiding superovulation. Occurrence of ovulation was determined by observation of fresh ovulation points at laparotomy 48 to 120 hours following HCG. In subsequent cycles animals were treated with indomethacin (treatment days 4 through 10) together with the established dose of HMG-HCG. In 8 cycles indomethacin 5 mg/kg was given i.m. once daily; in 9 cycles 10 mg/kg i.m. was administered in 2 divided doses. Following this, PGF2alpha (3 mg t.i.d. s.c.) was administered for 3 days together with indomethacin 10 mg/kg and HMG-HCG, beginning on the day prior to HCG. Determinations of progesterone were performed by RIA on treatment days 4, 7, 10, and 11. Eleven of the 13 control cycles were ovulatory. With indomethacin 5 mg/kg/day, 5 of 8 cycles were ovulatory but ovulation was delayed in 2 instances. Of 9 cycles using indomethacin 10 mg/kg/day only 1 was ovulatory. When PGF2alpha was administered in susequent cycles along with indomethacin (10 mg/kg) and HMG-HCG, ovulation occurred in 13 of 19 cycles. These data suggest that local ovarian PGF2alpha may be essential in the mechanics of follicle rupture in gonadotropin-treated rhesus monkeys.