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A new method for the synthesis of tartary buckwheat protein (TBP) and its effect on the cholesterol metabolism in rats and mice fed cholesterol-enriched diet are studied. The analysis reveals that TBP can be used as a potential source of functional food ingredients.

Dynapenia and multimorbidity are common health problems affecting older adults. However, few studies have systematically reviewed the association between dynapenia and multimorbidity. Therefore, this systematic review aimed to provide a comprehensive overview of studies on the association between these conditions. We searched four electronic databases for relevant articles published in July 2023. The main inclusion criteria were the following: a description of dynapenia, which indicates loss of muscle strength, and a description of multimorbidity with two or more chronic diseases. Five studies met these inclusion criteria. In all five of these studies, the participants were community-dwelling older adults. All the studies showed an association between dynapenia and multimorbidity. The prevalence of dynapenia and multimorbidity ranged from 16% to 25.9%. The results of our systematic review demonstrated that dynapenia in older adults increases the risk of multimorbidity. We propose that interventions and reversible changes in dynapenia can prevent multimorbidity. (PROSPERO Registration No. CRD42023443282).

Neutrophil extracellular trap (NET) formation is a unique self-defense mechanism of neutrophils; however, it is also involved in many diseases, including atherosclerosis. Resveratrol and catechin are antioxidants with anti-atherosclerotic properties. Here, we examined the effects of resveratrol, catechin, and other related compounds on NET formation. HL-60-derived neutrophils were pretreated with resveratrol and other compounds before stimulation with phorbol-myristate acetate (PMA). DNA and myeloperoxidase released from neutrophils were determined. Resveratrol suppressed the DNA release from neutrophils in a dose-dependent manner. NET formation was enhanced by 1-palmitoyl-2-oxovaleroyl phosphatidylcholine (POVPC), a truncated form of oxidized phospholipid, and resveratrol suppressed NET formation induced by POVPC and PMA. Furthermore, we designed several analogs of resveratrol or catechin whose conformation was restricted by the inhibition of the free rotation of aromatic rings. The conformationally constrained analogs were more effective at inhibiting NET formation; however, their inhibitory function decreased when compound was a large, hydrophobic analog. The most potent compounds, planar catechin and resveratrol, suppressed myeloperoxidase release from activated neutrophils. In addition, these compounds suppressed DNA release from neutrophils stimulated with calcium ionophore. These results suggest that resveratrol, catechin and their analogs exert anti-NET effects, and that constraining the geometry of these compounds enhanced their inhibitory effects., Competing Interests: No potential conflicts of interest were disclosed., (Copyright © 2024 JCBN.)

Background: Although the relationship between medication status, symptomatology, and outcomes has been evaluated, data on the prevalence of polypharmacy and potentially inappropriate medications (PIMs) and the association of polypharmacy and PIMs with swallowing function during follow-up are limited among hospitalized patients aged ≥65 years with dysphagia., Methods: In this 19-center cohort study, we registered 467 inpatients aged ≥65 years and evaluated those with the Food Intake LEVEL Scale (FILS) scores ≤8 between November 2019 and March 2021. Polypharmacy was defined as prescribing ≥5 medications and PIMs were identified based on the 2023 Updated Beers Criteria. We applied a generalized linear regression model to examine the association of polypharmacy and PIMs with FILS score at discharge., Results: We analyzed 399 participants (median age, 83.0 years; males, 49.8%). The median follow-up was 51.0 days (interquartile range, 22.0-84.0 days). Polypharmacy and PIMs were present in 67.7% of and 56.1% of patients, respectively. After adjusting for covariates, neither polypharmacy (β = 0.05; 95% confidence interval [CI], -0.04-0.13, p=0.30) nor non-steroidal anti-inflammatory medications (β = 0.09; 95% CI, -0.02-0.19; p=0.10) were significantly associated with FILS score at discharge., Conclusion: The results of this study indicated a high proportion of polypharmacy and PIMs among inpatients aged ≥65 years with dysphagia. Although these prescribed conditions were not significantly associated with swallowing function at discharge, our findings suggest the importance of regularly reviewing medications to ensure the appropriateness of prescriptions when managing older inpatients.

Objectives: To clarify the relationship between Phase of Illness at the time of admission to palliative care units and symptoms of patients with advanced cancer., Methods: This study was a secondary analysis of the East Asian collaborative cross-cultural Study to Elucidate the Dying process. Palliative physicians recorded data, including Phase of Illness, physical function and the Integrated Palliative care Outcome Scale. We used multinomial logistic regression to analyse ORs for factors associated with Phase of Illness. Twenty-three palliative care units in Japan participated from January 2017 to September 2018., Results: In total, 1894 patients were analysed-50.9% were male, mean age was 72.4 (SD±12.3) years, and Phase of Illness at the time of admission to the palliative care unit comprised 177 (8.9%) stable, 579 (29.2%) unstable, 921 (46.4%) deteriorating and 217 (10.9%) terminal phases. Symptoms were most distressing in the terminal phase for all items, followed by deteriorating, unstable and stable (p<0.001). The stable phase had lower association with shortness of breath (OR 0.73, 95% CI 0.57 to 0.94) and felt at peace (OR 0.73, 95% CI 0.56 to 0.90) than the unstable phase. In the deteriorating phase, weakness or lack of energy (OR 1.20, 95% CI 1.02 to 1.40) were higher, while drowsiness (OR 0.82, 95% CI 0.71 to 0.97) and felt at peace (OR 0.81, 95% CI 0.71 to 0.94) were significantly lower., Conclusion: Our study is reflective of the situation in palliative care units in Japan. Future studies should consider the differences in patients' medical conditions and routinely investigate patients' Phase of Illness and symptoms., Trial Registrarion Number: UMIN000025457., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Neutrophil extracellular traps (NETs) are found in patients with various diseases, including cardiovascular diseases. We previously reported that copper-oxidized low-density lipoprotein (oxLDL) promotes NET formation of neutrophils, and that the resulting NETs increase the inflammatory responses of endothelial cells. In this study, we investigated the effects of high-density lipoproteins (HDL) on NET formation. HL-60-derived neutrophils were treated with phorbol 12-myristate 13-acetate (PMA) and further incubated with oxLDL and various concentrations of HDL for 2 h. NET formation was evaluated by quantifying extracellular DNA and myeloperoxidase. We found that the addition of native HDL partially decreased NET formation of neutrophils induced by oxLDL. This effect of HDL was lost when HDL was oxidized. We showed that oxidized phosphatidylcholines and lysophosphatidylcholine, which are generated in oxLDL, promoted NET formation of PMA-primed neutrophils, and NET formation by these products was completely blocked by native HDL. Furthermore, we found that an electronegative subfraction of LDL, LDL(-), which is separated from human plasma and is thought to be an in vivo oxLDL, was capable of promoting NET formation. These results suggest that plasma lipoproteins and their oxidative modifications play multiple roles in promoting NET formation, and that HDL acts as a suppressor of this response.

The accuracy of body mass index (BMI) for sarcopenic dysphagia diagnosis, which remains unknown, was evaluated in this study among patients with dysphagia. We conducted a 19-site cross-sectional study. We registered 467 dysphagic patients aged ≥ 20 years. Sarcopenic dysphagia was assessed using a reliable and validated diagnostic algorithm. BMI was assessed using the area under the curve (AUC) in the receiver operating characteristic analysis to determine diagnostic accuracy for sarcopenic dysphagia. The study included 460 patients (median age, 83.0 years (76.0−88.0); men, 49.8%). The median BMI was 19.9 (17.3−22.6) kg/m2. Two hundred eighty-four (61.7%) patients had sarcopenic dysphagia. The AUC for sarcopenic dysphagia was 0.60−0.62 in the overall patients, male, female, and patients aged ≥ 65 years The BMI cut-off value for sarcopenic dysphagia diagnosis was 20.1 kg/m2 in the overall patients (sensitivity, 58.1%; specificity, 60.2%) and patients aged ≥ 65 years (sensitivity, 59.8%; specificity, 61.8%). Conclusion: Although the AUC, sensitivity and specificity of BMI for sarcopenic dysphagia diagnosis was approximately 0.6, BMI < 20.0 kg/m2 might be a predictor for sarcopenic dysphagia. In clinical settings, if patients with dysphagia have a BMI < 20.0 kg/m2, then sarcopenic dysphagia should be suspected as early as possible after admission.

Background: People receiving palliative care have complex, wide-ranging, and changing needs, not just physical distress, but also psychosocial, practical, and spiritual. Influences on complexity in palliative care are different among healthcare providers and may depend on diverse aspects of the patient's condition, time, and environment. Therefore, this study aimed to integrate and describe the perspective of complexity in palliative care., Methods: We used an integrative review, which is a method of compiling, summarizing, and analyzing existing insights from previous studies. We conducted an electronic literature search in MEDLINE (Ovid), PsycINFO (EBSCOhost), Web of Science Core Collection, and CINAHL (EBSCOhost), examining literature from May 1972 to September 2020 and updated in December 2020. Subsequently, synthesis without meta-analysis of the findings was completed., Results: We identified 32 peer-reviewed articles published in English. The included literature mainly originated in Europe and the United States. The research methods included quantitative studies (n=13), qualitative studies (n=12), case studies (n=3), and reviews (n=4). We identified 29 that influenced complexity in palliative care, 25 perceptions of the patient, including background and physical, psychological, social, and spiritual; two perceptions in the healthcare setting; and two perceptions in the socio-cultural setting. Above all, the perceptions of complexity in palliative care included younger age, prognosis, and spirituality. In addition, we added the identified perceptions of complexity with references to the complexity model in palliative care., Conclusions: Although this review was limited in its search strategy and some data sources may have been overlooked, it still provided perceptions that influenced complexity in palliative care. These complex influencing perceptions are necessary for patients to receive appropriate palliative care at the right time and for health care providers to conduct a multi-disciplinary team approach. Furthermore, longitudinal prospective data are needed to examine the changes and relationships among complexity over time.

Surface functionalized mesoporous silicates, MCM-41s, having 3-(2-pyridylmethylideneimino)propyl group (PI-MCM-41) or 3-(2-quinolylmethylideneimino)propyl group (QI-MCM-41) were prepared via Schiff base reaction, and the adsorption behavior of metal ions onto the modified MCM-41s was investigated. The function groups on the modified MCM-41 surface were confirmed by Fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), and elemental analysis. The metal ions examined, Co 2+ , Ni 2+ , Cu 2+ , Zn 2+ , Cd 2+ , and Pb 2+ , were quantitatively adsorbed on the PI-MCM-41 and QI-MCM-41, except for Mn 2+ . In the complexation with these metal ions, it was suggested that imine-N and heterocyclic-N atoms act as donor atoms. In addition, it was considered that the hydrophobicity derived from the organo-functional groups modified on MCM-41 contributed to improving the adsorption ability.

The function of oxidatively modified low-density lipoprotein (oxLDL) in the progression of cardiovascular diseases has been extensively investigated and well-characterized with regards to the activation of multiple cellular responses in macrophages and endothelial cells. Although accumulated evidence has revealed the presence of neutrophils in vascular lesions, the effect of oxLDL on neutrophil function has not been properly investigated. In the present decade, neutrophil extracellular traps (NETs) gained immense attention not only as a primary response against pathogenic bacteria but also due to their pathological roles in tissue damage in various diseases, such as atherosclerosis and thrombosis. In this study, we investigated if oxLDL affects NET formation and if it is a risk factor for inflammatory reactions in endothelial cells. HL-60-derived neutrophils were stimulated with phorbol 12-myristate 13-acetate (PMA) for 30 min to induce NET formation, followed by incubation with 20 μg/mL native or oxidized LDL for additional 2 h. Culture media of the stimulated cells containing released NETs components were collected to evaluate NET formation by fluorometric quantitation of released DNA and detection of myeloperoxidase (MPO) by western blot analysis. NET formation of HL-60-derived neutrophils induced by PMA was significantly enhanced by additional incubation with oxLDL but not with native LDL. Treatment of HL-60-derived neutrophils with oxLDL alone in the absence of PMA did not induce NET formation. Furthermore, the culture media of HL-60-derived neutrophils after NET formation were then transferred to human aortic endothelial cell (HAECs) culture. Treatment of HAECs with the culture media containing NETs formed by HL-60-derived neutrophils increased the expression of metalloproteinase-1 protein in HAECs when HL-60-derived neutrophils were incubated with native LDL, and the expression was accelerated in the case of oxLDL. In addition, the culture media from NETs formed by HL-60-derived neutrophils caused the elongation of HAECs, which was immensely enhanced by coincubation with native LDL or oxLDL. These data suggest that oxLDL may act synergistically with neutrophils to form NETs and promote vascular endothelial inflammation.

Protein phosphatase 1gamma, a serine/threonine phosphatase, is a metalloprotein that coordinates two Mn(2+) in the active site when expressed in Escherichia coli in a buffer containing MnCl(2). Herein, we report on the oxidatively induced copper for manganese exchange in protein phosphatase 1gamma, thus enabling firm confirmation of the four histidine (His) amino acid residues (His66, His125, His173, and His248) involved in metal coordination. By exchanging manganese with copper the oxidation yields for the peptides increased dramatically, thus simplifying detection of the oxidized peptides and analysis of the oxidation sites within the oxidized peptides. We also found that when copper was added during the oxidation process a new metal coordination center was formed at cysteine 39, 105, 140, and 155.

Herein we describe the further improvement of our in-house developed firefly bioluminescence assay system for the determination of inhibition of protein phosphatase (PP). The advantage with the new system is higher sensitivity as well as being time and sample efficient. The inhibition activity of tautomycin with PP1gamma was determined using the upgraded test system and Ki was found to be 4.5 nM, which compare favorably with the activity reported previously by others using different methods. The test system was then used in order to determine the activity of nine tautomycin (TTM) photoaffinity probes. One of the TTM photoaffinity probes (anti-10) was found to possess higher activity than the natural product itself with a Ki of 3.4 nM, while the remaining photoaffinity probes were found to possess Ki in the range of 8.0-213 nM.

Objective: This study evaluated the physiologic properties of high protein buckwheat flour (PBF) by examining its effects on serum cholesterol and body fat in rats and on cholesterol gallstone formation in mice., Methods: Animals were fed experimental diets that contained casein, buckwheat protein extract (BWP), or PBF as a protein source (net protein content 200 g/kg)., Results: In experiment 1, consumption of PBF and BWP for 10 d caused 33% and 31% decreases, respectively, in serum cholesterol of rats fed cholesterol-enriched diets when compared with consumption of casein (P < 0.05). Dietary PBF caused a significant decrease in liver cholesterol, whereas dietary BWP caused only a slight decrease (P > 0.05). Fecal excretion of neutral and acidic steroids in the PBF group was significantly higher than those in the BWP and casein groups. In experiment 2, consumption of PBF for 10 d significantly suppressed adipose tissue weight and hepatic activity of fatty acid synthase in rats fed cholesterol-free diets compared with consumption of casein (P < 0.05), whereas that of BWP for this period caused only a slight decrease in adipose tissue weight (P > 0.05). In experiment 3, dietary PBF and BWP significantly decreased the incidence of cholesterol gallstones and lithogenic index in mice fed cholesterol-enriched diets for 27 d, which was associated with increased fecal excretion of acidic steroids., Conclusion: This study demonstrated that PBF has strong activities against hypercholesterolemia, obesity, and gallstone formation, suggesting a potential usefulness of PBF as functional ingredient.

In the ciliary muscle, the tonic contraction requires a sustained influx of Ca2+ through the cell membrane. However, little has hitherto been known about the route(s) of Ca2+ influx in this tissue that lacks voltage-gated Ca2+ channels. To identify ion channels as the Ca2+ entry pathway we studied the effects of carbachol (CCh) on freshly isolated bovine ciliary muscle cells by whole-cell voltage clamp. Experiments were carried out using pipettes filled with K+ -free solution containing 100 mm caesium aspartate, 5 mm BAPTA and 180 microm GTP (pH 7.0; the intracellular free Ca2+ concentration, [Ca2+]i = 70 nm). CCh evoked an inward current showing polarity reversal at a holding potential near 0 mV. Analysis of the current noise distinguished two types of non-selective cation channel (NSCCL and NSCCS) with widely different unitary conductances (35 pS and 100 fS). The ratios of the permeabilities to Li+, Na+, Cs+, Mg2+, Ca2+, Sr2+ and Ba2+, estimated by cation replacement procedures, were 0.9: 1.0: 1.5: 0.2: 0.3: 0.4: 0.5 for NSCCL, and 1.0: 1.0: 1.8: 2.5: 2.6: 3.2: 5.0 for NSCCS. NSCCS, but not NSCCL, was strongly inhibited by elevation of [Ca2+]i. Both NSCCL and NSCCS were dose-dependently inhibited by 1-100 microm SKF96365, La3+ and Gd3+, which also inhibited the tonic component of the contraction produced in muscle bundles by CCh without markedly affecting the initial phasic component. NSCCL and/or NSCCS may serve as a major Ca2+ entry pathway required for sustained contraction of the bovine ciliary muscle. RT-PCR experiments in the bovine ciliary muscle (whole tissue) detected mRNAs of several transient receptor potential (TRP) channel homologues (TRPC1, TRPC3, TRPC4 and TRPC6), which are now regarded as possible molecular candidates for receptor-operated cation channels.

Repeat immobilization-stressed rats are leaner and have improved cold tolerance due to enhancement of brown adipose tissue (BAT) thermogenesis. This process likely involves stress-induced sympathetic nervous system activation and adrenocortical hormone release, which dynamically enhances and suppresses uncoupling protein 1 (UCP1) function, respectively. To investigate whether repeated immobilization influences UCP1 thermogenic properties, we assessed UCP1 mRNA, protein expression, and activity (GDP binding) in BAT from immobilization-naive or repeatedly immobilized rats (3 h daily for 4 weeks) and sham operated or adrenalectomized (ADX) rats. UCP1 properties were assessed before (basal) and after exposure to 3 h of acute immobilization. Basal levels of GDP binding and UCP1 expression was significantly increased (140 and 140%) in the repeated immobilized group. Acute immobilization increased GDP binding in both naive (180%) and repeated immobilized groups (220%) without changing UCP1 expression. In ADX rats, basal GDP binding and UCP1 gene expression significantly increased (140 and 110%), and acute immobilization induced further increase. These data demonstrate that repeated immobilization resulted in enhanced UCP1 function, suggesting that enhanced BAT thermogenesis contributes to lower body weight gain through excess energy loss and an improved ability to maintain body temperature during cold exposure.

It has been shown that norepinephrine (NE) can mediate vasodilatation by stimulating the production of nitric oxide (NO) in brown adipose tissue (BAT), resulting in an increase in BAT blood flow. We speculated that constitutive NO synthase (NOS) is involved in this NO production. However, it is not known whether constitutive NOS is expressed in BAT. To answer this question, we assessed the expression of two types of constitutive NOS, endothelial (eNOS) and neuronal NOS (nNOS), in BAT of rats. eNOS was abundantly expressed in both BAT and isolated brown adipocytes, whereas nNOS was not. Cold exposure, which is known to stimulate NE release from sympathetic nerve terminals in BAT, led to a significant increase in eNOS mRNA in this tissue. In contrast, very low levels of inducible NOS (iNOS) mRNA were expressed, and cold stimulation failed to increase iNOS mRNA levels in BAT. These results suggest that eNOS is the primary isoform that is responsible for NO production in BAT and that its expression may be under sympathetic control.

Membrane phospholipids are known for their role in the regulation of membrane structures and functions. Membrane phospholipid fatty acid docosahexaenoic acid (DHA) has been recently indicated to be important for the regulation of cellular activities, including metabolic regulation. Our previous studies have indicated the involvement of DHA in the regulation of brown adipose tissue (BAT) thermogenesis. The objective of the present study is to examine the changes in BAT phospholipid fatty acid composition including DHA and thermogenic activity in fasted and refed rats. Phospholipid content per microgram DNA was decreased in rats fasted for 72 h and it was not restored to the control level by refeeding for 72 h. Phospholipid fatty acid composition of BAT, as expressed by mol%, was modified in the fasted rats. Most notably, DHA, which constituted about 89% of the n-3 polyunsaturated fatty acids, was decreased concomitant with the increase in the n-6 polyunsaturated fatty acid arachidonic acid. The monounsaturated to saturated fatty acid ratio, which is an index of Delta(9)-desaturase activity and membrane fluidity, was decreased. Thermogenesis, as measured by the in vitro oxygen consumption of BAT, was suppressed in the fasted rats. All of the above changes were restored to normal levels after refeeding the fasted rats for 72 h. In vitro oxygen consumption correlated with the level of DHA and monounsaturated to saturated fatty acid ratio. These results indicate that the modification of phospholipid fatty acid composition, especially the modification of n-3 polyunsaturated fatty acid DHA, and membrane fluidity are related to BAT thermoregulation in fasted and refed rats.

The effects of hyperthyroidism and hypothyroidism on brown adipose tissue (BAT) thermogenesis and phospholipid fatty acid composition were investigated in rats. Chronic triiodothyronine (T3) treatment (hyperthyroidism) increased the interscapular BAT pad weight, its triacylglycerol content, and its DNA content. It did not affect basal and noradrenaline-stimulated in vitro oxygen consumption of BAT expressed per microg DNA, although it significantly increased the oxygen consumption of the whole BAT pad. T3 treatment had little effect on phospholipid content and phospholipid fatty acid composition. In contrast, chronic methimazole treatment (hypothyroidism) decreased the BAT pad weight and the triacylglycerol content, but did not significantly change the DNA content in comparison with the control. It significantly decreased the noradrenaline-stimulated BAT oxygen consumption expressed per microg DNA and per BAT pad, but did not change the basal oxygen consumption. Methimazole treatment significantly affected phospholipid content and phospholipid fatty acid composition. Among the major fatty acids of BAT, it decreased docosahexaenoic acid (DHA), arachidonic acid, palmitic acid, palmitoleic acid, and oleic acid, and it increased linoleic acid, stearic acid, and eicosapentaenoic acid. A regression analysis revealed a positive relationship between in vitro respiration and DHA levels in phospholipids (r = 0.404, p<0.05). These results suggest that thyroid hormones have trophic action on BAT and are necessary for BAT thermogenic activity. This study also suggests that DHA is involved in the regulation of BAT thermogenic activity, as we previously indicated.

Brown adipose tissue (BAT) is a major organ of nonshivering thermogenesis during cold acclimation, overfeeding, and nonthermal restraint stress. An increased unsaturation of fatty acids of membrane phospholipid in BAT has been shown to be closely associated with an enhanced function of this tissue as reported in other tissues. In the previous study, we found that among fatty acids detected, the n-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) in BAT phospholipid is the only fatty acid that altered concomitantly with a change in the in vitro thermogenic capacity for nonshivering thermogenesis of BAT from cold acclimated and restrained rats. To examine the effect of dietary DHA on fatty acid composition of phospholipid and in vitro BAT thermogenic activity, rats were fed a standard diet supplemented with purified DHA for 4 or 16 weeks. Dietary DHA for 4 or 16 weeks increased DHA level in BAT phospholipid, and it also decreased arachidonic acid. The unsaturation index increased and remained unchanged under DHA feeding for 4 and 16 weeks, respectively. In vitro thermogenic activity of BAT remained unchanged and decreased under dietary DHA for 4 and 16 weeks, respectively. These findings indicate that dietary DHA could increase DHA level in phospholipid of BAT, but it could not improve in vitro BAT thermogenic response.

The effects of 4-week cold exposure on capillary geometry, particularly in terms of the distribution of arteriolar and venular capillaries and their capillary domain areas, were studied for different types of skeletal muscles in male Wistar rats. Morphological data for capillaries and muscles were obtained from muscle cross-sections exposed to a double-staining method that distinguishes arteriolar portions from venular portions of capillaries. In soleus (SOL; type I fibers) and the deep portion of gastrocnemius (GASd; type I and IIa fibers) muscles, total capillary density and density of arteriolar capillary were significantly greater in cold-acclimated (CA) rats than in warm control (WC) rats (p < 0.05). In the superficial portion of the gastrocnemius (GASs; type IIb fibers), however, these changes in capillarity were not observed. After cold acclimation, fiber cross-sectional area was significantly decreased by 21, 28 and 15% in SOL, GASd and GASs (p < 0.05), respectively. In SOL and GASd but not in GASs, capillary domain areas of arteriolar, intermediate and venular portions in CA were significantly smaller than those of the respective portions in WC (p < 0.05). Succinate dehydrogenase activity was significantly increased after cold acclimation, by 31% in SOL and 21% in GASd (p < 0.05). In GASs, however, the activity remained unchanged after cold acclimation. These results suggest that adaptive changes in the oxygen transport system were observed after cold acclimation in the skeletal muscles that are mainly composed of oxidative fibers. These adaptive changes may improve the effective oxygen supply to muscle tissues that contribute to thermogenesis in a cold atmosphere.

We investigated the effects of nitric oxide synthase inhibition on the phospholipid fatty acid composition of brown adipose tissue by administering chronic N omega-nitro-L-arginine methyl ester (L-NAME) (0.01%) to rats via drinking water. L-NAME caused a significant decrease in C14:0, C16:0, C16:1 and C18:1, with a concomitant increase in C18:0 and C18:2, L-NAME treatment elicited an increase in the unsaturation index. There were no significant changes in other fatty acids including the polyunsaturated fatty acids docosahexaenoate (DHA, C22:6) and arachidonate (C20:4). Polyunsaturated fatty acid linoleate (C18:2) was increased and the arachidonate index decreased in the L-NAME-treated group, suggesting a diminished conversion of C18:2 to C20:4.

This study was undertaken to examine the possible role of nitric oxide (NO) on brown adipose tissue (BAT) thermogenesis in rats. The chronic administration of N omega-nitro-L-arginine methyl ester (L-NAME; NO synthase inhibitor) in drinking water given to rats decreased interscapular BAT (IBAT) weight as well as DNA content in a warm environment (25 +/- 1 degrees C; 2 and 4 weeks), and inhibited the cold-stimulated (5 +/- 1 degrees C; 2 weeks) increase in IBAT weight and DNA content. L-Arginine administration (4 weeks in a warm environment) increased the DNA content of IBAT. Chronic L-NAME administration (2 weeks in a warm environment) eliminated the NE-stimulated increase in in vivo oxygen consumption (VO2), caused hypothermia in acute cold exposure (0 degree C), and suppressed the NE-stimulated increase in in vitro IBAT VO2. In vitro incubation of native IBAT with L-NAME suppressed the basal and NE-stimulated increase in in vitro VO2. In vitro incubation of IBAT with methylene blue (soluble guanylate cyclase inhibitor and a scavenger of free NO) eliminated the NE-stimulated increase in in vitro IBAT VO2. These results suggest that the nitric oxide and NO-cGMP signaling systems are involved in the regulation of BAT cellularity and thermogenesis in rats.

The effects of cold acclimation and repetitive intermittent immobilization were examined on fatty acid (FA) compositions in phospholipids of rat interscapular brown adipose tissue (BAT) and plasma. As previously reported, cold acclimation and intermittent immobilization increased the degree of unsaturation as a whole in FAs of BAT but not in plasma. N-3 polyunsaturated docosahexaenoic acid (22-6; DHA) decreased in cold acclimation but increased in intermittent immobilization in phospholipids of BAT. DHA was decreased in phospholipids of plasma in both groups. Considering our previous findings that the in vitro thermogenic response of BAT was suppressed in cold acclimation and enhanced in intermittent immobilization, it was inferred that DHA in BAT is involved in the regulation of thermogenic function of this tissue.

Interference by lipids with fluorometric assay of DNA in adipose tissues using Hoechst 33258 was investigated. Mixed glycerides shifted the emission maximum of standard DNA and induced a dose-dependent increase in fluorescence intensity. Glycerides in the samples containing a known concentration of DNA yielded erroneously higher DNA concentrations. The DNA concentrations obtained from acetone-defatted white and brown adipose tissues (WAT and BAT) were lower than those of non-defatted ones, while DNA content did not differ in low lipid-containing skeletal muscle between defatted and non-defatted samples, indicating that large amounts of lipids interfere with DNA measurement using Hoechst 33258 and that acetone defatting is a simple method to avoid this interference. Using this defatting method, the cellularity of WAT and BAT was estimated in rats under various experimental conditions. Cold-acclimation and repetitive immobilization stress decreased the body weight gain and the epididymal WAT weight. Sucrose overfeeding increased WAT weight but not body weight. These treatments of 4 weeks' duration did not induce any significant difference in WAT cell number from controls, while cold-acclimation increased the tissue cell number as well as the BAT weight.

N omega-nitro-L-arginine methyl ester (L-NAME) (80 mg), a NO synthase blocker, completely abolished noradrenaline (NA)-induced increase in blood flow through brown adipose tissue (BAT) in the urethan-anesthetized rat. L-NAME suppressed NA-induced increase in blood flow dose-dependently. L-arginine (80 mg), but not D-arginine (80 mg), reversed the inhibitory effect of L-NAME (0.8 mg). NA-induced increase in BAT temperature was also decreased by L-NAME. These results suggest that nitric oxide mediates NA-induced increase in BAT blood flow.

The ventral blood islands (VBI) of Xenopus laevis embryos are known as the hemopoietic site where the initial erythropoiesis takes place at st. 28. To determine the site of postembryonic erythropoiesis, larvae were induced anemic by phenylhydrazine (PHZ) at st. 31 and 40, and the tissue distribution of regenerating erythrocytes was determined with an anti-larval hemoglobin (LHb) monoclonal antibody. Three days after total anemia induction, the LHb + cells were detected first in the liver and the digestive tract, followed by the appearance of a few LHb + cells in the blood vessels. The lavae which had been hepatectomized and cardiectomized before the PHZ treatment showed a remarkable reduction in recovery of the LHb + cells. Induction of anemia in the chimeric individuals containing cytogenetically labelled VBI tissues demonstrated that the VBI-derived cells contribute to the regenerating LHb + cells in all experimental individuals. These results suggest that the larval liver is the major site where the VBI-derived hemopoietic cells reside and differentiate into erythrocytes.

Previous immunohistochemical observations using the monoclonal antibody (XL-1) which recognizes all types of leukocytes in Xenopus laevis revealed the occurrence of XL-1+ cells in the mesenchyme throughout the early larval body, before the appearance of any lymphocytes. The present experiments were performed to determine whether these leukocytes originate, like lymphocytes and red blood cells (RBCs), in the ventral blood islands (VBI) or the dorsolateral plate (DLP). For tracing the derivation of cells, a specific staining by quinacrine to nuclei of X. laevis and Xenopus borealis hybrid (LB) cells was used to distinguish them from X. laevis (LL) cells. Orthotopic graftings of VBI tissue from st.22-23 LB embryos to the stage-matched LL embryos and examinations at st.44-45 before differentiation of the lymphocytes showed that the proportion of XL-1+ LB cells was always significantly lower than that of RBCs with the same marker in all experimental larvae. The head (LB)-body (LL) chimeras from st.22-23 embryos and culture of the head-portions as VBI- and DLP-free explants from st.14-23 embryos both demonstrated that a significant number of XL-1+ cells which had originated in the head portions had begun to differentiate by st.42-43. These results indicate that there is a significant population of larval nonlymphoid leukocytes (mostly macrophages) that do not originate from either the VBI or DLP region, and are distributed in the mesenchyme throughout the body.

A method for large-scale culture of isolated blastomeres of sea urchin embryos in spinner flasks was developed. Micromeres and meso-, macromeres isolated from sea urchin embryos at the 16-cell stage were cultured by this method and the patterns of protein synthesis by their descendants were examined by two-dimensional gel electrophoresis of [ 35 S] methionine-labeled proteins. Six distinct proteins with molecular weights of 140-kDa, 105-kDa, 43-kDa, 32-kDa, and 28-kDa (two components) were specifically synthesized by differentiating micromeres. Quantitative analysis of the two-dimensional gel patterns demonstrated that all these proteins, except the 32-kDa protein, appeared at the time of ingression of primary mesenchyme cells (PMC's) in vivo, several hours earlier than the onset of spicule formation. The synthesis of 32-kDa protein was paralleled to active spicule formation and the uptake of Ca 2+ . Cell-free translation products directed by poly (A) + RNAs isolated from descendant cells of micromeres and meso-, macromeres were compared by two-dimensional gel electrophoresis. Several spots specific to the micromere lineage were detected. However, none of them comigrated with the proteins synthesized specifically by the cultured micromeres. The results suggest that the expression of these proteins specific to differentiating micromeres may involve post-translational modification.