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Climate change is expected to have detrimental consequences on fragile ecosystems, threatening biodiversity, as well as food security of millions of people. Trees are likely to play a central role in mitigating these impacts. The microclimatic conditions below tree canopies usually differ substantially from the ambient macroclimate as vegetation can buffer temperature changes and variability. Trees cool down their surroundings through several biophysical mechanisms, and the cooling benefits occur also with trees outside forest. The aim of this study was to examine the effect of canopy cover on microclimate in an intensively modified Afromontane landscape in Taita Taveta, Kenya. We studied temperatures recorded by 19 microclimate sensors under different canopy covers, as well as land surface temperature (LST) estimated by Landsat 8 thermal infrared sensor. We combined the temperature records with high-resolution airborne laser scanning data to untangle the combined effects of topography and canopy cover on microclimate. We developed four multivariate regression models to study the joint impacts of topography and canopy cover on LST. The results showed a negative linear relationship between canopy cover percentage and daytime mean (R2=0.65) and maximum (R2=0.75) temperatures. Any increase in canopy cover contributed to reducing temperatures. The average difference between 0 % and 100 % canopy cover sites was 5.2 ∘C in mean temperatures and 10.2 ∘C in maximum temperatures. Canopy cover (CC) reduced LST on average by 0.05 ∘C per percent CC. The influence of canopy cover on microclimate was shown to vary strongly with elevation and ambient temperatures. These results demonstrate that trees have a substantial effect on microclimate, but the effect is dependent on macroclimate, highlighting the importance of maintaining tree cover particularly in warmer conditions. Hence, we demonstrate that trees outside forests can increase climate change resilience in fragmented landscapes, having strong potential for regulating regional and local temperatures.

Evapotranspiration (ET) of Amazon forests is a main driver of regional climate patterns and an important indicator of ecosystem functioning. Despite its importance, the seasonal variability of ET over Amazon forests, and its relationship with environmental drivers, is still poorly understood. In this study, we carry out a water balance approach to analyse seasonal patterns in ET and their relationships with water and energy drivers over five sub-basins across the Amazon Basin. We used in situ measurements of river discharge, and remotely sensed estimates of terrestrial water storage, rainfall, and solar radiation. We show that the characteristics of ET seasonality in all sub-basins differ in timing and magnitude. The highest mean annual ET was found in the northern Rio Negro basin (∼ 1497 mm year−1) and the lowest values in the Solimões River basin (∼ 986 mm year−1). For the first time in a basin-scale study, using observational data, we show that factors limiting ET vary across climatic gradients in the Amazon, confirming local-scale eddy covariance studies. Both annual mean and seasonality in ET are driven by a combination of energy and water availability, as neither rainfall nor radiation alone could explain patterns in ET. In southern basins, despite seasonal rainfall deficits, deep root water uptake allows increasing rates of ET during the dry season, when radiation is usually higher than in the wet season. We demonstrate contrasting ET seasonality with satellite greenness across Amazon forests, with strong asynchronous relationships in ever-wet watersheds, and positive correlations observed in seasonally dry watersheds. Finally, we compared our results with estimates obtained by two ET models, and we conclude that neither of the two tested models could provide a consistent representation of ET seasonal patterns across the Amazon.

The seasonal climate drivers of the carbon cycle in tropical forests remain poorly known, although these forests account for more carbon assimilation and storage than any other terrestrial ecosystem. Based on a unique combination of seasonal pan-tropical data sets from 89 experimental sites (68 include aboveground wood productivity measurements and 35 litter productivity measurements), their associated canopy photosynthetic capacity (enhanced vegetation index, EVI) and climate, we ask how carbon assimilation and aboveground allocation are related to climate seasonality in tropical forests and how they interact in the seasonal carbon cycle. We found that canopy photosynthetic capacity seasonality responds positively to precipitation when rainfall is −1 (water-limited forests) and to radiation otherwise (light-limited forests). On the other hand, independent of climate limitations, wood productivity and litterfall are driven by seasonal variation in precipitation and evapotranspiration, respectively. Consequently, light-limited forests present an asynchronism between canopy photosynthetic capacity and wood productivity. First-order control by precipitation likely indicates a decrease in tropical forest productivity in a drier climate in water-limited forest, and in current light-limited forest with future rainfall −1.

Fractional tree cover (Fcover) is an important biophysical variable for measuring forest degradation and characterizing land cover. Recently, atmospherically corrected Landsat data have become available, providing opportunities for high-resolution mapping of forest attributes at global-scale. However, topographic correction is a pre-processing step that remains to be addressed. While several methods have been introduced for topographic correction, it is uncertain whether Fcover models based on vegetation indices are sensitive to topographic effects. Our objective was to assess the effect of topographic correction on the accuracy of Fcover modelling. The study area was located in the Eastern Arc Mountains of Kenya. We used C-correction as a digital elevation model (DEM) based correction method. We examined if predictive models based on normalized difference vegetation index (NDVI), reduced simple ratio (RSR) and tasseled cap indices (Brightness, Greenness and Wetness) are improved if using topographically corrected data. Furthermore, we evaluated how the results depend on the DEM by correcting images using available global DEM (ASTER GDEM, SRTM) and a regional DEM. Reference Fcover was obtained from wall-to-wall airborne LiDAR data. Landsat images corresponding to minimum and maximum sun elevation were analyzed. We observed that topographic correction could only improve models based on Brightness and had very small effect on the other models. Cosine of the solar incidence angle (cos i) derived from SRTM DEM showed stronger relationship with spectral bands than other DEMs. In conclusion, our results suggest that, in tropical mountains, predictive models based on common vegetation indices are not sensitive to topographic effects.

Background and Purpose: Hypophysitis is one of the well-known adverse effects of immune checkpoint inhibitors. Immune checkpoint inhibitor-induced hypophysitis frequently causes irreversible hypopituitarism, which requires long-term hormone replacement. Despite the high frequency and clinical significance, characteristic MR imaging findings of immune checkpoint inhibitor-induced hypophysitis have not been established. In the present study, we aimed to review and extract the MR imaging features of immune checkpoint inhibitor-induced hypophysitis., Materials and Methods: This retrospective international multicenter study comprised 20 patients with melanoma who were being treated with immune checkpoint inhibitors and clinically diagnosed with immune checkpoint inhibitor-induced hypophysitis. Three radiologists evaluated the following MR imaging findings: enlargement of the pituitary gland and stalk; homogeneity of enhancement of the pituitary gland; presence/absence of a well-defined poorly enhanced area and, if present, its location, shape, and signal intensity in T2WI; and enhancement pattern in contrast-enhanced dynamic MR imaging. Clinical symptoms and hormone levels were also recorded., Results: Enlargement of the pituitary gland and stalk was observed in 12 and 20 patients, respectively. Nineteen patients showed poorly enhanced lesions (geographic hypoenhancing lesions) in the anterior lobe, and 11 of these lesions showed hypointensity on T2WI. Thyrotropin deficiency and corticotropin deficiency were observed in 19/20 and 12/17 patients, respectively, which persisted in 12/19 and 10/12 patients, respectively, throughout the study period., Conclusions: Pituitary geographic hypoenhancing lesions in the anterior lobe of the pituitary gland are characteristic and frequent MR imaging findings of immune checkpoint inhibitor-induced hypophysitis. They reflect fibrosis and are useful in distinguishing immune checkpoint inhibitor-induced hypophysitis from other types of hypophysitis/tumors., (© 2020 by American Journal of Neuroradiology.)

Study Question: What are the effects of fertility education on knowledge, childbearing desires and anxiety?, Summary Answer: Providing fertility information contributed to greater knowledge, but increased anxiety., What Is Known Already: Past studies have found that exposure to educational material improved fertility awareness and changed desires toward childbearing and its timing. Existing educational websites with evidence-based medical information provided in a non-judgmental manner have received favorable responses from reproductive-aged men and women., Study Design, Size, Duration: This three-armed (one intervention and two control groups), randomized controlled trial was conducted using online social research panels (SRPs) in Japan in January 2015., Participants/materials, Setting, Methods: A total of 1455 participants (726 men and 729 women) between 20 and 39 years of age who hoped to have (more) children in the future were block-randomized and exposed to one of three information brochures: fertility education (intervention group), intake of folic acid during pregnancy (control group 1) or governmental financial support for pregnancy and childbirth (control group 2). Fertility knowledge was measured with the Japanese version of the Cardiff Fertility Knowledge Scale (CFKS-J). Knowledge, child-number and child-timing desires, subjective anxiety (i.e. whether participants felt anxiety [primary outcome]), and scores on the State-Trait Anxiety Inventory were assessed immediately after exposure. Non-inferiority comparisons were performed on subjective anxiety with non-inferiority declared if the upper limit of the two-sided 95% confidence interval (CI) for risk difference did not exceed a margin of 0.15. This test for non-inferiority was only performed for subjective anxiety; all the other variables were tests of superiority., Main Results and the Role of Chance: Posttest scores on the CFKS-J (mean, SD) were higher in the intervention group than that of the control groups: intervention versus Control 1 and versus Control 2: 52.8 (28.8) versus 40.9 (26.2) (P< 0.001) versus 45.1 (27.1) (P = 0.003) among men and 64.6 (26.0) versus 50.8 (26.9) (P< 0.001) versus 53.0 (26.4) (P< 0.001) among women.The percentage of participants who felt subjective anxiety after exposure to the intervention brochure was significantly higher than that of the control groups: intervention versus Control 1 and versus Control 2: 32.6 versus 17.8% (risk difference [RD] = 0.149, 95% CI: 0.073-0.225) versus 14.5% (RD = 0.182, 95% CI: 0.108-0.256) among men, and 50.2 versus 26.3% (RD = 0.239, 95% CI: 0.155-0.322) versus 14.0% (RD = 0.362, 95% CI: 0.286-0.439) among women. Non-inferiority of the intervention was inconclusive (i.e. the CI included 0.15) among men whereas inferiority was declared among women. The incidence of anxiety was higher in the intervention group than that of the control groups especially among men aged 30 and older and among women aged 25 and older. No difference existed in childbearing desires between groups after exposure., Limitations, Reasons for Caution: The possibility of selection bias associated with the use of SRPs (higher socioeconomic status and education) and volunteer bias toward those more interested in fertility may limit the generalizability of these findings., Wider Implications of the Findings: In addition to education targeting a younger generation, psychological approaches are needed to alleviate possible anxiety caused by fertility information., Study Funding/competing Interests: This study was funded by National Center for Child Health and Development, Seiiku Medical Study Grant (24-6), the Daiwa Foundation Small Grants and Grant-in-Aid for JSPS Fellows (26-1591). No competing interest declared., Trial Registration Number: UMIN Clinical Trials Registry. Trial registration number, 000016168., Trial Registration Date: 13 January 2015., Date of First Patient's Enrolment: 15 January 2015., (© The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.)

Unlabelled: Enterohemorrhagic Escherichia coli O157 (O157) strains can be classified in clades by single nucleotide polymorphisms (SNPs), but this analysis requires significant laboratory effort. As the distribution of insertion sequence (IS) 629 insertions has been reported to be biased among different clades, O157 isolates can be putatively classified in clades by comparison with an IS629 distribution database. A database of the IS629 distribution in O157 strains isolated in Chiba Prefecture and their classification in clades was determined by SNP analysis and IS-printing, an easy and quick analytical tool for IS629 in the O157 genome. The IS629 distribution in O157 strains isolated in Fukuoka and Yamagata Prefectures was determined by IS-printing. These strains were putatively classified in clades by Relative Likelihood calculations that compared the IS-printing data and the IS629 distribution database. Concordance Ratios were calculated, which compared the number of strains putatively classified in a clade by Relative Likelihood to the number of strains classified in that clade by SNP analysis. For the Fukuoka and Yamagata strains, the Concordance Ratios for clades 3, 6 and 8 were 97-100%, for clade 7 about 88%, and for clades 2 and 12 over 90%. In conclusion, O157 clade 2, 3, 6, 7, 8 and 12 strains could be putatively classified by IS-printing., Significance and Impact of the Study: This study demonstrated that enterohemorrhagic E. coli O157 (O157) strains could be putatively classified in clades using an IS-printing system. IS-printing was previously developed as a relatively quick and easy tool for analysis of insertion sequence 629 in the O157 genome. Since most local government public health institutes in Japan carry out IS-printing for early detection of O157 outbreaks, these data should be useful for putative classification of O157 strains in each area., (© 2015 The Society for Applied Microbiology.)

Aims: The genetic differences of enterohaemorrhagic Escherichia coli O157 (O157) strains isolated from humans in three widely-separated areas in Japan were analysed to provide information on possible geographic aspects of O157 pathogenicity., Methods and Results: Epidemiologically unlinked O157 strains were isolated in Chiba (300 strains), Fukuoka (260 strains) and Yamagata (81 strains) prefectures. These strains were classified in clades by single nucleotide polymorphism in seven loci and lineage-specific polymorphism assay-6, and differences between the strains in each clade were compared by population genetic analyses using the IS-printing system. Analysis of the clades from the three areas showed linkage disequilibrium of the strains in each clade. Comparison of the genetic differences of strains from the three areas in each clade, from calculated ΦPT values, indicated that the strains in each clade were the same population in all three areas, except possibly the clade 12 strains., Conclusions: Population genetics analyses confirmed that the distribution of O157 strains in the clades isolated in three areas in Japan were similar and stable., Significance and Impact of the Study: The pathogenicity of O157 strains infecting humans was comparable due to the similar, stable geographic distribution of O157 clades., (© 2014 The Society for Applied Microbiology.)

Late-onset noninfectious pulmonary complications (LONIPCs) are life threatening for allogeneic hematopoietic SCT (allo-HSCT) recipients. However, the impact of LONIPCs on survival has not been properly evaluated and little is known about treatment efficacy. We retrospectively investigated 290 allo-HSCT recipients in our institute and reviewed the clinical aspects of 44 patients who had been diagnosed with LONIPCs. LONIPCs were significantly associated with higher rates of chronic GVHD (P<0001) and nonrelapse mortality (P=0.013), and lower rates of relapse (P=0.009). As a result of these effects, OS was significantly worse in those with LONIPCs (P=0.003). This result differs from a previous report. We then assessed short-term treatment response and final outcome. These results were defined by radiological findings, subjective symptoms, oxygen requirement and survival. Use of inhaled and systemic steroids did not affect either short-term response or final outcomes. However, administration of systemic corticosteroids earlier than at 21 days (median interval of time from onset of symptoms to systemic corticosteroids administration) was associated with a better outcome (P=0.054 for short-term response, and 0.016 for final outcome). Our study indicates that LONIPCs reduce OS, and early intervention with systemic corticosteroids may be effective.

Various splenic inflammatory pseudotumours are reported to be infected with Epstein-Barr virus (EBV), which is thought to be associated with the pathogenesis of the lesion. The term "inflammatory pseudotumour (IPT)-like follicular dendritic cell tumour", all cases of which are also EBV positive, has recently been proposed. Here, we describe the imaging findings of these splenic tumours and present the cases of an IPT-like follicular dendritic cell tumour and two EBV-positive inflammatory pseudotumours in two female patients and one male patient. These splenic lesions were found incidentally on pre-operative or post-operative screening or at medical check-up. CT performed on all three patients revealed low-density solitary masses in the spleen. MRI was performed on one patient; the solitary mass demonstrated isointensity on T(1) weighted images and low intensity on T(2) weighted images relative to the surrounding splenic parenchyma. Dynamic MRI study revealed that the mass did not enhance on the early phase but enhanced to the same degree as the surrounding splenic parenchyma on the delayed phase. The imaging findings are almost identical to those found in conventional IPT because the morphology is similar in both cases; however, attention should be paid to this new entity in the diagnosis of splenic lesions because of its neoplastic nature. Longer follow-up is also necessary for these patients compared with those with conventional IPT.

The allele frequencies for a Pro12-->Ala substitution in peroxisome proliferator-activated receptor-gamma differ among ethnic groups, and its relationship with diabetes and associated diseases is controversial. The prevalence of this polymorphism and its effects on clinical characteristics have now been evaluated with a large number of Japanese individuals with type 2 diabetes (n = 2,201) and normal control subjects (n = 1,212) recruited by 10 institutions located in seven different cities in Japan. The allele frequency for the Ala12 variant was significantly lower in the type 2 diabetic group than in the control group (2.39 vs. 4.13%, P = 0.000054). However, compared with subjects without the Ala12 variant, the diabetic subjects with this variant exhibited a significantly higher serum concentration of total cholesterol (P = 0.001), manifested a reduced capacity for insulin secretion as evaluated by homeostasis model assessment (P = 0.007), and tended to possess a higher level of HbA1c. These data suggest that the Ala12 variant is associated with a reduced risk for the development of diabetes in the general population, but that it may be also a risk factor for insulin deficiency and disease severity in individuals with type 2 diabetes.

Aims/hypothesis: The cumulative effects of several thrifty factors could contribute to the pathogenesis of Type II (non-insulin-dependent) diabetes mellitus. We screened the human UCP1 gene (UCP1) for polymorphisms associated with susceptibility to Type II diabetes., Methods: By using PCR and single-strand conformation polymorphism analysis, UCP1 were screened for mutations in 25 Type II diabetic subjects and 25 healthy control subjects. The allele frequencies of the detected polymorphisms were determined by PCR and restriction fragment length polymorphism analysis in 320 diabetic subjects and 250 control subjects., Results: An A-->C transition in the 5' untranslated region (UTR) of exon 1 (112 bp upstream of the translation initiation codon) and a Met229-->Leu variant were detected. The allele frequencies for the C variant and for the Leu229 variant were higher in the Type II diabetic group than in the control group (p = 0.017 and p = 0.038, respectively). These polymorphisms were in linkage disequilibrium (p < 0.00001). Luciferase assay showed that the former variant in the 5' UTR may affect the promoter activity of UCP1., Conclusion/interpretation: Both the A-->C polymorphism and the Met229-->Leu polymorphism of UCP1 are in linkage disequilibrium and could be one of the diabetes associated single nucleotide polymorphisms (SNPs).

Primary hyperoxaluria Type 1 (PH1) is caused by a functional deficiency of a liver enzyme, serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT), which catalyzes transamination between L-serine or l-alanine as an amino acid substrate and glyoxylate or pyruvate as an alpha-keto acid substrate. A high affinity for glyoxylate is a notable feature of this enzyme, suggesting a role in glyoxylate metabolism in vivo. Another conspicuous feature of SPT/AGT is its species-specific and food habit-dependent subcellular distribution. Thus, the enzyme is located in peroxisomes in herbivores and man, largely in mitochondria in carnivores, and in both the organelles in rodents. The mechanism of the species-specific dual organelle localization of SPT/AGT is either transcription of the gene from two different start sites or loss of the upstream translation initiation ATG codon by mutations. It appears that the mitochondrial versus peroxisomal distribution of SPT/AGT in different animal species is indispensable in meeting the metabolic needs caused by their respective food habits. As for the peroxisomal localization, glycolate is contained in plants much more than in animal tissues, and when ingested, it is converted to glyoxylate, an immediate precursor of oxalate, in liver peroxisomes. Therefore, peroxisomal localization of SPT/AGT may be indispensable for herbivores to convert the glyoxylate formed in peroxisomes into glycine in situ rather than forming oxalate. On the other hand, our recent studies showed that SPT/AGT contributed substantially to serine metabolism in rabbit, human, and dog livers; i.e., irrespective of its mitochondrial or peroxisomal localization. Thus, the mitochondrial localization of SPT/AGT was not a prerequisite for the metabolism of L-serine. Another source of glyoxylate is the metabolism of L-hydroxyproline, and in this case, the enzyme responsible for the glyoxylate formation has been reported to be a mitochondrial matrix enzyme. Collagen accounts for about 30% of total animal proteins and contains about 13% (w/w) hydroxyproline. It is therefore possible that both mitochondrial and peroxisomal SPT/AGT contribute to the metabolism of glyoxylate and serine, but the subcellular site for glyoxylate metabolism is different in herbivores and carnivores.

An enzymatic assay was developed for the spectrophotometric determination of glycolate in urine and plasma. Glycolate was first converted to glyoxylate with glycolate oxidase, and the glyoxylate formed was condensed with phenylhydrazine. The glyoxylate phenylhydrazone formed was then oxidized with K(3)Fe(CN)(6) in the presence of excess phenylhydrazine, and A(515) of the resulting 1, 5-diphenylformazan was measured. Since glycolate oxidase also acts on glyoxylate and L-lactate, the incubation of samples with glycolate oxidase was carried out in 120-170 mM Tris-HCl (pH 8.3) to obtain glyoxylate as its adduct with Tris. The pyruvate formed from lactate was removed by subsequent brief incubation with alanine aminotransferase in the presence of L-glutamate, and alpha-ketoglutarate formed was converted back to L-glutamate by glutamate dehydrogenase and an NADPH generating system. Thus the specificity of the assay relies principally on the substrate specificity of glycolate oxidase, and high sensitivity is provided by the high absorbance of 1,5-diphenylformazan at 515-520 nm. Plasma was deproteinized with perchloric acid, and then neutralized with KOH. Plasma and urine samples were then incubated with approximately 5 mM phenylhydrazine, and then treated with stearate-deactivated activated charcoal to remove endogenous keto and aldehyde acids as their phenylhydrazones. The normal plasma glycolate and urinary glycolate/creatinine ratio for adults determined by this method are approximately 8 microM and approximately 0.036, respectively.

Glyoxylate is an immediate precursor of oxalate, but in its metabolism the conversion into glycine catalyzed by serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT) appears to be the main route. When SPT/AGT is missing as in the case of primary hyperoxaluria type 1 (PH1) more glyoxylate is used for the oxalate production, resulting in calcium oxalate urolithiasis and finally systemic oxalosis. SPT/AGT is a unique enzyme of species-specific dual organelle localization; it is located largely in mitochondria in carnivores and entirely in peroxisomes in herbivores and man. For herbivores, the peroxisomal localization of SPT/AGT is indispensable to avoid massive production of oxalate, probably because liver peroxisomes are the main site of glyoxylate production from glycolate, and plants contain glycolate much more than animal tissues. Recently, we took charge of laboratory examination for 8 cases of primary hyperoxaluria in Japan, and felt that symptoms of some of the Japanese PH1 patients are apparently milder than those of Western patients. The reason of this is not clear, but from the above mentioned seemingly indispensable association of grass-eating with the peroxisomal localization of SPT/AGT it may be related, at least in part, to the food habit of Japanese, especially that of old generation, that they prefer boiled greens rather than frying or raw vegetables.

A full-term baby was born to an epileptic mother treated with two anti-epileptic drugs, sodium valproate and phenobarbital, throughout the pregnancy. She was given no information about the risk of teratogenesis of these drugs. At birth the patient was hypotonic and had clinical features specific for the fetal valproate syndrome. After a viral infection at three months of age, he had intractable and persistent wheezing. Suspecting the presence of congenital respiratory tract abnormality, we performed tracheobronchoscopy, which revealed a relatively big submucosal tumor on the left trachial wall below the vocal cord. Dyspnea and wheezing were remarkably improved by tracheolaryngotomy, but he died suddenly and unexplainedly at seven months of age.

The first victim of four serial murders occurring in Hiroshima in 1996 was an unidentified young female when she was found. In the lower jaw, the left and right deciduous second molars were identified, and the left and right permanent second premolars were revealed to be congenitally defective by X-ray examination. Comparison of the dental findings and study model after death with those before death identified the victim as a 16-year old female.

Networks of cultured cortical neurones exhibit regular, synchronized, propagating bursts which are synaptically mediated, and which are hypothesized to play a part in activity-dependent formation of connections during development in vivo. The relationship between the strength of synaptic connections and the characteristics of synchronized propagating bursting, however, is unclear. Modification of synchronized activity in cortical cultures in response to electrical stimulation was examined using multisite electrode array recording. By measuring the response of the network to weak, localized, test stimulation (TS), we observed a potentiation of activity following a relatively stronger inducing stimulation (IS). This potentiation was evident as an increased probability of eliciting bursts by TS, an increased frequency of spontaneous bursts and number of spikes per burst, and increased speed of burst propagation, and it lasted for at least 20 min. Changing the parameters of IS revealed that high frequency tetanic stimulation is not necessary to induce potentiation, while it is essential for IS to produce a regeneratively propagating burst. The results provide a direct demonstration of modification of both the spatial and temporal characteristics of synchronized network activity, and suggest an important physiological role for propagating synchronized bursting, as a mechanism for inducing plastic modifications in the developing cortex.

The present study was conducted in order to examine the role of low-density lipoprotein (LDL)-receptor activity in very-low-density lipoprotein (VLDL) triglyceride metabolism in vivo. Fructose-feeding (10% in drinking water) for 2 weeks resulted in elevated plasma triglyceride in heterozygote of Watanabe heritable hyperlipidemic (WHHL) rabbit (WHHLH) associated with suppressed fractional catabolic rate (FCR) of plasma triglyceride, whereas Japanese white (JW) rabbit with normal LDL receptor activity showed no remarkable change in plasma triglyceride turnover after fructose-feeding, suggesting an involvement of LDL receptor activity on triglyceride metabolism. Thereafter, in order to stimulate cellular LDL receptor activity, fluvastatin, a new 3-hydroxy-3-methylglutaryl-coenzyme-A (HMG-CoA) reductase inhibitor, was administered orally (1.52 +/- 0.26 mg/kg) to fructose-fed WHHLH. Significant suppression of triglyceride secretion rate (TGSR) was observed after treatment. However, since plasma triglyceride level was markedly suppressed, FCR of plasma triglyceride was significantly elevated by fluvastatin. Thus, it is speculated from the present data that LDL receptor activity is significantly involved in VLDL triglyceride metabolism in rabbits.

Experimental investigation of the dynamics of biological networks is a fundamental step towards understanding how the nervous system works. Spontaneous activity in cultured networks of cortical neurons has been investigated by using a multisite recording technique with planar electrode arrays. In these networks, the spatiotemporal firing patterns were studied in the presence of different extracellular solutions. Transitions from asynchronous firing dynamics to synchronous firing dynamics were observed when the extracellular Ca2+ concentration was increased from 0.1 mM to 1 mM. Addition of extracellular Mg2+ reduced the spontaneous activity at any Ca2+ concentration, and an increase in the extracellular K+ concentration enhanced the frequency of periodical synchronous bursts. N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptor antagonists inhibited synchronous activity. A spatiotemporal analysis of the data has been performed, and the properties of the network such as the synchronization and the periodicity have been quantified in order to clarify how variations of intrinsic parameters of the network can induce structural transitions in the neural dynamics. This experimental study is a possible approach to investigate the computational properties of a neuronal network.

We examined the effect of long-term (6 months) hyperinsulinemia on VLDL-triglyceride turnover in male Wistar rats. Hyperinsulinemia was induced in rats by daily s.c. injection of Ultralente insulin (6 U/day at 19:00). Fructose (F) or glucose (G) was supplied in the drinking water (10%) in order to prevent hypoglycemia. The rats were divided into 5 groups: (1) hyperinsulinemia with F water: group F + I; (2) hyperinsulinemia with G water: group G + I; (3) F water alone: group F; (4) G water alone: group G; and (5) control rats without sugar water group C. After 6 months of daily insulin injection triglyceride secretion rate (TGSR) was estimated using Triton WR1339 in all the rats. Groups F + I and G + I were obese and hypoglycemic compared to the other groups. Fasting plasma glucose level of group F was higher than any other group value. TGSR of group F + I was significantly higher than that of the control group, while that of group G + I was not, indicating that long-term hyperinsulinemia can stimulate hepatic triglyceride production when the rats were supplemented only with fructose. On the other hand, the rats in group G + I showed the lowest plasma free fatty acid level of all and their postheparin lipolytic activity was significantly elevated compared to that of the control rats. Moreover, they had suppressed plasma triglyceride levels and its fractional catabolic rate was significantly increased, suggesting that hyperinsulinemia can still stimulate triglyceride removal from the circulation of glucose supplemented rats even at month 6. In conclusion, exogenous hyperinsulinemia can stimulate hepatic triglyceride secretion even after 6 months duration when supplemented with fructose, while its stimulating effect on triglyceride removal from the circulation can be seen only with glucose supplementation. Thus, the effect of long-term hyperinsulinemia on plasma triglyceride turnover differs depending on the supplemented monosaccharides.

The recently cloned factor thrombopoietin (TPO) has been shown to exhibit megakaryocyte colony-stimulating activity in vitro. In this investigation, to further evaluate the action of TPO on megakaryocyte progenitor cells (colony-forming units-megakaryocyte [CFU-MK]), GpIIb/IIIa+ and GpIIb/IIIa- populations of CFU-MK were prepared from rat bone marrow cells based on their reactivity with P55 antibody, a monoclonal antibody against rat GpIIb/IIIa, and their responsiveness to recombinant human TPO (rhTPO) and recombinant rat interleukin-3 (rrIL-3) was examined using a megakaryocyte colony-forming assay (Meg-CSA). rhTPO supported only megakaryocyte colony growth from both fractions in a dose-dependent fashion. The mean colony size observed with the GpIIb/IIIa+ population was smaller than that seen with the GpIIb/IIIa- population. With the optimal concentration of either rhTPO or rrIL-3, similar numbers of megakaryocyte colonies were formed from the GpIIb/IIIa+ population previously shown to be highly enriched for CFU-MK. In contrast, the maximum number of megakaryocyte colonies from the GpIIb/IIIa- population stimulated by rhTPO was only 24.2% of that achieved with rrIL-3. Morphologic analysis of rhTPO-promoted megakaryocyte colonies from the GpIIb/IIIa+ population showed that the average colony size was smaller but that the mean diameter of individual megakaryocytes was larger than in megakaryocyte colonies promoted with rrIL-3. rhTPO plus rrIL-3, each at suboptimal concentrations, had an additive effect on proliferation of CFU-MK in the GpIIb/IIIa+ fraction, whereas rhTPO plus murine IL-6 or murine granulocyte-macrophage colony-stimulating factor (mG-M-CSF) modestly but significantly reduced megakaryocyte colony growth. These results indicate that TPO preferentially acts on GpIIb/IIIa+ late CFU-MK with lower proliferative capacity and interacts with some other cytokines in CFU-MK development.

Structure of rabbit lecithin:cholesterol acyltransferase (LCAT) and molecular basis for the effects of cholesterol feeding on LCAT expression were investigated by cloning and sequencing LCAT cDNA from rabbit. The rabbit and human sequences are 91% identical at the nucleotide level and 93% identical at the amino acid level. The interfacial substrate active site, asparagine-linked glycosylation sites, and sites at which rare mutations cause human familial LCAT deficiency are all highly conserved in the rabbit protein. The apparent molecular mass of rabbit LCAT, as determined by immunoblot analysis, was approximately equal to that of human LCAT. Rabbits showed 2.6- and 5.5-fold increases in serum LCAT activity 3 and 6 weeks, respectively, after switching to a cholesterol-enriched diet. Northern blot analysis revealed that the abundance of LCAT mRNA in liver increased 1.6- and 2.8-fold after 3 and 6 weeks, respectively, of cholesterol feeding. The marked temporal relation between the increase in serum LCAT activity and the liver LCAT mRNA abundance suggest that the regulation of LCAT activity in vivo may be primarily determined by changes in the amount of LCAT mRNA.

Long-term recording of spontaneous activity in cultured cortical neuronal networks was carried out using substrates containing multi-electrode arrays. Spontaneous uncorrelated firing appeared within the first 3 days and transformed progressively into synchronized bursting within a week. By 30 days from the establishment of the culture, the network exhibited a complicated non-periodic, synchronized activity pattern which showed no changes for more than 2 months and thus represented the mature state of the network. Pharmacological inhibition of activity only during the period when regular synchronized bursting was observed was capable of producing a different mature activity pattern from the control. These results suggest that periodic synchronized bursting plays a critical role in the development of synaptic connections.

Recently, we purified rat thrombopoietin (TPO) from plasma of irradiated rats (XRP) by measuring its activity that stimulated the production of megakaryocytes from megakaryocyte progenitor cells (CFU-MK) in vitro. We then cloned the cDNAs for rat and human TPO. In this study, we found the production of TPO by hepatocytes isolated with the collagenase perfusion method from both normal and thrombocytopenic rats, by a two-step fractionation of hepatocyte culture medium (CM). Subsequently, CM of rat hepatoma cell lines was screened for the presence of TPO; three cell lines, H4-II-E, McA-RH8994, and HTC, were found to produce TPO. According to the purification procedure for TPO from XRP, TPO was partially purified from 2 L CM of each of three cell lines with a six-step procedure. In the final reverse-phase column, TPO from each cell line was eluted with the same retention time as that from XRP, and the TPO fraction exhibited megakaryocyte colony-stimulating activity (Meg-CSA). TPO-active fraction eluted from the final reverse-phase column was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), extracted from the gel, and assayed. TPO activity from each cell line was found in the respective molecular weight region, indicating the heterogeneity of the TPO molecule. Using reverse transcriptase-polymerase chain reaction (RT-PCR), we detected the expression of TPO mRNA in hepatocytes, three hepatoma cell lines, normal rat liver, and X-irradiated rat liver. Northern blot analysis showed that TPO mRNA was expressed mainly in liver among the various organs tested. These data demonstrate that TPO is produced by rat hepatocytes and hepatoma cell lines and suggest that liver may be the primary organ that produces TPO.

We investigated the genetic defects in two patients with familial lecithin:cholesterol acyltransferase (LCAT) deficiency. Their clinical manifestations including corneal opacities, anemia, proteinuria, and hypoalphalipoproteinemia were identical for familial LCAT deficiency. Their LCAT activities and the cholesterol esterification rate (CER) were nearly zero, and their LCAT masses were below 10% of normal control values. Sequence analysis of the amplified DNA of case 1 revealed one base deletion of G at base 873 (first position of Val264) in exon 6, leading to a premature termination by frameshift. Sequence analysis of amplified DNA of case 2 revealed a single G to A converting Gly (GGT) to Ser (AGT) substitution at residue 344. When COS-1 cells were transfected with these mutants, LCAT activity in the medium was nearly zero, and the LCAT mass was undetectable (< 0.01 microgram/ml). In contrast, LCAT activity in the medium of COS-1 cells, transfected with wild-type LCAT, was 1.7 nmol/h per ml and the LCAT mass was 0.09 micrograms/ml. The LCAT mass in the cell lysates of the mutants was less than 12% of control for case 1 and 18% of control for case 2. Northern blot analysis of the mRNA of COS-1 cells transfected with the mutants showed the same amounts of LCAT mRNA as compared with wild-type LCAT. Biosynthesis of mutant LCATs was analyzed by pulse-chase and immunocytochemistry in transfected baby hamster kidney cells. SDS-PAGE/fluorography demonstrated that wild-type LCAT was synthesized as a high-mannose type of 56 kDa, which was very slowly converted to a mature form of 67 kDa and was secreted into the media. In contrast to the wild-type LCAT, the mutant precursors were not processed into the mature form but slowly degraded along with chase times. On steady and continuous labeling in the case of wild-type LCAT, the mature 67 kDa form was observed in both the cell lysate and media, whereas no mature form was detected in the cell lysates and media which were transfected mutant LCATs. These data suggest that the mutant LCATs are actually synthesized in an amount comparable to that of wild-type, but they are slowly degraded without being processed into the mature form. The immunocytochemistry revealed that mutant LCATs were mainly retained in the endoplasmic reticulum. These data suggest that these two mutations may disrupt the mutant LCATs' transport from the endoplasmic reticulum into Golgi apparatus, resulting in LCAT deficiency.

We measured the lactate concentration in whole blood of healthy infant with use of the recently developed assay system consisting of an enzyme-coated electrode and a small meter. The mean values of blood lactate concentration at each age in days were not showed the significant difference between male and female infants. The trace of lactate concentration change in days showed the peak at the age in 1 days and then, gradually decreased to the almost constant level at the age in 6 days after. At the age in 6-7 days, the concentration mean values of infants, born in 38-40 gestational weeks were lower than that of 37 and 41 weeks one. It was assumed by mathematically that these equilibrium constant level of the lactate concentration in early neonatal period was arrived at 6.20 days and the equilibrium level was estimated to 1.29 mmol/l. As the results, the normal ranges were calculated to the value of 0.49-3.95 mmol/l as initial (age in 0-3 days) and that of 0.29-2.92 mmol/l as final (age in 6-7 days) period, by the truncation method, respectively.

Plantlets were regenerated from 5-year subcultured compact callus derived from petiole tissues of wild viola (Viola patrinii DC.) but not from 5-year subcultured friable callus. Regeneration occurred most efficiently on medium that contained two-fold diluted basal salts of Murashige and Skoog's (MS) medium, 5 × 10(-6) M 1-naphthaleneacetic acid and 10(-6) M kinetin. The effect of dilution of MS basal salts could also be achieved solely by two-fold dilution of the potassium dihydrogen phosphate in the mixture.The present study revealed that dilution of MS basal salts, in particular of potassium dihydrogen phosphate, was important for the regeneration of wild viola. Moreover, although the callus had been subcultured for 5 years, regeneration of plantlets from callus was still possible. In addition, scanning electron microscopy revealed that details of the process of plant regeneration from subcultured callus varied with the age and source of callus and differed from that reported in rice.

A new and quantitative liquid culture system has been developed to measure the production of megakaryocytes from megakaryocyte progenitor cells (colony-forming units-megakaryocyte [CFU-MK]). The system uses as a target population a glycoprotein (Gp) IIb/IIIa+ subpopulation of rat bone marrow cells previously demonstrated to be highly enriched for CFU-MK. GpIIb/IIIa+ cells were cultured at 5 x 10(4) cells/mL (10(4) cells/well) with test samples in 96-well tissue culture plates for 4 days at 37 degrees C. During the final 3 hours of incubation, the cells were pulsed with [14C]5-hydroxytryptamine creatinine sulfate (14C-serotonin). After incubation, the plates were washed and the cell pellets were lysed with Triton-X 100. The cell lysate was infiltrated into a commercially available solid scintillator and dried, and radioactivity was measured. In this assay system, rat interleukin-3 (IL-3) was found to be the most potent among known cytokines tested. Murine granulocyte-macrophage colony-stimulating factor (GM-CSF), human erythropoietin (Epo), human IL-6, and murine stem cell factor (SCF) each alone stimulated megakaryocyte growth but were much less active than rat IL-3. Plasma of rats rendered thrombocytopenic by injection of monoclonal antirat platelet GpIIb/IIIa antibody exhibited significant activity, and the active protein fractions partially purified from the plasma showed much higher activity, but normal rat plasma had no effect. This liquid culture system allows the measurement of a large number of test samples--including a wide variety of cytokines and unknown growth factors, alone or in combinations--and provides a simple method for evaluating the early proliferative events involving CFU-MK in the megakaryocyte differentiation pathway.

The characteristics and mechanisms of synchronized firing in developing networks of cultured cortical neurons were studied using multisite recording through planar electrode arrays (PEAs). With maturation of the network (from 3 to 40 d after plating), the frequency and propagation velocity of bursts increased markedly (approximately from 0.01 to 0.5 Hz and from 5 to 100 mm/sec, respectively), and the sensitivity to extracellular magnesium concentration (0-10 mM) decreased. The source of spontaneous bursts, estimated from the relative delay of onset of activity between electrodes, varied randomly with each burst. Physical separation of synchronously bursting networks into several parts using an ultraviolet laser, divided synchronous bursting into different frequencies and phases in each part. Focal stimulation through the PEA was effective at multiple sites in eliciting bursts, which propagated over the network from the site of stimulation. Stimulated bursts exhibited both an absolute refractory period and a relative refractory period, in which partially propagating bursts could be elicited. Periodic electrical stimulation (at 1 to 30 sec intervals) produced slower propagation velocities and smaller numbers of spikes per burst at shorter stimulation intervals. These results suggest that the generation and propagation of spontaneous synchronous bursts in cultured cortical neurons is governed by the level of spontaneous presynaptic firing, by the degree of connectivity of the network, and by a distributed balance between excitation and recovery processes.

A thrombopoietic factor, termed thrombopoietin (TPO), was highly purified directly from the plasma of sublethally irradiated 1,100 rats by measuring the production of megakaryocytes from a highly enriched population of rat megakaryocyte progenitor cells (CFU-MK). The rat plasma TPO is a glycoprotein and strongly hydrophobic. The total activity and purification yields obtained were about 29% and 1.49 x 10(8), respectively. The amino acid sequences of the two peptide fragments prepared from the purified 19 kDa TPO were analyzed, and used for the cloning of rat and human TPO cDNAs. It was found that the 19 kDa TPO was truncated but comprised at least 163 amino acids. The sequence of human TPO cDNA revealed that the TPO was identical to the c-Mpl ligand. Both rat and human TPOs expressed in COS-1 cells exhibited significant activity toward the CFU-MK in vitro, and were active in stimulating platelet production in mice. These results indicate that a thrombopoietic factor originally found in the irradiated rat plasma is a ligand for the rat c-Mpl.

Diffuse axonal injury (DAI) is defined as widespread damage to axons in the white matter of the brain without focal injury such as contusion and acute subdural hematoma. A case of traumatic subdural hematoma from arterio-venous malformation accompanied by DAI is reported. A 58-year-old man was assaulted, and immediately lost consciousness, and remained unconscious during about 44 hours until his death. The autopsy revealed acute subdural hematoma (about 160 g) on left temporal lobe and left cingular, uncal and cerebellar tonsillar herniation, and tear and hemorrhage of the corpus callosum. Under this subdural hematoma, gray-whitish vascular lesion with subarachnoid hemorrhage was found. Histologically, this lesion was diagnosed as the arterio-venous malformation. Neuropathological examination of the corpus callosum, dorsolateral part of midbrain and superior cerebellar peduncle revealed DAI findings, such as swelling and ballooning of the myelin fibers, swelling and waving of axons, and retraction balls. Axon degenerations were also observed immunohistochemically by anti-200 kD neurofilament antibody. From the results, his unconsciousness from the moment of impact might be occurred from not only subdural hematoma but also DAI.

We present findings in the ninth known Japanese family with lecithin:cholesterol acyltransferase (LCAT) deficiency. A 54-year-old man (proband) and his 58-year-old brother presented with corneal opacity. Both subjects showed a marked decrease in serum high density lipoprotein (HDL)-cholesterol and in the cholesteryl ester ratio. Although apo A-I and A-II were low, apo E tended to be high. Serum LCAT activity and mass were not detectable. Urinary examination showed microhematuria or proteinuria. Renal function was normal and no anemia was demonstrated, but blood smears showed poikilocytosis with target cells. The serum LCAT activity of the proband's three sons, obligate heterozygotes of LCAT deficiency, was about one-half the normal level, and HDL-cholesterol and apo A-I levels were low normal.

To investigate the markers useful for evaluating the long-term efficacy of interferon (IFN) therapy, the quantity of hepatitis C virus (HCV) RNA and two anti-HCV antibody titers (anti-N14 and anti-C-100-3 antibody) in 21 chronic hepatitis C patients were determined. In all complete responders, a sustained clearance of the virus and reductions in the anti-HCV antibody titers were observed during and after therapy. In most of the temporary responders, reductions in the HCV RNA levels and in both anti-HCV antibody titers were observed temporarily during the therapy, and relapse followed. In nonresponders, although the HCV RNA levels and anti-N14 antibody titer tended to remain unchanged or increased during and after therapy, the anti-C-100-3 antibody titers showed no tendency. These results demonstrate that the monitoring of the HCV RNA level and anti-N14 antibody titer is clinically useful for following the patient's response to IFN therapy for chronic hepatitis C.

Background: Our previous observation revealed that probucol, a well known cholesterol-lowering drug, can improve impaired catabolism of plasma triglyceride in streptozotocin-diabetic rats without reducing plasma glucose. The present study was conducted using Triton WR1339 and endogenously radiolabeled lipoproteins in order to examine the mechanisms whereby the drug can stimulate triglyceride removal from the circulation in rats independent of glucose metabolism., Methods: The short- and long-term effects of probucol on triglyceride turnover were examined in rats. Male Wistar rats received a diet containing probucol (1%) for 2 weeks and 4 months. Triglyceride turnover was estimated in rats using Triton WR1339., Results: After 2 weeks of probucol administration, triglyceride and cholesterol concentrations in plasma and in the very-low-density lipoprotein (VLDL) fraction were suppressed slightly but significantly in the probucol-administered rats compared with standard chow-fed control rats. However, triglyceride secretion rates were similar in the two groups. Probucol-administered rats showed markedly suppressed triglyceride concentration in plasma and the VLDL fraction compared with chow-fed control rats after 4 months of administration. However, triglyceride secretion rate was significantly increased in rats given probucol. The radioisotope experiment revealed that probucol-administered rats can remove VLDLs (lipoproteins either from probucol-administered or control rats) from their system faster than normal rats and that probucol-containing VLDL can be removed from the circulation of both groups of rats more rapidly than normal VLDL. Therefore, probucol-administered rats as well as probucol-containing VLDL (lipoprotein from probucol-administered rats) are responsible for accelerated catabolism of VLDL-triglyceride., Conclusion: No significant differences in the lipid composition of newly-secreted VLDL particles between control rats and those given probucol were noted after Triton injection. The precise mechanism behind increased triglyceride secretion rate in rats after long-term administration of probucol is not known. However, we concluded from the present data that long-term administration of probucol results in markedly accelerated VLDL-triglyceride turnover without affecting lipid composition of newly secreted VLDL particles in normal rats.

Diabetes mellitus is an important risk factor for coronary heart disease and cerebral vascular disease. Moreover, diabetic patients are frequently hyperlipidemic (our recent study has revealed that approximately 70% of diabetic patients are dyslipidemic) and are at a higher risk when complicated with hyperlipidemia. In addition, abnormalities in lipoprotein metabolism, which may promote arteriosclerosis, are seen even in normolipidemic diabetics. One such alteration is an increased cholesterol level in intermediated-density lipoproteins from normolipidemic diabetic patients. Recently, abnormal particles in low-density lipoprotein (LDL) fraction, glycated-LDL and small, dense LDL, are regarded as atherogenic lipoproteins. Several factors promoting arteriosclerosis in diabetics, such as abnormal lipoprotein metabolism, increase of lipoprotein (a), hyperinsulinemia, enhancement of polyol pathway, dysfunction of platelet, are described here.

This study was conducted to determine whether overproduction of triglyceride-rich lipoprotein is an obligatory factor for experimental hypertriglyceridemia in nephrotic rats. Nephrosis was induced in male Wistar rats by administration of 150 mg/kg puromycin aminonucleoside. Nephrotic rats had slightly increased triglyceride secretion rate (TGSR) estimated using Triton WR1339 (0.53 +/- 0.05 vs. 0.45 +/- 0.04 mg/min, P < 0.05 vs. control rats) and marked hypertriglyceridemia (330.4 +/- 78.6 mg/dl). Rats made diabetic by 40 mg/kg streptozotocin were normotriglyceridemic (66.3 +/- 12.1 mg/dl) but had suppressed TGSR (0.33 +/- 0.09 mg/min). Experimental nephrosis was induced in diabetic rats. Their TGSR remained suppressed (0.35 +/- 0.06 mg/min) but they had marked hypertriglyceridemia (296.6 +/- 72.4 mg/dl) suggesting further impairment of triglyceride removal from the circulation in diabetic rats caused by nephrosis. Endogenously radiolabeled very low density lipoprotein (VLDL)-triglyceride from donor rats was reinjected into normal recipient rats. [3H]VLDL from the experimental groups (the rats with nephrosis, diabetes with nephrosis, and diabetes alone) were more slowly cleared by normal rats than VLDL from normal rats. These results suggest that circulating insulin is essential for increased triglyceride secretion in experimental nephrosis and that nephrotic hypertriglyceridemia can be induced only by a triglyceride removal defect. Therefore, hypersecretion of triglyceride-rich lipoprotein is not an obligatory factor for nephrotic hypertriglyceridemia.

The authors isolated a specific cDNA clone (clone 14) for non-A, non-B hepatitis virus infection. In this study, we developed an enzyme-linked immunosorbent assay (ELISA) using a synthetic oligopeptide encoded by clone 14 and examined its usefulness for detecting hepatitis C virus (HCV) antibody in 181 patients with chronic NANB hepatitis, 88 with cirrhosis and 24 with hepatocellular carcinoma associated with NANB hepatitis virus. Anti-clone 14 antibody was detected in 75% of patients with chronic NANB hepatitis, 57% of cirrhotic patients and 58% hepatocellular carcinoma patients. Anticlone 14 and anti-C-100 antibody assayed using a commercial kit were found in serum from 199 (69%) and 205 (70%) of these 294 patients, respectively. Approximately 85% of the patients showed the presence of anticlone 14 and/or anti-C-100 antibodies. We compared the presence of these antibodies and the second generation anti-HCV antibody using ELISA and HCV RNA by the polymerase chain reaction assay, in the same blood samples from 49 patients with chronic liver disease who had anti-clone 14 and/or anti-C-100 antibody. HCV RNA was detected in 38 of 40 (95%) plasma samples containing anti-clone 14 antibody, the prevalence of which was similar to that for anti-C-100 antibody (41/42, 98%) and the second generation anti-HCV antibody (46/47, 98%). Furthermore, 6 of 7 plasma samples containing anti-clone 14 antibody and lacking anti-C-100 antibody were positive for the second generation anti-HCV antibody and HCV RNA.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of simvastatin (MK-733, Banyu, Tokyo, Japan), a new inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase on triglyceride kinetics, was studied in chronically streptozocin-diabetic rats. Diabetic rats, in whom diabetes was induced by a single intravenous injection of streptozotocin (40 mg/kg body weight) 2 months before the experiment, had significantly higher plasma glucose, triglyceride, and cholesterol levels compared with nondiabetic control rats, but triglyceride secretion rate was not increased. Both triglyceride secretion rate and plasma triglyceride level were significantly suppressed under nonfasting conditions in diabetic rats fed a (0.1%) simvastatin-containing diet for 10 days. After an overnight fast, their triglyceride secretion rate was not suppressed by this diet. However, their plasma triglyceride level was significantly (50%) suppressed, suggesting improved triglyceride removal from the circulation. In diabetic rats, the newly secreted triglyceride-rich lipoprotein particles were significantly cholesterol-enriched, but simvastatin had no effect on their lipid composition. These results suggest that the hypertriglyceridemia seen in chronically diabetic rats is mainly due to a triglyceride-removal defect, and that simvastatin reduces plasma triglyceride levels in these rats both by stimulating triglyceride removal and by reducing its entry into the circulation.

The present study was conducted to see the effect of probucol on streptozotocin diabetes in rats. After 2 weeks of a 1% probucol diet, 35 or 50 mg/kg of streptozotocin were intravenously injected into male Wistar rats. All the rats became diabetic 2 days after treatment. Thereafter, in order to see the effect of probucol on spontaneous recovery from streptozotocin diabetes, 25 mg/kg of streptozotocin was injected into rats after two weeks of probucol diet and the diet was continued for additional two weeks. All the rats with a standard diet (group CS, n = 13) and 12 of 13 rats with probucol diet (group PS) became diabetic 2 days after streptozotocin injection. One rate from group PS did not develop diabetes. Two weeks after injection, only 4 of 13 rats in groups CS showed recovery, while 11 of 12 rats in group PS showed recovery from streptozotocin diabetes (p less than 0.05). The average blood glucose levels in group PS were significantly lower than group CS (10.5 +/- 4.6 vs 18.5 +/- 0.6 mM, p less than 0.05). In addition, the pancreatic insulin content of group PS was 8 times greater than that of group CS (0.75 +/- 0.24 vs 0.09 +/- 0.03 mmol/pancreas, p less than 0.01). Thus, the in vivo diabetogenic action of streptozotocin could not be reduced by pretreatment with probucol. However, recovery from streptozotocin diabetes was induced by subsequent treatment with probucol. The precise mechanisms for this phenomenon were not known; but the present findings suggest the protective effect of probucol on beta-cell damage induced by small dose of streptozotocin.

Very-low-density lipoprotein (VLDL) triglyceride turnover was examined in mildly streptozotocin (25 mg/kg)-diabetic rats, using Triton WR1339. Diabetic rats fed standard rat chow showed mild hyperglycemia and suppressed levels of plasma insulin. Their triglyceride secretion was significantly suppressed despite an elevated level of plasma free fatty acids. However, the plasma triglyceride level of these diabetic rats was significantly elevated compared with nondiabetic controls. This suggested that the removal of triglyceride from the circulation, as well as its entry into the circulation, was impaired in mildly insulin-deficient rats. Glucose or fructose supplementation (10% in drinking water for 14 days) significantly increased the triglyceride secretion rate of diabetic rats. Especially, fructose supplementation increased plasma insulin to normal levels, but resulted in markedly elevated plasma triglyceride levels (three times higher than glucose-supplemented or chow-fed diabetic rats) despite similar triglyceride secretion rates between the two types of sugar-supplemented diabetic rat groups. This suggested an impairment of triglyceride removal by dietary fructose. The result obtained from chow-fed diabetic rats indicates that mild but significant insulin deficiency resulted in mild hypertriglyceridemia, linked to impaired triglyceride removal rather than to an overproduction of VLDL-triglyceride, despite elevated levels of plasma free fatty acids. Furthermore, fructose feeding induced prominent hypertriglyceridemia not only by stimulating triglyceride secretion, but also by suppressing triglyceride removal from the circulation of mildly diabetic rats.

The effect of long-term (4 months) insulin deficiency on triglyceride turnover was examined using Triton WR1339 in rats. Triglyceride secretion rate was estimated in rats 2 weeks and 4 months after induction of diabetes with 40 mg/kg of streptozotocin. By the second week diabetic rats showed prominent hyperglycemia and the plasma insulin level was very low. In spite of a lower triglyceride secretion rate compared to non-diabetic control rats, diabetic rats showed normotriglyceridemia. Thus, the estimated fractional catabolic rate for plasma triglyceride was decreased in the diabetic rats of 2 weeks duration. By the fourth month diabetic rats still showed a suppressed triglyceride secretion rate but plasma triglyceride was markedly higher than in the non-diabetic control rats. Therefore, their estimated fractional catabolic rate for plasma triglyceride was severely suppressed. They also showed hyperglycemia and hypercholesterolemia. The triglyceride-rich lipoprotein particles obtained after Triton injection in long-term diabetic rats were significantly cholesterol-enriched and triglyceride-depleted compared to control rats. These changes were already present in 2-week diabetic rats but the magnitude was significantly smaller that those in long-term diabetic rats. All of these abnormalities (including triglyceride turnover and the particle composition) were almost normalized by 2 weeks of insulin treatment (6 units/day). Thus, it was concluded from the present data that duration of insulin deficiency is an important determinant of triglyceride removal rate from the circulation in rats. Further modification of lipid composition of triglyceride-rich lipoprotein particles by long-term insulin-deficiency could be one of the reasons for this removal defect.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of alminoprofen (AP), a non-steroidal anti-inflammatory agent, were investigated using several experimental gouty models. AP (3-30 mg/kg, p.o.) dose-dependently inhibited urate crystal-induced rat paw edema. AP (3-30 mg/kg, p.o.) inhibited the accumulation of exudate and decreased the total counts of leukocytes and the amount of PGE2 in a dose-dependent manner in sodium urate crystal-induced pleuritic rats. AP (0.3-10 mg/kg, p.o.) showed a dose-related analgesic activity on the pain response in sodium urate crystal-induced arthritic rats. AP (10(-5)-10(-3)M) inhibited the sodium urate crystal-induced beta-glucuronidase release from guinea pig neutrophils at more than 10(-4) M. AP (10(-5)-10(-3)M) did not inhibit the sodium urate crystal-induced production of O2- from guinea-pig neutrophils. AP (10(-6)-10(-4) M) inhibited dose-dependently the chemotaxis of leukocytes induced by chemotactic factors from guinea pig neutrophils stimulated with sodium urate crystals. AP (10(-6)-10(-4) M) inhibited the sodium urate crystal-induced production of PGE2 from rat peritoneal leukocytes in a dose-related manner. These results suggest that AP has a potent anti-inflammatory and analgesic activity in sodium urate crystal-induced inflammations, and these effects are exerted through its combined inhibitions of PGE2 synthesis, leukocyte chemotaxis and lysosomal enzyme release.

The anti-pyretic activity of alminoprofen (AP), a non-steroidal anti-inflammatory agent, and its mode of action were investigated in conscious febrile rabbits. A fever was evoked by i.v. injection of lipopolysaccharide (LPS), intracisternal (i.c.) injection of leukocytic pyrogen (LP) or i.c. injection of arachidonic acid (AA). The amount of PGE2 or AP in the cerebrospinal fluid (CSF) after i.v. LPS was estimated using an RIA or HPLC method. AP (3-30 mg/kg, p.o.) dose-dependently inhibited the LPS (0.5 micrograms/kg, i.v.)-induced fever; AP, ibuprofen, indomethacin and pranoprofen had ED50 values of 9.64, 26.45, 4.41 and 11.91 mg/kg, p.o., respectively. PGE2 in the CSF was markedly increased during the elevation of body temperature after i.v. LPS (0.5 microgram/kg). AP (30 mg/kg, p.o.) markedly inhibited the increase in PGE2 that was observed in the CSF during fever developed in response to i.v. LPS (0.5 micrograms/kg). The AP concentration in the CSF 2 hr after AP (30 mg/kg, p.o.) was 2.86 x 10(-6) (1.15-4.57 x 10(-6) M, a concentration too low to inhibit PG synthesis. A dose-dependent fever was observed after i.c. LP (1-8 unit) or AA (10-100 micrograms). AP (30 mg/kg, p.o.) shifted the dose-response curves for the i.c. LP-induced fever to the right, but did not have any effect on the i.c. AA-induced fever. These results suggest that AP has a relatively potent anti-pyretic activity, and its mechanism of action involves competition with LP at a site in the CNS, but does not involve an inhibition of cyclooxygenase at a central site, which has been considered as an anti-pyretic mechanism of nonsteroidal anti-inflammatory drugs.

The plasma enzyme, human lecithin-cholesterol acyltransferase (LCAT) is responsible for the majority of cholesterol ester formation in human plasma and is a key enzyme of the reverse transport of cholesterol from peripheral tissue to the liver. We sequenced genomic DNA of the LCAT gene from a Japanese male patient who was clinically and biochemically diagnosed as a familial LCAT deficiency. Analysis of all exons and exon-intron boundaries revealed only a single G to A transition within the sixth exon of both allele of the gene, leading to the substitution of methionine for isoleucinle at residue 293 of the mature enzyme. This mutation creates a new hexanucleotide recognition site for the restriction endonuclease Ndel. Familial study of Ndel digestion of the genomic DNA and determination of plasma LCAT activity established that the patient and his sister whose plasma LCAT activity were extremely reduced were homozygous and his children whose plasma LCAT activity were about half of normal controls were heterozygous for this mutation.

The long-term effect of probucol on triglyceride turnover was examined in streptozotocin (40 mg/kg) diabetic rats. Two diabetic groups were prepared: one group received a probucol-containing (1%) diet (probucol-treated diabetic) and the other standard diet (diabetic control). After 4 months of probucol diet, triglyceride turnover was estimated using Triton WR1339. In diabetic control rats, glucose, triglyceride and cholesterol concentrations in plasma and in the very low density lipoprotein (VLDL) fraction were markedly elevated and plasma insulin was suppressed compared to non-diabetic control rats. There was no significant difference in body weight, plasma glucose and insulin between the 2 diabetic groups. However, the probucol-treated diabetic group showed significantly suppressed levels of triglyceride and cholesterol in total plasma and in the VLDL fraction compared to each corresponding diabetic control value. On the other hand, there were no significant differences in triglyceride secretion rate between the 2 diabetic groups. Newly secreted VLDL particles after Triton injection from diabetic control rats were significantly cholesterol-enriched and triglyceride-depleted compared to those from non-diabetic control rats. However, the composition of those from probucol-treated diabetic rats was similar to that of non-diabetic control group. Prominent hypertriglyceridemia without increase in triglyceride secretion rate in diabetic control group indicates triglyceride removal defect in diabetic rats. Significant suppression of plasma triglyceride level without changes in the triglyceride secretion rate in the probucol-treated diabetic group suggests that probucol stimulated triglyceride removal in diabetic rats. Thus, probucol might normalize VLDL composition, thereby contributing to accelerated triglyceride removal from the circulation of streptozotocin diabetic rats without affecting glucose metabolism.