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The chemical constituents from Cornus officinalis were isolated and purified by various techniques such as macroporous adsorption resin, silica gel, octadecylsilyl(ODS), Sephadex LH-20 column chromatography and preparative high-performance liquid chromatography(HPLC). The structures of the isolates were determined by a combination of spectroscopic techniques such as high-resolution electrospray ionization mass spectrometry(HR-ESI-MS), one-dimensional(1D) and two-dimensional(2D) nuclear magnetic resonance(NMR) spectroscopy. Ten compounds were isolated from the aqueous extract of C. officinalis and identified as(±)-cornuscone(1),(-)-(Z)-4-hydroxy-3-methoxyphenylpropene 4-O-β-L-xylopyranosyl-(1→6)-β-D-glucopyranoside(2), kaempferol 3-O-β-D-glucopyranoside(3), kampferol(4), myricetin(5), trifolin(6), quercetin 3-O-β-D-glucopyranoside(7), quercetin 3-O-β-D-glucuronide-6″-methyl ester(8), quercetin 3-O-β-D-glucuronide-6″-ethyl ester(9) and pyrogallol(10). Compound 1 is a new secoiridoid, named(±)-cornuscone with a rare methyl substitution at the C-1 position. The anti-inflammatory activity of 1 was evaluated in lipopolysaccharide(LPS)-induced RAW264.7 cells in mice. The results showed the median inhibition concentration(IC_(50)) of 1 was(31.15±1.29)μmol·L~(-1), which demonstrated that the anti-inflammatory activity of 1 was significantly superior to that of indomethacin [IC_(50) value of(48.32±1.66)μmol·L~(-1)].

Grazing and climate change both contribute to diversity loss and productivity fluctuations. Sensitive climate conditions and long-term grazing activities have a profound influence on community change, particularly in high-altitude mountain grassland ecosystems. However, knowledge about the role of long-term continuous grazing management on diversity, productivity and the regulation mechanisms in fragile grassland ecosystems is still rudimentary. We conducted a long-term grazing experiment on an alpine typical steppe in the Qilian Mountains to assess effects of grazing intensity on soil, diversity, productivity and the regulation mechanisms. Plants and soil were sampled along grazing gradients at different distances from the pasture entrance (0, 0.3, 0.6, 0.9, 1.2 and 1.5 km) under the non-growing (WP) and the growing season grazing pasture (SAP). The results revealed that community diversity and biomass did not change significantly on a time scale, while the concentration of soil organic carbon and total phosphorus increased significantly. Heavy grazing (0-0.3 km) decreased community diversity and biomass. Grazing increased soil chemical properties in heavy grazed areas of WP, while the opposite was recorded in SAP. Soil chemical properties explained the largest variances in community diversity and community biomass. The prediction model indicates that grazing in WP mainly affects community diversity through soil chemical properties, and promotes a positive correlation between community diversity and community biomass; in SAP, the direct effect of grazing gradients on community diversity and biomass is the main pathway, but not eliminating the single positive relationship between diversity and biomass, which means that diversity can still be used as a potential resource to promote productivity improvement. Therefore, we should focus on the regulation of soil chemical properties in WP, such as the health and quality of soil, strengthening its ability to store water, sequester carbon and increase nutrients; focus on the management of livestock in SAP, including providing fertilizer and sowing to increase diversity and production in heavily grazed regions and reducing grazing pressure through regional rotational grazing. Ultimately, we call for strengthening the stability and sustainability of ecosystems through targeted and active human intervention in ecologically sensitive areas to cope with future grazing pressures and climate disturbances., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Hypothesis: In situ grown layered double hydroxide (LDH) is commonly used one of the anticorrosion ways for metal materials; Due to the dense growth of LDH on the metal surface, its special layered structure can effectively delay the corrosion rate of metal., Methods: In this study, we use a hydrothermal method to successfully grow Mg-Fe LDH film on steel substrates based on self-supplied Fe 3+ ions. The films were characterized by X-ray diffraction, scanning electron microscopy, and X-ray energy dispersive spectrometry. The potential corrosion resistance of the obtained Mg-Fe LDH film was confirmed using electrochemical impedance spectroscopy and polarization curves., Findings: After systematic adjustment and parameter optimization, it was found that Mg-Fe LDH film exhibited the best growth morphology and comprehensive performance with an initial pH value of 10, Mg 2+ /urea ratio of 1:4 and reaction time of 12 h. The SEM and electrochemical results further demonstrated that Mg-Fe LDH film play a good protection effect on carbon steel surface. This study provides an important reference for the processing of anticorrosion LDHs film., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Cornusdiridoid A-F (1-6), six unusual cornuside-morroniside secoiridoid dimers, and their possible new biogenetic precursor, 3″,5″-dehydroxycornuside (7), together with four known secoiridoids (8-11), were obtained from the fruits of Cornus officinalis. Their structures were elucidated on the basis of various spectroscopic and chemical methods. A plausible biosynthetic pathway of compounds 1-11 was proposed. The α-glucosidase inhibitory, antioxidant and anti-inflammatory activities of these isolates were evaluated. Some of them emerged out as potent antidiabetic, anti-inflammatory and free radical scavenging agents. Molecular docking was also carried out for antidiabetic target α-glucosidase to investigate the possible binding modes of the most potent α-glucosidase inhibitor, vincosamide (9). These results revealed that the secoiridoids from C. officinalis fruits may be served as new potential antidiabetic agents to prevent and treat type 2 diabetes., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Ethnopharmacological Relevance: Euphorbia fischeriana Steud. (Euphorbiaceae) is a perennial herb distributed in grassland, hill slopes or gravel hillside, with average altitude of 100-600 m. The whole grass of E. fischeriana is toxic with roots used as folk medicine to treat Zhushui, dyspepsia, abdominal distension, abdominal pain, cough, as well as external applications such as cure of scabies and tuberculosis of lymph nodes., Aim of the Review: This systematic review aims to provide a detailed and in-depth summary about the reported advances in traditional uses, clinical applications, phytochemistry, pharmacology and toxicity of E. fischeriana, so as to offer fresh ideas and broader vision and insights for subsequent studies., Materials and Methods: Various scientific data bases such as CNKI, Elsevier, Google Scholar, Pubmed, Science Direct, SciFinder Scholar and Web of Science were searched to collect information about E. fischeriana. Other relevant literatures were searched in 'Flora of China Editorial Committee', ancient books, Ph.D and Masters' Dissertation to get more data of E. fischeriana., Results: A total of 241 chemical constituents have been identified from the roots of E. fischeriana, including diterpenoids, triterpenoids, meroterpenoids, acetophenones, flavonoids, coumarins, steroids, phenolic acids, tannins, etc. Various pharmacological activities have been demonstrated, especially anti-tumor, antibacterial, anti-inflammatory, antiviral and anti-leukemia activities. Moreover, different investigations about clinical uses and toxicology of E. fischeriana indicated that attention should be paid to its usage and dosage., Conclusion: The researches of E. fischeriana are excellent, but gap still remains. As a poisonous traditional Chinese medicine, there are not enough studies on the toxicity of E. fischeriana. In addition, scholars' research on the pharmacological mechanism of E. fischeriana focuses more on the anti-tumor activity, which can be broadened in the future. Presumably, chemical constituents and biological activities of diterpenoids and trace meroterpenoids in E. fischeriana deserve further research in-depth in the future, in order to provide low toxicity and high efficiency lead compounds. Meanwhile, further studies on other medicinal aspects may lay a foundation for the comprehensive development and utilization of E. fischeriana., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Phenylketonuria (PKU) is an autosomal recessive genetic disease, caused by the phenylalanine hydroxylase (PAH) deficiency in the metabolic pathway, which prevents phenylalanine from being converted into tyrosine, leading to a large amount of phenylalanine discharged from the urine. Therefore, it is necessary to establish a simple, fast, accurate and reliable PKU molecular diagnostic method for clinical diagnosis., Methods: We established a novel diagnostic method by combining a single-tube multiplex PCR technique with molecular hybridization technique. The method was verified by DNA sequencing technology. The established new technology successfully detected 9 common PAH gene mutations in the Chinese population., Results: Double-blind analysis indicated that the diagnostic accuracy and specificity of the PKU sample were all 100%. Frequencies of single mutation R111X, R176X, Ex6-96A, R241C, R243Q, R252Q, Y356X, V399 V and R413P genotypes were 8, 0.5, 16.5, 1.5, 27, 4.5, 13, 10.5, 8.5% respectively., Conclusions: The established method of combing single-tube multiplex PCR with molecular hybridization technology can accurately and rapidly detect PAH gene mutations in Chinese and is suitable for screening of large PKU populations with clinical samples.

Fatty liver is a major lipid metabolic disease in perinatal dairy cows and is characterized by high blood levels of non-esterified fatty acid (NEFA) and insulin resistance. Berberine (BBR) has been reported to improve insulin sensitivity in mice with hepatic steatosis. Mitochondrial dysfunction is considered a causal factor that induces insulin resistance. This study investigates the underlying mechanism and the beneficial effects of BBR on mitochondrial and insulin signaling in bovine hepatocytes. Revised quantitative insulin sensitivity check index (RQUICKI) of cows with fatty liver was significantly lower than that of healthy cows. Importantly, the Akt and GSK3β phosphorylation levels, protein levels of PGC-1α and four of the five representative subunits of oxidative phosphorylation (OXPHOS) were significantly decreased in cows with fatty liver using Western Blot analysis. In bovine hepatocytes, 1.2 mmol/L NEFA reduced insulin signaling and mitochondrial respiratory chain function, and 10 and 20 umol/L BBR restored these changes. Furthermore, activation of PGC-1α played the same beneficial effects of BBR on hepatocytes treated with NEFA. BBR treatment improves NEFA-impaired mitochondrial respiratory chain function and insulin signaling by increasing PGC-1α expression in hepatocytes, which provides a potential new strategy for the prevention and treatment of fatty liver in dairy cows.

Silk glands are specialized in the synthesis of several secretory proteins. Expression of genes encoding the silk proteins in Bombyx mori silk glands with strict territorial and developmental specificities is regulated by many transcription factors. In this study, we have characterized B. mori sage, which is closely related to sage in the fruitfly Drosophila melanogaster. It is termed Bmsage; it encodes transcription factor Bmsage, which belongs to the Mesp subfamily, containing a basic helix-loop-helix motif. Bmsage transcripts were detected specifically in the silk glands of B. mori larvae through RT-PCR analysis. Immunoblotting analysis confirmed the Bmsage protein existed exclusively in B. mori middle and posterior silk gland cells. Bmsage has a low level of expression in the 4th instar molting stages, which increases gradually in the 5th instar feeding stages and then declines from the wandering to the pupation stages. Quantitative PCR analysis suggested the expression level of Bmsage in a high silk strain was higher compared to a lower silk strain on day 3 of the larval 5th instar. Furthermore, far western blotting and co-immunoprecipitation assays showed the Bmsage protein interacted with the fork head transcription factor silk gland factor 1 (SGF1). An electrophoretic mobility shift assay showed the complex of Bmsage and SGF1 proteins bound to the A and B elements in the promoter of fibroin H-chain gene(fib-H), respectively. Luciferase reporter gene assays confirmed the complex of Bmsage and SGF1 proteins increased the expression of fib-H. Together, these results suggest Bmsage is involved in the regulation of the expression of fib-H by being together with SGF1 in B. mori PSG cells.

A large and sudden increase in radiocarbon ((14)C) around AD 773 are documented in coral skeletons from the South China Sea. The (14)C increased by ~ 15‰ during winter, and remain elevated for more than 4 months, then increased and dropped down within two months, forming a spike of 45‰ high in late spring, followed by two smaller spikes. The (14)C anomalies coincide with an historic comet collision with the Earth's atmosphere on 17 January AD 773. Comas are known to have percent-levels of nitrogen by weight, and are exposed to cosmic radiation in space. Hence they may be expected to contain highly elevated (14)C/(12)C ratios, as compared to the Earth's atmosphere. The significant input of (14)C by comets may have contributed to the fluctuation of (14)C in the atmosphere throughout the Earth's history, which should be considered carefully to better constrain the cosmic ray fluctuation.

We have designed and built a microfluidic liquid cell capable of high-resolution atomic force microscope (AFM) imaging and force spectroscopy. The liquid cell was assembled from three molded poly(dimethylsiloxane) (PDMS) pieces and integrated with commercially purchased probes. The AFM probe was embedded within the assembly such that the cantilever and tip protrude into the microfluidic channel. This channel is defined by the PDMS assembly on the top, a PDMS gasket on all four sides, and the sample substrate on the bottom, forming a liquid-tight seal. Our design features a low volume fluidic channel on the order of 50 nl, which is a reduction of over 3-5 orders of magnitude compared to several commercial liquid cells. This device facilitates testing at high shear rates and laminar flow conditions coupled with full AFM functionality in microfluidic aqueous environments, including execution of both force displacement curves and high resolution imaging.

Accumulated evidence suggests that social support is influenced by genetic and environmental factors. However, there are little data that examine this issue from Asian samples. We reported results from a preliminary study that examined familial effects on social support in a Chinese adult twin sample. We administered a 10-item social support instrument that measures three dimensions of social support (i.e., objective support, subjective support, and utilization of support) developed for the Chinese population. Two hundred forty-two same-sex twin pairs, where both members of the pair completed the personal interview, were included in the final analysis. Structural equation modeling was used to estimate additive genetic (A), shared environmental (C), and nonshared environmental (E) effects on each dimension of social support. Familial factors (A+C) explained 56.63% [95% CI = 45.48-65.72%] and 42.42% [95% CI = 29.93-53.25%] of the total phenotypic variances of subjective support and utilization of support, respectively. For the objective support, genetic effects did not exist, but common environmental effect explained 37.56% [95% CI = 26.17-48.28%] of the total phenotypic variances. Neither gender nor age effects were seen on any dimension of social support. Except for objective support, genetic factors probably influence variation in subjective support and utilization of support. Shared environmental factors may influence all dimensions of social support.

Objective: To investigate the feasibility of multi-modalities in the reconstruction of circumferential defects after resection of cancers in pharyngoesophageal regions, and to compare the pros and cons between different surgical procedures., Methods: According to the nature and extend of defects, five different methods including pectoralis major myocutaneous flap, laryngeal tube replacement, free jejunum, free forearm flap and gastric pull-up were used to reconstruct the circumferential pharyngoesophageal defects in 72 patients. Function of deglutition and restoration of swallowing was regularly followed up and objectively evaluated., Results: A total of 35 cases of pectoralis major myocutaneous (PM) flaps, 8 cases of laryngeal tube replacement, 12 cases of free jejunum, 12 cases of free forearm flaps and 16 cases of gastric pull-up were performed. Different complications including wound infection, pharyngeal fistula, partial necrosis of PM flap, partial necrosis of gastric wall, stricture of anastomotic site were encountered in 15 cases. All patients survived the operation except one due to partial necrosis of the gastric wall. Two of 4 patients who developed anastomotic stricture can ingest half-liquid food, the remaining cases regained normal deglutition function. The mean postoperative follow-up time was 1. 6 years with 2-year survival rate of 45.3%., Conclusions: Circumferential defects resulting from resection of carcinomas in pharyngoesophageal region can be reconstructed with different operative techniques depending on the nature and extend of the defects. Once the operative indications are properly selected, the good reconstructive results are to be achieved.

Tissue factor (TF) has been implicated in the pathogenesis of various thrombotic disorders. Monoclonal antibodies (mAb) that specifically target TF may have potential as antithrombotic therapy. We designed a unique TF peptide (TFP) that was specific for the binding site to factor X (FX). This peptide was used to develop TF mAb that block the coagulation cascade by interfering with the combination of FX with the TF/FVIIa complex. Chemically synthesized TFP coupled to polylysine matrix was used as multiple antigenic peptide (TF-MAP) and this was used to immunize Balb/c mice for the preparation of hybridomas. One hybridoma cell line released an antibody, named TF4A12, which had high anticoagulant potency (by dilute prothrombin time assay). Western blotting showed that TF4A12 could bind TF-MAP and the soluble TF extracellular domain (sTF(1-219)). Results of FX activation assay and amidolytic activity assay showed that the anticoagulant ability of TF4A12 is due to blocking FX, but not FVII, binding to TF. Our study identified an efficient method of developing TF mAb that could block the coagulation cascade.

Objective: To clone the human tissue factor pathway inhibitor-2 (hTFPI-2) gene and express it by using prokaryotic expression system., Methods: The hTFPI-2 coding region was obtained by RT-PCR from human placenta total RNA. The coding fragment was then inserted into prokaryotic expression vector pET19b and expressed in E. coli BL21 by IPTG induction. The produced inclusion bodies were dissolved by denaturalizing chemicals, purified by ion exchange chromatograph, and refolded in air to form proper disulfide bonds. Chromogenic and gelatin zymography methods were used to evaluate the inhibiting effects of hTFPI-2 on trypsin, plasmin and MMPs individually. The inhibitory activity of hTFPI-2 on fabrisarcoma was investigated by matrigel., Results: The coding fragment of hTFPI-2 was cloned successfully and the protein was expressed as inclusion bodies which account for 20% - 30% of total host protein. The refolded hTFPI-2 could inhibit the invasive ability of fibrisarcoma HT-1080 as well as activity of plasmin, trypsin and MMPs., Conclusions: The activated hTFPI-2 is obtained by using prokaryotic expressed system effectively.

Photo-Fenton method, the combination of Fenton reagent with light, is an efficient method for waste water treatment. In the present paper, the degradation of great green SF using this method was studied. Great green SF is a kind of permanent and nondegradable dye. Through numerous experiments, the influences of various parameters including the UV absorption curve of great green dye SF, the concentration-absorbency curve of great green dye SF, dosage of Fe2+, dosage of H2O2, initial pH, different light sources, and cation-exchange resin on the degradation were researched intensively. The optimum condition for dye SF degradation was given. Under the experiment condition, the sun light can promote this reaction apparently. The reaction time can greatly be shortened too. After the cation-exchange resin was introduced into the Fenton system, the activation of H2O2 can be enhanced to a great extent. The dosage of H2O2 will be decreased. The degradation effect of the great green SF is better. Under optimal conditions, the overall color removal is more than 96.7% within 40 min; COD can be removed effectively at the same time.

The relationship between efferents of the hypothalamic suprachiasmatic nucleus (SCN) and neurons of the thalamic paraventricular nucleus (PVT) projecting to the amygdala was investigated in the rat using tract tracing in light and electron microscopy. Biotinylated dextran amine was used to label anterogradely SCN efferents. These fibers were found to reach the thalamic midline, terminating in PVT, through three pathways: anterodorsally through the preoptic region, dorsally through the periventricular hypothalamus, and through the contralateral medial hypothalamic and preoptic areas after crossing the midline in the optic chiasm. Preterminal and terminal-like elements labeled from the SCN were distributed throughout the rostrocaudal extent of PVT, with an anteroposterior gradient of density. Labeled terminal elements were densest in the dorsal portion of PVT beneath the ependymal lining and some of them entered the ependyma. Anterograde tracing of SCN fibers was combined with injections of retrograde tracers in the amygdala. Numerous retrogradely labeled cell bodies were seen throughout PVT, with a prevalence in its anterodorsal portion. Overlap was detected between puncta labeled from the SCN and retrogradely labeled neurons, especially in the anterodorsal sector of PVT, where numerous puncta were in close apposition to thalamo-amygdaloid cells. Electron microscopy revealed that boutons labeled from the SCN established synaptic contacts with dendritic profiles of PVT neurons labeled from the amygdala. The findings demonstrate that information processed in the biological clock is conveyed to the amygdala through PVT, indicating that this nucleus plays a role in the transfer of circadian timing information to the limbic system.

It has been repeatedly reported that Fos is spontaneously induced in several brain structures, including the cerebral cortex, during wakefulness. To ascertain whether cortical interneurons are involved in this state-dependent oscillation of gene regulation, we combined Fos immunocytochemistry with immunostaining of either parvalbumin or calbindin, known markers of cortical interneurons. Immunopositive neurons were examined in the sensorimotor and cingulate cortex. In rats perfused in basal conditions, a minor proportion (around 8%) of Fos-immunoreactive neurons in the parietal cortex were also parvalbumin- or calbindin-immunoreactive; these double immunostained cells accounted for 13% of the parvalbumin- and 34% of the calbindin-labeled neurons. Colocalization of Fos with either calcium-binding protein was instead not observed in the cingulate cortex. In rats stimulated by novel environmental cues during the period of wakefulness preceding perfusion, Fos-positive neurons increased markedly relative to unstimulated animals, and involved the majority of the calbindin- or parvalbumin-labeled cell populations (60-75% and over 95%, respectively). In the neuronal populations in which Fos was induced by exposure to the enriched environment, the proportion of calbindin- and parvalbumin-labeled cells was larger than in the unstimulated cases, and the increment was statistically significant in the cingulate cortex. The results demonstrate that Fos induction occurring in the cortex during undisturbed wakefulness in a familiar environment involves a minor proportion of interneurons. Furthermore, the findings indicate that the addition of novel environmental stimuli results in an increase of Fos-expressing neurons whose recruitment, at least in the cingulate cortex, involves a higher proportion of interneurons than of projection neurons.

Expression of inducible nitric oxide synthase (iNOS) protein was studied in the brain after intracerebroventricular injections of interferon (IFN)-gamma, and IFN-gamma combined with lipopolysaccharide (LPS) or tumor necrosis factor (TNF)-alpha, compared to ovalbumin as control. Wild-type mice and mice with targeted deletion of the IFN-gamma receptor gene were used. Findings based on iNOS immunoreactivity were evaluated at 1, 2, 4 and 7 days post-injection, using also quantitative image analysis and double labeling with glial cell markers. IFN-gamma administration induced iNOS immmunostaining in activated microglia and macrophages in the parenchyma surrounding the ventricular system, several cortical fields and fiber tracts. IFN-gamma-elicited iNOS immunoreactivity was down-regulated after 1 day. The number of iNOS-immunopositive cells was significantly enhanced by co-administration of LPS or TNF-alpha; IFN-gamma+TNF-alpha injections also resulted in longer persistence of iNOS immunoreactivity. No immunopositive cells were seen in the brain of IFN-gamma receptor knockout mice after IFN-gamma administration; very few immunostained macrophages were detected in these cases, mostly around the injection needle track, after co-administration of LPS or TNF-alpha. Western blot analysis confirmed a marked iNOS induction in the brain of wild-type mice 24 h after IFN-gamma+LPS injections. The findings show that inflammatory mediators circulating in the cerebrospinal fluid induce in vivo iNOS in the brain with topographical selectivity and temporal regulation. The data also demonstrate that the signaling cascade activated by IFN-gamma binding to its receptor is critical for iNOS induction, and the synergistic action of LPS and TNF-alpha as iNOS inducers in brain cells is largely mediated by the receptor-regulated action of IFN-gamma.

Microglial cells play a key role in the events triggered by infection, injury or degeneration in the central nervous system not only as scavenger cells but also as immune effector elements. We analyzed the features and distribution of cells of the microglia/macrophage lineage with OX-42 and ED-1 immunohistochemistry in the brain of experimental rats infected with the extracellular parasite Trypanosoma brucei. Such experimental infection provides a rat model of sleeping sickness or African trypanosomiasis, and is hallmarked in its advanced stages by severe alterations of the animals' sleep structure. In infected rats a remarkable activation of microglia, revealed by OX-42 immunoreactivity, became evident in the 3rd week post-infection in periventricular and subpial brain regions, with a prevalence in the hypothalamus. These features were concomitant with the onset of sleep anomalies, monitored with electroencephalographic recordings. Microglia activation increased in the following weeks, paralleling the progressive alterations of sleep parameters, and was most marked in the terminal stages of the infection, corresponding to the 6th-7th weeks. In addition, ED-1-immunoreactive macrophages and ramified microglia, confined to hypothalamic periventricular and basal regions, were evident after 4 weeks of disease. Degeneration of neuronal perikarya was not detected histologically in the infected brains at any time point. These data provide evidence for a reaction of microglia and macrophages in the brain of trypanosome-infected rats, and point out a selective distribution of these activated cells. The findings are discussed in relation to the animals' sleep disorder during trypanosome infection., (Copyright 1999 Elsevier Science B.V.)

The response to intracerebroventricular administration of interferon (IFN)-gamma was examined in the adult Wistar rat brain: major histocompatibility complex (MHC) antigens class I and II, CD8 and CD4 antigens, and the macrophage/microglia antigen OX42 were analyzed in respect to saline-injected cases over 1 week. The glial cell type expressing MHC antigens was characterized with double labeling. IFN-gamma was thus found to induce MHC class I and II expression in microglia, identified by tomato lectin histochemistry, and not in GFAP-immunostained astrocytes. MHC antigen-expressing microglia was detected in the periventricular parenchyma, several fields of the cerebral cortex, cerebellum, major fiber tracts, and brainstem superficial parenchyma. Different gradients of density and staining intensity of the MHC-immunopositive elements were observed in these regions, in which MHC class I antigens persisted up to 1 week, when MHC class II induction had declined. Quantitative analysis pointed out the proliferation of OX42-immunoreactive cells in periventricular and basal brain regions. CD8+ T cells were observed in periventricular regions, basal forebrain, and fiber tracts 3 days after IFN-gamma injection and their density markedly increased by 7 days. CD4+ T cells were also seen and they were fewer than CD8+ ones. However, numerous CD4+ microglial cells were observed in periventricular and subpial regions, especially 1 week after IFN-gamma injection. Our data indicate that this proinflammatory cytokine mediates in vivo microglia activation and T cell infiltration in the brain and that the cells involved in this immune response display a regional selectivity and a different temporal regulation of antigen expression., (Copyright 1998 Academic Press.)

The parasites Trypanosoma brucei cause African trypanosomiasis (sleeping sickness), a severe neuropsychiatric disease with marked disturbances of sleep-wake alternation. The sites of brain lesions are not well characterized. The present experimental investigation is focused on the hypothalamic suprachiasmatic nuclei, which play a role of a biological clock entraining endogenous rhythms in the mammalian brain. The electrophysiological properties of these neurons were analyzed in slice preparations from trypanosome-infected rats. The neuronal spontaneous activity, which shows a circadian oscillation, was markedly altered in the infected animals, displaying a reduced firing rate and phase advance of its circadian peak. The direct retinal fibers, which play a pivotal role in entrainment of the circadian pacemaker, displayed a normal density and distribution in the suprachiasmatic nuclei of infected animals after intraocular tracer injections in vivo. At the postsynaptic level, immunohistochemistry and Western blotting revealed in the suprachiasmatic nuclei of infected rats a selective decrease of the expression of glutamate AMPA GluR2/3 and NMDAR1 receptor subunits that gate retinal afferents. These data disclose an impairment of the neuronal functions in the biological clock in African trypanosomiasis, and may serve to unravel functional and molecular mechanisms behind endogenous rhythm disturbances.

Background: Nitric oxide has an important role in the pathophysiology of the gastric mucosa. However, to date, it is not clear if nitric oxide plays a cytoprotective or cytotoxic role in the pathogenesis of mucosal lesion., Methods: We have used the NADPH-diaphorase histochemistry that selectively stains cells containing nitric oxide synthase, the enzyme that catalyzes the production of nitric oxide on the antral gastric mucosa of children with antral gastritis., Results: In the lamina propria of the mucosa, the presence of the enzymatic activity was found in perivascular round cells and nerve fibers. In the epithelium, focal positivity to NADPH-diaphorase was found in superficial cells, mainly located in the extrusive zones. The epithelial cells in the pits and glands were negative, in the mucous layer, Helicobacter pylori were also stained by NADPH-diaphorase. A single H. pylori-infected child who was also examined after eradication of the H. pylori showed during the control examination absence of microorganisms and reduction of the NADPH-diaphorase-positive cells in the mucosa., Conclusions: Our results demonstrate that, in gastric mucosa, endogenous and exogenous structures express a NADPH-diaphorase activity.

The facial nerve was transected in rats at different postnatal ages, from birth to early adulthood. NADPH-diaphorase histochemistry was performed to analyze the induction of nitric oxide synthase, the synthetic enzyme of the free radical nitric oxide, in injured facial motoneurons. In addition, in situ nick-end labeling of DNA fragmentation (TUNEL technique) was performed after axotomy at birth, to verify the occurrence of apoptosis in the damaged facial motoneurons. A striking age-dependency was found in the induction of nitric oxide synthase activity in axotomized facial motoneurons. NADPH-diaphorase positivity was not detectable in these neurons 1 and 2 days after axotomy at birth, when apoptotic changes were evident and marked. In addition, NADPH-diaphorase staining was hardly detectable in the facial nucleus 4 days after axotomies at birth, when extensive motoneuron loss was evident. NADPH-diaphorase positivity was instead induced in the facial motoneurons axotomized from the end of the first postnatal week to adulthood, when the nerve cell loss was less severe than in newborns. However, the time course of the enzyme activity induction varied considerably in relation to the animals' age. These findings are discussed in relation to the role of nitric oxide in motoneuron death or protective response to injury and of oxidative stress in neurodegeneration.

Induction of NADPH-diaphorase (NDP) activity in the rat cerebral cortex was studied after autologous blood injection into the internal capsule as experimental model of intracerebral hemorrhage. The potential inhibitory effect on NDP induction by Nao Yi An (NYA), a complex derived from materials of animal and plant origin used in the treatment of intracerebral hemorrhage in traditional Chinese medicine, was also investigated. In animals without therapeutic treatment 2 and 4 days after injection of autologous blood, NDP activity was highly induced in pyramidal neurons in the neocortex, piriform, and entorhinal cortices, in astrocytes and in phagocytes in the hematoma and the area surrounding it, as well as in the subcortical white matter, and in endothelial cells in both the cortex and subcortical white matter bilaterally. Oral administration of NYA failed to inhibit NDP induction in endothelial cells but demonstrated a strong inhibitory effect on NDP activity induced in pyramidal neurons and astrocytes. NDP induction in phagocytes was also inhibited by the administration of NYA. Altogether the present results suggest that intracerebral hemorrhage in the internal capsule may induce nitric oxide synthase activity in different cell populations in the cortex and that administration of NYA can selectively inhibit such induction and, thus, potentially play a neuroprotective role.

Induction of NADPH-diaphorase (NDP) following ischemic infarction was studied by means of histochemistry in the rat cerebral cortex 1,2,7, and 14 days after distal occlusion of the right middle cerebral artery (MCA). The fine structure of cells in the penumbra region of the necrotic center was also investigated. MCA distal occlusion resulted in ischemic lesion of the frontoparietal cortex of variable extent; NDP induction was detected in neurons, astrocytes, macrophages, and endothelial cells, with regional specificity and a temporal gradient. One, two, and seven days after MCA occlusion, weak NDP positivity was consistently induced in some pyramidal neurons in cortical areas neighboring the necrotic area; NDP induction was also seen in pyramidal neurons of the ipsilateral anterior cingulate and infralimbic cortices and in the tenia tecta. In addition, numerous NDP-positive pyramidal neurons were detected in the contralateral frontoparietal cortex after relatively large ischemic lesions. Two weeks after MCA occlusion, NDP induction in neurons was only evident in the deep cortical layers near the lesion. NDP histochemistry combined with glial fibrillary acidic protein immunofluorescence, performed 7 days after MCA occlusion, indicated that the astrocytes at the periphery of the necrotic area were hypertrophic and some of them were also NDP-positive. One and two days after MCA occlusion, numerous macrophages displaying NDP positivity of variable intensity were seen at the periphery of the necrotic area and in the external capsule of the ischemic cerebral hemisphere. Many endothelial cells in the cortex and subcortical white matter were consistently NDP-positive in all rats. Electron microscopic studies indicated that the area adjacent to the necrotic center was composed of fibrous astrocytes, with the morphological characteristics of proliferation, and numerous lysosome-filled macrophages. Altogether the present results suggest that focal cerebral ischemia may induce in different cell types nitric oxide synthase, which is equivalent to NDP in fixed tissue. The induction of nitric oxide synthase may be related to (1) blood-flow regulation at relatively early postischemic stages, which may decline when collateral circulation is established, and/or (2) cytotoxic or neuroprotective mechanisms.

Neurons that contain nitric oxide synthase (NOS) type I and the calcium binding proteins calbindin D28k or parvalbumin were simultaneously visualized by means of double immunohistofluorescence in the cerebral cortex of Wistar and Sprague-Dawley rats. All the three immunoreactive cell populations were primarily represented by nonpyramidal neurons. NOS-immunoreactive cells were less numerous than the calbindin- or parvalbumin-immunoreactive ones, and were intermingled with the neurons containing these calcium binding proteins. NOS-immunoreactive cells were separate from the parvalbumin-immunoreactive ones, whereas a minor proportion of them was found to be colocalized with calbindin. The cortical neurons in which NOS and calbindin coexisted were more numerous in the Sprague-Dawley than in the Wistar rats, and displayed an anteroposterior gradient of density, with the highest concentration in the medial prefrontal, frontal, and cingulate cortices. Double NOS-calbindin-immunoreactive neurons prevailed in the deep cortical layers and they were relatively numerous in the cingulate cortex. The present data indicate a selectivity in the expression of NOS vs. calbindin and parvalbumin in cortical cells, and further support the chemical heterogeneity of GABAergic interneurons in the cerebral cortex.

The expression of Fos-related protein, encoded by the proto-oncogene c-fos, was investigated by means of immunohistochemistry in the paraventricular nucleus of the thalamic midline (PV) during nighttime and daytime in rats entrained to a 12-h light/12-h dark cycle. In the first step of this study the animal's physiological state preceding perfusion was monitored with electro-encephalographic recording. It was thus detected that the PV contained a considerable number of Fos-like-immunostained neurons during the hours of darkness, when the rats had been awake, and that the number of Fos-like-immunoreactive neurons was significantly lower during the hours of light, after a period of sleep. In the second step of this study Fos immunohistochemistry was combined with the retrograde transport of a gold-labeled tracer injected either in the amygdala or in the nucleus accumbens. This strategy enabled us to determine that in the rats perfused during nighttime Fos-related protein was spontaneously induced in PV cells projecting to these targets, with a significant prevalence of neurons projecting to the amygdala in the anterior portion of the PV and of neurons projecting to the nucleus accumbens in the posterior part of the nucleus. In addition, a significant reduction of Fos-like-immunoreactive cells was detected in the PV ipsilaterally to the injection, indicating that tracer administration and axonal transport may interfere with c-fos expression in neurons. Altogether the present data indicate that Fos-related protein expression undergoes a marked oscillation in the PV during 24 h in basal conditions, and that c-fos is induced in the PV relay neuronal subsets when the animal is awake.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunostaining of Fos, the nuclear protein encoded by the immediate early gene c-fos, is widely used to reveal the functional activation of neurons. The chemical identity of cells that express c-fos can be investigated with double immunohistochemistry. We report the usefulness of a sequential two-color avidin-biotin-immunoperoxidase method that provides a highly sensitive double immunostaining and allows long-term storage of the sections. In this protocol, metal intensification of diaminobenzidine (int-DAB) resulted in dark brown/black Fos immunostaining of the neuronal nucleus. The use of alpha-naphthol/pyronin reaction product yielded pink immunostaining of a second antigen in the cytoplasm. This combination produced higher contrast than that produced by int-DAB Fos immunostaining combined with conventional DAB light brown cytoplasmic staining. The sensitivity of the use of int-DAB and alpha-naphthol/pyronin was verified in different experimental paradigms, combining the immunocytochemical detection of Fos with that of the p75 nerve growth factor receptor, or parvalbumin, or calbindin D28k.

Neuronal interferon-gamma (N-IFN-gamma), recently isolated from the nervous system, has a molecular weight distinct from that of lymphocyte-derived IFN-gamma, but crossreacts immunologically and shares certain bioactivities with this cytokine. In the rat brain N-IFN-gamma-immunoreactive perikarya were concentrated in the hypothalamic tuberomammillary nuclei; some immunostained neurones were also detected in the dorsal pontine tegmentum. Immunopositive nerve fibres were profusely distributed through the periventricular hypothalamus and midline thalamus. Scattered fibres occurred diffusely through the brain, ramified in the subpial layer and also surrounded intrathecal vessels. A dense concentration of puncta was detected in the suprachiasmatic nuclei and in the molecular layer of the hippocampal dentate gyrus. A role of N-IFN-gamma in immunological reactions and in modulation of selective brain functions is suggested.

The mammalian suprachiasmatic nuclei of the hypothalamus (SCN) serve as pacemaker for circadian rhythms and the immediate-early gene c-fos is known to be induced by photic stimulation in the SCN of rodents. We studied the induction of Fos-related protein following a light pulse in rats infected with Trypanosoma brucei. This parasite causes in humans African sleeping sickness, a neuropsychiatric syndrome that involves changes of endogenous biological rhythms. Fos-like immunoreactivity after photic stimulation was dramatically reduced in the SCN of trypanosome- infected rats during the subjective night. These findings indicate that the photic entrainment of the biological clock may be altered during the infection.

Induction of an interferon-gamma-like molecule, previously isolated from neurons (N-IFN-gamma), and of the neuronal isoform I of the synthetic enzyme of the free radical nitric oxide, nitric oxide synthase I, as well as of NADPH-diaphorase, were examined in axotomized dorsal motor vagal and hypoglossal neurons. Unilateral transection of the vagal and hypoglossal nerves was performed in the same rat and an induction of N-IFN-gamma and nitric oxide synthase I immunostaining as well as NADPH-diaphorase histochemical positivity was observed in the ipsilateral motoneurons after 2-4 days. The immuno- and enzyme-histochemical positivities were much stronger in the dorsal motor vagal neurons than in hypoglossal neurons. Two and 4 weeks after axotomy N-IFN-gamma immunoreactivity and NADPH-diaphorase positivity persisted in the former, but started to decrease in the latter neurons. Previous data have shown that 23 weeks after nerve transection the majority of the dorsal motor vagal neurons are lost, while the majority of the hypoglossal neurons survive. The high and persistent expression of N-IFN-gamma and nitric oxide synthase I after axotomy in the dorsal motor vagal neurons, that are largely destined to die, indicates that the co-induction of these two molecules may be implicated in the pathogenesis of neuronal degeneration.

A newly isolated interferon-gamma (IFN-gamma) immunoreactive molecule, "neuronal IFN-gamma", and recombinant lymphocyte-derived IFN-gamma were injected intracerebroventricularly (i.c.v.) through a previously implanted cannula into adult male rats during both the light and the dark phases of the light/dark cycle. The two molecules caused a reduction in both frequency and duration of rearing and locomotion during the dark, but not the light, phase. A molecule isolated from Trypanosoma brucei brucei, a parasite of the same subspecies of trypanosomes which causes African sleeping sickness, can induce production and release of IFN-gamma and "neuronal IFN-gamma" from lymphocytes and neurons, respectively. I.c.v. injection of this factor also reduced rearing during the dark period, but to a less extent. Thus, "neuronal IFN-gamma" appears to have effects on animal behaviour in common with lymphocyte-derived IFN-gamma. This study highlights the potential role of these cytokines in behaviour disturbances.

Rats infected with the parasite Trypanosoma brucei brucei showed selective changes of c-fos expression in the suprachiasmatic nucleus of the hypothalamus (SCN) during spontaneous sleep (S) and wakefulness (W) under a basal 12 h/12 h light-dark (L-D) cycle. In the vast majority of W (D phase) control animals the SCN was devoid of cells displaying Fos-related immunopositivity, while Fos-like-immunoreactive (ir) neurones were detected in most S (L phase) control rats. In most infected animals, on the other hand, Fos-ir neurones were detected in the SCN during W, but not during the S period, with a significant difference between control and infected S rats. Thus, these data indicate that the basal c-fos expression in the SCN during the L-D and S-W cycles is considerably altered in experimental trypanosomiasis. This is the first observation of a selective change in the SCN in trypanosome-infected rat brains. Since the SCN plays an important role as a pace-maker for biological rhythms, this finding may provide a basis for understanding the pathogenesis behind endogenous rhythm dyregulation and changes in sleeping pattern in human trypanosomiasis (African sleeping sickness).

The relationship of neurons that express nitric oxide synthase type I (NOS-I), the synthetic enzyme of the free radical nitric oxide (NO), with cells that contain the low affinity p75 nerve growth factor receptor (NGFr) was examined in the basal forebrain region of the rat using double immunohistofluorescence. NOS and NGFr were found to be co-localized in a neuronal population that displayed a selective distribution. Double immunopositive neurons were evident in the medial septum and in rostral levels of the diagonal band nuclei where the vast majority (more than 80%) of NOS-immunoreactive cells were also NGFr-positive; the two cell populations were separate at more caudal levels of the basal forebrain. These findings support the chemical heterogeneity of septal and basal forebrain neurons which contain NGFr and indicate that in the rat a subset of neurons of the septum-diagonal band complex may release NO.

Intracerebroventricular (icv) injection of propidium iodide (PI) in the rat results in a transient movement disorder characterized by nystagmus, ataxia, and shaking. In the present study we used c-Fos as a marker for neuronal activation to investigate the neural substrate underlying this movement disorder. PI was injected into the lateral cerebral ventricle of freely moving rats through a previously implanted cannula. Animals were perfused 3 h after the injection and the brains were processed for c-Fos immunocytochemistry. Paired control animals were injected with saline. After PI injection, a significant Fos expression was seen in the cerebral cortex, thalamic midline nuclei, thalamic intralaminar nuclei, hypothalamus, central gray, pontine nuclei, locus coeruleus, vestibular complex, inferior olive, ventrolateral medulla, nucleus of solitary tract, and deep cerebellar nuclei. Few or no Fos immunoreactive cells were seen in the above structures of the control animals. The present study indicates that a large number of neurons located in many different neural structures are activated following icv injection of PI. Second, consistent with the cerebellar feature of the movement disorder, a major Fos expression was found in the cerebellar circuitry (deep cerebellar nuclei, pontine nuclei, vestibular complex, and inferior olive). It reinforces further the assumption that the movement disorder is due to cerebellar dysfunction caused by PI.

The rest-activity and body temperature 24 hours cycles, as well as the pattern of spontaneous sleep, were investigated in rats after infection with Trypanosoma brucei brucei. In the infected rats, which were entrained to a 12 hours/12 hours photoperiod, a considerable hypokinesia was detected during the hours of darkness. In most of the infected animals, the body temperature cycle displayed a lower amplitude and an advance of about 3 hours in respect to control rats; in addition, the body temperature rhythm was not significant in some infected rats. The relative proportion of slow wave synchronized sleep, as well as the rapid-eye movement (REM) latency, were significantly reduced in the infected animals, in which sleep was considerably fragmented. The induction of Fos (the protein encoded by the immediate early gene c-fos), in response to light stimulation during the early subjective night, was severely impaired in the hypothalamic suprachiasmatic nuclei in trypanosome-infected rats. Altogether these data point out a disruption of locomotor activity and body temperature 24 hours cycle and a major disorganization of sleep during experimental trypanosomiasis. In addition, our findings indicate that the molecular and functional correlates of the synchronizing action of the suprachiasmatic nuclei, which play a major role of biological clock of endogenous biological rhythms, could be altered during trypanosome infection.

The distribution of Fos, the protein product of the immediate early gene c-fos, was studied with immunocytochemistry in the adult male rat brain after nerve growth factor (NGF) administration. NGF was injected in the lateral cerebral ventricle through a previously implanted cannula. The total number of Fos-immunoreactive (ir) neurons in the brain was 2-3 times higher after NGF administration than in control animals (untreated or injected with cytochrome c). With respect to control rats, in the NGF-treated cases Fos-ir cells were more numerous in the anterior olfactory nucleus, in the medial prefrontal and anterior cingulate cortices, in the basal forebrain, in the preoptic and ventromedial nuclei of the hypothalamus, as well as anterior hypothalamic area, in the thalamic midline nuclei, and in some brainstem structures, such as the parabrachial nucleus. The relative quantitative increase of Fos-ir neurons varied in the different structures. In addition, Fos-ir neurons were evident after NGF administration in areas devoid of immunopositive cells in control animals. These included: frontoparietal and occipital cortical fields, the hypothalamic arcuate nucleus, and many brainstem structures, such as the dorsal nucleus of the lateral lemniscus, posterodorsal tegmental, medial and lateral vestibular, ventral cochlear, and prepositus hypoglossal nuclei. These findings demonstrate that the intracerebroventricular administration of NGF can induce c-fos expression in neurons in vivo. The distribution of Fos-ir neurons indicates that NGF can induce activation of functionally and chemically heterogeneous neuronal subsets in the brain.

Deer tooth samples from Zhoukoudian, the Peking Man Cave, were studied. Non-linear fitting is used to determine AD, from which ESR ages were calculated by both the disequilibria model (DU) and linear uptake model (LU). Comparison of ESR ages with those from U-series, FT and TL methods show that the enamel of deer teeth is a suitable material for ESR dating.

Surgery of the skull base tumor has been undertaken in 19 patients, 15 with anterior skull base tumor and 4 lateral skull base tumor. Among them, 16 cases had malignant tumors and 3 had benign tumors. The dura was exposed in 15 patients and resected in 5 patients due to tumor invasion. The dura and bony defects could be repaired with fascia lata, galea pericranial flaps and scalp flaps. Cautery was useful for treating minimally involved duras. Three out of 16 patients with malignant tumors were free of tumors for 4-14 years and 5 for 1.5-3 years postoperatively; 7 cases died of tumor recurrence.

Crude extracts of both fresh and dry ginger induced the perfused rat hindlimb to consume oxygen in association with increases in perfusion pressure and lactate production. The principles responsible for these observations, the gingerols and shogaols, were isolated and tested for relative thermogenic activity. The gingerol homologues possessed greater molar potency than their shogaol counterparts. (6)-Gingerol was the most potent principle isolated, causing a mean maximal increase in oxygen consumption of 1.4 +/- 0.1 mumol/g/h (21%), an increase in lactate efflux of 4.7 +/- 0.6 mumol/g/h (87%) with a perfusion pressure increase of 7.7 +/- 0.7 mmHg (30%). Increases in alkyl chain length within each homologous series led to decreased molar potency. Specific nitro-vasodilation using glyceryl trinitrate demonstrated that thermogenesis was at least partly associated with vasoconstriction. Concurrent infusion of alpha or beta antagonists showed that neither adrenergic receptors nor secondary catecholamine release were responsible for the observed effects. Increasing doses of the ginger principles ultimately led to inhibition of steady state oxygen consumption, although perfusion pressure continued to increase. Removal of high ginger principle doses was followed by apparent increases in oxygen uptake unaccompanied by elevated perfusion pressure. As a consequence, the effective concentration ranges of the ginger principles were relatively narrow. The cause of high dose effects is as yet undetermined but may have been due in part to disruption of mitochondrial function.

The distribution and morphological characteristics of catecholamine (CA)-containing cell bodies in the diencephalon of 7-9-month-old fetuses were studied by means of the Faglu histofluorescence method. Although the general distribution of CA cell bodies in human diencephalon observed in the present study is similar to that in other mammals, there are some significant differences. A great number of CA neurons lie within the lateroventral hypothalamic zone. There are more numerous larger CA neurons distributed outside the periventricular gray in the human diencephalon.