Dissecting the impact of dietary fiber type on atherosclerosis in mice colonized with different gut microbial communities.

Dietary fiber consumption has been linked with improved cardiometabolic health, however, human studies have reported large interindividual variations in the observed benefits. We tested whether the effects of dietary fiber on atherosclerosis are influenced by the gut microbiome. We colonized germ-free ApoE-/- mice with fecal samples from three human donors (DonA, DonB, and DonC) and fed them diets supplemented with either a mix of 5 fermentable fibers (FF) or non-fermentable cellulose control (CC) diet. We found that DonA-colonized mice had reduced atherosclerosis burden with FF feeding compared to their CC-fed counterparts, whereas the type of fiber did not affect atherosclerosis in mice colonized with microbiota from the other donors. Microbial shifts associated with FF feeding in DonA mice were characterized by higher relative abundances of butyrate-producing taxa, higher butyrate levels, and enrichment of genes involved in synthesis of B vitamins. Our results suggest that atheroprotection in response to FF is not universal and is influenced by the gut microbiome.

Impact of administration route on nanocarrier biodistribution in a murine colitis model.

The incidence of inflammatory bowel disease (IBD) is increasing worldwide. Although current diagnostic and disease monitoring tests for IBD sensitively detect gut inflammation, they lack the molecular and cellular specificity of positron emission tomography (PET). In this proof-of-concept study, we use a radiolabeled macrophage-targeted nanocarrier probe (64Cu-NOTA-D500) administered by oral, enema, and intraperitoneal routes to evaluate the delivery route dependence of biodistribution across healthy and diseased tissues in a murine model of dextran sodium sulfate (DSS)-induced colitis. High inter-subject variability of probe uptake in intestinal tissue was reduced by normalization to uptake in liver or total intestines. Differences in normalized uptake between healthy and DSS colitis animal intestines were highest for oral and IP routes. Differences in absolute liver uptake reflected a possible secondary diagnostic metric of IBD pathology. These results should inform the preclinical development of inflammation-targeted contrast agents for IBD and related gut disorders to improve diagnostic accuracy.

Integrated Label-Free and 10-Plex DiLeu Isobaric Tag Quantitative Methods for Profiling Changes in the Mouse Hypothalamic Neuropeptidome and Proteome: Assessment of the Impact of the Gut Microbiome.

Gut microbiota can regulate host physiological and pathological status through gut-brain communications or pathways. However, the impact of the gut microbiome on neuropeptides and proteins involved in regulating brain functions and behaviors is still not clearly understood. To address the problem, integrated label-free and 10-plex DiLeu isobaric tag-based quantitative methods were implemented to compare the profiling of neuropeptides and proteins in the hypothalamus of germ-free (GF)- vs conventionally raised (ConvR)-mice. A total of 2943 endogenous peptides from 63 neuropeptide precursors and 3971 proteins in the mouse hypothalamus were identified. Among these 368 significantly changed peptides (fold changes over 1.5 and a p-value of <0.05), 73.6% of the peptides showed higher levels in GF-mice than in ConvR-mice, and 26.4% of the peptides had higher levels in ConvR-mice than in GF-mice. These peptides were mainly from secretogranin-2, phosphatidylethanolamine-binding protein-1, ProSAAS, and proenkephalin-A. A quantitative proteomic analysis employing DiLeu isobaric tags revealed that 282 proteins were significantly up- or down-regulated (fold changes over 1.2 and a p-value of <0.05) among the 3277 quantified proteins. These neuropeptides and proteins were mainly involved in regulating behaviors, transmitter release, signaling pathways, and synapses. Interestingly, pathways including long-term potentiation, long-term depression, and circadian entrainment were involved. In the present study, a combined label-free and 10-plex DiLeu-based quantitative method enabled a comprehensive profiling of gut microbiome-induced dynamic changes of neuropeptides and proteins in the hypothalamus, suggesting that the gut microbiome might mediate a range of behavioral changes, brain development, and learning and memory through these neuropeptides and proteins.

[The clinical value of China Savin pollen extract used for skin prick test].

Objective:The aim of this study is to evaluate the effectiveness and safety of China Savin pollen extract which was used for skin prick test (SPT) in the diagnosis of China Savin pollen allergy. Method:Patients with diagnosis of allergic diseases were collected from Allergy Department of Peking Union Medical College Hospital. All patients were given SPT with China Savin pollen extract, and the mean wheal diameter (MWD) was measured after 15 minutes. Receiver operating characteristic curve (ROC) analysis was performed based on the results of serum specific immunoglobulin E (sIgE). The effectiveness of SPT in the diagnosis of China Savin pollen allergy was evaluated under different diagnostic cutoff values. Adverse events were also recorded to evaluate the safety. Result:A total of 1 029 patients were enrolled in this study without drop out case. There were 1 007 patients in full analysis set (FAS) and 765 patients in per protocol analysis set (PPS). The elimination rate was 25.66%. The area under the ROC curve of FAS is 0.814 (95%CI: 0.788-0.839); which of PPS is 0.829 (95%CI: 0.801-0.857). Based on the ROC curve of PPS, the optimal and the 95% specificity diagnostic cutoff values of MWD were 3.25 mm and 4.75 mm respectively. Based on different diagnostic cutoff value (3.00, 3.25 and 4.75 mm), the sensitivities of SPT with China Savin pollen extract were 0.740 0 (95%CI: 0.701 6-0.778 4), 0.700 (95%CI: 0.659 8-0.740 2) and 0.532 (95%CI: 0.488 3-0.575 7) respectively, whereas the specificity was gradually increased in sequence, which was 0.769 8 (95%CI: 0.719 1-0.820 5), 0.826 4 (95%CI: 0.780 8-0.872 0) and 0.950 9 (95%CI: 0.924 9-0.976 9) respectively. There were 7 adverse events observed among 6 patients (rate: 0.583%, 6/1 029). The manifestation was mild. There was no severe adverse event. Conclusion:SPT with China Savin pollen extract is an effective and safe tool for the diagnosis of China Savin pollen allergy. The effectiveness of diagnosis could be improved based on integration of medical history and different diagnostic threshold values of SPT.

Study on the anti-endotoxin effect of sinomenine using an Agilent genome array.

BACKGROUND: Endotoxin is a significant contributing factor underlying the occurrence of fever, diarrhea, inflammation, edema, coagulation, shock and other syndromes associated with gram-negative bacterial infections. To date, there is no effective treatment for endotoxemia. AIM: The aim of this study was to characterize differentially expressed genes in sinomenine-treated and lipopolysaccharide (LPS)-induced endothelial cells by microarray analysis and to determine the potential pharmacological activity of sinomenine. DESIGN: The cultured cells of five treatment groups (n = 3) were collected. Participants: total RNA was extracted and subjected to Agilent Porcine 4 × 44 K whole genome microarray. METHODS: Kyoto encyclopedia of genes and genomes and gene ontology software were applied to screen and analyze differentially regulated genes. RESULTS: The results showed that 723 differentially regulated genes were identified including 410 up-regulated genes and 313 down-regulated genes in therapy group vs. LPS group. Ten genes may be key controlled genes in the pathogenesis of LPS, including five up-regulated genes (ARG1, TLR2, IL1A, VCAM1, DKK3) and five down-regulated genes (HABP2, ID1, CHDH, GPX3, PTGFR), which primarily contribute to biological processes such as inflammatory response, vascular lesion, metabolic process and cell cycle. IL1A and FMO3 were considered as potent target genes. CONCLUSION: Global gene expression profile analysis showed that sinomenine might effectively be useful to regulate inflammatory responses as part of future anti-endotoxin therapies.

Soy Improves Cardiometabolic Health and Cecal Microbiota in Female Low-Fit Rats.

Phytoestrogen-rich soy is known to ameliorate menopause-associated obesity and metabolic dysfunction for reasons that are unclear. The gut microbiota have been linked with the development of obesity and metabolic dysfunction. We aimed to determine the impact of soy on cardiometabolic health, adipose tissue inflammation, and the cecal microbiota in ovariectomized (OVX) rats bred for low-running capacity (LCR), a model that has been previously shown to mimic human menopause compared to sham-operated (SHM) intact control LCR rats. In this study, soy consumption, without affecting energy intake or physical activity, significantly improved insulin sensitivity and body composition of OVX rats bred for low-running capacity. Furthermore, soy significantly improved blood lipid profile, adipose tissue inflammation, and aortic stiffness of LCR rats. Compared to a soy-free control diet, soy significantly shifted the cecal microbial community of LCR rats, resulting in a lower Firmicutes:Bacteroidetes ratio. Correlations among metabolic parameters and cecal bacterial taxa identified in this study suggest that taxa Prevotella, Dorea, and Phascolarctobacterium may be taxa of interest. Our results suggest that dietary soy ameliorates adiposity, insulin sensitivity, adipose tissue inflammation, and arterial stiffness and exerts a beneficial shift in gut microbial communities in a rat model that mimics human menopause.

Preclinical in vivo and in vitro comparison of the translocator protein PET ligands [18F]PBR102 and [18F]PBR111.

PURPOSE: To determine the metabolic profiles of the translocator protein ligands PBR102 and PBR111 in rat and human microsomes and compare their in vivo binding and metabolite uptake in the brain of non-human primates (Papio hamadryas) using PET-CT. METHODS: In vitro metabolic profiles of PBR102 and PBR111 in rat and human liver microsomes were assessed by liquid chromatography-tandem mass spectrometry. [18F]PBR102 and [18F]PBR111 were prepared by nucleophilic substitution of their corresponding p-toluenesulfonyl precursors with [18F]fluoride. List mode PET-CT brain imaging with arterial blood sampling was performed in non-human primates. Blood plasma measurements and metabolite analysis, using solid-phase extraction, provided the metabolite profile and metabolite-corrected input functions for kinetic model fitting. Blocking and displacement PET-CT scans, using PK11195, were performed. RESULTS: Microsomal analyses identified the O-de-alkylated, hydroxylated and N-de-ethyl derivatives of PBR102 and PBR111 as the main metabolites. The O-de-alkylated compounds were the major metabolites in both species; human liver microsomes were less active than those from rat. Metabolic profiles in vivo in non-human primates and previously published rat experiments were consistent with the microsomal results. PET-CT studies showed that K1 was similar for baseline and blocking studies for both radiotracers; VT was reduced during the blocking study, suggesting low non-specific binding and lack of appreciable metabolite uptake in the brain. CONCLUSIONS: [18F]PBR102 and [18F]PBR111 have distinct metabolic profiles in rat and non-human primates. Radiometabolites contributed to non-specific binding and confounded in vivo brain analysis of [18F]PBR102 in rodents; the impact in primates was less pronounced. Both [18F]PBR102 and [18F]PBR111 are suitable for PET imaging of TSPO in vivo. In vitro metabolite studies can be used to predict in vivo radioligand metabolism and can assist in the design and development of better radioligands.

The reproductive toxicity of melamine in the absence and presence of cyanuric acid in male mice.

Melamine, a chemical compound, was used widely in the manufacture of amino resins and plastics. Cyanuric acid related structurally to melamine was used as a water stabilizer in swimming pools. The combination of melamine and cyanuric acid was thought to be responsible for renal impairment in mammals. In the present work, we investigated the reproductive toxicity of melamine in the absence and presence of cyanuric acid in male mice. Pathological damages in different degrees were observed in the testis of male mice treated with different doses of both melamine alone and combination of melamine and cyanuric acid in a dose-dependent manner. Based on the TUNEL assay, the mice treated with high dose of melamine (50 mg/kg/day) had a significant increase in apoptotic index of spermatogenic cells (p<0.05) compared with the control group. Sperm abnormality test indicated that melamine alone resulted in abnormal sperm morphology. The mice from co-administration groups of melamine and cyanuric acid were not eating, and were most likely in renal failure. The combined exposure to melamine and cyanuric acid was revealed to have certain toxic effects on testis of male mice at a relative low dose (each at 1 mg/kg/day). Also, in comparison to melamine treated groups, more severe apoptosis was observed in co-administration groups of melamine and cyanuric acid with both middle (each at 5 mg/kg/day) and high doses (each at 25 mg/kg/day). However, all mice administrated with combination of melamine and cyanuric acid (each at 206, 412, or 824 mg/kg/day) died before day 6 from which no data were obtained on sperm abnormality. These results from this study demonstrated that melamine had certain toxic effects on testes of male mice, especially when ingested in high concentration. These results might be useful in evaluating the toxicity of melamine on reproductive system of male animal, and they also would be a supplement to the existing toxic profile of melamine.

Ginsenoside Rg1 restores the impairment of learning induced by chronic morphine administration in rats.

Rg1, as a ginsenoside extracted from Panax ginseng, could ameliorate spatial learning impairment. Previous studies have demonstrated that Rg1 might be a useful agent for the prevention and treatment of the adverse effects of morphine. The aim of this study was to investigate the effect of Rg1 on learning impairment by chronic morphine administration and the mechanism responsible for this effect. Male rats were subcutaneously injected with morphine (10 mg/kg) twice a day at 12 hour intervals for 10 days, and Rg1 (30 mg/kg) was intraperitoneally injected 2 hours after the second injection of morphine once a day for 10 days. Spatial learning capacity was assessed in the Morris water maze. The results showed that rats treated with Morphine/Rg1 decreased escape latency and increased the time spent in platform quadrant and entering frequency. By implantation of electrodes and electrophysiological recording in vivo, the results showed that Rg1 restored the long-term potentiation (LTP) impaired by morphine in both freely moving and anaesthetised rats. The electrophysiological recording in vitro showed that Rg1 restored the LTP in slices from the rats treated with morphine, but not changed LTP in the slices from normal saline- or morphine/Rg1-treated rats; this restoration could be inhibited by N-methyl-D-aspartate (NMDA) receptor antagonist MK801. We conclude that Rg1 may significantly improve the spatial learning capacity impaired by chonic morphine administration and restore the morphine-inhibited LTP. This effect is NMDA receptor dependent.

Association of methylenetetrahydrofolate reductase polymorphism and the risk of squamous cell carcinoma in renal transplant patients.

The relative risk of developing cutaneous squamous cell carcinoma (SCC) is significantly increased after organ transplantation. We investigated the genetic association of SCC in two pathways associated with cancer risks, with the potential for modification by vitamin supplementation. A total of 367 renal transplant recipients (117 with SCC and 250 without any skin cancer) were genotyped for key polymorphisms in the folate pathway (methylene tetrahydrofolate reductase; MTHFR:C677T), and the vitamin D pathway (vitamin D receptor: Intron8G/T;). Individuals carrying the MTHFR 677T allele had a marked increase in risk of SCC (adjusted odds ratio=2.54, P=0.002, after adjustment for age, ender, skin type, sun exposure score, and immunosuppression duration; lower 95% confidence boundary odds ratio of 1.41). In contrast, vitamin D receptor polymorphisms were not significantly associated. Folate-sensitive pathways may play a critical role in the elevated rate of SCC in renal transplant recipients.

Age-related changes in cochlear gene expression in normal and shaker 2 mice.

The vertebrate cochlea is a complex organ optimized for sound transduction. Auditory hair cells, with their precisely arranged stereocilia bundles, transduce sound waves to electrical signals that are transmitted to the brain. Mutations in the unconventional myosin XV cause deafness in both human DFNB3 families and in shaker 2 (sh2) mice as a result of defects in stereocilia. In these mutant mice, hair cells have relatively normal spatial organization of stereocilia bundles but lack the graded, stair-step organization. We used sh2 mice as an experimental model to investigate the molecular consequences of the sh2 mutation in the Myo15 gene. Gene expression profiling with Affymetrix GeneChips in deaf homozygous (sh2/sh2) mice at 3 weeks and 3 months of age, and in age-matched, normal-hearing heterozygotes (+/sh2) identified only a few genes whose expression was affected by genotype, but a large number with age-associated changes in expression in both normal mice and sh2/sh2 homozygotes. Microarray data analyzed using Robust Multiarray Average identified Aim1, Dbi, and Tm4sf3 as genes with increased expression in sh2/sh2 homozygotes. These increases were confirmed by quantitative reverse transcription-polymerase chain reaction. Genes exhibiting altered expression with age encoded collagens and proteins involved in collagen maturation, extracellular matrix, and bone mineralization. These results identified potential cellular pathways associated with myosin XV defects, and age-associated molecular events that are likely to be involved in maturation of the cochlea and auditory function.

Noise overstimulation induces immediate early genes in the rat cochlea.

In mammals, exposure to intense noise produces a permanent hearing loss called permanent threshold shift (PTS), whereas a moderate noise produces only a temporary threshold shift (TTS). Little is known about the molecular responses to such high intensity noise exposures. In this study we used gene arrays to examine the early response to acoustic overstimulation in the rat cochlea. We compared cochlear RNA from noise-exposed rats with RNA from unexposed controls. The intense PTS noise induced several immediate early genes encoding both transcription factors (c-FOS, EGR1, NUR77/TR3) and cytokines (PC3/BTG2, LIF and IP10). In contrast, the TTS noise down-regulated the gene for growth hormone. The response of these genes to different noise intensities was examined by quantitative RT-PCR 2.5 h after the 90-min noise exposure. For most genes, the extent of induction correlates with the intensity of the noise exposure. Three proteins (EGR1, NUR77/TR3, and IP10) were detected in many regions of the unexposed cochlea. After exposure to 120 dB noise, these proteins were present at higher levels or showed extended expression in additional regions of the cochlea. LIF was undetectable in the cochlea of unexposed rats, but could be seen in the organ of Corti and spiral ganglion neurons following noise. NUR77/TR3 was a nuclear protein before noise, but following noise translocated to the cytoplasm. These studies provide new insights into the molecular response to noise overstimulation in the mammalian cochlea.

[Effect of calcium hydroxide on human pulp cell intracellular calcium ion in vitro].

OBJECTIVE: To investigate the influence of calcium hydroxide to intracellular calcium ion of human pulp cells in vitro. METHODS: The sixth generation of human pulp cells were maintained in RPMI 1640 medium supplemented with Fluo-3 probe and Ca (OH)2 for restimulation. The intracellular calcium ion concentration was detected by laser confocal microscope. RESULTS: The intracellular calcium ion level in Ca (OH)2 pretreatment group increased after Ca (OH)2 stimulation, while it didn't response to Ca (OH)2 stimulus in group without Ca (OH)2, CaCl2 or NaHCO4 pretreatment. CONCLUSION: It is important to pretreat the pulp cells with Ca(OH)2 in order to increase the intracellular calcium.

[Studies on separation and identification of Rhizoma coptidis brugs based on microemulsion thin-layer chromatography].

OBJECTIVE: To investigate the use of microemulsion TLC for the separation and identification of Rhizoma Coptidis drugs. METHOD: Thirteen Rhizoma Coptidis drugs were separated and identified with six kinds of SDS/n-C4H9OH/n-C7H16/H2O microemulsions as mobile phase on polyamide film. Effects of microemulsions on separation were investigated. RESULTS: The best mobile phase was the O/W microemulsion(containing 75% water). Compared with general mobile phases, the separation and detection could be improved and the test conditions were more tolerant. CONCLUSION: This new method is simple, accurate and efficient for the separation and identification of Rhizoma Coptidis drugs.

[Experimental research on inhibitory effect of alcohol extracts from Loranthus yadoriki Sieb. on coxsackie B3 virus].

OBJECTIVE: To investigate the antiviral effect of two components (B and C) in the alcohol extracts from Loranthus yadoriki Sieb for development of new antiviral drugs for coxsackie B3 virus(CVB3). METHOD: Using ribavirin as positive control, the plaque reduction assay was adopted for pharmadynamic detection. RESULTS: In the HEp-2 cell system, with respect to direct virucidal activity and the inhibition on CVB3 infection and multiplication, ED50s of component B were 2.32 micrograms.ml-1, 0.24 microgram.ml-1 and 1.91 micrograms.ml-1, respectively and those of component C, 1.44 micrograms.ml-1, 2.06 micrograms.ml-1 and 3.70 micrograms.ml-1 respectively. For the inhibition on multiplication of CVB3, ED50 of ribavirin was 7.55 micrograms.ml-1. CONCLUSION: Treatment indexes (TI) of the direct virucidal activity and inhibitory effects of component B on CVB3 infection and multiplication 22.6, 219 and 27.5 respectively; component C, 115, 165 and 21.6; inhibition of ribavirin on CVB3 is 38, which is comparable to those of components B and C. It is thus suggested that the anti-CVB3 action of components B and C in the alcohol extracts from Loranthus yadoriki Sieb deserves to be exploited.

[Establishment and its application of a reporter-based screening model for discovering new ligands of estrogen receptor alpha subtypes].

AIM: To establish a sensitive and efficient reporter gene based screening model and use it to screen compounds for discovering new ligands of estrogen receptor alpha subtype. METHODS: A recombinant Epstein-Barr virus episomal vector (pMT/ERE-CAT) was constructed by inserting a synthetic sequence composed of five estrogen response elements upstream of promoter and a chloramphenicol acetyltransferase (CAT) gene downstream of promoter. pMT/ERE-CAT was transfected into HepG2 cells expressing estrogen receptor alpha subtype (ER +HepG2). Hygromycin (200 micrograms.mL-1) was added 48 h after transfection for selection. One stably transfected clone was isolated and used to screen compounds for activity of stimulating CAT gene expression using colorimetric CAT assay. RESULTS: In the ER +HepG2 cells, the expression of CAT gene was induced by estradiol. A dose-dependent expression of CAT gene with half-maximal induction at 0.07 nmol.L-1 was observed. The ER +HepG2 cell was used to screen compounds for activity of stimulating CAT gene expression. Resveratrol was found to produce a maximal level of induction (1.75 times of estradiol). In vitro radiation survival experiment showed that the radioprotection activity of resveratrol (D0 = 3.18 Gy) is stronger than that of estradiol (D0 = 2.59 Gy). CONCLUSION: Vector pMT/ERE-CAT was used to generate stably transfected ER +HepG2 cell lines. The cell lines can be used to screen compounds for estrogen activity by testing extracts of cells grown in microtiter wells directly using colorimetric CAT assay. This system should provide an efficient method for screening and analyzing the activity of large numbers of ligands of estrogen receptor.

[Clinical and experimental study on antiviral activity of reduqing against human cytomegalovirus].

OBJECTIVE: To investigate the therapeutic efficacy of traditional Chinese medicine Reduqing (RDQ) against human cytomegalovirus (HCMV) in clinical and its antiviral activity in vitro. METHODS: In clinical practice, HCMV antibody was detected to determine if the case was activity infected with HCMV. Fourteen patients were found and treated with RDQ. The drug was orally administered one dose, three times a day, for 18-30 days as one therapeutic course. And the efficacy was evaluated by ELISA, PCR and other methods. The in vitro inhibitory activity of RDQ against HCMV AD169 was carried out on human embryo lung fibroblasts (HEL) by cytopathic effect inhibition method. Ganciclovir (GCV) was used for positive control. RESULTS: Fourteen pre- or during pregnant women with HCMV infection were treated with RDQ. After 18-30 days of treatment, all of them showed HCMV-IgM negative conversion, HCMV DNA negative conversion in 7/10 cases, and virus excretion by urine and cervix secretion was inhibited in 4/4 and 1/1 case. Five women gave birth to 5 normal newborns at term after treatment, among them 2 were asymptomatic virus carrier, the other 3 were uninfected. Experimental study in vitro showed that the maximal tolerance dosage (TD0) of RDQ was 20 micrograms/L, the minimal therapeutic concentration (MTC) was 5 micrograms/L, 50% inhibitory concentration (IC50) was 5 micrograms/L and the therapeutic index (TI) was 4. It suggested that RDQ had anti-HCMV activity in vitro and the effect increased with its concentration. CONCLUSION: RDQ is a safe, valuable drug for inhibiting HCMV infection especially during pregnancy.

[The experimental and clinical study of inhibitory effects of Re-Du-Qing on human cytomegalovirus (HCMV)].

OBJECTIVE: To study the theurapeutic effect of Chinese medicine Re-Du-Qing on HCMV. METHODS: The expression of the HCMV late-mRNA in infected cells was measured by quantitative RT-PCR and the development of cytopathic effect (CPE) caused by HCMV was observed before and after the treatment of Chinese medicine "Re-Du-Qing". On the basis of its anti-HCMV activity study in vitro, Re-Du-Qing was applied to the child-bearing aged women and to pregnant women who were HCMV-IgM positive which indicated the active HCMV infection, meanwhile the serum levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-2 (IL-2) were measured before and after the treatment by the ELISA method. RESULTS: The results showed that the Re-Du-Qing had strong inhibition effect both on HCMV-mRNA and CPE. The serum level of the TNF-alpha increased and IL-2 decreased significantly in HCMV-IgM positive cases when compared with normal women. After being treated with Re-Du-Qing, the serum TNF-alpha and IL-2 in patients returned to normal level, and 72.9% of the HCMV-IgM positive cases turned negative which was significantly higher than that (13.6%) in control group. CONCLUSION: It was suggested that serum TNF-alpha level is closely related to the activity of HCMV which has harmful effect on immune system. Re-Du-Qing can enhance immunity of the body and has an obvious inhibition on HCMV in Vitro and Vivo, and can thus serve as safe and effective medicine for treatment of active HCMV infection, especially for the pregnant women.

NMR studies of internal dynamics of serine proteinase protein inhibitors: Binding region mobilities of intact and reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor (CMTI)-III of the squash family and comparison with those of counterparts of CMTI-V of the potato I family.

Serine proteinase protein inhibitors follow the standard mechanism of inhibition (Laskowski M Jr, Kato I, 1980, Annu Rev Biochem 49:593-626), whereby an enzyme-catalyzed equilibrium between intact (I) and reactive-site hydrolyzed inhibitor (I*) is reached. The hydrolysis constant, Khyd, is defined as [I*]/[I]. Here, we explore the role of internal dynamics in the resynthesis of the scissile bond by comparing the internal mobility data of intact and cleaved inhibitors belonging to two different families. The inhibitors studied are recombinant Cucurbita maxima trypsin inhibitor III (rCMTI-III; Mr 3 kDa) of the squash family and rCMTI-V (Mr approximately 7 kDa) of the potato I family. These two inhibitors have different binding loop-scaffold interactions and different Khyd values--2.4 (CMTI-III) and 9 (CMTI-V)--at 25 degrees C. The reactive-site peptide bond (P1-P1') is that between Arg5 and Ile6 in CMTI-III, and that between Lys44 and Asp45 in CMTI-V. The order parameters (S2) of backbone NHs of uniformly 15N-labeled rCMTI-III and rCMTI-III* were determined from measurements of 15N spin-lattice and spin-spin relaxation rates, and [1H]-15N steady-state heteronuclear Overhauser effects, using the model-free formalism, and compared with the data reported previously for rCMTI-V and rCMTI-V*. The backbones of rCMTI-III [(S2) = 0.71] and rCMTI-III* [(S2) = 0.63] are more flexible than those of rCMTI-V [(S2) = 0.83] and rCMTI-V* [(S2) = 0.85]. The binding loop residues, P4-P1, in the two proteins show the following average order parameters: 0.57 (rCMTI-III) and 0.44 (rCMTI-III*); 0.70 (rCMTI-V) and 0.40 (rCMTI-V*). The P1'-P4' residues, on the other hand, are associated with (S2) values of 0.56 (rCMTI-III) and 0.47 (rCMTI-III*); and 0.73 (rCMTI-V) and 0.83 (rCMTI-V*). The newly formed C-terminal (Pn residues) gains a smaller magnitude of flexibility in rCMTI-III* due to the Cys3-Cys20 crosslink. In contrast, the newly formed N-terminal (Pn' residues) becomes more flexible only in rCMTI-III*, most likely due to lack of an interaction between the P1' residue and the scaffold in rCMTI-III. Thus, diminished flexibility gain of the Pn residues and, surprisingly, increased flexibility of the Pn' residues seem to facilitate the resynthesis of the P1-P1' bond, leading to a lower Khyd value.