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Biomedical subjects

Ying Zhou

Publications and source records attributed to Ying Zhou.

At least 19 recordsLinked to original sources
PubMed2026

Effects of Short-Term Energy Limitation at Different Levels With Normal Protein Intake on Hepatic Lipid Metabolism and the Gut Microbiota in Overweight/Obese Mice.

This study investigated the sex-specific effects of graded short-term energy limitation (EL) with normal protein intake on hepatic lipid metabolism and the gut microbiota in overweight/obese mice. Mice were allocated to a normal control (NC) group, a high-fat diet model (MC) group, and groups receiving 20%, 30% or 40% EL (n&#x2009;=&#x2009;8 per group). All mice underwent blood biochemistry, liver biochemistry, histological, liver metabolomic, and fecal microbiota community genomic analyses. Relative to the NC group, both male and female MC mice developed varying degrees of insulin resistance, dyslipidaemia, and sex hormone dysregulation. However, disrupted hepatic lipid metabolism was detected solely in male mice, in association with changes in key lipid-metabolizing enzymes and metabolites; female mice showed only disturbed total cholesterol (TC) metabolism, which was linked to alterations in the cholesterol synthesis rate-limiting enzyme HMGCR. With normal protein intake, 20%, 30%, and 40% EL reduced hepatic triglyceride and TC synthesis in overweight/obese male mice by suppressing the expression of the key lipogenic enzyme DGAT and the activities of ACC and HMGCR (p&#x2009;<&#x2009;0.05). An effect on the lipolytic enzymes CPT1 and CYP7A1 was detected only at 30% EL (p&#x2009;<&#x2009;0.05), and hepatic metabolite profiles varied with the degree of EL. In female mice, only 40% EL significantly decreased TC synthesis by inhibiting both HMGCR expression and enzymatic activity (p&#x2009;<&#x2009;0.05). Furthermore, irrespective of sex, short-term EL (all levels) with normal protein intake reduced the gut Firmicutes/Bacteroidetes ratio in overweight/obese mice (p&#x2009;<&#x2009;0.05). In conclusion, graded short-term EL with adequate protein intake exerts differential effects on hepatic lipid metabolism and the gut microbiota in overweight/obese mice, with pronounced sex-specific differences.

energy limitation↗
PubMed2026

Digital Structured Education With Behavioral Nudge Tools for Adults With Type 2 Diabetes: Multicenter Randomized Controlled Trial.

BACKGROUND: Digital interventions offer scalable alternatives to traditional face-to-face diabetes education, but often face challenges related to inconsistent clinical effectiveness, and declining user engagement. However, whether a digital structured education program integrated with behavioral nudge tools can improve metabolic, behavioral, and psychological outcomes in adults with type 2 diabetes remains unclear. OBJECTIVE: This study aimed to evaluate the effectiveness of a digital structured education program integrated with behavioral nudge tools in improving metabolic, behavioral, and psychological outcomes among adults with type 2 diabetes. METHODS: This multicenter randomized controlled trial was conducted in the endocrinology departments of 4 hospitals in China. Adults with type 2 diabetes were randomly assigned to an intervention group receiving a digital structured education program integrated with behavioral nudge tools (n=146) or a control group receiving standard digital diabetes education (n=147). Assessments were conducted at baseline and 12-week follow-up. The primary outcome was hemoglobin A1c (HbA1c) at 12 weeks, adjusted for baseline HbA1c, and study center. Secondary outcomes included fasting blood glucose (FBG), weight, BMI, waist circumference, blood pressure, lipid profiles, self-management behaviors, self-efficacy, and habit strength. RESULTS: Among 293 participants (mean age 49.19, SD 10.02 y), 287 (97.9%) completed follow-up. At 12 weeks, the intervention group demonstrated significantly greater improvements than the control group in HbA1c (adjusted mean difference -0.38%, 95% CI -0.68% to -0.09%; P=.01), FBG (adjusted mean difference -0.75, 95% CI -1.27 to -0.44 mmol/L; P<.001), weight (adjusted mean difference -0.84, 95% CI -1.61 to -0.07 kg; P=.03), BMI (adjusted mean difference -0.38, 95% CI -0.65 to -0.11 kg/m&#xb2;; P=.01), systolic blood pressure (adjusted mean difference -2.71, 95% CI -4.62 to -0.79 mm Hg; P=.01), diastolic blood pressure (adjusted mean difference -2.92, 95% CI -4.47 to -1.37 mm Hg; P<.001), and total cholesterol (adjusted mean difference -0.27, 95% CI -0.48 to -0.05 mmol/L; P=.02). The intervention was also associated with significantly greater improvements in self-management behaviors, self-efficacy, and habit strength (all P<.05). CONCLUSIONS: Digital structured education integrated with behavioral nudge tools improved metabolic outcomes and strengthened psychological and behavioral determinants of self-management among adults with type 2 diabetes over a 12-week period. These findings suggest that a digital structured education program integrated with behavioral nudge tools may enhance diabetes self-management beyond standard digital diabetes education. Further studies with longer follow-up and real-world implementation are warranted to evaluate the sustainability, generalizability, and long-term clinical impact of this integrated intervention.

Humans↗
PubMed2026

Construction of an infectious clone of Spodoptera frugiperda densovirus and its biological characteristics.

Densoviruses are highly pathogenic to their insect hosts and have great potential for biocontrol. Spodoptera frugiperda densovirus (SfDV) was isolated from diseased larvae of Spodoptera frugiperda, while its biological functions remain unclear. Herein, we successfully constructed an infectious clone of SfDV. The S. frugiperda larvae transfected with the infectious clone exhibited anorexia, stunted growth, and reduced activity. Histopathological analysis further showed that the epidermis, fat body and trachea were infected instead of muscle and midgut tissues. Transmission electron microscopy (TEM) revealed that numerous virions of about 22&#x202f;nm were distributed within both the nucleoplasm and cytoplasm of epidermal cells. Moreover, many virions were also found contained within vesicles in the cytoplasm. The replication kinetics of the rescued SfDV (rSfDV) was similar to that of the parental SfDV. The median lethal dose (LD50) and median lethal time (LT50) values of rSfDV were 6.63&#x202f;&#xd7;&#x202f;107 viral genome copies (vgc), 5.23&#x202f;d, respectively, which were also comparable to those of the parental SfDV. Taken together, the infectious clone of SfDV provides an important tool for further exploring the genome function, pathogenesis, and interactions with its hosts.

Animals↗
PubMed2026

Pseudotargeted peptidomics approach for identifying species-specific biomarkers in fermented Cordyceps products: A case study on Bailing Capsule.

Bailing Capsule, a fermented Cordyceps product used as an adjunctive treatment for chronic kidney disease and chronic bronchitis, is a crucial substitute for wild Ophiocordyceps. However, its current quality control relies on non-specific small-molecule markers, making it hard to distinguish from other analogous products. In this study, a pseudotargeted peptidomics strategy was developed to discover characteristic peptides for differentiation. Firstly, digested peptides were obtained by trypsin digestion (37 &#xb0;C, 24 h, enzyme/protein ratio 1: 50) and profiled by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS). Subsequently, a multiple reaction monitoring (MRM) transition list was generated from the Q-TOF data using a "MRM-Ion Pair Finder" tool. These transitions were validated by ultra-high performance liquid chromatography triple-quadrupole mass spectrometry (UPLC-QQQ-MS) with 48 batches of samples from Bailing Capsule and four other analogous products. As a result, a total of 150 characteristic transitions specific to Bailing Capsule were identified, of which 76 peptide sequences were elucidated, and 12 peptide biomarkers were confirmed using synthetic standards. This pseudotargeted approach exhibits broad coverage and good sensitivity. Without requiring prior knowledge of peptide sequence, the transition-driven strategy reduces redundancy and enables focused, transition-based peptide differentiation. Compared to conventional small-molecule biomarkers, this peptide-based approach offers species-specific identification. This study demonstrates that a pseudotargeted peptidomics strategy may aid in the quality control of fermented Cordyceps products.

Cordyceps↗
PubMed2026

FuFiHLA: a tool for full-field HLA typing from long-read data.

MOTIVATION: Allele typing for Human Leukocyte Antigen (HLA) genes has many important clinical applications. Popular short-read typing can only accurately distinguish alleles at the coding sequence level, which potentially limit our understanding of the effect of variants in non-coding region. Long read data has been proved to be useful in typing HLA alleles in full resolution, but only a few tools are publicly available and with significant limitations in practical application. RESULTS: We developed FuFiHLA, a lightweight open-source software, to type HLA alleles. Currently it supports typing alleles of six HLA genes (HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQA1, and HLA-DQB1) from long reads. Evaluation using 233 PacBio HiFi WGS samples from HPRC shows that FuFiHLA achieves 99.6% accuracy in the full field allele typing and QV as 51.8 for consensus allele sequence construction. Additional testing on four Nanopore R10 reads demonstrates slightly reduced accuracy in the fourth field. AVAILABILITY: FuFiHLA is available at https://github.com/jingqing-hu/FuFiHLA under MIT License.

Humans↗
PubMed2025

A complete diploid human genome benchmark for personalized genomics.

Human genome resequencing typically involves mapping reads to a reference genome to call variants; however, this approach suffers from both technical and reference biases, leaving many duplicated and structurally polymorphic regions of the genome unmapped. Consequently, existing variant benchmarks, generated by the same methods, fail to assess these complex regions. To address this limitation, we present a telomere-to-telomere genome benchmark that achieves near-perfect accuracy (i.e. no detectable errors) across 99.4% of the complete, diploid HG002 genome. This benchmark adds 701.4 Mb of autosomal sequence and both sex chromosomes (216.8 Mb), totaling 15.3% of the genome that was absent from prior benchmarks. We also provide a diploid annotation of genes, transposable elements, segmental duplications, and satellite repeats, including 39,144 protein-coding genes across both haplotypes. To facilitate application of the benchmark, we developed tools for measuring the accuracy of sequencing reads, phased variant call sets, and genome assemblies against a diploid reference. Genome-wide analyses show that state-of-the-art de novo assembly methods resolve 2-7% more sequence and outperform variant calling accuracy by an order of magnitude, yielding just one error per 100 kb across 99.9% of the benchmark regions. Adoption of genome-based benchmarking is expected to accelerate the development of cost-effective methods for complete genome sequencing, expanding the reach of genomic medicine to the entire genome and enabling a new era of personalized genomics.

Journal Article↗
PubMed2025

SNORA47 affects stemness and chemotherapy sensitivity via EBF3/RPL11/c-Myc axis in luminal A breast cancer.

Chemotherapy sensitivity is an important factor that restricts the prognosis of breast cancer, and breast cancer stem cells (BCSCs) are the root cause of chemotherapy sensitivity. SNORA47, a member of the small nucleolar RNAs, has not been documented in the context of breast cancer, although it has been reported in lung cancer. In this study, high SNORA47 expression was linked to unfavorable survival outcomes among patients with Luminal A breast cancer in The Cancer Genome Atlas (TCGA). Among Luminal A patients, an elevated expression of SNORA47 correlated with high TNM stage (P&#x2009;=&#x2009;0.049). SNORA47 was strongly associated with breast cancer stemness phenotype and tumor sensitivity in vivo and in vitro. Our findings demonstrated that SNORA47, through its interaction with early B-cell factor 3(EBF3), facilitated the translocation of ribosomal protein L11(RPL11), which as a modulator that subsequently regulates the expression levels of the oncogene c-Myc. These discoveries provided novel insights into the molecular mechanisms of breast cancer progression and suggested potential therapeutic targets for overcoming drug sensitivity by disrupting the SNORA47-EBF3-RPL11 axis.

Humans↗
PubMed2025

IL-7-mediated expansion of autologous lymphocytes increases CD8+ VLA-4 expression and accumulation in glioblastoma models.

The efficacy of T cell-activating therapies against glioma is limited by an immunosuppressive tumor microenvironment and tumor-induced T cell sequestration. We investigated whether peripherally infused non-antigen specific autologous lymphocytes (ALT) could accumulate in intracranial tumors. We observed that non-specific autologous CD8+ ALT cells can indeed accumulate in this context, despite endogenous T cell sequestration in bone marrow. Rates of intratumoral accumulation were markedly increased when expanding lymphocytes with IL-7 compared to IL-2. Pre-treatment with IL-7 ALT also enhanced the efficacy of multiple tumor-specific and non-tumor-specific T cell-dependent immunotherapies against orthotopic murine and human xenograft gliomas. Mechanistically, we detected increased VLA-4 on mouse and human CD8+ T cells following IL-7 expansion, with increased transcription of genes associated with migratory integrin expression (CD9). We also observed that IL-7 increases S1PR1 transcription in human CD8+ T cells, which we have shown to be protective against tumor-induced T cell sequestration. These observations demonstrate that expansion with IL-7 enhances the capacity of ALT to accumulate within intracranial tumors, and that pre-treatment with IL-7 ALT can boost the efficacy of subsequent T cell-activating therapies against glioma. Our findings will inform the development of future clinical trials where ALT pre-treatment can be combined with T cell-activating therapies.

Cell migration/adhesion↗
PubMed2007

Tfb5 interacts with Tfb2 and facilitates nucleotide excision repair in yeast.

TFIIH is indispensable for nucleotide excision repair (NER) and RNA polymerase II transcription. Its tenth subunit was recently discovered in yeast as Tfb5. Unlike other TFIIH subunits, Tfb5 is not essential for cell survival. We have analyzed the role of Tfb5 in NER. NER was deficient in the tfb5 deletion mutant cell extracts, and was specifically complemented by purified Tfb5 protein. In contrast to the extreme ultraviolet (UV) sensitivity of rad14 mutant cells that lack any NER activity, tfb5 deletion mutant cells were moderately sensitive to UV radiation, resembling that of the tfb1-101 mutant cells in which TFIIH activity is compromised but not eliminated. Thus, Tfb5 protein directly participates in NER and is an accessory NER protein that stimulates the repair to the proficient level. Lacking a DNA binding activity, Tfb5 was found to interact with the core TFIIH subunit Tfb2, but not with other NER proteins. The Tfb5-Tfb2 interaction was correlated with the cellular NER function of Tfb5, supporting the functional importance of this interaction. Our results led to a model in which Tfb5 acts as an architectural stabilizer conferring structural rigidity to the core TFIIH such that the complex is maintained in its functional architecture.

Amino Acid Sequence↗
PubMed2006

Surgical management to conjoined twins in Shanghai area.

Conjoined twins are very rare congenital malformation. The aim of this study was to summarize our experiences of surgical separation on seven sets of conjoined twins, and improve the treatment of conjoined twins in the future. A retrospective review of surgical separation included data of prenatal diagnosis, associated malformation, timing of separation, intra- and postoperative management, and follow-up of six sets of conjoined twins at Shanghai Xin-Hua Hospital from 1980 to 2005 and one set at Shanghai Children's Hospital in 2002. Surgical separation was performed on seven sets of conjoined twins; six sets of thoracopagus-omphalapagus (including four sets of xipho-omphalopagus) and one set of ischiopagus. All sets presented varying degrees of severity of congenital cardiac malformations. Four sets were diagnosed prenatally by ultrasonography. Two sets of conjoined twins (case 2 and case 3) required emergent separation within 7 days after birth; both members of case 2 died within 2 days post operation, one member of case 3 died during operation while the other member survived. Five sets had scheduled separation undertaken more than 30 days after birth. One member of a set (case 6B) died 13 days after operation due to severe congenital cardiovascular anomalies. All other members of conjoined twins survived. Case 6A had a severe defect of the anterior thoracic cage and prosthesis of titanium alloy scaffold filled with silicone rubber was used to repair the defects successfully. Following up from 1980 to 2005, one member of a set (case 1A) died 4 years after operation due to pneumonia. Contact was lost to the surviving member of case 3 (ischiopagus). Other survivors of the separations had normal development. (1) Timing of operation and separation plan should be given according to the circumstances and the nature of the organ shared in each individual set of twins. (2) Prosthesis of titanium alloy scaffolds filled with silicone rubber may become one of viable methods for repairing severe defects of the thoracic cage.

Abnormalities, Multiple↗
PubMed2006

Flocculation mechanism induced by cationic polymers investigated by light scattering.

Three cationic polymers with molecular weights and charge densities of 3.0 x 10(5) g/mol and 10%, 1.1 x 10(5) g/mol and 40%, and 1.2 x 10(5) g/mol and 100% were chosen as flocculants to aggregate silica particles (90 nm), under various conditions, including change in polymer dosage, particle concentration, background electrolyte concentration, and shear rate. The size and structure of flocs produced were determined using the static light scattering technique. On the basis of measurements of polymer adsorption and its effect on the zeta potential and floc properties, it has been found that the polymer charge density plays an important role in determining the flocculation mechanism. Polymers with a 10% charge density facilitate bridging, 40% charged polymers bring about either a combination of charge neutralization and bridging or bridging, depending on the polymer dosage, and polymers with the charge density of 100% induce electrostatic patch flocculation mechanism at the optimum polymer dosage and below but bring about bridging mechanism at the polymer dosage approaching the adsorption plateau value. Bridging aggregation can readily be affected by the particle concentration, and an increase in particle concentration results in the formation of larger but looser aggregates, whereas electrostatic patch aggregation is independent of particle concentration. The addition of a background electrolyte aids in bridging aggregation while it is detrimental to electrostatic patch aggregation. It has also been found that the effect of shear rate on the mass fractal dimension depends on polymer charge density.

Journal Article↗
PubMed2006

Vi antigen biosynthesis in Salmonella typhi: characterization of UDP-N-acetylglucosamine C-6 dehydrogenase (TviB) and UDP-N-acetylglucosaminuronic acid C-4 epimerase (TviC).

Vi antigen, the virulence factor of Salmonella typhi, has been used clinically as a molecular vaccine. TviB and TviC are two enzymes involved in the formation of Vi antigen, a linear polymer consisting of alpha-1,4-linked N-acetylgalactosaminuronate. Protein sequence analysis suggests that TviB is a dehydrogenase and TviC is an epimerase. Both enzymes are expected to be NAD(+) dependent. In order to verify their functions, TviB and TviC were cloned, expressed in Escherichia coli, and characterized. The C-terminal His(6)-tagged TviB protein, purified from soluble cell fractions in the presence of 10 mM DTT, shows UDP-N-acetylglucosamine 6-dehydrogenase activity and is capable of catalyzing the conversion of UDP-N-acetylglucosamine (UDP-GlcNAc) to UDP-N-acetylglucosaminuronic acid (UDP-GlcNAcA) with a k(cat) value of 15.5 +/- 1.0 min(-)(1). The K(m) values of TviB for UDP-GlcNAc and NAD(+) are 77 +/- 9 microM and 276 +/- 52 microM, respectively. TviC, purified as C-terminal hexahistidine-tagged protein, shows UDP-GlcNAcA 4-epimerase and UDP-N-acetylgalactosamine (UDP-GalNAc) 4-epimerase activities. The K(m) values of TviC for UDP-GlcNAcA and UDP-N-acetylgalactosaminuronic acid (UDP-GalNAcA) are 20 +/- 1 microM and 42 +/- 2 microM, respectively. The k(cat) value for the conversion of UDP-GlcNAcA to UDP-GalNAcA is 56.8 +/- 0.5 min(-)(1), while that for the reverse reaction is 39.1 +/- 0.6 min(-)(1). These results show that the biosynthesis of Vi antigen is initiated by the TviB-catalyzed oxidation of UDP-GlcNAc to UDP-GalNAc, followed by the TviC-catalyzed epimerization at C-4 to form UDP-GalNAcA, which serves as the building block for the formation of Vi polymer. These results set the stage for future in vitro biosynthesis of Vi antigen. These enzymes may also be drug targets to inhibit Vi antigen production.

Amino Acid Sequence↗
PubMed2006

Action of beta-amyloid peptide₁₋₄₀ on I(HVA) and its modulation by ginkgolide B.

Whole-cell patch clamp recording was used to investigate the action of beta-amyloid peptide(1-40) (Abeta(1-40)) on high voltage-activated calcium channel current (I(HVA)) in acutely isolated hippocampal CA1 pyramidal neurons in rats and observe its modulation by ginkgolide B (GB). Drug was applied by extracellular bath or adding in the pipette solution, and its effect was determined by comparing the amplitude of I(HVA) before and after the drug application. Bath application of aggregated Abeta(1-40) at concentrations of 0.01~30 mumol/L increased the amplitude of I(HVA) in a dose-dependent manner by (5.43+/-3.01)% (n=8, P>0.05), (10.49+/-4.13) % (n=11, P>0.05), (40.69+/-8.01) % (n=16, P<0.01), (58.32+/-4.85) % (n=12, P<0.01), and (75.45+/-5.81) % (n=6, P<0.01), respectively, but had no effect on the I-V curve of I(HVA); fresh Abeta(1-40) almost had no effect on I(HVA) (n=5, P>0.05). L-type calcium channel antagonist nifedipine abolished the increase of I(HVA)by Abeta(1-40). The increase of I(HVA) by Abeta(1-40) (1.0 mumol/L) was enhanced to (66.19+/-5.74) % (P<0.05) by 8-Br-cAMP (membrane permeable analogue of cAMP) and to (73.21+/-6.90) % (P<0.05) by forskolin, an adenylyl cyclase (AC) agonist, and reduced to (20.08+/-2.18) % (P<0.05) by H-89, cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) antagonist. GB effectively inhibited the increase of I(HVA) by Abeta(1-40). The results indicate that Abeta(1-40) leads to an intracellular calcium overload by increasing I(HVA) via AC-cAMP-PKA. This may be one of the mechanisms for its neurotoxicity. GB can prevent neurons from neurotoxicity by inhibiting abnormal calcium influx caused by Abeta(1-40).

Amyloid beta-Peptides↗
PubMed2006

Estrogen down-regulates nicotine-induced adhesion molecule expression via nongenomic signal pathway in endothelial cells.

Although gonadal hormone mostly causes genotropic actions through the members of nuclear receptor family, it also can regulate these actions via membrane receptor. To explore the possibility of plasma membrane estrogen receptors (mER) mediating genotropic events, we have investigated estrogen's effect on nicotine-stimulated adhesion molecule expression and evaluated whether this effect depends on calcium, MAPK signal pathway. Fluorescence Spectroscopy analysis of Ca2+ from human umbilical vein endothelial cells (HUVECs) showed through mER, estrogen induced a rapid rise of intracellular free Ca2+ concentration and this rise could not be inhibited by tamoxifen (classic ER inhibitor). In the context of nicotine stimulating, however, estrogen attenuated phosphorylation of mitogen-activated protein kinase (MAPK) family members, extracellular signal regulated kinase 1/2 (ERK1/2), p38 but not c-Jun-N-terminal kinase (JNK) in HUVECs and this effect could not still be prevented by tamoxifen. In the meantime, estrogen also down-regulated surface/soluble vascular cell adhesion molecule (VCAM-1, sVCAM-1) and endothelial selectin (E-selectin, sE-selectin) levels, which was not abolished by tamoxifen either. Moreover, calcium chelator BAPTA, ERK1/2 inhibitor PD98059, p38 inhibitor SB203580 significantly reduced the production of nicotine-activated surface/soluble VCAM-1 and E-selectin and both of the remained levels were no longer regulated by estrogen. Our study here provides the information of decrease effect of mER-mediated estrogen through Ca2+ and ERK1/2, p38 MAPK signaling pathway on nicotine-stimulated expression of surface/soluble VCAM-1 and E-selectin in HUVECs.

Calcium Signaling↗
PubMed2006

Doublecortin-like kinase controls neurogenesis by regulating mitotic spindles and M phase progression.

The mechanisms controlling neurogenesis during brain development remain relatively unknown. Through a differential protein screen with developmental versus mature neural tissues, we identified a group of developmentally enriched microtubule-associated proteins (MAPs) including doublecortin-like kinase (DCLK), a protein that shares high homology with doublecortin (DCX). DCLK, but not DCX, is highly expressed in regions of active neurogenesis in the neocortex and cerebellum. Through a dynein-dependent mechanism, DCLK regulates the formation of bipolar mitotic spindles and the proper transition from prometaphase to metaphase during mitosis. In cultured cortical neural progenitors, DCLK RNAi Lentivirus disrupts the structure of mitotic spindles and the progression of M phase, causing an increase of cell-cycle exit index and an ectopic commitment to a neuronal fate. Furthermore, both DCLK gain and loss of function in vivo specifically promote a neuronal identity in neural progenitors. These data provide evidence that DCLK controls mitotic division by regulating spindle formation and also determines the fate of neural progenitors during cortical neurogenesis.

Animals↗
PubMed2006

Evidence that the promoter can influence assembly of antitermination complexes at downstream RNA sites.

The N protein of phage lambda acts with Escherichia coli Nus proteins at RNA sites, NUT, to modify RNA polymerase (RNAP) to a form that overrides transcription terminators. These interactions have been thought to be the primary determinants of the effectiveness of N-mediated antitermination. We present evidence that the associated promoter, in this case the lambda early P(R) promoter, can influence N-mediated modification of RNAP even though modification occurs at a site (NUTR) located downstream of the intervening cro gene. As predicted by genetic analysis and confirmed by in vivo transcription studies, a combination of two mutations in P(R), at positions -14 and -45 (yielding P(R-GA)), reduces effectiveness of N modification, while an additional mutation at position -30 (yielding P(R-GCA)) suppresses this effect. In vivo, the level of P(R-GA)-directed transcription was twice as great as the wild-type level, while transcription directed by P(R-GCA) was the same as that directed by the wild-type promoter. However, the rate of open complex formation at P(R-GA) in vitro was roughly one-third the rate for wild-type P(R). We ascribe this apparent discrepancy to an effect of the mutations in P(R-GCA) on promoter clearance. Based on the in vivo experiments, one plausible explanation for our results is that increased transcription can lead to a failure to form active antitermination complexes with NUT RNA, which, in turn, causes failure to read through downstream termination sites. By blocking antitermination and thus expression of late functions, the effect of increased transcription through nut sites could be physiologically important in maintaining proper regulation of gene expression early in phage development.

Artificial Gene Fusion↗
PubMed2006

The effects of five alkaloids from Bulbus Fritillariae on the concentration of cAMP in HEK cells transfected with muscarinic M(2) receptor plasmid.

The aim of this study was to investigate the effects of five alkaloids, namely verticine, verticinone, imperialine, imperialine-3beta-D-glucoside, and puqietinone, purified from Bulbus Fritillariae and used as an antitussive drug in traditional Chinese medicine, on their antimuscarinic M(2) function and the cAMP level of HEK cells transfected with muscarinic M(2) receptor plasmid. By transfecting the HEK cells with the method of calcium phosphate co-precipitation and screening with G418, the cells stably expressing M(2) receptor were identified. The expression of M(2) receptor in HEK cells was confirmed by both RT-PCR and western blot. The cAMP level in the treated cells was analyzed with RIA method ((125)I-cAMP KIT). And the results suggested that the five alkaloids could significantly elevate the cAMP concentration in the HEK cells transfected with muscarinic M(2) receptor plasmid (p < 0.01).

Alkaloids↗