[Unsupervised clustering analysis based on multidimensional features reveals distinct clinical characteristics and associated factors of different phenotypes in patients with chronic rhinosinusitis with nasal polyp].

Objective: To utilize routinely available clinical parameters to uncover the clinical features of different clusters in patients with chronic rhinosinusitis with nasal polyp (CRSwNP) through unsupervised clustering analysis. Methods: The clinical data from 155 CRSwNP patients undergoing nasal endoscopic surgery at Renmin Hospital of Wuhan University from 2021 to 2023 were prospectively collected, including 112 males and 43 females, aged from 7 to 87 years. Unsupervised clustering analysis was conducted using various clinical parameters, including age, gender, smoking and drinking history, local eosinophil (EOS) and neutrophil (NEU) counts, comorbid allergic rhinitis (AR), comorbid asthma, recurrence status, serum-specific IgE, total IgE, cytokine levels, peripheral blood EOS count and percentage, Lund-Mackay CT score, the ratio of CT scores for the maxillary sinus and ethmoid sinus (E/M ratio), visual analogue scale (VAS) score, Lund-Kennedy endoscopic score, and other common clinical indicators to elucidate the clinical characteristics of each cluster. Statistical analysis was conducted using GraphPad Prism 9.5 software. Results: Hierarchical clustering analysis identified four main clusters (Cluster A1-A4), showcasing distinct characteristics such as mild nasal polyps with higher peripheral blood cytokines levels, nasal polyps accompanied by allergies and asthma, a subtype of nasal polyps with high recurrence rates dominated by neutrophils, and nasal polyps with high eosinophil levels. Further subset clustering revealed two clusters of mild polyps (Cluster B1-B2) featuring high cytokine expression and comorbid AR; and two clusters of severe polyps (Cluster B3-B4) presented with severe symptoms, high Lund-Mackay CT score, and high Lund-Kennedy endoscopic score. Variations between Cluster B3 and B4 included symptom complexity, the degree of eosinophil infiltration, and the probability of comorbid asthma. Further clustering analysis for eosinophilic nasal polyps revealed a cluster characterized by highly neutrophilic infiltration and recurrent nasal polyps. The comprehensive analysis of multi-index correlations demonstrated valuable insights into the relationships between common clinical parameters of nasal polyps, providing valuable information for a deeper understanding of the pathogenesis of CRSwNP. Conclusion: The clustering analysis in this study categorizes CRSwNP patients into different clusters based on clinical features and disease outcomes, providing a new perspective for more precise clinical treatment strategies.

[Experimental study of dopamine ameliorating the inflammatory damage of olfactory bulb in mice with allergic rhinitis].

Objective: To investigate the effects of dopamine on olfactory function and inflammatory injury of olfactory bulb in mice with allergic rhinitis (AR). Methods: AR mouse model was established by using ovalbumin (OVA), and the mice were divided into two groups: olfactory dysfunction (OD) group and without OD group through buried food pellet test (BFPT). The OD mice were randomly divided into 2 groups, and OVA combined with dopamine (3, 6, 9 and 12 days, respectively) or OVA combined with an equal amount of PBS (the same treatment time) was administered nasally. The olfactory function of mice was evaluated by BFPT. The number of eosinophils and goblet cells in the nasal mucosa were detected by HE and PAS staining. Western blotting, immunohistochemistry or immunofluorescence were used to detect the expression of olfactory marker protein (OMP) in olfactory epithelium, the important rate-limiting enzyme tyrosine hydroxylase (TH) of dopamine, and the marker proteins glial fibrillary acidic protein (GFAP) and CD11b of glial cell in the olfactory bulb. TUNEL staining was used to detect the damage of the olfactory bulb. SPSS 26.0 software was used for statistical analysis. Results: AR mice with OD had AR pathological characteristics. Compared with AR mice without OD, the expression of OMP in olfactory epithelium of AR mice with OD was reduced (F=26.09, P<0.05), the expression of GFAP and CD11b in the olfactory bulb was increased (F value was 38.95 and 71.71, respectively, both P<0.05), and the expression of TH in the olfactory bulb was decreased (F=77.00, P<0.05). Nasal administration of dopamine could shorten the time of food globule detection in mice to a certain extent, down-regulate the expression of GFAP and CD11b in the olfactory bulb (F value was 6.55 and 46.11, respectively, both P<0.05), and reduce the number of apoptotic cells in the olfactory bulb (F=25.64, P<0.05). But dopamine had no significant effect on the number of eosinophils and goblet cells in nasal mucosa (F value was 36.26 and 19.38, respectively, both P>0.05), and had no significant effect on the expression of OMP in the olfactory epithelium (F=55.27, P>0.05). Conclusion: Dopamine can improve olfactory function in mice with AR to a certain extent, possibly because of inhibiting the activation of glial cells in olfactory bulb and reducing the apoptotic injury of olfactory bulb cells.

[The expression and significance of TRPM8 among chronic rhinosinusitis with nasal polyps].

Objective: To compare the expression and difference of melastatin-related transient receptor potential 8(TRPM8) among chronic rhinosinusitis, nasal polyps and normal mucosa tissues. And to explore the significant expression of TRPM8 among CRSwNP. Methods: Fifty-one patients underwent endoscopic sinus surgery in the Department of Otorhinolaryngology Head and Neck Surgery of Renmin Hospital of Wuhan University from February 2019 to January 2020 were recruited, including 33 males and 18 females, aged from 14 to 65 years old (34.55±1.689).Immunohistochemistry was used to detected the expression of TRPM8 protein among CRSsNP(17),CRSwNP (17) and control tissuses(17). In addition, the correlation between the expression of TRPM8 protein in CRSwNP patients and preoperative CT Lund-Mackay scores and preoperative VAS scores and sinonasal outcome test-20 scores was analyzed, respectively. The primary human nasal epithelial cells were cultured in vitro and the expression of TRPM8 was detected by quantitative real-time PCR and western blotting. The tissue in control group, chronic rhinosinusitis without nasal polyps (CRSsNP) group and the CRSwNP group were collected and grinded into tissue homogenized. The expression of TRPM8 protein was detected by western blotting after 24 h stimulation after homogenate was added into the medium of RPMI 2650 and primary nasal epithelial cells. Results: Compared with the control, the expression of TRPM8 was significantly up-regulated in nasal polyps (t=6.852, P<0.05). TRPM8 was mainly expressed in epithelial cells. The expression of TRPM8 in the epithelial cells of CRSsNP had no difference with the control group (t=1.980, P>0.05). In addition, the expression of TRPM8 in CRSwNP patients was positively correlated with the preoperative CT Lund-Mackay scores and VAS scores and SNOT-20 scores (r=0.512, P<0.05;r=0.853, P<0.01;r=0.814, P<0.01). After cultured primary epithelial cells in vitro, the expression level of TRPM8 in epithelial cells derived from nasal polyp was significantly higher than that in control group (t=8.845, P<0.05). By adding the homogenization of control and CRSsNP and CRSwNP tissues, the expression of TRPM8 in RPMI 2650 cells and primary nasal epithelial cells was changed and that was significantly increased after adding the homogenization of the group of CRSwNP. Conclusion: TRPM8 is highly expressed in nasal polyps epithelial cells, suggesting that TRPM8 may be involved in the pathogenesis of nasal polyps regulated by nasal epithelial cells.

[The sodium salicylate affects the expression of NMDA receptor and GABAa receptor subunits in spiral ganglion neurons of the cochlea through DA receptor].

Objective:To investigate mRNA expression of dopamine receptor subtypes in the rat cochlear spiral ganglion neurons (SGN) following exposure to the sodium salicylate. In addition, we observed the effect of sodium salicylate on N methyl-D-aspartic acid (NMDA) receptor subunit NR1 and gamma aminobutyric acid (GABA)a receptor subunit GABRα2 mRNA under the circumstance of DR activation or blocking. Moreover, we also focused on the the interaction between receptors mediated by SS.Method:Immunofluorescence techniques were applied to detect DR (DR1 and DR2) expression in cultured rat SGN. Moreover, RT-PCR was performed to assess NR1 and GABRα2 subunit mRNA.Result:Immunofluorescence images showed co-localization of DR1/DR2 and ßⅢ-tubulin protein in SGN bodies and axons. RT-PCR results illustrated that ①DR subtypes of DRd1-5 were detected in the SGN. ②The mRNA expression of all subtypes of DR and GABRα2, NR1 subunits were obviously upregulated except DRd3 after treatment with sodium salicylate. Among them, DRd1 expression increased 34.64%(t=-5.123,P=0.007),DRd2 expression increased 34.60%(t=-5.206,P=0.006),DRd4 expression increased 20.87%(t=-3.337,P=0.029),DRd5 expression increased 26.42%(t=-6.054,P=0.004),GABRα2 expression increased 30.41%(t=-2.839,P=0.047),NR1 expression increased 39.22%(t=-6.243,P=0.003).③After exposure to sodium salicylate (5 mmol/L), dopamine (100 µmol/L), DR1 agonist (SKF38393,20 µmol/L), DR2 agonist (Quinpirole,20 µmol/L), GABRα2 expression increased 21.78%,27.45%,33.02%,33.42% respectively (F=12.399,P=0.001),and NR1 expression increased 28.70%,26.82%,29.03%,35.05%(F=50.395,P=0.000) respectively.④Compared with the group of sodium salicylate treatment alone, both sodium salicylate + DR1 antagonist (SCH23390,20 µmol/L) group and sodium salicylate + DR2 antagonists (Eticlopride,20 µmol/L) group had a suppression on GABRα2 and NR1 mRNA expression.GABRα2 mRNA reduced 29.56%,37.10%(F=22.101,P=0.000) and NR1 mRNA expression decreased 37.62%,32.83% respectively(F=72.933,P=0.000).Conclusion:Most of the DR subtypes mRNA expression in SGN were increased following exposure to sodium salicylate. DR may be involved in the effect of sodium salicylate on GABAaR and NMDAR mRNA expression.

Short communication: Pheromonicin-SA affects mRNA expression of toll-like receptors, cytokines, and lactoferrin by Staphylococcus aureus-infected bovine mammary epithelial cells.

Pheromonicin-SA (Ph-SA) is a newly developed, engineered multidomain peptide that has a bactericidal effect against Staphylococcus aureus. The objective of this study was to characterize innate immune responses by Staph. aureus-stimulated bovine mammary epithelial cells (BMEC) following treatment with Ph-SA. Primary BMEC from one lactating Holstein cow were isolated and exposed to Staph. aureus for 2 h, and then treated with rifampicin or Ph-SA. Total RNA was isolated from BMEC at 0, 2, 6, 12, and 24 h postinfection, and the mRNA expression of selected genes, including toll-like receptor (TLR)2 and TLR4, IL-1ß, IL-6, IL-8, tumor necrosis factor α (TNF-α), and lactoferrin, was quantified by real-time PCR. In the rifampicin group, increases in the expression of mRNA for TNF-α, IL-1ß, IL-6, IL-8, and lactoferrin were observed at 6 h postinfection and in the expression of mRNA for TLR2 but not for TLR4 at 12 h postinfection. In the Ph-SA group, increases in the mRNA expression of TLR2, TNF-α, IL-1ß, IL-6, IL-8, and lactoferrin were observed at 6 h postinfection, and an increase in TLR4 mRNA expression was observed at 24 h postinfection. At 24 h postinfection, the mRNA expression of TLR4, TNF-α, IL-1ß, IL-6, IL-8, and lactoferrin was higher in the Ph-SA group than in the rifampicin group. In conclusion, Ph-SA might promote the expression of mRNA for TLR2, TLR4, the pro-inflammatory cytokines IL-1, IL-6, and TNF-α, the chemotactic factor IL-8, and lactoferrin in Staph. aureus-infected BMEC. Moreover, Ph-SA may be of value as an antibiotic in promoting innate immune responses by Staph. aureus-infected bovine mammary epithelial cells.

Serum concentration and mRNA expression in milk somatic cells of toll-like receptor 2, toll-like receptor 4, and cytokines in dairy cows following intramammary inoculation with Escherichia coli.

The objective of the current study was to investigate the toll-like receptors (TLR), including the soluble forms sTLR2 and sTLR4, involved in innate immune responses of dairy cows to experimentally induced Escherichia coli mastitis. Six clinically healthy Holstein dairy cows received an intramammary inoculation of E. coli O111:K58 between 63 and 83 d postpartum. Concentrations of sTLR2 and sTLR4, the proinflammatory cytokines IL-6 and tumor necrosis factor-α (TNF-α), and acute phase proteins serum amyloid A (SAA) and haptoglobin (Hp) in blood were measured by ELISA. Furthermore, 10mL of milk was collected from challenged quarters immediately before inoculation and at 6, 12, 24, 48, and 72 h after inoculation, and mRNA expression of selected genes, including TLR2, TLR4, IL-1ß, IL-6, TNF-α, and IL-8, was quantified by real-time PCR. Escherichia coli intramammary infection elicited a decrease in the circulating levels of leukocytes. Rectal temperature was elevated at 6h postinoculation (PI). Similarly, the serum concentrations of TNF-α, IL-6, and SAA increased at 6h PI. However, serum concentrations of sTLR2, sTLR4, and Hp did not differ after challenge. The mRNA expression of TLR2, IL-1ß, and IL-8 in milk somatic cells increased at 12h PI, whereas a decreased IL-6 mRNA expression was detected from 6 to 48 h PI. In conclusion, we found that TLR2 mRNA expression increased in milk somatic cells collected from infected quarters of cows challenged with E. coli, whereas the concentrations of sTLR2 and sTLR4 remained unchanged after challenge. Thus, sTLR2 and sTLR4 may protect the host by sequestrating pathogen-associated molecular patterns during E. coli mastitis.

Mesenchymal stem cells from Chinese Guizhou minipig by hTERT gene transfection.

INTRODUCTION: To provide standard implemental cells for biomedical research, an immortalized mesenchymal stem cell (MSC) lineage was established from the bone marrow of Chinese Guizhou minipigs. We investigated the characteristics of the MSC transfected with hTERT. METHODS: MSCs isolated from bone marrow (BM) were steadily transfected using a plasmid containing human telomerase reverse transcriptase gene (pCI-neo-hTERT) at population doubling (PD) time of 3. The expression of hTERT was analyzed by reverse transcriptase polymerase chain reaction; the morphology of the cells, by phase contrast microscope; cell growth curve, by MTS assay, and phenotype, flow cytometry and immunofluorescence. Karyotype and osteogenic differentiation of transfected cells were compared with normal MSCs. RESULTS: After transfection with the hTERT gene, the MSCs showed vigorous proliferation activity undergoing more than 60 PDs. Expression of the hTERT gene was detected in these cells. Transfected MSCs showed a prolonged life span, maintaining similar morphology and karyotype compared with normal MSCs. The apoptotic rate of transfected MSCs at PD60 was far below that of the normal MSCs at PD15. More than 99% of transfected MSCs were positive for stem cell markers, including SH-2, SH-3, SB-21, and CD29, and negative for CD34, CD45, and SLA-II. Transfected MSCs possessed the ability to differentiate into osteogenic as same as that of normal MSCs. CONCLUSION: The hTERT gene-induced immortalization prolonged the life span of MSCs, maintaining the overall properties identical to the original cells, findings that will be useful for basic research.

Pharmacokinetic characterization of hydroxylpropyl-beta-cyclodextrin-included complex of cryptotanshinone, an investigational cardiovascular drug purified from Danshen (Salvia miltiorrhiza).

1. The study aimed to investigate the pharmacokinetics of cryptotanshinone in a hydroxylpropyl-beta-cyclodextrin-included complex in dogs and rats. 2. Animals were administrated the inclusion complex of cryptotanshinone and the concentrations of cryptotanshinone and its major metabolite tanshinone IIA were determined by a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. 3. Cryptotanshinone in inclusion complex was absorbed slowly after an oral dose, and the C(max) and AUC(0-)(t) were dose-proportional. The bioavailability of cryptotanshinone in rats was (6.9% +/- 1.9%) at 60 mg kg(-1) and (11.1% +/- 1.8%) in dogs at 53.4 mg kg(-1). The t(1/2) of the compound in rats and dogs was 5.3-7.4 and 6.0-10.0 h, respectively. Cryptotanshinone showed a high accumulation in the intestine, lung and liver after oral administration, while the lung, liver and heart had the highest level following intravenous dose. Excretion data in rats showed that cryptotanshinone and its metabolites were mainly eliminated from faeces and bile, and the dose recovery rate was 0.02, 2.2, and 14.9% in urine, bile, and faeces, respectively. 4. The disposition of cryptotanshinone in an inclusion complex was dose-independent and the bioavailability was increased compared with that without cyclodextrin used to formulate the drug. Cryptotanshinone was distributed extensively into different organs. Excretion of cryptotanshinone and its metabolites into urine was extremely low, and they were mainly excreted into faeces and bile.

Preclinical factors affecting the pharmacokinetic behaviour of tanshinone IIA, an investigational new drug isolated from Salvia miltiorrhiza for the treatment of ischaemic heart diseases.

Tanshinone IIA (TSIIA) is a major active triterpenoid isolated from Salvia miltiorrhiza. The purposes of this study were to investigate various preclinical factors that determined the pharmacokinetics of TSIIA. After oral dosing at 6.7, 20, and 60 mg kg(-1), TSIIA was detected mainly as glucuronidated conjugate (TSIIAG) with only small amounts of the unchanged in the plasma. TSIIA was predominantly excreted into the bile and faeces as TSIIAG, and urine to a minor extent. The C(max) and AUC(0-)(t) of TSIIAG after i.p. administration were significantly lower than those after intragastric administration. The plasma concentration-time profiles of TSIIA following oral dosing of TSIIA showed multiple peaks. The C(max) and AUC(0-)(t) of TSIIA and its glucuronides in rats with intact bile duct were significantly lower than those of rats with bile duct cannulation. Studies from the linked-rat model and intraduodenal injection of bile containing TSIIA and its metabolites indicate that TSIIA glucuronides underwent hydrolysis and the aglycone was reabsorbed from the gut and excreted into the bile as conjugates. TSIIA had a wide tissue distribution, with a very high accumulation in the lung, but very limited penetration into the brain and testes. TSIIA was metabolized by rat CYP2C, 3A and 2D, as ticlopidine, ketoconazole and quinidine all inhibited TSIIA metabolism in rat liver microsomes. Taken collectively, these findings indicate that multiple factors play important roles in determining the pharmacokinetics of TSIIA.

Ponicidin inhibits cell growth on hepatocellular carcinoma cells by induction of apoptosis.

BACKGROUND AND AIM: Ponicidin is recently reported to have anti-tumour effects on a large variety of cancers. The present study was undertaken to investigate the anti-proliferation effects of ponicidin on hepatocellular carcinoma cells and its mechanism. METHODS: Two hepatocellular carcinoma cell lines, QGY-7701 and HepG-2 cells, were used. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell apoptosis was assessed by flow cytometry and deoxyribonucleic acid fragmentation analysis. Cell morphology was observed by Hoechst 33258 staining. Reverse transcriptase-polymerase chain reaction and Western blot analysis were used to detect Survivin as well as Bax and Bcl-2 expressions. RESULTS: Ponicidin could inhibit the growth of QGY-7701 and HepG-2 cells significantly by induction of apoptosis. Marked morphological changes of apoptosis were observed clearly. Both Survivin and Bcl-2 expressions were down-regulated remarkably while Bax expression up-regulated when apoptosis occurred. CONCLUSIONS: Ponicidin has significant anti-proliferation effects by inducing apoptosis on hepatocellular carcinoma cells in vitro, down regulation of Survivin and Bcl-2 as well as upregualation of Bax expressions may be the important apoptotic inducing mechanisms.

Hypercholesterolemia accelerates vascular calcification induced by excessive vitamin D via oxidative stress.

Hypercholesterolemia plays an important role in the initiation and progression of atherosclerosis and has a positive correlation with cardiovascular disease. Calcification is a common feature of atherosclerotic lesions and contributes to cardiovascular dysfunctions. The present study investigated the role of hypercholesterolemia in vascular calcification and its potential mechanism. Models of vascular calcification were established by administering vitamin D2 (VD) to rats alone or combined with a high-cholesterol diet (HCD) and by treating rat aorta smooth muscle cells (RASMCs) with beta-glycerophosphate (GP) alone or combined with oxidized low-density lipoprotein (oxLDL) in vitro. In rats, the combination of VD with HCD significantly enhanced vessel calcium deposition and the activity and mRNA expression of vessel alkaline phosphatase (ALP) compared to treatment with VD alone. This combination also enhanced serum levels of total cholesterol, oxLDL, and malondialdehyde as well as vascular production of superoxide anion, while it reduced the vascular activity of superoxide dismutase. Both simvastatin, a cholesterol-lowering agent, and antioxidant vitamin E antagonized the effects of the above combination. In RASMCs, oxLDL accumulation dependently accelerated calcium deposition in cell layers initiated by GP alone. Also, oxLDL stimulated ALP activity and mRNA expression in RASMCs in a concentration-dependent manner. Taken together, these results suggest that acceleration of vascular calcification by hypercholesterolemia might be attributed to oxidative stress and such calcification may be another target of statin or antioxidant action in antiatherosclerosis.

Crocetin improves endothelium-dependent relaxation of thoracic aorta in hypercholesterolemic rabbit by increasing eNOS activity.

Our previous studies have proven that crocetin (CCT), extracted from Gardenia jasminoides Ellis, possesses the anti-atherosclerotic effect. Because endothelial dysfunction strongly contributes to the initiation and progression of atherosclerosis, the present study aims to investigate whether CCT is capable of improving this dysfunction and to explore the possible mechanisms. Endothelial dysfunction was induced by in vivo feeding high cholesterol diet (HCD) to rabbit and by in vitro treating bovine aortic endothelial cells (BAECs) with oxidized LDL (oxLDL). Endothelium-dependent relaxation (EDR) evoked by acetylcholine (Ach) and endothelium-independent relaxation (RIDR) mediated by sodium nitroprusside (SNP) of thoracic aorta isolated from rabbit were measured. The results indicated that the EDR in HCD alone treated rabbits was seriously impaired and the maximal relaxation induced by Ach (10(-5.5) M) was only 54% that in control rabbit fed with regular diet. Oral complementation with CCT (15, 30 mg/kg) dose-dependently improved this impairment and restored the maximal relaxation to 68% and 80% that in control group, respectively. However, the EIDR maintained comparable in all groups. Complementation with CCT (15, 30 mg/kg) simultaneously increased serum level of nitric oxide (NO), upregulated vessel activity and mRNA expression of endothelial NO synthase (eNOS) as well as vessel cyclic GMP (cGMP) content compared with those in rabbit treated with HCD alone. Inducible NOS (iNOS) activity remained unchangeable in all groups. In BAECs, oxLDL treatment decreased NO production, downregulated both activity and mRNA expression of eNOS. While those decrease or downregulation were inhibited by co-treatment with CCT (0.1, 1, 10 microM) in a dose-dependent manner. These findings suggested that CCT significantly restored the EDR of thoracic aorta in hypercholesterolemic rabbit, which might be explained by its action to increase the vessel eNOS activity, leading to elevation of NO production.

Targeted regulation of imprinted genes by synthetic zinc-finger transcription factors.

Epigenetic control of transcription is essential for mammalian development and its deregulation causes human disease. For example, loss of proper imprinting control at the IGF2-H19 domain is a hallmark of cancer and Beckwith-Wiedemann syndrome, with no targeted therapeutic approaches available. To address this deficiency, we engineered zinc-finger transcription proteins (ZFPs) that specifically activate or repress the IGF2 and H19 genes in a domain-dependent manner. Importantly, we used these ZFPs successfully to reactivate the transcriptionally silent IGF2 and H19 alleles, thus overriding the natural mechanism of imprinting and validating an entirely novel avenue for 'transcription therapy' of human disease.

Purification and characterization of the membrane-bound complex of an ABC transporter, the histidine permease.

The bacterial histidine permease, an ABC transporter, from Salmonella typhimurium is composed of a membrane-bound complex, HisQMP2, comprising two hydrophobic subunits (HisQ and HisM), two copies of an ATP-hydrolyzing subunit, HisP, and a soluble receptor, HisJ. We describe the purification and characterization of HisQMP2 using a 6-histidines extension at the carboxy terminus of HisP [HisQMP2(his6)]. The purification is rapid and effective, giving a seven-fold purification with a yield of 85 and 98% purity. Two procedures are described differing in the detergent used (decanoylsucrose and octylglucoside, respectively) and in the presence of phospholipid. HisQMP2(his6) has ATPase and transport activities upon reconstitution into proteoliposomes (PLS). HisQMP2(his6) has a low level ATPase activity (intrinsic activity), which is stimulated to a different extent by the receptor--liganded and unliganded. Its pH optimum is 7.8-8.0, it requires a cation for activity and it displays cooperativity for ATP. The effect of various ATP analogs was analyzed. Determination of the molecular size of HisQMP2(his6) indicates that it is a monomer. The permeability properties of two kinds of reconstituted PLS preparations are described.

[Effect of aldosterone on the secretion of endothelin by ventricular fibroblasts].

Using cell culture, radioimmunoassay for endothelin and RT-PCR, the effect of aldosterone on the endothelin secretion of ventricular fibroblasts was studied. The results showed that aldosterone (1 x 10(-7) mol/L) promoted the expression of ppET-1 mRNA, which began to increase in 2 hours and attained the highest level in 4 hours, thereafter decreased; aldosterone increased the endothelin level in ventricular fibroblasts and fibroblast conditioned growth medium (FCGM) as well, which was blocked by spironolactone (1 x 10(-6) mol/L), an aldosterone receptor antagonist. The results suggest that aldosterone can increase endothelin secretion by ventricular fibroblasts, which can be inhibited by its receptor antagonist spironolactone.

[Role of conditioned growth medium for ventricular fibroblasts in promoting fibroblast collagen synthesis and proliferation].

Ventricular fibroblasts were cultured using conditioned growth medium for ventricular fibroblasts (FCGM). The rate of the total collagen synthesis of ventricular fibroblasts was measured by assaying the incorporation rate of [3H]-proline, whereas the proliferation of ventricular fibroblasts was assessed by determining the incorporation rate of [3H]-TdR and the expression of c-fos genes. FCGM significantly increased the [3H]-proline incorporation rate and [3H]-TdR incorporation rate of fibroblasts in a dose-dependent manner. Furthermore, FCGM promoted the c-fos gene expression of fibroblasts, which attained its maximum in 1 h. BQ123, an ETA receptor antagonist, partially blocked the above effects of FCGM, but AT1 receptor antagonist CV11974 and alpha-adrenergic receptor antagonist regitin did not. It is suggested that the ventricular fibroblast has an autorine function in promotion of collagen synthesis and proliferation of fibroblasts by secreting endothelin and other bioactive substances.

[Role of mitogen-activated protein kinase in the inhibition of myocardial hypertrophy by nitric oxide in renovascular hypertensive rats].

The aim of this study was to examine the effects of L-arginine, a nitric oxide (NO) precursor, on protein expression of endothelial nitric oxide (eNOS), nitrite/nitrate content, protein expression of mitogen-activated protein kinase phosphatase-1 (MKP-1) and the activity of mitogen-activated protein kinase (MAPK) in cardiac tissues in renovascular hypertensive rats (RHR). The Goldblatt renovascular hypertensive model was established by two-kidney one clip method. The rats were divided into four groups, respectively treated with 50, 150 and 450 mg/kg L-arginine and 150 mg/kg L-arginine plus 10 mg/kg L-NAME (an eNOS inhibitor) (i.p.). Another group did not receive specific treatment from the 5th week after renal artery constriction. Control group was sham-operated. Mean arterial blood pressure (MABP) and the ratio of left ventricular weight to body weight (LVW/BW) were measured 8 weeks after treatment. eNOS protein expression, nitrite/nitrate content, MKP-1 protein expression and MAPK activity in cardiac tissues were detected using Western blot analysis, enzyme-reduction method and substrate in-gel kinase assay, respectively. It was found that L-arginine significantly inhibited the increase of MABP and LVW/BW, attenuated the activity of MAPK, increased protein expression of eNOS and MKP-1 and potentiated production of NO in cardiac tissue with the most effective dosage of 150 mg/kg, and these effects of L-arginine could be inhibited by L-NAME. These results suggest that MKP-1 may play an important role in the NO-induced inhibition of myocardial hypertrophy. The anti-hypertrophic effects of L-arginine may involve increase of eNOS protein expression and NO production, potentiation of MKP-1 protein expression, and inhibition of MAPK activity in the cardiac tissue of RHR.

Regulation of an endogenous locus using a panel of designed zinc finger proteins targeted to accessible chromatin regions. Activation of vascular endothelial growth factor A.

We have mapped conserved regions of enhanced DNase I accessibility within the endogenous chromosomal locus of vascular endothelial growth factor A (VEGF-A). Synthetic zinc finger protein (ZFP) transcription factors were designed to target DNA sequences contained within the DNase I-hypersensitive regions. These ZFPs, when fused to either VP16 or p65 transcriptional activation domains, were able to activate expression of the VEGF-A gene as assayed by mRNA accumulation and VEGF-A protein secretion through a range exceeding that induced by hypoxic stress. Importantly, multiple splice variants of VEGF-A mRNA with defined physiological functions were induced by a single engineered ZFP transcription factor. We present evidence for an enhanced activation of VEGF-A gene transcription by ZFP transcription factors fused to VP16 and p65 targeted to two distinct chromosomal sites >500 base pairs upstream or downstream of the transcription start site. Our strategy provides a novel approach for dissecting the requirements for gene regulation at a distance without altering the DNA sequence of the endogenous target locus.

[Down-regulation of ETA receptor of vascular smooth muscle cells by 17 beta-estradiol].

In the present study, the effects of 17 beta-estradiol on vascular reactivity of ovariectomized rats and proliferation of cultured rat aortic smooth muscle cells were studied. The vascular reactivity was significantly increased in ovariectomized rats compared with the sham-operated animals. The selective ETA receptor antagonist BQ123 inhibited the increase in [3H]-TdR incorporation in response to ET-1 on vascular smooth muscle cells (VSMCs). 17 beta-estradiol also attenuated the ET-1 effects in a dose-dependent manner. The results of RT-PCR and Western blot show that expression of ETA receptor was decreased after treatment with 17 beta-estradiol. The effect of 17 beta-estradiol was partially inhibited by estrogen receptor antagonist tamoxifen. The above results demonstrate that proliferation of VSMCs stimulated by ET-1 was mainly mediated through ETA receptor. Due to the down-regulation of ETA receptor and mediation of estrogen receptor, 17 beta-estradiol inhibits the ET-1-induced proliferation of VSMCs and decreases the vascular reactivity of ovariectomized rats.