A new anthraquinone and a new naphthoquinone from the whole plant of Spermacoce latifolia.

A phytochemical study on the whole plant of Spermacoce latifolia led to the isolation of a new anthraquinone, 1,2,6-trihydroxy-5-methoxy-9,10-anthraquinone (1), and a new naphthoquinone, (2R)-6-hydroxy-7-methoxy-dehydroiso-α-lapachone (2), together with three known anthraquinones (3-5). Their structures were established on the basis of detailed spectroscopic analysis, including one- and two-dimensional NMR, ESI-MS, and HR-ESI-MS techniques. All the compounds were isolated from S. latifolia for the first time. Compounds 1, 2, 4, and 5 showed significant antibacterial activity toward Bacillus subtilis with MIC values ranging from 0.9 to 31.2 µg/ml, and compound 4 aslo exhibited antibacterial activity against Bacillus cereus with a MIC value 62.5 µg/ml. Compound 1 was further revealed to show significant in vitro α-glucosidase inhibitory activity with IC50 value of 0.653 mM.

Exploring key genes associated with neutrophil function and neutrophil extracellular traps in heart failure: a comprehensive analysis of single-cell and bulk sequencing data.

Background: Heart failure (HF) is a complex and heterogeneous manifestation of multiple cardiovascular diseases that usually occurs in the advanced stages of disease progression. The role of neutrophil extracellular traps (NETs) in the pathogenesis of HF remains to be explored. Methods: Bioinformatics analysis was employed to investigate general and single-cell transcriptome sequencing data downloaded from the GEO datasets. Differentially expressed genes (DEGs) associated with NETs in HF patients and healthy controls were identified using transcriptome sequencing datasets and were subsequently subjected to functional enrichment analysis. To identify potential diagnostic biomarkers, the random forest algorithm (RF) and the least absolute shrinkage and selection operator (LASSO) were applied, followed by the construction of receiver operating characteristic (ROC) curves to assess accuracy. Additionally, single-cell transcriptome sequencing data analysis identified key immune cell subpopulations in TAC (transverse aortic constriction) mice potentially involved in NETs regulation. Cell-cell communication analysis and trajectory analysis was then performed on these key cell subpopulations. Results: We identified thirteen differentially expressed genes (DEGs) associated with NET through differential analysis of transcriptome sequencing data from HF (heart failure) samples. Utilizing the Random Forest and Lasso algorithms, along with experimental validation, we successfully pinpointed four diagnostic markers (CXCR2, FCGR3B, VNN3, and FPR2) capable of predicting HF risk. Furthermore, our analysis of intercellular communication, leveraging single-cell sequencing data, highlighted macrophages and T cells as the immune cell subpopulations with the closest interactions with neutrophils. Pseudo-trajectory analysis sheds light on the differentiation states of distinct neutrophil subpopulations. Conclusion: In this study, we conducted an in-depth investigation into the functions of neutrophil subpopulations that infiltrate cardiac tissue in TAC mice. Additionally, we identified four biomarkers (CXCR2, FCGR3B, VNN3, and FPR2) associated with NETs in HF. Our findings enhance the understanding of immunology in HF.

Evaluation of the immunogenicity of a single chain chimeric peptide composed of hCGß and oLHα for inhibition of the growth of hCGß-expressing cancer cells.

Human chorionic gonadotropin (hCG) is a membrane-associated protein highly expressed in several types of human cancer cells. The expression in the cancer cells indicates that hCG may be a potential target molecule for cancer immunotherapy. The objective of this study was to develop a novel immunogenic molecule, which can efficiently induce the neutralizing antibody against hCG and which is also suitable for mass production. The immunogenicity of the recombinant single chain chimeric protein of hCGß-oLHα expressed by yeast was examined. Additionally, the inhibitory effects of the anti-hCGß-oLHα antibody on the growth of hCG-positive cancer cells were determined. It was found that hCGß-oLHα yielded high titers of anti-hCG rabbit antibody that could effectively neutralize the bioactivity of hCG. The rabbit anti-hCGß-oLHα IgG inhibited the proliferation of hCG-expressing human colorectal cancer cells (LS-174, HCT-116, HCT-15 and KM-12) in a dose-dependent manner. Furthermore, an intact anti-tumor vaccine was prepared by conjugating hCGß-oLHα with tetanus toxoid (TT) and this was used to immunize Balb/c mice bearing hCG-expressing SP2/0 tumor cells. The progression of tumors in these immunized mice was remarkably inhibited. These results suggest that hCGß-oLHα is a new promising immunogenic molecule for the development of an anti-hCG-based cancer vaccine.

Technical standards of a foldable capsular vitreous body in terms of mechanical, optical, and biocompatible properties.

We previously proposed a new strategy to fabricate a novel foldable capsular vitreous body (FCVB) as a vitreous substitute and found that the FCVB was a very good replacement for closely mimicking the morphology and restoring the physiologic function of the rabbit vitreous body. The aim of this article was to assess the mechanical, optical, and biocompatible properties of a FCVB made from liquid silicone rubber. The mechanical properties show that the shore hardness is 37.80 degrees, the tear strength is 47.14 N/mm, the tensile strength is more than 7.28 MPa, and the elongation ratio is more than 1200%; in addition, the FCVB has 300 nm mili apertures in the capsule. The optical properties reveal that transmittances are 92%, hazes are 5.74%, and spectral transmittance is 97%. The transmittance mission is 2.3% and can sustain a 1500 mW, 0.2 s, 532 nm green laser. The biocompatible properties are shown in the stable extracts experiment, no significant fever, good genetic safety, and no structural abnormality or apoptosis in the cornea, ciliary body, and retina over a 6-month observation period. These results indicate that the FCVB has good mechanical, optical, and biocompatible properties, and the assessment results can be recommended as the FCVB technical standards for industrial manufacturing and inspection.

A single intrauterine injection of the serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride reversibly inhibits embryo implantation in mice.

BACKGROUND: The study was conducted to investigate the inhibitory effect of 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) on embryo implantation in mice with a view to identifying whether it might be a suitable agent for postcoital contraception. STUDY DESIGN: The anti-implantation efficacy of AEBSF was determined by counting the number of visible implanted embryos on Day 8 of pregnancy following a single intrauterine injection of AEBSF at doses of 30, 300 and 3000 microg per mouse uterine horn on Day 3 of pregnancy. The reversibility of the inhibitory effect of AEBSF on implantation was further evaluated by observing the outcome of a subsequent pregnancy without AEBSF treatment. RESULTS: A dose-dependent inhibitory effect of AEBSF on embryo implantation in vivo was observed. Morphological analysis revealed no significant cytotoxicity of AEBSF on the mouse uterine epithelia. Furthermore, the anti-implantation effect of AEBSF was reversible. CONCLUSION: Intrauterine administration of AEBSF at an appropriate dose might provide a basis for the development of novel contraception.

Effect of chronic treatment of ohmefentanyl stereoisomers on cyclic AMP formation in Sf9 insect cells expressing human mu-opioid receptors.

The binding affinity of ohmefentanyl stereoisomers for mu-opioid receptors and the effect of chronic ohmefentanyl stereoisomers pretreatments on intracellular cAMP formation were investigated in Sf9 insect cells expressing human mu-opioid receptors (Sf9-mu cells). Competitive assay of [3H]ohmefentanyl binding revealed that these isomers had high affinity for micro-opioid receptors in Sf9-mu cells. Isomer F9204 had the highest affinity for mu-opioid receptors with the Ki value of 1.66 +/- 0.28 nM. After pretreated Sf9-mu cells with increasing concentrations of these isomers for 6 h, addition of naloxone (1 microM) precipitated an overshoot of foskolin-stimulated cAMP accumulation. The ability of these isomers to induce cAMP overshoot differed greatly with the order of F9202>F9205>F9208>F9206>F9204>F9207. Of these isomers, F9202 was 2.7-fold less potent than F9204 in receptor binding affinity, but 71.5-fold more potent in ability to induce cAMP overshoot. These results suggested that there was a significant stereo-structural difference among ohmefentanyl stereoisomers in ability to induce naloxone-precipitated cAMP overshoot in Sf9-mu cells.

Structure prediction and activity analysis of human heme oxygenase-1 and its mutant.

AIM: To predict wild human heme oxygenase-1 (whHO-1) and hHO-1 His25Ala mutant (delta hHO-1) structures, to clone and express them and analyze their activities. METHODS: Swiss-PdbViewer and Antheprot 5.0 were used for the prediction of structure diversity and physical-chemical changes between wild and mutant hHO-1. hHO-1 His25Ala mutant cDNA was constructed by site-directed mutagenesis in two plasmids of E. coli DH5alpha. Expression products were purified by ammonium sulphate precipitation and Q-Sepharose Fast Flow column chromatography, and their activities were measured. RESULTS: rHO-1 had the structure of a helical fold with the heme sandwiched between heme-heme oxygenase-1 helices. Bond angle, dihedral angle and chemical bond in the active pocket changed after Ala25 was replaced by His25, but Ala25 was still contacting the surface and the electrostatic potential of the active pocket was negative. The mutated enzyme kept binding activity to heme. Two vectors pBHO-1 and pBHO-1(M) were constructed and expressed. Ammonium sulphate precipitation and column chromatography yielded 3.6-fold and 30-fold higher purities of whHO-1, respectively. The activity of delta hHO-1 was reduced 91.21% after mutation compared with whHO-1. CONCLUSION: Proximal His25 ligand is crucial for normal hHO-1 catalytic activity. delta hHO-1 is deactivated by mutation but keeps the same binding site as whHO-1. delta hHO-1 might be a potential inhibitor of whHO-1 for preventing neonatal hyperbilirubinemia.

Analysis of heme oxygenase isomers in rat.

AIM: To purify and identify heme oxygenase (HO) isomers which exist in rat liver, spleen and brain treated with hematin and phenylhydrazine and in untreated rat liver and to investigate the characteristics of HO isomers, to isolate and confirm the rat HO-1 cDNA that actually encodes HO-1 by expressing cDNA in monkey kidney cells (COS-1 cells), to prepare the rat heme oxygenase-1 (HO-1) mutant and to detect inhibition of HO-1 mutated enzyme. METHODS: First, rat liver, spleen and brain microsomal fractions were purified by DEAE-Sephacel and hydroxylapatite. The characteristics including activity, immunity and inducibility of two isomers (HO-1 and HO-2), and their apparent molecular weight were measured by detecting enzymatic activities, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis, respectively. Second, plasmid pcDNA3HO1 containing native rat HO-1 cDNA and pcDNA3HO1D25 carrying mutated rat HO-1 cDNA (His25Ala) were constructed by site-directed mutagenesis. COS-1 cells transfected with pcDNA3HO1 and pcDNA3HO1D25 were collected and disrupted by sonication, the microsomes were prepared by ultracentrifugation. Third, the inhibition of rat HO-1 mutant was analyzed. RESULTS: Two isomers were purified and identified in treated rat liver, spleen, brain and untreated rat liver. HO-1 was the predominant form with a ratio of 2.0:1 and 3.2:1 of HO-1 and HO-2 in liver and spleen, respectively, but only the activity of HO-2 in the brain and untreated liver could be detected. The apparent molecular weights of HO-1 and HO-2 were about M(r)30 000 and M(r) 36 000 under reducing conditions, respectively. The antiserum against liver HO-2 was employed in Western blotting analysis, the reactivity of HO-1 in the liver was not observed. The plasmid pcDNA3HO1 was highly expressed in endoplasmic reticulum of transfected COS-1 cells. The specific activity was -5-fold higher than that of the control. However, the enzyme activity of mutated HO-1 declined. While an equal amount of mutant was added to the enzyme reaction system, the levels of bilirubin decreased 42 %. CONCLUSION: The studies suggest that HO-1 and HO-2 exist in the hematin and phenylhydrazine treated rat liver and spleen, but only HO-2 in the brain and untreated liver. Two constitutive forms are different in molecular weight, inducibility and immunochemical properties. The activity of expressed HO-1 in COS-1 cells is higher than that of purified enzyme from rat spleen tissue. It suggests that this clone has an insert of 1030 base-pairs encodes HO-1. His25Ala mutant reduced the formation of bilirubin and it suggests that the mutant could completely bind the heme with native enzyme.

Human &mgr; Opioid Receptor Expressed in Sf9 Insect Cells was Functionally Coupled to Endogenous G Protein.

Human &mgr; opioid receptor(H&mgr;OR) with a tag of six consecutive histidines at its carboxyl terminus overexpressed in recombinant baculovirus infected Sf9 insect cells was demonstrated to be functionally coupled to endogenous G proteins. Na(+) and GTP could reduce the affinity binding of etorphine and Ohmefentanyl(Ohm) to H&mgr;OR overexpressed in Sf9. The binding of (35)S GTPgammaS to Sf9 cell membranes containing H&mgr;OR was stimulated by DAGO and Ohm. This stimulation could be blocked by naloxone or the pretreatment of PTX. Also, DAGO and Ohm could inhibit the increasing of intracellular cAMP stimulated by forskolin. The results showed H&mgr;OR expressed in Sf9 insect cells were functionally coupled to endogenous PTX sensitive G(i/o) proteins.

Cloning, Expression and Antibody Production of the Disintegrin Domain of Human Fertilin beta.

A cDNA for the disintegrin domain (hf279) was isolated by PCR from human testis cDNAs. DNA sequencing indicated that hf279 cDNA encoded 93 amino acid residues, and it was identical with the reported sequence of fertilin beta. An expression plasmid, pGEX hf279, was constructed by inserting hf279 cDNA into plasmid pGEX-4T-2 containing gst gene. The expression plasmid was introduced into E.coli BL21(DE3) cells and a substantial amount of soluble fused protein GST-HF93 was obtained by the expression strain HF93/BL21 induced with IPTG. SDS-PAGE analysis revealed that the GST-HF93 fusion protein had an apparent molecular weight of 38 kD and accumulated up to 50% of bacterial soluble proteins. The fusion protein was purified by glutathione S-transferase (GST) Sepharose 4B column (purity 90%) and digested by thrombin to obtain the purified HF93 peptide (purity 80%). Polyclonal antibodies were obtained from the serum of miceimmunized with purified HF93 which was isolated by GST Sepharose 4B column and SDS-PAGE. ELISA and Western blot analysis showed its specificity to HF93. Therefore this antibody can be used in further studies on the function of HF93.

[Cloning, expression, and antibody production of mouse ISP2].

A cDNA encoding mouse implantation serine proteinase 2 ISP2 was amplified from total cDNAs of mouse uterus implantation sites on D4.5 of pregnancy by PCR, and sequenced GenBank accession No. A442918 . DNA sequencing indicated that the ISP2 cDNA had an unreported 204 bp sequence at 3' untranslated region besides the open reading frame encoding 279 amino acid residues, which was identical with literature. In order to obtain recombinant ISP2 rISP2 an expression plasmid pGEX-4T-2/ISP2 was constructed and transformed into E.coli BL21(DE3) strain. Expressed fusion protein GST-ISP2 was purified by SDS-PAGE and digested with thrombin, and the digestion mixture was subjected to SDS-PAGE again to recover rISP2. Rabbits were immunized using rISP2 as immunogen, and the polyclonal anti-ISP2 antisera were obtained. Immunohistochemical analysis using this antisera showed specific and high expression of ISP2 in mouse endometrial gland epithelium in early pregnancy.

Heme oxygenase-1-mediated CD4+CD25high regulatory T cells suppress allergic airway inflammation.

Heme oxygenase-1 (HO-1) has anti-inflammatory effects in asthma. CD4+CD25(high) regulatory T cells (Treg) are a potent immunoregulator that suppresses the immune response. We studied the effects of HO-1-mediated CD4+CD25(high) Treg on suppression of allergic airway inflammation by comparing mice treated with hemin, OVA, Sn-protoporphyrin (SnPP), and hemin plus SnPP. Airway responsiveness, airway eosinophil infiltration, the level of OVA-specific IgE, and the numbers of cells in general and eosinophils in particular in bronchial alveolar lavage fluid were lower in the hemin group than in the OVA, SnPP, and hemin plus SnPP groups. The expressions of HO-1 mRNA and protein in the lung were increased by repeated administrations of hemin and SnPP. However, the activity of HO-1 was highest in hemin mice. The percentage and suppressive function of CD4+CD25(high) Treg and the expression of Foxp3 mRNA were obviously enhanced after treatment with hemin. This increase was diminished by the administration of SnPP. The concentration of serum IL-10 was higher in the hemin group than in the other groups, whereas the level of serum TGF-beta did not significantly differ across groups. Furthermore, the ratio of IFN-gamma/IL-4 mRNA in the lung was higher in hemin-treated mice than in OVA and SnPP mice. The suppressive capacity of CD4+CD25(high) Treg was not enhanced in the IL-10-deficient mice treated with hemin. In conclusion, our experiments in the animal model demonstrated that HO-1 has anti-inflammatory effects, probably via enhancement of the secretion of IL-10 and promotion of the percentage of CD4+CD25(high) Treg.

Expression of dopamine D1 receptor in Sf9 insect cells and agonism of l-12-chloroscoulerine on recombinant D1 receptor.

AIM: To express dopamine D1 receptor in baculovirus-Sf9 cell system, and to investigate the effects of l-12-chloroscoulerine (l-CSL) on the recombinant D1 receptor (D1R). METHODS: The recombinant baculovirus, Autographa californica nuclear polyhedrosis virus bearing D1R (AcNPV- D1R) was generated, and then was used to produce recombinant D1R in Sf9 insect cells. Expression of D1R in Sf9 cells was monitored by [3H]SCH23390 binding assay. The effects of l-CSL on recombinant D1R were investigated by [3H]SCH23390 binding assay and cAMP assay. RESULTS: The recombinant baculovirus AcNPV bearing D1R cDNA was generated, and was successfully expressed in Sf9 insect cells. The expression level of (Bmax) was (0.94+/-0.06) nmol/g protein. The Kd value of [3H]SCH23390 was (1.9+/-0.3) nmol/L, which was consistent with the previous results from calf striatum tissues. l-CSL had a high affinity to recombinant D1R with Ki value of (6.3+/-1.4) nmol/L, and increased the intracellular cAMP level in a concentration-dependent manner with EC50 value of 0.72 micromol/L and 95 % confidence limit was 0.67-0.77 micromol/L. Thus l-CSL has the D1 receptor agonism. CONCLUSION: An efficient baculovirus-Sf9 insect cell system for dopamine D1 receptor was constructed and l-CSL presented the D1 receptor agonism on cellular-molecular level directly.

Binding affinity to and dependence on some opioids in Sf9 insect cells expressing human mu-opioid receptor.

AIM: To investigate the receptor binding affinity and naloxone-precipitated cAMP overshoot of dihydroetorphine, fentanyl, heroin, and pethidine in Sf9 insect cells expressing human mu-opioid receptor (Sf9-mu cells). METHODS: Competitive binding assay of [3H]ohmefentanyl was used to reveal the affinity for mu-opioid receptor in Sf9-mu cells. [3H]cAMP RIA was used to determine cAMP level. Antinociceptive activity was evaluated using degree 55 mouse hot plate test. Naloxone-precipitated withdrawal jumping was used to reflect physical dependence in mice. RESULTS: All drugs displayed antinociceptive activity and produced physical dependence in mice. The K(i) values of dihydroetorphine, fentanyl, heroin, and pethidine in competitive binding assay were (0.85+/-0.20) nmol, (59.1+/-11.7) nmol, (0.36+/-0.13) micromol, and (12.2+/-3.8) micromol respectively. The binding affinities of these drugs for mu-opioid receptor in Sf9-mu cells were paralleled to their antinociceptive activities in mice. After chronic pretreatment with these drugs, naloxone induced cAMP withdrawal overshoot in Sf9-mu cells. The dependence index in Sf9-mu cells was calculated as K(i) value in competitive binding assay over EC(50) value in naloxone-precipitated cAMP assay. The physical dependence index in mice was calculated as antinociceptive ED(50)/withdrawal jumping cumulative ED(50). There was a good linear correlation between dependence index in Sf9-mu cells and physical dependence index in mice. CONCLUSION: The Sf9-mu cells could be used as a cell model to evaluate the receptor binding affinity and physical dependent liability of analgesic agents.