MiR-140-5p inhibits larynx carcinoma invasion and angiogenesis by targeting VEGF-A.

OBJECTIVE: Larynx carcinoma is a common head-neck malignant tumor. Recent investigations showed the involvement of microRNA (miR) in regulation of multiple tumors. miR-140-5p showed decreased expression in various cancers, but without knowledge regarding its expression in larynx carcinoma and effects on cell invasion and angiogenesis. MATERIALS AND METHODS: Real-time quantitative PCR was firstly employed to measure miR-140-5p expression in larynx carcinoma and controlled tumor adjacent tissues. In larynx cancer cell line, agomir or antagomir of miR-140-5p was applied to up-regulate or down-regulate miR-140-5p, respectively. Western blot was used to evaluate vascular endothelial growth factor A (VEGF-A) expression, and cell proliferation was modified by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H- tetrazolium bromide (MTT) approach. Transwell approach was used to measure cell invasion, and angiogenesis assay was used to detect the effect on angiogenesis. Luciferase report assay (LRA) measured targeting binding between miR-140-5p and VEGF-A. RESULTS: Comparing to tumor adjacent tissues, larynx carcinoma cells showed significantly decreased miR-140-5p expression. Agomir up-regulated miR-140-5p expression and weakened proliferation and invasion potency, and inhibited angiogenesis. Antagomir down-regulated miR-140-5p and presented the opposite results. Finally, LRA confirmed direct binding between miR-140-5p and VEGF-A. CONCLUSIONS: MiR-140-5p can target VEGF-A in larynx carcinoma cell line to inhibit cell invasion and angiogenesis. MiR-140-5p thus may work as the direct molecular target of larynx carcinoma.

LncRNA PCAT-1 regulates the proliferation, metastasis and invasion of cervical cancer cells.

OBJECTIVE: To investigate the effects of long non-coding RNA (lncRNA) PCAT-1 on the proliferation, metastasis, and invasion of cervical cancer cells. MATERIALS AND METHODS: LncRNA PCAT-1 small interfering RNA (siRNA) and negative siRNA were transfected into cervical cancer cell lines and the expression of lncRNA PCAT-1 in cells was confirmed by Real-time quantitative polymerase chain reaction (qPCR). Cell counting kit-8 (CCK-8) and colony formation assay were applied to detect the effect of lncRNA PCAT-1 on cell proliferation. The wound-healing assay was applied to test the effect of lncRNA PCAT-1 on cell metastasis. Matrigel cell invasion assay was performed to detect the impact of lncRNA PCAT-1 expression on invasion. RESULTS: After transfected with the long non-coding PCAT-1 siRNA into cervical cancer cell lines for 48 h, the lncRNA PCAT-1 cells were significantly down-regulated. The results of CCK-8, clonogenic and wound-healing assay showed that the decreased expression of lncRNA PCAT-1 attenuated the proliferation and metastasis of cells. The results of matrigel cell invasion assay manifested that the decreased expression of lncRNA PCAT-1 could reduce the invasion ability. The up-regulation of lncRNA PCAT-1 was associated with poor prognosis of patients with cervical cancer. CONCLUSIONS: LncRNA PCAT-1 siRNA transfected into cervical cancer cell lines can effectively lower the expression of lncRNA PCAT-1, while lncRNA PCAT-1 expression can inhibit the proliferation, metastasis and invasion abilities of cervical cancer cells.

[Preterm ovarian hyperstimulation syndrome: a case report and literature review].

OBJECTIVE: To discuss the clinical characteristics, pathogenesis, diagnosis and treatment of preterm ovarian hyperstimulation syndrome(POHS). METHOD: The process of diagnosis and treatment of a test-tube female baby were summarized. She was deliveried at 32(+ 2) weeks of gestation, diagnosed with POHS, and born in the First Affiliated Hospital of Wenzhou Medical University(in 2015). Retrieval of related literature in PubMed database and Wanfang database was performed using the key words"ovarian hyperstimulation syndrome"and"preterm infants or newborns"from 1980 to 2015. RESULT: The patient developed labial hyperemia and edema, ectropion of vaginal mucosa and plica, swelling of the hypogastrium and upper legs at 41 days (38(+ 1) weeks post-conception). The child was continuously observed because diagnosis was not clear. The pelvic and abdominal ultrasonography examinations revealed a cyst in the right ovary and laboratory evaluation of the baby showed high concentrations of gonadotropin and estradiol at 49 days (39(+ 2) weeks post-conception), and thus the baby was diagnosed with POHS. With no special intervention measures, the baby became normal at 169 days (4 months post-conception). Six papers from foreign literature were retrieved and none from Chinese literature, which reported 12 cases of POHS. They all characterized prematurity, ovarian cyst/cysts, labial hyperemia and edema, swelling of the hypogastrium and upper leg, high serum gonadotropin and estradiol levels at 35 to 39 weeks post-conception, including 3 cases with breast enlargement, 1 case with vaginal bleeding and 1 case with ectropion of vaginal mucosa and plica. The treatments included in 1 case combined surgery with pharmacological intervention, in another case only pharmacological intervention, and in the others no interventional measures were taken but were only followed up. As for the results, the baby with the surgical treatment had recurrence, but the symptoms, ovarian cyst and hormone concentration of the other babies gradually became normal in 4-5 months. CONCLUSION: POHS is a rare and self-limiting disease. The common clinical features of this disease are prematurity, ovarian cyst or cysts, labia hyperemia and edema, swelling in the hypogastrium and upper legs, high serum gonadotropin and estradiol levels at 35 to 39 weeks post-conception. It does not require treatment if there is no complication, but follow-up is necessary.

The effects of food on the pharmacokinetics of mitiglinide tablets in healthy volunteers and a novel mass-spectrometric (UPLC-MS/MS) method for such studies.

WHAT IS KNOWN AND OBJECTIVE: Mitiglinide (MGN) is a new insulinotropic agent of the glinide class with rapid onset. The effects of food intake on the pharmacokinetic (PK) profile of mitiglinide tablets after single oral administration have not yet been reported in healthy adults. We aimed to assess the effects of food intake on the PK properties of mitiglinide (MGN) tablets, using a novel analytical method, after single escalating oral doses in healthy Chinese volunteers. METHODS: In this open-label, randomized, single-dose (three distinct doses), two-way crossover PK study, three doses of MGN 5, 10 or 20 mg were administered to healthy adult volunteers after an overnight fast (fasted condition) or low-fat breakfast (fed condition) (period 1). After 7 days, the participants received the same dose under the opposite fed/fasted condition (period 2). Serial blood samples were obtained before and through 8 h after study drug administration. Concentrations of MGN in plasma were determined using UPLC-MS/MS. Adverse events (AEs) were monitored and recorded on each in-clinic day. RESULTS AND DISCUSSION: Twenty-four Chinese volunteers (eight [four men, four women] volunteers per group) were enrolled in the study. The extent of absorption of MGN was similar in both fed and fasted conditions at single doses in the range 5-20 mg. Food intake was associated with decreases in C(max) by 60·4% to 65·2% in the three dose groups and greatly delayed T(max) [0·36(Standard deviation 0·16) vs. 1·75(0·92) hours with 5 mg, 0·29(0·19) vs. 1·97(0·81) hours with 10 mg and 0·30(0·10) vs. 1·18(0·68) hours with 20 mg; all, P < 0·05]. t(1/2) , CL/F and V/F (P > 0·05) were unaffected. MRT(0-8) at the 5 and 10-mg doses, but not at the 20-mg dose, were markedly lower in fasted volunteers than fed volunteers (P < 0·05). WHAT IS NEW AND CONCLUSIONS: Using a novel UPLC-MS/MS method, we showed that food intake affected the rate but not the extent of absorption of MGN within the 5- to 20-mg dose range. Gender did not appear to affect the PK properties of MGN in either fasted or fed states. MGN should be preferably taken before food.

Validated LC-MS/MS assay for the quantitative determination of nalbuphine in human plasma and its application to a pharmacokinetic study.

A solid-phase extraction-liquid chromatographic-tandem mass spectrometry method for the determination of nalbuphine concentrations in human plasma has been developed. Samples (1 mL) were extracted using a Strata™-X solid phase extraction cartridges. Chromatographic separation of nalbuphine and naloxone (internal standard) was achieved on a Phenomenex Kinetex PFP (2.6 µm, 100 A, 100 × 2.1 mm) column using a mobile phase consisting of 0.1% formic acid, 15 mM ammonium acetate in deionized water and acetonitrile (60:40, v/v). The flow rate was 0.3 mL/min and the total run time was 2 min. Detection of the analytes was achieved using positive ion electrospray ionization via multiple reactions monitoring mode. The mass transitions were m/z 358 → 340 for nalbuphine and m/z 328 → 310 for naloxone. The assay was linear over the concentration range 0.50-500.00 ng/mL, with correlation coefficients ≥0.995. The lower limit of quantitation was set at 0.5 ng/mL plasma based on an average signal-to-noise ratio of 44.79. The intra- and inter-day precision was less than 8.07% in terms of relative standard deviation and accuracy ranged from 94.97 to 106.29% at all quality control levels. The method was applied successfully to determine nalbuphine concentrations in human plasma samples obtained from subjects receiving intravenous administration of nalbuphine. The method is rapid, sensitive, selective and directly applicable to human pharmacokinetic studies involving nalbuphine.

Roles of disulfide bridges in scorpion toxin BmK M1 analyzed by mutagenesis.

The unique fold of scorpion toxins is a natural scaffold for protein engineering, in which multiple disulfide bonds are crucial structural elements. To understand the respective roles of these disulfide bridges, a mutagenesis analysis for the four disulfide bonds, 12-63, 16-36, 22-46 and 26-48, of a representative toxin BmK M1 from the scorpion Buthus martensii Karsch was carried out. All cysteines were replaced by serine with double mutations. The recombinant mutants were expressed in the Saccharomyces cerevisiae S-78 system. Toxic activities of the expressed mutants were tested on ICR mice in vivo and on neuronal Na+ channels (rNav1.2) by electrophysiological analysis. Recombinant variants M1 (C22S,C46S) and M1 (C26S,C48S) were not expressed at all; M1 (C16S,C36S) could be expressed at trace levels but was extremely unstable. Variant M1 (C12S,C63S) could be expressed in an amount comparable with that of unmodified rBmK M1, but had no detectable bioactivities. The results indicated that among the four disulfide bonds for long-chain scorpion toxins, loss of either bridge C22-C46 or C26-C48 is fatal for the general folding of the molecule. Bridge C16-C36 mainly contributes to the global stability of the folded scaffold, and bridge C12-C63 plays an essential role in the functional performance of scorpion toxins.

Overexpression, purification, crystallization and preliminary X-ray diffraction analysis of Cu,Zn superoxide dismutase from Peking duck.

The cDNA encoding Peking duck Cu,Zn superoxide dismutase (dSOD) was cloned and sequenced. The recombinant enzyme was overexpressed in Escherichia coli, purified to homogeneity and crystallized using the sitting-drop vapour-diffusion technique. Trigonal crystals of dSOD were obtained at 278 K at low ionic strength and around neutral pH. These crystals belong to space group P3(2)21, with unit-cell parameters a = 124.4, c = 163.5 A, gamma = 120 degrees. The asymmetric unit contains four dimers (eight monomers of Cu,Zn dSOD) and has a 56% solvent content, with a V(M) of 2.8 A(3) Da(-1). On a Rigaku R-AXIS IIc image-plate area-detector system, the crystal diffracted to 2.9 A. Unusual supermolecular double-helix packing with 9(2)2 non-crystallographic symmetry in crystals has been observed in the initial structural analysis.

Expression and purification of the BmK M1 neurotoxin from the scorpion Buthus martensii Karsch.

The gene encoding a neurotoxin (BmK M1) from the scorpion Buthus martensii Karsch was expressed in Saccharomyces cerevisiae at a high level with the alcohol dehydrogenase promoter. SDS-PAGE of the culture confirmed expression and showed secretion into medium from yeast. Recombinant BmK M1 was purified rapidly and efficiently by ion exchange and gel filtration chromatography to homogeneity, produced a single band on tricine-SDS-PAGE, and processed the homologous N-terminus. Amino acid analysis and N-terminal sequencing demonstrated that the recombinant toxin was processed correctly from the alpha-mating factor leader sequence and was chemically identical to the native form. The expressed recombinant BmK M1 was toxic for mice, which indicated that it was biologically active. Quantitative estimation showed that recombinant BmK M1 had an LD(50) similar to that of the native toxin.

A new antifungal peptide from the seeds of Phytolacca americana: characterization, amino acid sequence and cDNA cloning.

An antifungal peptide from seeds of Phytolacca americana, designated PAFP-s, has been isolated. The peptide is highly basic and consists of 38 residues with three disulfide bridges. Its molecular mass of 3929.0 was determined by mass spectrometry. The complete amino acid sequence was obtained from automated Edman degradation, and cDNA cloning was successfully performed by 3'-RACE. The deduced amino acid sequence of a partial cDNA corresponded to the amino acid sequence from chemical sequencing. PAFP-s exhibited a broad spectrum of antifungal activity, and its activities differed among various fungi. PAFP-s displayed no inhibitory activity towards Escherichia coli. PAFP-s shows significant sequence similarities and the same cysteine motif with Mj-AMPs, antimicrobial peptides from seeds of Mirabilis jalapa belonging to the knottin-type antimicrobial peptide.

Determination of amitriptyline and nortriptyline in human liver microsomes with reversed-phase HPLC in vitro.

AIM: To develop a method for simultaneous determinations of amitriptyline (Ami) and its metabolite nortriptyline (Nor) in human liver microsomes. METHODS: An incubation buffer containing microsomes, NADPH-generating system, and Ami, after termination of enzyme reaction and desipramine (Des) as internal standard (IS), was extracted with diethy ether and separated on a reversed-phase ODS column. Detection was achieved at 242 nm by ultraviolet detector. RESULTS: No potential interfering peaks were found. Ami and Nor gave rapid elution and baseline resolution. The linear curves of both analyses ranged 0.02-10 nmol and the limit of detection was 0.01 nmol. The recovery (94%-101%) had good precision with relative s of < 8.3%. CONCLUSION: This method is rapid, sensitive, and simple for studying the metabolism of Ami and Nor.

Pharmacological characterization of the cloned kappa opioid receptor as a kappa 1b subtype.

Substantial pharmacological evidence in vitro and in vivo has suggested the existence of subtypes of the kappa opioid receptor. Quantitative radioligand binding techniques resolved the presence of two high affinity binding sites for the kappa 1 ligand [3H]U69,593 in mouse brain membranes, termed kappa 1a and kappa 1b, respectively. Whereas the kappa 1a site has high affinity for fedotozine and oxymorphindole and low affinity for bremazocine and alpha-neoendorphin, site kappa 1b has high affinity for bremazocine and alpha-neoendorphin and low affinity for fedotozine and oxymorphindole. CI-977 and U69,593 bind equally well at both sites. To determine the relationship between these kappa 1 receptor subtypes and the recently cloned mouse kappa 1 receptor (KOR), we examined [3H]U69,593 binding to the KOR in stably transfected cells (KORCHN-8). Competition of [3H]U69,593 binding to the KOR by bremazocine, alpha-neoendorphin, fedotozine and oxymorphindole resolved a single class of binding sites at which these agents had binding affinities similar to that of the kappa 1b site present in mouse brain. These results suggest that the cloned KOR corresponds to the kappa 1 site in mouse brain defined as kappa 1b.

High level synthesis of biologically active recombinant trichosanthin in Escherichia coli.

Two forms of recombinant trichosanthin (rTCS) were synthesized in high levels in Escherichia coli by putting the TCS cDNA under the control of a T7 RNA polymerase-directed promoter. Purification schemes were developed to isolate the recombinant protein from both soluble and insoluble fractions. Form I rTCS possessed the mature TCS sequence and had similar biological activities as the natural protein. Its IC50 was approximately 0.13 nM in an in vitro rabbit reticulocyte translational system and a dose of around 35 micrograms protein per 25 g body weight was sufficient to induce complete abortion in mice. Form II rTCS had a propeptide of 19 aa at the C-terminus and was five times less active than Form I in inhibiting protein synthesis by a rabbit reticulocyte lysate.

Cloning of trichosanthin cDNA and its expression in Escherichia coli.

Several cDNA clones coding for trichosanthin (TCS) have been isolated from a cDNA library prepared from the poly(A)+RNA of the root tuber of Trichosanthes kirilowii Maximowicz. The nucleotide sequence codes for a protein of 289 amino acids (aa) including a putative signal peptide of 23 aa and an extra 19 aa at the C terminus; the latter two have not been found in TCS obtained from the natural product [Collins et al., J. Biol. Chem. 265 (1990) 8665-8669]. Recombinant TCS (reTCS) was synthesized in Escherichia coli, in which the cDNA without the signal sequence was expressed under the control of the trc promoter; reTCS was detected by a rabbit anti-TCS antiserum.