Characterization and Favorable in Vivo Properties of Heterodimeric Soluble IL-15·IL-15Rα Cytokine Compared to IL-15 Monomer

Interleukin-15 (IL-15), a 114-amino acid cytokine related to IL-2, regulates immune homeostasis and the fate of many lymphocyte subsets. We reported that, in the blood of mice and humans, IL-15 is present as a heterodimer associated with soluble IL-15 receptor α (sIL-15Rα). Here, we show efficient production of this noncovalently linked but stable heterodimer in clonal human HEK293 cells and release of the processed IL-15·sIL-15Rα heterodimer in the medium. Purification of the IL-15 and sIL-15Rα polypeptides allowed identification of the proteolytic cleavage site of IL-15Rα and characterization of multiple glycosylation sites. Administration of the IL-15·sIL-15Rα heterodimer reconstituted from purified subunits resulted in sustained plasma IL-15 levels and in robust expansion of NK and T cells in mice, demonstrating pharmacokinetics and in vivo bioactivity superior to single chain IL-15. These identified properties of heterodimeric IL-15 provide a strong rationale for the evaluation of this molecule for clinical applications.     

Design,synthesis, anticancer evaluation,and molecular modelling studies of novel tolmetin derivatives as potential VEGFR-2 inhibitors and apoptosis inducers

Novel tolmetin derivatives 5a–f to 8a–c were designed, synthesised, and evaluated for antiproliferative activity by NCI (USA) against a panel of 60 tumour cell lines. The cytotoxic activity of the most active tolmetin derivatives 5b and 5c was examined against HL-60, HCT-15, and UO-31 tumour cell lines. Compound 5b was found to be the most potent derivative against HL-60, HCT-15, and UO-31 cell lines with IC₅₀ values of 10.32 ± 0.55, 6.62 ± 0.35, and 7.69 ± 0.41 µM, respectively. Molecular modelling studies of derivative 5b towards the VEGFR-2 active site were performed. Compound 5b displayed high inhibitory activity against VEGFR-2 (IC₅₀ = 0.20 µM). It extremely reduced the HUVECs migration potential exhibiting deeply reduced wound healing patterns after 72 h. It induced apoptosis in HCT-15 cells (52.72-fold). This evidence was supported by an increase in the level of apoptotic caspases-3, -8, and -9 by 7.808-, 1.867-, and 7.622-fold, respectively. Compound 5b arrested the cell cycle in the G0/G1 phase. Furthermore, the ADME studies showed that compound 5b possessed promising pharmacokinetic properties.     

LF-15 & T7, Synthetic Peptides Derived from Tumstatin,Attenuate Aspects of Airway Remodelling in a Murine Model of Chronic OVA-Induced Allergic Airway Disease

Background

Tumstatin is a segment of the collagen-IV protein that is markedly reduced in the airways of asthmatics. Tumstatin can play an important role in the development of airway remodelling associated with asthma due to its anti-angiogenic properties. This study assessed the anti-angiogenic properties of smaller peptides derived from tumstatin, which contain the interface tumstatin uses to interact with the αVβ3 integrin.

Methods

Primary human lung endothelial cells were exposed to the LF-15, T3 and T7 tumstatin-derived peptides and assessed for cell viability and tube formation in vitro. The impact of the anti-angiogenic properties on airways hyperresponsiveness (AHR) was then examined using a murine model of chronic OVA-induced allergic airways disease.

Results

The LF-15 and T7 peptides significantly reduced endothelial cell viability and attenuated tube formation in vitro. Mice exposed to OVA+ LF-15 or OVA+T7 also had reduced total lung vascularity and AHR was attenuated compared to mice exposed to OVA alone. T3 peptides reduced cell viability but had no effect on any other parameters.

Conclusion

The LF-15 and T7 peptides may be appropriate candidates for use as novel pharmacotherapies due to their small size and anti-angiogenic properties observed in vitro and in vivo.     

Effects of (15S)-15-methyl prostaglandin F2 methyl ester and estrogens upon the corpus luteum and conceptus of the rhesus monkey

The corpus luteum inhibiting properties of eighteen 15-methyl prostaglandin analogs were determined in the rhesus monkey during concomitant stimulation of the corpus luteum with chorionic gonadotropin. The methyl ester of (15S)-15-methyl PGF_2^α (15M-PGF_2^α, 12.5 mg/monkey) lowered serum progesterone to 12% of pretreatment values within 24 hours, however progesterone returned to normal limits within 48 hours. Elongation of the top side-chain by two carbons (2a,2b-dihomo-15M-PGF_2^α methyl ester, 13 mg/monkey), substitution of a hydroxymethyl group at carbon 1 (2-decarboxy-2-hydroxymethyl-15M-PGF_2^α, 12 mg/monkey), or the formation of the carbon 1 amide (15M-PGF_2^α amide, 12.5 mg/monkey) improved the inhibitory activity of 15M-PGF_2^α; serum progesterone for these 3 analogs was depressed to 15–30% of pretreatment levels within 24 hours, and did not return to control values. Luteal function was not inhibited (12 or more mg/monkey) when the 15-methyl group was placed in the R configuration, the top side chain was shortened by two carbons, an amino group was substituted for carbon 1, the 5-oxa modification was added, or the 1,9-lactone was formed. Some other modifications of 15M-PGF_2^α were also inactive, although not all were tested at equivalent doses: 2,2-difluoro; 4,5-cis-didehydro; 9,11-dideoxy-9α,11α-dichloro; 11-deoxy; 17-phenyl; 1,15-lactone; and the p-benzamidophenyl ester of 2a,2b-dihomo-15M-PGF_2^α. (15S)-15-Methyl PGE₂ methyl ester (1 mg/monkey) depressed serum progesterone concentrations to 42% of pretreatment values within 24 hours; 2a,2b-dihomo-11-deoxy-(15S)-15-methyl PGE₂ methyl ester was inactive (5 mg/monkey). A corpus luteum inhibiting action of certain 15-methyl prostaglandins can be demonstrated in the rhesus monkey.     

Oligonucleotides containing pyrazolo[3,4-d]pyrimidines: 8-Aza-7-deazaadenines with bulky substituents in the 2- or 7-position

The synthesis of the 2'-deoxyadenosine analogues 1b, 2b, and 3c modified at the 7- and/or 2-position is described. The effect of 7-chloro and 2-methylthio groups on the duplex stability is evaluated. For that, the nucleosides 1b, 2b, and 3c were converted to the corresponding phosphoramidites 15, 19, and 22, which were employed in the solid-phase oligonucleotide synthesis. In oligonucleotide duplexes, compound 1b forms stable base pairs with dT, of which the separated 1b-dT base pairs contribute stronger than that of the consecutive base pairs. Compound 2b shows universal base pairing properties while its N8 isomer 3c forms duplexes with lower stability.     

The posttranslational modification of β-endorphin in the intermediate pituitary of the toad, Bufo marinus, includes processing at a monobasic cleavage site

Fractionation of an acid extract of 15 B. marinus intermediate pituitaries by a combination of gel filtration chromatography and cation exchange chromatography revealed one major and five minor forms of β-endorphin in this tissue. Based on reversed-phase HPLC and immunological properties, as well as amino acid composition and primary sequence analysis, it was deduced that the sequence of the major form of B. marinus β-endorphin is N-acetyl-YGGFMTPE. Overall, the steady-state analyses of the minor forms of β-endorphin indicated that the posttranslational processing of β-endorphin in the toad intermediate pituitary includes endoproteolytic cleavage at both paired basic and monobasic cleavage sites.     

Retracted: Upregulated microRNA-15b alleviates ovarian cancer through inhitbition of the PI3K/Akt pathway by targeting LPAR3

Ovarian cancer characterizes as the fourth leading consequence of death associated with cancer for women. Accumulating evidence underscores the vital roles of microRNAs (miRNAs) in preventing ovarian cancer development. Besides, induction of the phosphatidylinositol-3 kinase/serine/threonine kinase (PI3K/Akt) pathway associated with the ovarian cancer cell migration and invasion. The study aims to examine the effects of miR-15b on the proliferation, apoptosis, and senescence of human ovarian cancer cells by binding to lysophosphatidic acid receptor 3 (LPAR3) with the involvement of the PI3K/Akt pathway. The positive expression of LPAR3 protein was detected by immunohistochemistry. Then the interaction between miR-15b and LPAR3 was examined. The possible role of miR-15b in ovarian cancer was explored using gain- and loss-of-function experiments. Subsequently, the functions of miR-15b on PI3K/Akt pathway, proliferation, migration, invasion, senescence and apoptosis of ovarian cancer cells were assessed. Furthermore, in vivo tumorigenicity assay in nude mice was performed. LPAR3 was overexpressed, whereas miR-15b was poorly expressed in ovarian cancer tissues. LPAR3 is a direct target of miR-15b. Restored miR-15b promoted Bax expression, apoptosis, and senescence, inhibited expression of LPAR3 and Bcl-2, the extent of PI3K and Akt phosphorylation, as well as ovarian cancer cell proliferation, migration, and invasion. Further, tumor growth was observed to be prevented by miR-15b overexpression. Collectively, our study demonstrates that miR-15b represses the proliferation and drives the senescence and apoptosis of ovarian cancer cells through the suppression of LPAR3 and the PI3K/Akt pathway, highlighting an antitumorigenic role of miR-15b.     

Gastric Bleeding and Benorylate,a New Aspirin

Benorylate (4-acetamidophenyl 2-acetoxybenzoate) is a new esterified aspirin preparation whose antirheumatic properties are reported to be as good as those of aspirin. Gastrointestinal blood loss, measured with ⁵¹Cr-labelled red cells, during benorylate therapy was compared with that during therapy with soluble aspirin in 15 subjects, a simplified crossover procedure being used. Mean blood loss during benorylate therapy was 1·7 ml/day which was significantly less than that during therapy with soluble aspirin (5·1 ml/day; P <0·001). In 12 of the 15 patients blood loss with benorylate was less than 2·5 ml/day. Benorylate appears to be a definite improvement on current formulations of aspirin and should be a useful drug for the treatment of patients with chronic rheumatic disorders.     

Studies on the disulfide bridges of sarafotoxins. Chemical synthesis of sarafotoxin S6B and its homologue with different disulfide bridges

Sarafotoxin S6b, a strong vasocontractile peptide with 21 amino acid residues containing two sets of disulfide bridges, was chemically synthesized. The retention time on a reversed-phase HPLC, lethal and vasocontractile activities of natural sarafotoxin S6b agree with those of the synthetic [Cys1-15, Cys3-11]-sarafotoxin S6b. The combination of the disulfide bridges of sarafotoxin S6b is the same as that of endothelin-1, a mammalian vasocontractile peptide, which shows a high degree of sequence homology and shares several common pharmacological properties with sarafotoxins.     

Validity and Reliability of the Chinese Version of the Care Transition Measure

BackgroundThe 15-item care transition measure (CTM-15) is a reliable and valid instrument assessing the quality of care transition from patients’ perspectives. The aim of this study was to evaluate the psychometric properties of the CTM-15 and the CTM-3 (a 3-item short version of the CTM-15) in Mainland China.Methodology/FindingsThis was a cross-sectional study with a convenience sample of 646 patients in a general tertiary-level hospital in Chengdu, China. The results indicated that the Cronbach’s α values of the Chinese version of the two measures were 0.90 and 0.56, and the test-retest reliability values were 0.91 and 0.87, respectively. Three factors were extracted for the CTM-15 in Chinese populations. The CTM-15 and the CTM-3 scores discriminated well between patients with and without re-hospitalization for their index condition. The CTM-15 and the CTM-3 had significant positive relationships with self-rated health status. The CTM-3 score was significantly related to the CTM-15 score, and the CTM-3 score accounted for 64.23% of the variance of the CTM-15 score.Conclusions/SignificanceThis study has demonstrated the psychometric properties of the CTM-15 and the CTM-3 in Mainland China. Although the Cronbach’s α value of the CTM-3 is suboptimal, it has exhibited high test-retest reliability, convergent validity and criterion validity. Therefore, the CTM-3 can substitute the CTM-15 as a performance measurement tool when the sample size is large enough to compensate its suboptimal reliability or the reduced response burden is a concern.     

Construction and Properties of Escherichia coli Strains Exhibiting α-Complementation of β-Galactosidase Fragments In Vivo

In vivo α-complementation of β-galactosidase was demonstrated in 16 Z gene terminator (nonsense) mutant strains of Escherichia coli upon introduction of the episome F′M15 which specifies production of a mutant Z gene polypeptide containing a small deletion in the N-terminal region of the enzyme monomer. Genetic and biochemical analyses of the merodiploids showed that restoration of enzyme activity was due to their terminator/F′M15 genetic constitution resulting in the production of two enzymatically inactive polypeptides which associate in vivo to reconstitute active, stable β-galactosidase. The prematurely terminated polypeptide fragments known to be rapidly degraded in haploid cells were shown by phenotypic and biochemical studies to be stabilized (i.e., protected) in merodiploids by formation of complemented enzyme complexes with the M15 protein. Phenotypic properties of complementing diploids are described and are discussed in relation to in vitro determination of β-galactosidase activity.     

Chiroptical properties of heterotrimetallic ‘wing-tip’ butterflies; synthesis and crystal structure of (μ-H)Ru2Fe2PdC(CO)12 (η-ß-C10H15)

HRu₂Fe₂PdC(CO)₁₂ (η³-ß-C₁₀H₁₅) cluster was prepared in the reaction of (Et₄N) [HFe₂Ru₂C(CO)₁₂] with [Pd(η³-ß-C₁₀H₁₅)Cl]₂. X-ray structural study of HRu₂Fe₂PdC(CO)₁₂ (η³-ß-C₁₀H₁₅) (where ß-C₁₀H₁₅ is ß-pinenyl) revealed a wing-tip butterfly geometry of the metal core and (1R, 2S, 3S, 5R) absolute configuration for both crystallography independent molecules in the crystal. Chiroptical properties of this cluster are compared with other clusters containing a Pd(η³-ß-C₁₀H₁₅) fragment and discussed.     

COMPARISON OF THE CYTOPLASMIC AND PERIPLASMIC PRODUCTION OF RETEPLASE IN Escherichia coli

Reteplase is the recombinant type of tissue plasminogen activator variant. In this study, preplasmic and cytoplasmic (as inclusion body: IBs) production and activity of recombinant reteplase in E. coli were investigated and compared using a pET system (pET22b and pET15b). The cDNA of reteplase was cloned by polymerase chain reaction (PCR) amplification, sequenced, inserted into the vector pET 22b and pET15b, and expressed using isopropyl β-D-1-thiogalactopyranoside (IPTG). The recombinant plasmid was expressed in the form of inclusion body in pET 15b and in periplasmic space in pET22b. The obtained results of inclusion body extraction from recombinant pET22b (rpET22b) and recombinant pET15b (rpET15b) plasmids using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed a band of ～39 kD. However, the obtained results of periplasmic space extraction from rpET22b plasmid showed a very weak band, while cytoplasmic expression of reteplase (pET15b) produced a strong protein band confirmed with Western blotting. Consequently, our results demonstrated that the cytoplasmic expression system is efficient for the production of reteplase protein in prokaryote systems and a high amount of reteplase was obtained from the expressed proteins in the form of IBs. The obtained activity of rpET15b plasmid showed a higher enzyme absorbance in comparison to rpET22b plasmid. This suggests rpET15b as an appropriate candidate for reteplase production.     

Myxoma Virus Expressing a Fusion Protein of Interleukin-15 (IL15) and IL15 Receptor Alpha Has Enhanced Antitumor Activity

Myxoma virus, a rabbit poxvirus, can efficiently infect various types of mouse and human cancer cells. It is a strict rabbit-specific pathogen, and is thought to be safe as a therapeutic agent in all non-rabbit hosts tested including mice and humans. Interleukin-15 (IL15) is an immuno-modulatory cytokine with significant potential for stimulating anti-tumor T lymphocytes and NK cells. Co-expression of IL15 with the α subunit of IL15 receptor (IL15Rα) greatly enhances IL15 stability and bioavailability. Therefore, we engineered a new recombinant myxoma virus (vMyx-IL15Rα-tdTr), which expresses an IL15Rα-IL15 fusion protein plus tdTomato red fluorescent reporter protein. Permissive rabbit kidney epithelial (RK-13) cells infected with vMyx-IL15Rα-tdTr expressed and secreted the IL15Rα-IL15 fusion protein. Functional activity was confirmed by demonstrating that the secreted fusion protein stimulated proliferation of cytokine-dependent CTLL-2 cells. Multi-step growth curves showed that murine melanoma (B16-F10 and B16.SIY) cell lines were permissive to vMyx-IL15Rα-tdTr infection. In vivo experiments in RAG1^-/- mice showed that subcutaneous B16-F10 tumors treated with vMyx-IL15Rα-tdTr exhibited attenuated tumor growth and a significant survival benefit for the treated group compared to the PBS control and the control viruses (vMyx-IL15-tdTr and vMyx-tdTr). Immunohistological analysis of the subcutaneous tumors showed dramatically increased infiltration of NK cells in vMyx-IL15Rα-tdTr treated tumors compared to the controls. In vivo experiments with immunocompetent C57BL/6 mice revealed a strong infiltrate of both NK cells and CD8⁺ T cells in response to vMyx-IL15Rα-tdTr, and prolonged survival. We conclude that delivery of IL15Rα-IL15 in a myxoma virus vector stimulates both innate and adaptive components of the immune system.     

Knockout of the 15 kDa Selenoprotein Protects against Chemically-Induced Aberrant Crypt Formation in Mice

Evidence suggests that selenium has cancer preventive properties that are largely mediated through selenoproteins. Our previous observations demonstrated that targeted down-regulation of the 15 kDa selenoprotein (Sep15) in murine colon cancer cells resulted in the reversal of the cancer phenotype. The present study investigated the effect of Sep15 knockout in mice using a chemically-induced colon cancer model. Homozygous Sep15 knockout mice, and wild type littermate controls were given four weekly subcutaneous injections of azoxymethane (10 mg/kg). Sep15 knockout mice developed significantly (p<0.001) fewer aberrant crypt foci than controls demonstrating that loss of Sep15 protects against aberrant crypt foci formation. Dietary selenium above adequate levels did not significantly affect aberrant crypt foci formation in Sep15 knockout mice. To investigate molecular targets affected by loss of Sep15, gene expression patterns in colonic mucosal cells of knockout and wild type mice were examined using microarray analysis. Subsequent analyses verified that guanylate binding protein-1 (GBP-1) mRNA and protein expression were strongly upregulated in Sep15 knockout mice. GBP-1, which is expressed in response to interferon-γ, is considered to be an activation marker during inflammatory diseases, and up-regulation of GBP-1 in humans has been associated with a highly significant, increased five-year survival rate in colorectal cancer patients. In agreement with these studies, we observed a higher level of interferon-γ in plasma of Sep15 knockout mice. Overall, our results demonstrate for the first time, that Sep15 knockout mice are protected against chemically-induced aberrant crypt foci formation and that Sep15 appears to have oncogenic properties in colon carcinogenesis in vivo.     

An endosomally localized isoform of Eps15 interacts with Hrs to mediate degradation of epidermal growth factor receptor

Down-regulation of activated and ubiquitinated growth factor (GF) receptors by endocytosis and subsequent lysosomal degradation ensures attenuation of GF signaling. The ubiquitin-binding adaptor protein Eps15 (epidermal growth factor receptor [EGFR] pathway substrate 15) functions in endocytosis of such receptors. Here, we identify an Eps15 isoform, Eps15b, and demonstrate its expression in human cells and conservation across vertebrate species. Although both Eps15 and Eps15b interact with the endosomal sorting protein Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) in vitro, we find that Hrs specifically binds Eps15b in vivo (whereas adaptor protein 2 preferentially interacts with Eps15). Although Eps15 mainly localizes to clathrin-coated pits at the plasma membrane, Eps15b localizes to Hrs-positive microdomains on endosomes. Eps15b overexpression, similarly to Hrs overexpression, inhibits ligand-mediated degradation of EGFR, whereas Eps15 is without effect. Similarly, depletion of Eps15b but not Eps15 delays degradation and promotes recycling of EGFR. These results indicate that Eps15b is an endosomally localized isoform of Eps15 that is present in the Hrs complex via direct Hrs interaction and important for the sorting function of this complex.     

Clinical manufacturing of recombinant human interleukin 15. I. Production cell line development and protein expression in E. coli with stop codon optimization

Interleukin 15 (IL-15) has shown remarkable biological properties of promoting NK- and T-cell activation and proliferation, as well as enhancing antitumor immunity of CD8(+) T cells in preclinical models. Here, we report the development of an E. coli cell line to express recombinant human Interleukin-15 (rhIL-15) for clinical manufacturing. Human IL-15 cDNA sequence was inserted into a pET28b plasmid and expressed in several E. coli BL21 strains. Through product quality comparisons among several E. coli strains, including E. coli BL21(DE3), BL21(DE3)pLysS, BLR(DE3)pLysS, and BL21-AI, E. coli BL21-AI was selected for clinical manufacturing. Expression optimization was carried out at shake flask and 20-L fermenter scales, and the product was expressed as inclusion bodies that were solubilized, refolded, and purified to yield active rhIL-15. Stop codons of the expression construct were further investigated after 15-20% of the purified rhIL-15 showed an extraneous peak corresponding to an extra tryptophan residue based on peptide mapping and mass spectrometry analysis. It was determined that the presence of an extra tryptophan was due to a stop codon wobble effect, which could be eliminated by replacing TGA (opal) stop codon with TAA (ochre). As a novel strategy, a simple method of demonstrating lack of tRNA suppressors in the production host cells was developed to validate the cells in this study. The E. coli BL21-AI cells containing the rhIL-15 coding sequence with a triplet stop codon TAATAATGA were banked for further clinical manufacturing.     

14alpha-Hydroxyoestrone, a new oestrogen metabolite

1. Incubation of oestrone with ox adrenal glands leads to the formation of two main metabolites, one of which has recently been identified as 15α-hydroxyoestrone; the second metabolite (Oe-5) has now been obtained in crystalline form and identified as the hitherto unknown 14α-hydroxyoestrone. 2. The identity of compound Oe-5 with 14α-hydroxyoestrone was confirmed conclusively by comparison with 14α-hydroxyoestrone that was synthesized by pyrolytic aromatization of 14α-hydroxyandrosta-1,4-diene-3,17-dione. 3. The physical and chemical properties as well as the oestrogenic activity of 14α-hydroxyoestrone are described. 4. The biogenesis and metabolism of 14α-hydroxyoestrone were studied in various tissue preparations.