Insect Trypsin Modulating Oostatic Factor (Neb-TMOF) and Its Analogs: Preliminary Structure/Biological Function Relationship Studies

Neb-TMOF, the trypsin modulating oostatic factor of gray fleshfly Neobellieria bullata, is a hexapeptide with the following sequence: H-Asn-Pro-Thr-Asn-Leu-His-OH. It has been isolated from vitellogenic ovaries in 1994. TMOF, the newly discovered insect peptide, inhibits trypsin biosynthesis in the gut, lowers yolk polypeptide concentration in the hemolymph and strongly inhibits ecdysone biosynthesis by larval ring glands. It is interesting that this short non-protected peptide contains in its molecule two Asn residues at positions 1 and 4 and His at its C-terminus. To obtain information about the role of the His-6 and Asn-4 residues we synthesised two series of Neb-TMOF analogs, modified: (1) in position 6 by D-His (I), His(Bzl) (II) and Phe(p-X) derivatives, where X = NH₂ (III), NO₂ (IV), OEt (V) and OH (VI) and (2) in position 4 by such amino acid residues as Ser (VII), Thr (VIII), Gly (IX), Asp (X), Glu (XI) and D-Asn (XII). The influence of these peptides on trypsin biosynthesis in N. bullata was determined in vivo. In preliminary investigations, we found that Neb-TMOF, [Phe(NH₂)⁶], and [Phe(NO₂)⁶]-Neb-TMOF inhibited trypsin biosynthesis, whereas [D-His)⁶]- and [D-His(Bzl)⁶]-Neb-TMOF were inactive. In further biological studies performed in vitro on heart of Tenebrio molitor we found that Neb-TMOF and [Phe(p-NH₂)⁶-Neb-TMOF showed weak cardioexcitatory activity, about 30% of the cardioexcitatory activity of proctolin, an insect neuromodulating peptide.     

Hyperspectral and Thermal Imaging of Oilseed Rape (Brassica napus) Response to Fungal Species of the Genus Alternaria

In this paper, thermal (8-13 µm) and hyperspectral imaging in visible and near infrared (VNIR) and short wavelength infrared (SWIR) ranges were used to elaborate a method of early detection of biotic stresses caused by fungal species belonging to the genus Alternaria that were host (Alternaria alternata, Alternaria brassicae, and Alternaria brassicicola) and non-host (Alternaria dauci) pathogens to oilseed rape (Brassica napus L.). The measurements of disease severity for chosen dates after inoculation were compared to temperature distributions on infected leaves and to averaged reflectance characteristics. Statistical analysis revealed that leaf temperature distributions on particular days after inoculation and respective spectral characteristics, especially in the SWIR range (1000-2500 nm), significantly differed for the leaves inoculated with A. dauci from the other species of Alternaria as well as from leaves of non-treated plants. The significant differences in leaf temperature of the studied Alternaria species were observed in various stages of infection development. The classification experiments were performed on the hyperspectral data of the leaf surfaces to distinguish days after inoculation and Alternaria species. The second-derivative transformation of the spectral data together with back-propagation neural networks (BNNs) appeared to be the best combination for classification of days after inoculation (prediction accuracy 90.5%) and Alternaria species (prediction accuracy 80.5%).     

Englerin A Agonizes the TRPC4/C5 Cation Channels to Inhibit Tumor Cell Line Proliferation

Englerin A is a structurally unique natural product reported to selectively inhibit growth of renal cell carcinoma cell lines. A large scale phenotypic cell profiling experiment (CLiP) of englerin A on ¬over 500 well characterized cancer cell lines showed that englerin A inhibits growth of a subset of tumor cell lines from many lineages, not just renal cell carcinomas. Expression of the TRPC4 cation channel was the cell line feature that best correlated with sensitivity to englerin A, suggesting the hypothesis that TRPC4 is the efficacy target for englerin A. Genetic experiments demonstrate that TRPC4 expression is both necessary and sufficient for englerin A induced growth inhibition. Englerin A induces calcium influx and membrane depolarization in cells expressing high levels of TRPC4 or its close ortholog TRPC5. Electrophysiology experiments confirmed that englerin A is a TRPC4 agonist. Both the englerin A induced current and the englerin A induced growth inhibition can be blocked by the TRPC4/C5 inhibitor ML204. These experiments confirm that activation of TRPC4/C5 channels inhibits tumor cell line proliferation and confirms the TRPC4 target hypothesis generated by the cell line profiling. In selectivity assays englerin A weakly inhibits TRPA1, TRPV3/V4, and TRPM8 which suggests that englerin A may bind a common feature of TRP ion channels. In vivo experiments show that englerin A is lethal in rodents near doses needed to activate the TRPC4 channel. This toxicity suggests that englerin A itself is probably unsuitable for further drug development. However, since englerin A can be synthesized in the laboratory, it may be a useful chemical starting point to identify novel modulators of other TRP family channels.     

Differential expression of Snail1 transcription factor and Snail1-related genes in alveolar and embryonal rhabdomyosarcoma subtypes

Rhabdomyosarcoma (RMS) represents the most common sarcoma of soft tissue among children. Two main RMS subtypes are alveolar (ARMS) and embryonal (ERMS). The major goal of this study was to find differentially expressed genes between RMS subtypes that could explain higher metastatic potential in ARMS and would be useful for the differential diagnosis. Using RQ-PCR analysis we compared expression of Snail1 and Snail-related genes among 7 ARMS and 8 ERMS patients' samples obtained from the primary tumors and among 2 alveolar and 2 embryonal cell lines. Our results show that Snail1 is highly expressed both in ARMS patients' samples and the alveolar cell lines. We also found that the expression of E-Cadherin was downregulated and the expression of Matrix Metalloproteinases 2 and 9 (MMP-2 and MMP-9) was upregulated in ARMS. We assume that, as in many tumors, also in RMS Snail1 acts as a regulator for pathways known for their role in cells' metastasis and that Snail1 activity results in increased MMPs and decreased E-Cadherin expression. Our findings may explain higher ARMS aggressiveness. Moreover, we suggest that further studies should be performed to verify if Snail1 can be considered as a potential target for ARMS therapy.     

Spatial,temporal and interindividual epigenetic variation of functionally important DNA methylation patterns

DNA methylation is an epigenetic modification that plays an important role in gene regulation. It can be influenced by stochastic events, environmental factors and developmental programs. However, little is known about the natural variation of gene-specific methylation patterns. In this study, we performed quantitative methylation analyses of six differentially methylated imprinted genes (H19, MEG3, LIT1, NESP55, PEG3 and SNRPN), one hypermethylated pluripotency gene (OCT4) and one hypomethylated tumor suppressor gene (APC) in chorionic villus, fetal and adult cortex, and adult blood samples. Both average methylation level and range of methylation variation depended on the gene locus, tissue type and/or developmental stage. We found considerable variability of functionally important methylation patterns among unrelated healthy individuals and a trend toward more similar methylation levels in monozygotic twins than in dizygotic twins. Imprinted genes showed relatively little methylation changes associated with aging in individuals who are >25 years. The relative differences in methylation among neighboring CpGs in the generally hypomethylated APC promoter may not only reflect stochastic fluctuations but also depend on the tissue type. Our results are consistent with the view that most methylation variation may arise after fertilization, leading to epigenetic mosaicism.     

Studies of insect peptides alloferon,Any‐GS and their analogues. Synthesis and antiherpes activity

The subject of these studies was synthesis and determination of biological properties of a series of insect peptides, such as alloferon, Any‐GS and their analogues. The synthesis of 14 peptides was performed by the solid‐phase method. Biological effect of these peptides was evaluated by the antiviral test against Human Herpes Virus type 1 (HHV‐1) in vitro using a Vero cell line. It was found that the investigated peptides inhibit the replication of HHV‐1 in Vero cells. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Identification of novel chromosomal regions associated with airway hyperresponsiveness in recombinant congenic strains of mice

Airway responsiveness is the ability of the airways to respond to bronchoconstricting stimuli by reducing their diameter. Airway hyperresponsiveness has been associated with asthma susceptibility in both humans and murine models, and it has been shown to be a complex and heritable trait. In particular, the A/J mouse strain is known to have hyperresponsive airways, while the C57BL/6 strain is known to be relatively refractory to bronchoconstricting stimuli. We analyzed recombinant congenic strains (RCS) of mice generated from these hyper- and hyporesponsive parental strains to identify genetic loci underlying the trait of airway responsiveness in response to methacholine as assessed by whole-body plethysmography. Our screen identified 16 chromosomal regions significantly associated with airway hyperresponsiveness (genome-wide P ≤ 0.05): 8 are supported by independent and previously published reports while 8 are entirely novel. Regions that overlap with previous reports include two regions on chromosome 2, three on chromosome 6, one on chromosome 15, and two on chromosome 17. The 8 novel regions are located on chromosome 1 (92–100 cM), chromosome 5 (>73 cM), chromosome 7 (>63 cM), chromosome 8 (52–67 cM), chromosome 10 (3–7 cM and >68 cM), and chromosome 12 (25–38 cM and >52 cM). Our data identify several likely candidate genes from the 16 regions, including Ddr2, Hc, Fbn1, Flt3, Utrn, Enpp2, and Tsc.     

Cranioectodermal Dysplasia, Sensenbrenner Syndrome, Is a Ciliopathy Caused by Mutations in the IFT122 Gene

Cranioectodermal dysplasia (CED) is a disorder characterized by craniofacial, skeletal, and ectodermal abnormalities. Most cases reported to date are sporadic, but a few familial cases support an autosomal-recessive inheritance pattern. Aiming at the elucidation of the genetic basis of CED, we collected 13 patients with CED symptoms from 12 independent families. In one family with consanguineous parents two siblings were affected, permitting linkage analysis and homozygosity mapping. This revealed a single region of homozygosity with a significant LOD score (3.57) on chromosome 3q21-3q24. By sequencing candidate genes from this interval we found a homozygous missense mutation in the IFT122 (WDR10) gene that cosegregated with the disease. Examination of IFT122 in our patient cohort revealed one additional homozygous missense change in the patient from a second consanguineous family. In addition, we found compound heterozygosity for a donor splice-site change and a missense change in one sporadic patient. All mutations were absent in 340 control chromosomes. Because IFT122 plays an important role in the assembly and maintenance of eukaryotic cilia, we investigated patient fibroblasts and found significantly reduced frequency and length of primary cilia as compared to controls. Furthermore, we transiently knocked down ift122 in zebrafish embryos and observed the typical phenotype found in other models of ciliopathies. Because not all of our patients harbored mutations in IFT122, CED seems to be genetically heterogeneous. Still, by identifying CED as a ciliary disorder, our study suggests that the causative mutations in the unresolved cases most likely affect primary cilia function too.     

The effect of the leucopyrokinin analogue: [2-8]-leucopyrokinin on central opioid receptors in rats

The antinociceptive effect of intracerebroventricular injections of [2–8]-leucopyrokinin (LPK), a truncated leucopyrokinin analogue, was determined in rats, by means of a tail immersion test. We found a significant antinociceptive effect of three i.c.v. doses of [2–8]-LPK: 1, 5 and 10 nmol. Pre-treating animals with naloxone hydrochloride (1 mg/kg i.p.) completely blocked the effect of two high doses of [2–8]-LPK. To determine the sub-types of opioid receptors involved in [2–8]-leucopyrokinin-induced analgesia we injected specific blockers of μ-, δ- and κ-receptors namely, β-funaltrexamine hydrochloride, naltrindole hydrochloride and nor-binaltorphimine dihydrochloride, respectively, prior to [2–8]-leucopyrokinin at equimolar doses. We conclude that the antinociceptive effect of [2–8]-leucopyrokinin is mediated mainly by central μ- and δ-opioid receptors.