Differential Distribution of Gonadotropin-Releasing Hormone Variants in the Brain of Hydrochaeris hydrochaeris (Mammalia, Rodentia)

1.In a previous paper we reported evidence for the presence of mGnRH- and sGnRH-like peptides in the preoptic–hypothalamic region of the capybara Hydrochaeris hydrochaeris (Montaner et al., 1998). In that study, the presence of a cGnRH-II like molecule in olfactory bulb extracts was suggested.2.The capybara, the largest living rodent in the world, belongs to the order Hystricomorpha, which is considered to be one of the oldest groups of rodents. Some authors consider that this group is the ancestor of all remaining rodents.3.In this study we have characterized GnRH molecular variants found in extracts from the olfactory bulbs and the mesencephalic region of capybara. These regions represent the two GnRH neuronal systems: the terminal nerve–septopreoptic and the midbrain systems.4.An indirect method combining reverse-phase high-performance liquid chromatography (RP-HPLC) and radioimmunoassay (RIA) was used to characterize GnRH variants. The analysis of both extracts with two different RIA systems revealed three immunoreactive GnRH peaks, coeluting with mGnRH, cIIGnRH, and sGnRH synthetic standards. These results were additionally supported by serial dilution studies with specific antisera.5.To our knowledge this the first report on the presence of three GnRH variants in the brain of an eutherian mammal. These results suggest that, similarly to other vertebrates, the expression of multiple GnRH variants may also be a common pattern in mammals.     

Quantitative determination of nuclear pore complexes in cycling cells with differing DNA content

The number of pore complexes per nucleus was determined for a wide variety of cultured cells selected for their variable DNA content over a range of 1-5,6000. The pore number was compared to DNA content, nuclear surface area, and nuclear volume. Values for pore frequency (pores/square micrometer) were relatively constant in the species studied. When the pore to DNA ratio was plotted against the DNA content, there was a remarkable correlation which decreased exponentially for the cells of vertebrae origin. Exceptions were the heteroploid mammalian cells which had the same ratio as the diploid mammalian cells despite higher DNA content. The results are interpreted to mean that neither the nuclear surface, the nuclear volume, nor the DNA content alone determines the pore number of the nucleus, but rather an as yet undetermined combination of different factors. The surface and volume of vertebrate nuclei do not decrease with decreasing DNA content below a given value. The following speculation is suggested to account for the anomalous size changes of the nucleus relative to DNA content in vertebrates. Species with small DNA complements have a relatively large proportion of active chromatin which determines the limits of the physical parameters of the nucleus. The amount of active chromatin maybe the same for at least the vertebrates with low DNA content, At high DNA content, the nuclear parameters may be determined by the relatively high proportion of inactive condensed chromatin which increases the nuclear surface and volume.     

Systematic epitope analysis of the p26 EIAV core protein

The major core protein of equine infectious anemia virus (EIAV), p26, is one of the primary immunogenic structural proteins during a persistent infection of horses and is highly conserved among antigenically variants of viral isolates. In order to investigate its immune profile in more detail for a better diagnostic, an epitope mapping was carried out by means of two libraries of overlapping peptide fragments prepared by simultaneous and parallel SPPS on derivatized cellulose membranes (SPOT synthesis). Polyclonal equine sera from infected horses were used for the biological assay. Particularly two promising continuous epitopes (NAMRHL and MYACRD) were localized on the C-terminal extreme of p26, region 194-222. A cyclic synthetic fragment of 29 amino acid residues containing the identified epitopes was designed and studied. A significant conformational change towards a helical structure was observed when the peptide was cyclized by a bridge between Cys198 and Cys218. This observation correlated with an improvement of its ability to be recognized by specific antibodies in an EIA (Enzyme-linked Immunosorbent assay). These results suggest that the conformationally restricted synthetic antigen adequately mimics the native structure of this region of p26 core protein.     

Molecular design and synthesis of novel peptides from amphibians skin acting as inhibitors of cholinesterase enzymes

Cholinesterases are a family of enzymes that catalyze the hydrolysis of neurotransmitter acetylcholine. There are two types of cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), which differ in their distribution in the body. Currently, cholinesterase inhibitors (ChEI) represent the treatment of choice for Alzheimer's disease (AD). In this paper, we report the synthesis and inhibitory effect on both enzymes of four new peptides structurally related to P1‐Hp‐1971 (amphibian skin peptide found in our previous work. Sequence: TKPTLLGLPLGAGPAAGPGKR‐NH₂). The bioassay data and cytotoxicity test show that some of the compounds possess a significant AChE and BChE inhibition and no toxic effect. The present work demonstrates that diminution of the size of the original peptide could potentially result in new compounds with significant cholinesterase inhibition activity, although it appears that there is an optimal size for the sequence. We also conducted an exhaustive molecular modeling study to better understand the mechanism of action of these compounds by combining docking techniques with molecular dynamics simulations on BChE. This is the first report about amphibian peptides and the second one of natural peptides with ChE inhibitory activity. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Effects of EDTA treatment upon the protein subunit composition and mechanical properties of mammalian single skeletal muscle fibers

Considerable interest has been focused on the role of myosin light chain LC(2) in the contraction of vertebrate striated muscle. A study was undertaken to further our investigations (Moss, R.L., G.G. Giulian, and M.L. Greaser, 1981, J. Biol. Chem., 257:8588-8591) of the effects of LC(2) removal upon contraction in skinned fibers from rabbit psoas muscles. Isometric tension and maximum velocity of shortening, V(max), were measured in fiber segments prior to LC(2) removal. The segments were then bathed at 30 degrees C for up to 240 min in a buffer solution containing 20 mM EDTA in order to extract up to 60 percent of the LC(2). Troponin C (TnC) was also partially removed by this procedure. Mechanical measurements were done following the EDTA extraction and the readditions of first TnC and then LC(2) to the segments. The protein subunit compositions of the same fiber segments were determined following each of these procedures by SDS PAGE of small pieces of the fiber. V(max) was found to decrease as the LC(2) content of the fiber segments was reduced by increasing the duration of extraction. EDTA treatment also resulted in substantial reductions in tension due mainly to the loss of TnC, though smaller reductions due to the extraction of LC(2) were also observed. Reversal of the order of recombination of LC(2) and TnC indicated that the reduction in V(max) following EDTA treatment was a specific effect of LC(2) removal. These results strongly suggest that LC(2) may have roles in determining the kinetics and extent of interaction between myosin and actin.     

Inhibition of phosphodiesterase 4 modulates cytokine induction from toll like receptor activated,but not rhinovirus infected,primary human airway smooth muscle

Background

Virus-induced exacerbations of Chronic Obstructive Pulmonary Disease (COPD) are a significant health burden and occur even in those receiving the best current therapies. Rhinovirus (RV) infections are responsible for half of all COPD exacerbations. The mechanism by which exacerbations occur remains undefined, however it is likely to be due to virus-induced inflammation. Given that phophodiesterase 4 (PDE₄) inhibitors have an anti-inflammatory effect in patients with COPD they present a potential therapy prior to, and during, these exacerbations.

Methods

In the present study we investigated whether the PDE₄ inhibitor piclamilast (10^-6 M) could alter RV or viral mimetic (5 μg/mL of imiquimod or poly I:C) induced inflammation and RV replication in primary human airway smooth muscle cells (ASMC) and bronchial epithelial cells (HBEC). The mediators IL-6, IL-8, prostaglandin E₂ and cAMP production were assayed by ELISA and RV replication was assayed by viral titration.

Results

We found that in ASMCs the TLR3 agonist poly I:C induced IL-8 release was reduced while induced IL-6 release by the TLR7/8 agonist imiquimod was further increased by the presence of piclamilast. However, in RV infected ASMCs, virus replication and induced mediator release were unaltered by piclamilast, as was also found in HBECs. The novel findings of this study reveal that although PDE inhibitors may not influence RV-induced cytokine production in ASMCs and replication in either ASMCs or HBECs, they have the capacity to be anti-inflammatory during TLR activation by modulating the induction of these chemotactic cytokines.

Conclusion

By extrapolating our in vitro findings to exacerbations of COPD in vivo this suggests that PDE₄ inhibitors may have beneficial anti-inflammatory properties when patients are infected with bacteria or viruses other than RV.     

Nine new polymorphic microsatellite loci for the amplification of archived otolith DNA of Atlantic cod,Gadus morhua L.

Nine out of 22 microsatellite primers tested were successfully amplified on three samples of cod Gadus morhua L. (two contemporary and one archived otolith samples). All loci were polymorphic (5–23 alleles/locus). The average observed heterozygosity across loci and samples was 0.625, ranging from 0.294 to 0.895 at each locus. All loci were under Hardy–Weinberg equilibrium, except PGmo56 that showed significant excess of heterozygotes in all studied samples. The isolated loci were suitable for degraded DNA and therefore useful for conducting a long‐term temporal study with DNA obtained from archived otoliths of cod.     

A synthetic analog of plantaricin 149 inhibiting food-borne pathogenic bacteria:evidence for alpha-helical conformation involved in bacteria-membrane interaction.

Plantaricin-149 is a bacteriocin produced by Lactobacillus plantarum NRIC 149 (a LAB isolated from pineapple), which consists of a peptidic chain made up of 22 amino acid residues [Kato et al. J. Ferment. Bioeng. 1994; 77: 277-282]. In this work, a synthetic C-terminal amidated peptide analog denoted Pln149a was prepared by SPPS-Fmoc chemistry and the antagonistic activity against gram-positive and gram-negative bacteria was tested. The secondary structure was studied by circular dichroism (CD) and the vicinity of the tyrosine residue by fluorescence spectroscopy under different conditions. We report the results of the interaction of Pln149a with reverse micelles prepared from the amphiphilic AOT in cyclohexane.Synthetic plantaricin was active against one strain of Staphylococcus aureus and four strains of Listeria genus at pH 5.5 and 7.4 and, like its natural variant, inhibited L. plantarum ATCC 8014.The data derived from spectroscopic measurements in presence of AOT reverse micelles suggest that the secondary structure of the peptide upon interaction is an alpha-helix. In this membrane model, the hydrophobic side of the alpha-helix is inserted into the micelles, leaving the lysines exposed to the solvent and interacting with the polar moieties of AOT. The fluorescence data point out that the N-terminal tyrosine residue is close to the micellar interface.     

Mangafodipir a Selective Cytoprotectant — with Special Reference to Oxaliplatin and Its Association to Chemotherapy-Induced Peripheral Neuropathy (CIPN)

Oxaliplatin, in combination with 5-fluorouracil plus folinate (or capecitabine), has increased survival substantially in stage III colorectal cancer and prolonged life in stage IV patients, but its use is compromised because of severe toxicity. Chemotherapy-induced peripheral neuropathy (CIPN) is the most problematic dose-limiting toxicity of oxaliplatin. Oncologists included for years calcium and magnesium infusion as part of clinical practice for preventing CIPN. Results from a phase III prospective study published in 2014, however, overturned this practice. No other treatments have been clinically proven to prevent this toxicity. There is a body of evidence that CIPN is caused by cellular oxidative stress. Clinical and preclinical data suggest that the manganese chelate and superoxide dismutase mimetic mangafodipir (MnDPDP) is an efficacious inhibitor of CIPN and other conditions caused by cellular oxidative stress, without interfering negatively with the tumoricidal activity of chemotherapy. MnPLED, the metabolite of MnDPDP, attacks cellular oxidative stress at several critical levels. Firstly, MnPLED catalyzes dismutation of superoxide (O₂^??), and secondly, having a tremendous high affinity for iron (and copper), PLED binds and disarms redox active iron/copper, which is involved in several detrimental oxidative steps. A case report from 2009 and a recent feasibility study suggest that MnDPDP may prevent or even cure oxaliplatin-induced CIPN. Preliminary results from a phase II study (PLIANT) suggest efficacy also of calmangafodipir, but these results are according to available data obscured by a surprisingly low number of adverse events and a seemingly lower than expected efficacy of FOLFOX.     

Secondary structure-improved bioaffinity correlation in elongated and modified synthetic epitope peptides from p24 HIV-1 core protein

Summary In order to study the correlation between secondary structure and bioaffinity of long and modified sequences of p24 protein from HIV-1, three peptides containing the minimal size epitope from region 208–217 (EAAEWDRVHP) were prepared. It was found that peptide p24-1n, an elongated native sequence, has the lowest K_D and a predominant α-helix structure in the presence of trifluoroethanol. Three peptides containing another epitope from region 293–302 (FRDYVDRFK) were also synthesized. We have observed that modifications on the native sequence p24-2n (region 287–308) increased the α-helix content and this was correlated with an improvement of the recognition by antibodies in ELSA. Abbreviations: CD, circular dichroism; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; ELSA, enzyme linked immunosorbent assay; EDT, 1,2-ethanedithiol; Fmoc, fluorenylmethoxycarbonyl; FAB-MS, fast atom bombardment mass spectrometry; HPLC, high pressure liquid chromatography; MeCN, acetonitrile; NMM, N-methylmorpholine; PyBOP, benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate; SELCOM, self consistent method; TMB, 3,3′,5,5′-tetramethylbenzidine; TFA, trifluoroacetic acid; TFE, trifluoroethanol; Aminoacid symbols denote the L-configurations. Abbreviations for amino acids and nomenclature of peptides follow the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature (Eur. J. Biochem., 138 (1984) 9).