A comprehensive proteomic analysis of the accessory sex gland fluid from mature Holstein bulls

The expression of proteins in accessory sex gland fluid (AGF) of proven, high use mature Holstein bulls was evaluated. Thirty-seven bulls with documented fertility based on their non-return rates were studied. AGF was obtained by artificial vagina after bulls were surgically equipped with cannulae in the vasa deferentia. Samples of AGF were evaluated by two-dimensional SDS-PAGE, gels stained with Coomassie blue and polypeptide maps analyzed by PDQuest software. A master gel generated by the software representing the best pattern of spots in the AGF polypeptide maps was used as a reference for protein identification. Proteins were identified by Western blots and capillary liquid chromatography-nanoelectrospray ionization tandem-mass spectrometry (CapLC-MS/MS). The product ion spectra were processed using Protein Lynx Global Server 2.1 prior to database search with both PLGS and MASCOT (Matrix Science) software. The entire NCBI database was considered for mass fingerprint matching. An average of 52+/-5 spots was detected in the AGF 2D gels, which corresponded to proteins potentially involved in capacitation (bovine seminal plasma protein-BSP-A1/A2 and A3, BSP 30 kDa, albumin); sperm membrane protection, prevention of oxidative stress, complement-mediated sperm destruction and anti-microbial activity (albumin, clusterin, acidic seminal fluid protein--aSFP, 5'-nucleotidase--5'-NT, phospholipase A2--PLA2); acrosome reaction and sperm-oocyte interaction (PLA2, osteopontin); interaction with the extracellular matrix (tissue inhibitor of metalloproteinase 2, clusterin) and sperm motility (aSFP, spermadhesin Z13, 5'-NT). The 20 spots distinguished in all gels were matched to proteins associated with these functions. Proteins identified by tandem mass spectrometry as ecto-ADP-ribosyltransferase 5 and nucleobindin, never described before in the accessory sex gland secretions, were also detected. In summary, we identified a diverse range of components in the accessory sex gland fluid of a select group of Holstein bulls with documented fertility. Known characteristics of these proteins suggest that they play important roles in sperm physiology after ejaculation.     

Melatonergic Receptors (Mt1/Mt2) as a Potential Additional Target of Novel Drugs for Depression

Neurochemical Research - A complex pathogenesis involving several physiological systems is theorized to underline the development of depressive disorders. Depression is accompanied by circadian...     

Cholinesterase inhibition and its association with hematological,biochemical and oxidative stress markers in chronic pesticide exposed agriculture workers

The present study investigated the pesticide induced adverse health effects, hematological and biochemical alterations among agriculture workers. A cross sectional study of 51 agriculture workers and 54 unexposed subjects was carried out to evaluate hematological and biochemical alterations in blood. Pesticide exposed individuals were reported adverse clinical outcomes, including tingling, muscle pain, headache, skin disease, etc. A significant alterations in the level of hematological parameters, liver and renal dysfunctions markers and lipid profile suggested hematological, hepatic and renal dysfunctions. A significant decrease in the activity of acetylcholinesterase, reduced glutathione, superoxide dismutase, catalase and increased level of lipid peroxidation was also observed in these agriculture workers. Correlation coefficient analysis showed a positive correlation of chronic exposure with most of the hematological and biochemical parameters. The results demonstrate that the chronic exposure of pesticides cause reduction in the acetylcholinesterase activity and enhanced the risk of adverse clinical outcomes in agriculture workers.     

Similarities in mRNA expression of peripheral blood drug metabolizing enzymes and cancer marker genes with biopsy samples of head and neck cancer patients

Purpose: To develop peripheral blood mRNA expression profiles of drug metabolizing enzymes (DMEs) as a surrogate to monitor tobacco induced head and neck squamous cell carcinoma (HNSCC), attempts were made to investigate (i) similarities in alterations with the cancer marker genes in biopsy samples and (ii) if alterations similar to that seen in biopsy samples are reflected in peripheral blood.

Methods: Total RNA from eight soft gingival tissues and eight biopsy samples of HNSCC patients and total DNA and RNA from blood of healthy controls (n?=?150) and HNSCC patients (n?=?150) was processed for expression and genotyping studies. Blood from patients receiving chemo-radiotherapy was processed for follow-up study.

Results: qRT-PCR revealed significant increase in mRNA expression of DMEs in biopsy and blood samples of HNSCC patients when compared to controls. Similar alterations were observed in cancer marker genes in these samples. Patients with variant genotypes of DMEs showed greater magnitude of alterations in mRNA expression when compared to wild type controls. Responders of chemo-radiotherapy showed significant decline in induction of mRNA expression of DMEs and cancer marker genes

Conclusions: The data suggest that peripheral blood expression profiles could be used to monitor tobacco-induced HNSCC as well as the treatment response.     

Explication of bovine serum albumin binding with naphthyl hydroxamic acids using a multispectroscopic and molecular docking approach along with its antioxidant activity

In the present investigation, the protein‐binding properties of naphthyl‐based hydroxamic acids (HAs), N‐1‐naphthyllaurohydroxamic acid ( 1 ) and N‐1‐naphthyl‐p‐methylbenzohydroxamic acid ( 2 ) were studied using bovine serum albumin (BSA) and UV–visible spectroscopy, fluorescence spectroscopy, diffuse reflectance spectroscopy–Fourier transform infrared (DRS–FTIR), circular dichroism (CD), and cyclic voltammetry along with computational approaches, i.e. molecular docking. Alteration in the antioxidant activities of compound 1 and compound 2 during interaction with BSA was also studied. From the fluorescence studies, thermodynamic parameters such as Gibb's free energy (ΔG), entropy change (ΔS) and enthalpy change (ΔH) were calculated at five different temperatures (viz., 298, 303, 308, 313 or 318 K) for the HAs–BSA interaction. The results suggested that the binding process was enthalpy driven with dominating hydrogen bonds and van der Waals’ interactions for both compounds. Warfarin (WF) and ibuprofen (IB) were used for competitive site‐specific marker binding interaction and revealed that compound 1 and compound 2 were located in subdomain IIA (Sudlow's site I) on the BSA molecule. Conclusions based on above‐applied techniques signify that various non‐covalent forces were involved during the HAs–BSA interaction. Therefore the resulted HAs–BSA interaction manifested its effect in transportation, distribution and metabolism for the drug in the blood circulation system, therefore establishing HAs as a drug‐like molecule.     

Intracerebroventricular Injection of the Glutamatergic Receptors Antagonist Affects N/OFQ-Induced Hyperphagia in Neonatal Broilers: Role of NMDA and AMPA Receptors

International Journal of Peptide Research and Therapeutics - This study was designed to determine possible interaction of the central nociceptin/orphanin FQ (N/OFQ) and glutamatergic system on...     

Cysteine-specific ubiquitination protects the peroxisomal import receptor Pex5p against proteasomal degradation

Peroxisomal matrix protein import is mediated by dynamic import receptors, which cycle between the peroxisomal membrane and the cytosol. Proteins with a type 1 peroxisomal targeting signal (PTS1) are bound by the import receptor Pex5p in the cytosol and guided to the peroxisomal membrane. After cargo translocation into the peroxisomal matrix, the receptor is released from the membrane back to the cytosol in an ATP-dependent manner by the AAA-type ATPases Pex1p and Pex6p. These mechanoenzymes recognize ubiquitinated Pex5p-species as substrates for membrane extraction. The PTS1-receptor is either polyubiquitinated via peptide bonds at two certain lysines and results in proteasomal degradation or monoubiquitinated via a thioester-bond at a conserved cysteine, which enables the recycling of Pex5p and further rounds of matrix protein import. To investigate the physiological relevance of the conserved N-terminal cysteine of Pex5p, the known target amino acids for ubiquitination were substituted by site-directed mutagenesis. In contrast with Pex5p_C6A, Pex5p_C6K turned out to be functional in PTS1 import and utilization of oleic acid, independent of the lysines at position 18 and 24. In contrast with wild-type Pex5p, Pex5p_C6K displays an ubiquitination pattern, similar to the polyubiquitination pattern of Pex4p or Pex22p mutant strains. Moreover, Pex5p_C6K displays a significantly reduced steady-state level when the deubiquitinating enzyme Ubp15p is missing. Thus, our results indicate that not the cysteine residue but the position of ubiquitination is important for Pex5p function. The presence of the cysteine prevents polyubiquitination and rapid degradation of Pex5p.     

Cytotoxicity and genotoxicity of zingiberene on different neuron cell lines in vitro

The main objective of this study is to investigate the cytotoxic, genotoxic and antioxidant properties of zingiberene (ZBN) in an in vitro rat brain cell culture study. The cytotoxic effect was determined against the rat neuron and N2a neuroblastoma (N2a-NB) cell lines using the 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, while the antioxidant activity was assessed using the total antioxidant capacity (TAC) and total oxidative stress (TOS) assays. The effects on DNA damage were also evaluated in this study by the single cell gel electrophoresis assay. The results indicated that ZBN has an anti-proliferative activity suppressing the proliferation of N2a-NB cells at concentrations over 50 mg L⁻¹ and neuron cells at concentrations over 150 mg L⁻¹. In addition, ZBN treatments at higher doses (≤50 mg L⁻¹) led to increases of TOS levels in N2a-NB cell cultures. However 25 mg L⁻¹ of ZBN treatment caused increases of TAC levels in cultured neuron and N2a-NB cell cultures while ZBN at doses of 10–400 mg L⁻¹ did not increase the number of total damage score in both cell lines. This study clearly indicates that ZBN has a significant potential to be used as a natural anticancer agent in cultured N2a-NBs.     

Less is more: extreme genome complexity reduction with ddRAD using Ion Torrent semiconductor technology

Massively parallel sequencing a small proportion of the whole genome at high coverage enables answering a wide range of questions from molecular evolution and evolutionary biology to animal and plant breeding and forensics. In this study, we describe the development of restriction‐site associated DNA (RAD) sequencing approach for Ion Torrent PGM platform. Our protocol results in extreme genome complexity reduction using two rare‐cutting restriction enzymes and strict size selection of the library allowing sequencing of a relatively small number of genomic fragments with high sequencing depth. We applied this approach to a common freshwater fish species, the Eurasian perch (Perca fluviatilis L.), and generated over 2.2 MB of novel sequence data consisting of ~17 000 contigs, identified 1259 single nucleotide polymorphisms (SNPs). We also estimated genetic differentiation between the DNA pools from freshwater (Lake Peipus) and brackish water (the Baltic Sea) populations and identified SNPs with the strongest signal of differentiation that could be used for robust individual assignment in the future. This work represents an important step towards developing genomic resources and genetic tools for the Eurasian perch. We expect that our ddRAD sequencing protocol for semiconductor sequencing technology will be useful alternative for currently available RAD protocols.