A novel hypothesis for histone-to-protamine transition in Bos taurus spermatozoa

DNA compaction with protamines in sperm is essential for successful fertilization. However, a portion of sperm chromatin remains less tightly packed with histones, which genomic location and function remain unclear. We extracted and sequenced histone-associated DNA from sperm of nine ejaculates from three bulls. We found that the fraction of retained histones varied between samples, but the variance was similar between samples from the same and different individuals. The most conserved regions showed similar abundance across all samples, whereas in other regions, their presence correlated with the size of histone fraction. This may refer to gradual histone-protamine transition, where easily accessible genomic regions, followed by the less accessible regions are first substituted by protamines. Our results confirm those from previous studies that histones remain in repetitive genome elements, such as centromeres, and added new findings of histones in rRNA and SRP RNA gene clusters and indicated histone enrichment in some spermatogenesis-associated genes, but not in genes of early embryonic development. Our functional analysis revealed significant overrepresentation of cGMP-dependent protein kinase G (cGMP-PKG) pathway genes among histone-enriched genes. This pathway is known for its importance in pre-fertilization sperm events. In summary, a novel hypothesis for gradual histone-toprotamine transition in sperm maturation was proposed. We believe that histones may contribute structural information into early embryo by epigenetically modifying centromeric chromatin and other types of repetitive DNA. We also suggest that sperm histones are retained in genes needed for sperm development, maturation and fertilization, as these genes are transcriptionally active shortly prior to histone-to-protamine transition.Peer reviewe

High-throughput mRNA sequencing of stromal cells from endometriomas and endometrium

The aetiology of endometriosis is still unclear and to find mechanisms behind the disease development, it is important to study each cell type from endometrium and ectopic lesions independently. The objective of this study was to uncover complete mRNA profiles in uncultured stromal cells from paired samples of endometriomas and eutopic endometrium. High-throughput mRNA sequencing revealed over 1300 dysregulated genes in stromal cells from ectopic lesions, including several novel genes in the context of endometriosis. Functional annotation analysis of differentially expressed genes highlighted pathways related to cell adhesion, extracellular matrix–receptor interaction and complement and coagulation cascade. Most importantly, we found a simultaneous upregulation of complement system components and inhibitors, indicating major imbalances in complement regulation in ectopic stromal cells. We also performed in vitro experiments to evaluate the effect of endometriosis patients’ peritoneal fluid (PF) on complement system gene expression levels, but no significant impact of PF on C3, CD55 and CFH levels was observed. In conclusion, the use of isolated stromal cells enables to determine gene expression levels without the background interference of other cell types. In the future, a new standard design studying all cell types from endometriotic lesions separately should be applied to reveal novel mechanisms behind endometriosis pathogenesis

Purification, structure and function of bioactive peptides

Peptides are vitally important molecules and many evoke cellular responses. The completion of several genome sequencing projects has revealed a number of new genes. However, as functional peptides often contain posttranslational modifications and/or occur at various lengths, it is of great importance to detect, purify and characterize novel bioactive peptides. To achieve these goals, new methods for peptide detection, isolation and functional characterization have to be developed. In the present thesis, a broad range of different techniques for detection, purification, structural and functional characterization of new peptides, the production of recombinant peptides, their reconstitution with metals and functional characterization was utilized. A detection method was developed to screen for receptor ligands from tissue extracts using Chinese hamster ovary (CHO) cells with a Cytosensor. As a proof-of-concept, an active component in tissue extracts was purified by a seven-step protocol. The structure was determined by mass spectrometry (MS), which identified it as insulin-like growth factor-I (IGF-I). A novel ligand for an orphan G-protein coupled receptor, GPCR135, was detected in porcine brain extracts by guanosine 5`-O-(3-thiotriphosphate) (GTPγS) incorporation assay. The ligand was purified by a six-step procedure. Matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry analyses demonstrated that the ligand is relaxin 3, which is composed of two peptide chains linked together with disulfide bridges similarly to insulin-relaxin family of peptides. Pharmacological experiments showed that relaxin-3 is the only ligand that activates GPCR135. An elongated form of peptide histidine isoleucine amide (PHI), PHI-42, was detected in porcine intestinal extracts by cAMP assays on IMR-32 neuroblastoma cells. PHI-42 was purified from the mixture by a four-step procedure. The structure was determined by MALDI MS and Edman degradation, which showed that the 12-residue sequence of porcine PHI-42 is unique and links together PHI-27 and vasoactive intestinal polypeptide (VIP) in the precursor. A novel posttranslationally modified form of neurotensin (NT) was identified from porcine intestine and purified by a five-step procedure. Linear ion trap-Fourier transform, MALDI, and ESI quadropole time-of-flight (Q-TOF) mass spectrometries identified the posttranslational modification as an Arg-residue coupled to the gamma-carboxyl group of Glu-4 by an isopeptide bond. In addition, NT(2-13) and NT(3-13) together with a fragment (22-38) of dopamine- and adenosine 3 ,5 -monophosphate-regulated phosphoprotein (DARPP-32) were also detected and studied by MALDI MS and Edman degradation. All the NT forms were active in intracellular Ca2+ release assay. Human brain-specific metallothionein-3 (MT-3) was produced by recombinant technology and studied by ESI MS. The protein was purified as apo-MT-3 by a new three-step procedure. Titration of MT-3 with Zn2+ and Cd2+ ions and the stability of metal complexes were followed by ESI MS and the results were compared with data for common MT-s. We demonstrated that MT-3, in contrast to common MTs, binds divalent metals in a non-cooperative manner and at higher capacity than common MTs. The combination of bioassays, high performance liquid chromatography, and mass spectrometry has enabled us to detect, purify and characterize novel peptides from biological sources, yielding new knowledge about their localization, structure, processing and functions

Peptide-Based Glioma-Targeted Drug Delivery Vector gHoPe2

Gliomas are therapeutically challenging cancers with poor patient prognosis. New drug delivery strategies are needed to achieve a more efficient chemotherapy-based approach against brain tumors. The current paper demonstrates development of a tumor-targeted delivery vector that is based on a cell-penetrating peptide pVEC and a novel glioma-targeting peptide sequence gHo. The unique tumor-homing peptide gHo was identified using <i>in vitro</i> phage display technology. The novel delivery vector, which we designated as gHoPe2, was constructed by a covalent conjugation of pVEC, gHo, and a cargo; the latter could be either a labeling moiety (such as a fluorescent marker) or a cytostatic entity. Using a fluorescent marker, we demonstrate efficient uptake of the vector in glioma cells and selective labeling of glioma xenograft tumors in a mouse model. This is the first time that we know where <i>in vitro</i> phage display has yielded an efficient, <i>in vivo</i> working vector. We also demonstrate antitumor efficacy of the delivery vector gHoPe2 using a well-characterized chemotherapeutic drug doxorubicin. Vectorized doxorubicin proved to be more efficient than the free drug in a mouse glioma xenograft model after systemic administration of the drugs. In conclusion, we have characterized a novel glioma-homing peptide gHo, demonstrated development of a new and potential glioma-targeted drug delivery vector gHoPe2, and demonstrated the general feasibility of the current approach for constructing cell-penetrating peptide-based targeted delivery systems