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Nosemosis is one of the most widespread honeybee diseases. Its epidemical state can be determined as panzootic. The infectious agents are the microsporidia Nosema apis and N. ceranae. Numerous substances and preparations were tested in order to find a way to combat this disease. However, methodology used in artificial infection experiments is not unique; concentrations of N. ceranae spores in inoculum vary as well as the age of honey bees when they are infected. In addition, the disease itself is still relatively poorly understood. This makes the interpretation of such research difficult. The aim of this study is to investigate the effect of bee age and inoculum concentration on the development of N. ceranae infection. Honeybee workers were collectively infected at the age of 2 and 10 days post-emergence with concentrations of 104, 5 × 104, and 105 spores/bee. While the results indicate a significant effect of both tested factors on the development of N. ceranae, the relationship is not simple, and age alters the pattern of nosemosis development in response to the given concentrations. The course and the effect of Nosema ceranae infection differed depending on bee age at the moment of infection. The development of the infection differed depending on concentration of N. ceranae spores in inoculum. Concentration of N. ceranae spores had an effect on mortality and syrup intake in 10-day-old bees. [ABSTRACT FROM AUTHOR]

Eight synthetic histidine analogues of oxytocin and vasopressin are subject of investigations. The spectra of the peptides have been investigated in the terahertz band. The results are obtained in the terahertz time-domain spectroscopy arrangement. [ABSTRACT FROM AUTHOR]

Positive selection of cortical CD4+8+ thymocytes represents crucial and mysterious process in T cell development whereby short-lived precursors are rescued from programmed cell death and induced to differentiate towards long-lived CD4 and CD8 T cells. One reason that this process is not fully understood is that the inventory of genes changing their expression in positively selected CD4+8+ thymocytes is not yet complete. In this work Affymetrics GeneChip cDNA microarrays and cDNA-Representational Difference Analysis were used to search for unknown and known genes that were not identified before as being involved in positive selection. Comparison of transcriptosome of nonstimulated with transcriptosome of PMA/ionomycin stimulated thymoma cell line resembling CD4+8+ thymocytes and subtraction of cDNA of extrathymic tissues from cDNA of purified CD4+8+ thymocytes resulted in identification of 36 genes, which have not been previously reported to change their expression during positive selection. One of them represents a novel, third evolutionarily conserved gene within RAG locus. [ABSTRACT FROM AUTHOR]

Background: Autosomal dominant tubulointerstitial kidney disease (ADTKD) results from mutations in various genes, including REN, UMOD, MUC1, and HNF1B. ADTKD due to REN mutations (ADTKD-REN) is often characterized as a proteinopathy that triggers the endoplasmic reticulum stress (ERS) cascade, potentially sharing similarities with ADTKD-UMOD and ADTKD-MUC1 at the cellular level. This study, inspired by a patient harboring a W17R mutation, investigates ERS activation by this mutation alongside two other renin variants, W10R and L381P., Methods: We established stable cell lines expressing both wild-type and mutated renin forms (W17R, W10R, and L381P). Using luciferase reporter assays, RT-qPCR, and confocal microscopy, we evaluated ERS activation, determined the cellular localization of the renin variants, and characterized the mitochondrial network in the W17R line., Results: The L381P line exhibited ERS activation, including transcriptional upregulation of MANF and CRELD2. No ERS activation was observed in the W17R line, while the W10R line exhibited intermediate characteristics. Notably, the W17R variant was misrouted to the mitochondria resulting in changes of the mitochondrial network organisation., Conclusions: ERS activation is not a universal response to different renin mutations in ADTKD-REN. The pathogenesis of the W17R mutation may involve mitochondrial dysfunction rather than the ER pathway, albeit further research is needed to substantiate this hypothesis fully. Testing CRELD2 and MANF as targeted therapy markers for a specific subgroup of ADTKD-REN patients is recommended. Additionally, fludrocortisone treatment has shown efficacy in stabilizing the renal function of our patient over a four-year period without significant side effects., (© 2024. The Author(s), under exclusive licence to International Pediatric Nephrology Association.)

There are many artificial sources of radiofrequency electromagnetic field (RF-EMF) in the environment, with a value between 100 MHz and 6 GHz. The most frequently used signal is with a frequency of around 900 MHz. The direction of these changes positively impacts the quality of life, enabling easy communication from almost anywhere in the world. All living organisms in the world feel the effects of the electromagnetic field on them. The observations regarding the influence of a RF-EMF on honey bees, describing the general impact of RF-EMF on the colony and/or behavior of individual bees, such as reduction in the number of individuals in colonies, extended homing flight duration, decrease in breeding efficiency, changes in flight direction (movement of bees toward the areas affected by RF-EMF), increase in the intensity and frequency of sounds characteristic for those announcing the impending danger. In this work, we describe the changes in the levels of some of the stress-related markers in honey bees exposed to varying intensities and duration of RF-EMF. One-day-old honeybee worker bees were used for the study. The bees were randomly assigned to 9 experimental groups which were exposed to the following 900 MHz EMF intensities: 12 V/m, 28 V/m, and 61 V/m for 15 min, 1 h and 3 h. The control group was not exposed to the RF-EMF. Each experimental group consisted of 10 cages in which were 100 bees. Then, hemolymph was collected from the bees, in which the activity was assessed AST, ALT, ALP, GGTP, and level of nonenzymatic antioxidants albumin, creatinine, uric acid, and urea. Bees were also collected for the analysis of rps5, ppo, hsp10, hsp70, hsp90, and vitellogenin gene expression. Our study shows that exposure to a 900 MHz electromagnetic field induces a response in the honey bees that can be detected in the level of enzyme activity and the expression of stress-related genes. The response is similar to the one previously described as a result of exposition to UVB irradiation and most likely cannot be attributed to increased temperature., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Migdal et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The complementary sex determiner (csd) gene is responsible for controlling the sex-determination molecular switch in western honey bees (Apis mellifera): bees that are heterozygous for csd develop into females, whereas bees that are hemizygous or homozygous develop into males. The homozygous diploid males are destroyed at an early stage of their development. It has been proposed that the minimal number of amino acid differences between two csd alleles needed to fully determine femaleness is five and it has also been shown that smaller differences may result in forming an evolutionary intermediate that is not fully capable of female determination, but has increased fitness compared to the homozygous genotype. In this study, we have implemented a terminal restriction length polymorphism-based method of identifying and distinguishing paternal alleles in a given bee colony and assigning them to a particular maternal allele in order to gather information on large number of functional csd pairs and also to identify, to some extent, genotypes that are underrepresented or absent in bee colonies. The main finding of this study is the identification of a fully functional genotype consisting of csd alleles that differed from each other by a one amino acid position. The individuals carrying this genotype expressed only female-specific transcripts of feminizer and double-sex genes. By comparing the sequences differences between the csd pair identified in our study with those described earlier, we conclude that functional heterozygosity of the csd gene is dependent not only on the number of the amino acid differences but also on the sequence context and position of the change. The discovery of a functional allele pair differing by a single amino acid also implies that the generation of a new csd specificity may also occur during a single mutation step with no need for evolutionary intermediates accumulating further mutations., Competing Interests: The authors have declared that no competing interests exist.

Argireline-containing cosmetics attract public interest due to their confirmed reduction of facial wrinkles. Argireline is a peptide that works by inhibiting the release of neurotransmitters in the neuromuscular junction, producing a botox-like effect. Therefore, it is used as a safe needle-free alternative to botox treatment. In this work we investigated the presence of Argireline in cosmetic creams and sera by application of reversed phase liquid chromatography and tandem mass spectrometry (RP-HPLC/MS and MS/MS). The analysis revealed the presence of argireline and its oxidized form in several different cosmetics. The methionine residue in Argireline sequence was indicated as oxidation point according to neutral loss MS studies. The developed sample preparation strategy minimizes and monitors methionine oxidation, bringing to our attention the question of impact of ingredients on the stability of cosmetic product., (© 2021 Wiley-VHCA AG, Zurich, Switzerland.)

Mass spectrometry methods are commonly used in the identification of peptides and biomarkers. Due to a relatively low abundance of proteins in biological samples, there is a need for the development of novel derivatization methods that would improve MS detection limits. Hence, novel fluorescent N -hydroxysuccinimide esters of dihydro-[1,2,4]triazolo[4,3- a ]pyridin-2-ium carboxylates ( Safirinium P dyes) have been synthesized. The obtained compounds, which incorporate quaternary ammonium salt moieties, easily react with aliphatic amine groups of peptides, both in solution and on the solid support; thus, they can be applied for derivatization as ionization enhancers. Safirinium tagging experiments with ubiquitin hydrolysate revealed that the sequence coverage level was high (ca. 80%), and intensities of signals were enhanced up to 8-fold, which proves the applicability of the proposed tags in the bottom-up approach. The obtained results confirmed that the novel compounds enable the detection of trace amounts of peptides, and fixed positive charge within the tags results in high ionization efficiency. Moreover, Safirinium NHS esters have been utilized as imaging agents for fluorescent labeling and the microscopic visualization of living cells such as E. coli Top10 bacterial strain.

(1) Background : Vitamin D receptor (VDR) is present in multiple types of blood cells, and its ligand, 1,25-dihydroxyvitamin D (1,25D), is important for the proper functioning of the immune system. Activity of VDR is higher in hematopoietic stem and progenitor cells than in fully differentiated blood cells of mice and humans. In some human acute myeloid leukemia (AML) blasts, the expression of the VDR gene is also high. The mechanism of silencing the VDR gene expression during differentiation of blood cells has been addressed in this work. (2) Methods : The cells have been obtained using fluorescence activated sorting from murine tissues and from human umbilical cord blood (UCB). Then, the expression of the VDR gene and transcriptional activity of the VDR protein has been tested in real-time polymerase chain reaction (PCR). Eventually, the methylation of VDR promoter regions was tested using bisulfite sequencing. (3) Results : The CpG islands in VDR promoters were not methylated in the cells studied both in mice and in humans. The use of hypomethylating agents had no effect toward expression of human VDR transcripts, but it increased expression of the VDR-target gene, CYP24A1 . (4) Conclusions : The expression of the VDR gene and transcriptional activity of the VDR protein varies at successive stages of hematopoietic differentiation in humans and mice, and in blasts from AML patients. The experiments presented in this case indicate that methylation of the promoter region of the VDR gene is not the major mechanism responsible for these differences.

Microsporidia Nosema are transferred among bees via the faecal-oral route. Nosema spp. spores have been detected on flowers and transferred to hives along with the bee pollen. The aim of the present study was to determine whether Nosema microsporidia are transferred by air in an apiary, in a control area (without the presence of bee colonies), and/or in a laboratory during cage experiments with artificially infected bees. The novel way of transmission by air was investigated by the volumetric method using a Hirst-type aerobiological sampler located on the ground in the apiary, in the Botanical Garden and on the laboratory floor. Concurrently, the mean rate of Nosema infections in the foragers in the apiary was estimated with the Bürker haemocytometer method. Spore-trapping tapes were imaged by means of light microscopy, Nomarski interference contrast microscopy and scanning electron microscopy. The highest concentration of Nosema spores per 1m 3 of air (4.65) was recorded in August, while the lowest concentration (2.89) was noted in July. This was confirmed by a Real-Time PCR analysis. The presence of N. apis as well as N. ceranae was detected in each of the tested tapes from the apiary. The average copy number of N. apis was estimated at 14.4 × 10 4 copies per 1 cm 2 of the tape; whereas the number of N. ceranae was 2.24 × 10 4 copies per tape per 1 cm 2 . The results indicate that Nosema microsporidia were transferred by the wind in the apiary, but not in the Botanical Garden and laboratory by air. This was confirmed by genetic analyses. DNA from immobilised biological material was isolated and subjected to a PCR to detect the Nosema species. A fragment of the 16S rRNA gene, characteristic of Nosema apis and N. ceranae, was detected. Our research adds knowledge about the transfer of Nosema spp. microsporidia in the natural environment and indicates the season associated with the greatest risk of a bee colony infection with Nosema spp.

NWC is an uncharacterised protein containing three strongly conserved domains not found in any other known protein. Previously, we reported that the NWC protein is detected in cells in the germinal layer in murine testes (strain: C57BL/6), and its knockout results in no obvious phenotype. We determined the NWC expression pattern during spermatogenesis, and found this protein in spermatocytes and round spermatids, but not in epididymal sperm. Although NWC knockout males are fertile, we further characterised their reproductive potential employing non-standard mating that better simulates the natural conditions by including sperm competition. Such an approach revealed that the sperm of knockout males fail to successfully compete with control sperm. After analysing selected characteristics of the male reproductive system, we found that NWC knockout sperm had a reduced ability to fertilize cumulus-intact eggs during IVF. This is the first report describing a subtle phenotype of NWC knockout mice that could be detected under non-standard mating conditions. Our results indicate that NWC plays an important role in spermatogenesis and its deficiency results in the production of functionally impaired sperm., Competing Interests: The authors have declared that no competing interests exist.

The RAG-1 and RAG-2 genes form a recombinase complex that is indispensable for V(D)J recombination, which generates the diversity of immunoglobulins and T-cell receptors. It is widely accepted that the presence of RAGs in the genomes of jawed vertebrates and other lineages is a result of the horizontal transfer of a mobile genetic element. While a substantial amount of evidence has been gathered that clarifies the nature of the RAG transposon, far less attention has been paid to the genomic site of its integration in various host organisms. In all genomes of the jawed vertebrates that have been studied to date, the RAG genes are located in close proximity to the NWC gene. We have previously shown that the promoter of the murine NWC genes exhibits a bidirectional activity, which may have facilitated the integration and survival of the RAG transposon in the host genome. In this study, we characterise the promoters of the NWC homologues that are present in the representatives of other jawed vertebrates (H. sapiens, X. tropicalis and D. rerio). We show that the features that are characteristic for promoters as the hosts of a successful transposon integration (in terms of the arrangement, bidirectional and constitutive activity and the involvement of the Zfp143 transcription factor in the promoter regulation) are evolutionarily conserved, which indicates that the presence of RAG genes in jawed vertebrates is a direct result of a successful transposon integration into the NWC locus., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Embryonal carcinoma (EC) cells are pluripotent stem cells extensively used for studies of cell differentiation. Although retinoic acid (RA) is a powerful inducer of neurogenesis in EC cells, it is not clear what specific neuronal subtypes are generated and whether different RAR isotypes may contribute to such neuronal diversification. Here we show that RA treatment during EC embryoid body formation is a highly robust protocol for generation of striatal-like GABAergic neurons which display molecular characteristics of striatopallidal medium spiny neurons (MSNs), including expression of functional dopamine D2 receptor. By using RARα, β and γ selective agonists we show that RARγ is the functionally dominant RAR in mediating RA control of early molecular determinants of MSNs leading to formation of striatopallidal-like neurons. In contrast, activation of RARα is less efficient in generation of this class of neurons, but is essential for differentiation of functional dopaminergic neurons, which may correspond to a subpopulation of inhibitory dopaminergic neurons expressing glutamic acid decarboxylase in vivo.

Vitamin D receptor (VDR) is present in multiple blood cells, and the hormonal form of vitamin D, 1,25-dihydroxyvitamin D (1,25D) is essential for the proper functioning of the immune system. The role of retinoic acid receptor α (RARα) in hematopoiesis is very important, as the fusion of RARα gene with PML gene initiates acute promyelocytic leukemia where differentiation of the myeloid lineage is blocked, followed by an uncontrolled proliferation of leukemic blasts. RARα takes part in regulation of VDR transcription, and unliganded RARα acts as a transcriptional repressor to VDR gene in acute myeloid leukemia (AML) cells. This is why we decided to examine the effects of the combination of 1,25D and all- trans -retinoic acid (ATRA) on VDR gene expression in normal human and murine blood cells at various steps of their development. We tested the expression of VDR and regulation of this gene in response to 1,25D or ATRA, as well as transcriptional activities of nuclear receptors VDR and RARs in human and murine blood cells. We discovered that regulation of VDR expression in humans is different from in mice. In human blood cells at early stages of their differentiation ATRA, but not 1,25D, upregulates the expression of VDR . In contrast, in murine blood cells 1,25D, but not ATRA, upregulates the expression of VDR . VDR and RAR receptors are present and transcriptionally active in blood cells of both species, especially at early steps of blood development., Competing Interests: The authors declare no conflicts of interest.

The complementary sex determiner (csd) gene determines the sex of the western honey bee (Apis mellifera L.). Bees that are heterozygous at the csd locus develop into females; whereas hemizygous bees develop into males. The co-occurrence of two identical csd alleles in a single diploid genome leads to the genetic death of the bee. Thus, the maintenance of csd diversity in the population is favoured. The number and distribution of csd alleles is particularly interesting in light of the recent decline in the honey bee population. In this study, we analysed the distribution of csd alleles in two Polish populations separated by about 100 km. We analysed the maternal alleles of 193 colonies and found 121 different alleles. We also analysed the distribution and frequency of the alleles, and found that they are distributed unevenly. We show that the methods that have been used so far to estimate the total worldwide number of csd alleles have significantly underestimated their diversity. We also show that the uneven distribution of csd alleles is caused by a large number of infrequent alleles, which most likely results from the fact that these alleles are generated very frequently.

Two new somatostatin analogs with a characteristic part of the sequence -c(Cys-Phe-Trp-Lys-Thr-Cys)- and with two histidine and two aspartic acid moieties in their structures were synthesized and analyzed in terms of their coordination abilities with copper (II) ions. Both peptides bind Cu(II) effectively. Ligands form 4N complexes with [Formula: see text] binding mode in a basic range of pH. But in spite of very similar sequences of the two peptides a significant difference in the effectiveness of the binding of copper (II) ions was observed.

NWC is a third gene within recombination activating gene (RAG) locus, which unlike RAG genes is ubiquitously expressed and encodes a unique protein containing three strongly evolutionarily conserved domains not found in any other known protein. To get insight into its function we identified several proteins co-immunoprecipitating with NWC protein and generated new NWC-deficient mice. Here, we present evidence that unlike many other ubiquitously expressed evolutionarily conserved proteins, functional inactivation of NWC does not cause any gross developmental, physiological or reproductive abnormalities and that under physiological conditions NWC may be involved in assembling and functioning of cilia, cell surface organelles found on nearly every eukaryotic cell.

Acute myeloid leukemia (AML) is the predominant acute leukemia among adults, characterized by an accumulation of malignant immature myeloid precursors. A very promising way to treat AML is differentiation therapy using either all-trans-retinoic acid (ATRA) or 1,25-dihydroxyvitamin D3 (1,25D), or the use of both these differentiation-inducing agents. However, the effect of combination treatment varies in different AML cell lines, and this is due to ATRA either down- or up-regulating transcription of vitamin D receptor (VDR) in the cells examined. The mechanism of transcriptional regulation of VDR in response to ATRA has not been fully elucidated. Here, we show that the retinoic acid receptor α (RARα) is responsible for regulating VDR transcription in AML cells. We have shown that a VDR transcriptional variant, originating in exon 1a, is regulated by RARα agonists in AML cells. Moreover, in cells with a high basal level of RARα protein, the VDR gene is transcriptionally repressed as long as RARα agonist is absent. In these cells down-regulation of the level of RARα leads to increased expression of VDR. We consider that our findings provide a mechanistic background to explain the different outcomes from treating AML cell lines with a combination of ATRA and 1,25D., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Isotope exchange at the histidine C2 atom of imidazole in D2O solution is well known to occur at a significantly slower rate than the exchange of amide protons. Analysis of the kinetics of this isotope-exchange reaction is proposed herein as a method of detecting histidine phosphorylation. This modification of His-containing peptides is challenging to pinpoint because of its instability under acidic conditions as well as during CID-MS analysis. In this work, we investigated the effect of phosphorylation of the histidine side chain in peptides on deuterium-hydrogen exchange (DHX) in the imidazole. The results demonstrate that phosphorylation dramatically slows the rate of the DHX reaction. This phenomenon can be applied to detect phosphorylation of peptides at the histidine residue (e.g., in enzymatic digests). We also found that the influence of the peptide sequence on the exchange kinetics is relatively small. A CID fragmentation experiment revealed that there was no detectable hydrogen scrambling in peptides deuterated at C2 of the imidazole ring. Therefore, MS/MS can be used to directly identify the locations of deuterium ions incorporated into peptides containing multiple histidine moieties.

Improving the sensitivity of detection and fragmentation of peptides to provide reliable sequencing of peptides is an important goal of mass spectrometric analysis. Peptides derivatized by bicyclic quaternary ammonium ionization tags: 1-azabicyclo[2.2.2]octane (ABCO) or 1,4-diazabicyclo[2.2.2]octane (DABCO), are characterized by an increased detection sensitivity in electrospray ionization mass spectrometry (ESI-MS) and longer retention times on the reverse-phase (RP) chromatography columns. The improvement of the detection limit was observed even for peptides dissolved in 10 mM NaCl. Collision-induced dissociation tandem mass spectrometry of quaternary ammonium salts derivatives of peptides showed dominant a- and b-type ions, allowing facile sequencing of peptides. The bicyclic ionization tags are stable in collision-induced dissociation experiments, and the resulted fragmentation pattern is not significantly influenced by either acidic or basic amino acid residues in the peptide sequence. Obtained results indicate the general usefulness of the bicyclic quaternary ammonium ionization tags for ESI-MS/MS sequencing of peptides., (Copyright © 2014 John Wiley & Sons, Ltd.)

Recombination activating gene-2 (RAG-2) and NWC are strongly evolutionarily conserved overlapping genes which are convergently transcribed. In non-lymphoid cells the NWC promoter is active whereas in lymphocytes it is inactive due to the DNA methylation. Analysing the mechanism responsible for lymphocyte-specific methylation and inactivation of NWC promoter we found that Ikaros, a lymphocyte-specific transcription factor, acts as a repressor of NWC promoter--thus identifying a new Ikaros target--but is insufficient for inducing its methylation which depends on the antisense transcription driven by RAG-2 promoter. Possible implications of these observations for understanding evolutionary mechanisms leading to lymphocyte specific expression of RAG genes are discussed.

The concept of differentiation therapy of cancer is ~40 years old. Despite many encouraging results obtained in laboratories, both in vitro and in vivo studies, the only really successful clinical application of differentiation therapy was all-trans-retinoic acid (ATRA)-based therapy of acute promyelocytic leukemia (APL). ATRA, which induces granulocytic differentiation of APL leukemic blasts, has revolutionized the therapy of this disease by converting it from a fatal to a curable one. However, ATRA does not work for other acute myeloid leukemias (AMLs). Since 1,25-dihydroxyvitamin D3 (1,25D) is capable of inducing monocytic differentiation of leukemic cells, the idea of treating other AMLs with vitamin D analogs (VDAs) was widely accepted. Also, some types of solid cancers responded to in vitro applied VDAs, and hence it was postulated that VDAs can be used in many clinical applications. However, early clinical trials in which cancer patients were treated either with 1,25D or with VDAs, did not lead to conclusive results. In order to search for a molecular basis of such unpredictable responses of AML patients toward VDAs, we performed ex vivo experiments using patient's blast cells. Experiments were also performed using 1,25D-responsive and 1,25D-non-responsive cell lines, to study their mechanisms of resistance toward 1,25D-induced differentiation. We found that one of the possible reasons might be due to a very low expression level of vitamin D receptor (VDR) mRNA in resistant cells, which can be increased by exposing the cells to ATRA. Our considerations concerning the molecular mechanism behind the low VDR expression and its regulation by ATRA are reported in this paper.

A series of novel cyclic ureidopeptides, analogues of dermorphine/deltorphine tetrapeptide, were synthesized by solid phase peptide synthesis and/or in solution. The antinociceptive activity of N-substituted amides 1-10 was evaluated using hot-plate and tail-flick tests. Analogue 1 showed significant, stronger than morphine, antinociceptive effect after systemic applications. All analogues were also tested for their in vitro resistance to proteolysis by means of mass spectroscopy and it was found that all substituted amides 1-10 showed full stability during incubation with large excess of chymotrypsin and pepsin. Compound 1 is a lead molecule for further evaluation., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

In this paper we present the studies on coordination abilities of two short-chain analogues of somatostatin with free N-terminal and protected amino group towards copper (II) ions. The octreotide is the most popular analogue of the somatostatin (peptide hormone) used in medicine. Somatostatin analogues are used in diagnosis and treatment of the neuroendocrine tumors. Both analyzed analogues are characterized by the presence of two His instead of Cys residues in characteristic fragment of native peptide. We characterize coordination abilities of the ligands using potentiometric and spectroscopic methods. His-analogues of somatostatin are effective ligands for copper (II) ions. Both peptides are able to form the complexes with the cyclic structure., (Copyright © 2012 Elsevier Inc. All rights reserved.)

In the present study, the coordination abilities of ubiquitin 50-59 fragment and its analog containing βAsp residue are discussed. The analysis is provided based on the results of potentiometric and spectroscopic measurements supported by quantum-chemical calculations. Interesting differences in the coordination of the metal cation by modified and unmodified peptides are reported. Moreover, in order to further characterize experimentally observed species, we performed quantum-chemical calculations for structures mimicking ubiquitin 50-59 fragment as a step toward a better understanding of structural and energetical aspects related to the coordination abilities of ubiquitin., (Copyright © 2011. Published by Elsevier Inc.)

The recombination-activating genes (RAG-1 and RAG-2) encode a V(D)J recombinase responsible for rearrangements of antigen-receptor genes during T and B cell development, and RAG expression is known to correlate strictly with the process of rearrangement. In contrast to RAG-1, the expression of RAG-2 was not previously detected during any other stage of lymphopoiesis or in any other normal tissue. Here we report that the CpG island-associated promoter of the NWC gene (the third evolutionarily conserved gene in the RAG locus), which is located in the second intron of RAG-2, has bidirectional activity and is responsible for the detectable transcription of RAG-2 in some non-lymphoid tissues. We also identify evolutionarily conserved promoter fragments responsible for this bidirectional activity, and show that it is activated by transcription factor ZFP143. The possible implications of our findings are briefly discussed.

Nwc represents a mysterious third evolutionarily conserved gene within the Rag locus. Here, we analyzed the phenotype of Nwc(tmpro1) mice, in which the Rag2 intragenic region containing the previously identified promoter responsible for initiating transcription of Nwc in all cells except lymphocytes was deleted by homologous recombination. Despite strong nonlymphocyte-specific inhibition of Nwc transcription which runs through the regulatory region of Rag genes, their expression remained suppressed, and no developmental, morphological, anatomical, functional, physiological, or cellular defects in Nwc(tmpro1) mice could be observed. However, careful analysis of the Rag2 intergenic region uncovered a second evolutionarily conserved Nwc promoter region from which a previously unknown Nwc transcript can be generated in nonlymphocytes of Nwc(tmpro1) and normal mice. The above results reveal an unexpected additional complexity of transcriptional regulation within the Rag/Nwc locus and show that strong inhibition of Nwc transcription in nonlymphoid cells is well tolerated. Complete inactivation of Nwc is necessary to get insight into its function at transcriptional and posttranscriptional levels.

The coordination abilities of tetrapeptides containing beta-amino acids towards Cu(II) ions are presented. The studied tetrapeptides were: Ac-betaAlaHisGlyHis, betaAlaHisGlyHis, Ac-betaAspHisGlyHis, betaAspHisGlyHis, Ac-betaAspHisGly-dHis and betaAspHisGly-dHis. Thorough potentiometric titrations were carried out to establish the stoichiometry of the resulting metal-ligand complexes and the role of free -alphaCOO(-) side chain group in metal binding. The copper(II) coordination mode of the complexes was investigated by performing detailed spectroscopic analyses (UV-Vis, EPR, CD) in strict correlation with potentiometric measurements.

A replacement of both Cys residues by His in oxytocin (OXT) sequence allows for the formation of the stable complex with the {NH(2), N(Im), N(Im(macrochelate))} binding mode at the physiological pH. The detailed potentiometric and spectroscopic studies on the Cu(II) complexes of [His(1,6)]OXT, together with high resolution NMR investigations on 3D structures of Cu(II) complexes with [His(1,6)]OXT and [His(1,6)]AVP analogues are presented and discussed. Exchange of the Cys-S-S-Cys bridge by the His-Cu(II)-His motif is very promising, because the resulting complexes retain topological similarity to the native S-S bridged AVP and OXT at pH values corresponding to the physiological pH.

We have characterized the interaction between the Zn(2+) ions and the histidine analogues of oxytocin and arginine-vasopressin. Potentiometric methods were used for the determination of the stoichiometry of the complexes formed and the calculation of their stability constants. The NMR measurements revealed detailed structures of the complexes and confirmed the binding mode at physiological pH.

Recently, ubiquitin was suggested as a promising anti-inflammatory protein therapeutic. We found that a peptide fragment corresponding to the ubiquitin(50-59) sequence (LEDGRTLSDY) possessed the immunosuppressive activity comparable with that of ubiquitin. CD and NMR spectroscopies were used to determine the conformational preferences of LEDGRTLSDY in solution. The peptide mixture, obtained by pepsin digestion of ubiquitin, was even more potent than the intact protein. Although the peptide exhibited a well-defined conformation in methanol, its structure was distinct from the corresponding 50-59 fragment in the native ubiquitin molecule. (c) 2009 Wiley Periodicals, Inc. Biopolymers 91: 423-431, 2009.

Recently, a third evolutionarily conserved gene, NWC, was discovered within the recombination activating gene (RAG) locus, known to contain the RAG1 and RAG2 genes. Here, we identify and characterize the murine endogenous NWC protein which has no homology to any known protein and is ubiquitously expressed. In the cell, the NWC protein which has been suggested to function as a transcriptional repressor, is found in the cytoplasm as well as in the nucleus.

A new vasopressin analogue, [His1,6]AVP, was synthesized and characterized by potentiometric measurements as well as by UV-Vis, CD and EPR spectroscopy. At the physiological pH the peptide forms a stable complex with Cu2+ ions which is characterized by the {NH2, NIm, NIm(macrochelate)} binding mode. The replacement of both Cys by His residues in the vasopressin sequence results in a very significant increase in the efficiency of Cu2+ binding.

NWC, third evolutionarily conserved gene within RAG locus is transcribed at high level in all cells except mature T and B lymphocytes and their RAG negative progenitors. It is so, because in lymphocytes expression of NWC is regulated by RAG-1 promoter, while in other cells it is controlled by RAG-2 intragenic promoter which in T and B lymphocytes is silent. Here we show that lymphocyte-specific inactivation of NWC promoter is caused by CpG island hypermethylation accompanied by site-specific blocking of chromatin accessibility, which in contrast to RAG promoters, is not accompanied by expected posttranslational modifications of histone H3. These results indicate that accessibility of NWC promoter and RAG promoters to trans-acting factors is regulated by different epigenetic mechanisms. The implications of our findings for understanding mechanisms regulating transcription within RAG/NWC locus in different cells are discussed and the model of epigenetic control of this locus is proposed.

Molecular cloning of targeting vectors (TgVs) is a prerequisite procedure for gene disruption in embryonic stem cells. In cases where target genes display complex features (e.g., gene overlap, alternative exon usage), TgVs must mediate deletions with very high precision to prevent unwanted effects. This is often difficult to achieve by procedures using restriction endonucleases and DNA ligases. Therefore, to prepare TgVs for inactivation of two complex genes of immunological interest: PTPRF and NWC, we employed an alternative method, which involves engineering bacterial artificial chromosomes (BACs) by inducible, plasmid encoded "Red/ET recombinase" expression system. Here, we report rapid and efficient construction of PTPRF and NWC TgVs without using restriction endonucleases.

Cyclolinopeptide A (CLA), a cyclic nonapeptide from linseed, possesses strong immunosuppressive and antimalarial activity along with the ability to inhibit cholate uptake into hepatocytes. The structure of the peptide was studied extensively in solution as well as in the solid state. It is postulated that both the Pro-Pro cis-amide bond and an 'edge-to-face' interaction between the aromatic rings of two adjacent Phe residues are important for biological activity. Structure-activity relationship studies of many linear and cyclic analogues of CLA suggest that the Pro-Xxx-Phe sequence and the flexibility of the peptide are important for the immunosuppressive activity.

This paper describes the first potent and selective p300 histone acetyltransferase (HAT) inhibitor which is effective in live cells. This compound 7 is a coenzyme A analogue conjugated to a cell permeabilizing oligoArg peptide via disulfide linkage. This compound was shown to block cellular histone acetylation and transcription using a p300-sensitive reporter. It should thus be broadly useful for dissecting the role of p300 HAT activity in physiologic and disease states.

DEK is a mammalian protein that has been implicated in the pathogenesis of autoimmune diseases and cancer, including acute myeloid leukemia, melanoma, glioblastoma, hepatocellular carcinoma, and bladder cancer. In addition, DEK appears to participate in multiple cellular processes, including transcriptional repression, mRNA processing, and chromatin remodeling. Sub-nuclear distribution of this protein, with the attendant functional ramifications, has remained a controversial topic. Here we report that DEK undergoes acetylation in vivo at lysine residues within the first 70 N-terminal amino acids. Acetylation of DEK decreases its affinity for DNA elements within the promoter, which is consistent with the involvement of DEK in transcriptional repression. Furthermore, deacetylase inhibition results in accumulation of DEK within interchromatin granule clusters (IGCs), sub-nuclear structures that contain RNA processing factors. Overexpression of P/CAF acetylase drives DEK into IGCs, and addition of a newly developed, synthetic, cell-permeable P/CAF inhibitor blocks this movement. To our knowledge, this is the first reported example of acetylation playing a direct role in relocation of a protein to IGCs, and this may explain how DEK can function in multiple pathways that take place in distinct sub-nuclear compartments. These findings also suggest that DEK-associated malignancies and autoimmune diseases might be amenable to treatment with agents that alter acetylation.

Histone acetyltransferase (HAT) activities of proteins such as p300, CBP, and P/CAF play important roles in activation of gene expression. We now show that the HAT activity of p300 can also be required for down-regulation of transcription by a DNA binding repressor protein. Promyelocytic leukemia zinc finger (PLZF), originally identified as a fusion with retinoic acid receptor alpha in rare cases of all-trans-retinoic acid-resistant acute promyelocytic leukemia, is a transcriptional repressor that recruits histone deacetylase-containing corepressor complexes to specific DNA binding sites. PLZF associates with p300 in vivo, and its ability to repress transcription is specifically dependent on HAT activity of p300 and acetylation of lysines in its C-terminal C2-H2 zinc finger motif. An acetylation site mutant of PLZF does not repress transcription and is functionally deficient in a colony suppression assay despite retaining its abilities to interact with corepressor/histone deacetylase complexes. This is due to the fact that acetylation of PLZF activates its ability to bind specific DNA sequences both in vitro and in vivo. Taken together, our results indicate that a histone deacetylase-dependent transcriptional repressor can be positively regulated through acetylation and point to an unexpected role of a coactivator protein in transcriptional repression.

Recombination-activating gene (RAG)1 and RAG2 encode T and B lymphocyte-specific endonucleases indispensable for rearrangements of antigen-receptor gene segments but also capable of causing deleterious chromosome rearrangements. The mechanisms regulating RAG expression and repression are not clear. Here we identify NWC, a third evolutionarily conserved gene within the RAG locus, and show that it is ubiquitously expressed, with the notable exception of RAG-nonexpressing immature and mature T and B lymphocytes because in lymphocytes it is regulated by the RAG1 promoter and transcribed as RAG1-NWC hybrid mRNA molecules. We also show that in all other cells NWC is controlled by the RAG2 intragenic promoter, which in immature and mature T and B lymphocytes is silent. The possible implications of these findings for understanding the activation and inactivation of RAG genes in lymphocytes and their repression in other cells are discussed.

The orphan nuclear receptors Nur77 and Nurr1 are the members of the Nur77 family of transcription factors. We demonstrate that transcription of the Nur77 family genes was upregulated in PC12 cells following incubation with Ca2+ ionophore as well as cyclic AMP (cAMP) analog. On the other hand, cAMP analog induced strong increase, while Ca2+ ionophore induced weak increase in the transactivation activity of Nur77. We found that Nur77 and Nurr1 proteins were expressed in the nucleus following stimulation with cAMP analog but not after stimulation with Ca2+ ionophore. However, expression of Nur77 protein was increased in the cytoplasm of cells treated with Ca2+ ionophore. In conclusion, our results suggest that cAMP-induced and Ca2+-induced processes may differentially regulate activity of Nur77 at the level of translocation of Nur77 protein from the cytoplasm into the nucleus.

The review presents three hypotheses concerning the amino acid complementarity: 1) the Mekler-Blalock antisense hypothesis; 2) the Root-Bernstein approach based on stereochemical complementarity of amino acids and anti-amino acids coded by anticodons read in parallel with the coding DNA strand; 3) Siemion hypothesis resulting from the periodicity of the genetic code. The current state of knowledge as well as the results of the implementations of these hypotheses are compared. A special attention is given to Root-Bernstein and Siemion hypotheses, which differ in only few points of the complementarity prediction. We describe methods of investigation of peptide-antipeptide pairing, including circular dichroism, mass spectrometry, affinity chromatography and other techniques. The biological applications of complementarity principle are considered, such as search for bioeffector-bioreceptor interaction systems, the influence of peptide-antipeptide pairing on the activity of peptide hormones, and the application of antipeptides in immunochemistry. The possible role of amino acid-anti-amino acid interactions in the formation of the spatial structures of peptides, proteins and protein complexes is discussed. Such problems as the pairing preferences of protein-protein interfaces, the role of the pairing in the creation of disulfide bonds and the possible appearance of such interactions in beta-structure are also examined. The main intention of the paper is to bring the complementarity problem to the attention of the scientific community, as a possible tool in proteomics, molecular design and molecular recognition.

Using cDNA-Representational Difference Analysis it was found that expression of Opg, Ctse, Krt2-4, Fut-2, 24p3 and Wif-1 genes was elevated in intestinal adenomas as compared to normal epithelial cells of Apc(Min/+) mutant mice. Expression of Wif-1, which encodes Wnt inhibitory factor-1 was also detected in a number of tumor cell lines of epithelial cell origin including two human colon adenocarcinoma cell lines. The possible role of Wif-1 over-expression in the etiology of colorectal cancer is discussed.

Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with cancers in immunocompromised populations. Antiviral drugs targeted against lytic viral replication have limited efficacy in these disease settings. EBV infection of peripheral blood mononuclear cells induces growth proliferation and the EBV latency Epstein-Barr virus-encoded nuclear antigen (EBNA)2 transcriptional transactivator (TAT) is essential for this response. EBNA2 targets the cellular DNA-binding protein CBF1 to mimic activated Notch signaling. A 10-aa peptide from the CBF1 interaction domain of EBNA2 was synthesized as a fusion with the protein transduction domain of HIV-1 TAT. The EBNA2-TAT peptide blocked EBNA2-CBF1 interaction in an in vitro GST affinity assay and labeling with fluorescein confirmed that the EBNA2-TAT peptide efficiently entered cultured B cells. Neither EBNA2-TAT, nor a mutant peptide with a 2-aa substitution that was unable to block the EBNA2-CBF1 interaction, significantly affected the growth of non-EBNA2-expressing EBV(-) B cells or Burkitt's lymphoma Akata cells. However, treatment of an EBV-immortalized lymphoblastoid cell line with the EBNA2-TAT peptide stopped cell growth and reduced cell viability. RT-PCR analyses of gene expression in the peptide-treated lymphoblastoid cell line cultures revealed that EBNA2-TAT treatment down-regulated the EBNA2-responsive viral LMP1 and LMP2 genes and cellular CD23, intercellular adhesion molecule 1, BATF, and Cdk1 genes while up-regulating expression of the cyclin-dependent kinase inhibitor p21. EBV-induced outgrowth of B cells from cultured peripheral blood mononuclear cells was also blocked in a dose-responsive manner by the EBNA2-TAT peptide. This study suggests that cell-permeable EBNA2 peptides may have potential as novel anti-EBV therapeutics.