Comparative analysis of superantigen genes in Staphylococcus xylosus and Staphylococcus aureus isolates collected from a single mammary quarter of cows with mastitis

The purpose of this study was to analyze and compare genes encoding superantigens (SAgs) in Staphylococcus xylosus and Staphylococcus aureus isolates collected simultaneously from milk of the same cows with clinical mastitis. Genes encoding staphylococcal enterotoxins and enterotoxin-like proteins (sea-selu), toxic shock syndrome toxin 1 (tst-1) and exfoliative toxins (eta and etd) were investigated. It was found that among 30 isolates of S. xylosus, 16 (53.3%) harbored from 1 to 10 SAg genes. In total, in 16 SAg positive S. xylosus, 11 different enterotoxin genes were detected: sec, sed, seg, seh, sei, selm, seln, selo, selp, ser, selu and one etd gene encoding exfoliative toxin D. The most prevalent genes were ser, selu, and selo. Among all the positive isolates of S. xylosus, a total of 14 different SAg gene combinations were detected. One combination was repeated in 3 isolates, whereas the rest were detected only once. However, in the case of S. aureus all the 30 isolates harbored the same combination of SAg genes: seg, sei, selm, seln, selo and on the basis of PFGE analysis all belonged to the same clonal type. Also noteworthy was the observation that SAg genes detected in S. aureus have also been found in S. xylosus. The findings of this study further extend previous observations that SAg genes are present not only in S. aureus but also in coagulase-negative staphylococci, including S. xylosus. Therefore, taking into account that the SAg genes are encoded on mobile genetic elements it is possible that these genes can be transferred between different species of coexisting staphylococci.     

Sustained Receptor Stimulation Leads to Sequestration of Recycling Endosomes in a Classical Protein Kinase C- and Phospholipase D-dependent Manner

Considerable insight has been garnered on initial mechanisms of endocytosis of plasma membrane proteins and their subsequent trafficking through the endosomal compartment. It is also well established that ligand stimulation of many plasma membrane receptors leads to their internalization. However, stimulus-induced regulation of endosomal trafficking has not received much attention. In previous studies, we showed that sustained stimulation of protein kinase C (PKC) with phorbol esters led to sequestration of recycling endosomes in a juxtanuclear region. In this study, we investigated whether G-protein-coupled receptors that activate PKC exerted effects on endosomal trafficking. Stimulation of cells with serotonin (5-hydroxytryptamine (5-HT)) led to sequestration of the 5-HT receptor (5-HT_2AR) into a Rab11-positive juxtanuclear compartment. This sequestration coincided with translocation of PKC as shown by confocal microscopy. Mechanistically the observed sequestration of 5-HT_2AR was shown to require continuous PKC activity because it was inhibited by pretreatment with classical PKC inhibitor Gö6976 and could be reversed by posttreatment with this inhibitor. In addition, classical PKC autophosphorylation was necessary for receptor sequestration. Moreover inhibition of phospholipase D (PLD) activity and inhibition of PLD1 and PLD2 using dominant negative constructs also prevented this process. Functionally this sequestration did not affect receptor desensitization or resensitization as measured by intracellular calcium increase. However, the PKC- and PLD-dependent sequestration of receptors resulted in co-sequestration of other plasma membrane proteins and receptors as shown for epidermal growth factor receptor and protease activated receptor-1. This led to heterologous desensitization of those receptors and diverted their cellular fate by protecting them from agonist-induced degradation. Taken together, these results demonstrate a novel role for sustained receptor stimulation in regulation of intracellular trafficking, and this process requires sustained stimulation of PKC and PLD.The protein kinase C (PKC)² family of enzymes comprises 11 isoforms of serine/threonine kinases (1, 2) implicated in regulation of cell growth, differentiation, apoptosis, secretion, neurotransmission, and signal transduction (3–5). During the course of studying PKC, we showed that sustained stimulation of PKC with phorbol esters leads to translocation of classical PKC (cPKC) to a pericentrosomal region (6, 7). This sequestration was shown to be PLD-dependent (8, 9) and negatively regulated by ceramide formed from the salvage pathway (10). Ceramide inhibits autophosphorylation of cPKC, which was also found to be required for this novel translocation (11). Importantly sustained activation of cPKC also resulted in significant effects on recycling components and their sequestration to the same region, dubbed the pericentrion (defined as the cPKC-dependent subset of recycling endosomes). On the other hand, components and markers of the endolysosomal compartment were not sequestered to the pericentrion upon PKC stimulation (7). Functionally it was also shown that pericentrion formation and sequestration of PKC requires clathrin-dependent endocytosis. Most importantly, formation of the pericentrion is dynamic and reversible and requires continuous activation of PKC.G-protein-coupled receptors (GPCRs) are the largest family of integral membrane receptors. They contain seven transmembrane domains (12), are coupled to heterotrimeric G-proteins, and are activated by a vast number of ligands. They regulate many cellular processes and serve as targets for at least half of the therapeutics currently present on the market. Upon agonist binding, conformational changes in the receptor lead to coupling with G-proteins (composed of α, β, and γ subunits). This leads to dissociation of α and β/γ subunits that mediate downstream signaling (13). Interestingly PKC serves as one of the downstream targets of GPCRs. Thus, it became critical to determine whether persistent stimulation of receptors that couple to cPKC exerts effects on recycling endosomes. We focused on the serotonin (5-HT) 5-HT_2A receptor (5-HT_2AR) and the angiotensin II receptor (AT1AR) as two GPCRs that couple to G_q, which in turn activates phospholipase Cβ and then PKC (14, 15).In this study, we show that sustained stimulation of those receptors led to their sequestration in a PKC- and PLD-dependent manner. Most importantly, this led to global sequestration of endosomes with profound effects on other membrane receptors. Epidermal growth factor receptor (EGFR) and protease activated receptor-1 (PAR-1) are known to be targeted into a degradative pathway upon their agonist treatment (16–18). Interestingly 5-HT induced co-sequestration of those receptors with 5-HT_2AR and protected them from degradation upon their own agonist treatment. The implications of these results on regulation of trafficking by GPCRs are discussed.     

IGF-I: from diagnostic to triple-helix gene therapy of solid tumors

Alterations in the expression of growth factors and their receptors are associated with the growth and development of human tumors. One such growth factor is IGF-I (insulin-like growth factor I ), a 70-amino-acid polypeptide expressed in many tissues, including brain. IGF-I is also expressed at high levels in some nervous system-derived tumors, especially in glioblastoma. When using IGF-I as a diagnostic marker, 17 different tumors are considered as expressing the IGF-I gene. Malignant glioma, the most common human brain cancer, is usually fatal. Average survival is less than one year. Our strategy of gene therapy for the treatment of gliomas and other solid tumors is based on: 1) diagnostic using IGF-I gene expression as a differential marker, and 2) application of "triple-helix anti-IGF-I" therapy. In the latter approach, tumor cells are transfected with a vector, which encodes an oligoribonucleotide--an RNA strand containing oligopurine sequence which might be capable of forming a triple helix with an oligopurine and/or oligopyrimidine sequence of the promotor of IGF-I gene (RNA-IGF-I DNA triple helix). Human tumor cells transfected in vitro become down-regulated in the production of IGF-I and present immunogenic (MHC-I and B7 expression) and apoptotic characteristics. Similar results were obtained when IGF-I antisense strategy was applied. In both strategies the transfected cells reimplanted in vivo lose tumorigenicity and elicit tumor specific immunity which leads to elimination of established tumors.     

Oligo(A) and double-stranded segments in polyadenylated and non-polyadenylated RNA from cytoplasm and nuclei of chick embryo

Chick embryonic RNA was fractionated by affinity chromatography on oligo(dT)-cellulose and poly(U)-Sepharose into three classes: poly(A)+RNA containing poly(A) segments of 100 and more residues, poly(A)-oligo(A)+RNA containing oligo(A) segments of about 25 residues, and poly(A)-oligo(A)-RNA which bound to neither of the beds used and which contained double-stranded segments of 300 and more base pairs. These three classes of RNA were found in cytoplasmic as well as in heterogeneous nuclear RNA. Double-stranded segments in hnRNA, unlike those in cytoplasmic RNA, were intermolecular in nature; this may explain the occurrence of "giant" molecules in hnRNA.     

Fungi inhabiting knotwood of Pinus sylvestris infected by Porodaedalea pini

Abundance and diversity of fungi in naturally formed knots of Pinus sylvestris affected by Porodaedalea pini were investigated. Samples were taken from trees that were (i) affected, with internal heartwood decay and no conks, (ii) affected, with internal heartwood decay and conks and (iii) controls. The Illumina sequencing technology was used for amplification of DNA, sequencing and analysis. In total, 566,279 raw sequences were obtained from six samples. Sequences included 74% of culturable and 8.4% of non‐culturable fungi and 17.6% of organisms with no reference sequences in NCBI. Abundance of organisms in knotwood, measured as number of OTUs, ranged from 36,272 (29,506 for fungi) to 178,535 (177,484 for fungi) and differed significantly between two trees in a stand and between stands. The highest and lowest average number of fungal OTUs occurred in infected trees with no conks and in trees with conks, respectively. Number of taxa ranged from 171 to 415 and often differed significantly between two trees in one stand and between stands. Greatest diversity occurred in control trees. The number of fungal taxa shared by two trees in one stand was 67–152 and that shared by two stands was 51–141. The majority of fungi were Ascomycota. Those most common in pines affected by P. pini were Coniochaeta hoffmannii and C. fodinicola (19.65%–59.92%). Infundichalara microchona, Leotiomycetes spp. and Rhinocladiella atrovirens were also present. Another common species, Lecanora conizaeoides, occurred most often in control trees (0.30%–8.82%). Porodaedalea pini was detected only sporadically. Non‐culturable fungi were most frequent in the control trees. The greater average abundance and smaller average diversity of fungi in knots of trees infected by P. pini suggest that the pathogen successfully competes with some fungal species and does not inhibit the growth of survivors. Some fungi detected may contribute to production of natural biocides.     

Effect of cellularity on the sensitivity of detecting squamous lesions in liquid-based cervical cytology

OBJECTIVE: To evaluate the effect of cellularity on the sensitivity of both screening and diagnosis in a liquid-based cervical sample. STUDY DESIGN: SurePath samples (TriPath Imaging Inc., Burlington, North Carolina, U.S.A.) with known diagnoses were selected, including 18 negative, 16 low grade squamous intraepithelial lesion (LSIL) and 12 high grade squamous intraepithelial lesion (HSIL) cases. Through a serial dilution technique, samples of varying cellularity were prepared. The 275 slides were assigned random numbers and were routinely screened by 1 of 2 senior cytotechnologists, blinded to the reference diagnosis. Specimens with a screening diagnosis of atypical squamous cells of undetermined significance (ASCUS) or higher were reviewed by two pathologists, resulting in a final consensus diagnosis. Using a grid counting system, cellularity was determined for each slide. RESULTS: There was a clear demarcation in sensitivity between specimens with a cellularity of < 5,000 or > or = 5,000 squamous cells. This applied to both the sensitivity for screening and to the final consensus diagnosis. For cases with a reference diagnosis of LSIL+, at a cytotechnologist screening level of ASCUS or greater, sensitivity increased from 72.8% (< 5,000 cells) to 98.1% (> or = 5,000 cells) and for a reference diagnosis of HSIL from 85.7% to 100%, respectively. Similarly, for the consensus diagnosis, sensitivity rose from 78.5% (< 5,000 cells) to 96.6% (> or = 5,000 cells) for LSIL+ and from 82.9% to 100%, respectively, for HSIL. These differences were statistically significant (P < .001). CONCLUSION: A minimum cellularity of 5,000 squamous cells is recommended for SurePath liquid-based cervical preparations.     

Effect of salt and osmotic stress on changes in polyamine content and arginine decarboxylase activity in Lupinus luteus seedlings

The effects of NaCl (260 mM) and sorbitol (360 mM) isoosmotic stresses on polyamine titers in lupin (Lupinus luteus L. var. Ventus) in relation to organ-specific responses were investigated. Analysis showed that during the first few hours (4 h) of salt and osmotic stress higher amounts of putrescine (Put) and spermidine (Spd) were accumulated in the roots and leaves of lupin seedlings. After exposing the plants to a longer duration (24 h) of exposure to NaCl, the level of free Put decreased in roots and cotyledons by about 48% and 54%, respectively, and increased in hypocotyls and leaves by about 27% and 73%, respectively. The Level of free Spd also decreased in roots by about 50%, in contrast to the increase of Spd observed in hypocotyls and leaves by about 50% and 70%, respectively. The effect of non-ionic stress on the level of Put and Spd in studied organs of lupin was similar to that of NaCl. Free spermine was at an undetectable level in examined organs. However, in the roots of lupin growing for 24 h in the presence of NaCl and/or sorbitol, the activity of arginine decarboxylase (ADC) (EC 4.1.1.19) increased by about 66% and 80%, respectively. ADC activity in leaves was similar to that observed in the control. Additionally, in the roots and leaves of lupin growing under the stress condition (NaCl or sorbitol), a higher level of polyamines (PAs) bound to microsomal membranes was observed. It is probable that PAs bound to microsomal membranes prevent stress-induced damage. We conclude that both stresses induce biosynthesis of Put and other PAs in the roots, as well as Put accumulation in the leaves, and this may indicate translocation of Put from the roots to the shoot. The possible role of PAs in adaptive mechanisms to stress is discussed.     

Effect of Rho-associated kinase inhibition on actin cytoskeleton structure and calcium response in glioma C6 cells

The role of actin cytoskeleton functional state in glioma C6 cell morphology and calcium signaling was investigated through modification of myosin II activity by blocking Rho-associated kinase with the specific inhibitor Y-27632. Treatment of glioma C6 cells with ROCK inhibitor resulted in actin cytoskeleton reorganization and also in the changed shape and distribution of mitochondria. Changes in the distribution of ER, the main calcium store in glioma C6 cells, were not visible. The inhibition of myosin II activity influences the first phase of calcium signaling evoked by agonist, and both phases of thapsigargin-evoked calcium response. We suggest that the observed increase in Ca2+ release from intracellular stores induced by IP3 formation as well as inhibition of SERCA ATPase is at least in part related to severely affected mitochondria. Enhancement of capacitative calcium entry evoked by thapsigargin is probably associated with the reorganization of the acto-myosin II system. ATP-induced calcium response presents no changes in the second phase. We observed that ATP stimulation of Y-27632 pretreated cells leads to immediate morphological rearrangement of glioma C6 cells. It is a consequence of actin cytoskeleton reorganization: formation of stress fibers and relocation of phosphorylated myosin II to actin filaments. It seems that the agonist-evoked strong calcium signal may be sufficient for myosin II activation and the stress fiber organization. This is the first work showing the dependence between the functional state of the acto-myosin II system and calcium signaling stressing the reversible character of this relationship.     

The effect of new non-cross resistant antitumour agents on the energy state of human erythrocytes

Multidrug resistance (MDR) of tumour cells is related to the overexpression of ATP-dependent pumps responsible for the active efflux of antitumour agents out of resistant cells. Benzoperimidine and anthrapyridone compounds exhibit comparable cytotoxic activity against sensitive and MDR tumour cells. They diffuse extremely rapidly across the plasma membrane and render the ATP-dependent efflux inefficient. Such uptake could disturb an energy metabolism of normal cells possessing an elevated level of ATP-dependent proteins, especially erythrocytes having a high level of the MRP1, MRP4 and MRP5 proteins. In this study the effect of five antitumour agents: benzoperimidine (BP1), anthrapyridones (CO1, CO7) and reference drugs used in the clinic: doxorubicin (DOX) and pirarubicin (PIRA), on the energetic state in human erythrocytes has been examined. These compounds have various types of structure and kinetics of cellular uptake (slow--DOX, CO7, moderate--PIRA, fast--BP1, CO1) resulting in their different ability to saturate ATP-dependent transporters. The energetic state of erythrocytes was examined by determination of purine nucleotide contents (ATP, ADP, AMP), NAD(+) and values of adenylate energy charge (AEC) using an HPLC method. It was found that the level of nucleotides as well as the AEC value of erythrocytes were not changed during 24 h of incubation with these agents independently of their structure and ability to saturate ATP-dependent pumps. This is a very promising result in view of their potential use in the clinic as antitumour drugs against multidrug resistant cancers.     

Biochemical characterization and structural insight into aliphatic β‐amino acid adenylation enzymes IdnL1 and CmiS6

Macrolactam antibiotics such as incednine and cremimycin possess an aliphatic β‐amino acid as a starter unit of their polyketide chain. In the biosynthesis of incednine and cremimycin, unique stand‐alone adenylation enzymes IdnL1 and CmiS6 select and activate the proper aliphatic β‐amino acid as a starter unit. In this study, we describe the enzymatic characterization and the structural basis of substrate specificity of IdnL1 and CmiS6. Functional analysis revealed that IdnL1 and CmiS6 recognize 3‐aminobutanoic acid and 3‐aminononanoic acid, respectively. We solved the X‐ray crystal structures of IdnL1 and CmiS6 to understand the recognition mechanism of these aliphatic β‐amino acids. These structures revealed that IdnL1 and CmiS6 share a common recognition motif that interacts with the β‐amino group of the substrates. However, the hydrophobic side‐chains of the substrates are accommodated differently in the two enzymes. IdnL1 has a bulky Leu220 located close to the terminal methyl group of 3‐aminobutanoate of the trapped acyl‐adenylate intermediate to construct a shallow substrate‐binding pocket. In contrast, CmiS6 possesses Gly220 at the corresponding position to accommodate 3‐aminononanoic acid. This structural observation was supported by a mutational study. Thus, the size of amino acid residue at the 220 position is critical for the selection of an aliphatic β‐amino acid substrate in these adenylation enzymes. Proteins 2017; 85:1238–1247. © 2017 Wiley Periodicals, Inc.