Topical application of local anesthetics to melanoma increases the efficacy of anti-PD-1 therapy.

Experimental and clinical data have shown that the nervous system can significantly stimulate the initiation and progression of melanoma. In support of this, approaches that reduce the transmission of signals from peripheral nerves to effector tissues reduce the recurrence of melanoma. Therefore, we investigated the effect of topical application of the local anesthetic Pliaglis (7% lidocaine and 7% tetracaine) on the growth of melanoma induced by intradermal application of B16F0 cells in mice without treatment and in mice treated with the anti-PD-1 antibody. We found that application of Pliaglis to melanoma significantly reduced its growth and this effect was even pronounced in mice treated with the anti-PD-1 antibody. To determine the mechanisms and pathways responsible for the observed effect, the in vitro effect of incubating melanoma cells with lidocaine and/or tetracaine and the in vivo gene expression of cancer and immune-related factors, percentage of immune cells, gene expression of selected neurotransmitter receptors and nerve growth factors in melanoma tissue were studied. We found that lidocaine and tetracaine significantly reduced the viability of B16F0 cells in vitro. In mice with melanoma, Pliaglis potentiated the effect of anti-PD-1 antibody on gene expression of COX-2, IL-1ß, IL-6, CCL11, F4/80, CD206, and NCR1. In addition, Pliaglis increased the gene expression of α9nACHR and 5-HT2a receptors and decreased the gene expression of nerve growth factor receptor (p75NTR) and p53. We also observed Pliaglis-mediated changes in myeloid populations. Topical application of this local anesthetic cream decreased the CD11b+Gr1- population and increased the CD11b+Gr1high population. Our data suggest that Pliaglis reduces melanoma growth through a direct effect on melanoma cells as well as through modulation of the immune response. The involvement of nervous system-related signaling in the inhibitory effect of Pliaglis on melanoma is inconclusive from our data.

Sperm proteins ODF2 and PAWP as markers of fertility in breeding bulls.

Low fertility is the single most important factor limiting livestock reproductive performance, adversely affecting the cattle industry and causing millions of dollars of economic loss. In the livestock industry, male fertility is of crucial importance for the reproductive performance of livestock. However, there is a lack of reliable biomarkers to predict bull fertility in artificial insemination service. The objective of this study was to identify sperm proteins as biomarkers for bull fertility. To discover candidate sperm quality biomarkers, sperm proteome profiling was conducted in extreme high- and extreme low-fertile bulls selected from a pool of 1000 AI sires with varied fertility. Thirty-two differentially expressed proteins were identified. Among them, high levels of sperm outer dense fiber of sperm tails 2 (ODF2) and post-acrosomal assembly of sperm head protein (PAWP/WBP2NL) represented the most extreme differences in quantity between high- and low-fertility bulls. Protein immunodetection and flow cytometry used to validate these putative fertility markers in a combined cohort of 154 AI sires. Both ODF2 and PAWP correlated significantly with fertility. In conclusion, ODF2 and PAWP can be used to assess semen quality and predict sire fertility.

Effect of rapamycin on repeated immobilization stress-induced immune alterations in the rat spleen.

Chronic stress modulates immune system functions via neuroendocrine pathways. Rapamycin inhibits activity of immune cells through the mTOR signaling pathway. We investigated the effect of rapamycin (15 mg/kg, 3-times/week) on neuroimmune-endocrine system in the spleen of rats exposed to 42 cycles of 2-h immobilization. Rapamycin enhanced the activity of hypothalamic-pituitary-adrenocortical axis induced by stress exposure, prevented stress-induced expression of natural killer cell markers while reversed stress-evoked decline of Th2 immune response markers. Overall, our findings suggest that rapamycin may act on immune functions not only directly by inhibiting of mTOR in immune cells but also indirectly via modulation of neuroendocrine system.

Exposure to a single immobilization or lipopolysaccharide challenge increases expression of genes implicated in the development of Alzheimer's disease in the mice brain cortex.

OBJECTIVES: Despite extensive research efforts, mechanisms participating on development of Alzheimer's disease (AD) are covered only partially. Data from the last decades indicate that various stressors, as etiological factors, may play a role of in the AD. Therefore, we investigated the effect of two acute stressors, immobilization (IMO) and lipopolysaccharide (LPS), on the AD-related neuropathology. METHODS: Adult C57BL/6J mice males were exposed to a single IMO stress or a single intraperitoneal injection of LPS (250 µg/kg body weight). After terminating the experiments, the brains were removed and their cortices isolated. Gene expression of pro-inflammatory cytokines, as well as expression of genes implicated in the AD neuropathology were determined. In addition, mediators related to the activation of the microglia, monocytes, and perivascular macrophages were determined in brain cortices, as well. RESULTS: In comparison with the control animals, we found increased gene expression of proinflammatory mediators in mice brain cortex in both IMO and LPS groups. In stressed animals, we also showed an increased expression of genes related to the AD neuropathology, as well as positive correlations between genes implicated in AD development and associated neuroinflammation. CONCLUSIONS: Our data indicate that acute exposure to a strong IMO stressor, composed of the combined physical and psychological challenges, induces similar inflammatory and other ADrelated neuropathological changes as the immune LPS treatment. Our data also indicate that cytokines are most likely released from the peripheral immune cells, as we detected myeloid cells activity, without any microglia response. We hypothesize that stress induces innate immune response in the brain that consequently potentiate the expression of genes implicated in the AD-related neuropathology.

Repeated Stress Exaggerates Lipopolysaccharide-Induced Inflammatory Response in the Rat Spleen.

Spleen is an immune organ innervated with sympathetic nerves which together with adrenomedullary system control splenic immune functions. However, the mechanism by which prior stress exposure modulates the immune response induced by immunogenic challenge is not sufficiently clarified. Thus, the aim of this study was to investigate the effect of a single (2 h) and repeated (2 h daily for 7 days) immobilization stress (IMO) on the innate immune response in the spleen induced by lipopolysaccharide (LPS, 100 µg/kg). LPS elevated splenic levels of norepinephrine and epinephrine, while prior IMO prevented this response. LPS did not alter de novo production of catecholamines, however, prior IMO attenuated phenylethanolamine N-methyltransferase gene expression. Particularly repeated IMO exacerbated LPS-induced down-regulation of α1B- and ß1-adrenergic receptors (ARs), while enhanced α2A- and ß2-AR mRNAs. Elevated expression of inflammatory mediators (iNOS2, IL-1ß, IL-6, TNF-α, IL-10) was observed following LPS and repeated IMO again potentiated this effect. These changes were associated with enhanced Ly6C gene expression, a monocyte marker, and elevated MCP-1, GM-CSF, and CXCL1 mRNAs suggesting an increased recruitment of monocytes and neutrophils into the spleen. Additionally, we observed increased Bax/Bcl-1 mRNA ratio together with reduced B cell numbers in rats exposed to repeated IMO and treated with LPS but not in acutely stressed rats. Altogether, these data indicate that repeated stress via changes in CA levels and specific α- and ß-AR subtypes exaggerates the inflammatory response likely by recruiting peripheral monocytes and neutrophils to the spleen, resulting in the induction of apoptosis within this tissue, particularly in B cells. These changes may alter the splenic immune functions with potentially pathological consequences.

Exaggerated phosphorylation of brain tau protein in CRH KO mice exposed to repeated immobilization stress.

Neuroendocrine and behavioral stress responses are orchestrated by corticotropin-releasing hormone (CRH) and norepinephrine (NE) synthesizing neurons. Recent findings indicate that stress may promote development of neurofibrillary pathology in Alzheimer's disease. Therefore, we investigated relationships among stress, tau protein phosphorylation, and brain NE using wild-type (WT) and CRH-knockout (CRH KO) mice. We assessed expression of phosphorylated tau (p-tau) at the PHF-1 epitope and NE concentrations in the locus coeruleus (LC), A1/C1 and A2/C2 catecholaminergic cell groups, hippocampus, amygdala, nucleus basalis magnocellularis, and frontal cortex of unstressed, singly stressed or repeatedly stressed mice. Moreover, gene expression and protein levels of tyrosine hydroxylase (TH) and CRH receptor mRNA were determined in the LC. Plasma corticosterone levels were also measured. Exposure to a single stress increases tau phosphorylation throughout the brain in WT mice when compared to singly stressed CRH KO animals. In contrast, repeatedly stressed CRH KO mice showed exaggerated tau phosphorylation relative to WT controls. We also observed differences in extent of tau phosphorylation between investigated structures, e.g. the LC and hippocampus. Moreover, CRH deficiency leads to different responses to stress in gene expression of TH, NE concentrations, CRH receptor mRNA, and plasma corticosterone levels. Our data indicate that CRH effects on tau phosphorylation are dependent on whether stress is single or repeated, and differs between brain regions. Our findings indicate that CRH attenuates mechanisms responsible for development of stress-induced tau neuropathology, particularly in conditions of chronic stress. However, the involvement of central catecholaminergic neurons in these mechanisms remains unclear and is in need of further investigation.

Tauopathy in transgenic (SHR72) rats impairs function of central noradrenergic system and promotes neuroinflammation.

BACKGROUND: Brain norepinephrine (NE) plays an important role in the modulation of stress response and neuroinflammation. Recent studies indicate that in Alzheimer's disease (AD), the tau neuropathology begins in the locus coeruleus (LC) which is the main source of brain NE. Therefore, we investigated the changes in brain NE system and also the immune status under basal and stress conditions in transgenic rats over-expressing the human truncated tau protein. METHODS: Brainstem catecholaminergic cell groups (LC, A1, and A2) and forebrain subcortical (nucleus basalis of Meynert), hippocampal (cornu ammonis, dentate gyrus), and neocortical areas (frontal and temporal association cortices) were analyzed for NE and interleukin 6 (IL-6) mRNA levels in unstressed rats and also in rats exposed to single or repeated immobilization. Moreover, gene expression of NE-biosynthetic enzyme, tyrosine hydroxylase (TH), and several pro- and anti-inflammatory mediators were determined in the LC. RESULTS: It was found that tauopathy reduced basal NE levels in forebrain areas, while the gene expression of IL-6 was increased in all selected areas at the same time. The differences between wild-type and transgenic rats in brain NE and IL-6 mRNA levels were observed in stressed animals as well. Tauopathy increased also the gene expression of TH in the LC. In addition, the LC exhibited exaggerated expression of pro- and anti-inflammatory mediators (IL-6, TNFα, inducible nitric oxide synthases 2 (iNOS2), and interleukin 10 (IL-10)) in transgenic rats suggesting that tauopathy affects also the immune background in LC. Positive correlation between NE and IL-6 mRNA levels in cornu ammonis in stressed transgenic animals indicated the reduction of anti-inflammatory effect of NE. CONCLUSIONS: Our data thus showed that tauopathy alters the functions of LC further leading to the reduction of NE levels and exaggeration of neuroinflammation in forebrain. These findings support the assumption that tau-related dysfunction of LC activates the vicious circle perpetuating neurodegeneration leading to the development of AD.

Effect of a single and repeated stress exposure on gene expression of catecholamine biosynthetic enzymes in brainstem catecholaminergic cell groups in rats.

Brainstem catecholaminergic neurons significantly participate in the regulation of neuroendocrine system activity, particularly during stressful conditions. However, so far the precise quantitative characterisation of basal and stress-induced changes in gene expression and protein levels of catecholaminergic biosynthetic enzymes in these neurons has been missing. Using a quantitative reverse transcription-polymerase chain reaction method, we investigated gene expression of catecholamine biosynthetic enzymes in brainstem noradrenergic and adrenergic cell groups in rats under resting conditions as well as in acutely and repeatedly stressed animals. For the first time, we described quantitative differences in basal levels of catecholamine biosynthetic enzyme mRNA in brainstem catecholaminergic ascending and descending projecting cell groups. Moreover, we found and defined some differences among catecholaminergic cell groups in the time-course of mRNA levels of catecholaminergic enzymes following a single and especially repeated immobilisation stress. The data obtained support the assumption that brainstem catecholaminergic cell groups represent a functionally differentiated system, which is highly (but specifically) activated in rats exposed to stress. Therefore, potential interventions for the treatment of stress-related diseases need to affect the activity of brainstem catecholaminergic neurons not uniformly but with some degree of selectivity.

Sperm protamine-status correlates to the fertility of breeding bulls.

During fertilization, spermatozoa make essential contributions to embryo development by providing oocyte activating factors, centrosomal components, and paternal chromosomes. Protamines are essential for proper packaging of sperm DNA; however, in contrast to the studies of oocyte-related female infertility, the influence of sperm chromatin structure on male infertility has not been evaluated extensively. The objective of this study was to determine the sperm chromatin content of bull spermatozoa by evaluating DNA fragmentation, chromatin maturity/protamination, PRM1 protein status, and nuclear shape in spermatozoa from bulls with different fertility. Relationships between protamine 1 (PRM1) and the chromatin integrity were ascertained in spermatozoa from Holstein bulls with varied (high vs. low) but acceptable fertility. Sperm DNA fragmentation and chromatin maturity (protamination) were tested using Halomax assay and toluidine blue staining, respectively. The PRM1 content was assayed using Western blotting and in-gel densitometry, flow cytometry, and immunocytochemistry. Fragmentation of DNA was increased and chromatin maturity significantly reduced in spermatozoa from low-fertility bulls compared to those from high-fertility bulls. Field fertility scores of the bulls were negatively correlated with the percentage of spermatozoa displaying reduced protamination and fragmented DNA using toluidine blue and Halomax, respectively. Bull fertility was also positively correlated with PRM1 content by Western blotting and flow cytometry. However, detection of PRM1 content by Western blotting alone was not predictive of bull fertility. In immunocytochemistry, abnormal spermatozoa showed either a lack of PRM1 or scattered localization in the apical/acrosomal region of the nuclei. The nuclear shape was distorted in spermatozoa from low-fertility bulls. In conclusion, we showed that inadequate amount and localization of PRM1 were associated with defects in sperm chromatin structure, coinciding with reduced fertility in bulls. These findings are highly significant because they reveal molecular and morphological phenotypes of mammalian spermatozoa that influence fertility.

Stress-induced activation of the sympathoadrenal system is determined by genetic background in rat models of tauopathy.

Stress may accelerate onset of neurodegenerative diseases in vulnerable subjects and, vice versa, neurodegeneration affects the responsiveness to stressors. We investigated the neuroendocrine response to immobilization stress in normotensive Wistar-Kyoto rats (WKY), spontaneously hypertensive rats (SHR), and transgenic rats of respective WKY and SHR strains overexpressing human truncated tau protein. Plasma levels of epinephrine, norepinephrine, and corticosterone were determined. An immobilization-induced elevation of epinephrine and norepinephrine was significantly reduced in WKY transgenic rats compared to WKY wild-type rats, while no differences were seen between SHR transgenic and SHR wild-type animals. Our data have shown that sympathoadrenal system response to stress strongly depends on both tau protein-induced neurodegeneration and genetic background of experimental animals.

Protein expression pattern of PAWP in bull spermatozoa is associated with sperm quality and fertility following artificial insemination.

Post-acrosomal WW-domain binding protein (PAWP) is a signaling molecule located in the post-acrosomal sheath (PAS) of mammalian spermatozoa. We hypothesized that the proper integration of PAWP in the sperm PAS is reflective of bull-sperm quality and fertility. Cryopreserved semen samples from 298 sires of acceptable, but varied, fertility used in artificial insemination services were analyzed using immunofluorescence microscopy and flow cytometry for PAWP protein. In normal spermatozoa, PAWP fluorescence formed a regular band around the proximal PAS. Anomalies of PAWP labeling in defective spermatozoa were reflected in flow cytometry by varied intensities of PAWP-induced fluorescence. Distinct sperm phenotypes were also identified, including morphologically normal and some defective spermatozoa with moderate levels of PAWP; grossly defective spermatozoa with low/no PAWP; and defective spermatozoa with high PAWP. Analysis by ImageStream flow cytometry confirmed the prevalence of abnormal sperm phenotypes in the spermatozoa with abnormal PAWP content. Live/dead staining and video recording showed that some abnormal spermatozoa are viable and capable of progressive motility. Conventional flow-cytometric measurements of PAWP correlated significantly with semen quality and fertility parameters that reflect the sires' artificial insemination fertility, including secondary sperm morphology, conception rate, non-return rate, and residual value. A multiplex, flow-cytometric test detecting PAWP, aggresomes (ubiquitinated protein aggregates), and acrosomal integrity (peanut-agglutinin-lectin labeling) had a predictive value for conception rate, as demonstrated by step-wise regression analysis. We conclude that PAWP correlates with semen/fertility parameters used in the cattle artificial insemination industry, making PAWP a potential biomarker of bull fertility.

Glutamic acid decarboxylase gene disruption reveals signalling pathway(s) governing complex morphogenic and metabolic events in Trichoderma atroviride.

Glutamate decarboxylase (GAD) catalyses decarboxylation of glutamate to gamma-aminobutyrate (GABA) in a metabolic pathway connected to citrate cycle and known as GABA shunt. The gene (gad) was disrupted in Trichoderma atroviride CCM F-534 and viable mutants were characterized. Two of them were found to arise by homologous recombination and were devoid of both GAD activity and GABA. Mutants grew slower as compared to the wild type (F534). In the submerged culture, mutants developed less CO2 and consumed less O2 than the F534 without changing their respiratory quotients. Hyphae of mutants were more ramified than those of F534. Their ramification, in contrast to F534, was not increased by cyclosporin A, a drug causing hyphae ramification of several fungi and which is a calcineurin/cyclophilin inhibitor, or by FK506. Rapamycin, which is a cyclophilin but not calcineurin inhibitor, had a different effect on hyphae ramification in F534 and mutants. To examine the presence of GABA receptors in the fungus the effect of mammalian GABA-receptor modulators, such as bicuculline, gabapentin or carbamazepine on fungal morphology were investigated. Conidia of mutants germinated in a multipolar manner more frequently (up to 80 %) than those of F534. This trait was modified with cyclosporine A, FK506 and GABA receptor modulators in a different manner. Transport of chlorides, an intimate feature of GABA-regulated receptors/channels in animal cells, was measured in vegetative mycelia by means (36)Cl(-) uptake. It was significantly reduced in gad mutants. The results suggest that T. atroviride possesses a signalling pathway that involves GABA, putative GABA receptor(s), calcineurin, target of rapamycin and chloride transporter(s) to regulate physiological functions.

Pmch-deficiency in rats is associated with normal adipocyte differentiation and lower sympathetic adipose drive.

The orexigenic neuropeptide melanin-concentrating hormone (MCH), a product of Pmch, is an important mediator of energy homeostasis. Pmch-deficient rodents are lean and smaller, characterized by lower food intake, body-, and fat mass. Pmch is expressed in hypothalamic neurons that ultimately are components in the sympathetic nervous system (SNS) drive to white and interscapular brown adipose tissue (WAT, iBAT, respectively). MCH binds to MCH receptor 1 (MCH1R), which is present on adipocytes. Currently it is unknown if Pmch-ablation changes adipocyte differentiation or sympathetic adipose drive. Using Pmch-deficient and wild-type rats on a standard low-fat diet, we analyzed dorsal subcutaneous and perirenal WAT mass and adipocyte morphology (size and number) throughout development, and indices of sympathetic activation in WAT and iBAT during adulthood. Moreover, using an in vitro approach we investigated the ability of MCH to modulate 3T3-L1 adipocyte differentiation. Pmch-deficiency decreased dorsal subcutaneous and perirenal WAT mass by reducing adipocyte size, but not number. In line with this, in vitro 3T3-L1 adipocyte differentiation was unaffected by MCH. Finally, adult Pmch-deficient rats had lower norepinephrine turnover (an index of sympathetic adipose drive) in WAT and iBAT than wild-type rats. Collectively, our data indicate that MCH/MCH1R-pathway does not modify adipocyte differentiation, whereas Pmch-deficiency in laboratory rats lowers adiposity throughout development and sympathetic adipose drive during adulthood.

Catecholamine production is differently regulated in splenic T- and B-cells following stress exposure.

OBJECTIVES: Stress is accompanied also by a rise in splenic catecholamines (CAs). However, indications about endogenous CA production in the spleen exist but there are no data about the cellular source of this production and possible modification by stress. Therefore, our aim was to investigate whether splenic T- and B-cells are one of main sources in the spleen expressing tyrosine hydroxylase (TH), enzyme crucial for CA biosynthesis, and phenylethanolamine N-methyltransferase (PNMT) which is necessary for epinephrine production. We also investigated whether stress is able to modify expression of both enzymes and CA levels within these cell fractions as well as tried to explain functional consequences of changes observed. RESULTS: T-cells contain higher levels of TH mRNA than B-cells although protein levels appeared similar. On contrary, the PNMT mRNA and protein were higher in B-cells, which appeared to be the main source of PNMT in the spleen. T-cells increased TH and PNMT expression after acute stress while similar rise was observed in B-cells after repeated stress, most probably as a consequence of higher CA turnover in both cell populations. The rise in TH and PNMT was accompanied by an elevation of Bax/Bcl-2 mRNA ratio, number of apoptotic cells and also by a decline of IFN-γ mRNA in both cell types. Reduction of IL-2 and IL-4 mRNA was also observed in B-cells. CONCLUSION: Stress-induced stimulation of endogenous CA biosynthesis in lymphocytes is dependent on the type of lymphocyte population and duration of stressor and leads to attenuated IFN-γ expression and induction of apoptosis. These changes might contribute to dysregulation of specific immune functions involving T- and B-cells and may decrease the ability to cope with intracellular agents following stress situations.

Repeated immobilization stress induces catecholamine production in rat mesenteric adipocytes.

Catecholamines (CATs), the major regulator of lipolysis in adipose tissue, are produced mainly by the sympathoadrenal system. However, recent studies report endogenous CAT production in adipocytes themselves. This study investigated the effects of single and repeated (7-14 times) immobilization (IMO) stress on CAT production in various fat depots of the rat. Single IMO quickly induced a rise of norepinephrine (NE) and epinephrine (EPI) concentration in mesenteric and brown adipose depots. Adaptive response to repeated IMO included robust increases of NE and EPI levels in mesenteric and subcutaneous adipose tissue. These changes likely reflect the activation of sympathetic nervous system in fat depots by IMO. However, this process was also paralleled by an increase in tyrosine hydroxylase gene expression in mesenteric fat, suggesting regulation of endogenous CAT production in adipose tissue cells. Detailed time-course analysis (time course 10, 30, and 120 min) clearly showed that repeated stress led to increased CAT biosynthesis in isolated mesenteric adipocytes resulting in gradual accumulation of intracellular EPI during IMO exposure. Comparable changes were also found in stromal/vascular fractions, with more pronounced effects of single than repeated IMO. The potential physiological importance of these findings is accentuated by parallel increase in expression of vesicular monoamine transporter 1, indicating a need for CAT storage in adipocyte vesicles. Taken together, we show that CAT production occurs in adipose tissue and may be activated by stress directly in adipocytes.

Acute stress differently modulates ß1, ß2 and ß3 adrenoceptors in T cells, but not in B cells, from the rat spleen.

OBJECTIVES: Stress-induced rise in circulating catecholamines (CAs), followed by modulation of ß-adrenergic receptors (adrenoceptors, ARs), is one of the pathways involved in the stress-mediated effects of immune functions. The spleen is an organ with a high number of lymphocytes and provides a unique microenvironment in which they reside. Thus, lymphocytes may respond differently to CAs in the spleen than in the circulation. No reports exist concerning the involvement of ß-ARs in stress-mediated effects on T and B cells isolated from the spleen. Therefore, our aim was to investigate the effect of single stress exposure on gene expression and cellular localization of ß-adrenoceptor subtypes in splenic T and B cells. We tried to correlate changes in adrenoceptors with the expression of apoptotic proteins. METHODS: Immobilization (IMMO) was used as a stress model. T and B cells were isolated from rat spleen using magnetically labeled antibodies. The gene expression of individual adrenoceptors and apoptotic proteins was evaluated by real-time PCR. Immunofluorescence was used to evaluate localization and adrenoceptor expression. RESULTS: We have found T cells to be more vulnerable to stress compared to B cells, because of increased ß1-, ß2- and ß3-ARs after a single IMMO. Moreover, ß2-ARs translocated from the nucleus to the plasma membrane in T cells after IMMO. The rise in ß-ARs most probably led to the rise of Bax mRNA and Bax to Bcl-2 mRNA ratio. This might suggest the induction of an apoptotic process in T cells. CONCLUSION: Higher susceptibility of T cells to stress via modulation of ß-ARs and apoptotic proteins might shift the immune responsiveness in the spleen.

Adipocytes as a new source of catecholamine production.

Catecholamines are an important regulator of lipolysis in adipose tissue. Here we show that rat adipocytes, isolated from mesenteric adipose tissue, express genes of catecholamine biosynthetic enzymes and produce catecholamines de novo. Administration of tyrosine hydroxylase inhibitor, alpha-methyl-p-tyrosine, in vitro significantly reduced concentration of catecholamines in isolated adipocytes. We hypothesize that the sympathetic innervation of adipose tissues is not the only source of catecholamines, since adipocytes also have the capacity to produce both norepinephrine and epinephrine.

Repeated stress down-regulates ß(2)- and α (2C)-adrenergic receptors and up-regulates gene expression of IL-6 in the rat spleen.

Catecholamines are among first compounds released during stress, and they regulate many functions of the organism, including immune system, via adrenergic receptors (ARs). Spleen, as an immune organ with high number of macrophages, possesses various ARs, from which ß(2)-ARs are considered to be the most important for the modulation of immune functions. Nevertheless, little is known about the regulation and involvement of ARs in the splenic function by stress. Therefore, the aim of this work was to measure the gene expression of ARs and several cytokines in the spleen of rats exposed to a single and repeated (14×) immobilization stress (IMO). We have found a significant increase in ß(2)-AR mRNA after a single IMO, but a significant decrease in ß(2)-AR mRNA and protein level after repeated (14×) IMO. The most prominent decrease was detected in the gene expression of the α(2A)- and α(2C)-AR after repeated IMO. However, changes in mRNA were translated into protein levels only for the α(2C)-subtype. Other types of ARs remained unchanged during the stress situation. Since we proposed that these ARs might affect production of cytokines, we measured gene expression of pro-inflammatory (TNF-α, IL-1ß, IL-6 and IL-18) and anti-inflammatory (IL-10 and TGF-ß1) cytokines. We detected changes only in IL-6 and IL-10 mRNA levels. While IL-6 mRNA was increased, IL-10 mRNA dropped after repeated IMO. According to these results we suggest that changes of ß(2)- and α(2C)-ARs participate in IL-6-mediated processes in the spleen, especially during chronic stress situations.

Light accelerates the splicing of srh1 homologue gene transcripts in aerial mycelia of Trichoderma viride.

The expression of the Tvsrh1 gene encoding conidial hydrophobin was investigated during the development of surface-cultivated Trichoderma viride mycelia under different illumination regimes. Three transcripts of the whole gene amplified from the total mRNA were found with lengths of 400, 323 and 272 bp. The 400-bp transcript was slowly converted to the shorter forms in the dark. Light-pulse dramatically increased the rate of conversion, and a permanent illumination of mycelia was most efficient in this process. The sequencing of transcripts revealed that the 400 bp transcript contains two introns, whereas the intermediate one contains only one intron located distally from the 5'-end. The shortest transcript was without introns. The sum of all transcripts remained almost unchanged in the dark and increased upon the light pulse but decreased during development under permanent illumination. The appearance of conidia coincided with the complete conversion of the transcripts. The results showed that the splicing of the two introns was not random but sequential, and that it did not follow the cotranscriptional mechanism. Furthermore, they suggested that mRNA processing could represent another regulation level of gene expression by light during the photo-induced conidiation in T. viride.

Developmental changes in Trichoderma viride enzymes abundant in conidia and the light-induced conidiation signalling pathway.

The expression of glutamic acid decarboxylase gene and the laccase activity were measured during the development of surface-cultivated Trichoderma viride mycelia in order to examine their up-regulation by light. The results show that the changes in activity of GAD induced by light observed previously are caused by transcriptional regulation of gad gene expression in both submerged mycelia and aerial mycelia after photoinduction. The expression of tga gene encoding a T. viride G(alpha) protein was found not to be up-regulated by light and was also present in the non-conidiating mutant of T. viride suggesting that this protein is not involved in the regulation of conidiation in this fungus, or that it plays a role is in later stages of conidia development. The activity of laccase was also not light-inducible and may be related to the maturation of conidia.