Bálint Szalontai

Protective effects of the neuropeptide PACAP in diabetic retinopathy

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with highly potent neurotrophic and neuroprotective effects. PACAP and its receptors occur in the retina and PACAP has been applied in animal models of metabolic retinal disorders to reduce structural and functional damage. Furthermore, PACAP has been implicated as a potential anti-diabetic peptide. Our aim has been to investigate, by using a complex morphological, immunochemical and molecular biological approach, whether PACAP attenuates diabetic retinopathy. Diabetes was induced in rats with a single streptozotocin injection. PACAP was injected intravitreally into one eye (100 pmol) three times during the last week of a 3-week survival period. Retinas were processed for the following procedures: routine histology, immunohistochemistry (single and double labeling, whole-mount), quantitative reverse transcription with the polymerase chain reaction and Western blotting. Cone photoreceptors and dopaminergic amacrine and ganglion cells degenerated in diabetic retinas and glial fibrillary acidic protein were upregulated in Müller glial cells. The number of cones, the length of their outer segments and the cell number in the ganglion cell layer were decreased. PACAP ameliorated these structural changes. Moreover, PACAP increased the levels of PAC1-receptor and tyrosine-hydroxylase as detected by molecular biological methods. Thus, PACAP has significant protective effects in the diabetic retina. PACAP treatment attenuates neuronal cell loss in diabetic retinopathy, the protective effects of PACAP probably being mediated through the activation of PAC1-receptor. These results suggest that PACAP has a therapeutic potential in diabetic retinopathy.

Estrogen-dependent up-regulation of TRPA1 and TRPV1 receptor proteins in the rat endometrium.

Transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) receptors expressed predominantly in sensory nerves are activated by inflammatory stimuli and mediate inflammation and pain. Although they have been shown in the human endometrium, their regulation and function are unknown. Therefore, we investigated their estrogen- and progesterone-dependent alterations in the rat endometrium in comparison with the estrogen-regulated inflammatory cytokine macrophage migration inhibitory factor (MIF). Four-week-old (sexually immature) and four-month-old (sexually mature) female rats were treated with the non-selective estrogen receptor (ER) agonist diethylstilboestrol (DES), progesterone and their combination, or ovariectomized. RT-PCR and immunohistochemistry were performed to determine mRNA and protein expression levels respectively. Channel function was investigated with ratiometric [Ca(2+)]i measurement in cultured primary rat endometrial cells. Both TRP receptors and MIF were detected in the endometrium at mRNA and protein levels, and their localizations were similar. Immunostaining was observed in the immature epithelium, while stromal, glandular and epithelial positivity were observed in adults. Functionally active TRP receptor proteins were shown in endometrial cells by activation-induced calcium influx. In adults, Trpa1 and Trpv1 mRNA levels were significantly up-regulated after DES treatment. TRPA1 increased after every treatment, but TRPV1 remained unchanged following the combined treatment and ovariectomy. In immature rats, DES treatment resulted in increased mRNA expression of both channels and elevated TRPV1 immunopositivity. MIF expression changed in parallel with TRPA1/TRPV1 in most cases. DES up-regulated Trpa1, Trpv1 and Mif mRNA levels in endometrial cell cultures, but 17β-oestradiol having ERα-selective potency increased only the expression of Trpv1. We provide the first evidence for TRPA1/TRPV1 expression and their estrogen-induced up-regulation in the rat endometrium in correlation with the MIF.

Tall Wheatgrass Cultivar Szarvasi-1 (Elymus elongatus subsp. ponticus cv. Szarvasi-1) as a Potential Energy Crop for Semi-Arid Lands of Eastern Europe

By 2020, proportion of renewable energy sources should be around 20 per cent of the total energy consumption in the European Union, according to the new treaty signed by European leaders in 2009. This vast amount of renewable energy can be sourced from hydroelectric, geothermal, wind, solar power and, of course, from biofuels. To achieve this ambitious target, new technologies must be invented to exploit energy from the abiotic source of renewables and new energy plant species should be developed and produced, serving as source for solid, liquid biofuels and for biogas production. The most intensively studied and used bioenergy crops include miscanthus, reed canary grass, willows and poplars. We already have considerable knowledge about these energy plants from their taxonomical relations to their detailed crop technologies. In this chapter, we introduce a novel energy plant that has been cultivated for more than a century in many parts of the world for numerous purposes (e.g. land remediation, erosion control, forage), but its potential for energy production has not yet been realized. Tall wheatgrass, a new energy crop (Elymus elongatus subsp. ponticus cv. Szarvasi-1) has recently been introduced to cultivation in Hungary to provide biomass for solid biofuel energy production. The cultivar was developed in Hungary. The main goal of our research was to investigate the performance of Szarvasi-1 energy grass under different growing conditions (e.g. soil types, nutrition supply). We focused on the ecological background, biomass yield, weed composition, morphology, ecophysiology and the genetics of the plant.

Accelerated retinal aging in PACAP knock-out mice.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neurotrophic and neuroprotective peptide. PACAP and its receptors are widely distributed in the retina. A number of reports provided evidence that PACAP is neuroprotective in retinal degenerations. The current study compared retina cell type-specific differences in young (3-4months) and aged adults (14-16months), of wild-type (WT) mice and knock-out (KO) mice lacking endogenous PACAP production during the course of aging. Histological, immunocytochemical and Western blot examinations were performed. The staining for standard neurochemical markers (tyrosine hydroxylase for dopaminergic cells, calbindin 28 kDa for horizontal cells, protein kinase Cα for rod bipolar cells) of young adult PACAP KO retinas showed no substantial alterations compared to young adult WT retinas, except for the specific PACAP receptor (PAC1-R) staining. We could not detect PAC1-R immunoreactivity in bipolar and horizontal cells in young adult PACAP KO animals. Some other age-related changes were observed only in the PACAP KO mice only. These alterations included horizontal and rod bipolar cell dendritic sprouting into the photoreceptor layer and decreased ganglion cell number. Also, Müller glial cells showed elevated GFAP expression compared to the aging WT retinas. Furthermore, Western blot analyses revealed significant differences between the phosphorylation state of ERK1/2 and JNK in KO mice, indicating alterations in the MAPK signaling pathway. These results support the conclusion that endogenous PACAP contributes to protection against aging of the nervous system.

Presence of pituitary adenylate cyclase-activating polypeptide (PACAP) in the plasma and milk of ruminant animals

Milk contains a variety of proteins and peptides that possess biological activity. Growth factors, such as growth hormone, insulin-like, epidermal and nerve growth factors are important milk components which may regulate growth and differentiation in various neonatal tissues and also those of the mammary gland itself. We have recently shown that pituitary adenylate cyclase-activating polypeptide (PACAP), an important neuropeptide with neurotrophic actions, is present in the human milk in much higher concentration than in the plasma of lactating women. Investigation of growth factors in the milk of domestic animals is of utmost importance for their nutritional values and agricultural significance. Therefore, the aim of the present study was to determine the presence and concentration of PACAP in the plasma and milk of three ruminant animal species. Furthermore, the presence of PACAP and its specific PAC1 receptor were investigated in the mammary glands. Radioimmunoassay measurements revealed that PACAP was present in the plasma and the milk of the sheep, goat and the cow in a similar concentration to that measured previously in humans. PACAP38-like immunoreactivity (PACAP38-LI) was 5-20-fold higher in the milk than in the plasma samples of the respective animals, a similar serum/milk ratio was found in all the three species. The levels did not show significant changes within the examined 3-month-period of lactation after delivery. Similar PACAP38-LI was measured in the homogenates of the sheep mammary gland samples taken 7 and 30 days after delivery. PAC1 receptor expression was detected in these udder biopsies by fluorescent immunohistochemistry suggesting that this peptide might have an effect on the mammary glands themselves. These data show that PACAP is present in the milk of various ruminant domestic animal species at high concentrations, the physiological implications of which awaits further investigation.

Phylogenetic relationships in the Neotropical tribe Hamelieae (Rubiaceae, Cinchonoideae) and comments on its generic limits

Our molecular phylogenetic analyses shed some light on the evolutionary relationships within the Hamelieae tribe. Phylogenetic reconstructions based on Internal Transcribed Spacer and trnL‐F sequence data revealed the presence of three distinct evolutionary lineages. The first clade includes Hamelia and Syringantha, the second clade includes Deppea s.l. (including Bellizinca, Csapodya, and Edithea), and the third clade includes Pinarophyllon, Deppeopsis, Hoffmannia, Pseudomiltemia, Plocaniophyllon, Omiltemia, and Renistipula. The phylogenetic analysis re‐evaluated some taxonomical combinations. The transfer of Deppeopsis taxa from Deppea s.l. is supported, but however, the monophyly of the genus is not. The transfer of Renistipula from Rondeletieae is also highly supported. Both Csapodya and Edithea species form a well‐defined group among Deppea s.l. with high posterior probabilities, allowing to reconsider the exclusion or integration of these taxa to Deppea.

Sensitive fluorescent hybridisation protocol development for simultaneous detection of microRNA and cellular marker proteins (in the retina)

Nowadays, increasing number of microRNAs are found to have crucial roles in various physiological processes through gene expression regulation via RNA silencing as a result of base pairing with complementary mRNA sequences. To reveal the spatial distribution of microRNA expression in tissues, in situ hybridisation is the only method developed to date. This work aims to provide a novel approach to obtain information on the possible involvement of microRNA-s in regulatory processes under experimental conditions by enhancing fluorescent detection of microRNA labelling. Developing Wistar rats were used as a model system to analyse retinal microRNA expression in the first 3 postnatal weeks. Using cryosections, the crucial elements of optimal labels were (1) the concentration and duration of proteinase K treatment, (2) hybridisation temperature of microRNA probes and (3) temperature of stringency washes. Further improvements made possible to combine our in situ hybridisation protocol with double-label immunofluorescence allowing for the simultaneous detection of microRNA-s with high sensitivity and a neuronal cell marker and/or a synaptic marker protein. Thus, the regulatory microRNA-s can be localised in an identified cell type along with its potential target protein. We believe that our protocol can be easily adapted for a variety of tissues of different origins, developmental stages and experimental conditions.

Local upregulation of transient receptor potential ankyrin 1 and transient receptor potential vanilloid 1 ion channels in rectosigmoid deep infiltrating endometriosis

Transient Receptor Potential Vanilloid 1 (TRPV1) and Transient Receptor Potential Ankyrin 1 (TRPA1) expressed mainly by primary sensory neurons function as major nociceptive integrators. They are also present on the rat endometrium in an oestrogen-regulated manner. TRPV1 is upregulated in peritoneal and ovarian endometriosis patients, but there is no information about TRPA1 and their pathophysiological significances. In this study, patients undergoing laparoscopic surgery were investigated: severe dysmenorrhoea due to rectosigmoid deep infiltrating endometriosis (n = 15), uterine fibroid-induced moderate dysmenorrhoea (n = 7) and tubal infertility with no pain (n = 6). TRPA1 and TRPV1 mRNA and protein expressions were determined by quantitative polymerase chain reaction and semi-quantitative immunohistochemistry from the endometrium samples taken by curettage. Results were correlated with the clinical characteristics including pain intensity. TRPA1 and TRPV1 receptors were expressed in the healthy human endometrium at mRNA and protein levels. Sparse, scattered cytoplasmic TRPA1 and TRPV1 immunopositivities were found in the stroma and epithelial layers. We detected upregulated mRNA levels in deep infiltrating endometriosis lesions, and TRPV1 gene expression was also elevated in autocontrol endometrium of deep infiltrating endometriosis patients. Histological scoring revealed significant TRPA1 and TRPV1 difference between deep infiltrating endometriosis stroma and epithelium, and in deep infiltrating endometriosis epithelium compared to control samples. Besides, we measured elevated stromal TRPV1 immunopositivity in deep infiltrating endometriosis. Stromal TRPA1 and TRPV1 immunoreactivities strongly correlated with dysmenorrhoea severity, as well TRPV1 expression on ectopic epithelial cells and macrophages with dyspareunia. Epithelial TRPA1 and stromal TRPV1 immunopositivity also positively correlated with dyschezia severity. We provide the first evidence for the presence of non-neuronal TRPA1 receptor in the healthy human endometrium and confirm the expression of TRPV1 channels. Their upregulations in rectosigmoid deep infiltrating endometriosis lesions and correlations with pain intensity suggest potential roles in pathophysiological mechanisms of the disease.