Human benign Leydig cell tumor - Biochemical evaluation.

Recently we reported expressional alterations in 219 genes and their transcripts in Leydig cell tumors but nowadays there is still a lack of full basic biochemical characteristics of these tumors. The discovery of potential biochemical markers for tumor management from early detection, treatments, and control of therapy results may markedly supplement genetic data. Leydig cell micronodules were obtained from patients with azoospermia who were qualified for testicular biopsy. The biochemistry of Leydig cell tumors was analyzed using histological staining and spectrophotometric measurements of total proteins, carbohydrates, lipids, and nucleic acids. In addition, the levels of calcium (Ca2 +), copper (Cu2 +), zinc (Zn2 +), and selenium (Se2 +) ions were measured. When compared to healthy testis we revealed, for the first time, that in the interstitial tissue with Leydig cell tumors, great amounts of proteins, carbohydrates, lipids, and acids were dislocated from the seminiferous tubules. Measurements of organic compounds showed a decrease (P < 0.05) only in the Cu2 + content in Leydig cell tumors which may be related to their altered biochemical structure. This specific result may be promising for designing further approaches to manage this tumor based on combining morphological and molecular data.

Effect of Diosmin on Selected Parameters of Oxygen Homeostasis.

Chronic venous disease (CVD) is a condition characterized by functional disturbances in the microcirculation of the superficial and deep veins, affecting up to 30% of the global population. Diosmin, a phlebotropic drug, is commonly used in the treatment of CVD, and its beneficial effects have been described in numerous clinical studies. However, the precise molecular mechanism underlying the activity of diosmin is not yet fully understood. Therefore, the objective of our study was to investigate whether diosmin has an impact on oxygen management, as cardiovascular diseases are often associated with hypoxia. In our study, patients were administered a daily dosage of 2 × 600 mg of diosmin for 3 months, and we evaluated several factors associated with oxygen management, angiogenesis, and inflammation using biochemical assays. Our findings indicate that diosmin reduced the levels of fibroblast growth factors (FGF) and vascular endothelial growth factor (VEGF-C), while increasing endostatin and angiostatin levels, suggesting a potential influence on angiogenesis regulation. Furthermore, diosmin exhibited anti-inflammatory properties by suppressing the levels of tumor necrosis factor-alpha (TNF-α), interleukin 1-beta (IL-1ß), and interleukin 6 (IL-6), while promoting the production of interleukin 12 (IL-12). Additionally, diosmin significantly decreased the levels of hypoxia-inducible factor (HIF), anion gap (AG), and lactate, indicating its potential influence on the hypoxia-inducible factor pathway. These findings suggest that diosmin may play a crucial role in modulating oxygen management and inflammation in the context of chronic venous disease.

Influence of Umbilical Cord Blood Biochemical Parameters and Disease Condition on the Expression of the TSG-6 Gene in Umbilical Mesenchymal Stem Cells.

BACKGROUND Mesenchymal stem cells (MSCs) are capable of secreting different substances, including the anti-inflammatory protein TSG-6, which can be useful in the treatment of diseases with inflammatory reactions. The main aim of this study was to evaluate the expression of the TSG-6 gene in MSCs derived from the umbilical cord. For better understanding of the anti-inflammatory properties of MSCs, we additionally assessed the expression of some interleukins (ILs). MATERIAL AND METHODS The study group included 45 patients after delivery, aged from 21 to 46 years; the average patient age was 33 years. MSCs were isolated enzymatically from umbilical cord Wharton's jelly, in vitro cultured, and characterized using flow cytometry; qPCR was performed to assess expression of the studied genes. The expression of genes of a number of pro-inflammatory ILs in MSCs was investigated in relation to the health of patients (coexistence of hypertension), the level of leukocytes, pCO2, and hemoglobin in the blood. RESULTS Our research showed that the expression of the TSG-6 gene in MSCs depends on coexisting diseases in the patient and the biochemical parameters of umbilical cord blood, including the important role of cord blood pH. We found that the levels of IL2 and IL6 expression were correlated with pCO2, and IL6 expression were correlated with pO2. CONCLUSIONS Our study suggests that maternal health status and cord blood biochemical parame-ters could affect the anti-inflammatory properties of MSCs; however, this needs to be confirmed in a future study.

Biological Potential of Carnivorous Plants from Nepenthales.

Since Charles Darwin and his book carnivorous plants have aroused interest and heated debate. In addition, there is growing interest in this group of plants as a source of secondary metabolites and in the application of their biological activity. The aim of this study was to trace the recent literature in search of the application of extracts obtained from families Droseraceae, Nepenthaceae, and Drosophyllaceae to show their biological potential. The data collected in the review clearly indicate that the studied Nepenthales species have great biological potential in terms of antibacterial, antifungal, antioxidant, anti-inflammatory, and anticancer use. We proposed that further investigations should include: (i) bioactivity-guided investigations of crude plant extract to connect a particular type of action with a specific compound or a group of metabolites; (ii) a search for new bioactive properties of carnivorous plants; (iii) establishment of molecular mechanisms associated with specific activity. Furthermore, further research should be extended to include less explored species, i.e., Drosophyllum lusitanicum and especially Aldrovanda vesiculosa.

Carnivorous Plants from Nepenthaceae and Droseraceae as a Source of Secondary Metabolites.

Carnivorous plants are able to attract small animals or protozoa and retain them in their specialized traps. Later, the captured organisms are killed and digested. The nutrients contained in the prey bodies are absorbed by the plants to use for growth and reproduction. These plants produce many secondary metabolites involved in the carnivorous syndrome. The main purpose of this review was to provide an overview of the secondary metabolites in the family Nepenthaceae and Droseraceae, which were studied using modern identification techniques, i.e., high-performance liquid chromatography or ultra-high-performance liquid chromatography with mass spectrometry and nuclear magnetic resonance spectroscopy. After literature screening, there is no doubt that tissues of species from the genera Nepenthes, Drosera, and Dionaea are rich sources of secondary metabolites that can be used in pharmacy and for medical purposes. The main types of the identified compounds include phenolic acids and their derivatives (gallic, protocatechuic, chlorogenic, ferulic, p-coumaric acids, gallic, hydroxybenzoic, vanillic, syringic caffeic acids, and vanillin), flavonoids (myricetin, quercetin, and kaempferol derivatives), including anthocyanins (delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, and cyanidin), naphthoquinones (e.g., plumbagin, droserone, and 5-O-methyl droserone), and volatile organic compounds. Due to the biological activity of most of these substances, the importance of the carnivorous plant as a pharmaceutical crop will increase.

Comparison of SOX2 and POU5F1 Gene Expression in Leukapheresis-Derived CD34+ Cells before and during Cell Culture.

Bone marrow is an abundant source of both hematopoietic as well as non-hematopoietic stem cells. Embryonic, fetal and stem cells located in tissues (adipose tissue, skin, myocardium and dental pulp) express core transcription factors, including the SOX2, POU5F1 and NANOG gene responsible for regeneration, proliferation and differentiation into daughter cells. The aim of the study was to examine the expression of SOX2 and POU5F1 genes in CD34-positive peripheral blood stem cells (CD34+ PBSCs) and to analyze the influence of cell culture on the expression of SOX2 and POU5F1 genes. The study material consisted of bone marrow-derived stem cells isolated by using leukapheresis from 40 hematooncology patients. Cells obtained in this process were subject to cytometric analysis to determine the content of CD34+ cells. CD34-positive cell separation was conducted using MACS separation. Cell cultures were set, and RNA was isolated. Real-time PCR was conducted in order to evaluate the expression of SOX2 and POU5F1 genes and the obtained data were subject to statistical analysis. We identified the expression of SOX2 and POU5F1 genes in the examined cells and demonstrated a statistically significant (p < 0.05) change in their expression in cell cultures. Short-term cell cultures (<6 days) were associated with an increase in the expression of SOX2 and POU5F1 genes. Thus, short-term cultivation of transplanted stem cells could be used to induce pluripotency, leading to better therapeutic effects.

Senescence and autophagy relation with the expressional status of non-canonical estrogen receptors in testes and adrenals of roe deer (Capreolus capreolus) during the pre-rut period.

The roe deer bucks represent a spontaneous model to study the synchronized testicular involution and recrudescence cycles. However, cellular processes and hormonal control of steroidogenic glands are scarcely known. For the present study testes and adrenal glands obtained from roe deer during the pre-rut season were used. We aimed to determine (i) senescence and autophagy involvement in testis atrophy (immunohistochemical analysis for tumor suppressor protein encoded by the cyclin-dependent kinase inhibitor 2A; p16 and microtubule-associated protein 1A/1B-light chain 3; LC3, respectively), (ii) the size of the adrenal cortex and medulla (morphometric analysis), (iii) G-protein coupled estrogen receptor (GPER) and estrogen-related receptors (ERRs; type α, ß, and Y) distribution and expression (qRT-PCR and immunohistochemical analyses) and (iv) serum testosterone and estradiol levels (immunoassay ELISA). This study revealed pre-rut characteristics of testis structure with the presence of both senescence and autophagy-positive cells and higher involvement of senescence, especially in spermatogenic cells (P < 0.05). In the adrenal cortex, groups of cells exhibiting shrinkage were observed. The presence of ERRs in cells of the seminiferous epithelium and interstitial Leydig cells and GPER presence distinctly in Leydig cells was revealed. In adrenals, these receptors were localized in groups of normal-looking cells and those with shrinkage. Morphometric analysis showed differences in cortex width which was smaller (P < 0.05) than that of the medulla. A weak immunohistochemical signal was observed for ERRß when compared to ERRα and ERRγ. The mRNA expression level of ERRα and ERRγ was lower (P < 0.001 and P < 0.05, respectively) while ERRß was higher (P < 0.001) in adrenals when compared to testes. mRNA GPER expression was similar in both glands. In the pre-rut season, the testosterone level was 4.89 ng/ml while the estradiol level was 0.234 ng/ml. We postulate that: (i) senescence and autophagy may be involved in both reinitiation of testis function and/or induction of abnormal processes, (ii) hormonal modulation of testis inactivity may affect adrenal cortex causing cell shrinkage, (iii) ERRs and GPER localization in spermatogenic cells and interstitial cells, as well as cortex cells, may maintain and control the morpho-functional status of both glands, and (iv) androgens and estrogens (via ERRs and GPER) drive cellular processes in the testis and adrenal pre-rut physiology.

The Localization of Cell Wall Components in the Quadrifids of Whole-Mount Immunolabeled Utricularia dichotoma Traps.

Utricularia (bladderworts) are carnivorous plants. They produce small hollow vesicles, which function as suction traps that work underwater and capture fine organisms. Inside the traps, there are numerous glandular trichomes (quadrifids), which take part in the secretion of digestive enzymes, the resorption of released nutrients, and likely the pumping out of water. Due to the extreme specialization of quadrifids, they are an interesting model for studying the cell walls. This aim of the study was to fill in the gap in the literature concerning the immunocytochemistry of quadrifids in the major cell wall polysaccharides and glycoproteins. To do this, the localization of the cell wall components in the quadrifids was performed using whole-mount immunolabeled Utricularia traps. It was observed that only parts (arms) of the terminal cells had enough discontinuous cuticle to be permeable to antibodies. There were different patterns of the cell wall components in the arms of the terminal cells of the quadrifids. The cell walls of the arms were especially rich in low-methyl-esterified homogalacturonan. Moreover, various arabinogalactan proteins also occurred. Cell walls in glandular cells of quadrifids were rich in low-methyl-esterified homogalacturonan; in contrast, in the aquatic carnivorous plant Aldrovanda vesiculosa, cell walls in the glandular cells of digestive glands were poor in low-methyl-esterified homogalacturonan. Arabinogalactan proteins were found in the cell walls of trap gland cells in all studied carnivorous plants: Utricularia, and members of Droseraceae and Drosophyllaceae.

Antioxidant Potential of Diosmin and Diosmetin against Oxidative Stress in Endothelial Cells.

Phlebotropic flavonoids, including diosmin and its aglycone diosmetin, are natural polyphenols widely used in the prevention and treatment of chronic venous insufficiency (CVI). As oxidative stress plays an important role in the development of pathophysiology of the cardiovascular system, the study aimed to investigate the protective effects of diosmin and diosmetin on hydrogen peroxide (H2O2)-induced oxidative stress in endothelial cells. The cells were pretreated with different concentrations of the flavonoid prior to the H2O2 exposure. The cell viability, the level of intracellular reactive oxygen species (ROS), the activity of cellular antioxidant enzymes-including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase GPx-and the malondialdehyde (MDA) level were assessed. It was found that the H2O2-induced oxidative stress was ameliorated by diosmin/diosmetin in a concentration-dependent manner. The flavonoids restored the activity of cellular antioxidant enzymes and lowered the MDA level upregulated by the H2O2 exposure. These results indicate that diosmin and diosmetin may prevent oxidative stress in endothelial cells; therefore, they may protect against the development and progression of oxidative-stress-related disorders.

Evaluation of the Impact of Pregnancy-Associated Factors on the Quality of Wharton's Jelly-Derived Stem Cells Using SOX2 Gene Expression as a Marker.

SOX2 is a recognized pluripotent transcription factor involved in stem cell homeostasis, self-renewal and reprogramming. It belongs to, one of the SRY-related HMG-box (SOX) family of transcription factors, taking part in the regulation of embryonic development and determination of cell fate. Among other functions, SOX2 promotes proliferation, survival, invasion, metastasis, cancer stemness, and drug resistance. SOX2 interacts with other transcription factors in multiple signaling pathways to control growth and survival. The aim of the study was to determine the effect of a parturient's age, umbilical cord blood pH and length of pregnancy on the quality of stem cells derived from Wharton's jelly (WJSC) by looking at birth weight and using SOX2 gene expression as a marker. Using qPCR the authors, evaluated the expression of SOX2 in WJSC acquired from the umbilical cords of 30 women right after the delivery. The results showed a significant correlation between the birth weight and the expression of SOX2 in WJSC in relation to maternal age, umbilical cord blood pH, and the length of pregnancy. The authors observed that the younger the woman and the lower the umbilical cord blood pH, the earlier the delivery occurs, the lower the birth weight and the higher SOX2 gene expression in WJSC. In research studies and clinical applications of regenerative medicine utilizing mesenchymal stem cells derived from Wharton's Jelly of the umbilical cord, assessment of maternal and embryonic factors influencing the quality of cells is critical.

Leydig Cells in Immunocastrated Polish Landrace Pig Testis: Differentiation Status and Steroid Enzyme Expression Status.

Porker immunocastration against gonadoliberin (GnRH) secretion has been utilized since 2009; however, consumers are still skeptical of it. This is due to not having full information available on the problem of a boar taint, as well as a lack of research on morphological and molecular changes that may occur in the animal reproductive system and other body systems. The present study aimed to explore the functional status of steroidogenic Leydig cells of the testicular interstitial tissue in immunocastrated Polish Landrace pigs. Analyses were performed using Western blot, immunohistochemistry for relaxin (RLN), insulin-like 3 protein (INSL3), pelleted growth factor receptor α (PDGFRα), cytochrome P450scc, 3ß- and 17ß-hydroxysteroid dehydrogenases (3ß-HSD, 17ß-HSD), cytochrome P450arom, and 5α-reductase (5α-RED). Immunoassay ELISA was used to measure the androstenone, testosterone, and estradiol levels in the testis and serum of immunocastrates. We revealed disturbances in the distribution and expression of (i) RLN, indicating an inflammatory reaction in the interstitial tissue; (ii) INSL3 and PDGFRα, indicating alterations in the differentiation and function of fetal, perinatal, or adult Leydig cell populations; (iii) P450scc, 3ß-HSD, 17ß-HSD, P450arom, and 5α-RED, indicating disturbances in the sex steroid hormone production and disturbed functional status of Leydig cells; as well as (iv) decreased levels of androstenone, testosterone, and estradiol in testicular tissue and serum, indicating the dedicated action of Improvac to reduce boar taint at both the hypothalamic-hypophysis-gonadal axis and local level (Leydig cells). In summary, our study provides a significant portion of knowledge on the function of Leydig cells after immunocastration, which is also important for the diagnosis and therapy of testis dysfunction due to GnRH action failure and/or Leydig cell differentiational-functional alterations.

The G-Protein-Coupled Membrane Estrogen Receptor Is Present in Horse Cryptorchid Testes and Mediates Downstream Pathways.

Cryptorchidism in horses is a commonly occurring malformation. The molecular basis of this pathology is not fully known. In addition, the origins of high intratesticular estrogen levels in horses remain obscure. In order to investigate the role of the G-protein-coupled membrane estrogen receptor (GPER) and establish histological and biochemical cryptorchid testis status, healthy and cryptorchid horse testes were subjected to scanning electron microscopy analysis, histochemical staining for total protein (with naphthol blue black; NBB), acid content (with toluidine blue O; TBO), and polysaccharide content (with periodic acid-Schiff; PAS). The expression of GPER was analyzed by immunohistochemistry and Western blot. GPER-mediated intracellular cAMP and calcium (Ca2+) signaling were measured immunoenzymatically or colorimetrically. Our data revealed changes in the distribution of polysaccharide content but not the protein and acid content in the cryptorchid testis. Polysaccharides seemed to be partially translocated from the interstitial compartment to the seminiferous tubule compartment. Moreover, the markedly decreased expression of GPER and GPER downstream molecules, cAMP and Ca2+, suggests their potential role in testis pathology. Increased estrogen levels in cryptorchid conditions may be linked to disturbed GPER signaling. We postulate that GPER is a prominent key player in testis development and function and may be used as a new biomarker of horse testis in health and disease.

The BIRC Family Genes Expression in Patients with Triple Negative Breast Cancer.

The BIRC (baculoviral IAP repeat-containing; BIRC) family genes encode for Inhibitor of Apoptosis (IAP) proteins. The dysregulation of the expression levels of the genes in question in cancer tissue as compared to normal tissue suggests that the apoptosis process in cancer cells was disturbed, which may be associated with the development and chemoresistance of triple negative breast cancer (TNBC). In our study, we determined the expression level of eight genes from the BIRC family using the Real-Time PCR method in patients with TNBC and compared the obtained results with clinical data. Additionally, using bioinformatics tools (Ualcan and The Breast Cancer Gene-Expression Miner v4.5 (bc-GenExMiner v4.5)), we compared our data with the data in the Cancer Genome Atlas (TCGA) database. We observed diverse expression pattern among the studied genes in breast cancer tissue. Comparing the expression level of the studied genes with the clinical data, we found that in patients diagnosed with breast cancer under the age of 50, the expression levels of all studied genes were higher compared to patients diagnosed after the age of 50. We observed that in patients with invasion of neoplastic cells into lymphatic vessels and fat tissue, the expression levels of BIRC family genes were lower compared to patients in whom these features were not noted. Statistically significant differences in gene expression were also noted in patients classified into three groups depending on the basis of the Scarff-Bloom and Richardson (SBR) Grading System.

The Correlation of Mutations and Expressions of Genes within the PI3K/Akt/mTOR Pathway in Breast Cancer-A Preliminary Study.

There is an urgent need to seek new molecular biomarkers helpful in diagnosing and treating breast cancer. In this elaboration, we performed a molecular analysis of mutations and expression of genes within the PI3K/Akt/mTOR pathway in patients with ductal breast cancer of various malignancy levels. We recognized significant correlations between the expression levels of the studied genes. We also performed a bioinformatics analysis of the data available on the international database TCGA and compared them with our own research. Studies on mutations and expression of genes were conducted using High-Resolution Melt PCR (HRM-PCR), Allele-Specific-quantitative PCR (ASP-qPCR), Real-Time PCR molecular methods in a group of women with ductal breast cancer. Bioinformatics analysis was carried out using web source Ualcan and bc-GenExMiner. In the studied group of women, it was observed that the prevalence of mutations in the studied PIK3CA and AKT1 genes was 29.63%. It was stated that the average expression level of the PIK3CA, PIK3R1, PTEN genes in the group of breast cancer patients is lower in comparison to the control group, while the average expression level of the AKT1 and mTOR genes in the studied group was higher in comparison to the control group. It was also indicated that in the group of patients with mutations in the area of the PIK3CA and AKT1 genes, the PIK3CA gene expression level is statistically significantly lower than in the group without mutations. According to our knowledge, we demonstrate, for the first time, that there is a very strong positive correlation between the levels of AKT1 and mTOR gene expression in the case of patients with mutations and without mutations.

Interstitial Leydig Cell Tumorigenesis-Leptin and Adiponectin Signaling in Relation to Aromatase Expression in the Human Testis.

Although epidemiological studies from the last years report an increase in the incidences of Leydig cell tumors (previously thought to be a rare disease), the biochemical characteristics of that tumor important for understanding its etiology, diagnosis, and therapy still remains not completely characterized. Our prior studies reported G-protein coupled estrogen receptor signaling and estrogen level disturbances in Leydig cell tumors. In addition, we found that expressions of multi-level-acting lipid balance- and steroidogenesis-controlling proteins including peroxisome proliferator-activated receptor are altered in this tumor. In order to get deeper into the other molecular mechanisms that regulate lipid homeostasis in the Leydig cell tumor, here we investigate the presence and expression of newly-described hormones responsible for lipid homeostasis balancing (leptin and adiponectin), together with expression of estrogen synthase (aromatase). Samples of Leydig cell tumors (n = 20) were obtained from patients (31-45 years old) and used for light and transmission electron microscopic, western blotting, and immunohistochemical analyses. In addition, body mass index (BMI) was calculated. In tumor mass, abundant lipid accumulation in Leydig cells and various alterations of Leydig cell shape, as well as the presence of adipocyte-like cells, were observed. Marked lipid content and various lipid droplet size, especially in obese patients, may indicate alterations in lipid homeostasis, lipid processing, and steroidogenic organelle function in response to interstitial tissue pathological changes. We revealed significantly increased expression of leptin, adiponectin and their receptors, as well as aromatase in Leydig cell tumors in comparison to control. The majority of patients (n = 13) were overweight as indicated by their BMI. Moreover, a significant increase in expression of phospholipase C (PLC), and kinases Raf, ERK which are part of adipokine transductional pathways, was demonstrated. These data expand our previous findings suggesting that in human Leydig cell tumors, estrogen level and signaling, together with lipid status, are related to each other. Increased BMI may contribute to certain biochemical characteristics and function of the Leydig cell in infertile patients with a tumor. In addition, altered adipokine-estrogen microenvironment can have an effect on proliferation, growth, and metastasis of tumor cells. We report here various targets (receptors, enzymes, hormones) controlling lipid balance and estrogen action in Leydig cell tumors indicating their possible usefulness for diagnostics and therapy.

Unusual developmental morphology and anatomy of vegetative organs in Utricularia dichotoma-leaf, shoot and root dynamics.

The terrestrial carnivorous species Utricularia dichotoma is known for a great phenotypic plasticity and unusual vegetative organs. Our investigation on 22 sources/populations revealed that after initiation of a leaf and two bladders on a stolon, a bud was formed in the proximal axil of the leaf, developing into a rosette with up to seven organs. The first two primordia of the bud grew into almost every possible combination of organs, but often into two anchor stolons. The patterns were generally not population specific. The interchangeability of organs increased with increasing rank in the succession of organs on stolon nodes. A high potential of switching developmental programs may be successful in a fluctuating environment. In this respect, we were able to show that bladders developed from anchor stolons experimentally when raising the water table. Anatomical structures were simple, lacunate and largely homogenous throughout all organs. They showed similarities with many hydrophytes, reflecting the plant's adaptation to (temporarily) submerged conditions. The principal component analysis was used in the context of dynamic morphology to illustrate correlations between organ types in the morphospace of U. dichotoma, revealing an organ specific patchwork of developmental processes for typical leaves and shoots, and less pronounced for a typical root. The concept and methods we applied may prove beneficial for future studies on the evolution of Lentibulariaceae, and on developmental morphology and genetics of unusual structures in plants.

Floral micromorphology and nectar composition of the early evolutionary lineage Utricularia (subgenus Polypompholyx, Lentibulariaceae).

Utricularia (Lentibulariaceae) is a genus comprising around 240 species of herbaceous, carnivorous plants. Utricularia is usually viewed as an insect-pollinated genus, with the exception of a few bird-pollinated species. The bladderworts Utricularia multifida and U. tenella are interesting species because they represent an early evolutionary Utricularia branch and have some unusual morphological characters in their traps and calyx. Thus, our aims were to (i) determine whether the nectar sugar concentrations and composition in U. multifida and U. tenella are similar to those of other Utricularia species from the subgenera Polypompholyx and Utricularia, (ii) compare the nectary structure of U. multifida and U. tenella with those of other Utricularia species, and (iii) determine whether U. multifida and U. tenella use some of their floral trichomes as an alternative food reward for pollinators. We used light microscopy, histochemistry, and scanning and transmission electron microscopy to address those aims. The concentration and composition of nectar sugars were analysed using high-performance liquid chromatography. In all of the examined species, the floral nectary consisted of a spur bearing glandular trichomes. The spur produced and stored the nectar. We detected hexose-dominated (fructose + glucose) nectar in U. multifida and U. tenella as well as in U. violacea. In both U. multifida and U. tenella, there were trichomes that blocked the entrance into the throat and spur. Because these trichomes were rich in chromoplasts and contained lipid droplets, they may form an additional visual attractant. Bearing in mind the phylogenetic hypothesis for the genus, we suggest that an early ancestor of Utricularia had a nectariferous spur flower with a lower lip that formed a wide landing platform for bee pollinators.

The Trap Architecture of Utricularia multifida and Utricularia westonii (subg. Polypompholyx).

Utricularia are carnivorous plants which have small hollow vesicles as suction traps that work underwater by means of negative pressure and watertightness of the entrance for capturing small animal prey. Utricularia multifida and U. westonii have specific thick-walled traps, which are triangular in a transverse section but their functioning is unclear. Some authors suggest that the trap door in U. multifida acts as a simple valve without a suction trapping mechanism. Our main aim was to check the anatomical trap characters that are responsible for possible water outflow and maintaining negative pressure as main functional parts of the active trap suction mechanism in both species. Using different microscopic techniques, we investigated the ultrastructure of external trap glands, quadrifids, glands near the entrance (bifids, monofids), and also pavement epithelium. Quadrifids of both species have a similar structure to those known in other species from the genus, which possess the suction trap mechanism. Glands near the entrance in U. multifida and U. westonii, which are responsible for water pumping in other species, are typically developed as in other species in the genus and have pedestal cells which are transfer cells. The transfer cells also occur in glands of the pavement epithelium, which is again typically developed as in other species in the genus. Simple biophysical tests did not confirm reliably neither the negative underpressure formation in the traps nor the watertightness of the entrance in both species. Our anatomical results indirectly support the hypothesis that both species have suction traps like all other Utricularia species, but the biophysical data rather suggest a passive valve mechanism.

The Structure and Occurrence of a Velum in Utricularia Traps (Lentibulariaceae).

Bladderworts (Utricularia, Lentibulariaceae, Lamiales) are carnivorous plants that form small suction traps (bladders) for catching invertebrates. The velum is a cuticle structure that is produced by specialized trichomes of the threshold pavement epithelium. It is believed that the velum together with the mucilage seals the free edge of the trap door and that it is necessary for correct functioning of the trap. However, recently, some authors have questioned the occurrence of a velum in the traps of the Utricularia from the various sections. The main aim of this study was to confirm whether velum occurs in the traps of the Utricularia species from the subgenera Polypompholyx, Bivalvaria, and Utricularia. The 15 species were examined from subg. Polypompholyx, subg. Bivalvaria, and subg. Utricularia. A velum was found in all examined Utricularia species. In the traps of the members of section Pleiochasia, there was an outer velum (forming a complete ring) and an inner velum. In the traps of Utricularia uniflora (Lasiocaules), there was only an inner velum. In these species, the formation of the velum was accompanied by intensive mucilage production, and as a result, when door was closed (set position), the mucilage and the velum touched the surface of the door. In members of both sections of Pleiochasia and Lasiocaules, the pavement epithelium had a more complicated structure (four to five zones) than in the members of the subgenera Bivalvaria and Utricularia in which three distinct zones occurred (an outer with a velum, a middle and an internal with the mucilage trichomes). Even in U. purpurea, where the threshold was a reduced pavement epithelium, it consisted of three functional zones and the presence of a velum. Two main types of velum have been proposed. A velum was present in Utricularia traps regardless of the trap type or the habitat (aquatic, epiphytic, and terrestrial species). We proposed broad definition of velum as cuticle membranes covered by mucilage; from a functional point of view, this definition is more useful and more reflects complexity of this structure.

Floral micromorphology of the bird-pollinated carnivorous plant species Utricularia menziesii R.Br. (Lentibulariaceae).

Background and Aims: Bird pollination is rare among species in the genus Utricularia, and has evolved independently in two lineages of this genus. In Western Australia, the Western Spinebill, Acanthorhynchus superciliosus, visits flowers of Utricularia menziesii (section Pleiochasia: subgenus Polypompholyx). This study aimed to examine the micromorphology of U. menziesii flowers to assess traits that might be linked to its pollination strategy. Methods: Light microscopy, histochemistry and scanning electron microscopy were used. Nectar sugar composition was analysed using high-performance liquid chromatography. Key Results: The flowers of U. menziesii fulfil many criteria that characterize bird-pollinated flowers: red colour, a large, tough nectary spur that can withstand contact with a hard beak, lack of visual nectar guides and fragrance. Trichomes at the palate and throat may act as tactile signals. Spur nectary trichomes did not form clearly visible patches, but were more frequently distributed along vascular bundles, and were small and sessile. Each trichome comprised a single basal cell, a unicellular short pedestal cell (barrier cell) and a multicelled head. These trichomes were much smaller than those of the U. vulgaris allies. Hexose-dominated nectar was detected in flower spurs. Fructose and glucose were present in equal quantities (43 ± 3.6 and 42 ± 3.6 g L-1). Sucrose was only detected in one sample, essentially at the limit of detection for the method used. This type of nectar is common in flowers pollinated by passerine perching birds. Conclusions: The architecture of nectary trichomes in U. menziesii was similar to that of capitate trichomes of insect-pollinated species in this genus; thus, the most important specializations to bird pollination were flower colour (red), and both spur shape and size modification. Bird pollination is probably a recent innovation in the genus Utricularia, subgenus Polypompholyx, and is likely to have evolved from bee-pollinated ancestors.