Differential Urinary Proteomic Analysis of Endometrial Cancer.

Endometrial cancer is one of the most frequent gynecological malignancies present in more than 95 % of all uterine cancers. In spite of that, screening of such disease is not commonly performed in clinical practice due to enormous costs and relatively low sensitivity. Therefore, developing an effective screening test to diagnose endometrial cancer at early stages is of great importance for the clinical area of investigation. In this work, we applied urinary proteomics (i.e., bottom-up proteomic approach followed by nano HPLC-ESI-MS/MS) in patients with endometrial cancer, with respect to find proteins aimed for the early diagnostics and screening. According to the results, the significant semi-quantitative changes were observed in urinary proteome of treated patients. The proteins that may be pivotal in pathogenesis of endometrial cancer, like cadherin-1 (CDH1), vitronectin (VTN) and basement membrane specific-heparan sulphate proteoglycan core protein (HSPG2) were down-regulated, when compared to the control group. Ultimately, it can be stated that urinary proteomics has a potential for the searching of cancer protein biomarkers based on their altered concentration.

Combining parallel pattern generation of electrohydrodynamic lithography with serial addressing.

Electrohydrodynamic lithography (EHDL) is a parallel patterning process which typically makes use of topographically structured electrodes to guide pattern formation along areas of higher electrical field strength. The main driving force for pattern formation is an electrostatic pressure acting on a thin film polymer surface caused by a voltage applied between a top and bottom electrode. We here demonstrate that the principle can be applied using an addressable electrode composed of interdigitated fingers. Depending on the applied voltages, line patterns with different periodicities were fabricated. Our proof-of-concept experiments pave the way for a parallel pattern replication process where a serially addressed master is used. We complement the experiments by modelling the potentials across the electrodes and electrostatic forces acting on the polymer surface using different addressing schemes. Numerical simulations of the experimental setup pointed to some critical issues we experienced during the design of the experiments.

Genome-wide screen for differentially methylated long noncoding RNAs identifies Esrp2 and lncRNA Esrp2-as regulated by enhancer DNA methylation with prognostic relevance for human breast cancer.

The majority of long noncoding RNAs (lncRNAs) is still poorly characterized with respect to function, interactions with protein-coding genes, and mechanisms that regulate their expression. As for protein-coding RNAs, epigenetic deregulation of lncRNA expression by alterations in DNA methylation might contribute to carcinogenesis. To provide genome-wide information on lncRNAs aberrantly methylated in breast cancer we profiled tumors of the C3(1) SV40TAg mouse model by MCIp-seq (Methylated CpG Immunoprecipitation followed by sequencing). This approach detected 69 lncRNAs differentially methylated between tumor tissue and normal mammary glands, with 26 located in antisense orientation of a protein-coding gene. One of the hypomethylated lncRNAs, 1810019D21Rik (now called Esrp2-antisense (as)) was identified in proximity to the epithelial splicing regulatory protein 2 (Esrp2) that is significantly elevated in C3(1) tumors. ESRPs were shown previously to have a dual role in carcinogenesis. Both gain and loss have been associated with poor prognosis in human cancers, but the mechanisms regulating expression are not known. In-depth analyses indicate that coordinate overexpression of Esrp2 and Esrp2-as inversely correlates with DNA methylation. Luciferase reporter gene assays support co-expression of Esrp2 and the major short Esrp2-as variant from a bidirectional promoter, and transcriptional regulation by methylation of a proximal enhancer. Ultimately, this enhancer-based regulatory mechanism provides a novel explanation for tissue-specific expression differences and upregulation of Esrp2 during carcinogenesis. Knockdown of Esrp2-as reduced Esrp2 protein levels without affecting mRNA expression and resulted in an altered transcriptional profile associated with extracellular matrix (ECM), cell motility and reduced proliferation, whereas overexpression enhanced proliferation. Our findings not only hold true for the murine tumor model, but led to the identification of an unannotated human homolog of Esrp2-as which is significantly upregulated in human breast cancer and associated with poor prognosis.

Changes in urine autofluorescence in ovarian cancer patients.

Ovarian cancer is the type of cancer with the highest mortality rate among gynaecologic malignancies. Due to lack of screening tools, this disease is mainly diagnosed at a progressed stage, when it is too late to adequate therapy. Despite many attempts, enough sensitive and specific biomarker was not still uncovered. Fluorescence spectroscopy has proven to be a useful diagnostic tool with high efficiency. Fluorescence detection has three major advantages over other light-based investigation methods: high sensitivity, high speed, and reliability. Biological materials consist of a number of intrinsic fluorescent compounds -autofluorophores, which are associated with cardinal metabolic pathways. It is well known, that cancerous tissue metabolism is altered compared to healthy one, what influence also intrinsic fluorophores composition of bodily fluids. Urine is one of the biological fluids that could be obtained most easily and displays a blue - green fluorescence that can change in case of pathological process. Analysis of urine autofluorescence is non invasive and simple technique. Using fluorescent spectroscopy, ovarian cancer patients and healthy control group were discerned with high significance, so we predict that fluorescence analysis of urine could be a potential means of ovarian cancer screening.

Effects of exercise stress on the endocannabinoid system in humans under field conditions.

The effects of physical exercise stress on the endocannabinoid system in humans are almost unexplored. In this prospective study, we investigated in a crossover design and under field conditions at different altitudes the effects of physical exercise on the endocannabinoid system (ECS) in 12 trained healthy volunteers. For determination of alterations on the ECS three different protocols were analyzed: Protocol A (physical exercise at lower altitude) involved strenuous hiking below 2,100 m, whereas Protocol B (physical exercise by active ascent to high altitude) involved hiking up to 3,196 m, an accommodation at the cottage and a descent the next day. Protocol C (passive ascent) included a helicopter ascent to 3,196 m, an overnight stay at this altitude and a flight back to the base camp the following day. The cumulative hiked altitude in Protocol A and B was comparable (~1,650 m). The blood EC concentrations of anandamide increased significantly in Protocol A/B from baseline (T0) 0.12 ± 0.01/0.16 ± 0.02 (mean ± SEM) to 0.27 ± 0.02/0.42 ± 0.02 after exercise (T1) (p < 0.05). Anandamide levels in Protocol C remained stable at 0.20 ± 0.02. We conclude that the ECS is activated upon strenuous exercise whereas the combination with hypoxic stress further increases its activity. The reduced partial pressure of oxygen at high altitude alone did not affect this system. In summary, physical exercise activates the endocannabinoid system, whereas the combination with high altitude enhances this activation. This discloses new perspectives to adaptation mechanisms to physical exercise.

Autonomic recovery following sprint interval exercise.

The autonomic nervous activity was assessed following supramaximal exercise through heart rate (HR) and blood pressure (BP) variability (HRV and BPV) and baroreflex sensitivity (BRS). The beat-to-beat HR and BP were recorded during the supine and standing states before (PRE) and at 60 (R60) and 120 min (R120) following single (one Wingate, 1W) and multiple sprint intervals (four Wingates interspersed with 4 min of light cycling, 4W). The supine low frequency (LF) component was increased (P<0.001) and the high frequency (HF) was reduced (P<0.01) at R60 (LF, 178.1 ± 11.0; HF, 74.8 ± 10.5) compared with PRE (LF, 140.2 ± 7.4; HF, 110.4 ± 7.2) after both exercises. Supine systolic BPV LF:HF was higher at R60 (4.6 ± 1.4) compared with PRE (6.8 ± 2.4) only after 4W (P=0.035). Supine BRS was lower (P<0.001) at R60 (6.8 ± 1.1) than at PRE (15.3 ± 1.8) and R120 (11.3 ± 1.3). BRS at R120 remained lower after 4W (P=0.02). Standing BRS was less (P<0.001) at R60 (2.3 ± 0.5) than at PRE (5.6 ± 0.8) or R120 (3.7 ± 0.6) and returned to PRE values only after 1W. We concluded that (a) autonomic balance is shifted to a greater sympathetic and less parasympathetic activation following both types of exercise, (b) it takes longer than 1 h to recover following supramaximal exercise and (c) the recovery is longer after 4W than 1W.

Mesencephalic dopamine neuron number and tyrosine hydroxylase content: Genetic control and candidate genes.

The mesotelencephalic dopamine system shows substantial genetic variation which fundamentally affects normal and pathological behaviors related to motor function, motivation, and learning. Our earlier radioenzyme assay studies demonstrated significantly higher activity of tyrosine hydroxylase (TH), the first and rate limiting enzyme in the biosynthesis of catecholamine neurotransmitters, in the substantia nigra-ventral tegmental area of BALB/cJ mice in comparison with that of C57BL/6ByJ mice. Here, using quantitative immunoblotting and immunocytochemistry, we tested the hypothesis that mesencephalic TH protein content and number of nigral TH-positive neurons show strain-dependent differences in C57BL/6ByJ and BALB/cJ parallel to those observed in the TH activity studies. Immunoblotting experiments detected significantly higher mesencephalic TH protein content in BALB/cJ in comparison to C57BL/6ByJ (P<0.05). Immunocytochemical studies demonstrated that the number of TH-positive cells in substantia nigra was 31.3% higher in BALB/cJ than that in C57BL/6ByJ (P<0.01), while the average dopamine neuron volume was not significantly different. In a search for candidate genes that modulate TH content and the size of mesencephalic dopamine neuron populations we also studied near-isogenic mouse sublines derived from the C57BL/6ByJ and BALB/cJ progenitor strains. A whole-genome scan with 768 single nucleotide polymorphism markers indicated that two sublines, C4A6/N and C4A6/B, were genetically very similar (98.3%). We found significantly higher mesencephalic TH protein content in C4A6/B in comparison to C4A6/N (P=0.01), and a tendency for higher number of dopamine neurons in the substantia nigra in C4A6/B in comparison to C4A6/N, which, however, did not reach statistical significance. To identify the genetic source of the TH content difference we analyzed the single nucleotide polymorphism (SNP) genotype data of the whole-genome scan, and detected two small differential chromosome segments on chr. 13 and chr. 14. Microarray gene expression studies and bioinformatic analysis of the two differential regions implicated two cis-regulated genes (Spock1 and Cxcl14, chr. 13), and two growth factor genes [bone morphogenetic protein 6 (Bmp6) (chr. 13), and fibroblast growth factor 14 (Fgf14) (chr. 14)]. Taken together, the results suggest that (1) nigral dopamine neuron number and TH protein content may be genetically associated but further studies are needed to establish unequivocally this linkage, and (2) Spock1, Cxcl14, Bmp6, and Fgf14 are novel candidates for modulating the expression and maintenance of TH content in mesencephalic dopamine neurons in vivo.

Oriented immobilisation of engineered single-chain antibodies to develop biosensors for virus detection.

Single chain variable fragment (scFv) molecules were selected from a synthetic phage display library then cloned into a generic vector for expression of the scFv fused to the light chain constant domain of human immunoglobulin with a C-terminal cysteine residue (scFvC(L)cys). A heterobifunctional maleimide linker was synthesised and a strategy for functionalization of gold with the scFvC(L)cys fusion proteins elaborated. Successful covalent attachment of functional scFvC(L)cys was demonstrated using a surface plasmon resonance-based sensor. The results showed that the immobilised scFvC(L)cys molecules were functional and specific binding curves (with response relative to the concentration of virus antigen) were obtained over more than 25 cycles of binding and dissociation. ScFv molecules lacking the C-terminal cysteine performed poorly in similar experiments. The work demonstrates the feasibility of using simple scFv selection and cloning procedures combined with oriented immobilisation of scFvC(L)cys fusion proteins for robust antigen sensing surfaces in immunosensor or other biotechnological applications.

Inhibitory effect of galanin on dopamine induced increased oxytocin secretion in rat neurohypophyseal tissue cultures.

The regulation of oxytocin (OT) release by galanin (GAL) at the neurohypophyseal (NH) nerve terminal is not adequately understood. The effect of GAL on the secretion of OT was studied in 13- to 14-day cultures of isolated rat NH tissue. By this time, the hormone content of the medium had become constant. The OT content of the supernatant medium was determined by RIA after a 1- or 2-h incubation. A significantly decreased content of OT was found following incubation with 10(-6)-10(-8) M doses of GAL. Dopamine (DA) and the DA-active drugs apomorphine (APM) and Pro-Lys-Gly (PLG) (10(-6) M in each medium) increased the OT synthesis of NH tissue cultures. This elevation of OT secretion could be blocked by the administration of GAL together with DA, APM or PLG. The DA-blocking effect of GAL was prevented by previous treatment with the GAL receptor antagonist galantid (M15). The results indicate that OT release from the NH is directly influenced by the GAL-ergic system. The GAL-ergic control of OT secretion from NH tissue in rats can occur at the level of the posterior pituitary.

Inhibitory effect of galanin on dopamine-induced enhanced vasopressin secretion in rat neurohypophyseal tissue cultures.

The effect of galanin (GAL) on vasopressin (VP) secretion was studied in 13-14-day cultures of isolated rat neurohypophyseal (NH) tissue. The VP content of the supernatant was determined by radioimmunoassay (RIA) after a 1- or 2-h incubation. A significantly decreased content of VP was detected following the administration of 10(-6)-10(-9) M doses of GAL. Dopamine (DA) and the DA-active drugs apomorphine (APM) and Pro-Lys-Gly (PLG) (10(-6) M in each medium) increased the VP level of NH tissue cultures. This VP concentration elevation could be blocked by the administration of GAL together with DA, APM or PLG. The DA-blocking effect of GAL was prevented by previous treatment with the GAL receptor antagonist galantid (M15). The results indicate that VP release is directly influenced by the GAL-ergic system. The GAL-ergic control of VP secretion from NH tissue in rats can occur independently of the hypothalamus, at the level of the posterior pituitary.

Circadian clock-regulated expression of phytochrome and cryptochrome genes in Arabidopsis.

Many physiological and biochemical processes in plants exhibit endogenous rhythms with a period of about 24 h. Endogenous oscillators called circadian clocks regulate these rhythms. The circadian clocks are synchronized to the periodic environmental changes (e.g. day/night cycles) by specific stimuli; among these, the most important is the light. Photoreceptors, phytochromes, and cryptochromes are involved in setting the clock by transducing the light signal to the central oscillator. In this work, we analyzed the spatial, temporal, and long-term light-regulated expression patterns of the Arabidopsis phytochrome (PHYA to PHYE) and cryptochrome (CRY1 and CRY2) promoters fused to the luciferase (LUC(+)) reporter gene. The results revealed new details of the tissue-specific expression and light regulation of the PHYC and CRY1 and 2 promoters. More importantly, the data obtained demonstrate that the activities of the promoter::LUC(+) constructs, with the exception of PHYC::LUC(+), display circadian oscillations under constant conditions. In addition, it is shown by measuring the mRNA abundance of PHY and CRY genes under constant light conditions that the circadian control is also maintained at the level of mRNA accumulation. These observations indicate that the plant circadian clock controls the expression of these photoreceptors, revealing the formation of a new regulatory loop that could modulate gating and resetting of the circadian clock.

Conditional circadian regulation of PHYTOCHROME A gene expression.

The phytochrome photoreceptors and the circadian clock control many of the same developmental processes, in all organs and throughout the growth of Arabidopsis plants. Phytochrome A (phyA) provides light input signals to entrain the circadian clock. The clock is known to rhythmically regulate its light input pathway, so we tested rhythmic regulation of phyA, using transgenic plants carrying a PHYA promoter fusion to the luciferase reporter (PHYA:LUC). We provide the first images of LUC activity with subcellular resolution in intact tissue. PHYA transcription and the accumulation of all three PHYA mRNAs were indeed clock controlled. PHYA is expressed throughout the seedling, so we tested whether circadian rhythms were observed in all PHYA-expressing organs and whether the rhythms were autonomously controlled by each organ. In contrast to our previous results using other clock controlled genes, the rhythmic pattern of PHYA expression varied markedly among isolated organs and between isolated organs and intact plants. High-amplitude rhythms were maintained for many days in isolated leaves in darkness, whereas the leaves of intact plants rapidly lost rhythmicity. Wounding the leaves of intact plants had no effect. The rhythmic pattern of PHYA expression is not organ autonomous but depends upon the physical continuity or isolation of the rhythmic tissues, consistent with the presence of a transmitted signal that controls the overt expression of circadian rhythms without necessarily affecting the underlying clock. A circadian system might be present in most, if not all, plant cells, but its effect on intracellular rhythms can be controlled by supracellular signaling.

Activation-induced apoptosis and cell surface expression of Fas (CD95) ligand are reciprocally regulated by retinoic acid receptor alpha and gamma and involve nur77 in T cells.

It has been previously shown that CD4+ T cells enter the apoptotic suicide program via the Fas ligand (FasL)/Fas-mediated pathway upon T cell receptor (TCR) stimulation. In Jurkat cells TCR stimulation regulates the de novo synthesis of FasL, while in the influenza hemagglutinin-specific CD4+ murine T cell hybridoma (IP-12-7) the cell surface appearance of a preformed FasL is initiated. Both processes are dependent on new mRNA and protein synthesis, involve up-regulation of nur77, and can be inhibited by retinoic acids (RA). Two groups of nuclear receptors for RA have been identified: retinoic acid receptors (RAR) and retinoid X receptors (RXR). In this study various synthetic retinoids were used to define which receptors regulate TCR-mediated apoptosis. It is demonstrated that the inhibition is mediated via RARalpha, while RARgamma enhances TCR-mediated apoptosis, and when both receptors are stimulated, the costimulation by RXR will promote the effect of RARalpha. Evidence is presented that these receptors affect the transcriptional activity of nur77 and consequently the expression of FasL. Our data suggest a complex interaction between the various isoforms of retinoid receptors in regulating T cell death and demonstrate that the target through which retinoids regulate TCR-mediated apoptosis is nur77.

A novel strategy for the expression of foreign genes from plant virus vectors.

Potato virus X (PVX)-based vector constructs were generated to investigate the use of an internal ribosome entry site (IRES) sequence to direct translation of a viral gene. The 148-nucleotide IREScp sequence from a crucifer-infecting strain of tobacco mosaic virus was used to direct expression of the PVX coat protein (CP). The IRES was inserted downstream of the gene encoding green fluorescent protein (GFP) and upstream of the PVX CP, in either sense or antisense orientation, such that CP expression depended on ribosome recruitment to the IRES. Stem-loop structures were inserted at either the 3'- or 5'-end of the IRES sequence to investigate its mode of action. In vitro RNA transcripts were inoculated to Nicotiana benthamiana plants and protoplasts: levels of GFP and CP expression were analysed by enzyme-linked immunosorbent assay and the rate of virus cell-to-cell movement was determined by confocal laser scanning microscope imaging of GFP expression. PVX CP was expressed, allowing cell-to-cell movement of virus, from constructs containing the IRES sequence in either orientation, and from the construct containing a stem-loop structure at the 5'-end of the IRES sequence. No CP was expressed from a construct containing a stem-loop at the 3'-end of the IRES sequence. Our results suggest that the IRES sequence is acting in vivo to direct expression of the 3'-proximal open reading frame in a bicistronic mRNA thereby demonstrating the potential of employing IRES sequences for the expression of foreign proteins from plant virus-based vectors.

Effects of dopamine and dopamine-active compounds on oxytocin and vasopressin production in rat neurohypophyseal tissue cultures.

The effects of dopamine (DA) or DA-active drugs on the synthesis of neurohypophyseal (NH) hormones were studied in 13-14 day cultures of isolated NH tissue from rats. The following DA-active compounds were used (10(-6) M in each medium): DA, apomorphine (APM), Pro-Lys-Gly (PLG), butaclamol (B), haloperidol (HP), chlorpromazine (CPZ) and sulpiride (SP). The oxytocin (OT) and vasopressin (VP) contents of the condensed media were determined by RIA after a 1 or 2 h incubation. Significantly increased contents of OT and VP were detected in the tissue culture media following DA, APM or PLG administration. This elevation of NH hormone production could be blocked by previous administration of B or the DA receptor antagonists HP, CPZ or SP. The application of B after DA agonists proved ineffective. The results indicate that NH hormone production can be directly influenced by the DA-ergic system. The DA-ergic control of NH hormone secretion in rats can occur independently of the hypothalamus, at the level of the posterior pituitary.

In situ derivatisation using pressurized liquid extraction to determine phenols, sterols and carboxylic acids in environmental samples and microbial biomasses.

Pressurized liquid extraction was combined with in-situ derivatisation to extract polar analytes such as phenols (including chlorophenols) sterols and carboxylic acids from environmental and microbial samples. This one-step protocol uses acetic anhydride as an acetylation agent, N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) as an silylation agent, and boron trifluoride-methanol, phenyltrimethyl ammoniumhydroxide and trimethyl sulfoniumhydroxide as methylation agents. It results in faster extraction rates and better or comparable extraction efficiencies when compared to classical approaches. The addition of a silylation agent also facilitates the extraction kinetics of analytes not accessible to silylation (e.g. polycyclic aromatic hydrocarbons or alkylbenzenes). This may be attributed to a dissociative action of the agent to weaken analyte-matrix interactions.

[Suckling-induced changes in oxytocin and alpha-melanocyte-stimulating hormone contents of the median eminence and various lobes of the pituitary gland]. / Az alpha-melanocyta-stimuláló hormon és oxytocin elválasztásának és lokális transzportjának vizsgálata szopási inger hatására a hypophysis különbözó régióiban és az eminencia medianaban.

Previous reports have implicated that pituitary-derived prolactin (PRL) is secreted from two distinct zones of mammotropes within the anterior lobe (AL). The inner zone (AL-IZ), located adjacent to the NIL, is supposed to be involved in the rapid and massive discharge of PRL from the pituitary gland due to suckling stimulus. Anatomically, the AL-IZ has an intimate contact with the NIL because the blood arriving from the posterior pituitary through the short portal vessels (SPV) baths it first. Based on these facts it would be hypothesized that the locally released and/or produced important compounds, like oxytocin (OXT) and alpha-melanocyte-stimulating hormone (alpha-MSH), can be delivered to the AL-IZ. Therefore, the purpose of this study was to examine the possible local transportation of these hormones into various regions of the pituitary gland (adenohypophysis inner-zone (AL-IZ), outer zone (AL-OZ), intermediate lobe (IL), neural lobe (NL)) and median eminence of lactating rats. We have measured the concentration of OXT and alpha-MSH from tissue samples of nonsuckled and suckled rats using specific RIA-s. There were no changes in the concentration of OXY and alpha-MSH in the AL-IZ and AL-OZ due to suckling stimulus. In contrast, our data provide compelling evidence that OXT is transported into the IL, which can be further increased by suckling stimulus. Our data have shown a lack of local delivery of either alpha-MSH or OXY into the AL that raises serious doubt about their possible role in PRL secretion during suckling stimulus.

Cell death in HIV pathogenesis and its modulation by retinoids.

Patients infected with the human immunodeficiency virus exhibit a progressive decline in the CD4 T-cell number, resulting in immunodeficiency and increased susceptibility to opportunistic infections and malignancies. Although CD4 T cell production is impaired in patients infected with HIV, there is now increasing evidence that the primary basis of T cell depletion is accelerated apoptosis of CD4 and CD8 T cells. The rate of lymphocyte apoptosis in HIV infection correlates inversely with the progression of the disease: it is low in long-term progressors and in patients undergoing highly active antiretroviral therapy. Interestingly, only a minor fraction of apoptotic lymphocytes are infected by HIV, indicating that the enhanced apoptosis does not necessarily always serve to remove the HIV+ cells and results from mechanisms other than direct infection. Thus, understanding and influencing the mechanisms of HIV-associated lymphocyte apoptosis may lead to new therapies for HIV disease. In this paper the potential effects of retinoids on CD4 T cell apoptosis is discussed.

Suckling-induced change in oxytocin but not in alpha-MSH concentrations of the median eminence, the neural-, intermediate- and anterior lobes of the pituitary gland.

Previous reports have implicated that pituitary-derived prolactin (PRL) is secreted from two distinct zones of mammotropes within the anterior lobe (AL). The inner zone (AL-IZ), located adjacent to the neuro-intermediate lobe (NIL), is supposed to be involved in the rapid and massive discharge of PRL from the pituitary gland due to suckling stimulus. Whereas the outer-zone (AL-OZ) gives the basal secretion and it does not play a role in the acute secretory response during nursing. Anatomically, the AL-IZ has an intimate contact with the NIL because the blood passing through the short portal vessels (SPV) bathes it first. Based on this fact it would be hypothesized that locally released and/or produced compounds, like OXY and alpha-MSH, can be delivered to the AL-IZ. In conjunction, OXY and alpha-MSH have already been implicated to play a role in the regulation of PRL release during suckling. Therefore, the purpose of this study was to examine the possible local transportation of these hormones into the median eminence and various regions of the pituitary gland of lactating rats. We have measured the concentrations of OXY and alpha-MSH from tissue samples of nonsuckled (NS) and 10 or 30 min after suckling (S) was initiated using specific RIAs. It has been shown that there are no changes in the concentration of OXY and alpha-MSH in theAL-IZ and AL-OZ due to suckling stimulus. In contrast, our data provide compelling evidence that OXY is transported into the IL, which can be further increased by suckling stimulus. These data suggest that blood transfusing NL passes through the IL before it is drained into the cavernous sinus, which opens the road for OXY into the general circulation. In addition, our data have unequivocally shown a lack of local delivery of either alpha MSH or OXY into the AL that raises serious doubt about their possible role in PRL secretion during suckling stimulus.

Light-induced nuclear import of phytochrome-A:GFP fusion proteins is differentially regulated in transgenic tobacco and Arabidopsis.

Phytochromes (phy) are a family of photoreceptors that control various aspects of light-dependent plant development. Phytochrome A (phyA) is responsible for the very low fluence response (VLFR) under inductive light conditions and for the high irradiance response (HIR) under continuous far-red light. We have recently shown that nuclear import of rice phyA:GFP is regulated by VLFR in transgenic tobacco. The import is preceded by very fast, light-induced formation of sequestered areas of phyA:GFP in the cytosol. Here we report that expression of the Arabidopsis phyA:GFP fusion protein in phyA-deficient Arabidopsis plants complements the mutant phenotype. In these transgenic Arabidopsis lines, both light-dependent cytosolic formation of sequestered areas of the phyA:GFP as well as VLFR or HIR-mediated nuclear import of the fusion protein was observed. By contrast, light-dependent nuclear import of the same fusion protein was induced only by continuous far-red light (HIR) but not by pulses of far-red light (VLFR) in transgenic tobacco. These results demonstrate that photoregulation of intracellular partitioning of the Arabidopsis phyA:GFP differs significantly in different genetic backgrounds.