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This paper presents the influence of varieties and different doses of applied nitrogen on grain yield and protein content of triticale. The experiment was set in the period from 2010 to 2012 in the North of Montenegro, in the vicinity of Bijelo Polje. The research included 5 varieties of winter triticale (Odyssey, Kg-20, Triumph, Rtanj and Tango) originating from different breeding houses and the following varieties of fertilization: control (without fertilization), only nitrogen in the amount of 60 kg ha-1 and nitrogen in the amount of 60 and 90 kg ha-1 in combination with the same amount of phosphorus and potassium (80 kg ha-1). The results of the study showed that the lowest average grain yield was obtained in the non-fertilizing variant - control. The use of fertilizers in all tested varieties has led to a very significant increase in yield in all variants compared to control. The Kg20 variety had the lowest average yield, and the Tango variety had the highest. The highest average protein content was achieved in the fertilizer variant where only nitrogen was used in the amount of 60 kg ha-1. Among the researched varieties, the Triumph variety had the highest protein content in the grains. The data on the achieved yields and the content of protein in grains, depending on the variety and the used doses of fertilizer, indicate the characteristics of individual varieties and can serve as a criterion for the selection of the most suitable variety for certain agroecological conditions. This is particularly important for cattle-oriented farms, where the main priority is to ensure a sufficient amount of quality food.

The photoselective, light-dispersive shade nets can be used as an alternative to protect crops from adverse environmental conditions such as; excessive solar radiation, heat and drought stress, wind and hail, birds, flying pests, thus improving crop’s production, yield and quality. The physiological parameters discussed in the review include: vegetable growth parameters (leaf area, leaf chlorophyll), tissue structure, fruit ripening, physiological disorders, pest and disease incidence, fruit quality parameters (soluble solids content and titratable acidity), bioactive compounds (antioxidant activity, ascorbic acid, carotenoid and flavonoid contents) and aroma volatile compounds at harvest. Also, it is evident in the reviewed literature that light quality influences the biosynthesis, accumulation and retention of vegetable phytochemicals, as well as the decay development during storage. These new strategies to modulate light quality should be conveyed to vegetable producing farmers, thus allowing them to preserve the freshness and post-harvest quality of vegetables for an extended period of time, and to meet the consumers demand for vegetables with high nutritional value all year round. Research on light manipulation in horticultural systems is necessary for a sustainable and market-oriented open field and greenhouse vegetable production in the future.

The experiments took into account four cultivars origin from different agroclimacteric regions, usually grown in our country (Prizrenski pogačar, Kupusinski jabučar, Trebinjska kapula and Holandski žuti), are The sowings were realized on 15 July (first term), 5 August (second) and 25th August (third). Sowings were carried out in rows 45cm apart and 5cm apart along the row (number of plants 44,8/m2). Experimen was split-plot designed with three replicaties. Gibberellic acid-GA3 sprays were applied with concentration at 50ppm on plants at the 6-7 leaves stage. Yield and its principal components were analyzed (percentage of flowering, capsules/umbel, seed/capsule, 1000 seed weight, germination energy, germination). Flowering percentage in the two years was affected by sowing date. Sowing postponement from July to August caused a reduction in the flowered plant from 90% to 40%, which produced only one umbel per plant. Sowing-flowering-seed ripening was reached almost simultaneously after 405-370 days. Seed production was highest in the first term sowing from 15 July for all cultivars. The cultivars showed differences in both years. Cultivar Kupusinski jabučar obtained the highest yield in the first term sowing - 440,4 kg/ha (in first year), while Holandski žuti obtained significantly lower in the third term from 25 August with only 140,6kg/ha (in first year).

The primary cilium is an important signaling organelle critical for normal development and tissue homeostasis. Its small dimensions and complexity necessitate advanced imaging approaches to uncover the molecular mechanisms behind its function. Here, we outline how single-molecule fluorescence microscopy can be used for tracking molecular dynamics and interactions and for super-resolution imaging of nanoscale structures in the primary cilium. Specifically, we describe in detail how to capture and quantify the 2D dynamics of individual transmembrane proteins PTCH1 and SMO and how to map the 3D nanoscale distributions of the inversin compartment proteins INVS, ANKS6, and NPHP3. This protocol can, with minor modifications, be adapted for studies of other proteins and cell lines to further elucidate the structure and function of the primary cilium at the molecular level., (Copyright © 2023 Elsevier Inc. All rights reserved.)

DNA replication is normally coupled with centriole duplication in the cell cycle. Trophoblast giant cells (TGCs) of the placenta undergo endocycles resulting in polyploidy but their centriole state is not known. We used a cell culture model for TGC differentiation to examine centriole and centrosome number and properties. Before differentiation, trophoblast stem cells (TSCs) have either two centrioles before duplication or four centrioles after. We find that the average nuclear area increases approximately eight-fold over differentiation, but most TGCs do not have more than four centrioles. However, these centrioles become disengaged, acquire centrosome proteins, and can nucleate microtubules. In addition, some TGCs undergo further duplication and disengagement of centrioles, resulting in substantially higher numbers. Live imaging revealed that disengagement and separation are centriole autonomous and can occur asynchronously. Centriole amplification, when present, occurs by the standard mechanism of one centriole generating one procentriole. PLK4 inhibition blocks centriole formation in differentiating TGCs but does not affect endocycle progression. In summary, centrioles in TGC endocycles undergo disengagement and conversion to centrosomes. This increases centrosome number but to a limited extent compared with DNA reduplication.

The Hedgehog-signaling pathway is an important target in cancer research and regenerative medicine; yet, on the cellular level, many steps are still poorly understood. Extensive studies of the bulk behavior of the key proteins in the pathway established that during signal transduction they dynamically localize in primary cilia, antenna-like solitary organelles present on most cells. The secreted Hedgehog ligand Sonic Hedgehog (SHH) binds to its receptor Patched1 (PTCH1) in primary cilia, causing its inactivation and delocalization from cilia. At the same time, the transmembrane protein Smoothened (SMO) is released of its inhibition by PTCH1 and accumulates in cilia. We used advanced, single molecule-based microscopy to investigate these processes in live cells. As previously observed for SMO, PTCH1 molecules in cilia predominantly move by diffusion and less frequently by directional transport, and spend a fraction of time confined. After treatment with SHH we observed two major changes in the motional dynamics of PTCH1 in cilia. First, PTCH1 molecules spend more time as confined, and less time freely diffusing. This result could be mimicked by a depletion of cholesterol from cells. Second, after treatment with SHH, but not after cholesterol depletion, the molecules that remain in the diffusive state showed a significant increase in the diffusion coefficient. Therefore, PTCH1 inactivation by SHH changes the diffusive motion of PTCH1, possibly by modifying the membrane microenvironment in which PTCH1 resides., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Super-resolution (SR) microscopy has been used to observe structural details beyond the diffraction limit of ∼250 nm in a variety of biological and materials systems. By combining this imaging technique with both computer-vision algorithms and topological methods, we reveal and quantify the nanoscale morphology of the primary cilium, a tiny tubular cellular structure (∼2-6 μm long and 200-300 nm in diameter). The cilium in mammalian cells protrudes out of the plasma membrane and is important in many signaling processes related to cellular differentiation and disease. After tagging individual ciliary transmembrane proteins, specifically Smoothened, with single fluorescent labels in fixed cells, we use three-dimensional (3D) single-molecule SR microscopy to determine their positions with a precision of 10-25 nm. We gain a dense, pointillistic reconstruction of the surfaces of many cilia, revealing large heterogeneity in membrane shape. A Poisson surface reconstruction algorithm generates a fine surface mesh, allowing us to characterize the presence of deformations by quantifying the surface curvature. Upon impairment of intracellular cargo transport machinery by genetic knockout or small-molecule treatment of cells, our quantitative curvature analysis shows significant morphological differences not visible by conventional fluorescence microscopy techniques. Furthermore, using a complementary SR technique, two-color, two-dimensional stimulated emission depletion microscopy, we find that the cytoskeleton in the cilium, the axoneme, also exhibits abnormal morphology in the mutant cells, similar to our 3D results on the Smoothened-measured ciliary surface. Our work combines 3D SR microscopy and computational tools to quantitatively characterize morphological changes of the primary cilium under different treatments and uses stimulated emission depletion to discover correlated changes in the underlying structure. This approach can be useful for studying other biological or nanoscale structures of interest., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

There are many opened questions about the precocious role of oxidative stress in the physiopathology of the early stage of transitory ischemic attack (TIA) and defined focal brain ischemia, as well as about its correlation with clinical severity, short-lasting and clinical outcome prediction in these conditions. The study evaluates the values of glutathione (GSH), glutathione peroxidase, and superoxide dismutase (SOD) in hemolysates and total thiol content (-SH), advanced oxidation protein products (AOPP), SOD, and malondialdehyde (MDA) in plasma, in TIA and stroke patients in the early stage of their neurological onset. The results are interpreted in view of the potential relationship between tested parameters and clinical severity and clinical outcome prediction. Better hemolysates' and total antioxidant profile with higher values of AOPP were observed in TIA compared to stroke patients (p < 0.05). The stroke patients with initially better clinical presentation showed better antioxidant profile with lower values of AOPP (p < 0.05). In TIA patients, this was observed for GSH, -SH content, and AOPP (p < 0.05), which correlated with a short risk for stroke occurrence in this group (p < 0.01). Beyond MDA values, all tested parameters showed correlation with clinical outcome in stroke patients (p < 0.05). The measurement of oxidative stress in TIA and stroke patients would be important for identifying patients' subgroups which might receive supporting therapy providing better neurological recovery and clinical outcome. That approach might give us an additional view of a short-lasting risk of stroke occurrence after TIA, and its clinical outcome and prognosis.

Alterations in Hedgehog (Hh) signaling lead to birth defects and cancers including medulloblastoma, the most common pediatric brain tumor. Although inhibitors targeting the membrane protein Smoothened suppress Hh signaling, acquired drug resistance and tumor relapse call for additional therapeutic targets. Here we show that phosphodiesterase 4D (PDE4D) acts downstream of Neuropilins to control Hh transduction and medulloblastoma growth. PDE4D interacts directly with Neuropilins, positive regulators of Hh pathway. The Neuropilin ligand Semaphorin3 enhances this interaction, promoting PDE4D translocation to the plasma membrane and cAMP degradation. The consequent inhibition of protein kinase A (PKA) enhances Hh transduction. In the developing cerebellum, genetic removal of Neuropilins reduces Hh signaling activity and suppresses proliferation of granule neuron precursors. In mouse medulloblastoma allografts, PDE4D inhibitors suppress Hh transduction and inhibit tumor growth. Our findings reveal a new regulatory mechanism of Hh transduction, and highlight PDE4D as a promising target to treat Hh-related tumors.

Accumulation of the signaling protein Smoothened (Smo) in the membrane of primary cilia is an essential step in Hedgehog (Hh) signal transduction, yet the molecular mechanisms of Smo movement and localization are poorly understood. Using ultrasensitive single-molecule tracking with high spatial/temporal precision (30 nm/10 ms), we discovered that binding events disrupt the primarily diffusive movement of Smo in cilia at an array of sites near the base. The affinity of Smo for these binding sites was modulated by the Hh pathway activation state. Activation, by either a ligand or genetic loss of the negatively acting Hh receptor Patched-1 (Ptch), reduced the affinity and frequency of Smo binding at the base. Our findings quantify activation-dependent changes in Smo dynamics in cilia and highlight a previously unknown step in Hh pathway activation.

DNA assembly techniques have developed rapidly, enabling efficient construction of complex constructs that would be prohibitively difficult using traditional restriction-digest based methods. Most of the recent methods for assembling multiple DNA fragments in vitro suffer from high costs, complex set-ups, and diminishing efficiency when used for more than a few DNA segments. Here we present a cycled ligation-based DNA assembly protocol that is simple, cheap, efficient, and powerful. The method employs a thermostable ligase and short Scaffold Oligonucleotide Connectors (SOCs) that are homologous to the ends and beginnings of two adjacent DNA sequences. These SOCs direct an exponential increase in the amount of correctly assembled product during a reaction that cycles between denaturing and annealing/ligating temperatures. Products of early cycles serve as templates for later cycles, allowing the assembly of many sequences in a single reaction. To demonstrate the method's utility, we directed the assembly of twelve inserts, in one reaction, into a transformable plasmid. All the joints were precise, and assembly was scarless in the sense that no nucleotides were added or missing at junctions. Simple, efficient, and low-cost cycled ligation assemblies will facilitate wider use of complex genetic constructs in biomedical research.

The Hedgehog (Hh) signaling pathway has been implicated in the most common childhood brain tumor, medulloblastoma (MB). Given the toxicity of post-surgical treatments for MB, continued need exists for new, targeted therapies. Based upon our finding that Neuropilin (Nrp) transmembrane proteins are required for Hh signal transduction, we investigated the role of Nrp in MB cells. Cultured cells derived from a mouse Ptch (+/-) ;LacZ MB (Med1-MB), effectively modeled the Hh pathway-related subcategory of human MBs in vitro. Med1-MB cells maintained constitutively active Hh target gene transcription, and consistently formed tumors within one month after injection into mouse cerebella. The proliferation rate of Med1-MBs in culture was dependent upon Nrp2, while reducing Nrp1 function had little effect. Knockdown of Nrp2 prior to cell implantation significantly increased mouse survival, compared to transfection with a non-targeting siRNA. Knocking down Nrp2 specifically in MB cells avoided any direct effect on tumor vascularization. Nrp2 should be further investigated as a potential target for adjuvant therapy in patients with MB.

The Hedgehog (Hh) pathway is essential for vertebrate embryogenesis, and excessive Hh target gene activation can cause cancer in humans. Here we show that Neuropilin 1 (Nrp1) and Nrp2, transmembrane proteins with roles in axon guidance and vascular endothelial growth factor (VEGF) signaling, are important positive regulators of Hh signal transduction. Nrps are expressed at times and locations of active Hh signal transduction during mouse development. Using cell lines lacking key Hh pathway components, we show that Nrps mediate Hh transduction between activated Smoothened (Smo) protein and the negative regulator Suppressor of Fused (SuFu). Nrp1 transcription is induced by Hh signaling, and Nrp1 overexpression increases maximal Hh target gene activation, indicating the existence of a positive feedback circuit. The regulation of Hh signal transduction by Nrps is conserved between mammals and bony fish, as we show that morpholinos targeting the Nrp zebrafish ortholog nrp1a produce a specific and highly penetrant Hh pathway loss-of-function phenotype. These findings enhance our knowledge of Hh pathway regulation and provide evidence for a conserved nexus between Nrps and this important developmental signaling system.

In animal cells, the primary cilium transduces extracellular signals through signaling receptors localized in the ciliary membrane, but how these ciliary membrane proteins are retained in the cilium is unknown. We found that ciliary membrane proteins were highly mobile, but their diffusion was impeded at the base of the cilium by a diffusion barrier. Septin 2 (SEPT2), a member of the septin family of guanosine triphosphatases that form a diffusion barrier in budding yeast, localized at the base of the ciliary membrane. SEPT2 depletion resulted in loss of ciliary membrane protein localization and Sonic hedgehog signal transduction, and inhibited ciliogenesis. Thus, SEPT2 is part of a diffusion barrier at the base of the ciliary membrane and is essential for retaining receptor-signaling pathways in the primary cilium.

Compartmentalization within cells provides spatial organization of signaling pathways and ensures the specificity of signaling. In vertebrates, the primary cilium, a tiny microtubule-based protrusion present on most cells, is essential for organizing events during Hedgehog signal transduction. When cells are stimulated with Hedgehog ligands, proteins in the pathway move in and out of the cilia. Protein kinase A (PKA), which is implicated in diverse cellular processes including protein trafficking, is a component of the Hedgehog signaling pathway. PKA has been localized near primary cilia, at a location suitable for regulating the localization of other proteins in the pathway.

The transcriptional program orchestrated by Hedgehog signaling depends on the Gli family of transcription factors. Gli proteins can be converted to either transcriptional activators or truncated transcriptional repressors. We show that the interaction between Gli3 and Suppressor of Fused (Sufu) regulates the formation of either repressor or activator forms of Gli3. In the absence of signaling, Sufu restrains Gli3 in the cytoplasm, promoting its processing into a repressor. Initiation of signaling triggers the dissociation of Sufu from Gli3. This event prevents formation of the repressor and instead allows Gli3 to enter the nucleus, where it is converted into a labile, differentially phosphorylated transcriptional activator. This key dissociation event depends on Kif3a, a kinesin motor required for the function of primary cilia. We propose that the Sufu-Gli3 interaction is a major control point in the Hedgehog pathway, a pathway that plays important roles in both development and cancer.

The function of primary cilia depends critically on the localization of specific proteins in the ciliary membrane. A major challenge in the field is to understand protein trafficking to cilia. The Hedgehog (Hh) pathway protein Smoothened (Smo), a 7-pass transmembrane protein, moves to cilia when a ligand is received. Using microscopy-based pulse-chase analysis, we find that Smo moves through a lateral transport pathway from the plasma membrane to the ciliary membrane. Lateral movement, either via diffusion or active transport, is quite distinct from currently studied pathways of ciliary protein transport in mammals, which emphasize directed trafficking of Golgi-derived vesicles to the base of the cilium. We anticipate that this alternative route will be used by other signaling proteins that function at cilia. The path taken by Smo may allow novel strategies for modulation of Hh signaling in cancer and regeneration.

The Hedgehog (Hh) signaling pathway controls growth, cell fate decisions, and morphogenesis during development. Damage to Hh transduction machinery can lead to birth defects and cancer. The transmembrane protein Smoothened (Smo) relays the Hh signal and is an important drug target in cancer. Smo enrichment in primary cilia is thought to drive activation of target genes. Using small-molecule agonists and antagonists to dissect Smo function, we find that Smo enrichment in cilia is not sufficient for signaling and a distinct second step is required for full activation. This 2-step mechanism--localization followed by activation--has direct implications for the design and use of anticancer therapeutics targeted against Smo.

Primary cilia are essential for transduction of the Hedgehog (Hh) signal in mammals. We investigated the role of primary cilia in regulation of Patched1 (Ptc1), the receptor for Sonic Hedgehog (Shh). Ptc1 localized to cilia and inhibited Smoothened (Smo) by preventing its accumulation within cilia. When Shh bound to Ptc1, Ptc1 left the cilia, leading to accumulation of Smo and activation of signaling. Thus, primary cilia sense Shh and transduce signals that play critical roles in development, carcinogenesis, and stem cell function.

Niemann-Pick type C is a neurodegenerative lysosomal storage disorder caused by mutations in either of two genes, npc1 and npc2. Cells lacking Npc1, which is a transmembrane protein related to the Hedgehog receptor Patched, or Npc2, which is a secreted cholesterol-binding protein, have aberrant organelle trafficking and accumulate large quantities of cholesterol and other lipids. Though the Npc proteins are produced by all cells, cerebellar Purkinje neurons are especially sensitive to loss of Npc function. Since Niemann-Pick type C disease involves circulating molecules such as sterols and steroids and a robust inflammatory response within the brain parenchyma, it is crucial to determine whether external factors affect the survival of Purkinje cells (PCs). We investigated the basis of neurodegeneration in chimeric mice that have functional npc1 in only some cells. Death of mutant npc1 cells was not prevented by neighboring wild-type cells, and wild-type PCs were not poisoned by surrounding mutant npc1 cells. PCs undergoing cell-autonomous degeneration have features consistent with autophagic cell death. Chimeric mice exhibited a remarkable delay and reduction of wasting and ataxia despite their substantial amount of mutant tissue and dying cells, revealing a robust mechanism that partially compensates for massive PC death., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

The preimplantation development of the mammalian embryo encompasses a series of critical events: the transition from oocyte to embryo, the first cell divisions, the establishment of cellular contacts, the first lineage differentiation-all the first subtle steps toward a future body plan. Here, we use microarrays to explore gene activity during preimplantation development. We reveal robust and dynamic patterns of stage-specific gene activity that fall into two major phases, one up to the 2-cell stage (oocyte-to-embryo transition) and one after the 4-cell stage (cellular differentiation). The mouse oocyte and early embryo express components of multiple signaling pathways including those downstream of Wnt, BMP, and Notch, indicating that conserved regulators of cell fate and pattern formation are likely to function at the earliest embryonic stages. Overall, these data provide a detailed temporal profile of gene expression that reveals the richness of signaling processes in early mammalian development.

Sonic hedgehog (Shh) directs the development of ventral cell fates, including floor plate and V3 interneurons, in the mouse neural tube. Here, we show that the transcription factors Gli2 and Gli3, mediators of Shh signaling, are required for the development of the ventral cell fates but make distinct contributions to controlling cell fates at different locations along the rostral-caudal axis. Mutants lacking Patched1 (Ptc1), the putative receptor of Shh, were used to analyze Gli functions. Ptc1(-/-) mutants develop floor plate, motor neuron, and V3 interneuron progenitors in lateral and dorsal regions, suggesting that the normal role of Ptc1 is to suppress ventral cell development in dorsal neural tube. The Ptc1(-/-) phenotype is rescued, with restoration of dorsal cell types, by the lack of Gli2, but only in the caudal neural tube. In triple mutants of Gli2, Gli3, and Ptc1, dorsal and lateral cell fates are restored in the entire neural tube. These observations suggest that Gli2 is essential for ventral specification in the caudal neural tube, and that in more rostral regions, only Gli3 can promote development of ventral cells if Gli2 is absent. Thus, Shh signaling is mediated by overlapping but distinct functions of Gli2 and Gli3, and their relative contributions vary along the rostral-caudal axis.

Mutations in mouse and human patched1 (ptc1) genes are associated with birth defects and cancer. Ptc1 is a receptor for Hedgehog (Hh) signaling proteins. Hh proteins activate transcription of target genes, including ptc1, and Ptc1 represses those genes, both by regulating the activity of Gli transcription factors. We have established mammalian cell lines with reduced Ptc1 function and a lacZ reporter to investigate Hh signal transduction. Embryonic fibroblasts were derived from mice, heterozygous or homozygous for a ptc1 mutation that inserts lacZ under the control of the ptc1 promoter (ptc1-lacZ). In heterozygous ptc1 cells, ptc1-lacZ was expressed at low levels but could be induced by Sonic Hedgehog (Shh) and Gli-1. Homozygous ptc1 cells expressed high levels of ptc1-lacZ without Hh stimulation. ptc1-lacZ expression was dependent on cell density in ptc1 homozygotes and Hh-stimulated heterozygotes but was independent of density when Gli1 was used to activate ptc1-lacZ. A wild-type ptc1 transgene introduced into homozygous ptc1 cells greatly reduced ptc1-lacZ expression. Expression of either half of Ptc1 alone resulted in improper maturation of the protein and a failure to complement the ptc1(-/-) cells. When co-expressed, both Ptc1 halves matured and had an activity similar to that of the intact protein. Three missense PTCH1 mutations exhibited significant functions in homozygous ptc1 cells. The missense mutants retained activity when expressed at about 10-fold lower levels and appeared as stable as wild-type Ptc1. These studies suggest that some tumors and disease phenotypes may arise from small reductions in PTCH1 activity.

Basal cell carcinoma, medulloblastoma, rhabdomyosarcoma and other human tumours are associated with mutations that activate the proto-oncogene Smoothened (SMO) or that inactivate the tumour suppressor Patched (PTCH). Smoothened and Patched mediate the cellular response to the Hedgehog (Hh) secreted protein signal, and oncogenic mutations affecting these proteins cause excess activity of the Hh response pathway. Here we show that the plant-derived teratogen cyclopamine, which inhibits the Hh response, is a potential 'mechanism-based' therapeutic agent for treatment of these tumours. We show that cyclopamine or synthetic derivatives with improved potency block activation of the Hh response pathway and abnormal cell growth associated with both types of oncogenic mutation. Our results also indicate that cyclopamine may act by influencing the balance between active and inactive forms of Smoothened.

The membrane protein Patched (Ptc) is a key regulator of Hedgehog (Hh) signaling in development and is mutated in human tumors. Ptc opposes Hh-induced gene transcription and sequesters Hh protein. To dissect these functions, we tested partially deleted forms of Ptc in Drosophila. Deletion of either half of Ptc abolishes all function while coexpression of the halves restores nearly full activity. Deletion of the final 156 residues of Ptc permits Hh sequestration but abolishes inhibition of Hh targets. This deletion has dominant-negative activity, promoting target gene activation in a ligand-independent manner. We observe little or no association of full-length or partially deleted Ptc with the membrane protein Smoothened in Drosophila cultured cells.

Thymosin alpha 1 (T alpha 1) is a well-characterized immunopotentiating polypeptide originally isolated from calf thymus. We have recently shown in vivo, probable hypothalamic effects of T alpha 1 to decrease the release of the pituitary hormones, TSH, PRL and ACTH from the pituitary gland. Therefore, in the present study we evaluated the effect of the peptide on the release of hypothalamic regulatory hormones: thyrotropin-releasing hormone (TRH) and corticotropin-releasing hormone (CRH), as well as somatostatin (SRIH), from medial basal hypothalamic (MBH) fragments incubated in vitro. After a preliminary time-course study indicated that a 30-min incubation period was optimal, it was used for all the other experiments. At the end of the incubation the tissue was still able to respond to a depolarizing K+ concentration for 15 min by a 4-fold increase of TRH concentration compared to control basal release during the preceding 30 min. T alpha 1 was shown to inhibit the release of TRH and CRH from MBH fragments incubated in vitro with a minimal effective dose (MED) of 10(-11) M. SRIH and CRH release was also inhibited but the MED for these peptides was 10(-9) M. The relative responsiveness to the action of T alpha 1 was TRH greater than CRH, which was greater than SRIH. This correlated with our previous in vivo results for pituitary hormone release, except in the case of SRIH since we previously did not detect any significant effect of the peptide on growth hormone release. Finally, we evaluated the possible involvement of other neurotransmitters in the effect of T alpha 1 on TRH release.(ABSTRACT TRUNCATED AT 250 WORDS)

The thymosins are a family of hormone-like products of epithelial cells of the thymus which are important in maintenance and function of the immune system. Thymosin fraction 5, a partially purified extract of calf thymus, can influence pituitary hormone release. We have studied the effects of thymosin alpha 1 (T alpha 1), the first peptide isolated from thymosin fraction 5, on thyrotropin (TSH), adrenocorticotropin (ACTH), prolactin (Prl) and growth hormone (GH) release. To evaluate its effect in vivo we injected the peptide into the third ventricle of conscious male rats and measured the concentration of the pituitary hormones in plasma at different times after the injection. Following third-ventricular injection of T alpha 1, there was a significant decrease in plasma TSH and ACTH concentrations in comparison with values of control groups injected with diluent. The decrease in plasma TSH was of longer duration and was obtained with a lower dose of T alpha 1 than that of ACTH. Also, a significant decrease in plasma Prl was observed, with the same dose as for TSH. On the other hand, there were no significant changes in plasma GH. To examine if there is any direct effect of T alpha 1 at the pituitary level, we incubated hemipituitaries from male rats in vitro with different concentrations of the peptide. In this system T alpha 1 evoked a dose-dependent release of TSH and ACTH, while there was no effect on the release of Prl and GH.(ABSTRACT TRUNCATED AT 250 WORDS)

For the majority of leukemic patients, leukapheresis represents emergency treatment aimed at reducing the number of white blood cells and producing an immediate improvement in the clinical picture. We have shown that leukapheresis procedures performed for the therapy of leukocytosis in 4 patients with acute leukemia (2 myelocytic; 1 lymphocytic; 1 monoblastic) resulted in marked reduction in the white blood cell count and a considerable reduction in symptomatology. Repeat removal of white blood cells applied in 20 instances for patients with chronic myelocytic leukemia also produced a significant decrease in the cell count and relief of symptoms such as sweating, malaise, and pain due to splenomegaly. In chronic lymphocytic leukemia (31 patients), intensive and frequent leukapheresis procedures were followed by a marked fall in white blood cell count, regression of splenomegaly/lymphadenopathy and resolution of many symptoms and signs induced by the large number of cells.

The aim of the present study was to evaluate the physiological significance of the rapid, short-loop, negative feedback of prolactin by passive immunization with antiserum to rat prolactin injected into the third cerebral ventricle (3V) of conscious, freely moving intact or castrated male rats. Blood samples for measurement of plasma prolactin concentrations were removed through implanted external jugular catheters. After injection of 3 microliters of undiluted antiserum, plasma levels of prolactin decreased rapidly (within 5 min) to values undetectable by RIA. Further study revealed that this dose of antiprolactin serum had combined with circulating prolactin, thus rendering it undetectable by RIA. To overcome this problem, we repeated the experiment injecting 2 microliters of diluted antiserum (dilution factors 20, 100, 200, 2,000) into the 3V. When compared to values of plasma prolactin in control rats injected with 2 microliters of normal rabbit serum, none of the dilutions of antiserum induced a significant change in prolactin concentrations for as long as 4 h after injection. Since there was no effect of intraventricularly injected antiprolactin serum on basal prolactin secretion, in the next experiment, intact as well as castrated male rats were subjected to ether stress 30 min after intraventricular injection of antiserum (dilution factor 100). The elevation of plasma prolactin which followed ether stress was significantly higher in male rats pretreated with antiprolactin serum than that which occurred in control rats. A similar enhancement of the increase in plasma prolactin following ether stress, but of longer duration, was obtained in castrated rats injected with antiprolactin serum.(ABSTRACT TRUNCATED AT 250 WORDS)

The possible involvement of arachidonic acid (AA) release in growth-hormone-releasing factor (GRF)-induced somatostatin (SRIF) release from the median eminence (ME) of the hypothalamus was evaluated in adult male rats using an in vitro incubation system. The MEs were preincubated with [14C]-AA, then washed and incubated with vehicle or test agents, and the release of SRIF and [14C]-AA into the medium was measured. In the experiments designed only to determine SRIF release, the MEs were first preincubated for 30 min. The medium was then discarded and replaced with fresh buffer or test substances and incubated for 10, 20 and/or 30 min. GRF (10(-10) M) stimulated both AA and SRIF release significantly within 20 min, with maximum release occurring at 30 min. The stimulatory effect of GRF on AA release was coincident with the release of SRIF. A phospholipase A2 inhibitor (10(-6) M, quinacrine) completely abolished the stimulatory effect of GRF on both AA and SRIF release. The release of SRIF induced by GRF was also inhibited by both indomethacin (10(-6) M, a cyclooxygenase inhibitor) and metyrapone (10(-6) M, a cytochrome P-450 inhibitor). On the other hand, nordihydroguaiaretic acid (10(-6) M, a lipoxygenase inhibitor) had no effect on GRF-evoked SRIF release. The data presented here suggest that an important GRF-mediated event leading to SRIF secretion is an elevated release of AA from ME fragments in vitro. In conclusion, our data are suggestive that the stimulatory effect of GRF on SRIF release is due, in part, to the release and subsequent metabolism of AA to one or more metabolites.

Neuropeptide Y (NPY) is a peptide found in a variety of hypothalamic loci which is frequently colocalized with catecholamines. It is also secreted into hypophyseal portal vessels. The injection of NPY into the third ventricle (3V) lowered plasma GH levels in conscious, freely moving male rats. To determine the physiological significance of the hypothalamic inhibitory action of the peptide, highly specific antiserum directed against NPY was injected into the 3V of conscious rats. 3V injection of the antiserum evoked a significant elevation of plasma GH within 2 h on comparison to values in normal rabbit serum-injected, ovariectomized rats. The difference increased and reached a maximum at 6 h after injection. On the other hand, there was no effect of the antiserum in ovariectomized, estrogen, progesterone-blocked rats. Intraventricular injection of the anti-NPY serum also caused a significant elevation of plasma GH within 2 h in normal male rats and the increases above values in normal rat serum-injected control animals became even more significant at 3 and 4 h. To determine the mechanism by which NPY lowers GH after its intraventricular injection, its effect on the release of somatostatin (SRIF) from median eminence fragments incubated in vitro was examined. NPY stimulated SRIF release with a highly significant effect at a concentration of 10(-9) M. Borderline stimulation was observed at doses as low as 10(-11) M. The curve was bell-shaped with a declining release at 10(-8) M and 10(-7) M. The releasing action of NPY was blocked by either the alpha 1-receptor blocker, prazosin (10(-6) M), or the beta-receptor blocker, propranolol (10(-6) M), but was not affected by the alpha 2-receptor blocker, yohimbine (10(-6) M). We conclude that NPY has a physiologically significant inhibitory action within the hypothalamus to suppress GH release in ovariectomized female and intact male rats by stimulation of SRIF release by alpha 1 and beta-adrenergic receptor-mediated mechanisms.

Neuropeptide Y (NPY) is a peptide originally isolated from porcine brain and subsequently shown to be widely distributed in the body of several species, including man. Neuropeptide Y is a circulating peptide; however, blood levels were higher in portal than peripheral blood of anesthesized rats. Earlier studies in ovariectomized and intact male rats have shown that intraventricular injection of NPY inhibits release of growth hormone (GH) and luteinizing hormone (LH) without producing significant modification of plasma follicle stimulating hormone (FSH) and thyrotropin stimulating hormone (TSH). In the male low doses of NPY elevate prolactin (PRL) whereas high doses suppress its release. To assess the physiologic significance of these actions, we injected a highly specific anti-NPY serum (aNPY) into the third cerebral ventricle (3V) of unrestrained male, ovariectomized, and ovariectomized, estrogen progesterone blocked rats and measured plasma GH, PRL, LH and TSH by blood sampling via indwelling jugular catheters. Third ventricular injection of aNPY (2 microliters of 1:10 dilution) caused a significant elevation of plasma GH levels after 3 and 4 h compared to the values in NRS (1:10)-injected rats. To determine if these changes were due to alterations in pituitary responsiveness to somatostatin, the rats were injected intravenously with a challenge dose of somatostatin (0.5 microgram) 2 h after previous injection of aNPY or NRS, and blood samples were taken every 10 min for 30 min. The responses did not differ in both groups which indicated that the antiserum was not acting directly on the pituitary gland.(ABSTRACT TRUNCATED AT 250 WORDS)