The effect of auxin (indole-3-acetic acid) on the growth rate and tropism of the sporangiophore of <Emphasis Type="Italic">Phycomyces blakesleeanus</Emphasis> and identification of auxin-related genes

The roles of fungal auxins in the regulation of elongation growth, photo-, and gravitropism are completely unknown. We analyzed the effects of exogenous IAA (indole-3-acetic acid), various synthetic auxins including 1-NAA (1-naphthaleneacetic acid) and 2,4-D (2,4-dichlorophenoxyacetic acid), and the auxin transport inhibitor NPA (N-1-naphtylphtalamic acid) on the growth rate and bending of the unicellular sporangiophore of the zygomycete fungus, Phycomyces blakesleeanus. Sporangiophores that were submerged in an aqueous buffer responded to IAA with a sustained enhancement of the growth rate, while 1-NAA, 2,4-D, and NPA elicited an inhibition. In contrast, sporangiophores kept in air responded to IAA with a 20 to 40% decrease of the growth rate, while 1-NAA and NPA elicited an enhancement. The unilateral and local application of IAA in the growing zone of the sporangiophore elicited in 30 min a moderate negative tropic bending in wild type C2 and mutant C148madC, which was, however, partially masked by a concomitant avoidance response caused by the aqueous buffer. Auxin transport-related genes ubiquitous in plants were found in a BLAST search of the Phycomyces genome. They included members of the AUX1 (auxin influx carrier protein 1), PILS (PIN-LIKES, auxin transport facilitator protein), and ABCB (plant ATP-binding cassette transporter B) families while members of the PIN family were absent. Our observations imply that IAA represents an intrinsic element of the sensory transduction of Phycomyces and that its mode of action must very likely differ in several respects from that operating in plants.     

Subcutaneously administrated genistein and daidzein decrease serum cholesterol and increase triglyceride levels in male middle-aged rats

Nutritional supplements containing soybean phytoestrogens, the isoflavones genistein (G) and daidzein (D), are increasingly used as alternative therapy for osteoporosis, cancer, and cardiovascular and other diseases with a frequency that increases with advancing age. In this study we examined the effects of subcutaneous administration of either G or D on serum lipid levels in orchidectomized (Orx) and intact (IA) middle-aged male rats, which are experimental models of andropause. Sixteen-month-old Wistar rats were treated with 10 mg/kg and 30 mg/kg of either G or D. The control groups received testosterone, estradiol, or vehicle for 3 weeks, after which the total serum cholesterol (TC), low-density lipoprotein (LDL-C), high-density lipoprotein (HDL-C), and total triglycerides (TT) were measured. Compared with the matching vehicle-treated controls, the higher doses of G and D and testosterone treatment significantly (P < 0.05) lowered the TC and lipoprotein cholesterol levels. The greatest effect was observed regarding LDL-C in both Orx and IA males after G and D treatments, in which LDL-C decreased by more than 30%. The lower isoflavone doses induced a significant cholesterol-lowering effect (P < 0.05) only in the Orx group. Like the estradiol treatment, the higher doses of G and D increased the TT levels in both rat models by more than 50% (P < 0.05). The lower doses of isoflavones increased TT only in the Orx group. In male middle-aged rats, injections of higher doses of G and D decreased the serum cholesterol levels, as did testosterone injection, and brought about an increase in serum triglycerides similar to that observed after estradiol treatment.     

Interaction of the CC-chemokine RANTES with glycosaminoglycans activates a p44/p42 mitogen-activated protein kinase-dependent signaling pathway and enhances human immunodeficiency virus type 1 infectivity

The interaction of the CC-chemokine RANTES with its cell surface receptors transduces multiple intracellular signals: low concentrations of RANTES (1 to 10 nM) stimulate G-protein-coupled receptor (GPCR) activity, and higher concentrations (1 microM) activate a phosphotyrosine kinase (PTK)-dependent pathway. Here, we show that the higher RANTES concentrations induce rapid tyrosine phosphorylation of multiple proteins. Several src-family kinases (Fyn, Hck, Src) are activated, as is the focal adhesion kinase p125 FAK and, eventually, members of the p44/p42 mitogen-activated protein kinase (MAPK) family. This PTK signaling pathway can be activated independently of known seven-transmembrane GPCRs for RANTES because it occurs in cells that lack any such RANTES receptors. Instead, activation of the PTK signaling pathway is dependent on the expression of glycosaminoglycans (GAGs) on the cell surface, in that it could not be activated by RANTES in GAG-deficient cells. We have previously demonstrated that RANTES can both enhance and inhibit infection of cells with human immunodeficiency virus type 1 (HIV-1). Here we show that activation of both PTK and MAPK is involved in the enhancement of HIV-1 infectivity caused by RANTES in cells that lack GPCRs for RANTES but which express GAGs.     

Cell surface delivery of the measles virus nucleoprotein: a viral strategy to induce immunosuppression

Although only a few blood cells are infected during measles, this infectious disease is followed by acute immunosuppression, associated with high infant mortality. Measles virus nucleoprotein has been suggested to contribute to virus-induced inhibition of the immune response. However, it has been difficult to understand how this cytosolic viral protein could leave an infected cell and then perturb the immune response. Here we demonstrate that intracellularly synthesized nucleoprotein enters the late endocytic compartment, where it recruits its cellular ligand, the Fcgamma receptor. Nucleoprotein is then expressed at the surfaces of infected leukocytes associated with the Fcgamma receptor and is secreted into the extracellular compartment, allowing its interaction with uninfected cells. Finally, cell-derived nucleoprotein inhibits the secretion of interleukin-12 and the generation of the inflammatory reaction, both shown to be impaired during measles. These results reveal nucleoprotein egress from infected cells as a novel strategy in measles-induced immunosuppression.     

Frequencies of mtDNA haplogroups in southeastern Europe--Croatians, Bosnians and Herzegovinians, Serbians, Macedonians and Macedonian Romani

Mitochondrial DNA polymorphisms were analyzed in of 1,610 randomly chosen adult men from 11 different regions from southeastern Europe (Croatians, Bosnians and Herzegovinians, Serbians, Macedonians and Macedonian Romani). MtDNA HVS-I region together with RFLP sites diagnostic for main Euroasian and African mtDNA haplogroups were typed to determine haplogroup frequency distribution. The most frequent haplogroup in studied populations was H with the exception of Macedonian Romani among whom the most frequent were South Asian (Indian) specific variants of haplogroup M. The multidimensional scaling plot showed two clusters of populations and two outliers (Macedonian Romani and the most distant from mainland Croatian island of Korcula). The first cluster was formed by populations from three Croatian islands (Hvar, Krk and Brac) and the second cluster was formed by Macedonians, Serbians, Croatians from mainland and coast, Herzegovinians, Bosnians, Slovenians, Poles and Russians. The present analysis does not address a precise evaluation of phylogenetic relations of studied populations although some conclusions about historical migrations could be noticed. More extended conclusions will be possible after deeper phylogenetic and statistical analyses.     

Viral Inhibition of BAK Promotes Murine Cytomegalovirus Dissemination to Salivary Glands

Apoptosis induction is an important host defense mechanism to control viral infection, which is antagonized by viral proteins. Murine cytomegalovirus m41.1 encodes a viral inhibitor of BAK oligomerization (vIBO) that blocks the mitochondrial apoptosis mediator BAK. However, its importance for viral fitness in vivo has not been investigated. Here, we show that an m41.1-deficient virus attains reduced titers in salivary glands of wild-type but not Bak1^−/− mice, indicating a requirement of BAK inhibition for optimal dissemination in vivo.     

Construction of improved vectors for gene cloning inMicromonospora melanosporea

Two vectors forMicromonospora melanosporea have been constructed with theStreptomyces plasmid pIJ702 along with thesgm gene (sisomicin-gentamicin resistance gene fromM. zionensis) as the second antibiotic resistance marker. These plasmids, containingsgm gene as the second selectable marker, may be an attractive alternative to pIJ702, which is incapable of conferring melanin production inM. melanosporea and consequently is not useful for insertional inactivation in this bacterium. The constructions remove restriction site forM. melanosporea restriction endonuclease and provide additional unique sites for the insertional inactivation of selectable markers, which enhance the use of these plasmids as general cloning vectors in bothM. melanosporea andS. lividans. On the other side, inS. lividans, plasmid pMK33-1 facilitates isolation and studies of promoters based on detection of extremely convenient phenotype of melanin production. This has been proved by shotgun cloning of chromosomal DNA fragments ofM. melanosporea and chromogenic identification ofS. lividans transformants which were capable of producing a melanin.     

Endogenous Gibberellin Profile During Christmas Rose (Helleborus niger L.) Flower and Fruit Development

Gibberellins (GAs) were identified and quantified during flower and fruit development in the Christmas rose (Helleborus niger L.), a native of southeastern Europe with a long international horticultural tradition. Physiologically, the plant differs from popular model species in two major respects: (1) following anthesis, the initially white or rose perianth (formed in this species by the sepals) turns green and persists until fruit ripening, and (2) the seed is shed with an immature embryo, a miniature endosperm, and a prominent perisperm as the main storage tissue. GA₁ and GA₄ were identified by full-scan mass spectra as the major bioactive GAs in sepals and fruit. LC-MS/MS system in accord with previously verified protocols also afforded analytical data on 12 precursors and metabolites of GAs. In the fruit, GA₄ peaked during rapid pericarp growth and embryo development and GA₁ peaked during the subsequent period of rapid nutrient accumulation in the seeds and continued pericarp enlargement. In the sepals, the flux through the GA biosynthetic pathway was highest prior to the light green stage when the photosynthetic system was induced. Unfertilized, depistillated, and deseeded flowers became less green than the seed-bearing controls; chlorophyll accumulation could be restored by applying GA₁, GA₄, and, less efficiently, GA₃ to the deseeded fruit. The sepals of unfertilized and depistillated flowers indeed contained very low levels of GA₄ and gradually decreasing levels of GA₁. However, the concentrations of their precursors and metabolites were less affected. These data suggest that a signal(s) from the fruit stimulates GA biosynthesis in the sepals resulting in greening. The fruit-derived GAs appear to be mainly involved in pericarp growth and seed development.     

Evaluation of Reference Genes for RT-qPCR Expression Studies in Hop (Humulus lupulus L.) during Infection with Vascular Pathogen Verticillium albo-atrum

Hop plant (Humulus lupulus L.), cultivated primarily for its use in the brewing industry, is faced with a variety of diseases, including severe vascular diseases, such as Verticillium wilt, against which no effective protection is available. The understanding of disease resistance with tools such as differentially expressed gene studies is an important objective of plant defense mechanisms. In this study, we evaluated twenty-three reference genes for RT-qPCR expression studies on hop under biotic stress conditions. The candidate genes were validated on susceptible and resistant hop cultivars sampled at three different time points after infection with Verticillium albo-atrum. The stability of expression and the number of genes required for accurate normalization were assessed by three different Excel-based approaches (geNorm v.3.5 software, NormFinder, and RefFinder). High consistency was found among them, identifying the same six best reference genes (YLS8, DRH1, TIP41, CAC, POAC and SAND) and five least stably expressed genes (CYCL, UBQ11, POACT, GAPDH and NADH). The candidate genes in different experimental subsets/conditions resulted in different rankings. A combination of the two best reference genes, YLS8 and DRH1, was used for normalization of RT-qPCR data of the gene of interest (PR-1) implicated in biotic stress of hop. We outlined the differences between normalized and non-normalized values and the importance of RT-qPCR data normalization. The high correlation obtained among data standardized with different sets of reference genes confirms the suitability of the reference genes selected for normalization. Lower correlations between normalized and non-normalized data may reflect different quantity and/or quality of RNA samples used in RT-qPCR analyses.     

Recurrent somatic embryogenesis and plant regeneration from immature zygotic embryos of cabbage (Brassica oleracea var. capitata) and cauliflower (Brassica oleracea var. botrytis)

A simple and rapid protocol was established for repetitive somatic embryogenesis and subsequent plant regeneration in two important Brassica oleracea varieties, cabbage and cauliflower. Direct regeneration of somatic embryos (SEs) was achieved from immature zygotic embryos cultured on B5 plant growth regulator (PGR)-free (B5-0) induction medium and on B5 medium supplemented with 1 mg l^?1 2,4-dichlorophenoxyacetic acid (2,4-D) (B5-D). Zygotic embryos of both cabbage and cauliflower at the cotyledonary (C) stage (1.8 mm long) incubated on B5-0 medium displayed the highest embryo-forming capacities (EFCs) of 11.84 and 11.95, respectively. Secondary somatic embryos (SSEs) appeared on the cabbage and cauliflower’s primary embryos at a high frequency (83.3 and 87.5 %, respectively), and this process continued in a repetitive way on PGR-free Murashige and Skoog (MS-0) medium. The embryogenic potential of the cultures with a gradual diminution was maintained for 10 months (ten cycles). A total of 20 % of the mature SSEs from cabbage and 55 % from cauliflower spontaneously regenerated plantlets on MS-0 medium. The addition of 1 mg l^?1 6-benzyladenine (BA) or 6-furfurylaminopurine (Kin) in the regeneration medium significantly improved somatic embryo conversion into plantlets by up to 56 % in cabbage and 79 % in cauliflower. Regenerated plants acclimated successfully to ex vitro conditions and displayed morphological and reproductive characteristics similar to seed-derived plants. Effective recurrent somatic embryogenesis may be an appropriate practical solution for clonal propagation and genetic modifications of cabbage and cauliflower.