Dimorphic and yeast-like mutants of the genusCephalosporium Cda

A series of mutants, in which the mycelial type of growth gradually changes to the dimorphic and permanent yeast-like forms, were isolated from cultures ofCephalosporium sp. subjected to UV radiation. The intermediate stage between the mycelial and dimorphic strains (mutants 2/29 and 2/R) is characterized by the absence of aerial hyphae, ability to form conidiophores inside agar and by polymorphism of conidia. The Y-M transformation of two dimorphic mutants obtained from the 2/R mutant depends on temperature. Another mutant isolated from the 2/29 strain was found to form the mycelial phase only when osmolarity of the medium increased. At 22°C the transformation of all three dimorphic strains was influenced by the carbon source: the Y phase predominated in glucose-containing media, the M phase predominated in media with amino acids or citrate serving as carbon sources. Another mutant (2/7R) was found to grow permanently in the Y phase and was not influenced by temperature, osmolarity of the medium and by the carbon source. It is assumed that the dimorphism of the mutants is caused by a conformational mutation inhibiting the apical growth. This mutation can be phenotypically reversed by some factors of the environment.     

Regulation of tissue inhibitor of metalloproteinases-1 in rat Sertoli cells: induction by germ cell residual bodies, interleukin-1alpha, and second messengers

In the testis, FSH has been shown to induce the expression and secretion of tissue inhibitor of metalloproteinases-1 (TIMP-1) from Sertoli cells in vitro. This study was performed to elucidate further the cellular origin of testicular TIMP-1 and its expression by hormonal and paracrine factors. This is the first report on the expression of testicular TIMP-1 in vivo. TIMP-1 mRNA in whole testis was decreased after hypophysectomy and strongly increased by the injection of FSH-S17 to hypophysectomized rats. Primary cultures of both peritubular and Sertoli cells showed basal expression of TIMP-1 mRNA. In contrast, we were unable to detect TIMP-1 mRNA in Leydig cells, freshly isolated immature germ cells (primary spermatocytes and spermatids), or residual bodies. We further show that treatment of Sertoli cells with 8-(4-chlorophenyl)thio-cAMP (8-CPTcAMP) in combination with 12-O-tetradecanoylphorbol 13-acetate (TPA) or Ca(2+) inducers (calcium ionophore A23187 or thapsigargin) had additive (TPA) and synergistic effects (Ca(2+)) on the level of TIMP-1 mRNA and secreted protein. We also show that both the level of TIMP-1 mRNA and secreted protein from Sertoli cells were strongly increased by residual bodies, as well as by the cytokine interleukin-1alpha. TIMP-1 was not up-regulated by either 8-CPTcAMP or interleukin-1alpha in peritubular cells. In contrast to the regulated secretory fraction of TIMP-1, we also detected constitutively expressed immunoreactive TIMP-1 in the nucleus of Sertoli cells, suggesting a role of nuclear TIMP-1 in these cells. In conclusion, our data show that secretion of TIMP-1 from Sertoli cells is highly regulated by hormonal and local processes in the testis, indicating that TIMP-1 is of physiological importance during both testicular development and spermatogenesis.     

Rotavirus VP4 and VP7-Derived Synthetic Peptides as Potential Substrates of Protein Disulfide Isomerase Lead to Inhibition of Rotavirus Infection

Rotavirus infection of MA104 cells has been shown to be inhibited by cell membrane-impermeant thiol/disulfide exchange inhibitors and anti-PDI antibodies. To characterise the amino acid sequences of rotavirus structural proteins potentially mediating cell surface PDI?Csubstrate interactions, rotavirus-derived peptides from VP4 and VP7 (RRV) and VP7 (Wa), and their modified versions containing serine instead of cysteine were synthesized. Cysteine-containing VP7 peptides corresponding to residues 189?C210 or 243?C263 caused an infectivity inhibitory effect of about 64 and 85?%, respectively, when added to cells. Changing cysteine to serine significantly decreased the inhibitory effect. A cysteine-containing peptide corresponding to VP4 residues 200?C219 and its scrambled version reduced infectivity by 92 and 80?%, respectively. A cysteine to serine change in the original VP4 200?C219 peptide did not affect its inhibitory effect. Non-rotavirus related sequences containing cysteine residues efficiently inhibited rotavirus infectivity. Antibodies against VP7 residues 189?C210 or 243?C263 significantly inhibited rotavirus infectivity only after virus attachment to cells had occurred, whereas those against VP4 200?C219 peptide inhibited infectivity irrespective of whether virus or cell-attached virus was antibody-treated. A direct PDI?Cpeptide interaction was shown by ELISA for cysteine-containing VP7 and VP4 peptides. Virus?Ccell attachment was unaffected by the peptides inhibiting virus infectivity. The results showed that even though cysteine residues in the peptides tested are important in both virus infectivity inhibition and in vitro PDI?Cpeptide interaction, the accompanying amino acid sequence also plays some role. As a whole, our findings further support our hypothesis that cell surface PDI from MA104 cells might be contributing to rotavirus entry at a post-attachment step.     

Effects of Pre-Treatments with Abscisic Acid and/or Benzyladenine on Gas Exchange of French Bean, Sugar Beet, and Maize Leaves During Water Stress and After Rehydration

Net photosynthetic rate (P_N), transpiration rate (E), and stomatal conductance (g_s) during water stress and after rehydration were measured in Phaseolus vulgaris, Beta vulgaris, and Zea mays. Immediately before imposition of water stress by cessation of watering, plants were irrigated with water (control), 100 M abscisic acid (ABA), and/or 10 M N⁶-benzyladenine (BA). In all three species, application of ABA decreased g_s, E, and P_N already 1 h after application. However, during water stress g_s, E, and P_N in plants pre-treated with ABA remained higher than in plants pre-treated with water. Positive effects of ABA application were observed also after rehydration. In contrast, the effects of pre-treatment with BA were species-specific. While in bean plants BA application ameliorated negative effect of water stress, only very slight effects were observed in maize, and in sugar beet BA even aggravated the effects of water stress.     

In silico studies of potential phosphoresidues in the human nucleophosmin/B23: its kinases and related biological processes

Human nucleophosmin/B23 is a phosphoprotein involved in ribosome biogenesis, centrosome duplication, cancer, and apoptosis. Its function, localization, and mobility within cells, are highly regulated by phosphorylation events. Up to 21 phosphosites of B23 have been experimentally verified even though the corresponding kinase is known only for seven of them. In this work, we predict the phosphorylation sites in human B23 using six kinase-specific servers (KinasePhos 2.0, PredPhospho, NetPhosK 1.0, PKC Scan, pkaPS, and MetaPredPS) plus DISPHOS 1.3, which is not kinase specific. The results were integrated with information regarding 3D structure and residue conservation of B23, as well as cellular localizations, cellular processes, signaling pathways and protein-protein interaction networks involving both B23 and each predicted kinase. Thus, all 40 potential phosphosites of B23 were predicted with significant score (>0.50) as substrates of at least one of 38 kinases. Thirteen of these residues are newly proposed showing high susceptibility of phosphorylation considering their solvent accessibility. Our results also suggest that the enzymes CDKs, PKC, CK2, PLK1, and PKA could phosphorylate B23 at higher number of sites than those previously reported. Furthermore, PDK, GSK3, ATM, MAPK, PKB, and CHK1 could mediate multisite phosphorylation of B23, although they have not been verified as kinases for this protein. Finally, we suggest that B23 phosphorylation is related to cellular processes such as apoptosis, cell survival, cell proliferation, and response to DNA damage stimulus, in which these kinases are involved. These predictions could contribute to a better understanding, as well as addressing further experimental studies, of B23 phosphorylation.     

Long-Term Biomonitoring of Soil Contamination Using Poplar Trees: Accumulation of Trace Elements in Leaves and Fruits

Phytostabilization aims to immobilize soil contaminants using higher plants. The accumulation of trace elements in Populus alba leaves was monitored for 12 years after a mine spill. Concentrations of As and Pb significantly decreased, while concentrations of Cd and Zn did not significantly over time. Soil concentrations extracted by CaCl₂ were measured by ICP-OES and results of As and Pb were below the detection limit. Long-term biomonitoring of soil contamination using poplar leaves was proven to be better suited for the study of trace elements. Plants suitable for phytostabilization must also be able to survive and reproduce in contaminated soils. Concentrations of trace elements were also measured in P. alba fruiting catkins to determine the effect on its reproduction potential. Cadmium and Zn were found to accumulate in fruiting catkins, with the transfer coefficient for Cd significantly greater than Zn. It is possible for trace elements to translocate to seed, which presents a concern for seed germination, establishment and colonization. We conclude that white poplar is a suitable tree for long-term monitoring of soil contaminated with Cd and Zn, and for phytostabilization in riparian habitats, although some caution should be taken with the possible effects on the food web.

Supplemental materials are available for this article. Go to the publisher's online edition of International Journal of Phytoremediation to view the supplemental file.     

Molecular characterization of American cockroach tropomyosin (Periplaneta americana allergen 7), a cross-reactive allergen

Inhalation of allergens produced by the American cockroach (Periplaneta americana) induces IgE Ab production and the development of asthma in genetically predisposed individuals. The cloning and expression in Escherichia coli of P. americana tropomyosin allergen have been achieved. The protein shares high homology with other arthropod tropomyosins (80% identity) but less homology with vertebrate ones (50% identity). The recombinant allergen was produced in E. coli as a nonfusion protein with a yield of 9 mg/l of bacterial culture. Both natural and recombinant tropomyosins were purified by isoelectric precipitation. P. americana allergen 1 (Per a 1) and Per a 7 (tropomyosin) are to date the only cross-reacting allergens found in cockroaches. ELISA and Western blot inhibition experiments, using natural and recombinant purified tropomyosins from shrimp and cockroach, showed that tropomyosin induced cross-reactivity of IgE from patients allergic to these allergens, suggesting that this molecule could be a common allergen among invertebrates.     

Flux analysis of recombinant Saccharomyces cerevisiae YPB-G utilizing starch for optimal ethanol production

Genetically modified Saccharomyces cerevisiae strain (YPB-G) which secretes a bifunctional fusion protein that contains both Bacillus subtilis -amylase and Aspergillus awamori glucoamylase activities was used for the direct conversion of starch into ethanol. Starch was either supplied initially to different nutrient media or added instantaneously to the reactor at various discrete time instants (pulse feeding). Stoichiometric modeling was used to investigate the effects of initial substrate concentration and growth rate of the recombinant yeast culture on ethanol production. Reaction stoichiometries describing both the anabolism and catabolism of the microorganism were used as an input to flux balance analysis (FBA), the preferred metabolic modeling approach since the constructed stoichiometric network was underdetermined. Experiments for batch and fed-batch systems at different substrate concentrations were analyzed theoretically in terms of flux distributions using ethanol production rate as the maximization criteria. Calculated ethanol rates were in agreement with experimental measurements, suggesting that this recombinant microorganism is sufficiently evolved to optimize its ethanol production. The function of the main pathways of yeast metabolism (PPP, EMP, TCA) are discussed together with the node analyses of glucose-6-P and pyruvate branch points. Theoretical node analysis revealed that if the split ratio in G6P branch point is changed by genetic manipulations, the ethanol yield would be affected considerably.     

Behavioural and cardiovascular responses to electrical stimulation of the medulla oblongata of the cat

Effects of stimulation of the nucleus tractus solitarii, the dorsal motor nucleus of the vagus, the nucleus reticularis paramedianus, and the nucleus cuneatus were studied in free-moving cats. Stimulation of the medullary nuclei that are known to be involved in the central nervous control of cardiovascular functions might activate preprogrammed motor responses such as licking and sniffing, and induce complex behavioural response patterns such as sleep or flight reaction. Moreover, both lever-pressing for rewarding brain stimulation, and eating in food deprived cats might be modulated by these stimulations. In a shuttle box the cats showed no tendency toward shuttling during stimulation, except the stimulation of the nucleus reticularis paramedianus which produced aversion. The cardiovascular and respiratory effects varied parallel with the behavioural responses. It is concluded that the medullary nuclei related to visceral functions are capable of affecting somatomotor behaviour either directly on the motor system, or by inducing complex response patterns in which somatomotor and visceral responses are integrated.     

Molecular and morphological diversity of Trebouxia microalgae in sphaerothallioid Circinaria spp. lichens1

Three vagrant (Circinaria hispida, Circinaria gyrosa, and Circinaria sp. ‘paramerae’) and one crustose (semi‐vagrant, Circinaria sp. ‘oromediterranea’) lichens growing in very continental areas in the Iberian Peninsula were selected to study the phycobiont diversity. Mycobiont identification was checked using nrITS DNA barcoding: Circinaria sp. ‘oromediterranea’ and Circinaria sp. ‘paramerae’ formed a new clade. Phycobiont diversity was analyzed in 50 thalli of Circinaria spp. using nrITS DNA and LSU rDNA, with microalgae coexistence being found in all the species analyzed by Sanger sequencing. The survey of phycobiont diversity showed up to four different Trebouxia spp. as the primary phycobiont in 20 thalli of C. hispida, in comparison with the remaining Circinaria spp., where only one Trebouxia was the primary microalga. In lichen species showing coexistence, some complementary approaches are needed (454 pyrosequencing and/or ultrastructural analyses). Five specimens were selected for high‐throughput screening (HTS) analyses: 22 Trebouxia OTUs were detected, 10 of them not previously known. TEM analyses showed three different cell morphotypes (Trebouxia sp. OTU A12, OTU S51, and T. cretacea) whose ultrastructure is described here in detail for the first time. HTS revealed a different microalgae pool in each species studied, and we cannot assume a specific pattern between these pools and the ecological and/or morphological characteristics. The mechanisms involved in the selection of the primary phycobiont and the other microalgae by the mycobiont are unknown, and require complex experimental designs. The systematics of the genus Circinaria is not yet well resolved, and more analyses are needed to establish a precise delimitation of the species.