Sexual differentiation in Mucor: Trisporic acid response mutants and mutants blocked in zygospore development

Spores of a minus strain of Mucor mucedo (Bref.) were treated with 1-methyl-[3-nitro]-1-nitro-soguanidine and mutants were isolated either by testing for zygophore induction with externally supplied trisporic acids (TA) or by mating with wild type plus colonies. Mutants were found defective (Tar^?) or temperature-sensitive (Tar-Ts) in their reaction towards trisporic acids, blocked or temperature-sensitive in their mating with plus strain (Mat^? or Mat-Ts) or temperature-sensitive in zygospore development (Zyg-Ts). The inability to react against externally supplied trisporic acids was not necessarily coupled with an inability to mate with plus strain (phenotype Tar^? Mat⁺). This indicated that the diffusion and uptake of trisporic acids is not a necessary prerequisite to the sexual interaction of Mucor mating types.     

A toolbox for class I HDACs reveals isoform specific roles in gene regulation and protein acetylation

The class I histone deacetylases are essential regulators of cell fate decisions in health and disease. While pan- and class-specific HDAC inhibitors are available, these drugs do not allow a comprehensive understanding of individual HDAC function, or the therapeutic potential of isoform-specific targeting. To systematically compare the impact of individual catalytic functions of HDAC1, HDAC2 and HDAC3, we generated human HAP1 cell lines expressing catalytically inactive HDAC enzymes. Using this genetic toolbox we compare the effect of individual HDAC inhibition with the effects of class I specific inhibitors on cell viability, protein acetylation and gene expression. Individual inactivation of HDAC1 or HDAC2 has only mild effects on cell viability, while HDAC3 inactivation or loss results in DNA damage and apoptosis. Inactivation of HDAC1/HDAC2 led to increased acetylation of components of the COREST co-repressor complex, reduced deacetylase activity associated with this complex and derepression of neuronal genes. HDAC3 controls the acetylation of nuclear hormone receptor associated proteins and the expression of nuclear hormone receptor regulated genes. Acetylation of specific histone acetyltransferases and HDACs is sensitive to inactivation of HDAC1/HDAC2. Over a wide range of assays, we determined that in particular HDAC1 or HDAC2 catalytic inactivation mimics class I specific HDAC inhibitors. Importantly, we further demonstrate that catalytic inactivation of HDAC1 or HDAC2 sensitizes cells to specific cancer drugs. In summary, our systematic study revealed isoform-specific roles of HDAC1/2/3 catalytic functions. We suggest that targeted genetic inactivation of particular isoforms effectively mimics pharmacological HDAC inhibition allowing the identification of relevant HDACs as targets for therapeutic intervention.     

Combined effect of cyprid age and lipid content on larval attachment and metamorphosis of Balanus amphitrite darwin

Various algal diets of different lipid content and composition were used to rear batches of naupliar larvae of Balanus amphitrite. The cyprids in these larval batches differed in lipid content and were used to investigate the combined effect of cyprid lipid content and cyprid age on attachment and metamorphosis. For this purpose, cyprids were aged for 0,3 and 6 d at 8°C prior to utilization in laboratory attachment assays. The percentage attachment of cyprids with similar lipid content differed significantly among the three age categories. A strong and a weak positive relationship between cyprid lipid content and attachment were monitored in young and old cyprids, respectively. A significant interaction (two‐way ANOVA) between cyprid age and lipid content was observed, indicating that these factors jointly affect larval attachment and metamorphosis in B. amphitrite from the beginning of the cyprid stage.     

Characterization of novel SF3b and 17S U2 snRNP proteins,including a human Prp5p homologue and an SF3b DEAD-box protein

Mass spectrometry was used to identify novel proteins associated with the human 17S U2 snRNP and one of its stable subunits, SF3b. Several additional proteins were identified, demonstrating that 17S U2 snRNPs are significantly more complex than previously thought. Two of the newly identified proteins, namely the DEAD-box proteins SF3b125 and hPrp5 (a homologue of Saccharomyces cerevisiae Prp5p) were characterized further. Immunodepletion experiments with HeLa nuclear extract indicated that hPrp5p plays an important role in pre-mRNA splicing, acting during or prior to prespliceosome assembly. The SF3b-associated protein SF3b125 dissociates at the time of 17S U2 formation, raising the interesting possibility that it might facilitate the assembly of the 17S U2 snRNP. Finally, immunofluorescence/FISH studies revealed a differential subnuclear distribution of U2 snRNA, hPrp5p and SF3b125, which were enriched in Cajal bodies, versus SF3b155 and SF3a120, which were not; a model for 17S U2 snRNP assembly based on these findings is presented. Taken together, these studies provide new insight into the composition of the 17S U2 snRNP and the potential function of several of its proteins.     

The human histamine H2-receptor couples more efficiently to Sf9 insect cell Gs-proteins than to insect cell Gq-proteins: limitations of Sf9 cells for the analysis of receptor/Gq-protein coupling

The human histamine H2-receptor (hH2R) couples to Gs-proteins to activate adenylyl cyclase and to Gq-proteins to activate phospholipase C, but phospholipase C activation has not consistently been observed. The aim of this study was to compare coupling of hH2R to insect and mammalian Gs- and Gq-proteins in Spodoptera frugiperda (Sf9) cells. Interaction of hH2R with mammalian G proteins was assessed with coexpressed proteins or receptor-Galpha fusion proteins that enhance coupling efficiency. hH2R efficiently coupled to insect Gs-proteins to activate adenylyl cyclase. However, hH2R poorly coupled to insect Gq-proteins as assessed by the lack of enhancement of histamine-stimulated steady-state GTP hydrolysis by regulators of G protein signaling (RGS proteins). In contrast, RGS-proteins efficiently enhanced GTP hydrolysis stimulated by the human platelet-activating factor receptor (PAFR) and the histamine H1-receptor (H1R) from man and guinea pig. The measurement of intracellular free Ca2+ concentration was not useful for studying receptor/Gq-protein coupling. hH2R also efficiently interacted with mammalian Gs-proteins, specifically with fused Gsalpha as assessed by guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS)-sensitive high-affinity agonist binding, agonist-stimulated [35S]GTPgammaS binding and adenylyl cyclase activation. In contrast, coupling of hH2R to coexpressed and fused mammalian Gqalpha was poor. However, our inability to reconstitute efficient coupling of PAFR and H1R to mammalian Gqalpha indicated that a large portion of the expressed G protein was functionally inactive. Taken together, our data show that hH2R couples more efficiently to insect cell Gs-proteins than to insect cell Gq-proteins. Unfortunately, there are significant limitations in the usefulness of Sf9 cells for comparing the coupling of receptors to mammalian Gs- and Gq-proteins and assessing Gq-mediated activation of effector systems.     

Neuregulin 1 type III/ErbB signaling is crucial for Schwann cell colonization of sympathetic axons

Analysis of Schwann cell (SC) development has been hampered by the lack of growing axons in many commonly used in vitro assays. As a consequence, the molecular signals and cellular dynamics of SC development along peripheral axons are still only poorly understood. Here we use a superior cervical ganglion (SCG) explant assay, in which axons elongate after treatment with nerve growth factor (NGF). Migration as well as proliferation and apoptosis of endogenous SCG-derived SCs along sympathetic axons were studied in these cultures using pharmacological interference and time-lapse imaging. Inhibition of ErbB receptor tyrosine kinases leads to reduced SC proliferation, increased apoptosis and thereby severely interfered with SC migration to distal axonal sections and colonization of axons. Furthermore we demonstrate that SC colonization of axons is also strongly impaired in a specific null mutant of an ErbB receptor ligand, Neuregulin 1 (NRG1) type III. Taken together, using a novel SC development assay, we demonstrate that NRG1 type III serves as a critical axonal signal for glial ErbB receptors that drives SC development along sympathetic axons.     

How does the eye breathe? Evidence for neuroglobin-mediated oxygen supply in the mammalian retina

Visual performance of the vertebrate eye requires large amounts of oxygen, and thus the retina is one of the highest oxygen-consuming tissues of the body. Here we show that neuroglobin, a neuron-specific respiratory protein distantly related to hemoglobin and myoglobin, is present at high amounts in the mouse retina (approximately 100 microm). The estimated concentration of neuroglobin in the retina is thus about 100-fold higher than in the brain and is in the same range as that of myoglobin in the muscle. Neuroglobin is expressed in all neurons of the retina but not in the retinal pigment epithelium. Neuroglobin mRNA was detected in the perikarya of the nuclear and ganglion layers of the neuronal retina, whereas the protein was present mainly in the plexiform layers and in the ellipsoid region of photoreceptor inner segment. The distribution of neuroglobin correlates with the subcellular localization of mitochondria and with the relative oxygen demands, as the plexiform layers and the inner segment consume most of the retinal oxygen. These findings suggest that neuroglobin supplies oxygen to the retina, similar to myoglobin in the myocardium and the skeletal muscle.     

mRNPs, polysomes or granules: FMRP in neuronal protein synthesis

mRNA localization and regulated translation play central roles in neurite outgrowth and synaptic plasticity. A key molecule in these processes is the Fragile X mental retardation protein, FMRP, which is involved in the metabolism of neuronal mRNAs. Absence or mutation of FMRP leads to spine dysmorphogenesis and impairs synaptic plasticity. Studies that have mainly been performed on the mouse and Drosophila models for Fragile X Syndrome showed that FMRP is involved in translational regulation at synapses, but even 15 years after discovery of the FMR1 gene, the precise working mechanisms remain elusive.     

Male-specific cell migration into the developing gonad

Background: The gene Sry acts as a developmental switch, initiating a pathway of gene activity that leads to the differentiation of testis rather than ovary from the indifferent gonad (genital ridge) in mammalian embryos. The early events following Sry expression include rapid changes in the topographical organization of cells in the XY gonad. To investigate the contribution of mesonephric cells to this process, gonads from wild-type mice (CD1), and mesonephroi from a transgenic strain ubiquitously expressing β-galactosidase (ROSA26), were grafted together in vitro. After culture, organs were fixed and stained for β-galactosidase activity to identify cells contributed from the mesonephros to the male or female gonad.Results: Migration of mesonephric cells occurred into XY but not XX gonads from 11.5–16.5 days post coitum (dpc). Somatic cells contributed from the mesonephros were distinguished by their histological location and by available cell-specific markers. Some of the migrating cells were endothelial; a second population occupied positions circumscribing areas of condensing Sertoli cells; and a third population lay in close apposition to endothelial cells.Conclusions: Migration from the mesonephros to the gonad is male specific at this stage of development and depends on an active signal that requires the presence of a Y chromosome in the gonad. The signals that trigger migration operate over considerable distances and behave as chemoattractants. We suggest that migration of cells into the bipotential gonad may have a critical role in initiating the divergence of development towards the testis pathway.     

In situ FT-IR microscopic study on enzymatic treatment of poplar wood cross-sections

The feasibility of Fourier transform infrared (FT-IR) microscopy to monitor in situ the enzymatic degradation of wood was investigated. Cross-sections of poplar wood were treated with cellulase Onozuka RS within a custom-built fluidic cell. Light-optical micrographs and FT-IR spectra were acquired in situ from normal and tension wood fibers. Light-optical micrographs showed almost complete removal of the gelatinous (G) layer in tension wood. No structural and spectral changes were observed in the lignified cell walls. The accessibility of cellulose within the lignified cell wall was found to be the main limiting factor, whereas the depletion of the enzyme due to lignin adsorption could be ruled out. The fast, selective hydrolysis of the crystalline cellulose in the G-layer, even at room temperature, might be explained by the gel-like structure and the highly porous surface. Young plantation grown hardwood trees with a high proportion of G-fibers thus represent an interesting resource for bioconversion to fermentable sugars in the process to bioethanol.