Mxi2 sustains ERK1/2 phosphorylation in the nucleus by preventing ERK1/2 binding to phosphatases

ERK1/2 (extracellular-signal-regulated kinase 1/2) MAPKs (mitogen-activated protein kinases) are tightly regulated by the cellular microenvironment in which they operate. Mxi2 is a p38α splice isoform capable of binding to ERK1/2 and ensuring their translocation to the nucleus. Therein Mxi2 sustains ERK1/2 phosphorylation levels and, as a consequence, ERK1/2 nuclear signals are enhanced. However, the molecular mechanisms underlying this process are still unclear. In the present study, we show that Mxi2 prevents nuclear but not cytoplasmic phosphatases from binding to and dephosphorylating ERK1/2, disclosing an unprecedented mechanism for the spatial regulation of ERK1/2 activation. We also demonstrate that the kinetics of ERK1/2 extranuclear signals can be significantly altered by artificially tethering Mxi2 to the cytoplasm. In this case, Mxi2 abolishes ERK1/2 inactivation by cytoplasmic phosphatases and potentiates ERK1/2 functions at this compartment. These results highlight Mxi2 as a key spatial regulator of ERK1/2 functions, playing a pivotal role in the balance between ERK1/2 nuclear and cytoplasmic signals.     

DNA extraction conditions fromPorphyra perforata using LiCl

A rapid and economical method of DNA extraction from a red seaweedPorphyra perforata J. Agardh has been developed by the use of lithium chloride. This paper describes the optimization of extraction conditions. Heat treatment of tissues in a solution (0.8 M LiCl, 0.6% Sarkosyl, 10 mM EDTA, 0.2% PVPP, 5% ß-mercaptoethanol, pH 9.0) at 55 °C for 10 min extracts DNA that is of sufficient quality to be used as a template for PCR amplification. Total DNA yield was approximately 30 to 50g g^–1 t of partially dried tissue. Total RNA yield was approximately 400g g^–1 of partially dried tissue. Carbohydrate was contained as approximately 40 to 90 mM (expressed as glucose equivalents) from 1 g tissue, and protein contamination calculated as the O.D. 260/280 ratio was in the range of 1.4 to 1.7. The DNA was characterized by high molecular weight larger than 50 kb.     

Anaerobic heterotrophic dark metabolism in the cyanobacterium Oscillatoria limnetica: Sulfur respiration and lactate fermentation

The cyanobacterium Oscillatoria limnetica, capable of anoxygenic photosynthesis in the light with sulfide as electron donor can anaerobically break down its intracellular polyglucose in the dark. In the absence of elemental sulfur, the organism carries out lactate fermentation; in its presence, anaerobic respiration occurs in which sulfur is reduced to sulfide. Induction of anoxygenic photosynthesis or synthesis of new proteins is not necessary for either process. Cells adapted in the dark to sulfur reduction are capable of anoxygenic photosynthesis during a subsequent light period, unless protein synthesis has been inhibited during the dark incubation period.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - FCCP Carbonylcyanide p-trifluoromethoxyphenylhydrazone - mgat milligramatom - OD optical density     

THE ULTRASTRUCTURE OF GALLS ON THE RED ALGA GRACILARIA EPIHIPPISORA1

A strain of Gracilaria epihippisora Hoyle produces gall-like cell proliferations in culture. These growths can be excised and grown separately, where they retain an undifferentiated morphology and reach 5mm in diameter. The gall tissue consists of a single morphological cell type without any differentiation between surface and internal cells as is characteristic of normal thallus tissue. Gall cells are typically 20–40 μm in diameter and contain the usual complement of organelles and a prominent vacuole, although there are several distinct features. The large multilobed plastids have an extensive proliferation of thylakoid membranes, which form an arrangement of loops and spirals. The thallus outer cell wall layer is highly reduced. The gall growths contain intracellular virus-like particles (ca. 80 nm in diameter) that occur in discrete groups.     

DEVELOPMENTAL STUDIES IN PORPHYRA (RHODOPHYCEAE). III. EFFECT OF CULTURE CONDITIONS ON WALL REGENERATION AND DIFFERENTIATION OF PROTOPLASTS1

Protoplasts isolated from thalli of four Porphyra species regenerated successfully into differentiated plantlets. The efficiency of protoplast isolation and the developmental patterns of the regenerating protoplasts depended on the type of tissues from which they were isolated. However, culture conditions greatly influenced the patterns of development at the cellular and organismal levels. Sorbitol, nitrogen, and agar concentration in the medium controlled rates of cell division, thickening of cell walls, development of rhizoids, and formation of calluses or differentiated blades. Agitation disturbed the attachment of the protoplasts to a substrate. Cells in agitated cultures produced suspensions of single cells and non-polarized small calluses. Calluses which developed from protoplasts survived in storage for over two years. The stored calluses, and cells and protoplasts that were isolated from them, were subcultured successfully. We forsee extensive use of Porphyra cell suspensions for strain selection and vegetative propagation of cultivars. This technology, which makes vegetative cloning of selected Porphyra plants possible, may eliminate the need for cultivation and storage of the conchocelis phase. Protoplasts are also being used as tools for studies in genetic engineering of these commercial species.     

Wall loss and origin of soluble carbohydrates during mating of Chlamydomonas reinhardi

In the mating reaction between gametes of the green alga Chlamydomonas reinhardi, a lytic factor which solubilizes the cell wall is released. It has been shown that carbohydrates accumulate in the supernatant of mating gametes. We present here data which support the notion that the released carbohydrates arise from solubilized gametic cell walls. The evidence is based, in part, on the comparison of the carbohydrates and amino acids in the acid hydrolysates of cell-free supernatants to the reported composition of isolated cell walls. In both cases the three predominant sugars are mannose, arabinose and galactose, and also, in both cases, large amounts of the amino acid hydroxyproline are present. In addition, it is shown that if gametic cell walls are removed prior to mating interactions by treatment with a preparation of lytic factor, much less carbohydrate is subsequently released into the supematant, when such ‘nake’ gametes are mated.     

Hsp90 and angiogenesis in bone disorders--lessons from the avian growth plate

Thiram-induced tibial dyschondroplasia (TD) and vitamin-D deficiency rickets are avian bone disorders of different etiologies characterized by abnormal chondrocyte differentiation, enlarged and unvascularized growth plates, and lameness. Heat-shock protein 90 (Hsp90) is a proangiogenic factor in mammalian tissues and in tumors; therefore, Hsp90 inhibitors were developed as antiangiogenic factors. In this study, we evaluated the association between Hsp90, hypoxia, and angiogenesis in the chick growth plate. Administration of the Hsp90 inhibitor to TD- and rickets-afflicted chicks at the time of induction resulted in reduction in growth-plate size and, contrary to its antiangiogenic effect in tumors, a major invasion of blood vessels occurred in the growth plates. This was the result of upregulation of the VEGF receptor Flk-1, the major rate-limiting factor of vascularization in TD and rickets. In addition, the abnormal chondrocyte differentiation, as characterized by collagen type II expression and alkaline phosphatase activity, and the changes in hypoxia-inducible factor-1α (HIF-1α) in both disorders were restored. All these changes resulted in prevention of lameness. Inhibition of Hsp90 activity reduced growth-plate size, increased vascularization, and mitigated lameness also in TD chicks with established lesions. In summary, this is the first reported demonstration of involvement of Hsp90 in chondrocyte differentiation and growth-plate vascularization. In contrast to the antiangiogenic effect of Hsp90 inhibitors observed in mammals, inhibition of Hsp90 activity in the unvascularized TD- and rickets-afflicted chicks resulted in activation of the angiogenic switch and reinstated normal growth-plate morphology.     

Effect of LiCl on compression and tension properties ofPophyra perforata tissue

The mechanical properties of various differentiated regions of thePorphyra perforata thallus and the effect of LiCl were studied by performing compression and tension tests. Among the various differentiated tissues, the holdfast area was high in its compressive modulus of elasticity and tensile modulus of elasticity, possibly related to its thick matrix. Vegetative non-dividing tissue and vegetative dividing tissue were the most flexible and strong, showing the highest percentage elongation at break and tensile strength. The patch area, which is a transition zone leading to sexually mature tissue, had moderate values of tensile properties. Meanwhile, sexually differentiated male and female tissues had the highest compressive modulus of elasticity and lowest tensile properties. Thes tisues tended to crumble easily. Treatments in LiCl, as used for DNA extraction, resulted in a decrease in both compressive modulus of elasticity (87%) and tensile modulus of elasticity (54%). After treatment of tissue for chromosome staining in a method using LiCl, there was a marked decrease in tensile modulus of elasticity (49%), while the compressive modulus of elasticity remained unchanged. Such mechanical changes verify the softening effect of LiCl on the seaweedP. perforata tissue.Author for correspondence     

An inactivation gate in the selectivity filter of KCNQ1 potassium channels

Inactivation is an inherent property of most voltage-gated K⁺ channels. While fast N-type inactivation has been analyzed in biophysical and structural details, the mechanisms underlying slow inactivation are yet poorly understood. Here, we characterized a slow inactivation mechanism in various KCNQ1 pore mutants, including L273F, which hinders entry of external Ba²⁺ to its deep site in the pore and traps it by slowing its egress. Kinetic studies, molecular modeling, and dynamics simulations suggest that this slow inactivation involves conformational changes that converge to the outer carbonyl ring of the selectivity filter, where the backbone becomes less flexible. This mechanism involves acceleration of inactivation kinetics and enhancement of Ba²⁺ trapping at elevated external K⁺ concentrations. Hence, KCNQ1 slow inactivation considerably differs from C-type inactivation where vacation of K⁺ from the filter was invoked. We suggest that trapping of K⁺ at s₁ due to filter rigidity and hindrance of the dehydration-resolvation transition underlie the slow inactivation of KCNQ1 pore mutants.     

Prolyl 4-hydroxylase from Volvox carteri. A low-Mr enzyme antigenically related to the alpha subunit of the vertebrate enzyme.

Prolyl 4-hydroxylase was isolated in a highly purified form from a multi-cellular green alga, Volvox carteri, by a procedure consisting of ion-exchange chromatography and affinity chromatography on poly(L-hydroxyproline) coupled to Sepharose. Two other affinity-column procedures were also developed, one involving 3,4-dihydroxyphenylacetate and the other 3,4-dihydroxyphenylpropionate linked to Sepharose. The Km values of the Volvox enzyme for the co-substrates and the peptide substrate, as well as the inhibition constants for selected 2-oxoglutarate analogues, were similar to those of the enzyme from Chlamydomonas reinhardii, except that the Km for 2-oxoglutarate with the Volvox enzyme was 6-fold greater. The temperature optimum of the Volvox enzyme was also 10 degrees C higher. The apparent Mr of the Volvox enzyme by gel filtration was about 40,000, being similar to that reported for the Chlamydomonas enzyme but markedly lower than that of the vertebrate enzymes. A similar apparent Mr of about 40,000 was also found for prolyl 4-hydroxylase from the green alga Enteromorpha intestinalis, whereas the enzyme from various vascular plants gave an apparent Mr greater than 300,000. SDS/polyacrylamide-gel electrophoresis demonstrated in the highly purified Volvox enzyme the presence of a major protein band doublet with a Mr of about 65,000 and a minor doublet of Mr about 55,000-57,000. A polyclonal antiserum, prepared against the Mr-65,000 doublet, stained in immunoblotting the Mr-65,000 doublet as well as the alpha subunit, but not the beta subunit, of the vertebrate prolyl 4-hydroxylase. An antiserum against the beta subunit of the vertebrate enzyme stained in immunoblotting a Mr-50,000 polypeptide in a partially purified Volvox enzyme preparation, but did not stain either the Mr-65,000 or the Mr-55,000-57,000 doublet of the highly purified enzyme. The data thus suggest that the active Volvox carteri prolyl 4-hydroxylase is an enzyme monomer antigenically related to the alpha subunit of the vertebrate enzyme.