The regulation of the energy-dependent phosphate uptake by the blue-green alga Anacystis nidulans

Investigations of the energy-dependent accumulation of orthophosphate by the blue-green alga Anacystis nidulans have established: 1. The transport through the cell membrane is the rate-limiting step in the incorporation of phosphate.-2. This transport is facilitated by a carrier that can be activated by Ca²⁺ and Mg²⁺ and inhibited by EDTA.-3. The activation of the carrier in the light is associated with changes of the cytoplasmic Mg²⁺ content.-4. Intracellular phosphate is shown to be present in bound form.-5. The energy-dependent accumulation of orthophosphate within the cell depends strictly on the cytoplasmic pH and not on the energy conversion at the thylakoid membrane which is responsible for the energy supply. The cytoplasmic pH is different in the light, in the dark, and in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). Orthophosphate accumulation can most readily be explained in terms of a pH dependent precipitation into a complex with bivalent cations rather than by an active transport against a concentration gradient.Abbreviation CCCP Carbonyl cyanide m-chlorophenylhydrazone     

Morphology and physiology of peripheral giant interneurons in the forelegs (whips) of the whip spider Heterophrynus elaphus Pocock (Arachnida: Amblypygi)

Summary The tarsi of the modified front legs (whips) of the whip spider Heterophrynus elaphus contain two afferent giant fibers, GN1 and GN2, with diameters at the tibia-tarsus joint of ca. 21 m and 14 m, respectively. The somata of these two neurons lie in the periphery, about 25 cm away from the CNS. These two neurons are interneurons which receive mechanoreceptive inputs from approximately 750 and 1500 bristles, respectively. The receptive fields of GN1 and GN2 overlap; they extend for 40 mm (GN1) and 90 mm (GN2) along the length of the tarsus. About 90% of the synapses onto the giant fibers are axo-axonic. Mechanical stimulation of a single bristle is sufficient to elicit action potentials in one or both interneurons. The response of the interneurons adapts quickly. Average conduction time from the soma to the CNS is 45 ms for GN1 and 55 ms for GN2. Mean conduction velocities are 5.5 and 4.2 m/s, respectively. Activity in the giant fibers does not elicit a motor response; hence the giant fibers do not mediate an escape response. Possible functions of these giant fibers are discussed and compared to those of giant fiber systems in other arthropods.Abbreviations GN giant neuron - S segment     

2-Methyl-6-phytylquinol and 2,3-dimethyl-5-phytylquinol as precursors of tocopherol synthesis in spinach chloroplasts

The incorporation of [Me-¹⁴C] from SAM-[Me-¹⁴C] into precursors indicates the following sequence of tocopherol synthesis in spinach: 2-methyl-6-phytylquinol (6-phytyltoluquinol) (1a) → 2,3-dimethyl-5-phytylquinol (phytylplastoquinol) (2a)→,γ-tocopherol (5a)→α-tocopherol (6). 1a is particularly preferred to 2-methyl-5-phytylquinol (1b) and 2-methyl-3-phytylquinol (1c). 1a only forms 2a. 2a is converted to 6 via 5a and, to a lesser extent, 2,5-dimethyl-6-phylquinol (2b) to 6 via β-tocopherol (5b). Trimethylphytylquinol (3) is not an intermediate in the formation of 6. All reactions are independent of light.     

Dimerization of DNA methyltransferase 1 is mediated by its regulatory domain

DNA methylation is a major epigenetic modification and plays a crucial role in the regulation of gene expression. Within the family of DNA methyltransferases (Dnmts), Dnmt3a and 3b establish methylation marks during early development, while Dnmt1 maintains methylation patterns after DNA replication. The maintenance function of Dnmt1 is regulated by its large regulatory N‐terminal domain that interacts with other chromatin factors and is essential for the recognition of hemi‐methylated DNA. Gelfiltration analysis showed that purified Dnmt1 elutes at an apparent molecular weight corresponding to the size of a dimer. With protein interaction assays we could show that Dnmt1 interacts with itself through its N‐terminal regulatory domain. By deletion analysis and co‐immunoprecipitations we mapped the dimerization domain to the targeting sequence TS that is located in the center of the N‐terminal domain (amino acids 310–629) and was previously shown to mediate replication independent association with heterochromatin at chromocenters. Further mutational analyses suggested that the dimeric complex has a bipartite interaction interface and is formed in a head‐to‐head orientation. Dnmt1 dimer formation could facilitate the discrimination of hemi‐methylated target sites as has been found for other palindromic DNA sequence recognizing enzymes. These results assign an additional function to the TS domain and raise the interesting question how these functions are spatially and temporarily co‐ordinated. J. Cell. Biochem. 106: 521–528, 2009. © 2009 Wiley‐Liss, Inc.     

Tocopherol and plastoquinone synthesis in spinach chloroplasts subfractions

Subfractions isolated from intact purified spinach chloroplasts are able to prenylate the aromatic moiety of α-tocopherol and plastoquinone-9 precursors. The biosynthesis of α-tocopherol and plastoquinone-9 is a compartmentalized process. The chloroplast envelope membranes are the only site of the enzymatic prenylation in α-tocopherol synthesis whereas the thylakoid membrane is also involved in the prenylation and methylation sequence of plastoquinone-9 biosynthesis. A very active kinase which forms phytyl-PP is localized in the stroma. Phytol but not geranylgeraniol is the polyprenol precursor of the side chain of α-tocopherol in spinach chloroplasts.     

Simian Virus 40 Large T Antigen Is Phosphorylated at Multiple Sites Clustered in Two Separate Regions

The phosphorylation sites of simian virus 40 large T antigen were determined within the primary structure of the molecule. Exhaustive digestion of ³²P-labeled large T antigen with trypsin generated six major phosphopeptides which could be separated in a newly developed isobutyric acid-containing chromatography system. By partial tryptic digestion, large T antigen was cleaved into an amino-terminal fragment of 17,000 daltons and overlapping fragments from the carboxy-terminal region ranging in size between 71,000 and 13,000 daltons. The location of the phosphopeptides was then determined by fingerprint analyses of individual fragments. Their physical properties were analyzed by sizing on polyacrylamide gels and by sequential digestion and peptide mapping; their amino acid composition was determined by differential labeling with various amino acids. The amino-terminal 17,000-dalton fragment gave rise to only one phosphopeptide (phosphopeptide 3) that contained half of the phosphate label incorporated into large T antigen. It contained phosphoserine and phosphothreonine sites, all of which were clustered within a small segment between Cys₁₀₅ and Lys₁₂₇. This segment contained five serines and two threonines. Among these, Ser₁₀₆, Ser₁₂₃, and Thr₁₂₄ were identified as phosphorylated residues; in addition, either one or both of Ser₁₁₁ and Ser₁₁₂ were phosphorylated. The neighboring residues, Ser₁₂₃ and Thr₁₂₄, were found in three different phosphorylation states in that either Ser₁₂₃ or Thr₁₂₄ or both were phosphorylated. Phosphopeptides 1, 2, 4, 5, and 6 were all derived from a single fragment extending 26,000 daltons upstream from the carboxy terminus of large T antigen. Phosphopeptide 6 was identical with the previously determined phosphothreonine peptide phosphorylated at Thr₇₀₁. Phosphopeptides 1, 2, 4, and 5 contained only serine-bound phosphate. Phosphopeptides 1, 2, and 4 represented overlapping peptides, all of which were phosphorylated at Ser₆₃₉ located next to a cluster of six acidic residues. In phosphopeptide 5, a large peptide ranging from Asn₆₅₃ to Arg₆₉₁, at least two of seven serines were phosphorylated. Thus, large T antigen contains at least eight phosphorylation sites. Their clustering within two separate regions might correlate with structural and functional domains of this protein.     

Microelectrophoretic studies with 2-bromo-α-ergocriptine on dopaminergic neurons in the feline caudate nucleus

The influence of microelectrophoretically applied dopamine and pressure released 2-Bromo-α-ergocriptine (CB 154) on the spontaneous activity of single neurones in the feline caudate nucleus was studied. Two types of dopamine sensitive neurones were found: the spike-rate of the first type was reduced by dopamine, the firing of the other type was facilitated by dopamine. Both types of neurones were influenced by pressure released CB 154. Neurones which were excited by dopamine were excited by CB 154, while with the other type CB 154 resulted only in an amplification of the inhibition by dopamine.     

Morphogenesis of digitate structures in hot spring silica sinters of the El Tatio geothermal field,Chile

In silica-rich hot spring environments, internally laminated, digitate sinter deposits are often interpreted as bio-mediated structures. The organic components of microbial communities (cell surfaces, sheaths and extracellular polymeric substances) can act as templates for silica precipitation, therefore influencing digitate sinter morphogenesis. In addition to biologic surface-templating effects, various microenvironmental factors (hydrodynamics, local pH and fluctuating wind patterns) can also influence silica precipitation, and therefore the morphology of resulting digitate sinters. Digitate sinter morphology thus depends on the dynamic interplay between microenvironmentally driven silica precipitation and microbial growth, but the relative contributions of both factors are a topic of continuing research. Here we present a detailed study of digitate silica sinters in distal, low-temperature regimes of the El Tatio geothermal field, Chile. This high-altitude geothermal field is extremely arid and windy, and has one of the highest silica precipitation rates found in the world. We find that digitate silica sinters at El Tatio always accrete into the prevailing eastward wind direction and exhibit laminar growth patterns coinciding with day–night cycles of wind- and thermally driven evaporation and rewetting. Subaerial parts of digitate sinters lack preserved organics and sinter textures that would indicate past microbial colonization, while filamentous cyanobacteria with resistant, silicified sheaths only inhabit subaqueous cavities that crosscut the primary laminations. We conclude that, although fragile biofilms of extremophile micro-organisms may have initially been present and templated silica precipitation at the tips of these digitate sinters, the saltation of sand grains and precipitation of silica by recurrent wind- and thermally driven environmental forcing at El Tatio are important, if not dominant factors shaping the morphology of these digitate structures. Our study sheds light on the relative contributions of biogenic and abiogenic factors in sinter formation in geothermal systems, with geobiological implications for the cautious interpretation of stromatolite-like features in ancient silica deposits on Earth and Mars.