Citrulline immunohistochemistry may not necessarily identify nitric oxide synthase activity: the pitfall of peptidylarginine deiminase.

Nitric oxide synthase (NOS) converts L-arginine as a substrate to form nitric oxide and the "by-product" citrulline. To characterize NOS activity in the nervous tissue at the single-cell level, citrulline immunostaining is considered to be a suitable means of working on the principle that in brain tissue, due to the incomplete urea cycle, citrulline is produced exclusively by NOS. This assumption is correct for free citrulline but it does not consider the conversion of arginine to citrulline residues of proteins by the calcium-dependent peptidylarginine deiminase (PAD). Using a polyclonal antiserum against citrulline we observed in cerebellar cell cultures immunopositivity in a few, mostly NOS-positive, neurons, in activated microglia, and in oligodendroglia (which under control conditions are in doubt to be able to express NOS), but not in astroglia. Treatment with the excitotoxin kainate substantially enhanced the staining intensity for citrulline in neurons and glial cells. To distinguish between free (NOS-related) and protein-bound (PAD-related) citrulline we blocked NOS activity by 7-nitroindazole or L-N5-(1-iminoethyl)lysine. The results provide evidence that citrulline immunolabeling results partly from PAD-mediated protein citrullination, enhanced pathophysiologically under stimulated conditions by exposure to kainate. Our immunocytochemical observations were corroborated by Western blot analysis showing several bands of citrulline-positive proteins, whose number and staining intensity depended on kainate treatment and calcium ions.     

Motifs,modules and games in bacteria

Global explorations of regulatory network dynamics, organization and evolution have become tractable thanks to high-throughput sequencing and molecular measurement of bacterial physiology. From these, a nascent conceptual framework is developing, that views the principles of regulation in term of motifs, modules and games. Motifs are small, repeated, and conserved biological units ranging from molecular domains to small reaction networks. They are arranged into functional modules, genetically dissectible cellular functions such as the cell cycle, or different stress responses. The dynamical functioning of modules defines the organism's strategy to survive in a game, pitting cell against cell, and cell against environment. Placing pathway structure and dynamics into an evolutionary context begins to allow discrimination between those physical and molecular features that particularize a species to its surroundings, and those that provide core physiological function. This approach promises to generate a higher level understanding of cellular design, pathway evolution and cellular bioengineering.     

Cholesterol favors phase separation of sphingomyelin

The phase behavior of mixed lipid dispersions representing the inner leaflet of the cell membrane has been characterized by X-ray diffraction. Aqueous dispersions of phosphatidylethanolamine:phosphatidylserine (4:1 mole/mole) have a heterogeneous structure comprising an inverted hexagonal phase H(II) and a lamellar phase. Both phases coexist in the temperature range 20-45 degrees C. The fluid-to-gel mid-transition temperature of the lamellar phase assigned to phosphatidylserine is decreased from 27 to 24 degrees C in the presence of calcium. Addition of sphingomyelin to phosphatidylethanolamine/phosphatidylserine prevents phase separation of the hexagonal H(II) phase of phosphatidylethanolamine but the ternary mixture phase separates into two lamellar phases of periodcity 6.2 and 5.6 nm, respectively. The 6.2-nm periodicity is assigned to the gel phase enriched in sphingomyelin of molecular species comprising predominantly long saturated hydrocarbon chains because it undergoes a gel-to-fluid phase transition above 40 degrees C. The coexisting fluid phase we assign to phosphatidylethanolamine and phosphatidylserine and low melting point molecular species of sphingomyelin which suppresses the tendency of phosphatidylethanolamine to phase-separate into hexagonal H(II) structure. There is evidence for considerable hysteresis in the separation of lamellar fluid and gel phases during cooling. The addition of cholesterol prevents phase separation of the gel phase of high melting point sphingomyelin in mixtures with phosphatidylserine and phosphatidylethanolamine. In the quaternary mixture the lamellar fluid phase, however, is phase separated into two lamellar phases of periodicities of 6.3 and 5.6 nm (20 degrees C), respectively. The lamellar phase of periodicity 5.6 nm is assigned to a phase enriched in aminoglycerophospholipids and the periodicity 6.3 nm to a liquid-ordered phase formed from cholesterol and high melting point molecular species of sphingomyelin characterized previously by ESR. Substituting 7-dehydrocholesterol for cholesterol did not result in evidence for lamellar phase separation in the mixture within the temperature range 20-40 degrees C. The specificity of cholesterol in creation of liquid-ordered lamellar phase is inferred.     

Simultaneous measurement of cellular respiration and acidification with a single CMOS ISFET

In vivo, the pH value and oxygen partial pressure are the most important physico-chemical parameters in the microenvironment of human tissues. In vitro, the extracellular acidification rate of cell cultures is an indicator of global cellular metabolism, while the rate of oxygen consumption is a measure of mitochondrial activity. Earlier approaches had the disadvantage that these two values had to be measured with two separate sensors at different loci within the tissue or cell culture. Furthermore, conventional Clark-type oxygen sensors are not very compatible for miniaturisation, making it impossible to measure at small cell volumes or even at the single cell level. We have, therefore, developed an ISFET based sensor structure which is able to measure both pH and oxygen partial pressure. This sensor structure was tested in vitro for simultaneous records of cellular acidification and respiration rates at the same site within the cell culture. This sensor is manufactured by a CMOS-process.     

Lithostratigraphy and carbonate microfacies across the Permian–Triassic boundary near Julfa (NW Iran) and in the Baghuk Mountains (Central Iran)

Permian–Triassic boundary sections in the Julfa (NW Iran) and Abadeh (Central Iran) regions display a succession of three characteristic rock units, (1) the Paratirolites Limestone with the mass extinction horizon at its top, (2) the ‘Boundary Clay’, and (3) the earliest Triassic Elikah Formation with the conodont P–Tr boundary at its base. The carbonate microfacies reveals a facies change, in the sections near Julfa, within the Paratirolites Limestone with an increasing number of intraclasts, Fe–Mn crusts, and biogenic encrustation. A decline in carbonate accumulation occurs towards the top of the unit with a sponge packstone in the sections, and finally resulting in a complete demise of the carbonate factory. The succession of the ‘Boundary Clay’ differs in the two regions; thin horizons of sponge packstone are present in the Julfa region and ‘calcite fans’ of probably inorganic origin in the Abadeh Region. The skeletal carbonate factory of the Late Permian was restored with the deposition of microbial carbonates at the base of the Elikah Formation, where densely laminated bindstone, floatstone with sparry calcite spheres, and oncoid wackestone/floatstone predominate.     

Developmental refinement of hair cell synapses tightens the coupling of Ca2+ influx to exocytosis

Cochlear inner hair cells (IHCs) develop from pre‐sensory pacemaker to sound transducer. Here, we report that this involves changes in structure and function of the ribbon synapses between IHCs and spiral ganglion neurons (SGNs) around hearing onset in mice. As synapses matured they changed from holding several small presynaptic active zones (AZs) and apposed postsynaptic densities (PSDs) to one large AZ/PSD complex per SGN bouton. After the onset of hearing (i) IHCs had fewer and larger ribbons; (ii) Ca_V1.3 channels formed stripe‐like clusters rather than the smaller and round clusters at immature AZs; (iii) extrasynaptic Ca_V1.3‐channels were selectively reduced, (iv) the intrinsic Ca²⁺ dependence of fast exocytosis probed by Ca²⁺ uncaging remained unchanged but (v) the apparent Ca²⁺ dependence of exocytosis linearized, when assessed by progressive dihydropyridine block of Ca²⁺ influx. Biophysical modeling of exocytosis at mature and immature AZ topographies suggests that Ca²⁺ influx through an individual channel dominates the [Ca²⁺] driving exocytosis at each mature release site. We conclude that IHC synapses undergo major developmental refinements, resulting in tighter spatial coupling between Ca²⁺ influx and exocytosis.     

Circular Dichroism Sensing of Chiral Compounds Using an Achiral Metal Complex as Probe

Coordination of a chiral substrate to (meso‐salen)cobalt(II) nitrate and subsequent oxidation generates a Co(III) complex exhibiting a strong chiroptical readout that is attributed to spontaneous substrate‐to‐ligand chirality imprinting. The characteristic circular dichroism (CD) response of the (salen)cobalt complex can be used for enantiomeric analysis of a variety of chiral substrates based on a simple CD measurement at low concentration and without additional purification steps. This chirality sensing approach has potential for high‐throughput enantiomeric excess (ee) screening applications and minimizes solvent waste production. Chirality 26:379–384, 2014. © 2014 Wiley Periodicals, Inc.     

The influence of bacteria on the arsenic species produced by laboratory cultures of the marine phytoplankton Dunaliella tertiolecta

To assess whether bacteria influence the biogeochemical cycling of arsenic by laboratory cultures of the marine phytoplankton Dunaliella tertiolecta, the arsenic species produced by D. tertiolecta were compared in “operationally sterile” and bacteria spiked cultures. It was observed that glycerol (Gly-) arsenoriboside (41–78 %), phosphate (PO₄?) arsenoriboside (7–38 %) and arsenate (As(V)) (15–21 %) were the major water-soluble arsenic species in all D. tertiolecta cultures irrespective of whether cultures were operationally sterile or contained added bacteria. PO₄-riboside (46–74 %) and Gly-riboside (24–36 %) were also the major arsenic species in hydrolysed lipid extracts of D. tertiolecta irrespective of whether cultures were operationally sterile or contained bacteria. In addition to similarities in the arsenic species produced, total arsenic concentrations and culture growth did not differ relative to whether cultures were operationally sterile or not. Similar bacterial strains were identified in all D. tertiolecta cultures irrespective of whether bacteria were added or not. Consequently, it is evident that the presence of “foreign” or “added” bacteria in D. tertiolecta has minimal influence on the metabolism and cycling of arsenic by phytoplankton. Thus, the use of laboratory phytoplankton cultures containing bacteria may be appropriate means to investigate arsenic biogeochemical cycling unlike previously believed.