C14 Assays and Autoradiographic Studies on the Rooster Comb

The distribution of C¹⁴ was studied in various parts of the rooster comb following treatment with testosterone. The value of gas-phase assay of C¹⁴ in tissue has been demonstrated and the results compared with those of autoradiographic studies on the same tissue. The results of these experiments showed that androgen treatment significantly increases the rate of incorporation of C¹⁴ in various parts of the comb. The specific activity of carbon in the comb, cornea, and liver differed, depending on which precursor, viz. glucose-6-C¹⁴, glucose-1-C¹⁴, and glucuronolactone-U-C¹⁴, was administered. The highest values were obtained after the administration of glucose-6-C¹⁴; glucuronolactone-U-C¹⁴ gave the lowest specific activity. The specific activity of carbon in different parts of the comb showed considerable variation. Carbon assay of serial sections of the comb cut at various planes showed that the specific activity of carbon was highest in the mucoid layer. Both C¹⁴ assays and autoradiograms indicate that C¹⁴ is also present in other parts of the comb. As seen in autoradiography, the concentration of C¹⁴ was highest in the epithelium, in the blood vessel walls, and in the avascular collagenous tissue. These results, and indications from previous studies, suggest that the high specific activity of carbon in the mucoid layer is due mainly to the presence of C¹⁴-labelled hyaluronic acid. Autoradiograms and PAS staining suggest that a significant amount of C¹⁴ is also incorporated into the glycoproteins associated with the collagen fibers.     

C14 assays and autoradiographic studies on the rooster comb

The distribution of C(14) was studied in various parts of the rooster comb following treatment with testosterone. The value of gas-phase assay of C(14) in tissue has been demonstrated and the results compared with those of autoradiographic studies on the same tissue. The results of these experiments showed that androgen treatment significantly increases the rate of incorporation of C(14) in various parts of the comb. The specific activity of carbon in the comb, cornea, and liver differed, depending on which precursor, viz. glucose-6-C(14), glucose-1-C(14), and glucuronolactone-U-C(14), was administered. The highest values were obtained after the administration of glucose-6-C(14); glucuronolactone-U-C(14) gave the lowest specific activity. The specific activity of carbon in different parts of the comb showed considerable variation. Carbon assay of serial sections of the comb cut at various planes showed that the specific activity of carbon was highest in the mucoid layer. Both C(14) assays and autoradiograms indicate that C(14) is also present in other parts of the comb. As seen in autoradiography, the concentration of C(14) was highest in the epithelium, in the blood vessel walls, and in the avascular collagenous tissue. These results, and indications from previous studies, suggest that the high specific activity of carbon in the mucoid layer is due mainly to the presence of C(14)-labelled hyaluronic acid. Autoradiograms and PAS staining suggest that a significant amount of C(14) is also incorporated into the glycoproteins associated with the collagen fibers.     

Process of comb division of reused nests in the Australian paper waspRopalidia plebeiana (Hymenoptera,Vespidae)

Summary InRopalidia plebeiana, combs made in the previous year are often reused by foundresses in the following spring and structurally divided into subnests by them. Close observations of the comb dividing process revealed that: 1) combs that had multiple brood areas (areas where cells had eggs) were frequently divided into subnests, while those with single brood areas were never divided; 2) groups of foundresses each occupied a particular brood area even before comb division started; and 3) frequency with which a foundress practiced comb cutting was independent of her social status or oviposition frequency. These observations suggest that the initial grouping of foundresses that colonize an old nest leads to subsequent comb division, rather than the major egg layers cutting combs to safeguard their oviposition territories from their rivals.     

A new hypothesis on the simultaneous direct and indirect proton pump mechanisms in NADH-quinone oxidoreductase (complex I)

Recently, Sazanov’s group reported the X-ray structure of whole complex I [Nature, 465, 441 (2010)], which presented a strong clue for a “piston-like” structure as a key element in an “indirect” proton pump. We have studied the NuoL subunit which has a high sequence similarity to Na⁺/H⁺ antiporters, as do the NuoM and N subunits. We constructed 27 site-directed NuoL mutants. Our data suggest that the H⁺/e⁻ stoichiometry seems to have decreased from (4H⁺/2e⁻) in the wild-type to approximately (3H⁺/2e⁻) in NuoL mutants. We propose a revised hypothesis that each of the “direct” and the “indirect” proton pumps transports 2H⁺ per 2e⁻.     

Combs and sexual selection in black grouse (Tetrao tetrix)

We studied supra-orbital combs in lekking black grouse (Tetrao tetrix) in relation to sexual selection at five leks in Finland1991-1998 and four leks in Sweden 1992-1995. Comb size wasestimated in two ways: by observing its natural size in thefield at different behaviors ("observed comb size"), and bymeasuring the comb size from captured birds ("measured combsize"). The size of combs is highly variable, and individualscan change it within seconds. Males express their larger combsduring display, as compared to other behaviors. Observed mean comb sizes were larger on leks with a higher number of malesand a higher number of copulations. Measured and observed combsizes and copulatory success did not significantly correlatewhen all males where analyzed, but a positive and significantrelationship between observed comb size and copulatory success was found within males that achieved copulations. Measured comblength correlated positively with the amount of testosterone.While females were present on the lek, displaying and successfulmales showed the largest observed comb size. When we comparedobserved comb size during fighting between successful and unsuccessfulmales and correlated comb size of pairs of fighting males withtheir fighting activity, no significant differences in combsize were found. The result that comb size correlated significantlywith an increase in testosterone level and that larger combsize, within successful males, predicted higher copulatorysuccess suggests that combs may be a cue for females to assessmale quality. The lack of a significant relationship betweenobserved comb size and fighting behavior suggests that combsize either has minor importance in male-male signaling onthe lek or that males may express similar-sized combs duringfighting to avoid serious fights and thus risk of comb injuries.     

Effect of caponisation on physicochemical and sensory characteristics of chickens

The meat fats content associated to nutritional and sensory characteristics are the most important concerns of consumers. To study the effect of caponisation on the meat quality of two different breed chickens, slaughtered at 18 weeks of age, raised under the same conditions, the physicochemical and sensory characteristics of capons (castrated males at 8 weeks of age) and roosters’ meat of native Amarela Portuguesa and native Pedrês Portuguesa breeds were evaluated. Forty Amarela (20 roosters and 20 capons), 40 Pedrês (20 roosters and 20 capons) chickens, and also, six free-range chicken and six broilers were evaluated. The pH, water-holding capacity, Warner-Bratzler shear force, moisture content, ash, myoglobin, collagen, CP, total fat and fatty acids profile were evaluated in breast and leg meat, according to standard procedures. Leg meat capon showed greater intramuscular fat content (P⩽0.05), monounsaturated fatty acids (MUFA) and CP (P⩽0.001) than leg roosters. Caponisation increased the content of myoglobin and MUFA (P⩽0.05) and reduced the moisture content in the leg (P⩽0.05). The main fatty acids found were oleic acid (C18:1), palmitic acid (C16:0) and linoleic acid (C18:2). The greatest value of C18:1 was observed in capon’s breast (P⩽0.01). Sensory analysis was made to compare the Amarela and Pedrês meat with a free-range chicken and a broiler. The sensory taste panel classified the capon’s meat (Amarela and Pedrês) as juicier, less fibrous and tougher than rooster’s meat. The broiler was in general juicier, tenderer and less fibrous than the other birds. The results of sensory analysis complement those obtained in physicochemical analysis, suggesting that caponisation promotes an overall improvement in meat quality.     

Bioluminescence of the arm light organs of the luminous squid Watasenia scintillans

The squid Watasenia scintillans emits blue light from numerous photophores. According to Tsuji [F.I. Tsuji, Bioluminescence reaction catalyzed by membrane-bound luciferase in the “firefly squid”, Watasenia scintillans, Biochim. Biophys. Acta 1564 (2002) 189–197.], the luminescence from arm light organs is caused by an ATP-dependent reaction involving Mg²⁺, coelenterazine disulfate (luciferin), and an unstable membrane-bound luciferase. We stabilized and partially purified the luciferase in the presence of high concentrations of sucrose, and obtained it as particulates (average size 0.6–2 µm). The ATP-dependent luminescence reaction of coelenterazine disulfate catalyzed by the particulate luciferase was investigated in detail. Optimum temperature of the luminescence reaction is about 5 °C. Coelenterazine disulfate is a strictly specific substrate in this luminescence system; any modification of its structure resulted in a very heavy loss in its light emission capability. The light emitter is the excited state of the amide anion form of coelenteramide disulfate. The quantum yield of coelenterazine disulfate is calculated at 0.36. ATP could be replaced by ATP-γ-S, but not by any other analogues tested. The amount of AMP produced in the luminescence reaction was much smaller than that of coelenteramide disulfate, suggesting that the reaction mechanism of the Watasenia bioluminescence does not involve the formation of adenyl luciferin as an intermediate.     

Ascorbate reverses high glucose- and RAGE-induced leak of the endothelial permeability barrier

High glucose concentrations due to diabetes increase leakage of plasma constituents across the endothelial permeability barrier. We sought to determine whether vitamin C, or ascorbic acid (ascorbate), could reverse such high glucose-induced increases in endothelial barrier permeability. Human umbilical vein endothelial cells and two brain endothelial cell lines cultured at 25 mM glucose showed increases in endothelial barrier permeability to radiolabeled inulin compared to cells cultured at 5 mM glucose. Acute loading of the cells for 30–60 min with ascorbate before the permeability assay prevented the high glucose-induced increase in permeability and decreased basal permeability at 5 mM glucose. High glucose-induced barrier leakage was mediated largely by activation of the receptor for advanced glycation end products (RAGE), since it was prevented by RAGE blockade and mimicked by RAGE ligands. Intracellular ascorbate completely prevented RAGE ligand-induced increases in barrier permeability. The high glucose-induced increase in endothelial barrier permeability was also acutely decreased by several cell-penetrant antioxidants, suggesting that at least part of the ascorbate effect could be due to its ability to act as an antioxidant.     

Ascorbic acid prevents high glucose-induced apoptosis in human brain pericytes

High glucose concentrations due to diabetes increase apoptosis of vascular pericytes, impairing vascular regulation and weakening vessels, especially in brain and retina. We sought to determine whether vitamin C, or ascorbic acid, could prevent such high glucose-induced increases in pericyte apoptosis. Culture of human microvascular brain pericytes at 25 mM compared to 5 mM glucose increased apoptosis measured as the appearance of cleaved caspase 3. Loading the cells with ascorbate during culture decreased apoptosis, both at 5 and 25 mM glucose. High glucose-induced apoptosis was due largely to activation of the receptor for advanced glycation end products (RAGE), since it was prevented by specific RAGE inhibition. Culture of pericytes for 24 h with RAGE agonists also increased apoptosis, which was completely prevented by inclusion of 100 μM ascorbate. Ascorbate also prevented RAGE agonist-induced apoptosis measured as annexin V binding in human retinal pericytes, a cell type with relevance to diabetic retinopathy. RAGE agonists decreased intracellular ascorbate and GSH in brain pericytes. Despite this evidence of increased oxidative stress, ascorbate prevention of RAGE-induced apoptosis was not mimicked by several antioxidants. These results show that ascorbate prevents pericyte apoptosis due RAGE activation. Although RAGE activation decreases intracellular ascorbate and GSH, the prevention of apoptosis by ascorbate may involve effects beyond its function as an antioxidant.     

Improvement of yield of Pleurotus eryngii var. eryngii by substrate supplementation and use of a casing overlay

Improved yield and biological efficiency (BE) of Pleurotus eryngii var. eryngii were achieved by supplementation of substrate with a commercial delayed-release nutrient and use of a casing overlay. Yield increases of 14% were achieved from cased substrates that were supplemented at time of casing with delayed-release nutrient (Remo’s). Use of a casing layer enhanced yield by 141% over non-cased substrates. When casing and substrate supplementation were combined, yield increased 179% over non-cased/non-supplemented substrates. Mushrooms harvested from cased substrates were darker in color and solids contents were lower compared to non-cased substrates. An additional break of mushrooms was harvested from non-cased “spent” substrate by fragmenting and re-supplementing the substrate prior to the application of a casing overlay. Three production methods were compared for their effect on mushroom yield: “standard”, “casing” and “casing after first break”. Casing of the substrate before first break (“casing” production method) resulted in the highest yield and biological efficiency.     

Analysis of protein aggregation kinetics using short amino acid peptide tags

Understanding protein solubility, and consequently aggregation, is an important issue both from an academic and a biotechnological application viewpoints. Here we report the effects of 10 representative amino acids on the aggregation kinetics of proteins. The effects were determined by measuring the solubility of a simplified bovine pancreatic trypsin inhibitor (BPTI) variant, to which short artificial tags containing the amino acid of interest were added at its C-terminus. We determined the solubility of the tagged variants as a function of equilibration time (20 min to 48 h) and total protein concentration ranging from 0.10 mg/ml to 25.0 mg/ml. We observed, as anticipated, that proteins precipitated when the total protein concentration exceeded a critical value. However, when the total protein concentration was further increased, the apparent solubility reached a concentration above the critical value, and slowly decreased to a value under the critical concentration upon increasing the equilibration period. We rationalized these observations by identifying three different solubility values, the “transient solubility (TS)”, the “aggregation initiation concentration (AIC)” and the “long-term solubility (LS)”. AIC and LS are parameters determined essentially by the amino acid types composing the tags and could be considered as an amino acid's intrinsic property. On the other hand, TS is an apparent solubility that is measured after some (20 min in our case) equilibration time and is often considered as the “solubility” of the protein. Similar aggregation kinetic patterns were observed with natural proteins, indicating the generality of the observations made using our model protein.     

The On-Off Switch in Regulated Myosins: Different Triggers but Related Mechanisms

In regulated myosin, motor and enzymatic activities are toggled between the on-state and off-state by a switch located on its lever arm domain, here called the regulatory domain (RD). This region consists of a long α-helical “heavy chain” stabilized by a “regulatory” light chain (RLC) and an “essential” light chain (ELC). The on-state is activated by phosphorylation of the RLC of vertebrate smooth muscle RD or by direct binding of Ca²⁺ to the ELC of molluscan RD. Crystal structures are available only for the molluscan RD. To understand in more detail the pathway between the on-state and the off-state, we have now also determined the crystal structure of a molluscan (scallop) RD in the absence of Ca²⁺. Our results indicate that loss of Ca²⁺ abolishes most of the interactions between the light chains and may increase the flexibility of the RD heavy chain. We propose that disruption of critical links with the C-lobe of the RLC is the key event initiating the off-state in both smooth muscle myosins and molluscan myosins.     

Codon sextets with leading role of serine create “ideal” symmetry classification scheme of the genetic code

The standard classification scheme of the genetic code is organized for alphabetic ordering of nucleotides. Here we introduce the new, “ideal” classification scheme in compact form, for the first time generated by codon sextets encoding Ser, Arg and Leu amino acids. The new scheme creates the known purine/pyrimidine, codon–anticodon, and amino/keto type symmetries and a novel A + U rich/C + G rich symmetry. This scheme is built from “leading” and “nonleading” groups of 32 codons each. In the ensuing 4 × 16 scheme, based on trinucleotide quadruplets, Ser has a central role as initial generator. Six codons encoding Ser and six encoding Arg extend continuously along a linear array in the “leading” group, and together with four of six Leu codons uniquely define construction of the “leading” group. The remaining two Leu codons enable construction of the “nonleading” group. The “ideal” genetic code suggests the evolution of genetic code with serine as an initiator.     

The state transition mechanism—simply depending on light-on and -off in Spirulina platensis

The state transition in cyanobacteria is a long-discussed topic of how the photosynthetic machine regulates the excitation energy distribution in balance between the two photosystems. In the current work, whether the state transition is realized by “mobile phycobilisome (PBS)” or “energy spillover” has been clearly answered by monitoring the spectral responses of the intact cells of the cyanobacterium Spirulina platensis. Firstly, light-induced state transition depends completely on a movement of PBSs toward PSI or PSII while the redox-induced one on not only the “mobile PBS” but also an “energy spillover”. Secondly, the “energy spillover” is triggered by dissociation of PSI trimers into the monomers which specially occurs under a case from light to dark, while the PSI monomers will re-aggregate into the trimers under a case from dark to light, i.e., the PSI oligomerization is reversibly regulated by light switch on and off. Thirdly, PSI oligomerization is regulated by the local H⁺ concentration on the cytosol side of the thylakoid membranes, which in turn is regulated by light switch on and off. Fourthly, PSI oligomerization change is the only mechanism for the “energy spillover”. Thus, it can be concluded that the “mobile PBS” is a common rule for light-induced state transition while the “energy spillover” is only a special case when dark condition is involved.     

Photosystem II reaction center with altered pigment-composition: reconstitution of a complex containing five chlorophyll a per two pheophytin a with modified chlorophylls

Pigment-depleted Photosystem II reaction centers (PS II-RCs) from a higher plant (pea) containing five chlorophyll a (Chl) per two pheophytin a (Phe), were treated with Chl and several derivatives under exchange conditions [FEBS Lett. 434 (1998) 88]. The resulting reconstituted complexes were compared to those obtained by pigment exchange of “conventional” PS II-RCs containing six Chl per two Phe. (1) The extraction of one Chl is fully reversible. (2) The site of extraction is the same as the one into which previously extraneous pigments have been exchanged, most likely the peripheral D1-H118. (3) Introducing an efficient quencher (Ni-Chl) into this site results in only 25% reduction of fluorescence, indicating incomplete energy equilibration among the “core” and peripheral chlorophylls.     

Bemerkenswerte endogene Farbveränderung der Eischale von Chioninia delalandii (Duméril & Bibron, 1839) (Reptilia: Sauria: Scincidae)

The paper describes an unusual endogenous eggshell colouration observed in an egg of the Cap Verde skink Chioninia delalandii.A female specimen, kept in a terrarium, laid three eggs. Two of them were considered as fertilized (oval germ-disk, weakly pink). They were embedded 1 cm deep in a layer of moistened clay granules (substrate/water 2:1) and kept under different temperatures (egg 1 “cool”, 26-27 °C; egg 2 “warm”, 29-30 °C). There was a normal embryonic development in both eggs from their volume-enlargement and characteristic allometric growth (egg wide > egg length). The young hatch after 51 and 56 days.A change in eggshell colour, however, occurred in the “warm” kept egg during the last third of its incubation period. It started with a small spot (2-3 mm) in the outer area of the animal egg pole and spread into a dark-violet colouration over the whole eggshell within 15 days. After hatching of the young the shells of both eggs were examined. In the non-coloured egg there was no great difference in colour between the inner and outer egg layer, while in the coloured egg there was a distinct difference between the inner part, which was dark violet-gray, and the pale gray calcareous deck-layer. From the macroscopic view along the edge of the eggshell it was not identifiable, if the colour pigment was infiltrated into protein fibrils of the condensed surface layer.A possible explanation for the eggshell colouration could be an unusual embryonic pigmentation. This assumption is based on the first appearance of a restricted, point-like coloured area and its further regular extension. It might be that dark pigments (melanophores?) reached the eggshell (membrana testacea) and infiltrated the border-area to the condensed surface layer.     

Mimicking SP-C palmitoylation on a peptoid-based SP-B analogue markedly improves surface activity

Hydrophobic lung surfactant proteins B and C (SP-B and SP-C) are critical for normal respiration in vertebrates, and each comprises specific structural attributes that enable the surface-tension-reducing ability of the lipid-protein mixture in lung surfactant. The difficulty in obtaining pure SP-B and SP-C on a large scale has hindered efforts to develop a non-animal-derived surfactant replacement therapy for respiratory distress. Although peptide-based SP-C mimics exhibit similar activity to the natural protein, helical peptide-based mimics of SP-B benefit from dimeric structures. To determine if in vitro surface activity improvements in a mixed lipid film could be garnered without creating a dimerized structural motif, a helical and cationic peptoid-based SP-B mimic was modified by SP-C-like N-terminus alkylation with octadecylamine. “Hybridized” mono- and dialkylated peptoids significantly decreased the maximum surface tension of the lipid film during cycling on the pulsating bubble surfactometer relative to the unalkylated variant. Peptoids were localized in the fluid phase of giant unilamellar vesicle lipid bilayers, as has been described for SP-B and SP-C. Using Langmuir-Wilhelmy surface balance epifluorescence imaging (FM) and atomic force microscopy (AFM), only lipid-alkylated peptoid films revealed micro- and nanostructures closely resembling films containing SP-B. AFM images of lipid-alkylated peptoid films showed gel condensed-phase domains surrounded by a distinct phase containing “nanosilo” structures believed to enhance re-spreading of submonolayer material. N-terminus alkylation may be a simple, effective method for increasing lipid affinity and surface activity of single-helix SP-B mimics.     

Hierarchical distribution of ascending slopes, nearly neutral networks, highlands, and local optima at the dth order in an NK fitness landscape

We obtained several structural features of an NK fitness landscape by analytical approach. Particularly, we focused on spatial distributions of “ascending slopes”, “highlands”, “nearly neutral networks”, and “local optima” along the fitness coordinate W, from the viewpoint of adaptive walks with step-width d , where d is the number of mutated sites (Hamming distance) after a generation. The parameter k governs the degree of the ruggedness on the NK landscape, and we handled cases where k is moderate against the sequence length. From the foot up to the middle region on the landscape, many ascending slopes exist (high evolvability) and these slopes extend up near the “highland”, which is mathematically defined as the specific region where the expectation of the fitness increment becomes zero. Denoting the standard deviation of the fitness change at by SD^*, we considered the existence of “nearly neutral networks”, which percolate in the fitness band between W-SD^* and W+SD^*. Our results suggest that the highland corresponds to a phase-transition threshold of the formation of the nearly neutral networks. Near or over the highland, “local optima at the dth order” appear drastically (low evolvability), where d means the radius of their basins. The value of increases with d increasing. Then, as the fitness (=altitude) becomes higher, the basin size of the local optima increases. This leads to a conclusion that it is very hard or impossible for walkers with step-width d to reach near the global peak when d is a realistic large value: d=1-6, and suggests that the region over the middle in real landscapes may be considerably smooth with small k-values to maintain high evolvability.     

Free radical fragmentation of cardiolipin by cytochrome c

The effect of cytochrome c (cyt c) on degradation of cardiolipin in its polar part was investigated in cardiolipin/phosphatidylcholine (CL/PC) liposomes incubated with cyt c/H₂O₂/and (or) ascorbate by high-performance thin layer chromatography and MALDI-TOF mass spectrometry. It has been shown that phosphatidic acid (PA) and phosphatidylhydroxyacetone (PHA) were formed in the system under conditions where hydrogen peroxide favours a release of heme iron from cyt c. The formation of PA and PHA occurs via an OH-induced fragmentation taking place in the polar moiety of cardiolipin. Formation of fragmentation products correlated with the loss of CL in CL/PC liposomes incubated with cyt c/H₂O₂/ascorbate or with Cu²⁺/H₂O₂/ascorbate.     

New Aspects of the Spontaneous Polymerization of Actin in the Presence of Salts

The mechanism of salt-induced actin polymerization involves the energetically unfavorable nucleation step, followed by filament elongation by the addition of monomers. The use of a bifunctional cross-linker, N,N′-(1,4-phenylene)dimaleimide, revealed rapid formation of the so-called lower dimers (LD) in which actin monomers are arranged in an antiparallel fashion. The filament elongation phase is characterized by a gradual LD decay and an increase in the yield of “upper dimers” (UD) characteristic of F-actin. Here we have used 90° light scattering, electron microscopy, and N,N′-(1,4-phenylene)dimaleimide cross-linking to reinvestigate relationships between changes in filament morphology, LD decay, and increase in the yield of UD during filament growth in a wide range of conditions influencing the rate of the nucleation reaction. The results show irregularity and instability of filaments at early stages of polymerization under all conditions used, and suggest that an earlier documented coassembling of LD with monomeric actin contributes to the initial disordering of the filaments rather than to the nucleation of polymerization. The effects of the type of G-actin-bound divalent cation (Ca²⁺/Mg²⁺), nucleotide (ATP/ADP), and polymerizing salt on the relation between changes in filament morphology and progress in G-actin-to-F-actin transformation show that ligand-dependent alterations in G-actin conformation determine not only the nucleation rate but also the kinetics of ordering of the filament structure in the elongation phase. The time courses of changes in the yield of UD suggest that filament maturation involves cooperative propagation of “proper” interprotomer contacts. Acceleration of this process by the initially bound MgATP supports the view that the filament-destabilizing conformational changes triggered by ATP hydrolysis and P_i liberation during polymerization are constrained by the intermolecular contacts established between MgATP monomers prior to ATP hydrolysis. An important role of contacts involving the DNase-I-binding loop and the C-terminus of actin is proposed.