Effect of CDP-choline on age-dependent modifications of energy- and glutamate-linked enzyme activities in synaptic and non-synaptic mitochondria from rat cerebral cortex

The effect of aging and CDP-choline treatment (20 mg kg⁻¹ body weight i.p. for 28 days) on the maximal rates (V_max) of representative mitochondrial enzyme activities related to Krebs’ cycle (citrate synthase, α-ketoglutarate dehydrogenase, malate dehydrogenase), glutamate and related amino acid metabolism (glutamate dehydrogenase, glutamate–oxaloacetate- and glutamate–pyruvate transaminases) were evaluated in non-synaptic and intra-synaptic “light” and “heavy” mitochondria from frontal cerebral cortex of male Wistar rats aged 4, 12, 18 and 24 months.     

An activation gating switch in Kv1.2 is localized to a threonine residue in the S2-S3 linker

The activation properties of Kv1.2 channels are highly variable, with reported half-activation (V_1/2) values ranging from ∼−40 mV to ∼+30 mV. Here we show that this arises because Kv1.2 channels occupy two distinct gating modes (“fast” and “slow”). “Slow” gating (τ_act = 90 ± 6 ms at +35 mV) was associated with a V_1/2 of activation of +16.6 ± 1.1 mV, whereas “fast” gating (τ_act = 4.5 ± 1.7 ms at +35 mV) was associated with a V_1/2 of activation of −18.8 ± 2.3 mV. It was possible to switch between gating modes by applying a prepulse, which suggested that channels activate to a single open state along separate “fast” and “slow” activation pathways. Using chimeras and point mutants between Kv1.2 and Kv1.5 channels, we determined that introduction of a positive charge at or around threonine 252 in the S2-S3 linker of Kv1.2 abolished “slow” activation gating. Furthermore, dialysis of the cytoplasm or excision of cell-attached patches from cells expressing Kv1.2 channels switched gating from “slow” to “fast”, suggesting involvement of cytoplasmic regulators. Collectively, these results demonstrate two modes of activation gating in Kv1.2 and specific residues in the S2-S3 linker that act as a switch between these modes.     

Characterisation of physiological and immunological differences between Pacific oysters (Crassostrea gigas) genetically selected for high or low survival to summer mortalities and fed different rations under controlled conditions

Within the framework of a national scientific program named “MORtalités ESTivales de l'huître creuse Crassostrea gigas” (MOREST), a family-based experiment was developed to study the genetic basis of resistance to summer mortality in the Pacific oyster, Crassostrea gigas. As part of the MOREST project, the second generation of three resistant families and two susceptible families were chosen and pooled into two respective groups: “R” and “S”. These two groups of oysters were conditioned for 6 months on two food levels (4% and 12% of oyster soft-tissue dry weight in algal dry weight per day) with a temperature gradient that mimicked the Marennes-Oléron natural cycle during the oyster reproductive period. Oyster mortality remained low for the first two months, but then rapidly increased in July when seawater temperature reached 19 °C and above. Mortality was higher in “S” oysters than in “R” oysters, and also higher in oysters fed the 12% diet than those fed 4%, resulting in a decreasing, relative order in cumulative mortality as follows; 12% “S” > 12% “R” > 4% “S” > 4% “R”. Although the observed mortality rates were lower than those previously observed in the field, the mortality differential between “R” and “S” oysters was similar. Gonadal development, estimated by tissue lipid content, followed a relative order yielding a direct, positive relationship between reproductive effort and mortality as we reported precedently by quantitative histology. Regarding hemocyte parameters, one of the most striking observations was that reactive oxygen species (ROS) production was significantly higher in “S” oysters than in “R” oysters in May and June, regardless of food level. The absence of known environmental stress under these experimental conditions suggests that the ROS increase in “S” oyster could be related to their higher reproductive activity. Finally, a higher increase in hyalinocyte counts was observed for”S” oysters, compared to “R” oysters, in July, just before mortality. Taken together, our results suggest an association of genetically based resistance to summer mortality, reproductive strategy and hemocyte parameters.     

Local spin dynamics in magnetic molecular chains studied by NMR and μSR

We present NMR and μ⁺SR study of spin dynamics in one-dimensional and quasi-one-dimensional molecular magnets of recent synthesis. In particular, we focus on the so called Gd(hfac)₃NIT-R and CoPhOMe magnetic chains families. For Gd-R helimagnets we show some differences between “weakly frustrated systems” and “fully frustrated systems”. The different behaviour is due to the different radical inserted in the chains (R = Me, Ph for “weakly frustrated systems” and R = iPr, Et for “fully frustrated systems”). The existence of different phase transitions, particularly to 3D long-range magnetic order in Gd-Ph and to chiral order in Gd-iPr, is remarked together with a comparison between results obtained from macroscopic and microscopic investigating techniques. As regards CoPhOMe slowly relaxing chain, the ¹H NMR measurements confirm the freezing of the spins at low temperature, which prevents the 3D long-range order, and display the presence of two relaxation mechanisms related to distinct contributions to the local spin relaxation.     

Antiparallel Conformation of Knob and Hole Aglycosylated Half-Antibody Homodimers Is Mediated by a CH2–CH3 Hydrophobic Interaction

Bispecific antibody and antibody-like molecules are of wide interest as potential therapeutics that can recognize two distinct targets. Among the variety of ways such molecules have been engineered is by creating “knob” and “hole” heterodimerization sites in the CH3 domains of two antibody heavy chains. The molecules produced in this manner maintain their biological activities while differing very little from the native human IgG sequence. To better understand the knob-into-hole interface, the molecular mechanism of heterodimerization, and to engineer Fc domains that could improve the assembly and purity of heterodimeric reaction products, we sought crystal structures of aglycosylated heterodimeric and homodimeric “knob” and “hole” Fc fragments derived from bacterial expression. The structure of the knob-into-hole Fc was determined at 2.64 Å. Except for the sites of mutation, the structure is very similar to that of the native human IgG1 Fc, consistent with a heterodimer interaction kinetic K_D of < 1 nM. Homodimers of the “knob” and “hole” mutants were also obtained, and their X-ray structures were determined at resolutions 2.5 Å and 2.1 Å, respectively. Both kinds of homodimers adopt a head-to-tail quaternary structure and thus do not contain direct knob/knob or hole/hole CH3 interactions. The head-to-tail arrangement was disfavored by adding site-directed mutations at F241 and F243 in the CH2 domains, leading to increases in both rate and efficiency of bispecific (heterodimer) assembly.     

Effect of temperature, pH and buffer presence on ethanol production from synthesis gas by "Clostridium ragsdalei"

Fermentation pH, incubation temperature, and presence or absence of media buffer can alter the activity of microorganisms. For instance, carbon monoxide and hydrogen components of syngas show decreased solubility with increasing temperature, Clostridium species preferentially switch from acetogenesis to solventogenesis phase at pH below 5.0, and morpholinoethanesulfonic acid (MES) added as media buffer has been shown to increase lag time for ethanol production. The objective of the present study was to determine the effects of temperature, pH and MES buffer on ethanol production by “Clostridium ragsdalei”. This study showed syngas fermentation using “Clostridium ragsdalei” at 32 °C with media without buffer was associated with higher ethanol concentration and reduced lag time in switching to solventogenesis. Temperature above 40 °C and pH below 5.0 were outside the optimal range for growth and metabolism of the bacteria.     

The "electrostatic-switch" mechanism: Monte Carlo study of MARCKS-membrane interaction

The binding of the myristoylated alanine-rich C kinase substrate (MARCKS) to mixed, fluid, phospholipid membranes is modeled with a recently developed Monte Carlo simulation scheme. The central domain of MARCKS is both basic (ζ = +13) and hydrophobic (five Phe residues), and is flanked with two long chains, one ending with the myristoylated N-terminus. This natively unfolded protein is modeled as a flexible chain of “beads” representing the amino acid residues. The membranes contain neutral (ζ = 0), monovalent (ζ = −1), and tetravalent (ζ = −4) lipids, all of which are laterally mobile. MARCKS-membrane interaction is modeled by Debye-Hückel electrostatic potentials and semiempirical hydrophobic energies. In agreement with experiment, we find that membrane binding is mediated by electrostatic attraction of the basic domain to acidic lipids and membrane penetration of its hydrophobic moieties. The binding is opposed by configurational entropy losses and electrostatic membrane repulsion of the two long chains, and by lipid demixing upon adsorption. The simulations provide a physical model for how membrane-adsorbed MARCKS attracts several PIP₂ lipids (ζ = −4) to its vicinity, and how phosphorylation of the central domain (ζ = +13 to ζ = +7) triggers an “electrostatic switch”, which weakens both the membrane interaction and PIP₂ sequestration. This scheme captures the essence of “discreteness of charge” at membrane surfaces and can examine the formation of membrane-mediated multicomponent macromolecular complexes that function in many cellular processes.     

Metabolic memory effect of the saturated fatty acid, palmitate, in monocytes

Hyperglycaemia has a deferred detrimental effect on glucose metabolism, termed “metabolic memory”. Elevated saturated fatty acids promote insulin resistance, hyperglycaemia and associated atherosclerotic complications, but their effect on “metabolic memory” is unknown. Therefore we investigated whether basal and insulin-stimulated (10⁻⁶ M for 12 h) glucose (2-deoxy-d-[³H]-glucose) uptake was affected by palmitate pre-treatment human THP-1 monocytes. Palmitate-induced a time-dependent and concentration-dependent inhibition of insulin-stimulated glucose uptake, showing almost complete abolition of the insulin-stimulatory effect with 300 μM palmitate. Basal glucose uptake was unaffected by palmitate. When palmitate was washed out, the inhibitory effect on insulin-stimulated glucose uptake persisted for at least 60 h.     

Recruitment abundance estimation: Role of glass eel (Anguilla anguilla L.) response to light

Most fish populations are declining worldwide and their management would benefit from a better estimation of recruitment. In glass eels, field studies suggest that estuarine migratory glass eels are sensitive enough to light to change their vertical location according to factors such as water turbidity and/or moon brightness. The response of glass eel (Anguilla anguilla L.) to light was tested in the laboratory using boxes where fish could choose between a lit and an unlit side. Responses were quantified as the proportion of glass eels remaining in the unlit chamber. Decreasing light levels were used and tested on different “age” glass eels (“age” in days since capture). In addition, measures of light at different depths of the water column were carried out in the Adour estuary (43°30′ N, 1°30′ W). The glass eel light avoidance level was lower in non-pigmented glass eel (less than 10 ^− 10 W cm ^− 2), than in pigmented ones (10 ⁻⁹-10 ^− 8 W cm ^− 2). These results and field data on the measurement of light energy in the water column of Adour estuary are compared with previously published data on the estuarine migration of glass eel.     

E-NTPDase and E-ADA activities in lymphocytes associated with the immune response of rats experimentally infected with Toxoplasma gondii

An investigation of E-NTPDase and E-ADA activities in lymphocytes from rats experimentally infected with Toxoplasma gondii was carried out in this study. For this purpose, twenty four adult male Wistar rats were divided in two groups/four subgroups (A1 and A2; B1 and B2–6 animal/each group), with “A” as uninfected and “B” inoculated with T. gondii (RH strain). Sampling was performed on days 5 and 10 post-infection (p.i.), with evaluation of hemogram, immunoglobulins (IgM and IgG) and activity of E-NTPDase and E-ADA in lymphocytes. Enzymes essays showed ATP hydrolysis increased on days 5 (P < 0.05) and 10 (P < 0.01) p.i., as well as an increase of ADP hydrolysis on day 10 (P < 0.01) p.i. E-ADA activity on lymphocytes was also increased in both evaluated periods (P < 0.01). Based on E-NTPDase and E-ADA increased activities observed on lymphocytes, it is possible to suggest their involvement in an anti-inflammatory response, consisting of a modulatory response, preventing excessive tissue damage caused by the infection with Toxoplasma gondii.     

High production of cold-tolerant chitinases on shrimp wastes in bench-top bioreactor by the Antarctic fungus Lecanicillium muscarium CCFEE 5003: Bioprocess optimization and characterization of two main enzymes

The Antarctic fungus Lecanicillium muscarium CCFEE-5003 was preliminary cultivated in shaken flasks to check its chitinase production on rough shrimp and crab wastes. Production on shrimp shells was much higher than that on crab shells (104.6 ± 9.3 and 48.6 ± 3.1 U/L, respectively). For possible industrial applications, bioprocess optimization was studied on shrimp shells in bioreactor using RSM to state best conditions of pH and substrate concentration. Optimization improved the production by 137% (243.6 ± 17.3). Two chitinolytic enzymes (CHI1 and CHI2) were purified and characterized. CHI1 (MW ca. 61 kDa) showed optima at pH 5.5 and 45 °C while CHI2 (MW ca. 25 kDa) optima were at pH 4.5 and 40 °C. Both enzymes maintained high activity levels at 5 °C and were inhibited by Fe⁺⁺, Hg⁺⁺ and Cu⁺⁺. CHI2 was strongly allosamidin-sensitive. Both proteins were N-acetyl-hexosaminidases (E.C. 3.2.1.52) but showed different roles in chitin hydrolysis: CHI1 could be defined as “chitobiase” while CHI2 revealed a main “eso-chitinase” activity.     

Cultivation of the first mesophilic representative ("mesotoga") within the order Thermotogales

Cultivated members of the order Thermotogales comprise only thermophilic to hyperthermophilic anaerobic microorganisms. However, based on molecular studies, the existence of mesophilic members (“mesotoga”) within this order has been postulated but has not been demonstrated by cultural approaches so far. A “mesotoga” (strain PhosAc3) that belonged to an uncultivated lineage distantly related to the thermophilic Kosmotoga genus has now been cultivated in axenic culture. It grew between 30 °C and 50 °C (optimum 40 °C) and oxidized lactate using elemental sulphur as a terminal electron acceptor. Further genomic and physiological characterization of strain PhosAc3 will be important not only for understanding bacterial adaptation to high and moderate temperatures at small evolutionary scales, but also because “mesotoga” might play a crucial ecological role in ecosystems polluted by aromatic compounds.     

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.     

Early senescence in heterozygous ABCA1 mutation skin fibroblasts: A gene dosage effect beyond HDL deficiency?

Purpose

Homozygous ABCA1 gene mutation causes Tangier disease (TD). The effects reported in heterozygous state regard plasma HDL, cell cholesterol efflux and coronary artery disease. We investigated whether in vitro replicative skin fibroblast senescence shown in TD proband (Hom), his father (Het), and in a healthy control might be induced in a “gene-dosage way”.

Methods

Senescence was evaluated by staining test for β-Galactosidase and telomere length (TL) on fibroblast DNA at different replicative stages. ABCG1 and LDLR (low density lipoprotein receptor) gene expression was also evaluated.

Results

Hom cells showed early senescent morphology and reduced growth at all passages in vitro. The cell positive percentage for β-Galactosidase test was highly increased in Hom compared to Het cells at late replicative status (66.1% vs 41.3% respectively). TL was significantly shorter at high stage either in Hom (p < 0.0001) or in Het (p < 0.005). At early replication cycles ABCG1 gene expression was about 3-fold higher in Hom compared to Het cells (0.44 vs 0.14 arbitrary unit).

Conclusions

ABCA1 gene mutation may have “gene-dosage way” effect on in vitro fibroblast senescence. Furthermore, increased ABCG1 and LDLR gene expression could highlight a role of ABCA1 on cytoskeleton regulation associated to cell cholesterol metabolism.     

Mutations in Desmin's Carboxy-Terminal “Tail” Domain Severely Modify Filament and Network Mechanics

Inherited mutations in the gene coding for the intermediate filament protein desmin have been demonstrated to cause severe skeletal and cardiac myopathies. Unexpectedly, some of the mutated desmins, in particular those carrying single amino acid alterations in the non-α-helical carboxy-terminal domain (“tail”), have been demonstrated to form apparently normal filaments both in vitro and in transfected cells. Thus, it is not clear if filament properties are affected by these mutations at all. For this reason, we performed oscillatory shear experiments with six different desmin “tail” mutants in order to characterize the mesh size of filament networks and their strain stiffening properties. Moreover, we have carried out high-frequency oscillatory squeeze flow measurements to determine the bending stiffness of the respective filaments, characterized by the persistence length l_p. Interestingly, mesh size was not altered for the mutant filament networks, except for the mutant DesR454W, which apparently did not form proper filament networks. Also, the values for bending stiffness were in the same range for both the “tail” mutants (l_p = 1.0-2.0 μm) and the wild-type desmin (l_p = 1.1 ± 0.5 μm). However, most investigated desmin mutants exhibited a distinct reduction in strain stiffening compared to wild-type desmin and promoted nonaffine network deformation. Therefore, we conclude that the mutated amino acids affect intrafilamentous architecture and colloidal interactions along the filament in such a way that the response to applied strain is significantly altered.In order to explore the importance of the “tail” domain as such for filament network properties, we employed a “tail”-truncated desmin. Under standard conditions, it formed extended regular filaments, but failed to generate strain stiffening. Hence, these data strongly indicate that the “tail” domain is responsible for attractive filament-filament interactions. Moreover, these types of interactions may also be relevant to the network properties of the desmin cytoskeleton in patient muscle.     

Combined effect of oxidative stress-related gene polymorphisms on atherosclerosis

It is well known that oxidative stress plays critical roles in the pathogenesis of atherosclerosis. In this study, we enrolled 1746 type 2 diabetic subjects, determined 4 common genetic variants related to oxidative stress (glutamate-cysteine ligase modifier subunit (GCLM) C-588T, myeloperoxidase G-463A, human paraoxonase 1 Gln192Arg and NAD(P)H oxidase p22phox C242T polymorphisms), and measured carotid intima-media thickness (IMT) as a surrogate marker for atherosclerosis. GCLM C-588T polymorphism was associated with average IMT (AveIMT) (r = 0.090, p = 0.0008), but the association between the other 3 polymorphisms and AveIMT did not reach the statistical significance. However, AveIMT was significantly greater as the total number of 4 concomitant “pro-oxidant alleles” in each subject was increased (r = 0.108, p < 0.0001). Furthermore, the number of “pro-oxidant alleles” was a risk factor for a high AveIMT independently of conventional risk factors (p = 0.0003). In conclusion, accumulation of oxidative stress-associated alleles was associated with carotid atherosclerosis in type 2 diabetic patients.     

Pitfalls in comparisons of genetic distances: a case study of the avian family Acrocephalidae

Genetic distances are increasingly being used for identification and species delimitation, especially since the introduction of “barcoding”. While for phylogenetic inferences great care is generally taken to choose the best-fit evolutionary model, this is usually neglected in calculating genetic distances. Moreover, distances obtained from others than best-fit models, different lengths of sequences, and even different loci are often freely compared. We examined the influence of different methods on calculating genetic distances using mitochondrial cytochrome b sequences for the passerine family Acrocephalidae.We found substantial differences between: (1) corrected distances based on the best-fit model (TrN + Γ) vs. uncorrected p-distances; (2) distances calculated based on different parts of the same gene; and (3) distances calculated using the methods of “complete deletion” vs. “pairwise deletion” for sequences that included uncertain nucleotides. All these methodological differences affected comparisons between species and potential taxonomical conclusions.We suggest that (1) different loci are incomparable. (2) Only perfectly homologous regions (same length, same part of locus) should be compared. (3) In the case of sequences with some uncertain nucleotides, only distances calculated by the method of “complete deletion” are fully comparable. (4) Only distances based on the optimal substitution model should be used. (5) Even within the same locus, corrected genetic distances are unique to the study in which they are calculated, as they are conditional on the particular dataset and model selected for that dataset.     

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.     

A structural investigation of complex I and I + III2 supercomplex from Zea mays at 11-13 Å resolution: Assignment of the carbonic anhydrase domain and evidence for structural heterogeneity within complex I

The projection structures of complex I and the I + III₂ supercomplex from the C₄ plant Zea mays were determined by electron microscopy and single particle image analysis to a resolution of up to 11 Å. Maize complex I has a typical L-shape. Additionally, it has a large hydrophilic extra-domain attached to the centre of the membrane arm on its matrix-exposed side, which previously was described for Arabidopsis and which was reported to include carbonic anhydrase subunits. A comparison with the X-ray structure of homotrimeric γ-carbonic anhydrase from the archaebacterium Methanosarcina thermophila indicates that this domain is also composed of a trimer. Mass spectrometry analyses allowed to identify two different carbonic anhydrase isoforms, suggesting that the γ-carbonic anhydrase domain of maize complex I most likely is a heterotrimer. Statistical analysis indicates that the maize complex I structure is heterogeneous: a less-abundant “type II” particle has a 15 Å shorter membrane arm and an additional small protrusion on the intermembrane-side of the membrane arm if compared to the more abundant “type I” particle. The I + III₂ supercomplex was found to be a rigid structure which did not break down into subcomplexes at the interface between the hydrophilic and the hydrophobic arms of complex I. The complex I moiety of the supercomplex appears to be only of “type I”. This would mean that the “type II” particles are not involved in the supercomplex formation and, hence, could have a different physiological role.