Cardiolipin Is Dispensable for Oxidative Phosphorylation and Non-fermentative Growth of Alkaliphilic Bacillus pseudofirmus OF4

Cardiolipin (CL), a membrane phospholipid in bacteria and mitochondria, has been hypothesized to facilitate movement of protons on the outer surface of membranes in support of respiration-dependent ATP synthesis, oxidative phosphorylation (OXPHOS). If so, the high levels of membrane CL found in alkaliphilic bacteria, such as Bacillus pseudofirmus OF4, might facilitate its robust OXPHOS at pH 10.5, where the bulk protonmotive (PMF) force is low. To address the role of CL in Bacillus pseudofirmus OF4, we studied strains in which genes (cls) potentially encoding a CL synthase (CLs) were deleted: three single (ΔclsA, ΔclsB, and ΔclsC), one double (ΔclsA/B), and one triple (ΔclsA/B/C) mutant. Two-dimensional thin layer chromatography analyses of lipid extracts from ³²P-labeled strains showed that the wild-type CL content was 15% of total phospholipids at pH 10.5 versus 3% at pH 7.5 during log phase. The % CL was higher (28–33%) at both pH values during stationary phase. The clsA gene plays a major role in CL biosynthesis as no detectable CL was found in ΔclsA-containing mutants, whereas the CL precursor phosphatidylglycerol was elevated. The ΔclsB mutant exhibited no significant reduction in CL, but clsB expression was up-regulated and appeared to support growth at pH 7.5. In the absence of detectable CL, the alkaliphile showed no significant deficits in non-fermentative growth, respiration-dependent ATP synthesis, or salt tolerance. Minor deficits in respiration and ATP synthase assembly were noted in individual mutants. In long term survival experiments, significant growth defects were found in ΔclsA strains and the ΔclsC strain at pH 10.5.     

Diel rhythmicity in amino acid uptake by Prochlorococcus

The marine cyanobacterium Prochlorococcus , the most abundant phototrophic organism on Earth, numerically dominates the phytoplankton in nitrogen (N)-depleted oceanic gyres. Alongside inorganic N sources such as nitrite and ammonium, natural populations of this genus also acquire organic N, specifically amino acids. Here, we investigated using isotopic tracer and flow cytometric cell sorting techniques whether amino acid uptake by Prochlorococcus is subject to a diel rhythmicity, and if so, whether this was linked to a specific cell cycle stage. We observed, in contrast to diurnally similar methionine uptake rates by Synechococcus cells, obvious diurnal rhythms in methionine uptake by Prochlorococcus cells in the tropical Atlantic. These rhythms were confirmed using reproducible cyclostat experiments with a light-synchronized axenic Prochlorococcus (PCC9511 strain) culture and ³⁵S-methionine and ³H-leucine tracers. Cells acquired the tracers at lower rates around dawn and higher rates around dusk despite >10⁴ times higher concentration of ammonium in the medium, presumably because amino acids can be directly incorporated into protein. Leucine uptake rates by cells in the S+G₂ cell cycle stage were consistently 2.2 times higher than those of cells at the G₁ stage. Furthermore, S+G₂ cells upregulated amino acid uptake 3.5 times from dawn to dusk to boost protein synthesis prior to cell division. Because Prochlorococcus populations can account from 13% at midday to 42% at dusk of total microbial uptake of methionine and probably of other amino acids in N-depleted oceanic waters, this genus exerts diurnally variable, strong competitive pressure on other bacterioplankton populations.     

Pacemaker and network mechanisms of rhythm generation: cooperation and competition

The origin of rhythmic activity in brain circuits and CPG-like motor networks is still not fully understood. The main unsolved questions are (i) What are the respective roles of intrinsic bursting and network based dynamics in systems of coupled heterogeneous, intrinsically complex, even chaotic, neurons? (ii) What are the mechanisms underlying the coexistence of robustness and flexibility in the observed rhythmic spatio-temporal patterns? One common view is that particular bursting neurons provide the rhythmogenic component while the connections between different neurons are responsible for the regularisation and synchronisation of groups of neurons and for specific phase relationships in multi-phasic patterns. We have examined the spatio-temporal rhythmic patterns in computer-simulated motif networks of H-H neurons connected by slow inhibitory synapses with a non-symmetric pattern of coupling strengths. We demonstrate that the interplay between intrinsic and network dynamics features either cooperation or competition, depending on three basic control parameters identified in our model: the shape of intrinsic bursts, the strength of the coupling and its degree of asymmetry. The cooperation of intrinsic dynamics and network mechanisms is shown to correlate with bistability, i.e., the coexistence of two different attractors in the phase space of the system corresponding to different rhythmic spatio-temporal patterns. Conversely, if the network mechanism of rhythmogenesis dominates, monostability is observed with a typical pattern of winnerless competition between neurons. We analyse bifurcations between the two regimes and demonstrate how they provide robustness and flexibility to the network performance.     

Fatty acid biosynthesis from glutamate and glutamine is specifically induced in neuronal cells under hypoxia

Hypoxia is involved in many neuronal and non‐neuronal diseases, and defining the mechanisms for tissue adaptation to hypoxia is critical for the understanding and treatment of these diseases. One mechanism for tissue adaptation to hypoxia is increased glutamine and/or glutamate (Gln/Glu) utilization. To address this mechanism, we determined incorporation of Gln/Glu and other lipogenic substrates into lipids and fatty acids in both primary neurons and a neuronal cell line under normoxic and hypoxic conditions and compared this to non‐neuronal primary cells and non‐neuronal cell lines. Incorporation of Gln/Glu into total lipids was dramatically and specifically increased under hypoxia in neuronal cells including both primary (2.0‐ and 3.0‐fold for Gln and Glu, respectively) and immortalized cultures (3.5‐ and 8.0‐fold for Gln and Glu, respectively), and 90% to 97% of this increase was accounted for by incorporation into fatty acids (FA) depending upon substrate and cell type. All other non‐neuronal cells tested demonstrated decreased or unchanged FA synthesis from Gln/Glu under hypoxia. Consistent with these data, total FA mass was also increased in neuronal cells under hypoxia that was mainly accounted for by the increase in saturated and monounsaturated FA with carbon length from 14 to 24. Incorporation of FA synthesized from Gln/Glu was increased in all major lipid classes including cholesteryl esters, triacylglycerols, diacylglycerols, free FA, and phospholipids, with the highest rate of incorporation into triacylglycerols. These results indicate that increased FA biosynthesis from Gln/Glu followed by esterification may be a neuronal specific pathway for adaptation to hypoxia.

     

Validated spectrofluorimetric and spectrophotometric methods for the determination of brimonidine tartrate in ophthalmic solutions via derivatization with NBD‐Cl. Application to stability study

Two simple, selective and accurate methods were developed and validated for the determination of brimonidine tartrate (BT) in pure state and pharmaceutical formulations. Both methods are based on the coupling of the drug with 4‐chloro‐7‐nitro‐2,1,3‐benzoxadiazole in borate buffer (pH 8.5) at 70 °C and measurement of the reaction product spectrophotometrically at 407 nm (method I) or spectrofluorimetrically at 528 nm upon excitation at 460 nm (method II). The calibration graphs were rectilinear over the concentration ranges of 1.0–16.0 and 0.1–4.0 µg/mL with lower detection limits of 0.21 and 0.03, and lower quantification limits of 0.65 and 0.09 µg/mL for methods I and II, respectively. Both methods were successfully applied to the analysis of commercial ophthalmic solution with mean recovery of 99.50 ± 1.00 and 100.13 ± 0.71%, respectively. Statistical analysis of the results obtained by the proposed methods revealed good agreement with those obtained using a comparison method. The proposed spectrofluorimetric method was extended to a stability study of BT under different ICH‐outlined conditions such as alkaline, acidic, oxidative and photolytic degradation. Furthermore, the kinetics of oxidative degradation of the drug was investigated and the apparent first‐order reaction rate constants, half‐life times and Arrhenius equation were estimated. The proposed methods are practical and valuable for routine applications in quality control laboratories for the analysis of BT. Copyright © 2014 John Wiley & Sons, Ltd.     

Prebiotic Syntheses Under Shock in the Water – Formamide – Potassium Bicarbonate – Sodium Hydroxide System

Origins of Life and Evolution of Biospheres - Syntheses under shock in nitrogen bubbled samples of the water – formamide – bicarbonate – sodium hydroxide system at...     

Kinetics of actin unfolding induced by guanidine hydrochloride.

The kinetics of actin unfolding induced by guanidine hydrochloride has been studied. On the basis of obtained experimental data a new kinetic pathway of actin unfolding was proposed. We have shown that the transition from native to inactivated actin induced by guanidine hydrochloride (GdnHCl) passes through essential unfolding of the protein. This means that inactivated actin should be considered as the off-pathway species rather than an intermediate conformation between native and completely unfolded states of actin, as has been assumed earlier. The rate constants of the transitions that give rise to the inactivated actin were determined. At 1.0-2.0 M GdnHCl the value of the rate constant of the transition from native to essentially unfolded actin exceeds that of the following step of inactivated actin formation. It leads to the accumulation of essentially unfolded macromolecules early in the unfolding process, which in turn causes the minimum in the time dependencies of tryptophan fluorescence intensity, parameter A, characterizing the intrinsic fluorescence spectrum position, and tryptophan fluorescence anisotropy.     

Infrastructure and the environment in the Anthropocene

For centuries, man‐made infrastructure has been viewed as separate from natural systems. Yet in the past few centuries, as the scale and scope of human activities have dramatically increased, there is accumulating evidence that natural systems are becoming increasingly, and in some cases entirely, managed by humans. The dichotomy between infrastructure and the environment is narrowing, and natural systems are increasingly becoming human design spaces. This is already apparent with the management of hydrologic systems for urban water supply, wildlife, agriculture, forests, and even the atmosphere, and we can expect management of the environment to become more so as human activities grow. Yet our infrastructure largely remains obdurate. They are designed to last for long times even as changes in the environment and technology accelerate. As such, our current infrastructure paradigms fail at the level of the complex, integrated systems and behaviors that characterize the Anthropogenic Earth. Infrastructure in the future will need to be designed for adaptive capacity and the complexities associated with techno‐environmental systems.     

Social correlates of stress in adult males of the great gerbil,Rhombomys opimus,in years of high and low population densities

The great gerbil, Rhombomys opimus, is the most social species in the Gerbillinae. The social structure consists of family groups that occupy isolated systems of burrows consisting of one breeding male, from one to seven females, and juveniles. During a year of peak density and one of density decline, we studied the influence of group size, group composition, local density, and distance to the nearest groups on fecal corticosterone and testosterone concentrations in breeding males. We also examined the relationship of hormone concentrations to the survival of males during the summer drought between the spring and the fall. We found that males differed in concentrations of steroid hormones. Concentrations of testosterone were lower whereas those of corticosterone tended to be higher in a year of high population densities compared with higher testosterone and lower corticosterone in a year with a lower density. This finding suggests that stress may be greater in higher densities because of increased social contact. Stepwise regression analysis revealed a positive and significant influence of the number of adult females in a family group on concentrations of fecal corticosterone and testosterone in adult males. Concentrations of corticosterone were also significantly higher in males that disappeared from family groups between the spring and the fall compared with males still alive in family groups in the fall. There was no change in concentrations of testosterone. These results suggest that social interactions within large family groups may be an important source of stress for adult males.