Light sensitivity of the ciliate Tetrahymena vorax induced by the fluorescent dye acridine orange

The ciliate Tetrahymena vorax is normally insensitive to light. However, after uptake of acridine orange, blue light evokes instant backward swimming. The dye accumulates mainly in posterior vacuoles, with half-maximal uptake after 1 min. Illumination for 10 s induced a depolarisation of approximately 15 mV lasting less than 2 s, followed by a sustained hyperpolarisation of approximately 20 mV. Deciliated cells displayed a similar response. The hyperpolarisation was linked to reduced membrane resistance, showed a reversal potential of approximately −55 mV and was blocked by 1 mmol l⁻¹ TEA. The rate of rise of electrically evoked Ca²⁺-spikes was reduced during the hyperpolarisation, which is compatible with elevated cytosolic Ca²⁺ concentration. This suggests that the hyperpolarisation may be caused by activation of Ca²⁺-sensitive K⁺ channels. The depolarisation was abolished in Ca²⁺-free medium, whereas the hyperpolarisation was unaffected. Illumination for 2 s, or prolonged stimulation restricted to the anterior part of the cell, induced depolarisation only. Illumination of the posterior part caused delayed hyperpolarisation with no preceding depolarisation. We conclude that the induced backward swimming is associated with Ca²⁺ influx through anterior channels, while Ca²⁺ released from intracellular stores activates K⁺ channels responsible for the delayed hyperpolarisation.     

Fast mass spectrometry-based enantiomeric excess determination of proteinogenic amino acids

A rapid determination of the enantiomeric excess of proteinogenic amino acids is of great importance in various fields of chemical and biologic research and industries. Owing to their different biologic effects, enantiomers are interesting research subjects in drug development for the design of new and more efficient pharmaceuticals. Usually, the enantiomeric composition of amino acids is determined by conventional analytical methods such as liquid or gas chromatography or capillary electrophoresis. These analytical techniques do not fulfill the requirements of high-throughput screening due to their relative long analysis times. The method presented allows a fast analysis of chiral amino acids without previous time consuming chromatographic separation. The analytical measurements base on parallel kinetic resolution with pseudoenantiomeric mass tagged auxiliaries and were carried out by mass spectrometry with electrospray ionization. All 19 chiral proteinogenic amino acids were tested and Pro, Ser, Trp, His, and Glu were selected as model substrates for verification measurements. The enantiomeric excesses of amino acids with non-polar and aliphatic side chains as well as Trp and Phe (aromatic side chains) were determined with maximum deviations of the expected value less than or equal to 10ee%. Ser, Cys, His, Glu, and Asp were determined with deviations lower or equal to 14ee% and the enantiomeric excess of Tyr were calculated with 17ee% deviation. The total screening process is fully automated from the sample pretreatment to the data processing. The method presented enables fast measurement times about 1.38 min per sample and is applicable in the scope of high-throughput screenings.     

Wildlife trade products available to U.S. military personnel serving abroad

Military personnel and affiliates have significant buying power that can influence demand for wildlife products. Purchase and transport of certain wildlife products violates United States laws, military regulations, and national country laws where the items were purchased. We surveyed military bazaars (n = 4) in Kabul, Afghanistan from June 2007 to March 2009 to observe which species were available to soldiers. In June 2008, we conducted a pilot survey of U.S. Army personnel (n = 371) stationed at Fort Drum, New York, USA, who had been deployed or stationed overseas including in Afghanistan and Iraq. Soldiers reported skins of wild felids and gray wolf Canis lupus as most commonly observed wildlife products available for sale in Afghanistan. Forty percent of respondents said they had either purchased or seen other members of the military purchase or use wildlife products. The U.S. military was willing to assist in curtailing supply and demand for wildlife products in order to protect soldiers from unknowingly breaking the law and to conserve wildlife in the countries where they serve. Regular, focused training of military personnel should be considered an important step to reducing trade in wildlife products by addressing both demand and market supply.     

The Role of Protein-Ligand Contacts in Allosteric Regulation of the Escherichia coli Catabolite Activator Protein

Allostery is a fundamental process by which ligand binding to a protein alters its activity at a distant site. Both experimental and theoretical evidence demonstrate that allostery can be communicated through altered slow relaxation protein dynamics without conformational change. The catabolite activator protein (CAP) of Escherichia coli is an exemplar for the analysis of such entropically driven allostery. Negative allostery in CAP occurs between identical cAMP binding sites. Changes to the cAMP-binding pocket can therefore impact the allosteric properties of CAP. Here we demonstrate, through a combination of coarse-grained modeling, isothermal calorimetry, and structural analysis, that decreasing the affinity of CAP for cAMP enhances negative cooperativity through an entropic penalty for ligand binding. The use of variant cAMP ligands indicates the data are not explained by structural heterogeneity between protein mutants. We observe computationally that altered interaction strength between CAP and cAMP variously modifies the change in allosteric cooperativity due to second site CAP mutations. As the degree of correlated motion between the cAMP-contacting site and a second site on CAP increases, there is a tendency for computed double mutations at these sites to drive CAP toward noncooperativity. Naturally occurring pairs of covarying residues in CAP do not display this tendency, suggesting a selection pressure to fine tune allostery on changes to the CAP ligand-binding pocket without a drive to a noncooperative state. In general, we hypothesize an evolutionary selection pressure to retain slow relaxation dynamics-induced allostery in proteins in which evolution of the ligand-binding site is occurring.     

JNK signaling is needed to tolerate chromosomal instability

Chromosomal instability (CIN), as a common feature of tumors, represents a potential therapeutic target if ways can be found to specifically cause apoptosis in unstably dividing cells. We have previously shown that if signaling through the JNK pathway is reduced, apoptosis is triggered in models of chromosomal instability induced by loss of the spindle checkpoint. Here we identify components upstream and downstream of JNK that are able to mediate this effect, and test the involvement of p53 and DNA damage in causing apoptosis when JNK signaling is reduced in CIN cells. We show that cell cycle progression timing has a strong effect on the apoptosis seen when JNK signaling is reduced in genetically unstable cells: a shortened G₂ phase enhances the apoptosis, while lengthening G₂ rescues the JNK-deficient CIN cell death phenotype. Our findings suggest that chromosomal instability represents a significant stress to dividing cells, and that without JNK signaling, cells undergo apoptosis because they lack a timely and effective response to DNA damage.     

Characterization of a Mycobacterium tuberculosis Nanocompartment and Its Potential Cargo Proteins

Mycobacterium tuberculosis has evolved various mechanisms by which the bacterium can maintain homeostasis under numerous environmental assaults generated by the host immune response. M. tuberculosis harbors enzymes involved in the oxidative stress response that aid in survival during the production of reactive oxygen species in activated macrophages. Previous studies have shown that a dye-decolorizing peroxidase (DyP) is encapsulated by a bacterial nanocompartment, encapsulin (Enc), whereby packaged DyP interacts with Enc via a unique C-terminal extension. M. tuberculosis also harbors an encapsulin homolog (CFP-29, Mt-Enc), within an operon with M. tuberculosis DyP (Mt-DyP), which contains a C-terminal extension. Together these observations suggest that Mt-DyP interacts with Mt-Enc. Furthermore, it has been suggested that DyPs may function as either a heme-dependent peroxidase or a deferrochelatase. Like Mt-DyP, M. tuberculosis iron storage ferritin protein, Mt-BfrB, and an M. tuberculosis protein involved in folate biosynthesis, 7,8-dihydroneopterin aldolase (Mt-FolB), have C-terminal tails that could also interact with Mt-Enc. For the first time, we show by co-purification and electron microscopy that mycobacteria via Mt-Enc can encapsulate Mt-DyP, Mt-BfrB, and Mt-FolB. Functional studies of free or encapsulated proteins demonstrate that they retain their enzymatic activity within the Mt-Enc nanocompartment. Mt-DyP, Mt-FolB, and Mt-BfrB all have antioxidant properties, suggesting that if these proteins are encapsulated by Mt-Enc, then this nanocage may play a role in the M. tuberculosis oxidative stress response. This report provides initial structural and biochemical clues regarding the molecular mechanisms that utilize compartmentalization by which the mycobacterial cell may aid in detoxification of the local environment to ensure long term survival.     

The ethics of ectogenesis-aided foetal treatment

In this paper, we aim to stimulate ethical debate about the morally relevant connection between ectogenesis and the foetus as a potential beneficiary of treatment. Ectogenesis could facilitate foetal interventions by treating the foetus independently of the pregnant woman and provide easier access to the foetus if interventions are required. The moral relevance hereof derives from the observation that, together with other developments in genetic technology and prenatal treatment, this may catalyse the allocation of a patient status to the foetus. The topic of foetal medicine is of growing interest to clinicians, and it also deserves due attention from an ethical perspective. To the extent that these developments contribute to the allocation of a patient status to the foetus (and to its respective interests for medical treatment), normative questions arise about how moral responsibilities towards foetal interests should be balanced against the interests of the pregnant woman. We conclude that, even if ectogenesis could facilitate foetal therapy, it is important to remain sensitive to the fact that it would not circumvent the key ethical concerns that come with in utero foetal treatment and that it may even exacerbate potential conflicts between directive treatment recommendations and the pregnant woman’s autonomous decision to the contrary.     

The solubilization of the L and M antigens from sheep red cell membranes

The Lp, L1 and M antigens from sheep red cells were solubilized using the non-ionic detergent Triton X-100 in the presence of dithiothreitol. Recovery rates were improved when membranes were sonicated at 4 degrees C in the presence of the detergent; values in the range 16-25% (M) and 9-17% (Lp and L1) were achieved for recovery.