Directed evolution of a highly active Yersinia mollaretii phytase

Phytase improves as a feed supplement the nutritional quality of phytate-rich diets (e.g., cereal grains, legumes, and oilseeds) by hydrolyzing indigestible phytate (myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate) and increasing abdominal absorption of inorganic phosphates, minerals, and trace elements. Directed phytase evolution was reported for improving industrial relevant properties such as thermostability (pelleting process) or activity. In this study, we report the cloning, characterization, and directed evolution of the Yersinia mollaretii phytase (Ymphytase). Ymphytase has a tetrameric structure with positive cooperativity (Hill coefficient was 2.3) and a specific activity of 1,073?U/mg which is ～10 times higher than widely used fungal phytases. High-throughput prescreening methods using filter papers or 384-well microtiter plates were developed. Precise subsequent screening for thermostable and active phytase variants was performed by combining absorbance and fluorescence-based detection system in 96-well microtiter plates. Directed evolution yielded after mutant library generation (SeSaM method) and two-step screening (in total ～8,400 clones) a phytase variant with ～20% improved thermostability (58°C for 20?min; residual activity wild type ～34%; variant ～53%) and increased melting temperature (1.5°C) with a slight loss of specific activity (993?U/mg).     

Bcl-2 is a monomeric protein: prevention of homodimerization by structural constraints

The pro-apoptotic activity of the Bcl-2 family member Bax has been shown to be facilitated by homodimerization. However, it is unknown whether Bcl-2 or Bcl-x(L) have to homodimerize to protect cells from apoptosis. Here we show by co-immunoprecipitation and FPLC analyses that while Bax multimerizes and forms heterodimers with Bcl-2, there is no evidence for Bcl-2 homodimerization, even in conditions under which Bcl-2 protects cells from apoptosis. Immunofluorescence studies confirmed that Bax can attract active, soluble Bcl-2 to mitochondrial membranes, but that nuclear/ER membrane-bound Bcl-2 was incapable of dislocating soluble Bcl-2. The failure of Bcl-2 to homodimerize is due to structural constraints as versions of Bcl-2 deleted or mutated in the BH1 and BH2 domains effectively dimerized with wild-type Bcl-2 and were dislocated by Bcl-2 inside cells. These data indicate that naturally occurring Bcl-2 does not expose protein domains that mediate homodimerization and therefore most likely acts as a monomer to protect cells from apoptosis.     

Molecular basis of bacterial resistance to chloramphenicol and florfenicol

Chloramphenicol (Cm) and its fluorinated derivative florfenicol (Ff) represent highly potent inhibitors of bacterial protein biosynthesis. As a consequence of the use of Cm in human and veterinary medicine, bacterial pathogens of various species and genera have developed and/or acquired Cm resistance. Ff is solely used in veterinary medicine and has been introduced into clinical use in the mid-1990s. Of the Cm resistance genes known to date, only a small number also mediates resistance to Ff. In this review, we present an overview of the different mechanisms responsible for resistance to Cm and Ff with particular focus on the two different types of chloramphenicol acetyltransferases (CATs), specific exporters and multidrug transporters. Phylogenetic trees of the different CAT proteins and exporter proteins were constructed on the basis of a multisequence alignment. Moreover, information is provided on the mobile genetic elements carrying Cm or Cm/Ff resistance genes to provide a basis for the understanding of the distribution and the spread of Cm resistance--even in the absence of a selective pressure imposed by the use of Cm or Ff.     

The association between exposure determined by radiofrequency personal exposimeters and human exposure: A simulation study

The selection of an adequate exposure assessment approach is imperative for the quality of epidemiological studies. The use of personal exposimeters turned out to be a reasonable approach to determine exposure profiles, however, certain limitations regarding the absolute values delivered by the devices have to be considered. Apart from the limited dynamic range, it has to be taken into account that these devices give only an approximation of the exposure due to the influence of the body of the person carrying the exposimeter, the receiver characteristics of the exposimeter, as well as the dependence of the measured value on frequency band, channel, slot configuration, and communication traffic. In this study, the relationship between the field strength measured close to the human body at the location of the exposimeter and the exposure, that is, the field strength at the location of the human body without the human body present, is investigated by numerical means using the Visible Human model as an anatomical phantom. Two different scenarios were chosen: (1) For FM, GSM, and UMTS an urban outdoor scenario was examined that included a transmitting antenna mounted on the roof of one of four buildings at a street crossing, (2) For WLAN an indoor scenario was investigated. For GSM the average degree of underestimation by the exposimeter (relation of the average field levels at the location of the exposimeter to the field level averaged over the volume of the human body without the body present) was 0.76, and for UMTS 0.87; for FM no underestimation was found, the ratio was 1. In the case of WLAN the degree of underestimation was more pronounced, the ratio was 0.64. This study clearly suggests that a careful evaluation of correction factors for different scenarios is needed prior to the definition of the study protocol. It has to be noted that the reference scenario used in this study does not allow for final conclusions on general correction factors. Bioelectromagnetics 31:535–545, 2010. © 2010 Wiley‐Liss, Inc.     

Multiple stressors on biotic interactions: how climate change and alien species interact to affect pollination

Global change may substantially affect biodiversity and ecosystem functioning but little is known about its effects on essential biotic interactions. Since different environmental drivers rarely act in isolation it is important to consider interactive effects. Here, we focus on how two key drivers of anthropogenic environmental change, climate change and the introduction of alien species, affect plant–pollinator interactions. Based on a literature survey we identify climatically sensitive aspects of species interactions, assess potential effects of climate change on these mechanisms, and derive hypotheses that may form the basis of future research. We find that both climate change and alien species will ultimately lead to the creation of novel communities. In these communities certain interactions may no longer occur while there will also be potential for the emergence of new relationships. Alien species can both partly compensate for the often negative effects of climate change but also amplify them in some cases. Since potential positive effects are often restricted to generalist interactions among species, climate change and alien species in combination can result in significant threats to more specialist interactions involving native species.     

The tarantula toxin psalmotoxin 1 inhibits acid-sensing ion channel (ASIC) 1a by increasing its apparent H+ affinity

Acid-sensing ion channels (ASICs) are ion channels activated by extracellular protons. They are involved in higher brain functions and perception of pain, taste, and mechanical stimuli. Homomeric ASIC1a is potently inhibited by the tarantula toxin psalmotoxin 1. The mechanism of this inhibition is unknown. Here we show that psalmotoxin 1 inhibits ASIC1a by a unique mechanism: the toxin increases the apparent affinity for H(+) of ASIC1a. Since ASIC1a is activated by H(+) concentrations that are only slightly larger than the resting H(+) concentration, this increase in H(+) affinity is sufficient to shift ASIC1a channels into the desensitized state. As activation of ASIC1a has recently been linked to neurodegeneration associated with stroke, our results suggest chronic desensitization of ASIC1a by a slight increase of its H(+) affinity as a possible way of therapeutic intervention in stroke.     

LC-MS/MS in the Clinical Laboratory – Where to From Here?

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has seen enormous growth in clinical laboratories during the last 10-15 years. It offers analytical specificity superior to that of immunoassays or conventional high performance/pressure liquid chromatography (HPLC) for low molecular weight analytes and has higher throughput than gas chromatography-mass spectrometry (GC-MS). Drug/Toxicology and Biochemical Genetics/Newborn Screening laboratories were at the vanguard of clinical LC-MS/MS use, but have been eclipsed by Endocrine laboratories. In USA reference/referral laboratories, most steroids and biogenic amines are now assayed by LC-MS/MS, and the technology has started to penetrate into smaller laboratories. Assays for mineralo- and gluco-corticoids and their precursors, sex steroids, metanephrines and 25-hydroxy vitamin D highlight the advantages of LC-MS/MS.However, several limitations of LC-MS/MS have become apparent, centring on the interacting triangle of sensitivity - specificity - throughput. While sample throughput is higher than for conventional HPLC or GC-MS, it lags behind automated immunoassays. Techniques which improve throughput include direct sample injection, LC-multiplexing and samplemultiplexing. Measures to improve specificity and sensitivity include sample clean-up and optimising chromatography to avoid interferences and ion suppression due to sample-matrix components. Next generation instrumentation may offer additional benefits.The next challenge for clinical LC-MS/MS is peptide/protein analysis. The quest for multi-biomarker profiles for various diseases has largely failed, but targeted peptide and protein testing by LC-MS/MS, directed at analytical and clinical questions that need to be answered, is proving highly successful. We anticipate that this will result in similar growth of clinical protein/peptide LC-MS/MS as has been seen for low molecular weight applications.