Molecular cloning and biochemical characterization of VIM-12, a novel hybrid VIM-1/VIM-2 metallo-beta-lactamase from a Klebsiella pneumoniae clinical isolate, reveal atypical substrate specificity

Metallo-beta-lactamases (MBLs) are considered an emerging family of Zn2+-dependent enzymes that significantly contribute to the resistance of many nosocomial pathogens against beta-lactam antimicrobials. Since these plasmid-encoded enzymes constitute specific molecular targets for beta-lactams, their exact mode of action is greatly important in deploying efficient anti-infective treatments and for the control of severe multi-resistant nosocomial infections, which becomes a global problem. A novel hybrid VIM-1/VIM-2-type beta-lactamase (named VIM-12) has recently been identified in a clinical isolate of Klebsiella pneumoniae in Greece. The sequence of this enzyme is highly similar with that of VIM-1 at its N-terminal region and with that of VIM-2 at its C-terminal region, raising the question of whether this sequence similarity reflects also a similar functional role. Moreover, the possible contribution of this novel beta-lactamase to the overall antibiotic resistance of this specific clinical isolate was investigated. The gene encoding VIM-12 was cloned and expressed, and the recombinant enzyme was used for detailed kinetic analysis, using a variety of beta-lactam antibiotics. VIM-12 was found to exhibit narrow substrate specificity, compared to other known beta-lactamases, limited mainly to penicillin and to a much lesser extent to imipenen. Interestingly, meropenem was found to act as a noncompetitive inhibitor of the enzyme, although the active site of VIM-12 exhibited complete conservation of residues among VIM enzymes. We conclude that VIM-12 represents a novel and unique member of the family of known metallo-beta-lactamases, exhibiting atypical substrate specificity.     

Impact of early applied upper limb stimulation: The EXPLICIT-stroke programme design

Background  

Main claims of the literature are that functional recovery of the paretic upper limb is mainly defined within the first month post stroke and that rehabilitation services should preferably be applied intensively and in a task-oriented way within this particular time window. EXplaining PLastICITy after stroke (acronym EXPLICIT-stroke) aims to explore the underlying mechanisms of post stroke upper limb recovery. Two randomized single blinded trials form the core of the programme, investigating the effects of early modified Constraint-Induced Movement Therapy (modified CIMT) and EMG-triggered Neuro-Muscular Stimulation (EMG-NMS) in patients with respectively a favourable or poor probability for recovery of dexterity.     

A systematic analysis of the factors that determine the strength of pre-mRNA splicing enhancers.

We find that the strength of splicing enhancers is determined by the relative activities of the bound serine-arginine (SR)-rich splicing factors, the number of SR proteins within the enhancer complex and the distance between the enhancer and the intron. Remarkably, the splicing activity of the bound SR proteins is directly proportional to the number of RS tetrapeptide sequences within the RS domain. Quantitative analysis of the effects of varying the distance between the enhancer and the intron revealed that the splicing efficiency is directly proportional to the calculated probability of a direct interaction between the enhancer complex and the 3' splice site. These data are consistent with a model in which splicing enhancers function by increasing the local concentration of SR proteins in the vicinity of the nearby intron through RNA looping.     

Characterization of the Ligand Binding Functionality of the Extracellular Domain of Activin Receptor Type IIB

The single transmembrane domain serine/threonine kinase activin receptor type IIB (ActRIIB) has been proposed to bind key regulators of skeletal muscle mass development, including the ligands GDF-8 (myostatin) and GDF-11 (BMP-11). Here we provide a detailed kinetic characterization of ActRIIB binding to several low and high affinity ligands using a soluble activin receptor type IIB-Fc chimera (ActRIIB.Fc). We show that both GDF-8 and GDF-11 bind the extracellular domain of ActRIIB with affinities comparable with those of activin A, a known high affinity ActRIIB ligand, whereas BMP-2 and BMP-7 affinities for ActRIIB are at least 100-fold lower. Using site-directed mutagenesis, we demonstrate that ActRIIB binds GDF-11 and activin A in different ways such as, for example, substitutions in ActRIIB Leu⁷⁹ effectively abolish ActRIIB binding to activin A yet not to GDF-11. Native ActRIIB has four isoforms that differ in the length of the C-terminal portion of their extracellular domains. We demonstrate that the C terminus of the ActRIIB extracellular domain is crucial for maintaining biological activity of the ActRIIB.Fc receptor chimera. In addition, we show that glycosylation of ActRIIB is not required for binding to activin A or GDF-11. Together, our findings reveal binding specificity and activity determinants of the ActRIIB receptor that combine to effect specificity in the activation of distinct signaling pathways.