Properties of acetylcholinesterase and non-specific cholinesterase in rat superior cervical ganglion and plasma

Amphiphile dependency, solubility in aqueous solutions, and sensitivity to proteolysis of acetylcholinesterase (AChE) and nonspecific cholinesterase (nsChE) in the rat superior cervical ganglion were studied and compared to properties of soluble plasma cholinesterases. Ganglion AChE shows strong amphiphile dependency: an amphyphilic substance must be present in the homogenizing medium in order to obtain maximal apparent enzyme activity. Apparent activity of AChE solubilized in Ringer's solution was also increased after subsequent addition of a detergent. The 4 S molecular form, predominant in this extract (corresponding to the fastest electrophoretic band), is very sensitive to papain proteolysis but can be protected by a detergent. This molecular form therefore carries an important hydrophobic domain and is probably membrane bound in situ. The 10 S form of ganglionic AChE, extracted in Ringer's solution, is probably a soluble enzyme since, like soluble plasma enzymes, it is not amphiphile dependent and is rather resistant to proteolysis. Ganglion nsChE is more water soluble, less amphiphile dependent and more protease resistant than AChE.     

The magnetic diver differential microgasometer

The magnetic differential microgasometer described here is basically an equalarm torsion balance for measuring the reduced weight of submerged objects. The changes of buoyancy (ΔV_gx;ΔRW) are compensated for by the magnetic force acting on the same arm where the change has occurred (substitution method of weighing). Two ampullae, one with the sample and the other with the blank, are placed into the loops mounted at the ends of the arms. The gas volume and the geometry of the ampullae are practically equal. Therefore, the instrument is stable and fairly insensitive to changes of barometric pressure and temperature, and it can be used for gasometric measurements in an open flotation vessel. The applicability of the method for experiments where high blanks cannot be avoided was demonstrated by respiration measurements of multiplying cells and by measurements of cholinesterase activity of muscle end-plate samples.     

Polyglutamylation is a post-translational modification with a broad range of substrates

Polyglutamylation is a post-translational modification that generates lateral acidic side chains on proteins by sequential addition of glutamate amino acids. This modification was first discovered on tubulins, and it is important for several microtubule functions. Besides tubulins, only the nucleosome assembly proteins NAP1 and NAP2 have been shown to be polyglutamylated. Here, using a proteomic approach, we identify a large number of putative substrates for polyglutamylation in HeLa cells. By analyzing a selection of these putative substrates, we show that several of them can serve as in vitro substrates for two of the recently discovered polyglutamylases, TTLL4 and TTLL5. We further show that TTLL4 is the main polyglutamylase enzyme present in HeLa cells and that new substrates of polyglutamylation are indeed modified by TTLL4 in a cellular context. No clear consensus polyglutamylation site could be defined from the primary sequence of the here-identified new substrates of polyglutamylation. However, we demonstrate that glutamate-rich stretches are important for a protein to become polyglutamylated. Most of the newly identified substrates of polyglutamylation are nucleocytoplasmic shuttling proteins, including many chromatin-binding proteins. Our work reveals that polyglutamylation is a much more widespread post-translational modification than initially thought and thus that it might be a regulator of many cellular processes.     

Leptin Acts via Leptin Receptor-Expressing Lateral Hypothalamic Neurons to Modulate the Mesolimbic Dopamine System and Suppress Feeding

The lateral hypothalamic area (LHA) acts in concert with the ventral tegmental area (VTA) and other components of the mesolimbic dopamine (DA) system to control motivation, including the incentive to feed. The anorexigenic hormone leptin modulates the mesolimbic DA system, although the mechanisms underlying this control have remained incompletely understood. We show that leptin directly regulates a population of leptin receptor (LepRb)-expressing inhibitory neurons in the LHA and that leptin action via these LHA LepRb neurons decreases feeding and body weight. Furthermore, these LHA LepRb neurons innervate the VTA, and leptin action on these neurons restores VTA expression of the rate-limiting enzyme in DA production along with mesolimbic DA content in leptin-deficient animals. Thus, these findings reveal that LHA LepRb neurons link anorexic leptin action to the mesolimbic DA system.     

Monoclonal antibodies for the structural analysis of the Na+/H+ antiporter NhaA from Escherichia coli

Since their advent some 25 years ago, monoclonal antibodies have developed into powerful tools for structural and functional analysis of their cognate antigens. Together with the respective antigen binding fragments, antibodies offer exclusive capacities in detection, characterization, purification and functional assays for every given ligand. Antibody-fragment mediated crystallization represents a major advance in determining the three-dimensional structure of membrane-bound protein complexes. In this review, we focus on the methods used to generate monoclonal antibodies against the NhaA antiporter from Escherichia coli as a paradigm of secondary transporters. We describe examples on how antibodies are helpful in understanding structure and function relationships for this important class of integral membrane proteins. The generated conformation-specific antibody fragments are highly valuable reagents for co-crystallization attempts and structure determination of the antiporter.     

The outcome of autologous chondrocyte transplantation treatment of cartilage lesions in the knee

Recent results of the clinical outcome of autologous chondrocyte transplantation (ACT) treatment in a group of 28 patients with focal femoral condyle cartilage lesions revealed a correlation trend with the quality of the in vitro cell culture matrix-protein synthesis. No impact of the patients' age and chondrocyte cryopreservation prior to implantation was observed. Further studies are needed to confirm the preliminary results.     

Inhibition of cysteine and aspartyl proteinases in the alfalfa weevil midgut with biochemical and plant-derived proteinase inhibitors

Proteolytic activities in alfalfa weevil (Hypera postica) larval midguts have been characterized. Effects of pH, thiol activators, low-molecular weight inhibitors, and proteinase inhibitors (PIs) on general substrate hydrolysis by midgut extracts were determined. Hemoglobinolytic activity was highest in the acidic to mildly acidic pH range, but was maximal at pH 3.5. Addition of thiol-activators dithiothreitol (DTT), 2-mercaptoethanol (2-ME), or L-cysteine had little effect on hemoglobin hydrolysis at pH 3.5, but enhanced azocaseinolytic activity two to three-fold at pH 5.0. The broad cysteine PI E-64 reduced azocaseinolytic activity by 64% or 42% at pH 5 in the presence or absence of 5 mM L-cysteine, respectively. Inhibition by diazomethyl ketones, Z-Phe-Phe-CHN(2) and Z-Phe-Ala-CHN(2), suggest that cathepsins L and B are present and comprise approximately 70% and 30% of the cysteine proteolytic activity, respectively. An aspartyl proteinase component was identified using pepstatin A, which inhibited 32% (pH 3.5, hemoglobin) and 50% (pH 5, azocasein) of total proteolytic activity. This activity was completely inhibited by an aspartyl proteinase inhibitor from potato (API), and is consistent with the action of a cathepsin D-like enzyme. Hence, genes encoding PIs with specificity toward cathepsins L, B and D could potentially be effective for control of alfalfa weevil using transgenic plants.     

Analysis of preincisional and postincisional treatment with alpha2-adrenoreceptor agonist clonidine regarding analgesic consumption and hemodynamic stability in surgical patients

Preemptive analgesia aims to prevent the sensitization of central nervous system, hence the development of pathologic pain after tissular injury. The aim of the study was to assess the effect of preincisional clonidine treatment on analgesic consumption and hemodynamic stability compared to clonidine administered at the end of the operation and control group. Ninety-one patients undergoing elective colorectal surgery were randomly assigned to four groups: peroral clonidine before operation, epidural clonidine before operation, epidural clonidine at the end of operation, and epidural saline before operation as a control group. After the operation, patient-controlled analgesia with epidural morphine was instituted. Analgesic consumption, blood pressure and heart rate were obtained at 1, 2, 6 and 24 h postoperatively, and the cumulative consumption of analgesics was assessed at the end of the study period. Significant differences (p < 0.05) in postoperative systolic blood pressure, with highest hemodynamic stability was observed at 1 h and 6 h in the group of patients administered epidural clonidine before operation. In this group of patients we found significant reduction in analgesic consumption during the study period (p < 0.05), compared to other groups. The cumulative consumption of analgesics assessed at the end of the study period was significantly reduced (p < 0.05) in the group of patients administered epidural clonidine before operation (8.40 +/- 3.74, respectively) as compared with the peroral clonidine before operation (16.79 +/- 5.75, respectively), epidural clonidine at the end of the operation (11.11 +/- 4.24, respectively) and control group of patients (18.00 +/- 6.45, respectively). Preincisional administration of epidural clonidine was associated with a significantly lower analgesic use, lower cumulative analgesic consumption and greater hemodynamic stability, in comparison with other groups.