Chimeric β-Lactamases: Global Conservation of Parental Function and Fast Time-Scale Dynamics with Increased Slow Motions

Enzyme engineering has been facilitated by recombination of close homologues, followed by functional screening. In one such effort, chimeras of two class-A β-lactamases – TEM-1 and PSE-4 – were created according to structure-guided protein recombination and selected for their capacity to promote bacterial proliferation in the presence of ampicillin (Voigt et al., Nat. Struct. Biol. 2002 9:553). To provide a more detailed assessment of the effects of protein recombination on the structure and function of the resulting chimeric enzymes, we characterized a series of functional TEM-1/PSE-4 chimeras possessing between 17 and 92 substitutions relative to TEM-1 β-lactamase. Circular dichroism and thermal scanning fluorimetry revealed that the chimeras were generally well folded. Despite harbouring important sequence variation relative to either of the two ‘parental’ β-lactamases, the chimeric β-lactamases displayed substrate recognition spectra and reactivity similar to their most closely-related parent. To gain further insight into the changes induced by chimerization, the chimera with 17 substitutions was investigated by NMR spin relaxation. While high order was conserved on the ps-ns timescale, a hallmark of class A β-lactamases, evidence of additional slow motions on the µs-ms timescale was extracted from model-free calculations. This is consistent with the greater number of resonances that could not be assigned in this chimera relative to the parental β-lactamases, and is consistent with this well-folded and functional chimeric β-lactamase displaying increased slow time-scale motions.     

Gut pH, redox conditions and oxygen levels in an aquatic caterpillar: potential effects on the fate of ingested tannins

Larvae of the freshwater moth Acentria ephemerella (Pyralidae, Lepidoptera) can fully develop on Myriophyllum spicatum, a submerged macrophyte containing 7-10% of its dry mass as tannins. We investigated the physicochemical gut parameters of larvae fed with M. spicatum or Potamogeton perfoliatus, a food plant lacking tannins, and the chemical fate of ingested polyphenols. Microelectrode studies revealed that larval midguts were slightly alkaline (pH 8) and had a positive redox potential. Whole guts were oxygen sinks owing to the oxygen demand of the gut contents. Oxygen penetrated the midgut up to 100 microm, but the centres of the foregut and midgut were always anoxic. The physicochemical parameters of the guts did not change with the food plant. The major tannin from M. spicatum, tellimagrandin II, was significantly depleted in the midgut and was not detected in faeces. In vitro studies indicated that tellimagrandin II is rapidly depleted mainly through oxidation, and hydrolysis might also occur. Our findings for A. ephemerella are compared with those for terrestrial Lepidoptera, and possible mechanisms for adaptations to tannin-rich food plants are discussed.     

Role of endogenous brain-derived neurotrophic factor and sortilin in B cell survival

Brain-derived neurotrophic factor (BDNF), a major neuronal growth factor, is also known to exert an antiapoptotic effect in myeloma cells. Whereas BDNF secretion was described in B lymphocytes, the ability of B cells to produce sortilin, its transport protein, was not previously reported. We studied BDNF production and the expression of its receptors, tyrosine protein kinase receptor B and p75 neurotrophin receptor in the human pre-B, mature, and plasmacytic malignant B cell lines under normal and stress culture conditions (serum deprivation, Fas activation, or their combination). BDNF secretion was enhanced by serum deprivation and exerted an antiapoptotic effect, as demonstrated by neutralization experiments with antagonistic Ab. The precursor form, pro-BDNF, also secreted by B cells, decreases under stress conditions in contrast to BDNF production. Stress conditions induced the membranous expression of p75 neurotrophin receptor and tyrosine protein kinase receptor B, maximal in mature B cells, contrasting with the sequestration of both receptors in normal culture. By blocking Ab and small interfering RNA, we evidenced that BDNF production and its survival function are depending on sortilin, a protein regulating neurotrophin transport in neurons, which was not previously described in B cells. Therefore, in mature B cell lines, an autocrine BDNF production is up-regulated by stress culture conditions and exerts a modulation of apoptosis through the sortilin pathway. This could be of importance to elucidate certain drug resistances of malignant B cells. In addition, primary B lymphocytes contained sortilin and produced BDNF after mitogenic activation, which suggests that sortilin and BDNF might be implicated in the survival and activation of normal B cells also.     

Feeding preference of Coptotermes formosanus (Isoptera: Rhinotermitidae) for gamma-irradiated wood impregnated with benzoylphenylurea compounds under laboratory conditions

The feeding preference of Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae) for 200-kGy gamma-irradiated Cryptomeria japonica D. Don (Japanese cedar) sapwood impregnated with benzoylphenylurea compounds such as hexaflumuron and noviflumuron was examined by three laboratory tests. Although termites were not deterred from feeding on gamma-irradiated wood samples that had been impregnated with hexaflumuron or noviflumuron, termite mortality was significantly higher compared with solvent controls in the no-choice test. All live termites were transferred to paper disks immediately after the no-choice test to investigate changes in mortality with time, and this test also confirmed the effects of hexaflumuron and noviflumuron on worker termites, which showed a significant feeding preference for gamma-irradiated wood. Only the 1480 ppm noviflumuron-impregnated gamma-irradiated wood specimens showed significant differences in mortality in the two-choice test. These results suggest that gamma-irradiated C. japonica wood, which is locally abundant in Japan, may have potential as a bait substrate for benzoylphenylurea compounds.     

Sardine early survival, physical condition and stress after introduction to captivity

The main factors that affect early survival, physical damage and stress reactions of sardine Sardina pilchardus after live capture and introduction to captivity were examined. A total of 2800 sardines were captured alive from commercial purse seiners in five trials off southern Portugal and monitored for 4 weeks in aquaculture tanks. Survival rates varied considerably between trials (from <20 to >80% after a month), with most deaths occurring in the first 5 days. Sardine early survival was affected by factors related to conditions at sea (catch composition, sea temperature and transportation density), during introduction to captivity (magnitude of thermal shock, land transportation duration and use of antibacterial treatment) and, possibly, their interaction. Physical damage was related to the probability of dying, with fish that died during the first week showing significantly higher scale loss and larger caudal fin erosion that those that were alive in the same period. For all stress variables measured (blood haematocrit, cortisol, glucose and ions in the plasma), the most extreme values were attained during introduction to captivity or in the first hours after. After 2 weeks in captivity, most variables had returned to levels close to those observed at the onset of purse-seine fishing, suggesting that maintenance conditions were adequate to permit a rapid recovery from fishing and transport stress.     

In vivo trafficking of endogenous opioid receptors

Several approaches have been taken for these in vivo studies. In many studies, the use of semi-quantitative immuno-electron microscopy is the approach of choice. Endogenous opioid receptors display differential subcellular distributions with mu opioid receptor (MOPR) being mostly present on the plasma membrane and delta-opioid receptor (DOPR) and kappa-opioid receptor (KOPR) having a significant intracellular pool. Etorphine and DAMGO cause endocytosis of the MOPR, but morphine does not, except in some dendrites. Interestingly, chronic inflammatory pain and morphine treatment promote trafficking of intracellular DOPR to the cell surface which may account for the enhanced antinociceptive effects of DOPR agonists. KOPR has been reported to be associated with secretory vesicles in the posterior pituitary and translocated to the cell surface upon salt loading along with the release of vasopressin. The study of endogenous opioid receptors using in vivo models has produced some interesting results that could not have been anticipated in vitro. In vivo studies, therefore, are essential to provide insight into the mechanisms underlying opioid receptor regulation.     

MMP-12 induces IL-8/CXCL8 secretion through EGFR and ERK1/2 activation in epithelial cells

Macrophage metalloelastase (MMP-12) is described to be involved in pulmonary inflammatory response. To determine the mechanisms linking MMP-12 and inflammation, we examined the effect of recombinant human MMP-12 (rhMMP-12) catalytic domain on IL-8/CXCL8 production in cultured human airway epithelial (A549) cells. Stimulation with rhMMP-12 resulted in a concentration-dependent IL-8/CXCL8 synthesis 6 h later. Similar results were also observed in cultured BEAS-2B bronchial epithelial cells. In A549 cells, synthetic matrix metalloproteinase (MMP) inhibitors prevented rhMMP-12-induced IL-8/CXCL8 release. We further demonstrated that in A549 cells, rhMMP-12 induced transient, peaking at 5 min, activation of ERK1/2. Selective MEK inhibitors (U0126 and PD-98059) blocked both IL-8/CXCL8 release and ERK1/2 phosphorylation. IL-8/CXCL8 induction and ERK1/2 activation were preceded by EGF receptor (EGFR) tyrosine phosphorylation, within 2 min, and reduced by selective EGFR tyrosine kinase inhibitors (AG-1478 and PD168393) by a neutralizing EGFR antibody and by small interfering RNA oligonucleotides directed against EGFR, implicating EGFR activation. In addition, we observed an activation of c-Fos in A549 cells stimulated by rhMMP-12, dependent on ERK1/2. Using small interfering technique, we showed that c-Fos is involved in rhMMP-12-induced IL-8/CXCL8 production. From these results, we conclude that one mechanism, by which MMP-12 induces IL-8/CXCL8 release from the alveolar epithelium, is the EGFR/ERK1/2/activating protein-1 pathway.