Characterization of aminopeptidase N from Torpedo marmorata kidney

Summary— A major antigen of the brush border membrane of Torpedo marmorata kidney was identified and purified by immunoprecipitation. The sequence of its 18 N terminal amino acids was determined and found to be very similar to that of mammalian aminopeptidase N (EC 3.4.11.2). Indeed aminopeptidase N activity was efficiently immunoprecipitated by monoclonal antibody 180K1. The purified antigen gives a broad band at 180 kDa after SDS-gel electrophoresis, which, after treatment by endoglycosidase F, is converted to a thinner band at 140 kDa. This antigen is therefore heavily glycosylated. Depending on solubilization conditions, both the antigen and peptidase activity were recovered either as a broad peak with a sedimentation coefficient of 18S (2% CHAPS) or as a single peak of 7.8S (1% CHAPS plus 0.2 % C₁₂E₉), showing that Torpedo aminopeptidase N behaves as an oligomer stabilized by hydrophobic interactions, easily converted into a 160 kDa monomer. The antigen is highly concentrated in the apical membrane of proximal tubule epithelial cells (600 gold particles/μm² of brush border membrane) whereas no labeling could be detected in other cell types or in other membranes of the same cells (basolatéral membranes, vacuoles or vesicles). Monoclonal antibodies prepared here will be useful tools for further functional and structural studies of Torpedo kidney aminopeptidase N.     

Homologous, homeologous, and illegitimate repair of double-strand breaks during transformation of a wild-type strain and a rad52 mutant strain of Saccharomyces cerevisiae.

Different modes of in vivo repair of double-strand breaks (DSBs) have been described for various organisms: the recombinational DSB repair (DSBR) mode, the single-strand annealing (SSA) mode, and end-to-end joining. To investigate these modes of DSB repair in Saccharomyces cerevisiae, we have examined the fate of in vitro linearized replicative plasmids during transformation with respect to several parameters. We found that (i) the efficiencies of both intramolecular and intermolecular linear plasmid DSB repair are homology dependent (according to the amount of DNA used during transformation [100 ng or less], recombination between similar but not identical [homeologous] P450s sequences sharing 73% identity is 2- to 18-fold lower than recombination between identical sequences); (ii) the RAD52 gene product is not essential for intramolecular recombination between homologous and homeologous direct repeats (as in the wild-type strain, recombination occurs with respect to the overall alignment of the parental sequences); (iii) in contrast, the RAD52 gene product is required for intermolecular interactions (the rare transformants which are obtained contain plasmids resulting from deletion-forming intramolecular events involving little or no sequence homology); (iv) similarly, sequencing data revealed examples of intramolecular joining within the few terminal nucleotides of the transforming DNA upon transformation with a linear plasmid with no repeat in the wild-type strain. The recombinant junctions of the rare illegitimate events obtained with S. cerevisiae are very similar to those observed in the repair of DSB in mammalian cells. Together, these and previous results suggest the existence of alternative modes for DSB repair during transformation which differ in their efficiencies and in the structure of their products. We discuss the implications of these results with respect to the existence of alternative pathways and the role of the RAD52 gene product.     

Autoradiographic Distribution and Characteristics of High- and Low-Affinity Polyamine-Sensitive [3H]Ifenprodil Sites in the Rat Brain: Possible Relationship to NMDAR2B Receptors and Calmodulin

Abstract: We have studied the regional distribution and characteristics of polyamine-sensitive [³H]ifenprodil binding sites by quantitative autoradiography in the rat brain. In forebrain areas ifenprodil displaced [³H]ifenprodil (40 nM) in a biphasic manner with IC₅₀ values ranging from 42 to 352 nM and 401 to 974 µM. In hindbrain regions, including the cerebellum, ifenprodil displacement curves were monophasic with IC₅₀ values in the high micromolar range. Wiping studies using forebrain slices (containing both high- and low-affinity sites) or cerebellar slices (containing only the low-affinity site) showed that high- and low-affinity ifenprodil sites are sensitive to spermine and spermidine, to the aminoglycoside antibiotics neomycin, gentamicin, and kanamycin, and to zinc. Two calmodulin antagonists, W7 and calmidazolium, also displaced [³H]ifenprodil from both sites. Other calmodulin antagonists, including trifluoperazine, prenylamine, and chlorpromazine, selectively displaced [³H]ifenprodil from its low-affinity site in hindbrain and forebrain regions. High-affinity [³H]ifenprodil sites, defined either by ifenprodil displacement curves or by [³H]ifenprodil binding in the presence of 1 mM trifluoperazine, were concentrated in the cortex, hippocampus, striatum, and thalamus with little or no labeling of hindbrain or cerebellar regions. This distribution matches that of NMDAR2B mRNA, supporting data showing that ifenprodil has a preferential action at NMDA receptors containing this subunit. Low-affinity [³H]ifenprodil sites have a more ubiquitous distribution but are especially concentrated in the molecular layer of the cerebellum. [³H]Ifenprodil was found to bind to calmodulin-agarose with very low affinity (IC₅₀ of ifenprodil = 516 µM). This binding was displaced by calmodulin antagonists and by polyamines, with a potency that matched their displacement of [³H]ifenprodil from its low-affinity site in brain sections. However, the localization of the low-affinity [³H]ifenprodil site does not strictly correspond to that of calmodulin, and its identity remains to be further characterized. The restricted localization of high-affinity [³H]ifenprodil binding sites to regions rich in NMDAR2B subunit mRNA may explain the atypical nature of this NMDA antagonist.     

Skewed inactivation of an X chromosome deleted at the dystrophin gene in an asymptomatic mother and her affected daughter

A girl with severe Becker muscular dystrophy and apparently normal chromosomes had a heterozygous deletion for exons 51, 52, and 53 of the dystrophin gene. This deletion was transmitted by her mother, who was unaffected. To differentiate the normal and the deleted X chromosomes, fluorescence in situ hybridization (FISH) was applied to metaphase chromosomes, using probes for both exons 51 and 52, which are only 388 and 113 base pairs long, respectively. FISH signals were observed in one or both chromatids of one chromosome, but never on both chromosomes, suggesting the lack of hybridization on the deleted X chromosome. Using 5-bromodeoxyuridine incorporation to differentiate the late (inactive) and the early replicating (active) X chromosomes, 77% of the signals were observed on the active X chromosomes in the mother. This percentage was only 18% in the daughter, suggesting that skewed inactivation of the X chromosomes was responsible for the phenotypic differences.     

The effect of a free radical scavenger and platelet-activating factor antagonist on FFA accumulation in post-ischemic canine brain

The effects of the platelet-activating factor antagonist BN 50739 and a free radical scavenger dimethyl sulfoxide on the accumulation of free fatty acids in post-ischemic canine brain are reported. Following 14 min of complete normothermic ischemia and 60 min of reperfusion, the total brain FFAs were approximately 150% higher than in the control group (p<0.05). Perfusion with the platelet-activating factor antagonist BN50739 in its diluent dimethyl sulfoxide during 60 min of post-ischemic reoxygenation resulted in a 61.8% (p<0.01) reduction in the total brain free fatty acid accumulation. Palmitic, stearic, oleic, linoleic, and arachidonic acids decreased by 53.8%, 63.5%, 69.0%, 47.4%, and 57.2%, respectively. Although dimethyl sulfoxide alone caused stearic and arachidonic acids to return to the normal concentration range, BN 50739 had a significant influence on recovery of palmitic, oleic, and linoleic acids and was previously shown to provide significant therapeutic protection against damage to brain mitochondria following an ischemic episode. Because free fatty acid accumulation is one of the early phenomena in cerebral ischemia, this study provides evidence to support the hypothesis that both platelet-activating factor and free radicals are involved in initiating cerebral ischemic injury.     

Decreased Electroconvulsive Shock-Induced Diacylglycerols and Free Fatty Acid Accumulation in the Rat Brain by Ginkgo biloba Extract (EGb 761): Selective Effect in Hippocampus as Compared with Cerebral Cortex

Abstract: The effect of Ginkgo biloba extract (EGb 761) treatment (100 mg/kg/day, per os, for 14 days) on electroconvulsive shock (ECS)-induced accumulation of free fatty acids (FFA) and diacylglycerols (DAG) was analyzed in rat cerebral cortex and hippocampus. EGb 761 reduced the FFA pool size by 33% and increased the DAG pool by 36% in the hippocampus. These endogenous lipids were unaffected in cerebral cortex. During the tonic seizure (10 s after ECS) the fast accumulation of FFA, mainly 20:4, was similar in sham- and EGb 761 -treated rats, in both the cerebral cortex and hippocampus. However, further accumulation of free 18:0 and 20:4, observed in the hippocampus of sham-treated rats during clonic seizures (30 s to 2 min after ECS), did not occur in EGb 761-treated animals. The rise in DAG content triggered in the cortex and hippocampus by ECS was delayed by EGb 761 treatment from 10 s to 1 min, when values similar to those in sham animals were attained. Moreover, in the hippocampus the size of the total DAG pool was decreased by 19% during the tonic seizure. At later times, DAG content showed a faster decrease in EGb 761-treated rats. By 2 min levels of all DAG acyl groups decreased to values significantly lower than in sham animals in both cortex and hippocampus. This study shows that EGb 761 treatment affects, with high selectivity, lipid metabolism and lipid-derived second messenger release and removal in the hippocampus, while affecting to a lesser extent the cerebral cortex.     

Coupling Among Energy Failure, Loss of Ion Homeostasis, and Phospholipase A2 and C Activation During Ischemia

Abstract— The objective of the present experiments was to correlate changes in cellular energy metabolism, dissipative ion fluxes, and lipolysis during the first 90 s of ischemia and, hence, to establish whether phospholipase A₂or phospholipase C is responsible for the early accumulation of phospholipid hydrolysis products. Ischemia was induced for 15–90 s in rats, extracellular K⁺ (K⁺_e) was recorded, and neocortex was frozen in situ for measurements of labile tissue metabolites, free fatty acids, and diacylglycerides. Ischemia of 15-and 30-s duration gave rise to a decrease in phosphocreatine concentration and a decline in the ATP/free ADP ratio. Although these changes were accompanied by an activation of K⁺ conductances, there were no changes in free fatty acids until after 60s, when free arachidonic acid accumulated. An increase in other free fatty acids and in total diacylglyceride content did not occur until after anoxic depolarization. The results demonstrate that the early functional changes, such as activation of K⁺ conductances, are unrelated to changes in lipids or lipid mediators. They furthermore suggest that the initial lipolysis occurs via both phospholipase A₂ and phospholipase C, which are activated when membrane depolarization leads to influx of calcium into cells.     

How drought events during the last century have impacted biomass carbon in Amazonian rainforests

During the last two decades, inventory data show that droughts have reduced biomass carbon sink of the Amazon forest by causing mortality to exceed growth. However, process-based models have struggled to include drought-induced responses of growth and mortality and have not been evaluated against plot data. A process-based model, ORCHIDEE-CAN-NHA, including forest demography with tree cohorts, plant hydraulic architecture and drought-induced tree mortality, was applied over Amazonia rainforests forced by gridded climate fields and rising CO₂ from 1901 to 2019. The model reproduced the decelerating signal of net carbon sink and drought sensitivity of aboveground biomass (AGB) growth and mortality observed at forest plots across selected Amazon intact forests for 2005 and 2010. We predicted a larger mortality rate and a more negative sensitivity of the net carbon sink during the 2015/16 El Niño compared with the former droughts. 2015/16 was indeed the most severe drought since 1901 regarding both AGB loss and area experiencing a severe carbon loss. We found that even if climate change did increase mortality, elevated CO₂ contributed to balance the biomass mortality, since CO₂-induced stomatal closure reduces transpiration, thus, offsets increased transpiration from CO₂-induced higher foliage area.     

Damage to living trees contributes to almost half of the biomass losses in tropical forests

Accurate estimates of forest biomass stocks and fluxes are needed to quantify global carbon budgets and assess the response of forests to climate change. However, most forest inventories consider tree mortality as the only aboveground biomass (AGB) loss without accounting for losses via damage to living trees: branchfall, trunk breakage, and wood decay. Here, we use ~151,000 annual records of tree survival and structural completeness to compare AGB loss via damage to living trees to total AGB loss (mortality + damage) in seven tropical forests widely distributed across environmental conditions. We find that 42% (3.62 Mg ha⁻¹ year⁻¹; 95% confidence interval [CI] 2.36–5.25) of total AGB loss (8.72 Mg ha⁻¹ year⁻¹; CI 5.57–12.86) is due to damage to living trees. Total AGB loss was highly variable among forests, but these differences were mainly caused by site variability in damage-related AGB losses rather than by mortality-related AGB losses. We show that conventional forest inventories overestimate stand-level AGB stocks by 4% (1%–17% range across forests) because assume structurally complete trees, underestimate total AGB loss by 29% (6%–57% range across forests) due to overlooked damage-related AGB losses, and overestimate AGB loss via mortality by 22% (7%–80% range across forests) because of the assumption that trees are undamaged before dying. Our results indicate that forest carbon fluxes are higher than previously thought. Damage on living trees is an underappreciated component of the forest carbon cycle that is likely to become even more important as the frequency and severity of forest disturbances increase.