Reversal of Ketamine/Xylazine combination anesthesia by Atipamezole in olive baboons (Papio anubis)

Background  The potential of Atipamezole (ATI) to reverse Ketamine/Xylazine (KET/XYL) anesthesia in the Olive baboon ( Papio anubis ) was studied.
Methods  Anesthesia was induced with 10 mg/kg KET and 0.5 mg/kg XYL intramuscularly. Mean arousal time (MAT), heart rate (HR), systolic arterial blood pressure (SAP), rectal temperature, respiratory rate (RR), and hemoglobin oxygen saturation (SpO₂) were monitored. Baboons were treated with: KET/XYL only, KET/XYL followed by 100 μg/kg ATI or by 200 μg/kg ATI administered 25 minutes after KET/XYL.
Results  Atipamezole rapidly reversed depressed HR and SAP (10 ± 5.2 minutes), RR (5 ± 2 minutes) and SpO₂ (3 ± 6 minutes) and significantly decreased MAT (13 ± 2.2 minutes) vs. KET/XYL alone (35 ± 5 minutes). Emesis was absent and salivation was observed after administration of 200 μg/kg ATI only.
Conclusions  Atipamezole at 100 μg/kg is sufficient for rapid and smooth reversal of KET/XYL anesthesia in the Olive baboon with minimal side effects.     

The non-competitive NMDA antagonists MK-801 and PCP, as well as the competitive NMDA antagonist SDZ EAA494 (D-CPPene), interact synergistically with clonidine to promote locomotion in monoamine-depleted mice

The present study shows that high doses of the non-competitive NMDA antagonist phencyclidine (PCP) as well as of the competitive NMDA antagonist SDZ EAA494 (D-CPPene) increase locomotion in monoamine-depleted mice. The pattern of movement produced following treatment with these agents is very similar to that previously observed following MK-801 administration to monamine-depleted mice. When subthreshold doses of MK-801, PCP and SDZ EAA494 were combined with the alpha-adrenergic agonist clonidine, a dramatic stimulation of locomotion was observed in monoamine-depleted mice; the gross appearance of the animals was similar with the three drug combinations. These results support our previous conclusion that suppression of glutamatergic neurotransmission promotes the locomotor stimulatory potential of other (e.g. adrenergic) transmitter systems. The present findings may be of relevance for future treatment strategies in (L-DOPA-resistant) Parkinson's disease.     

CDKN2B expression and subcutaneous adipose tissue expandability: Possible influence of the 9p21 atherosclerosis locus

Risk alleles within a gene desert at the 9p21 locus constitute the most prevalent genetic determinant of cardiovascular disease. Previous research has demonstrated that 9p21 risk variants influence gene expression in vascular tissues, yet the biological mechanisms by which this would mediate atherosclerosis merits further investigation. To investigate possible influences of this locus on other tissues, we explored expression patterns of 9p21-regulated genes in a panel of multiple human tissues and found that the tumor suppressor CDKN2B was highly expressed in subcutaneous adipose tissue (SAT). CDKN2B expression was regulated by obesity status, and this effect was stronger in carriers of 9p21 risk alleles. Covariation between expression of CDKN2B and genes implemented in adipogenesis was consistent with an inhibitory effect of CDKN2B on SAT proliferation. Moreover, studies of postprandial triacylglycerol clearance indicated that CDKN2B is involved in down-regulation of SAT fatty acid trafficking. CDKN2B expression in SAT correlated with indicators of ectopic fat accumulation, including markers of hepatic steatosis. Among genes regulated by 9p21 risk variants, CDKN2B appears to play a significant role in the regulation of SAT expandability, which is a strong determinant of lipotoxicity and therefore might contribute to the development of atherosclerosis.     

Driving membrane curvature in clathrin-dependent and clathrin-independent endocytosis

Cellular activity depends to a large extent on membrane bilayer dynamics. Many processes, such as organelle biogenesis and vesicular transport, rely on alterations in membrane structure and shape. It is now widely accepted that intracellular membrane curvature generation and remodelling is mediated and regulated by protein action, and the mechanisms behind the processes are currently being revealed. Here, we will briefly discuss the key principles of membrane deformation and focus on different endocytic events that use various kinds of proteins to shape the plasma membrane into transport carriers. The entry routes are adopted to make sure that a vast variety of molecules on the cell surface can be regulated by endocytosis. The principles for membrane sculpting of endocytic carriers can be viewed either from a perspective of rigid coat budding or of flexible opportunistic budding. We will discuss these principles and their implications, focusing on clathrin-dependent and -independent carrier formation and the proteins involved in the respective pathways.     

Characterization of oil and starch accumulation in tubers of Cyperus esculentus var. sativus (Cyperaceae): A novel model system to study oil reserves in nonseed tissues

? Premise of the study: Storage oil (triacylglycerol) accumulates in tissues such as the embryo and endosperm of seeds and the fruit mesocarp, but seldom in underground organs. As a rare exception, cultivated variants of yellow nutsedge (Cyperus esculentus) contain high amounts of both oil and starch in the mature tubers. ? Methods: Biochemical analyses and light and electron microscopy were used to study the accumulation patterns of storage nutrients in developing nutsedge tubers. ? Key results: During the initial phase of tuber development, the conducting rhizome tissue is transformed into a storage compartment, then massive storage reserves accumulate in the tuber. At the beginning of tuber development, a large sugar load coincided with the onset of starch accumulation. Oil accumulation started later, concomitant with a substantial drop in the sugar content. Initially, oil accumulated at a lower rate compared to starch, but the rate later increased; after 6 wk, oil made up 24% of tuber dry mass, while starch made up 32%. Protein concentration changed only a small amount throughout this development. Oil and starch accumulated in the same cells throughout the tubers in a sequential fashion during tuber development. ? Conclusions: The developmental pattern in the build up of storage nutrients in the tubers highlights nutsedge as a novel model plant, having potential to significantly widen our understanding on how synthesis of storage reserves, and in particular oils, is regulated and directed in nonseed tissues such as tubers and roots.     

Loss of glutathione peroxidase 3 expression is correlated with epigenetic mechanisms in endometrial adenocarcinoma

Glutathione peroxidase 3 (GPX3) is one of the key enzymes in the cellular defense against oxidative stress and the hepatocyte growth factor receptor, (MET) has been suggested to be influenced by the GPX3 gene expression. In a previous microarray study performed by our group, Gpx3 was identified as a potential biomarker for rat endometrial adenocarcinoma (EAC), since the expression was highly downregulated in rat EAC tumors. Herein, we have investigated the mRNA expression and Gpx3 and Met in rat EAC by real time quantitative PCR (qPCR), and the methylation status of Gpx3. In addition we have examined the expression of GPX3 and MET in 30 human EACs of different FIGO grades and 20 benign endometrial tissues. We found that the expression of GPX3 was uniformly down regulated in both rat and human EAC, regardless of tumor grade or histopathological subtype, implying that the down-regulation is an early event in EAC. The rate of Gpx3 promoter methylation reaches 91%, where biallelic methylation was present in 90% of the methylated tumors. The expression of the Met oncogene was slightly upregulated in EACs that showed loss of expression of Gpx3, but no tumor suppressor activity of Gpx3/GPX3 was detected. Preliminary results also suggest that the production of H₂O₂ is higher in rat endometrial tumors with down-regulated Gpx3 expression. A likely consequence of loss of GPX3 protein function would be a higher amount of ROS in the cancer cell environment. Thus, the results suggest important clinical implications of the GPX3 expression in EAC, both as a molecular biomarker for EAC and as a potential target for therapeutic interventions.     

N2 fixation and nitrogen allocation to above and below ground plant parts in red clover-grasslands

Leys, used for grazing or production of forage to be conserved as silage or hay, are very important crops in northern areas. In order to measure the N₂ fixation in leys of varying ages and during different parts of the season, detailed measurements were taken of yield, N₂ fixation and the amounts of N remaining in the field after harvesting red clover (Trifolium pratense L.)-grass leys at a site in northern Sweden, where they are generally harvested twice per growing season. Entire plants, including stubble and roots, were sampled at the time of first and second harvest and, in addition, at the end of the growing season in three neighbouring fields, carrying a first, a second and a third year ley, respectively. N₂ fixation was measured by both ¹⁵N isotope dilution (ID) and ¹⁵N natural abundance (NA) methods. The proportion of clover dry matter (DM) in the stands increased from the first to the second harvest, but the grasses dominated throughout the entire season, especially below ground. The N concentrations, in both herbage and whole plants, were about twice as high in the clover as in the grasses. Seasonal variations in N concentrations were minor, and total N contents followed the same trends as DM. The clover acquired nearly all of its N from N₂ fixation: the proportion of N in clover herbage derived from N₂ fixation was often >0.8 throughout the season. The variations in the amounts of N₂ fixed during the course of the season corresponded well to the seasonal changes in clover biomass. Amounts of fixed N₂ allocated to clover herbage during the whole season were in the range 4 to 6 g N m⁻² in this unusually rainy year. Calculations of daily N allocation rates to herbage showed that N uptake rates were similar, and high, in grasses during May–June and July–August, while N₂ fixation rates in clover were about 10-fold as high in July–August as in May–June, reflecting the need for N in clover growth. The proportion of N remaining in clover stubble and roots after the first and second harvests was about 60 and 25%, respectively, while about 60% of the N in grasses remained in stubble and roots after both harvests. The considerable amounts of biomass and N that were left in field after harvesting red clover-grass leys are important for re-growth of the plants and provide substantial N fertilization for the next crop in the crop rotation.     

Structures of Mycobacterium tuberculosis 1-deoxy-D-xylulose-5-phosphate reductoisomerase provide new insights into catalysis

Isopentenyl diphosphate is the precursor of various isoprenoids that are essential to all living organisms. It is produced by the mevalonate pathway in humans but by an alternate route in plants, protozoa, and many bacteria. 1-deoxy-D-xylulose-5-phosphate reductoisomerase catalyzes the second step of this non-mevalonate pathway, which involves an NADPH-dependent rearrangement and reduction of 1-deoxy-D-xylulose 5-phosphate to form 2-C-methyl-D-erythritol 4-phosphate. The use of different pathways, combined with the reported essentiality of the enzyme makes the reductoisomerase a highly promising target for drug design. Here we present several high resolution structures of the Mycobacterium tuberculosis 1-deoxy-D-xylulose-5-phosphate reductoisomerase, representing both wild type and mutant enzyme in various complexes with Mn(2+), NADPH, and the known inhibitor fosmidomycin. The asymmetric unit corresponds to the biological homodimer. Although crystal contacts stabilize an open active site in the B molecule, the A molecule displays a closed conformation, with some differences depending on the ligands bound. An inhibition study with fosmidomycin resulted in an estimated IC(50) value of 80 nm. The double mutant enzyme (D151N/E222Q) has lost its ability to bind the metal and, thereby, also its activity. Our structural information complemented with molecular dynamics simulations and free energy calculations provides the framework for the design of new inhibitors and gives new insights into the reaction mechanism. The conformation of fosmidomycin bound to the metal ion is different from that reported in a previously published structure and indicates that a rearrangement of the intermediate is not required during catalysis.     

Active site of epoxide hydrolases revisited: a noncanonical residue in potato StEH1 promotes both formation and breakdown of the alkylenzyme intermediate

The carboxylate of Glu35 in the active site of potato epoxide hydrolase StEH1 interacts with the catalytic water molecule and is the first link in a chain of hydrogen bonds connecting the active site with bulk solvent. To probe its importance to catalysis, the carboxylate was replaced with an amide through an E35Q mutation. Comparing enzyme activities using the two trans-stilbene oxide (TSO) enantiomers as substrates revealed the reaction with R,R-TSO to be the one more severely affected by the E35Q mutation, as judged by determined kinetic parameters describing the pre-steady states or the steady states of the catalyzed reactions. The hydrolysis of S,S-TSO afforded by the E35Q mutant was comparable with that of the wild-type enzyme, with only a minor decrease in activity, or a change in pH dependencies of kcat, and the rate of alkylenzyme hydrolysis, k3. The pH dependence of E35Q-catalyzed hydrolysis of R,R-TSO, however, exhibited an inverted titration curve as compared to that of the wild-type enzyme, with a minimal catalytic rate at pH values where the wild-type enzyme exhibited maximum rates. To simulate the pH dependence of the E35Q mutant, a shift in the acidity of the alkylenzyme had to be invoked. The proposed decrease in the pKa of His300 in the E35Q mutant was supported by computer simulations of the active site electrostatics. Hence, Glu35 participates in activation of the Asp nucleophile, presumably by facilitating channeling of protons out of the active site, and during the hydrolysis half-reaction by orienting the catalytic water for optimal hydrogen bonding, to fine-tune the acid-base characteristics of the general base His300.