Purification and properties of alcohol oxidase from Tanacetum vulgare

Alcohol oxidase (alcohol: O₂ oxidoreductase) from leaves of Tanacetum vulgare has been purified 5150-fold to homogeneity on disc electrophoresis and gel electrofocussing. The enzyme which is probably flavoprotein, has molecular weight 180 000 daltons and is comprised of two sub-units of 94 000 and 75 000 daltons. It is active over a broad range (pH 5–9) and best accepts primary aliphatic alcohols with 6 to 10 carbons, especially those with a 2-ene group. K_m values for hex-trans-2-ene-1-ol, geraniol (3,7-dimethylocta-trans-2,6-dien-1-ol) and n-octanol were 0.19, 1.56 and 0.49 mM respectively. The significance of the enzyme in the formation of leaf aldehyde (hex-trans-2-ene-1-al) and in terpene metabolism is discussed.     

Transferrin microheterogeneity as a probe in normal and disease states

Isoelectric focusing of iron saturated serum has been established as a convenient method for showing transferrin glycan microheterogeneity. In a clinical setting, the method is used in the detection of cerebrospinal fluid leakage, the screening for surreptitious alcohol abuse and in the diagnosis of the carbohydrate deficient glycoprotein syndrome. In normal physiological states it can also be used as a tool to probe for changes in N-glycosylation.     

Binding of lithium and boron to human plasma proteins

The binding of lithium and boron, at normal physiological levels, to plasma proteins has been investigated by the techniques of precipitation with ethyl alcohol and gel chromatography. Assays of lithium and boron were made by thermal neutron activation and mass spectrometric assay of³He and⁴He. Results of alcohol precipitation experiments for plasma from two apparently healthy donors showed that 13 ± 4% and 16 ± 3% of the lithium in plasma is protein bound, but essentially no boron is bound under the conditions used. We believe that because of denaturation of proteins which occurs during alcohol precipitation, these percentages represent lithium and boron tightly bound to protein molecules. The results of the gelchromatography experiment, on the other hand, showed that lithium and boron are bound to a wide range of plasma proteins, from low (∼ 60,000 amu) to high (∼ 1,000,000 amu) molecular weights, and to very low- (∼ 6000 amu) molecular-weight ligands. Although a clear identification of the specific proteins which bind lithium and boron cannot be made at present, some possibilities can be suggested. Certain commercial equipment, instruments, or materials are identified in this article to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose.     

Prenatal ethanol exposure persistently impairs NMDA receptor-dependent activation of extracellular signal-regulated kinase in the mouse dentate gyrus

The dentate gyrus (DG) is the central input region to the hippocampus and is known to play an important role in learning and memory. Previous studies have shown that prenatal alcohol is associated with hippocampal-dependent learning deficits and a decreased ability to elicit long-term potentiation (LTP) in the DG in adult animals. Given that activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling cascade by NMDA receptors is required for various forms of learning and memory, as well as LTP, in hippocampal regions, including the DG, we hypothesized that fetal alcohol-exposed adult animals would have deficits in hippocampal NMDA receptor-dependent ERK1/2 activation. We used immunoblotting and immunohistochemistry techniques to detect NMDA-stimulated ERK1/2 activation in acute hippocampal slices prepared from adult fetal alcohol-exposed mice. We present the first evidence linking prenatal alcohol exposure to deficits in NMDA receptor-dependent ERK1/2 activation specifically in the DG of adult offspring. This deficit may account for the LTP deficits previously observed in the DG, as well as the life-long cognitive deficits, associated with prenatal alcohol exposure.     

Disorders in memory and learning in offspring of alcoholized female rats,and a possibility for correction of these changes

We studied the effect of alcohol intoxication of albino female rats on the process of learning and memory of their adult (two-month-old) offspring and also a possibility for correction of the observed changes using dolivin. During pregnancy and lactation, female rat of the experimental group obtained 15% solution of ethanol instead of water. To estimate the successfulness of spatial learning of their offspring, we used a multiway elevated labyrinth; the level of consolidation of memory traces was estimated using a passive avoidance test in a chamber with dark and illuminated sections. In the tested offspring rats, prenatal exposure to ethanol induced dramatic drops in the indices of both the above tests. The use of dolivin simultaneously with ethanol exposure allowed us to demonstrate the clear protective properties of this complex preparation containing such antioxidants as hypoxen and vitamin E: disorders in behavior of the offspring of alcoholized females were smoothed significantly. Neirofiziologiya/Neurophysiology, Vol. 40, No. 2, pp. 130–136, March–April, 2008.     

Prenatal Ethanol Exposure Decreases GAP-43 Phosphorylation and Protein Kinase C Activity in the Hippocampus of Adult Rat Offspring

Abstract: Consumption of moderate quantities of ethanol during pregnancy produces deficits in long-term potentiation in the hippocampal formation of adult offspring. Protein kinase C (PKC)-mediated phosphorylation of the presynaptic protein GAP-43 is critical for the induction of long-term potentiation. We tested the hypothesis that this system is affected in fetal alcohol-exposed (FAE) rats by measuring GAP-43 phosphorylation and PKC activity in the hippocampus of adult offspring of rat dams that had consumed one of three diets throughout gestation: (a) a 5% ethanol liquid diet, which produced a maternal blood ethanol concentration of 83 mg/dl (FAE); (b) an isocalorically equivalent 0% ethanol diet (pair-fed); or (c) lab chow ad libitum. Western blot analysis using specific antibodies to PKC-phosphorylated GAP-43 revealed that FAE rats had an ∼50% reduction in the proportion of phosphorylated GAP-43. Similarly, we found that PKC-mediated incorporation of ³²P into GAP-43 was reduced by 85% in hippocampal slices from FAE rats compared with both control groups. FAE animals also showed a 50% reduction in total hippocampal PKC activity, whereas the levels of six major PKC isozymes did not change in any of the diet groups. These results suggest that GAP-43 phosphorylation deficits in rats prenatally exposed to moderate levels of ethanol are not due to alterations in the expression of either the enzyme or substrate protein, but rather to a defect in kinase activation.     

Asymmetric synthesis of a fluoxetine precursor with an artificial fusion protein of a ketoreductase and a formate dehydrogenase

Herein we describe the kinetic characterization of a fusion protein from the 3-ketoacyl-[acyl-carrier-protein]-reductase (KR) from Synechococcus PCC 7942 and a mutant formate dehydrogenase from Mycobacterium vaccae N10 (MycFDH). Upon purification, a specific proteolytic cleavage of the MycFDH was observed. The cleavage site was elucidated, which is ubiquitously spread among prokaryotic FDHs. After depletion of the cleavage site the correct, full length fusion protein was obtained. In asymmetric reductions of ethylbenzoyl acetate (EBA) this fusion protein performed equal or even better than the free enzymes, yielding up to 39% more of the fluoxetine precursor ethyl-(S)-3-hydroxy-3-phenylpropanoate ((S)-HPPE). The rate acceleration is due to an improved K_m,EBA of the KR subunit.     

Essential‐Oil Composition,Antileishmanial, and Toxicity Study of Artemisia abyssinica and Satureja punctata ssp. punctata from Ethiopia

Essential oils of Artemisia abyssinica and Satureja punctata ssp. punctata from Ethiopia were analyzed by GC and GC/MS, and screened for leishmanicidal activity against promastigote and axenic amastigotes of Leishmania donovani and L. aethiopica, including toxicity studies on human monocytic leukemia cells (THP‐1) and erythrocytes in vitro. GC/MS of A. abyssinica oil revealed 67 compounds (99.94%) with the major constituents yomogi alcohol (38.47%), artemisyl acetate (24.88%), and artemisia alcohol (6.70%), and oxygenated monoterpenes (84.00%) as the dominant group. The oil of S. punctata contained 67 compounds (99.49%) with the main constituents geranial (27.62%), neral (21.72%), α‐bisabolol (13.62%), and (E)‐nerolidol (4.82%), of which oxygenated mono‐ and sesquiterpenes (58.39 and 26.91%, resp.) showed highest abundance. Both oils showed effect on promastigotes (MIC 76.5 to 312.5 nl/ml) and amastigotes (EC₅₀ 4.06 to 131.00 nl/ml) of L. donovani and L. aethiopica, and varying toxicities on THP‐1 cells (CC₅₀ 0.013 to 350 nl/ml with selectivity index between 0.001 and 28) and erythrocytes (with LC₅₀ 0.35 to 1.52 μl/ml). S. punctata oil exerted highest activity against both Leishmania sp. and toxicity. The revealed antileishmanial activities support further isolation and investigation of oil constituents for in vitro/in vivo evaluation.     

Are all evening-types doomed? Latent class analyses of perceived morningness–eveningness,sleep and psychosocial functioning among emerging adults

An overwhelming amount of research has indicated that evening-types report more negative psychosocial functioning as well as more negative sleep characteristics (e.g. more sleep problems) relative to morning-types. Researchers also find a strong, consistent link between poor sleep characteristics and negative psychosocial functioning. These studies, however, have been based on a variable-centred approach, and thus were not able to assess possible individual differences within morning-types and evening-types with respect to their sleep characteristics prior to assessing differences in psychosocial functioning. Thus, it is not clear whether it is morningness–eveningness per se or sleep characteristics that explain the differences in psychosocial functioning found between morning-types and evening-types. The purpose of the present two-year longitudinal study was to employ a person-centred approach to determine whether there are subgroups within morning-types and evening-types based on 10-sleep characteristics (e.g. sleep problems and sleep duration). Then subgroups were compared on three indices of psychosocial functioning (i.e. academics, intrapersonal adjustment and alcohol consumption), both concurrently, as well as one year later. Participants were 780 (72.2% female; M?=?19.0 years, SD?=?0.90) emerging adults at a mid-sized university in Southern Ontario, who were either morning-types or evening-types. A latent class analysis (LCA) conducted for morning-types yielded two subgroups, classified as having good sleep characteristics (i.e. morning-good) and poor sleep characteristics (i.e. morning-poor). Results of a second LCA conducted for evening-types yielded three subgroups, classified as having good (i.e. evening-good), moderate (i.e. evening-moderate) and poor (i.e. evening-poor) sleep characteristics. Results comparing subgroups across the 10-sleep characteristics indicated that morning-good and evening-good individuals reported very similar scores, and both were characterized by the least sleep problems and longest sleep duration relative to the other subgroups. In terms of the three psychosocial functioning indices we found that academic achievement generally did not differ across the five subgroups (i.e. morning-good, morning-poor, evening-good, evening-moderate and evening-poor). With respect to intrapersonal adjustment, morning-good and evening-good subgroups reported significantly better intrapersonal adjustment relative to the other subgroups across time. Interestingly, evening-type subgroups generally reported higher alcohol consumption than morning-type subgroups. Overall, these results suggest that intrapersonal adjustment in particular appears to be associated more with differences in sleep characteristics (i.e. sleep problems and duration), than with morningness–eveningness per se, while the opposite is generally true for alcohol consumption. Lifestyle and personality factors likely also play a critical role. Importantly, our study is the first to identify a subgroup of evening-types who report good sleep characteristics and similar levels of intrapersonal adjustment and academic achievement to that of the majority of morning-types.