Evidence for Structural Dissimilarity in the Neurotransmitter Binding Region of Purified Acetylcholine Receptors from Human Muscle and Torpedo Electric Organ

Acetylcholine receptor (AChR) purified from human skeletal muscle affinity-alkylated with bromoacetyl[methyl-3H]choline bromide ([3H]BAC) in mildly reducing conditions to yield a specifically radiolabeled polypeptide, Mr 44,000, the alpha-subunit. The binding of [125I]alpha-bungarotoxin to AChR was completely inhibited by affinity-alkylation, indicating that the human AChR's binding site for alpha-bungarotoxin is closely associated with the alpha-subunit's acetylcholine binding site. Structures in the vicinity of the alpha-bungarotoxin binding sites of AChRs from human muscle and Torpedo electric organ were compared by varying the conditions of alkylation. Under optimal conditions of reduction and alkylation, both human and Torpedo AChR incorporated BAC in equivalence to the number of alpha-bungarotoxin binding sites. However, with limited conditions of reduction but sufficient BAC to alkylate 100% of the alpha-bungarotoxin binding sites of human AChR, only 71% of the Torpedo AChR's binding sites were alkylated. In optimal conditions of reduction but with the minimal concentration of BAC that permitted 100% alkylation of the human AChR's alpha-bungarotoxin sites, only 74% of the Torpedo AChR's binding sites were alkylated. These data suggest that the neurotransmitter binding region of human muscle AChR is structurally dissimilar from that of Torpedo electric organ, having a higher binding affinity for BAC and an adjacent disulfide bond that is more readily accessible to reducing agents.     

Human lymphocyte tubulin: Purification and characterization in normal and leukemic cells

Tubulin has been purified from human blood and tonsil lymphocytes. Using gel filtration, the molecular weight of human lymphocyte tubulin was estimated to be 119 000. The proteins was shown to consist of two subunits, with molecular weights of 61 000 and 58 000 comparable to the α and β polypeptides of human brain tubulin. A partial identity reaction was observed between lymphocyte tubulin and human tubulin when tested by double immunodiffusion against a rabbit anti-human brain tubulin antibody. In the presence of GTP, the purified protein polymerized to form microtubules. Tubulin was localized to the cell's juxtacentriolar region by immunofluorescence and electron microscopy. When assayed by a colchicine-binding assay corrected for time decay, the binding affinity was 1.50 ± 0.86 · 10⁶M^?1 and a level in normal lymphocytes of 1.21 · 10^?2 ± 0.79 g/g of soluble protein was determined. Since chronic lymphocytic leukemia lymphocytes have an anomalous capping behavior as well as an unusual susceptibility to colchicine toxicity, the properties and levels of tubulin were determined in these cells. Similar values were obtained for the level, decay rate, molecular weight, and K_a for colchicine as for normal lymphocytes. Chronic lymphocytic leukemia lymphocyte tubulin polymerized in a normal fashion. It thus appears that a decrease in the quantity or function of tubulin does not account for these anomalies in the chronic lymphocytic leukemia lymphocyte.     

Comparison of the relative sensitivity of human lymphocytes and mouse splenocytes to two spindle poisons

Aneugenic compounds act on non-DNA targets to exert genotoxicity via an indirect mechanism. In contrast to DNA-binding agents, these compounds are expected to possess threshold levels of activity. Therefore, the risk for adverse effects following human exposure to an aneugen could be minimal, if the threshold of activity has been clearly determined in vivo and in vitro and providing the human exposure level is below this threshold. Thus, the development of a single-cell model to allow comparisons between in vitro and in vivo threshold values for aneugenic compounds is of importance.The in vivo micronucleus test is one of the main assays used in genetic toxicology, and is often performed in the mouse. Thus, an extensive database is available in the literature. However, there are only few data concerning the in vitro micronucleus assay using mouse cells, as the majority of in vitro micronucleus assays have been performed using human lymphocytes. In addition, there is a lack of data concerning thresholds for any compound using this model.First, we evaluated whether the use of mouse splenocytes would be an acceptable alternative to that of human lymphocytes to identify aneugens. To allow valid comparisons, the two protocols were first harmonized. Thus, phytohemagglutinin (PHA) and concanavalin A were used as specific mitogens for human lymphocytes and mouse splenocytes, respectively, in order to achieve similar cell-proliferation rates. To achieve similar and sufficient numbers of binucleated cells, cytochalasin B was added 44 and 56 h after culture initiation of the human and mouse cells, respectively.Second, we compared the sensitivity of the mouse protocol with that of the human protocol by exposing the cells to the aneugens nocodazole and paclitaxel.There was good reproducibility of the cytotoxic/genotoxic responses of the two cell models following exposure to the aneugens. The sensitivity of the mouse splenocytes to paclitaxel was higher than that of the human lymphocytes. The two cell types were equally sensitive to nocodazole.     

Albumin stabilizes 14,15-leukotriene A4

14,15-Leukotriene A4 is a pivotal biosynthetic intermediate in 15-lipoxygenase initiated leukotriene biosynthesis. This compound hydrolyzes instantaneously in phosphate buffer at pH 7.4. However, addition of human or bovine albumin to otherwise identical buffer solutions increases its stability. Intact 14,15-leukotriene A4 then decomposes by first-order kinetics with rate constants inversely proportional to the albumin concentration. Stabilization of 14,15-leukotriene A4 under certain conditions may influence its proportionate transformation by enzymatic vs non-enzymatic processes.     

Specific localization of 2′,3′-cyclic nucleotide 3′-phosphohydrolase, (Ca2+/Mg2+)-ATPase,and acetylcholinesterase in human erythyrocyte membrane

A procedure was developed for the detection of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in myelin. This assay was sufficiently sensitive to detect the low levels of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in human erythrocytes. The 2′,3′-cyclic nucleotide 3′-phosphohydrolase of human erythrocytes was determined to be exclusively associated with the inner (cytosolic) side of the membrane. Leaky ghostsand resealed ghosts were assayed for 2′,3′-cyclic nucleotide 3′-phosphohydrolase, (Ca²⁺/Mg²⁺-ATPase, and acetylcholinesterase activity, and the 2′,3′-cyclic nucleotide 3′-phosphohydrolase profile is the same as that of the (Ca²⁺/Mg²⁺)-ATPase, an established inner membrane maker.     

Phytotoxic and antimicrobial activities and chemical analysis of leaf essential oil fromAgastache rugosa

Seasonal variations in the chemical composition of leaves fromAgastache rugosa were analyzed by gas chromatography-mass spectrometry (GC-MS). Phytotoxic and antimicrobial activities of its essential oil were investigated by characterizing 31 components, primarily methylchavicol (80.24%),dl-limonene (3.50%), linalool (4.23%), 5-methyl-2-(1-methylethylidene)-cyclohexanone (3.84%), and β-caryophyllene (2.39%). The contents of most of these chemical constituents varied significantly (P<0.05) over time, with the highest oil yield being early in the growing season (May). Their phytotoxic effects on the seed germination ofMajorana hortensis, Trifolium repens, Rudbeckia hirta, Chrysanthemum zawadskii var.latilobum, Melissa officinalis,Taraxacum platycarpum, andTagetes patula also were determined. These oils partially or completely inhibited germination, and prevented the growth of radicles and hypocotyls. Results of antimicrobial testing showed that the oils also entirely blocked the growth of four microorganisms (Staphylococcus aureus sub.aures, Escherichia coli, Salmonella enteritidis, andPseudomonas aeruginosa). These inhibitory effects might depend on the activity of methylchavicol, a main compound inA. rugosa.     

Chemical characterization of cytotoxic indole acetic acid derivative from mulberry fruit (Morus alba L.) against human cervical cancer

The fruit of the white mulberry tree (Morus alba L.) is a multiple fruit with a sweet flavor commonly consumed around the world. Chemical investigation of the fruits led to the isolation of two indole acetic acid derivatives (1 −2) including a new compound, which turned out to be an isolation artifact, 3S-(β-D-glucopyranosyloxy)-2,3-dihydro-2-oxo-1H-indole-3-acetic acid butyl ester (1), along with five known compounds (3 −7). Compounds 2 and 7 were newly identified from mulberry fruit. The new isolation artifact (1) exhibited cytotoxic effect on human cervical cancer Hela cells in a dose-dependent manner. Compound 1 activated caspase-8, caspase-9, and caspase-3, followed by cleavage of PARP, a substrate of caspase-3, in a dose-dependent manner. Simultaneous alterations in protein expression of mitochondrial factors Bax, BID and Bcl-2 were also observed. A comparison between compounds 1 and 2 led to a structure-activity relationship analysis of the cytotoxic effect. These results suggest that compound 1 could be beneficial in human cervical cancer treatment, and provide a theoretical basis for further application of compound 1.     

Trace elements in ancient Peruvian mummy hair: A preliminary report

Hair samples of 46 prehistoric mummies from five different pre-Inca sites in Peru were analyzed for Ca, Sr, Ni, Mn, and Ba by DCP/AES. The five sites displayed differing burial customs: whereas the dead of Cahuachi (n=13), Huayuri (n=2) and Brujo (n=5) had been interred unwrapped in simple stone pits, the mummies of Las Trancas (n=17) and Pacatnamu (n=9) had been granted protection by fabrics and wooden coffins. At the sites with no protection, the hair samples contained extremely high concentrations of Ca and Sr which by far exceeded the usual physiological ranges. This can probably be explained by contamination from the surrounding soil. In contrast, the protected depositions of Las Trancas and Pacatnamu exhibited Ca and Sr concentrations within the present range of variation. This may indicate that favourable conditions had prevented contamination in these cases. For Mn the same trend can be observed, both Cahuachi and Huayuri samples showed markedly increased values. For Ni and Ba on the other hand no correlation with the type of interment was found. In the two non-contaminated series, the mean values of Mn, Ba, and Ni were considerably higher in Las Trancas than in Pacatnamu. As Mn, Ni, and Ba tend to occur in higher concentrations in vegetable than in animal food items, these differences may indicate a differential availability of animal protein at the two locations     

An 11400 year paleoecological history of a British chalk grassland

The first broad-based, paleoecological analysis of a sedimentary sequence on the British chalk, dating to the terminal Pleistocene, reveals a history of climatic, vegetational and faunal change. The past co-occurrence of currently allopatric species among molluscs, beetles and plants supports hypotheses of the impermanence of communities. Modern pollen rain data are utilized to refine the ecological interpretation of the fossil pollen data. The presence of the Windermere Interstadial (Allerod) and the Loch Lomond re-advance (Younger Dryas) are represented by decreased abundances of arboreal taxa and increased representation of cold grassland elements. Open grassland habitats appear to have been a continuous landscape element, at least locally, since the late-glacial period on the northern British chal Mands, although their species composition has changed greatly in the last 11400 yr.