The three-dimensional structure of acarbose bound to glycogen phosphorylase

Acarbose, a pseudotetrasaccharide with a conduritol ring at the nonreducing terminus, is a naturally occurring inhibitor of amylases. It is shown here to be an inhibitor of glycogen phosphorylase and to bind more tightly to the enzyme than the equivalent malto-oligosaccharide substrate. X-ray crystallographic studies of the acarbose-phosphorylase a complex in the presence of glucose and caffeine reveal the structure of acarbose as bound to the storage site of phosphorylase. The acarbose binds in an orientation such that the conduritol ring makes no protein contacts. As with malto-oligosaccharides bound at this site, the observed conformation of acarbose is stabilized by O-2-O-3' hydrogen bonding and is similar to, but not identical with, that predicted by hard-sphere exo-anomeric effect calculations and justified by 1H nuclear magnetic resonance studies (Bock, K., and Pedersen, H. (1984) Carbohydr. Res. 132, 142-149). Intramolecular O2-O3' hydrogen bonds appear to play an important role in stabilizing the conformation observed in these studies, even for those residues closely associated with the protein.     

A freeze-preservation of synchronously dividing cultured cells of Acer pseudoplatanus L

A suspension culture of sycamore (Acer pseudoplatanus L.) was synchronised in division by release from nitrogen starvation. Cell samples, taken during the lag phase and synchronous growth, were examined cytologically and subjected to a freeze-preservation protocol. A high positive correlation was found between mitotic index (percentage of cells showing mitotic figures) and cell survival, as measured by fluorescein diacetate staining and reduction of 2,3,5-triphenyl tetrazolium chloride. Specific staining of lethally damaged cells and subsequent examination of the surviving cells demonstrated that the latter had a lower mitotic index and more consistent, low value of nuclear DNA than the total population. This indicated that it is cells, newly entered into G₁ phase of the cell cycle, which are particularly resistant to the stresses imposed by the freeze preservation protocol.     

Direct 1H n.m.r. determination of the stereochemical course of hydrolyses catalysed by glucanase components of the cellulase complex

The stereochemical courses of the hydrolyses catalysed by three glycosidases have been determined directly by 1H nmr. The anomeric configuration of the initially formed product was ascertained in each case by observation of the chemical shift and coupling constant of the anomeric proton at the new hemiacetal centre. Two of the enzymes investigated, an endo-glucanase and an exo-glucanase are components of the cellulase complex of Cellulomonas fimi. The third enzyme is the beta-glucosidase from almond emulsin. Two of these enzymes, the exo-glucanase and the almond beta-glucosidase catalysed hydrolysis with retention of anomeric configuration, in agreement with previous observations on the almond enzyme. The endo-glucanase catalysed hydrolysis with inversion of configuration, this result being confirmed by optical rotation measurements. This 1H nmr approach has several advantages over other techniques in that it is applicable to a wide variety of glycosidases and substrates and it is non-destructive, allowing recovery of the enzyme.     

1-Deoxy-d-galactonojirimycins with dansyl capped N-substituents as β-galactosidase inhibitors and potential probes for GM1 gangliosidosis affected cell lines

Two simple and reliably accessible intermediates, N-carboxypentyl- and N-aminohexyl-1-deoxy-d-galactonojirimycin were employed for the synthesis of a set of terminally N-dansyl substituted derivatives. Reaction of the terminal carboxylic acid of N-carboxypentyl-1-deoxy-d-galactonojirimycin with N-dansyl-1,6-diaminohexane provided the chain-extended fluorescent derivative. Employing bis(6-dansylaminohexyl)amine, the corresponding branched di-N-dansyl compound was obtained. Partially protected N-aminohexyl-1-deoxy-d-galactonojirimycin served as intermediate for two additional chain-extended fluorescent 1-deoxy-d-galactonojirimycin (1-DGJ) derivatives featuring terminal dansyl groups in the N-alkyl substituent. These new compounds are strong inhibitors of d-galactosidases and may serve as leads en route to pharmacological chaperones for GM1-gangliosidosis.     

Carrion flies of forensic interest: a study of seasonal community composition and succession in Lisbon,Portugal

Information on Diptera community, seasonality and successional patterns in every geographical region is fundamental for the use of flies as forensic indicators of time of death. In order to obtain these data from the Lisbon area (Portugal), experiments were conducted during the four seasons of the year, using piglet carcasses as animal models. Five stages were recognized during the decomposition process. The stages, besides visually defined, could be separated taking into account the occurrence and abundance of the specific groups of Diptera collected. In general, the bloated stage recorded higher abundance and species‐richness values. Seventy‐one species were identified, belonging to 39 families, in a total of 20 144 adult Diptera collected. Autumn yielded the highest values of species richness, whereas winter had the lowest. In all seasons of the year, Calliphoridae was the dominant family; Muscidae and Fanniidae were very abundant as well. Calliphora vicina Robineau‐Desvoidy, Calliphora vomitoria (Linnaeus), Chrysomya albiceps (Wiedemann), Lucilia ampullacea Villeneuve, Lucilia caesar (Linnaeus), Lucilia sericata (Meigen) (Calliphoridae), Hydrotaea ignava (Harris), Muscina prolapsa (Harris), Synthesiomyia nudiseta (van der Wulp) (Muscidae), Piophila megastigmata McAlpine, Stearibia nigriceps (Meigen) (Piophilidae) and Nemopoda nitidula (Fallén) (Sepsidae) were revealed to be very important members of the Diptera community collected. The necrophagous behaviour, demonstrated by their immatures, using carrion as a food source makes them useful forensic indicator species. Also of relevance is the presence of Chrysomya megacephala (Fabricius), S. nudiseta and P. megastigmata, foreign species established in the local carrion communities. This study also marks the first record of S. nudiseta in Portugal.     

Isolation and characterization of microsatellite markers from Pacific white shrimp (Litopenaeus vannamei)

We report the development of 11 polymorphic microsatellite loci in pacific white shrimp (Litopenaeus vannamei) using an unenriched genomic library. The number of the alleles ranged from two to 18 and observed hererozygosity ranged from 0.0286 to 0.9429, indicating that these markers will be useful for population studies and mapping in pacific white shrimp. Seven loci were detected deviated from Hardy–Weinberg, caused by deficiency of heterozygote, suggesting population genetic structure across the sampled population. No evidence for linkage disequilibrium was found.     

Characterisation of a 34 kD protein from potato cyst nematodes, using monoclonal antibodies with potential for species diagnosis

Two monoclonal antibodies, which differentially recognise the two species of potato cyst nematodes (PCN), Globodera pallida and G. rostochiensis, are described. They have been shown to have potential for quantification of these two species, recognising proteins of the same molecular weight (34 kD) in both species. Further investigation showed these proteins to have isoelectric points at pH values of 5.7 in G. pallida and 5.9 in G. rostochiensis, in common with the proteins used by Fleming & Marks (1983) to differentiate the species of PCN. They are likely to be structurally very similar, with the same physiological function (and therefore similar concentrations) in the two species. In cross-reactivity tests with a wide range of soil nematode species, the antibodies reacted strongly only with species of the genus Globodera, and thereby confirmed their potential as the basis of a quantitative immunoassay likely to be useful in management of PCN populations.     

Variability in host plant resistance of Sorghum to Striga Hermonthica infestation in West Africa

Fourteen elite sorghum lines were evaluated for their resistance to Striga hermonthica at three locations in Nigeria and Mali. Results showed that many of the lines especially MALISOR 84-1, SAMSORG 41, 97-SB-F5DT-64 (Keninkédié) and the check SRN 39 remained resistant to Striga in all locations with low emerged Striga counts, while SAMSORG 14 had the highest Striga infestation in all locations. Considerable variation in reaction to Striga infestation was observed on Séguètana, 97-SB-F5DT-63 (Wasa), 97-SB-F5DT-65, CMDT 38, CMDT 39 and CMDT 45 which were susceptible to Striga at Samaru, Nigeria but were resistant to Striga at both locations in Mali. Based on low Striga resistance and high grain yield, lines MALISOR 84-1, SAMSORG 41, 97-SB-F5DT-64, 97-SB-F5DT-65, CMDT 39 and SAMSORT 14 have been nominated for wider evaluation across more West African countries.