Cysteine-scanning mutagenesis and substituted cysteine accessibility analysis of transmembrane segment 4 of the Glut1 glucose transporter

A low resolution model has been proposed for the exofacial conformation of the Glut1 glucose transporter in which eight transmembrane segments form an inner helical bundle stabilized by four outer helices. The role of transmembrane segment 4, predicted to be an inner helix in this structural model, was investigated by cysteine-scanning mutagenesis in conjunction with the substituted cysteine accessibility method using the membrane-impermeant, sulfhydryl-specific reagent, p-chloromercuribenzenesulfonate (pCMBS). A functional, cysteine-less, parental Glut1 molecule was used to produce 21 Glut1 point mutants by individually changing each residue along transmembrane helix 4 to a cysteine. The single cysteine mutants were then expressed in Xenopus oocytes, and their expression levels, transport activities, and sensitivities to pCMBS were determined. In striking contrast to all of the other seven predicted inner helices, none of the 21 helix 4 single-cysteine mutants was demonstrably inhibited by pCMBS. However, cysteine substitution within helix 4 resulted in an unusually high number of severely transport-defective mutants. The low absolute transport activities of two of these mutants (G130C and G134C) were due to their extremely low levels of expression, presumably a result of structural instability and consequent degradation in oocytes, suggesting that these two residues play an important role in maintaining the native structure of Glut1. The other two transport-defective mutants (Y143C and E146C) exhibited low specific transport activities, implying that these two residues play an important role in the transport cycle. Based on these data, we conclude that the exoplasmic end of helix 4 lies outside the inner helical bundle in the exofacial configuration of Glut1.     

Structural and kinetic analysis of the substrate specificity of human fibroblast activation protein alpha

Fibroblast activation protein alpha (FAPalpha) is highly expressed in epithelial cancers and has been implicated in extracellular matrix remodeling, tumor growth, and metastasis. We present the first high resolution structure for the apoenzyme as well as kinetic data toward small dipeptide substrates. FAPalpha exhibits a dipeptidyl peptidase IV (DPPIV)-like fold, featuring an alpha/beta-hydrolase domain and an eight-bladed beta-propeller domain. Known DPPIV dipeptides are cleaved by FAPalpha with an approximately 100-fold decrease in catalytic efficiency compared with DPPIV. Moreover, FAPalpha, but not DPPIV, possesses endopeptidase activity toward N-terminal benzyloxycarbonyl (Z)-blocked peptides. Comparison of the crystal structures of FAPalpha and DPPIV revealed one major difference in the vicinity of the Glu motif (Glu(203)-Glu(204) for FAPalpha; Glu(205)-Glu(206) for DPPIV) within the active site of the enzyme. Ala(657) in FAPalpha, instead of Asp(663) as in DP-PIV, reduces the acidity in this pocket, and this change could explain the lower affinity for N-terminal amines by FAPalpha. This hypothesis was tested by kinetic analysis of the mutant FAPalpha/A657D, which shows on average an approximately 60-fold increase in the catalytic efficiency, as measured by k(cat)/K(m), for the cleavage of dipeptide substrates. Furthermore, the catalytic efficiency of the mutant is reduced by approximately 350-fold for cleavage of Z-Gly-Pro-7-amino-4-methylcoumarin. Our data provide a clear understanding of the molecular determinants responsible for the substrate specificity and endopeptidase activity of FAPalpha.     

Wardium canarisi n. sp. (Cestoda: Hymenolepididae) parasite de Arenaria melanocephala (Aves: Charadrii) d'Alaska

The authors describe and illustrate Wardium canarisi n. sp. (Cestoda: Hymenolepididae), an intestinal parasite of Arenaria melanocephala (Aves: Charadrii) from Alaska, characterised by a strobila 20–40 mm long, 10 aploparaksoid hooks 19–21 m long, a short cylindrical cirrus (40 m) covered with very minute spines (0.2 m), and a short (8–18 m) and extremely narrow (1 m) copulatory vagina. These characters have no equivalent, even approximate, among the 27 species of Wardium parasitic in the Charadrii which are reviewed. The genus Debrosia Spassky, 1987 appears to be justified.     

Agrobacterium-mediated sorghum transformation

Agrobacterium tumefaciens was used to genetically transform sorghum. Immature embryos of a public (P898012) and a commercial line (PHI391) of sorghum were used as the target explants. The Agrobacterium strain used was LBA4404 carrying a `Super-binary' vector with a bar gene as a selectable marker for herbicide resistance in the plant cells. A series of parameter tests was used to establish a baseline for conditions to be used in stable transformation experiments. A number of different transformation conditions were tested and a total of 131 stably transformed events were produced from 6175 embryos in these two sorghum lines. Statistical analysis showed that the source of the embryos had a very significant impact on transformation efficiency, with field-grown embryos producing a higher transformation frequency than greenhouse-grown embryos. Southern blot analysis of DNA from leaf tissues of T₀ plants confirmed the integration of the T-DNA into the sorghum genome. Mendelian segregation in the T₁ generation was confirmed by herbicide resistance screening. This is the first report of successful use of Agrobacterium for production of stably transformed sorghum plants. The Agrobacterium method we used yields a higher frequency of stable transformation that other methods reported previously.     

A general model of error-prone PCR

In this paper, we generalize a previously-described model of the error-prone polymerase chain reaction (PCR) reaction to conditions of arbitrarily variable amplification efficiency and initial population size. Generalisation of the model to these conditions improves the correspondence to observed and expected behaviours of PCR, and restricts the extent to which the model may explore sequence space for a prescribed set of parameters. Error-prone PCR in realistic reaction conditions is predicted to be less effective at generating grossly divergent sequences than the original model. The estimate of mutation rate per cycle by sampling sequences from an in vitro PCR experiment is correspondingly affected by the choice of model and parameters.     

Specific leaf area and dry matter content estimate thickness in laminar leaves

BACKGROUND AND AIMS: Leaf thickness plays an important role in leaf and plant functioning, and relates to a species' strategy of resource acquisition and use. As such, it has been widely used for screening purposes in crop science and community ecology. However, since its measurement is not straightforward, a number of estimates have been proposed. Here, the validity of the (SLA x LDMC)(-1) product is tested to estimate leaf thickness, where SLA is the specific leaf area (leaf area/dry mass) and LDMC is the leaf dry matter content (leaf dry mass/fresh mass). SLA and LDMC are two leaf traits that are both more easily measurable and often reported in the literature. METHODS: The relationship between leaf thickness (LT) and (SLA x LDMC)(-1) was tested in two analyses of covariance using 11 datasets (three original and eight published) for a total number of 1039 data points, corresponding to a wide range of growth forms growing in contrasted environments in four continents. KEY RESULTS AND CONCLUSIONS: The overall slope and intercept of the relationship were not significantly different from one and zero, respectively, and the residual standard error was 0.11. Only two of the eight datasets displayed a significant difference in the intercepts, and the only significant difference among the most represented growth forms was for trees. LT can therefore be estimated by (SLA x LDMC)(-1), allowing leaf thickness to be derived from easily and widely measured leaf traits.     

Fate of pathogens present in livestock wastes spread onto fescue plots

Fecal wastes from a variety of farmed livestock were inoculated with livestock isolates of Escherichia coli O157, Listeria monocytogenes, Salmonella, Campylobacter jejuni, and Cryptosporidium parvum oocysts at levels representative of the levels found in naturally contaminated wastes. The wastes were subsequently spread onto a grass pasture, and the decline of each of the zoonotic agents was monitored over time. There were no significant differences among the decimal reduction times for the bacterial pathogens. The mean bacterial decimal reduction time was 1.94 days. A range of times between 8 and 31 days for a 1-log reduction in C. parvum levels was obtained, demonstrating that the protozoans were significantly more hardy than the bacteria. Oocyst recovery was more efficient from wastes with lower dry matter contents. The levels of most of the zoonotic agents had declined to below detectable levels by 64 days. However, for some waste types, 128 days was required for the complete decline of L. monocytogenes levels. We were unable to find significant differences between the rates of pathogen decline in liquid (slurry) and solid (farmyard manure) wastes, although concerns have been raised that increased slurry generation as a consequence of more intensive farming practices could lead to increased survival of zoonotic agents in the environment.     

Yeast Ypt11 is targeted to recycling endosomes in mammalian cells

BACKGROUND INFORMATION: In yeast, Ypt11 or Ypt32 along with the highly homologous Ypt8 or Ypt31 has been reported to be an essential component of intra-Golgi trafficking and has been implicated in the budding of vesicles from the most distal Golgi compartment. RESULTS AND CONCLUSIONS: In the present study, we show that, in human cells, after heterologous expression of GFP-Ypt11 (where GFP stands for green fluorescent protein), the protein is targeted to transferrin-positive recycling endosomes. This compartment has been shown to form extensive tubular networks on applying the drug Brefeldin A. We also show, by confocal fluorescent microscopy, that these networks also contain Rab11 in cells expressing CFP-Rab11a (where CFP stands for cyan fluorescent protein) fusion protein and that these structures are identical with those targeted by GFP-Ypt11.     

Esterified astaxanthin levels in lobster epithelia correlate with shell colour intensity: potential role in crustacean shell colour formation

Carotenoids, particularly astaxanthin, are the primary pigment in crustacean shell colour. Sub-adults of the western rock lobster, Panulirus cygnus, moult from a deep red colour (termed the red phase) to a much paler colour (the white phase) at sexual maturation. We observe a 2.4-fold difference in the amount of total carotenoid present in the shell extracts of reds compared to whites, as might be expected. However, analysis of the underlying epithelium shows that there is no correlation with shell colour and the amount of free (unesterified) astaxanthin-the level of free astaxanthin in reds and whites is not significantly different. Instead, we observe a correlated two-fold difference in the amount of esterified astaxanthin present in the epithelium of red versus white individuals. These data suggest a role for esterified astaxanthin in regulating shell colour formation and suggest that esterification may promote secretion and eventual incorporation of unesterified astaxanthin into the exoskeleton.