Cloning and expression of a Streptomyces plicatus chitinase (chitinase-63) in Escherichia coli

4-Methylumbelliferyl (4-MU) glycosides of N-acetylglucosamine oligosaccharides were used as substrates to detect expression of a Streptomyces chitinase in Escherichia coli. Low levels of enzyme were detected when S. plicatus DNA was cloned into a bacteriophage lambda vector (EMBL-4). Subcloning into E. coli plasmids also gave low but detectable levels of enzyme expression. High level expression was achieved by resection of the cloned S. plicatus DNA with Bal31 followed by in-frame fusion to the amino-terminal peptide sequence of beta-galactosidase found in the pUC vectors. The Streptomyces chitinase was secreted into the periplasmic space of E. coli, and its signal sequence was removed. We characterized the activity of the cloned enzyme and compared it to three other purified Streptomyces plicatus chitinases with respect to hydrolysis of the 4-MU oligosaccharides. We found that two of the enzymes form 4-methylumbelliferone much more rapidly from the 4-MU disaccharide than from the trisaccharide. These same enzymes convert the 4-MU trisaccharide primarily to diacetylchitobiose and the 4-MU monosaccharide, a nonfluorescent product. The latter compound is not hydrolyzed appreciably by any of the enzymes. On the basis of these results, we suggest a new definition of "exo" and "endo" chitinase that differs from that found in the literature. We propose that exochitinase activity be defined as processive action starting at the nonreducing ends of chitin chains with release of successive diacetylchitobiose units, and that endochitinase activity be defined as random cleavage at internal points in chitin chains.     

An X-ray microanalytical azo dye technique for the localization of acid phosphatase activity

Summary A new method is described for the histochemical localization of acid phosphatase. Naphthol AS BI, enzymatically released from naphthyl AS BI phosphoric acid, is coupled with diazotized 2,5-dibromoaniline to produce a fine insoluble red azo dye. The histochemical and cytochemical localization of this final reaction product in rat liver is described. In the electron microscope, sites of the azo dye can be detected by X-ray microanalysis of ultrathin cryosections of reactive tissue.This research was supported by Scientific Research Council Grant No. B/RG/67527     

Relationships between land use, spatial scale and stream macroinvertebrate communities

1. The structure of lotic macroinvertebrate communities may be strongly influenced by land‐use practices within catchments. However, the relative magnitude of influence on the benthos may depend upon the spatial arrangement of different land uses in the catchment. 2. We examined the influence of land‐cover patterns on in‐stream physico‐chemical features and macroinvertebrate assemblages in nine southern Appalachian headwater basins characterized by a mixture of land‐use practices. Using a geographical information system (GIS)/remote sensing approach, we quantified land‐cover at five spatial scales; the entire catchment, the riparian corridor, and three riparian ‘sub‐corridors’ extending 200, 1000 and 2000 m upstream of sampling reaches. 3. Stream water chemistry was generally related to features at the catchment scale. Conversely, stream temperature and substratum characteristics were strongly influenced by land‐cover patterns at the riparian corridor and sub‐corridor scales. 4. Macroinvertebrate assemblage structure was quantified using the slope of rank‐abundance plots, and further described using diversity and evenness indices. Taxon richness ranged from 24 to 54 among sites, and the analysis of rank‐abundance curves defined three distinct groups with high, medium and low diversity. In general, other macroinvertebrate indices were in accord with rank‐abundance groups, with richness and evenness decreasing among sites with maximum stream temperature. 5. Macroinvertebrate indices were most closely related to land‐cover patterns evaluated at the 200 m sub‐corridor scale, suggesting that local, streamside development effectively alters assemblage structure. 6. Results suggest that differences in macroinvertebrate assemblage structure can be explained by land‐cover patterns when appropriate spatial scales are employed. In addition, the influence of riparian forest patches on in‐stream habitat features (e.g. the thermal regime) may be critical to the distribution of many taxa in headwater streams draining catchments with mixed land‐use practices.     

Five in situ observations of live oarfish Regalecus glesne (Regalecidae) by remotely operated vehicles in the oceanic waters of the northern Gulf of Mexico

As part of the SERPENT Project, five observations of apparently healthy oarfish Regalecus glesne by remotely operated vehicles are reported from the northern Gulf of Mexico. Regalecus glesne were observed between 2008 and 2011 at depths from within the epipelagic and mesopelagic zones. These observations include the deepest verified record of R. glesne (463–492 m) and the first record of an arthropod ectoparasite (isopod).     

Changes in ponderal index and body mass index across childhood and their associations with fat mass and cardiovascular risk factors at age 15

Background

Little is known about whether associations between childhood adiposity and later adverse cardiovascular health outcomes are driven by tracking of overweight from childhood to adulthood and/or by vascular and metabolic changes from childhood overweight that persist into adulthood. Our objective is to characterise associations between trajectories of adiposity across childhood and a wide range of cardiovascular risk factors measured in adolescence, and explore the extent to which these are mediated by fat mass at age 15.

Methods and Findings

Using data from the Avon Longitudinal Study of Parents and Children, we estimated individual trajectories of ponderal index (PI) from 0–2 years and BMI from 2–10 years using random-effects linear spline models (N = 4601). We explored associations between PI/BMI trajectories and DXA-determined total-body fat-mass and cardiovascular risk factors at 15 years (systolic and diastolic blood pressure, fasting LDL- and HDL-cholesterol, triglycerides, C-reactive protein, glucose, insulin) with and without adjustment for confounders. Changes in PI/BMI during all periods of infancy and childhood were associated with greater DXA-determined fat-mass at age 15. BMI changes in childhood, but not PI changes from 0–2 years, were associated with most cardiovascular risk factors in adolescence; associations tended to be strongest for BMI changes in later childhood (ages 8.5–10), and were largely mediated by fat mass at age 15.

Conclusion

Changes in PI/BMI from 0–10 years were associated with greater fat-mass at age 15. Greater increases in BMI from age 8.5–10 years are most strongly associated with cardiovascular risk factors at age 15, with much of these associations mediated by fat-mass at this age. We found little evidence supporting previous reports that rapid PI changes in infancy are associated with future cardiovascular risk. This study suggests that associations between early overweight and subsequent adverse cardiovascular health are largely due to overweight children tending to remain overweight.     

Shifts in allochthonous input and autochthonous production in streams along an agricultural land-use gradient

Relative contributions of allochthonous inputs and autochthonous production vary depending on terrestrial land use and biome. Terrestrially derived organic matter and in-stream primary production were measured in 12 headwater streams along an agricultural land-use gradient. Streams were examined to see how carbon (C) supply shifts from forested streams receiving primarily terrestrially derived C to agricultural streams, which may rely primarily on C derived from algal productivity. We measured allochthonous input, chlorophyll a concentration, and periphyton biomass in each stream, and whole-stream metabolism in six streams. Our results suggest a threshold between moderate- and heavy-agriculture land uses in which terrestrially derived C is replaced by in-stream algal productivity as the primary C source for aquatic consumers. A shift from allochthonous to autochthonous production was not evident in all heavy-agriculture streams, and only occurred in heavy-agriculture streams not impacted by livestock grazing. We then compared our findings to rates of allochthonous input and GPP in streams with minimal human influences in multiple biomes to assess how land-use practices influence C sources to stream ecosystems. The proportion of C derived from allochthonous versus autochthonous sources to heavy-agriculture streams was most similar to grassland and desert streams, while C sources to forested, light-, and moderate-agriculture streams were more similar to deciduous and montane coniferous forest streams. We show that C source to streams is dependent on land use, terrestrial biome, and degradation of in-stream conditions. Further, we suggest that within a biome there seems to be a compensation such that total C input is nearly equal whether it is from allochthonous or autochthonous sources.     

Isolation and sequence analysis of cDNA clones coding for rat skeletal muscle creatine kinase

A series of cDNA clones corresponding to 1494 bases of rat muscle creatine kinase mRNA has been isolated and characterized. The identity of these clones has been confirmed by DNA sequence analysis and by comparison of the predicted amino acid sequence with that determined for the purified protein. The cDNA sequence accounts for the entire coding sequence of the creatine kinase protein in addition to the complete 3' untranslated region and 68 bases of 5' noncoding region. Sequences corresponding to the active site region of the protein, the initiation codon, the termination codon, and poly(A) addition signal have been identified.