Sequence analysis and molecular characterization of the Lactococcus lactis temperate bacteriophage BK5-T

The Lactococcus lactis temperate bacteriophage BK5-T is one of twelve type phages that define L. lactis phage species. This paper describes the nucleotide sequence and analysis of a 21-kbp region of the BK5-T genome and completes the nucleotide sequence of the genome of this phage. The 40,003-nucleotide linear genome encodes 63 open reading frames. Sequence runoff experiments showed that the cohesive ends of the BK5-T genome contained a 12-bp 3' single-stranded overhang with the sequence 5'-CACACACATAGG-3'. Two major BK5-T structural proteins, of approximately 30 and 20 kDa, were identified, and N-terminal sequence analysis determined that they were encoded by orf7 and orf12, respectively. A 169-bp fragment containing a 37-bp direct repeat and several smaller repeat sequences conferred resistance to BK5-T infection when introduced in trans to the host cell and is likely a part of the BK5-T origin of replication (ori).     

Relations of mineral-soil C and N to climate and texture: regional differences within the conterminous USA

Soil is a prominent component of terrestrial C and N budgets. Soil C and N pools are influenced by, and may reciprocally influence, many environmental factors. Our objective was to determine the quantitative relations of surface mineral-soil organic C, N, and C/N ratios to climate and soil texture across seven ecological regions that make up the conterminous USA. Up to 608 soil profiles per region and their corresponding climates were evaluated with regression analysis. The organic C pool (kg C m⁻²) in the upper 20 cm of mineral soil was positively related to mean annual precipitation, evapotranspiration and clay content in all regions. It was negatively related to a temperature/precipitation index in all regions and negatively related to mean annual temperature, except in the northwest temperate forest region. Soil C/N ratios were negatively related to clay or silt content in all regions. These relations are consistent with concepts of moisture and temperature controls on detrital production, differential effects of temperature on detrital production and decomposition, and stabilization of organic matter by clay and silt. Differences in quantitative relations among regions may be related to vegetation-composition effects on soil organic matter processes, clay mineralogy, and faunal mixing of surface organic horizons with mineral soil. Regional differences also occurred in the importance of climate vs. soil texture in explaining the variability in soil C. The regional differences indicate the importance of using region-specific, rather than generalized, equations for projecting long-term soil responses to climate change and for conducting ecosystem-model calibration or validation.     

Stabilization of a supplemental digestive enzyme by post-translational engineering using chemically-activated polyethylene glycol

Many enzymes used as digestive aids exhibit, at best, moderate stability when incubated under gastrointestinal conditions. A supplemental β-galactosidase administered orally to treat lactose intolerance was conjugated to 40 kDa, branched polyethylene glycol (PEG). PEGylation increased the enzyme's relative activity at lower pH values (2.5-4.5) and doubled enzyme stability at pH 2.5. The PEGylated enzyme retained significantly more residual activity after exposure to simulated gastric conditions (52% versus 31%), a consequence of protection from both pepsin and low pH mediated inactivation. Conjugation also provided significant protection against the proteolytic component of pancreatin. Overall, the PEGylated enzyme retained over twice the levels of residual activity recorded for non-PEGylated enzyme after exposure to complete simulated gastrointestinal conditions. PEGylation also marginally improved the enzyme's kinetic characteristics. When using its physiological substrate (lactose), K(m) values recorded were slightly decreased (from 83 to 60 μM) and k(cat)/K(m) values (M(-1) s(-1)) were increased from 100 to 147. This appears to be the first report of the use of a conjugated PEG to stabilize a digestive enzyme and the first report of the ability of conjugated PEG to stabilize a protein at low pH.     

Partial cambial mortality in high-elevation Pinus aristata (Pinaceae)

Partial cambial mortality is a growth form that is characteristic of Pinus aristata trees. To better elucidate their cambial death pattern, tree size and aspect of cambial death data were gathered from three Pinus aristata forests in central Colorado, USA. Stripping frequency tended to be higher for larger diameter classes. Partial cambial mortality exhibits significant directionality within each stand. Furthermore, cambial death was measured to be most frequent on the wind-exposed side of stripped trees in two of the three study sites and appeared to be at the third. Data presented here support the hypothesis that wind plays a role in the occurrence of partial cambial mortality in Pinus aristata. The mechanisms by which wind causes cambial mortality remain unclear.     

Functional design space of single-veined leaves: role of tissue hydraulic properties in constraining leaf size and shape

BACKGROUND AND AIMS: Morphological diversity of leaves is usually quantified with geometrical characters, while in many cases a simple set of biophysical parameters are involved in constraining size and shape. One of the main physiological functions of the leaf is transpiration and thus one can expect that leaf hydraulic parameters can be used to predict potential morphologies, although with the caveat that morphology in turn influences physiological parameters including light interception and boundary layer thickness and thereby heat transfer and net photosynthesis. METHODS: An iterative model was used to determine the relative sizes and shapes that are functionally possible for single-veined leaves as defined by their ability to supply the entire leaf lamina with sufficient water to prevent stomatal closure. The model variables include the hydraulic resistances associated with vein axial and radial transport, as well as with water movement through the mesophyll and the leaf surface. KEY RESULTS: The four parameters included in the model are sufficient to define a hydraulic functional design space that includes all single-veined leaf shapes found in nature, including scale-, awl- and needle-like morphologies. This exercise demonstrates that hydraulic parameters have dissimilar effects: surface resistance primarily affects leaf size, while radial and mesophyll resistances primarily affect leaf shape. CONCLUSIONS: These distinctions between hydraulic parameters, as well as the differential accessibility of different morphologies, might relate to the convergent evolutionary patterns seen in a variety of fossil lineages concerning overall morphology and anatomical detail that frequently have evolved in linear and simple multi-veined leaves.     

Carbohydrate Metabolism in Taproots of Medicago sativa L. during Winter Adaptation and Spring Regrowth

Our objective was to identify amylases that may participate in starch degradation in alfalfa (Medicago sativa L.) taproots during winter hardening and subsequent spring regrowth. Taproots from field-grown plants were sampled at intervals throughout fall, winter, and early spring. In experiment 1, taproots were separated into bark and wood tissues. Concentrations of soluble sugars, starch, and buffer-soluble proteins and activities of endo- and exoamylase were determined. Starch concentrations declined in late fall, whereas concentrations of sucrose increased. Total amylolytic activity (primarily exoamylase) was not consistently associated with starch degradation but followed trends in soluble protein concentration of taproots. This was especially evident in spring when both declined as starch degradation increased and shoot growth resumed. Activity of endoamylase increased during periods of starch degradation, especially in bark tissues. In experiment 2, a low starch line had higher specific activity of taproot amylases. This line depleted its taproot starch by late winter, after which taproot sugar concentrations declined. As in experiment 1, total amylolytic activity declined in spring in both lines, whereas that of endoamylase increased in both lines even though little starch remained in taproots of the low starch line. Several isoforms of both amylases were distinguished using native polyacrylamide electrophoresis, with isoforms being similar in bark and wood tissues. The slowest migrating isoform of endoamylase was most prominent at each sampling. Activity of all endoamylase isoforms increased during winter adaptation and in spring when shoot growth resumed. Endoamylase activity consistently increased at times of starch utilization in alfalfa taproots (hardening, spring regrowth, after defoliation), indicating that it may serve an important role in starch degradation.     

Plant collecting spread and densities: their potential impact on biogeographical studies in Thailand

Aims To produce representative aggregate maps of plant collection locations in Thailand and discuss their impact on biogeographical studies in Thailand and the surrounding region. Location Thailand. Methods A representative data set comprising 6593 plant specimen records for Thailand has been assembled. The data set contains ± all known collections for fifteen representative plant families and further records for another 104. All records are localized to Changwat (province), 6441 to at least quarter degree square. Results Analysis shows that the spread of collecting activity in Thailand is markedly uneven; 20% of collections come from a single Changwat (Chiang Mai) and 53% of Changwat have fifty or fewer collections. The distribution of collections by Changwat and by quarter degree square is erratic with most squares and Changwat having few collections, both in proportionate and absolute terms. Some of the most densely forested Changwats and squares appear undercollected. Distribution maps for common, easily recognized tree species in the genus Syzygium show distributional gaps. Conclusions Thailand is defined as an undercollected country. Even within the few well‐collected quarter degree squares the spread of collecting is still poor; almost all collections being localized to one of three mountain ranges or their foothills. There are many gaps in collecting activity which make impossible a straightforward interpretation of biogeographical pattern. It is argued that targeted collecting activity is needed, that assembly of this type of data set is therefore essential and that our data set and its interpretation is a model for all countries in the region.     

Heterologous cross-linking of Lyt-2 (CD8) to the alpha beta-T cell receptor is more effective in T cell activation than homologous alpha beta-T cell receptor cross-linking

Ag recognition of Lyt-2 (CD8)-positive T lymphocytes requires the presentation by APC of a suitably processed Ag in association with MHC class I molecules. In previous studies we have obtained evidence that, for optimal activation, both the alpha beta-TCR and Lyt-2 have to participate in this recognition process. In the current study we investigate the functional consequences of limited cross-linking of these cell surface molecules by using soluble, dimeric hetero- and homoconjugates of mAb to Lyt-2 and to the TCR beta-chain (F23.1). Heterologous cross-linking of Lyt-2 to the TCR induced a vigorous, selective Lyt-2+ T cell proliferative response. Functionally active cytotoxic cells were generated, and a high frequency of responding cells was observed in limiting dilution analyses. In contrast, homologous TCR cross-linking initiated a less pronounced proliferation with a relatively low frequency of response, whereas Lyt-2 cross-linking resulted in no cellular proliferation. Significant T cell activation occurred with exposure to anti-Lyt-2: F23.1 mAb dimers at concentrations an order of magnitude lower than those required for stimulation by F23.1:F23.1 mAb dimers. The induction of proliferation by mAb dimers occurred in the absence of Fc components and in rigorously APC depleted, purified T cell preparations. Effective stimulation of resting T cells could be induced also by heterodimers of monovalent Fab fragments. Heterologous cross-linking of Lyt-2 to the TCR was superior to homologous TCR cross-linking primarily with respect to proliferation in IL-2 containing media and to IL-2R expression, whereas proliferation in response to other lymphokines and the production of IL-2 itself were similar under both cross-linking regimens. Thus, when linked to the TCR, Lyt-2 contributed a strong, positive signal toward IL-2-dependent growth of resting T cells. We assume that in the case of Ag-driven T cell activation, the class I MHC molecule acts as the physiologic cross-linking ligand for Lyt-2 and the TCR.     

Grizzly bear movements relative to roads: application of step selection functions

Access management is among the most important conservation actions for grizzly bears in North America. In Alberta, Canada, nearly all grizzly bear mortalities are caused by humans and occur near roads and trails. Consequently, understanding how bears move relative to roads is of crucial importance for grizzly bear conservation. We present the first application of step‐selection functions to model habitat selection and movement of grizzly bears. We then relate this to a step‐length analysis to model the rate of movement through various habitats. Grizzly bears of all sex and age groups were more likely to select steps closer to roads irrespective of traffic volume. Roads are associated with habitats attractive to bears such as forestry cutblocks, and models substituting cutblocks for roads outperformed road models in predicting bear selection during day, dawn, and dusk time periods. Bear step lengths increased near roads and were longest near highly trafficked roads indicating faster movement when near roads. Bear selection of roads was consistent throughout the day; however, time of day had a strong influence over selection of forest structure and terrain variables. At night and dawn, bears selected forests of intermediate age between 40 and 100 yr, and bears selected older forests during the day. At dawn, bears selected steps with higher solar radiation values, whereas, at dusk, bears chose steps that were significantly closer to edges. Because grizzly bears use areas near roads during spring and most human‐caused mortalities occur near roads, access management is required to reduce conflicts between humans and bears. Our results support new conservation guidelines in western North America that encourage the restriction of human access to roads constructed for resource extraction.