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The first gene of a family of prokaryotic proteases with a specificity for l,d‐configured peptide bonds has been identified in Escherichia coli. The gene named ldcA encodes a cytoplasmic l,d‐carboxypeptidase, which releases the terminal d‐alanine from l‐alanyl‐d‐glutamyl‐meso‐diaminopimelyl‐d‐alanine containing turnover products of the cell wall polymer murein. This reaction turned out to be essential for survival, since disruption of the gene results in bacteriolysis during the stationary growth phase. Owing to a defect in muropeptide recycling the unusual murein precursor uridine 5′‐pyrophosphoryl N‐acetylmuramyl‐tetrapeptide accumulates in the mutant. The dramatic decrease observed in overall cross‐linkage of the murein is explained by the increased incorporation of tetrapeptide precursors. They can only function as acceptors and not as donors in the crucial cross‐linking reaction. It is concluded that murein recycling is a promising target for novel antibacterial agents.
The cell envelope of most bacteria is stabilized by an exoskeleton made of the cross‐linked heteropolymer murein (peptidoglycan). Its basic building block, a peptidyl‐disaccharide, is polymerized by the formation of glycosidic and peptide bonds. Thereby, a bag‐shaped structure, the murein sacculus, is synthesized which completely encloses the cell (reviewed by Höltje, 1998). In the case of Escherichia coli the subunits of murein consist of the peptide d‐alanyl‐d‐glutamyl‐meso‐diaminopimelyl‐d‐alanyl‐d‐alanine (l‐Ala‐d‐Glu‐m‐A2pm‐d‐Ala‐d‐Ala) or truncated forms of this peptide, which are linked via an amide bond to the lactyl group of the muramic acid of the disaccharide N‐acetylglucosamine‐β‐1.4‐N‐acetylmuramic acid (GlcNAc‐MurNAc; van Heijenoort, 1994). Importantly, the peptide side chains consist of d‐ and l‐ (or meso‐) amino acids giving rise to the presence of unusual l,d‐, as well as d,d‐, peptide bonds in murein. Cross‐linkage of the murein strands is achieved by a head‐to‐tail cross‐linking of the peptides via a peptide bond between the carboxyl group of the terminal amino acid of one peptide moiety and the non‐alpha amino group present in a peptide side chain protruding from a neighboring glycan strand. In E.coli two types of cross‐links can be formed (Glauner et al., 1988). The majority is found between the carboxyl group of a terminal d‐Ala and the ω‐amino group at the d‐center of the meso‐diaminopimelic acid resulting in a cross‐linking d,d‐peptide bond. To a lesser extent also the carboxyl group at the l‐center of a terminal meso‐diaminopimelic acid present in a tripeptide stem peptide is linked to the ω‐amino group at the d‐center of the meso‐diaminopimelic acid of another peptide side chain giving rise to the formation of an l,d‐cross‐linking peptide bond.
Not surprisingly, l,d‐ and d,d‐peptide bonds are import ant targets for highly specific antibacterial agents since they are not found in eukaryotes. The most important group of antibiotics, the β‐lactams, inhibit the enzymes specifically involved in the formation and cleavage of the d,d‐peptide bonds (Waxman and Strominger, 1983). Since these enzymes bind penicillin covalently, they are referred to as penicillin‐binding proteins (PBPs; Spratt, 1975). In addition to transpeptidases and endopeptidases, carboxypeptidases have also been described (Izaki et al., 1966). Being PBPs many of the d,d‐enzymes from different sources have been characterized in some detail (Ghuysen, 1991).
By way of contrast, knowledge about enzymes with a specificity for l,d‐peptide bonds is limited. In particular, no gene encoding an l,d‐transpeptidase, an l,d‐endopeptidase or an l,d‐carboxypeptidase has been cloned to date and the physiological functions of these proteins are still under discussion (Templin and Höltje, 1998). The best characterized example of an enzyme of this group is an l,d‐carboxypeptidase isolated from E.coli (Metz et al., 1986a,b; Ursinus et al., 1992). The enzyme cleaves the d‐alanine in position 4 that is linked via an l,d‐peptide bond to the l‐center of the meso‐diaminopimelic acid in position 3 of the peptide side chains. Such an activity was shown earlier to oscillate during the cell cycle of E.coli with highest activity during cell division (Beck and Park, 1976, 1977). The function(s) of l,d‐carboxypeptidases remain(s) obscure, although an involvement in cell division has been proposed (Begg et al., 1990; Höltje, 1998). Here we report on the cloning, overexpression and deletion of a cytoplasmic l,d‐carboxypeptidase which turned out to be essential at the onset of the stationary phase of growth by specifically interfering with murein recycling.
Identification of the gene encoding the L,D‐carboxypeptidase A
Classical protein purification methods yielded a highly enriched fraction of an l,d‐carboxypeptidase, which could be used for biochemical characterization of the enzyme (Ursinus et al., 1992). Nevertheless, the amount of protein was not high enough to obtain a partial peptide sequence that could have been used for identification of the gene encoding this enzyme. Since a simple and fast assay for l,d‐carboxypeptidase using uridine 5′‐pyrophosphoryl N‐acetylmuramyl‐l‐alanyl‐d‐glutamyl‐meso‐diaminopimelyl‐d‐alanine (UDP‐MurNAc‐tetrapeptide) as a substrate, which can be used even with crude cell extracts, was available (Ursinus et al., 1992), we decided to screen an expression library for the corresponding gene. Therefore, the Kohara miniset library (Noda et al., 1991) was scored for phage‐based overexpression of l,d‐carboxypeptidase activity. Screening the 479 phages from the miniset library led to the identification of one clone with a reproducible increase in l,d‐carboxypeptidase activity. This phage (λ 245) carries a 17.4 kb insert containing 13 potential reading frames. Only one of the predicted proteins, f304 (Blattner et al., 1997) had a calculated molecular mass close to the experimentally determined value of the l,d‐carboxypeptidase (Ursinus et al., 1992). Therefore, this coding region was amplified from phage DNA by PCR and directly cloned into an expression vector. A transformant of MC1061 carrying this construct (pLD2) was induced to overproduce the cloned gene product by adding isopropyl‐thio‐β‐d‐galactoside (IPTG). A 4500‐fold increase in l,d‐carboxypeptidase activity was detected and it could be concluded that the gene for an l,d‐carboxypeptidase active on UDP‐MurNAc‐tetrapeptide had been identified. This high degree of overproduction had no effect on growth rate or cell viability. Since this is the first example of the cloning of a gene coding for an enzyme possessing l,d‐carboxypeptidase activity the gene was named ldcA (l,d‐carboxypeptidase A).
Sequence analysis suggests a prokaryotic protease family
The reading frame for the LdcA consists of 912 base pairs, coding for a protein with a predicted molecular mass of 33 572 Da. No apparent N‐terminal signal sequence could be detected by using the TopPred II algorithm (Claros and von Heijne, 1994); therefore, a cytosolic location was expected.
Interestingly, the ldcA gene and emtA, a recently identified gene coding for a lytic endo‐transglycosylase (Kraft et al., 1998), are found side‐by‐side (Figure 1). Whereas the genes are transcribed in opposite directions, the promotors for both genes seem to overlap (M.F.Templin, unpublished). A coordinated expression might be significant, since some of the muropeptides released by EmtA are substrates for the l,d‐carboxypeptidase.
Similarity searches with the predicted coding sequence were performed using the PSI‐BLAST algorithm (Altschul et al., 1997) and revealed the presence of homologs in different bacterial species including Synechocystis sp., Bacillus subtilis, Streptomyces coelicolor, Vibrio cholerae, Rickettsia prowazekii and Treponema pallidum (e values <10−63 in a PSI‐BLAST search after the second iteration). No significantly similar proteins were detected in any eukaryote. When extending the searches to the available 'unfinished bacterial genomes' (last update 15 March 1999; preliminary sequence data were obtained from the Institute for Genomic Research website at http://www.tigr.o..) further homologs of LdcA were detected in Salmonella typhi, Neisseria gonorrhoeae, Neisseria meningitidis, Bordetella pertussis, Chlorobium tepidum, Pseudomonas aeruginosa, Streptococcus pyogenes, Streptococcus mutans and Deinococcus radiodurans (e values in the tblastn search <10−5).
An alignment using the identified proteins shows three regions of high similarity (Figure 2). Therefore, the database searches suggest that these orthologs form a protease family present in a wide range of bacteria.
Subcellular localization of LdcA
Earlier experiments pointed to a periplasmic location of an l,d‐carboxypeptidase that could be released from whole cells by Tris–EDTA treatment (Beck and Park, 1977). However, the sequence analysis discussed above favors a cytoplasmic location for LdcA. Therefore, cell fractionation experiments were performed. Following the method of Witholt et al. (1979), cells were fractionated into periplasmic extract, membrane fraction and cytosolic extract. Using UDP‐MurNAc‐tetrapeptide as a substrate, enzyme activity was exclusively found in the cytoplasm in wild‐type E.coli MC1061 (Table I). Even upon high induction of expression of LdcA only traces of activity (>1%) were found in the membrane or in the periplasmic fraction (data not shown). The same result was obtained after Tris–EDTA treatment of the cells performed as described by Beck and Park, (1977). This shows that the LdcA is a cytoplasmic enzyme.
Substrate specificity and inhibition of the LdcA
With the construction of an N‐terminally His6‐tagged version of the enzyme it was possible to obtain sufficient amounts of highly enriched enzyme preparations (2.53×106 units/mg protein). This allowed us to determine the substrate specificity of the LdcA in some detail and to examine the sensitivity against different antibiotics. Cytoplasmic fractions containing 1.34 units of enzyme were incubated with a variety of murein derivatives carrying l‐Ala‐d‐Glu‐meso‐A2pm‐d‐Ala tetrapeptide side chains. As listed in Table II, LdcA does not interact with isolated high molecular mass murein sacculi or with cross‐linked muropeptides. Thus, only monomeric muropeptides, free tetrapeptide and UDP‐activated murein precursors are substrates for LdcA. This finding is in accordance with results obtained by others (Leguina et al., 1994), who showed that an l,d‐carboxypeptidase is present in E.coli that acts on low molecular mass muropeptides, but not on the native polymer murein. The specificity determined is consistent with the fact that LdcA is a cytoplasmic enzyme that cannot come into contact with the murein sacculus in the periplasm.
l,d‐carboxypeptidase activity had been shown to be inhibited competitively by β‐lactam antibiotics containing a d‐configured amino acid as a side chain (Hammes, 1978; Hammes and Seidel, 1978a; Ursinus et al., 1992). Metz et al. (1986b) reported an inhibition of the activity by penem antibiotics. The enriched enzyme fraction was used to test the senitivity of LdcA: little or no inhibition could be shown for Penicillin G and the penems Imipemem and CGP31608 (kindly supplied by Ciba‐Geigy, Basel, Switzerland); β‐lactams containing a d‐configured amino acid as a side chain including Cefminox, Cephalosporin C and Norcardicin A (kindly supplied by Eli‐Lilly, Indianapolis, IN) inhibited enzyme activity (Table II).
Construction and growth phenotype of a deletion mutant
To get an idea of the function of the ldcA, a plasmid‐coded deletion was constructed and transferred to the chromosome of E.coli MC1061 by λ‐phage‐mediated transduction (Kulakauskas et al., 1991). The obtained mutant named LD3 did not contain the coding sequence for the protein. No l,d‐carboxypeptidase activity could be detected in soluble extracts when UDP‐MurNAc‐tetrapeptide was used as a substrate (data not shown), suggesting that the identified enzyme is the only activity of this type present in E.coli.
Despite the fact that viable clones could be obtained, growth during the stationary phase was severely affected. After growth on Luria‐Bertani (LB) agar for 72 h at 37°C, only a small percentage of the cells remained viable. Liquid cultures of wild‐type bacteria and of the LdcA deletion mutant growing aerobically at 37°C had the same growth rate during the exponential phase (Figure 3). However, upon onset of the stationary phase, growth of the mutant culture seemed to slow down and the culture eventually started to lyse. Phase‐contrast microscopy (Figure 4) showed cells that had lost their shape and, in addition, many lysed cells were visible. The number of viable cells determined after 12 h of growth was reduced to 3.5×107 in the LdcA mutant culture compared with 1.8×109 in the control culture. Therefore, spontaneous autolysis occurred when the culture entered the stationary growth phase. This phenotype could be suppressed by a plasmid carrying the ldcA gene.
Changes in the murein composition of the mutant
Lysis of the LdcA deletion mutant in the stationary growth phase could be the result of some defects in the structure of the murein sacculus. Therefore, the composition of the murein of an exponentially growing culture (OD578 0.5) and a culture just entering the stationary phase (OD578 1.5) was analyzed. Isolated sacculi were digested completely using a muramidase and the degradation products (muropeptides) were separated and quantified by reversed‐phase high‐pressure liquid chromatography (RP‐HPLC) (Glauner, 1988). The results shown in Table III reveal significant differences in the relative amounts of the various muropeptides between the ldcA mutant and the parental strain. Whereas these differences were obvious in the murein from cells harvested in the exponential phase of growth, they were more pronounced for a culture grown to an OD578 of 1.5. In particular the overall cross‐linkage was reduced by 23% leading to an increase in the monomeric muropeptide disaccharide–tetrapeptide by 16% and disaccharide–tripeptide by 46%. Whereas the number of dimeric d,d‐cross‐bridges was reduced by 12%, an even more dramatic decrease was observed for the trimeric and tetrameric structures containing d,d‐cross‐links: the relative amounts decreased by 67 and 58%, respectively. Interestingly, the unusual l,d‐cross‐linkage between two m‐A2pm residues was increased. This finding is consistent with a number of reports showing a rise of l,d‐cross‐links when d,d‐cross‐links decrease for various reasons (Driehuis and Wouters, 1987; Kohlrausch and Höltje, 1991a). Therefore, formation of l,d‐cross‐bridges is considered to represent a salvage mechanism to keep the overall degree of cross‐linkage at a high level even under conditions where normal d,d‐cross‐links, which depend on the presence of sufficient pentapeptides, can no longer be formed. It is important to note that the number of chain ends, which reflects the average length of the glycan strands, did not increase in the mutant indicating that the impending lysis event was not caused by autolytic processes described previously (Kitano and Tomasz, 1979; Kohlrausch and Höltje, 1991a). We conclude that autolysis of the ldcA mutant is the result of a dramatically reduced overall murein cross‐linkage.
Determination of murein precursors and phenotypic suppression of ldcA mutants
Since LdcA is present in the cytoplasm it can interact only with cytoplasmic murein precursors and murein turnover products. The turnover products, however, are known to be recycled by their incorporation into the precursor molecules (Goodell, 1985). Therefore, the intracellular pools of the final UDP‐activated murein precursors were determined in an LdcA mutant and compared with the pools present in wild‐type cells. The precursors were radioactively labeled by growth of the cells in the presence of [3H]diaminopimelic acid, extracted with hot water and separated by HPLC. As shown in Figure 5, in wild‐type cells only UDP‐MurNAc‐pentapeptide could be found in the cytoplasm. By way of contrast in the deletion mutant HfrH‐ldcA only a small amount of pentapeptide is detected but there is a prominent peak corresponding to UDP‐MurNAc‐tetrapeptide which has never been detected in E.coli before. In addition, free tetrapeptide is also found.
With a possible role in recycling, it was interesting to investigate the effects of mutations in other genes coding for proteins involved in muropeptide recycling. It was particularly interesting to study the effect of an ampD mutation, which is known to cause a severe block in muropeptide recycling, since cytoplasmic hydrolysis of muropeptides into the sugar and peptide part is abolished (Höltje et al., 1994; Jacobs et al., 1995). It was therefore expected that a mutation in ampD would counteract the toxic effect of a lack in LdcA. Indeed, a mutant carrying deletions in both ampD and ldcA showed no lysis during stationary growth. Analysis of precursor pools in this mutant showed a decrease in the amounts of free tetrapeptide and UDP‐MurNAc‐tetrapeptide, whereas MurNAc‐pentapeptide was increased compared with the ldcA single mutant (Figure 5).
The observed phenotypic suppression by a mutation in ampD further supports the idea that LdcA takes part in the recycling pathway. But it should be noted that this type of phenotypic suppression might also occur in an in vivo situation when LdcA is inactivated by specific inhibitors. This would not only lead to restored viability, but in bacteria possessing a certain type of a chromosomally encoded β‐lactamase (e.g. Citrobacter freundii; Lindberg et al., 1987) this might even lead to a decreased sensitivity against penicillins or cephalosporins. A combination of the ldcA deletion with a mutation in the N‐acetylglucosaminidase, encoded by nagZ, which also participates in muropeptide recycling did not change the intracellular pool of UDP‐MurNAc‐pentapeptide and did not suppress bacteriolysis in the stationary growth phase (data not shown).
Recycling of the murein turnover products, which make up ∼50% per generation of the total murein (Goodell, 1985), is an important energy‐saving mechanism. Only a very small amount of turnover material, ∼4–7% per generation, is lost to the medium (Goodell and Schwarz, 1985). Therefore, it is not surprising to find that E.coli has a number of systems which make recycling as efficient as possible. Several uptake systems have been described, which are involved in the transportation of the murein turnover products from the periplasm back into the cytoplasm. Muropeptides are imported via the AmpG transporter (Jacobs et al., 1994), whereas peptides that are released from the murein or from muropeptides by the action of periplasmic amidases are accepted by the Opp (Goodell and Higgins, 1987) and Mpp (Park et al., 1998) permeases.
The fate of the major turnover products, the disaccharide‐anhydro tri‐ and tetrapeptides, has been of considerable interest in recent research. A close connection between the regulation of expression of certain chromosomally encoded β‐lactamases and murein recycling could be demonstrated (Jacobs et al., 1997). The β‐lactamase work showed that the amount of cell wall material released is used to sense the state of the murein sacculus. In a situation where the incorporation of new material is inhibited, e.g. treatment with β‐lactam antibiotics, the amount of released turnover products increases dramatically. Certain bacterial species exploit this increase of released muropeptides to induce β‐lactamase expression.
Actually, most of the known enzymes of the recycling pathway have been identified during studies of β‐lactamase induction. Not only has the uptake system AmpG been identified during a screen for genes involved in β‐lactamase induction, but also the cytoplasmic amidase AmpD, which releases the peptide moiety from cytosolic muropeptides (Lindberg et al., 1987). Other enzymes known to be involved in recycling are the cytoplasmic β‐N‐acetylglucosaminidase NagZ (Park, 1996) and the muropeptide ligase Mpl, which has been shown to catalyze the addition of the released peptide to the nucleotide‐activated MurNAc (Mengin‐Lecreulx et al., 1996). Whereas mutations in these genes mainly affect the efficiency of the recycling process, the enzyme identified in this study is essential for the survival of bacteria that have entered stationary phase.
The reaction catalyzed by the LdcA, the conversion of cytosolic tetrapeptide‐containing structures to tripeptide‐containing structures, is obviously needed for recycling. The formation of the murein precursor UDP‐MurNAc‐tripeptide occurs by the sequential addition of amino acids to the activated sugar moiety (van Heijenoort, 1994). Alternatively, the muropeptide ligase Mpl uses peptides released during recycling directly (Park et al., 1998). In the next step the general precursor for murein synthesis UDP‐MurNAc‐pentapeptide is made from UDP‐MurNAc‐tripeptide by the addition of the dipeptide d‐alanyl‐d‐alanine. Since a single d‐alanine cannot be added to a tetrapeptide, an activated tripeptide has to be present to facilitate the synthesis of the final precursor. In the absence of the l,d‐carboxypeptidase the hydrolysis of tetrapeptide‐conaining structures does not take place and the consequences are striking: the formation of UDP‐MurNAc‐tetrapeptide occurs, a dead‐end intermediate that cannot be converted into a pentapeptide precursor (Figure 6). When incorporated into the murein sacculus, it affects the number of cross‐links in the polymer since tetrapeptides can function only as acceptors and not as donors for transpeptidation. A decrease in the overall degree of cross‐linkage in the sacculus is the consequence of this failure to synthesize a sufficient supply of pentapeptide precursors. This leads not only to a weakened cell wall, but also results finally in autolysis. Therefore this work was able to identify not only the gene coding for this new type of carboxypeptidase, but also its physiological role during recycling of the cell wall polymer murein. With an enzyme required for survival and which seems to be present in a wide range of bacterial species, a potential target for antimicrobial agents has been identified. Antibiotically active substances inhibiting l,d‐carboxypeptidases have been known for quite a while (Hammes and Seidel, 1978b; Metz et al., 1986b). The best characterized example is an antibiotic called Nocardicin A. It has been shown that this monocyclic β‐lactam is able to bind to LdcA. This property has been exploited and has led to the purification of the enzyme by affinity chromatography (Ursinus et al., 1992). Nocardicin A is a bi‐headed structure: a β‐lactam with a d‐amino acid in its side chain. It interacts non‐covalently via its d‐amino acid residue with LdcA and, in addition, binds covalently with its β‐lactam ring to the classical PBPs. It is expected that completely different structures, not related to β‐lactams, may be picked up in a screen for specific inhibitors of LdcA and other l,d‐enzymes.
Interestingly, the lethal effect of the absence of LdcA seems to be restricted to the stationary phase of growth. This suggests that the contribution of muropeptide recycling, compared with de novo murein precursor synthesis, is of greater importance when the cells enter this part of their life cycle. Correspondingly, it has been shown that expression of the amidase AmiA, which is involved in periplasmic recycling, is under the control of the sigma factor RpoS (Troup et al., 1994; Jahn et al., 1996). First results indicate an increase in the expression of ldcA at this time, which is not dependent on RpoS (M.F.Templin, unpublished data). The phenotype of the ldcA mutant demonstrates that interference with murein recycling can result in autolysis. Thus a screen for compounds affecting recycling could lead to the identification of antimicrobial agents active against resting bacteria, which present a still unsolved problem in antibacterial chemotherapy.
Materials and methods
Bacterial strains, bacteriophages and plasmids
Escherichia coli MC1061 (Casabadan and Cohen, 1980) was used as a general cloning host and for the construction of a deletion mutant of ldcA. For the phage‐based overexpression assay E.coli D456 (Edwards and Donachie, 1993) was used. The Kohara miniset λ library (Noda et al., 1991) was employed for the screen to identify the gene encoding the l,d‐carboxypeptidase. Phage λ 245 (7C10) from this library was used as a DNA source for cloning the gene of interest and for PCR amplification of the flanking regions. The general cloning vector was pBluescriptII SK+ (Stratagene, La Jolla, CA); the kanamycin resistance determinant was taken from pUC4K (Pharmacia, Uppsala, Sweden). In order to construct an inducible expression system, the ldcA gene was cloned into plasmid pJFK118EH (Bishop and Weiner, 1993), an N‐terminally His6‐tagged version of the enzyme was obtained after cloning of the coding region into pQE30 (Qiagen, Hilden, Germany). For construction of a double mutant in ampDE and ldcA JRG582 (Guest and Stephens, 1980) and its isogenic parent strain HfrH were used. To test the influence of a nagZ mutation in a ldcA background, the ldcA deletion was transduced into E593 (Yem and Wu, 1976) and its parent AB1157.
Growth conditions and phase‐contrast microscopy
Bacteria were cultivated aerobically at 37°C in LB medium (Miller, 1972). Growth was monitored by determination of the optical density of the culture at 578 nm (OD578) in an Eppendorf photometer (Eppendorf, Hamburg, Germany). When required, the medium was supplemented with either 50 μg/ml kanamycin and/or 50 μg/ml ampicillin. Radioactively labeled murein precursors were prepared from bacteria, which were grown for at least four generations in LB medium supplemented with 10 μCi/ml meso‐2,6‐diamino‐[3,4,5‐3H]pimelic acid (A2pm; ARC, St Louis, MO) (Kohlrausch et al., 1989).
Phase‐contrast micrographs of bacterial cells were taken with a Zeiss Axiovert 135 TV microscope equipped with a Plan‐Neofluar 100× lens (Zeiss, Oberkochen, Germany) and a Photometrics Series 200 image‐grabbing system (Photometrics, Tucson, AZ). Pictures were edited using the IPLab Spectrum 3.0 software (Signal Analytics, Vienna, VA) and Adobe Photoshop 3.0 (Adobe Systems Inc., Mountain View, CA).
Screening of a λ phage library of E.coli W3110
The λ‐phage‐mediated overexpression assay as described by Henderson et al. (1995) was used to screen a chromosomal library of E.coli W3110. After infection of 3 ml of E.coli D456 with high titer lysates of individual λ clones from the Kohara miniset λ library (Noda et al., 1991), the bacteria were incubated for 45 min at 37°C. Infected bacteria (2 ml) were cooled rapidly on ice, harvested by centrifugation (2 min, 15 000 g), resuspended in 60 μl buffer (0.1 M Tris–HCl pH 8.0, 10 mM MgSO4, 1% Triton X‐100) and frozen. For the l,d‐carboxypeptidase assay 20 μl of the lysed cells were used in the standard enzyme assay.
Assay for L,D‐carboxypeptidase activity
The general assay to determine l,d‐carboxypeptidase activity employs RP‐HPLC and has been described in detail previously (Ursinus et al., 1992). The substrate used in the assays was UDP‐N‐acetylmuramyl‐l‐Ala‐d‐Glu‐m‐A2pm‐d‐Ala (UDP‐MurNAc‐tetrapeptide), which was prepared from UDP‐MurNAc‐pentapeptide, which contains an additional C‐terminal d‐Ala. UDP‐MurNAc‐pentapeptide was obtained from Bacillus cereus using a previously published method (Kohlrausch and Höltje, 1991b), converted to UDP‐MurNAc‐tetrapeptide by digestion with d,d‐carboxypeptidase (PBP5) from E.coli and purified using RP‐HPLC as described previously (Kohlrausch and Höltje, 1991b). Muramyl tetrapeptide was obtained after acid hydrolysis of UDP‐MurNAc‐tetrapeptide and purification by HPLC (Ursinus et al., 1992). Disaccharide–tetrapeptide (Tetra), anhydro‐disaccharide–tetrapeptide (Tetra‐anh), bis‐disaccharide–tetrapeptide (Tetra‐Tetra) and bis‐anhydro‐disaccharide–tetrapeptide (Tetra‐Tetra‐anh) were isolated from muramidase digests of murein sacculi and quantified using the method of Glauner (1988). Tetrapeptide was prepared from Tetra by digestion with purified human serum amidase (Harz et al., 1990).
The substrate concentration in the assays was generally 0.7 mM; to save substrate the concentration of UDP‐MurNAc‐tetrapeptide in the library screen was adjusted to 0.14 mM. The reactions were performed in a final volume of 100 μl 0.1 M Tris–HCl pH 8.0 and incubated for 45 min at 37°C. The reaction was terminated by boiling the samples for 3 min. After a brief centrifugation (15 000 g, 2 min), 90 μl of the supernatant was frozen or used directly for HPLC analysis. The separation and quantification of substrate and reaction product (UDP‐MurNAc‐tripeptide) was performed by RP‐HPLC on a 5 μm Hypersil ODS column (Bischoff, Leonberg, Germany) as described by Ursinus et al. (1992). However, slightly modified elution conditions were used. A linear gradient of 2–20% acetonitrile in 0.05% trifluoroacetic acid which was built up in 20 min was employed.
To determine l,d‐carboxypeptidase activity on high molecular mass murein, LdcA was incubated with isolated murein sacculi (Harz et al., 1990) for 2 h at 37°C and the reaction was stopped by boiling the samples for 3 min. Analysis of the murein was performed as described below.
DNA manipulations and PCR
Standard techniques were used to manipulate the plasmid DNA (Sambrook et al., 1989) and E.coli was transformed as described by Inoue et al. (1990). Restriction endonucleases were purchased from Boehringer Mannheim (Mannheim, Germany), oligonucleotides came from MWG‐Biotech (Ebersberg, Germany). PCR (Saiki et al., 1988) was performed using 1.25 units of Pfu polymerase (Stratagene, La Jolla, CA) in a volume of 50 μl of the supplied buffer with 0.5 μM of each primer and 2 μl of Kohara λ phage 245 lysate as a template. After initial denaturation for 3 min at 92°C, touchdown PCR (Don et al., 1991) was performed with 0.5 min of annealing, 1 min of extension at 72°C and 0.5 min of denaturation at 92°C. The annealing temperatures were 61, 59, 57, 55 and 53°C for three cycles each and finally 51°C for another 15 cycles.
Construction of an inducible expression system
The gene encoding the l,d‐carboxypeptidase was cloned into the plasmid pJFK118EH (Bishop and Weiner, 1993) to obtain an inducible expression system under the control of the tac promoter. The coding region was amplified from Kohara phage λ245 DNA by touchdown PCR by using the primers 5′‐TTGGAATTCCAACGGCACACCTAAGCGGC‐3′ and 5′‐AGAGTCGACTTTACTTAGCCCTGAAGCG‐3′, introducing an EcoRI restriction site and a SalI restriction site at the ends. The amplified product was purified and digested with EcoRI and SalI and ligated into pJFK118EH cut with these enzymes and dephosphorylated with shrimp alkaline phosphatase (Boehringer Mannheim). The resulting construct, pLD2, was transformed into E.coli MC1061. For induction of overproduction of LdcA 0.4 mM IPTG was added to a growing culture.
Construction of a His6–LdcA fusion protein
To allow a simple purification of the l,d‐carboxypeptidase, a fusion protein possessing a His6 tag at the N‐terminus of the LdcA was constructed. Using touchdown PCR with Kohara phage λ245 lysate as a template and 5′‐CGTGTCGACCGGTTAATGGAATTATCGGCG‐3′ and 5′‐CTCGAGCTCTCGACGACCAAAAAAGGCGAT‐3′ as primers the coding sequence of ldcA was amplified, cut (SalI, SacI) and cloned into pQE30 (digested with SalI, SacI). Transformation of LD3 with this construct (pHLD5) complemented the growth defect of the mutant, indicating that the fusion protein is functional. To purify the fusion protein, 100 ml MC1061 freshly transformed with pHLD5 was grown to an OD578 of 0.8 and harvested by centrifugation. After washing with 50 ml ice‐cold buffer (100 mM Tris–HCl pH 8.0), the cells were resuspended in 5 ml buffer and disrupted by one passage through a precooled French pressure cell at 15 000 lb/in2 and centrifuged (30 min, 300 000 g). l,d‐Carboxypeptidase activity in the supernatant was found to be 2.5×106 units/mg protein. To determine the substrate specificity and the sensitivity against different antibiotics, 1.3 units from this preparation were used.
Construction of a ldcA deletion mutant
The gene exchange method of Kulakauskas et al. (1991) was used to construct a deletion of the coding region of the ldcA gene. First, the flanking regions of the gene were amplified (750 base pairs upstream with 5′‐GAAGTCGACGCGCGACTGATTTACATCG‐3′ and 5′‐CAGGATGACCGAATTCCATGGCAATTCCTTGCTGAC‐3′; 800 base pairs downstream with 5′‐GAAGCGGCCGCAATGGATATAATCCGGCG‐3′ and 5′‐GAATTGCCATGGAATTCGGTCATCCTGTTCTTAAAATG‐3′), fused by PCR digested with SalI and NotI and cloned into pBluescriptII SK+ (cut with NotI and SalI). The introduction of a selectable marker [the EcoRI fragment containing the npt gene from pUC4K (Pharmacia, Uppsala, Sweden)] into the EcoRI site of this construct resulted in a plasmid‐borne deletion. This mutation was transferred to the chromosome of MC1061 by λ‐mediated transduction using Kohara phage λ245 as an intermediate as described (Kulakauskas et al., 1991). The obtained transductants were checked by PCR for the presence of the mutated copy of the ldcA gene. One of the clones LD3, showing no l,d‐carboxypeptidase activity on UDP‐MurNAc‐tetrapeptide, was chosen for further experiments. To examine the effect of the absence of LdcA in a ampDE mutant background, the described ldcA allele was transferred to HfrH and JRG by P1 transduction (Miller, 1972). The ldcA deletion was also transduced into E593, a strain which has been shown to lack a cytoplasmic N‐acetylglucosaminidase active on isolated muropeptides (Yem and Wu, 1976) and into its parent strain AB1157.
Cell fractionation
To determine the subcellular localization of LdcA, periplasmic proteins were isolated by preparing sphaeroplasts with Tris‐EDTA and lysozyme under osmotic protection (Witholt et al., 1979). The cytoplasmic fraction was obtained after disruption of the sphaeroplasts with glass beads and ultracentifugation (20 min, 300 000 g) to sediment the membranes. As an alternative method, periplasmic proteins were released by EDTA treatment (Levy and Leive, 1970; Beck and Park, 1977). Fractionation efficiency was determined by measuring acid phosphatase activity as a periplasmic marker enzyme (Atlung et al., 1989) and glucose‐6‐phosphate dehydrogenase as a specific cytoplasmic enzyme (Bergmeyer, 1974).
Murein structure analysis
Murein sacculi were isolated as described previously (Glauner, 1988). After digestion with α‐amylase and pronase, the murein was hydrolyzed with cellosyl (kindly provided by Hoechst‐Marion‐Roussel AG, Frankfurt, Germany). The resulting muropeptides were reduced with sodium borohydride and fractionated by RP‐HPLC as described by Glauner (1988).
Determination of soluble murein precursors
To analyze the precursor pools in different bacterial strains, the cells were grown in LB medium containing 10 μCi/ml meso‐2,6‐diamino‐[3,4,5‐3H] pimelic acid (20 Ci/mmol; ARC) for at least four generations. At an optical density of OD578 1.0 aliquots (1.8 ml) were withdrawn for analysis of the precursors. The cells were chilled rapidly on ice, centrifuged (3 min, 15 000 g), washed once with 1 ml ice‐cold medium and resuspended in 250 μl ice‐cold water. In order to extract the soluble murein precursors the sample was boiled for 10 min. After centrifugation (3 min, 15 000 g) the radiolabeled precursors from 100 μl of the supernatant were determined by RP‐HPLC on 5 μm Hypersil‐ODS (4.6 mm × 125 mm). Chromatography was performed at room temperature at a flow rate of 1 ml/min using 0.05% (w/v) trifluoracetic acid (buffer A) and 60% (w/v) acetonitrile in buffer A. Samples were injected in 1% buffer B and elution started with a gradient from 2 to 3% buffer B for 5 min which was followed by isogratic elution with 3% buffer B for 30 min. The column was washed with 100% buffer B for 5 min and then equilibrated.
We thank Uli Schwarz for his encouragement and support throughout these studies. We are grateful to Y.Kohara for supplying us with the miniset λ library. This work was supported by the European Commission (Project BIOU‐CT96‐0122).
- Copyright © 1999 European Molecular Biology Organization


text:
I ran across these verses in my devotions this morning and they totally jumped off my Bible at me. I hope they bless you. Let's make these verses our prayer?
yes, love this verse!! I've always been told it is a good one to post near the TV! 🙂
Our homes are where we can pour ourselves into our children and husbands and make such a difference! It is no coincidence that this is where most of our secret or not-so-secret annoyances come from – how many loads of laundry do I do and it is never finished? Why do you need a meal EVERY day? Who left the dirty glass here for me to pick up – am I a maid? It is good to remember that we need to walk in integrity in our own homes more than we need to 'look good' out in the world somewhere…
"Turn my eyes from worthless things." This is the Scripture verse that is on my bathroom mirror this week! Thanks for the post….a blessing indeed.

subset: commoncrawl


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MP Wildlife Special- Land of Mowgli (Online) | 4 Days 3 Nights
|Madhya Pradesh is known for its gurgling rivers, towering cliffs and lush jungles. For a chance to see all these jewels of nature, opt for this package by MakeMyTrip. To begin with, you go on a tour of Jabalpur, covering the majestic Dhuandhar Falls. These waterfalls are created as the Narmada River takes an around 30-metre plunge after crossing the Marble Rocks. You can also see these incredible cliffs on a motorboat cruise. Further, move to Bandhavgarh National Park, home to tigers, hyenas, wolves and several species of deer. You can enjoy a jungle safari here before this India getaway package takes you to Kanha National Park. The rich biodiversity of this protected territory inspired the famous Jungle Book by Rudyard Kipling. You will get ample time to shop in your sojourn.
MP Wildlife Special- Land of Mowgli (Online)- Joining Direct | 4 Days 3 Nights
|Madhya Pradesh is an all-round destination, with natural beauty and rich biodiversity being its biggest highlights. Opt for this India package by MakeMyTrip if you want to delve into both these aspects of the state. Your venture begins from Jabalpur, settled on the banks of the mighty Narmada River. While here, you will be amazed at the sight of the wide Dhuandhar Falls. These have been named so as the water creates a dense mist during the monsoon. Further upstream are the scenic Marble Rocks that you can see on a motorboat. You then move to Bandhavgarh National Park before heading to Kanha National Park. Both these protected territories are home to elephants, tigers, wolves, hyenas and gaurs along with several kinds of deer.
Wildlife Retreat to Taj Safari Lodge-Banjaar Tola (Online) | 3 Days 2 Nights
|- Want to get away from the city, but still do not want to give up on luxury? Then, pick this India travel package to Kanha National Park in Madhya Pradesh. On your sojourn, you stay at Taj Safaris Lodge - Banjaar Tola, known for blending modern amenities with naturalistic elements. All its ethnic-style tents feature a private deck and local timber furniture. Meals, served at the restaurant or in the forest, comprise dishes from the region and beyond. The retreat also has a library for you to read and pool to swim. Along with accommodation at this lodge, the package also includes a short walk along the Banjar River. The hike will give you an opportunity to see as well as learn about the jungle and its animals.
Tiger Safari Weekend in Kanha - Wildlife Special (Online) | 3 Days 2 Nights
|The very place, which inspired Rudyard Kipling to author his famous Jungle Book, Kanha is a great destination for wildlife enthusiast. This national park in the heart of Madhya Pradesh is home to tigers, gaurs, blackbucks, striped hyenas, wolves, sloth bears and wild boars. It is also visited by a number of migratory birds in the winter, the most significant among which are steppe eagles. The ecosystems here include dense woods as well as open meadows with tall grasses. Among the highlights of this India vacation package would be a private Jeep safari in the company of trained naturalists. During the excursion, they would tell you about the vegetation in the region as well as habits of animals. In your leisure time, you can take a walk in your surrounding areas.
Pachmarhi and Kanha Special (5 days)- Joining Direct | 5 Days 4 Nights
|Pachmarhi and Kanha together make a delightful holiday; a sojourn to the heart of India's natural heritage. The place is full of stories and anecdotes. Take this holiday for fresh air, wondrous landscape, and tiger spotting.


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In Search of the Giant Squid in All the Wrong Waters: Week 29
Friday, July 4, 2014, 9:27am
Today, I searched for the giant squid in Boneyard Creek which some of the locals, for the Independence Day weekend, have renamed—via cardboard sign decorated with flag stickers—Firecracker Creek. I have an opinion about these locals, but as I cast with my Stars 'n' Stripes lure baited with crumbs of the same brand of blood meal my mom used to grow cucumbers when I was a kid, I kept it to myself. They were bigger than me. Instead, I thought about slaughter, about the things that had to die so I could have creeping vine cucurbits on my romaine and my iceberg. I cast and the air felt nitrogen-rich, and full of vaporized bratwurst fat, capable of repelling even the most tenacious of rabbits. I caught no squid, but I did catch what appeared to be a bedbug, so far from any mattress. I took a step closer to the bank and, beneath my shoe, genocide again. Red glare. One of the locals said something about capital punishment and another local said something about molasses and anthills, and yet another said something about tar and cardinal feathers. I tried not to connect the dots. I tried not to associate patriotism with daddy-love, independence with eviction, Diaspora. The smoke bombs drew tentacles onto the air. I tried, but I am dependant on these other bodies for my immunity.


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RCMP officers have seized $1.15-million of counterfeit $100 bills at a Richmond apartment – one of the largest hauls in British Columbia history according to police.
The Federal Commercial Crime Section monitored the forgers for two months before they raided the residential property on Dec. 11. Officers seized the unfinished notes and money-making equipment. Four men in their 20s were arrested.
Sergeant Tony Farahbakhchian, Pacific region counterfeit co-ordinator for the unit, said officers were able to secure the notes before any were released into circulation.
Five stacks of the uncut sheets of bills, approximately six inches high, were on display at the press conference held at RCMP headquarters in Surrey, B.C.
"Just before the Christmas season would have been a good opportunity for these bad guys to try and pass a number of these notes when the tellers at the stores are busy," he said.
Sgt. Farahbakhchian said the counterfeiters were likely trying to rush out the fake $100 bills before the new polymer notes, in circulation since Nov. 14, became more common.
And, he said, the public is vulnerable as they don't take advantage of the security features on Canadian currency.
"The majority of people don't look for all the features like the holograms and the security threads," he said. "These guys know that most businesses don't look for all these features and so they try to make a half decent note in the hope that it will pass."


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I read about hub challenges. But I didn't get a clear idea. So my doubts are -
1. Is it just posting quality hubs every day or will the admin give any topic for a particular day?
2. Is there any formalities to enter this challenge?
3. What is the benefit of participating in this challenge?
4. Is there any restriction of experience in HP for this challenge?
Challenges are just a way to motivate yourself. There are no prizes, except personal satisfaction that you've achieved something you set out to do. By publishing your commitment on the HubChallenges forum, you are publicly saying, "I'm going to do this", so that other Hubbers can support you. That's all. They have always been like that.
Hi abidareacode,
I'm surprised to see that it says this forum is inactive, so I hope you receive my reply. I wanted to let you know that there is a more current thread with some helpful info about Challenges that some of us are embarking on. Please see this thread for more info:
You are also welcome to take a look at my challenge hub which I started on Friday:
http://easylearningweb.hubpages.com/hub … tivity-Log
Thanks and Good Luck to You!
Writer Fox....I read that full. There says about a commitment. 1. Where I have to place this commitment ?
2. What is the benefit then?
3. If I cannot complete the commitment what will happen?
4.Who will monitor the commitment?
5. What are those stellar hubs?
These questions are aroused in my mind.
The Challenge is something that people used to do years ago. No one from HP monitors anything. It was something Hubbers did to encourage each other to write more Hubs. What happened is that a lot of Hubs were written too quickly and they weren't that good. Here's a link to the stellar Hub suggestions:
http://hubpages.com/learningcenter/Elem … tellar-Hub
Thanks Writer Fox...So a hub with these attributes are called stellar hubs. But
will any accolade will appear on posting stellar hubs ?
Who decides it as stellar or not ?
Is hub challenge exists now?
You can still do a Hub Challenge if you want to, but you probably won't find many other people to participate with you,
No, you don't get any accolade for following the pattern suggested for a stellar Hub. Hubs are rated by the Quality Assessment Process and that's how you get the Hub Score beside each one on your Accounts page.
So it means these all things are now merely a concept. No one looks or supports ! Why should they be still alive in FAQ and other pages?
Some of the information on the site hasn't been updated in a while.
Yes....I am back here after four years. Like me many will be there. Also new hubbies will get confused as FAQ remains , but no one cares or updates. Who will do these?
I don't know. But, on each article in the Learning Center there is a date at the bottom showing the last time the page was updated.
Writer Fox- I just read about the Earning Program. I do not understand what it was saying that it will charge us. Please help. Thank you and God bless.
Victoria, where do you see the Earnings Program will charge you? They will not.
You cannot earn anything from the Earnings Program until you can get an Adsense account. So I suggest, don't worry about it yet.
I clicked on the link then I clicked terms for the program, and it said something about fees that I'm confused not sure if we are charged?
Thank you. God bless you.
Victoria, You won't get charged, only paid. You must have misunderstood something.
Oh ok thank you so very much God bless you.
HubChallenges are like resolutions - you make a promise, and announce it to the public.
Since it is public, it keeps you accountable to the public. But if you fail, then you only have shame and humiliation, but only if anyone else is paying attention. Not really - there isn't shame and humiliation - you are better off having made the challenge because even if you didn't fill your obligation fully, you at least made progress. You may even encourage others to join the challenge or find a buddy or two who will keep you motivated.
The problem with the challenge is that people tend to promise unrealistic things - i.e. I promise to write 30 hubs in 30 days or even I promise to write 100 hubs in 30 days. Since quality is more important than quantity, these types of challenges are a waste of time. Because of that, you may not find a lot of participation in your challenges.
People still do participate in challenges. The key is to choose a good challenge. Challenges that might be good (these haven't been tested):
1. Read all of the learning center articles - one a [day][week] and notify staff of outdated information
2. Improve all of my current hubs by end of the year
3. Read all of the winning hubs in the Hubbie awards.
4. Write only hubs that fit the definition of stellar this year
5. Improve my grammar by [taking a course] [reading this book]
6. Increase my knowledge base and creativity by trying something new each weekend this year
7. Read xx hubs about how to use amazon capsules that work and write one hub that incorporates the good advice. [you'll get some bad and mediocre advice in the mix but by reading many hubs, you may find a combination that is really good advice]
8. Increase my portfolio by taking xx photos per [day, week, month].
9. Organize my photos by loading them to my computer and labeling them so I can find them quickly to add to hubs.
10. Learn to take better photos by [reading hubs][taking acourse][reading xx book].
Millinair...Thanks for your helping reply. So I changed my challenge to a new direction. As you told I have been working in modifying my hubs written in 2010! Yes, it was successful from 2 featured hubs now I own more than 20 and whenever I get time I look for the same. The other ways you told also are practiced and happy to know that it is a right way of new hub challenges.
I would recommend doing one challenge at a time. Modifying your hubs that you wrote in 2010 is a great one. Now simply come up with a deadline or time frame of how many per week and you can start. You can even list links here to show which ones you have done.
Can I show links here ? Is it sure ? I am asking this because I was banned for the last four years from forum on account of self promotion.I didn't understand what I did wrong. So I afraid that giving links to my own hubs in forum discussion will be self promotion and will get banned again. So please clear my doubt.
by Simon Cook4 years ago
Take one of your old hubs, update it with new content, better photos, improved titles and add some additional modules (make it a stellar hub effectively) and then post it here for critique!
by Jennifer Arnett3 years ago
It has been such a fun exercise to participate in Hubpages author Annart's shorty story challenge. She posted a picture of her own painting and asked everyone to write a story from it. It has been amazing to...
by James Paterson8 years ago
Thought that this new thread would make it easier for everyone to share their hubs for the Hub Challenge.....jimmyhere is my first http://hubpages.com/hub/Ladies-Rolex-Watches
by Lois Ryan2 weeks ago
I haven't been actively writing on Hub Pages in a while and want to get back into this and this might get me motivated.I remember hearing about a 100 hub challenge some years ago and I know that I could not possibly...
by Amelia Griggs3 years ago
I am creating my own 30 Hubs in 30 Days Challenge and there seems a broken link in regards to information on hub challenges. On the http://hubpages.com/faq/ page, for "41. What is a Hub Challenge?" if I click...
by Mizzery Chick17 months ago
I'm just coming back after a several year hiatus, and one of the most exciting things that started when I was here was the HubChallenge. Originally, it was 30 hubs in 30 days. Now I see it's changed to either updating...
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text:
Macroevolutionary Processes— Radiations. Review. plant breeding systems span range from obligately selfing to obligately outcrossing various strategies have evolved to promote outcrossing; major ones are: self-incompatibility—chemical control of pollen germination on style
Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.
Reinthal & Meyer (1997)
reinforcement by competitive exclusion?
Givnish et al. (1997)
Photos: www.siamexotica.com/Brocchinia.html
Givnish et al. (1997)
Givnish et al. (1997)
Kauai = 5.1 my
Oahu = 3.7-2.6 my
Maui Nui complex =
1.9 my-800,000 y
Hawaii =
400,000-180,000 my
Alakai Swamp/Mt Waialeale
(wettest place on earth)
wet forest, cliffs
Waimea Canyon
(extremely arid)
Moist forest
Sandy or rocky
Coastal sites
Viola wailenalenae
(shrub, swamp)
Viola kauaiensis
(herb, open bog)
Viola tracheliifolia
(treelet, dry forest)
Ballard (unpubl.); Havran et al. (2009)
Principal Components Analysis of
Leaf Characters
Ballard et al. (unpubl.)
Leaf Anatomy of Violets on Kauai
V.kauaensis—thick UE & LE; tall palisade cells 1 layer thick
V.tracheliifolia—thick UE, 1-2 layers, thin LE; short palisade
cells, 1-2 layers thick; druses
V.wailenalenae—thick UE, thin LE; short palisade cells, 1-2
layers thick; druses
Havran (unpubl.)
Havran et al. (2009)
Source Areas for Wintering Hawaiian Songbirds
Ballard and Sytsma (2000)


text:
SUNDERLAND will face Spanish side Real Betis in the dress rehearsal for their Premier League campaign after the Black Cats confirmed a seventh pre-season friendly.
Betis, who finished bottom of La Liga last season, will face Sunderland at Bishop Auckland's Heritage Park on Thursday, August 7 (kick-off 7.30pm).
The game comes four days after Sunderland return from their pre-season trip to Portugal and is likely to be their final friendly before the Premier League gets underway on August 16.
Tickets for the game are priced at £15 for adults and £5 for concessions. Purchasing details will be announced in due course.


text:
Comment Re:Obligatory (Score 1) 630
Who is this ingenious "Kung Fu Monkey" that I may subscribe to his newsletter?
Thanks for the cough-inducing guffaw..
Who is this ingenious "Kung Fu Monkey" that I may subscribe to his newsletter?
Thanks for the cough-inducing guffaw..
I assume you mean something like what happened in South Africa? Of course, nobody was going to miss trading with them so much (quick: name 3 things in your house made in SA. Time's up....). Plus, apartheid was particularly odious to the "great and good" who run things, including especially the media that it was a very popular trade embargo.
The problem with China is not just that they are egregious offenders against the rights of their citizens (including those who'd really rather forgo the privilege, like the Tibetans), but that they have become indispensable to our own financial comfort. This makes any proposal at using trade leverage a non-starter.
There are two things that work in medicine. Surgery and antibiotics. Everything else treats symptoms or confirms you need either surgery or antibiotics.
This is nonsense, of course. There are a wide variety of medications for diabetes, hypertension, high cholesterol and a huge number of metabolic/physiological illnesses from Alzheimer's to Yaws (can't think of a "z" disease at the moment).
Stem cell therapies (even using embryo-derived cell lines) are not illegal. However, there are professional and ethical standards of treatment and research protocols that would prohibit this kind of "scientific voodoo" medicine. The sad fact is that this woman was desperate enough to try this snake oil treatment, but it was a serious error in judgment.
We don't know the details of her decision or of the medical issue that she faced. But there is no "policy" issue here; we can't command the science to provide a reliable treatment without careful study. Sadly, this patient either didn't have the time or the patience to wait and paid the price for that.
It seems (from the article) that the draconian punishment of exclusion from all federally-funded programs was not optional, thus necessitating the bogus "whipping-boy" subsidiary. This is stupid law, of the same stripe as large minimal sentences for drug possession and many other politically-motivated examples. That's just our lawmakers pandering to the thirst for vengeance exhibited by many other posters here who seem to want to punish the corporation, as if it exists as a real person.
The fact is that you can't really "punish" a corporation, since it isn't really a person (no matter what the law says on that). Neither can you really tax one since the cost of fines and/or taxes are just passed along in the price of products. Tax the income derived from the investment by the shareholders, by all means, but taxing a corporation's profits just makes it another tax collector and distorts operational and investment decisions.
What we have is bad law - treating corporations as persons for some purposes (and, inconsistently, not for some others), and then the one mandating a stupid punishment for crimes that should be attributed to individuals.
All of this further diminishes the rapidly fading sense of personal responsibility that was once such a defining characteristic of American culture. So long, American republic, it was nice while it lasted.
If you can count your money, you don't have a billion dollars. -- J. Paul Getty


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Message from CEO Eric S. Gordon
It's hard to believe we are already half way through the 2017-18 school year. As we approach the start of the second semester, we have much to reflect on, to be grateful for and to celebrate in the Cleveland Metropolitan School District.
Our state report card released at the start of the school year was a reminder of the continued challenges we face in meeting Ohio's new, more rigorous learning standards for our students. Their academic performance, gauged against the performance of their peers across the state, was also an affirmation of reforms in place as CMSD continues to build on the progress we are seeing under The Cleveland Plan.
Our students' improved performance shows that CMSD is now the fastest-improving urban school district in Ohio, having outperformed 88 other Ohio school districts in K-3 Literacy this year. With a 20% increase in our graduation rate, CMSD is the third-fasted growing district in the Ohio 8.
Strong parental engagement is key to the success we are seeing, our students' attendance continues to improve and CMSD students are graduating better prepared than ever for the colleges and careers that await them.
Even as we are seeing gains in every type of school we offer in a growing portfolio of school choices at CMSD, we are mindful of the many challenges our students and families face that can be barriers to success. Our caring and dedicated educators are as committed as ever to building on strategies that are working, and to increasing the partnerships we are forming with a growing number of community agencies dedicated to removing barriers and ensuring the success of every child in Cleveland.
Today and every day of the school year, I am grateful to those who are helping us reach that goal by ensuring those supports are in place throughout a child's K-12 school experience.
Congratulations on a great first semester at CMSD!
Sincerely,Eric S. GordonChief Executive Officer
Information about
CEO Eric Gordon


text: SCHOLARS HOLDINGS LIMITED has been working since 1/26/2016. The current status of the business is Active SCHOLARS HOLDINGS LIMITED located on 22 BACKBRAE STREET, KILSYTH, GLASGOW, LANARKSHIRE, UNITED KINGDOM, G65 0NHDirector Bedford Robert is known as a Director of the company. Director Bedford Dorothy is known as a Director of the company.


text: That's about the only way to describe the nuisance. The plant have been beautiful and have about 7 pepper that are in varying stages of size. Suddenly today, there is a white substance/spot, each about the size of a pin head, but also varying in size to a degree. They don't look like a bug, but if you put your fingernail up next to one, it moves. WHAT on earth is this short of using and insecticide, what can I do to eliminate them? I've never grown peppers before, so this is a first for me. For now, I'm going to try a soapy solution. Thanks for any information.


text:
Manifest Investing features an e-newsletter focusing on successful long-term investing including web-based investment research and features/tools for stock and fund screening as well as resources for portfolio design and management. Our focus is on demystifying investing — particularly when it comes to the caretaking of a portfolio — enabling investors to experience successful long-term investing with their personal investing or retirement plans. We endorse and encourage investment clubs as vehicles for support and group learning.
Mark Robertson (firstname.lastname@example.com) is founder and Managing Partner of Manifest Investing, served as senior contributing editor for Better Investing and has worked with successful investment clubs and individual investors since 1993. He has appeared on National Public Radio, CNBC and ABC to discuss long-term investing. He has also worked with Smart Money, Barron's, Money magazine and the Motley Fool and been covered by the Chicago Tribune, Wall Street Journal and MarketWatch as well as a number of local publications.
Kurt Kowitz (firstname.lastname@example.com) co-founder and Technology Manager, is responsible for web site development, operations and customer service. Kurt was largely responsible for the development of online data in support of software and web-based delivery at NAIC. As father to two young boys, Kurt is interested in long-term investing for purposes of college one day and retirement another: Responsibly Lazy Shopping
For more on getting started at Manifest Investing: Guided Tour — The Basics
Ken Kavula (firstname.lastname@example.com) has served the modern investment club movement in a wide variety of leadership volunteer positions. A retired educator, he is regarded as a small company champion and respected speaker nationwide. Ken also belongs to four investment clubs, including two Model clubs and a family club.
Little Nudges Can Make A Difference
Cy Lynch (firstname.lastname@example.com) is a respected and experienced long-term investor and educator. Cy is a frequent contributor at MANIFEST, providing regular educational topics and a knight of the Round Table series.
Hugh McManus (email@example.com) is a pharmaceutical scientist, successful long-term investor and renowned advocate at investment education conferences. A frequent contributor to MANIFEST, Hugh is also a knight, participating in our Round Table series.


text:
Frequently Asked Questions
CO-ADD is funded by the Wellcome Trust and The University of Queensland as a community initiative to fight drug-resistant micro-organisms.
This is a community based activity to build hits, leads and new knowledge in the antibiotic and anti fungal space. We ask that you release results to the community 2 years after you receive our primary screening report.
Samples will be screened primarily against 5 ESKAPE bacterial pathogens and 2 fungal/yeasts pathogens. If active, samples will be back screened for cytotoxicity and Quality Controlled.
We generally ask for 1-2 mg of pure compound which will be enough to do primary screening, hit confirmation and hit validation. We will accept a maximum of 5 mg. If your samples are in DMSO: 50 uL at 10mg/mL or 50 to 100 uL at 10 mM.
We require all compounds to be soluble in DMSO for our assays and dry material to be shipped in 1-2 mL Eppendorf tubes (or similar).
We do not ask for QC of compounds, but purity information can be provided. Some QC by HPLC-MS will be part of the free screening process, with 5% of samples analysed randomly. All active and confirmed hits will be analysed for purity before progressing to Hit validation.
Although not obligated to under the terms of the agreement to screen, we encourage participants to give us as much information as they can about their compounds, this includes stability, to ensure compounds are handled in the most appropriate manner for the compound type. When we have little information provided we do the best we can with our standard procedures and the QC pipeline should highlight most issues, if any.
All samples are kept in long term storage so that we can go back to them if a reason arises in the future. We do not advocate sending the remainder of the compounds back as standard protocol due to logistics, however, on special request we can handle this on a case by case basis and send the compounds back.
Once screening is complete your results will be sent to you in PDF form by email.
We are a determined group of scientific experts working together to discover new antibiotics that can kill the superbugs that cause millions of infections and deaths around the world. Meet the CO-ADD team.
Samples delivery requirements
- Pure synthetic compounds
- Pure natural products
- 1 mg of pure dry material (5 mg maximum)
- If samples are in DMSO: 50 uL at 10mg/mL or 50 to 100 uL at 10 mM
- In 1 to 2 mL Eppendorf tube or similar, or in plates (if needed we can send out bar-coded tubes)
- We require all compounds to be soluble in DMSO
- Please check the terms and conditions for provision of compounds & material to CO-ADD
CO-ADD may cover the cost of shipping where required.
CO-ADD we can send out bar-coded tubes - contact us for more information.
Once you supply CO-ADD with your sample details, importation paperwork will be provided for you and must accompany the package in the way described in the documentation in order to clear Australian quarantine and customs.
- No public information is released naming those involved with CO-ADD.
- Screening results shall not be provided to any third party for a period of at least 2 years after receipt of the primary screening report to allow the participant time to protect any intellectual property if appropriate.
- Please check the terms and conditions for provision of compounds & material to CO-ADD
- Two years after receipt of the primary screening report or as otherwise agreed between the participant and CO-ADD the participant agrees that the chemical structures and screening data shall be published in an open access database for use by the community (CO-ADD database or CO-ADD partner database such as ChEMBL).
- Please check the terms and conditions for provision of compounds & material to CO-ADD
Intellectual property
- The compound owner owns all IP during the free screening stages, CO-ADD makes no claim to IP. Results and data generated by screening the compound/s or material shall be the property of the participant.
- Please check the terms and conditions for provision of compounds & material to CO-ADD
The compound owner may file for IP or publish the results, CO-ADD asks that you acknowledge CO-ADD appropriately.
CO-ADD kindly requests that you acknowledge CO-ADD when you publish work using the following statement, "Antimicrobial screening was performed by CO-ADD (The Community for Antimicrobial Drug Discovery), funded by the Wellcome Trust (UK) and The University of Queensland (Australia)."


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Vu Data logger
Product Description
This robust, portable, remote datalogger, with a large LCD display, has 3 built-in sensors: Sound, Light and Temperature and 2 plug-in sensor sockets.
Connection to a computer is via USB. See and capture sensor readings wherever you are!
With an easy to use menu and just four buttons, this clever box of tricks makes data logging child's play!
Capturing data from an experiment has never been easier, simply start 'EasyLog' recording and data from all of the sensors is recorded automatically.
With logging durations being anything from a few seconds up to 14 days, its perfect for both classroom and field work.


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by William Westernhoff
(Shreveport LA)
LSU will win because they truly have a better team. They also have better athletes. OSU may have the better athletic facility, but WHO CARES!?? Les is going to lead them to victory. Why?? He has to. He won't let Michigan out do him with their win over Florida. OSU has never seen the speed overall on defense like LSU has this year. LSU by 10!
Comments for LSU will win!
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Join in and write your own page! It's easy to do. How? Simply click here to return to College Sports Discussion.
Please Visit our Amazon, Ebay, Etsy and Better Display Cases stores.


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A YOUNG girl has been flown to hospital after she was injured in a quad bike crash at Beaudesert this afternoon.
The RACQ CareFlight Rescue chopper was sent to the property just before 1pm after the girl came off the quad bike and fell about one metre on to the ground.
She had suspected head, spinal and abdominal injuries.
The girl, whose age is unknown, was assessed and treated by the on-board critical care doctor and flight intensive care paramedic before being flown to the Mater Children's Hospital in a stable condition, according to CareFlight Queensland.
Originally published as Girl hurt in quad bike fall


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Share and Share Alike ~ Give Credit Where Credit is Due
Information and ideas from Share2Learn may be freely used for non-commercial purposes. Use of the information constitutes acceptance of CreativeCommons terms as outlined on the copyright page. Credit must be given to the original author/contributor and/or to this website,
Please email me with any ideas, comments, or suggestions!
Share2Learn was designed and is maintained by me, Renée Goularte.
This website journey began as a way to gather and categorize my own and other teachers' lessons and ideas for the classroom. I created it in the early 1990s when a colleague gave me a copy of website software that she had won in a contest and didn't want. I thought, well... why not make a website about teaching? Over time, it has expanded to include photos of student work samples, teachers' stories from the classroom, a collection of Internet resource links for teachers and parents, and much more.
Share2Learn has always been an ongoing work in progress and process, with many teachers having contributed successful teaching ideas to the site. It has become a vast depository for the ideas and lessons of many people. Teachers and parents are invited to borrow and use any of the ideas found here, as long as credit is given to the original source, and to this website.
Share2Learn is a great resource, but for current goings-on, please visit my Facebook page and Pinterest boards, my Creating Art With Kids blog, and my TeachersPayTeachers store.
More about me...
I have been a teacher, a writer, and an artist, and have always been a perpetual student. I love learning and creating new things, and alternate my time between a number of assorted projects.
My professional teaching career began in San José, California in 1988 in a third grade classroom. I soon moved to 2/3 combination classes and then spent four years teaching in a Multiage 1-2-3 program, which I started with a colleague. I have also taught a 4/5 combination class and Kindergarten, and have worked specifically with GATE students, ELL students, and at-risk students in all elementary grades, In my last two years of teaching before I retired in 2010, I taught Art to Kindergarten, 1st, and 2nd grade students. For me, teaching Art was coming full circle. I have a BA in Art and English from San José State University, where I earned Standard Secondary and Multiple Subjects teaching credentials, as well as a Master's Degree in Elementary Education with a focus in Mathematics. I am also Montessori-certified.
Along with my classroom experience, I have written articles on teaching, written lessons professionally, maintained a number of websites and blogs, and facilated workshops for teachers and parents. Plus, I sell my own classroom-tested art lessons and other teaching resources on TeachersPayTeachers.com.
In addition to this website, I maintain three blogs: Creating Art With Kids highlights the Art lessons I've done with my students, peripheral visions is a collection of random thoughts mostly related to art-making and writing, and Art In Process specifically documents my own creative process. I also have a website that serves as an online gallery to showcase my watercolor, drawing, and mixed-media art work. I participate in a monthly poetry workshop group. Some of that poetry occasionally finds its way to my peripheral visions blog.
Creating Art With Kids is also a book, a collection of art activities for elementary classroom based on the elements of design -- many ideas I have used over the years in my classes. It is self-published, and can be purchased through me. Follow the links for more information about this book.
In addition to teaching, making art, and writing, I have presented Art, Math, and Language workshops for teachers at the California Math Council's yearly conference at Asilomar in Monterey, California and at the California Association of Bilingual Educators' yearly state conference in San José, California.I have also written lessons for Read-Write-Think and also for Santillana USA Publishing.
Click here for a list of some of my writings, art, and lessons online.
Have an idea? Comment? Suggestion?


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Leland is a small fishing town in Michigan with a big personality. Down by the boardwalk it looks like something out of Disney World. So when Melissa asked if we would meet her in Leland to capture her family's portraits we jumped at the opportunity. We drove up Leelanau Peninsula and met the family down by the harbor. We learned that Melissa's family was there celebrating her mom's birthday. The family flew in from all over the U.S. to join in the celebrations and they wanted to document them all being together.
The boardwalk was flooded with summer tourists so instead we walked to a nearby spot along the water to start. The stories between sisters and each other had us in stitches as we walked along with them. As we photographed them people were casting their lines into the water hoping to catch some fish and small boats passed by along the river. It truly showed how active this fishing town really is. As we walked near downtown it smelled like fish fry was being fried up at the local restaurants.
As the sun starting slowly setting we made our way to Leland's harbor. Sounds of seagulls and teenagers splashing around Lake Michigan filled the air. The docks were perfect to relax by and also capture the sunset from. It was truly a beautiful summer night in Northern Michigan. Big thanks again to Melissa and her family for allowing us to document their family gathering. We hope the rest of their celebrations were just as wonderful. Enjoy!
The ripples of waves off of the harbor and the family chatting on the dock makes this one of our favorite images from their session.
There was a group of teenagers jumping off into the warm summer waters of Lake Michigan.
That sunset was a fantastic ending to a Northern Michigan summer evening.


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Of the moment // 6.3.16
One of the reasons why I blog is simply to have the space to write: getting things "on paper" helps me to process my feelings and organize my thoughts. I also love having a record of my life over the past few years: a place that holds all of my stories, my photos, the ups and downs of travel. But sometimes, I also just want to remember and share the little things that brought me day-to-day joy. Every Friday, I'll give you a peek into the book I currently can't put down, a great restaurant discovery, a fun new product or an inspiring quote: a few things that I think you might enjoy before we all take off into the glory that is the weekend!
I talked about my career in two very different contexts this week: first, speaking to a group of eighth graders at a charter school's Career Day and second, about my love of routines and commitment to company culture on the Creating Your Own Path podcast. I was probably more nervous about the podcast (it's strange to think about listening to your own voice, isn't it?), but I swear, eighth graders are a tough crowd. Both were challenging in different ways, and I hope that they were helpful!
IT'S ROSE SEASON Y'ALL! I have made a personal pact with myself that I will stop and smell any roses I see walking down the street, and it's made every wander around the neighborhood that much more pleasant.
Summer uniform that goes from work to play pretty effortlessly: Everlane tank, American Apparel wrap skirt, neutral mules. p.s. how cute is Jillian with her baby bump?! It was so fun to do our third annual birthday drinks at a rooftop bar in the city!
And the vista from said birthday drinks: Mr. Purple has got to be the hippest new rooftop bar in New York City, I still can't get over this gorgeous downtown view. Bonus: there's a pool on this rooftop too!
I've been reading Almost Fearless for years now, and have read as Christine moved to Beijing, and then Beirut, and then Mexico, and then Barcelona, and had one kid, and then two. It's strange to be so aware of someone else's life without ever having met them (although we do have a fair amount of mutual friends). She just released her first book Mother Tongue, a recollection of her language learning adventures around the globe. I honestly wasn't sure how much I'd love the book since I already knew so much of the plotline, BUT I absolutely blew through it! Especially interesting if you speak another language or want to learn one.
"And so with the sunshine and the great bursts of leaves growing on the trees, just as things grow in fast movies, I had that familiar conviction that life was beginning over again with the summer." -F. Scott Fitzgerald
This weekend, I'm most excited to christen our rooftop with a bunch of friends, beers and homemade popsicles on Sunday! Also squeezing in drinks with coworkers, a brunch and a housewarming party, aka this will be the most social I've been in months.


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1 results containing "Stationery--1880-1890."
Larger image available anywhere | Larger image available only at the Library of Congress | Not Digitized |
[Blank sheet of stationery with elaborate watermark for the French papermakers Canson & Montgolfier, including an 1883 calendar surrounded by a decorative border. Border includes a balloon in flight, a coat of arms, and filigree]
1883] | 1 item (ephemera). | Canson & Montgolfier
LOT 13416, no. 48 [P&P] | LC-DIG-ppmsca-02577 (digital file from original item)


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One of the burliest full-size trucks, the Chevrolet Silverado 1500 not only offers a class-leading towing capacity, but also a powerful engine. The new Chevrolet Silverado 1500, offered in St. Clairsville, showcases a quiet yet upscale cabin space, which is brimming with latest infotainment options. Comfort and class are conjoined together under one roof. Whether you require a work truck, an adaptable family hauler or a weekend trooper on road or off road, this light-duty truck has got your back. Its unmatched fuel efficiency will leave St. Clairsville drivers spellbound at what this pick-up can achieve. Learn more about the new Chevrolet Silverado 1500 in St. Clairsville today! We also have a huge inventory of other new vehicles. Visit Whiteside of St. Clairsville Inc.!
Search used 2013 Chevrolet Silverado 1500 Vehicles in St. Clairsville, OH near Wheeling, WV
Your used 2013 Chevrolet Silverado 1500 Vehicle Resource Serving Wheeling
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text:
OCR Gateway GCSE Additional Science Student Book Mixed media product
by Graham Bone, Simon Broadley, Sue Hocking, Mark Matthews, Jim Newall, Angela Saunders, Nigel Saunders
OCR GCSE Gateway Science is designed to engage every student and stimulate their interest in science.
It also provides comprehensive exam preparation and assessment to help your students understand what is expected of them and improve their grades. The GCSE Additional Science Student Book places anemphasis on explaining, theorising and modelling science.
It provides a natural progression from GCSE Science and gives students a solid foundation to move on to study separate AS and A Level science.
- Format: Mixed media product
- Pages: 272 pages
- Publisher: Oxford University Press
- Publication Date: 19/05/2011
- Category: Sciences, general science
Free Home Delivery
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Pick up orders
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text: I have to say that I've been really good for quite awhile now and have been able to pass up PCE and other sales, but once I saw those threads about Legacy at the outlet (thanks so much Wander!), I figured I had to go. Legacy 06 is my all time favorite Coach style, and I got to the outlet pretty early--it was getting busier by the minute! I'd hate to be a husband at the Coach outlet this afternoon; he'd be well advised to bring his football helmet and a few defensive moves! They had mandy in white, black, whiskey and natural, satchels in whiskey and black, shoulder bags in whiskey and black, and a few alis. I even saw a clay 07 gigi tote that wasn't going to be lonely for long, KWIM? Here are my bargain basement buys from the outlet! Only the whiskey mandy is mine--the others are for a sister and friend who asked me to pick them up for them. Of course, looking at them all together makes me wanna keep all of 'em! I guess this means that 06 legacy is being deleted now? I never thought I'd see these bags at the outlet--it's always so hit and miss there--but I'm enjoying the huge savings and thanks to all your tpf ladies for these tips!


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On Saturday, 93.29 percent of Lake Michigan was covered by ice, the most in records dating back to 1973.
This extent bested the previous record of 93.1 percent set in 1979.
Related: Lake Michigan Ice Cover Reaches Record Coverage! (National Weather Service)
The ice levels rose steadily December and January before sharply falling in late February. But in early March, following an arctic blast, they surged to Saturday's peak level.
Ice cover across the Great Lakes collectively this winter has reached historic levels. On Thursday, ice covered 92.19 percent of the Great Lakes, second most on record.
This winter's Great Lakes ice cover runs counter to the long-term trend, which is in a steep decline. As Jeff Masters explains at Weather Underground:
The long-term trend in recent decades is sharply downward; Great Lakes ice cover declined 71% between 1973 – 2010. A 2012 study published in the Journal of Climate by researchers at NOAA's Great Lakes Environmental Research Laboratory found that the biggest loser of ice during the 1973 – 2010 time period was Lake Ontario, which saw an 88% decline in ice cover. During the same time period, Superior lost 79% of its ice, Michigan lost 77%, Huron lost 62%, and Erie lost 50%. The loss of ice is due to warming of the lake waters. Winter air temperatures over the lower Great Lake increased by about 2.7°F (1.5°C) from 1973 – 2010, and by 4 – 5°F (2.3 – 2.7°C) over the northern Lakes, including Lake Superior.
This winter's snow and ice bonanza in the Great Lakes is a positive for their water levels, which have been well below normal in recent years. The Associated Press discusses how rising waters may reverse some of the economic hardships associated with depleted levels over the last decade or so:
The prolonged slump hammered the shipping industry, forcing vessels to carry lighter loads to avoid scraping bottom in channels and ports. Marina owners lost money as slips were too shallow for boats to dock. Vegetation sprang up along waterfronts, frustrating hotel and cottage owners.
But the last 14 months have seen a long-awaited comeback, fueled by plentiful snow and rain. Superior and Michigan-Huron's seasonal rises were almost double their average gains in 2013.
And the signs continue pointing upward. The snow's water content is the highest in a decade on Lakes Superior, Michigan and Huron.
While water rises will benefit Great Lakes tourism and commerce, there's concern about possible flooding from rains and melting snow and ice this spring.
"The towering snowpack rimming the watershed will melt this spring and much of the water will flow into the lakes or the streams that feed them," reports the AP. "The runoff is expected to be so bountiful that some areas will be in danger of flooding, a prospect that could be worsened by ice jams on swollen rivers."
flying over frozen Lake Michigan was pretty cool. #ily Chicago pic.twitter.com/N9ff3DSXog— bri nelson (@feeldaBRIze) March 9, 2014


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Drawing on her own career as a Park Ranger, Annalisa Daughety has crafted a series about femal park rangers in various US state parks. Her second book, Love is Monumental, has just released. I love the actors she has picked for her characters – makes me want to read the story more!
Over to you, Annalisa:~
Brief physical description
Thirty year old Vickie is petite and has shoulder-length dark hair. Her lips are full and her eyes are green. When she's at work, she wears the required park ranger uniform, but when not on the job, she loves to wear dresses and heels. She's very fashion-conscious and is often the one her friends turn to for fashion advice. She was born and raised in Tennessee and her voice still carries a hint of a southern accent.
Thatcher Torrey:
Thatcher is tall with thick brown hair and brown eyes. At thirty-eight, his hair is just beginning to get some silvery strands. He tans well—spends a lot of time fishing in the spring and summer. When he isn't teaching, he might have a little bit of stubble—but not a full beard. He's the type of guy (typical bachelor) who gets so caught up in work and home improvement projects, he often forgets to get his hair cut or go shopping. By day, he wears typical college professor attire—button down shirts, tweed jackets and wire-rimmed glasses. Outside of work, he dresses casually—too casually if you ask Vickie.
Actor/famous person
Ginnifer Goodwin would be a good candidate for Vickie. And after seeing the Librarian movies, I definitely think Noah Wyle would make a great Thatcher.
Strengths and weaknesses
Strengths: Vickie has a way of making people feel at ease. She always knows how to handle social situations with grace and a smile—something she attributes to her Southern belle upbringing. She's the kind of person who does things by the book—and has a carefully planned out life.
Weaknesses: Vickie's quest for perfection is one of her biggest downfalls, especially where her personal life is concerned—because no one can ever live up to her high expectations.
Strengths: Thatcher is hard working and very intelligent. He has a reputation for being a fair teacher and always strives to earn the respect of his colleagues.
Weaknesses: Thatcher has spent so many years throwing himself into his work that he's a little socially awkward. (But in a bumbling, adorable kind of way.) He often says the wrong thing, not realizing how it might come across. Thatcher hides behind his work so he doesn't have time to be lonely.
Quirk (if any)
When Vickie tells her friends about a date she's gone on, she always uses a nickname instead of the guy's real name. Throughout the book we meet Chewbacca, Gold Medal Man, Mr. Potty Mouth, and the Professor. She also has an affinity for 80's movies, so much so that she named her cats Jake and Lloyd after characters in Sixteen Candles and Say Anything.
One of Thatcher's quirks is his aversion to technology. He doesn't own a cell phone or even a digital camera. He grumbles that while his students could win contests for quick text messages, they have trouble carrying on face-to-face conversations.
Your inspiration for the character
As with any character I write, I suppose there are pieces of myself—both good and bad—in Vickie. She's also a combination of many friends I've known through the years.
I wanted Thatcher's character to challenge Vickie. Where she's polished and always knows exactly what to say, he's rougher around the edges and often sticks his foot in his mouth.
Background to the story
The entire Walk in the Park series was inspired by my experiences as a national park ranger. I was a ranger at Shiloh National Military Park, a Civil War battlefield, which served as the setting for the first book in the series, Love is a Battlefield. For book two, I wanted a setting that was a contrast to the secluded small town of Shiloh. In my search through various parks, I came across the Washington Monument–and Love is Monumental was born. In each of the books in the series, it's almost as if the park is an additional character.
Thanks Annalisa. We will enjoy having you back in October for a spotlight in the Grand Canyon with Ainsley, Jake and Dustin from Love is Grand!
Relz Reviewz Extras
Visit Annalisa's website and blog
Buy Annalisa's books from Amazon or Koorong


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Download Dating Tips for Men apk 1.03 free for Android smartphone
Posted by PenniApps in Social | May 20, 2015 | 0 Comments
Apk file size: 6.3 MB
If pursuing the one that you really like has always been your biggest problem, or always been dump by the woman on your first date? What have gone wrong? Maybe now is the time that you seek advice and learn how to be a great man on that first date. And learning have never been this handy when you can be reminded of what you have to do. Learn from personal experiences of the other men out there, when you're just shy to ask them, then let them share it to you in here.
Dating Tips for Men gives you latest advises from love gurus around the world, and also personal experiences of other guys out there. Since dating changes over time, therefore you must cope up with the challenge and take that new step towards the boundaries of what you've already know and what you've already done.
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PenniApps part of our Social and have average installs from 100 to 500. Last Update May 20, 2015. Google play rating is 100.0. Current verison is 1.03. Actual size 6.3 MB.
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This is it. We will be basing the rest of our efforts in getting Justice 4 Cecil in how well we as a community can rally together. It's time to get the Lion listed as endangered, enact Cecil's Law and defeat the Laws that help create exceptions for Wealthy Trophy Hunters.
We've asked a legal consultant to look things over to help us see clearly through the language of this CITES proposal and the SHARE ACT and the BIPARTISAN SPORTSMAN ACT.
Here's the documentation referring to the proposed laws we reviewed:
- H.R. 2406: SHARE Act
- S. 556: Bipartisan Sportsmen's Act of 2015
- S. 405: Bipartisan Sportsmen's Act of 2015
- S. 2363 (113th): Bipartisan Sportsmen's Act of 2014
- S. 1996 (113th): Bipartisan Sportsmen's Act of 2014
- H.R. 3590 (113th): SHARE Act 2013
- S. 1660 (113th): SPORT Act 2013
- H.R. 2799 (113th): SHARE Act 2013
- H.R. 3197 (113th): SHARE Act of 2013
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