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Wild-type Escherichia coli grows more slowly on glucosamine (GlcN) than on N-acetylglucosamine (GlcNAc) as a sole source of carbon. Both sugars are transported by the phosphotransferase system, and their 6-phospho derivatives are produced. The subsequent catabolism of the sugars requires the allosteric enzyme glucosamine-6-phosphate (GlcN6P) deaminase, which is encoded by nagB, and degradation of GlcNAc also requires the nagA-encoded enzyme, N-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase. We investigated various factors which could affect growth on GlcN and GlcNAc, including the rate of GlcN uptake, the level of induction of the nag operon, and differential allosteric activation of GlcN6P deaminase. We found that for strains carrying a wild-type deaminase (nagB) gene, increasing the level of the NagB protein or the rate of GlcN uptake increased the growth rate, which showed that both enzyme induction and sugar transport were limiting. A set of point mutations in nagB that are known to affect the allosteric behavior of GlcN6P deaminase in vitro were transferred to the nagB gene on the Escherichia coli chromosome, and their effects on the growth rates were measured. Mutants in which the substrate-induced positive cooperativity of NagB was reduced or abolished grew even more slowly on GlcN than on GlcNAc or did not grow at all on GlcN. Increasing the amount of the deaminase by using a nagC or nagA mutation to derepress the nag operon improved growth. For some mutants, a nagA mutation, which caused the accumulation of the allosteric activator GlcNAc6P and permitted allosteric activation, had a stronger effect than nagC. The effects of the mutations on growth in vivo are discussed in light of their in vitro kinetics.
Escherichia coli is renowned for its ability to use a diverse array of organic compounds as sources of carbon and energy. However, different carbohydrates do not produce the same growth yield. One of the best carbon sources, after glucose, is N-acetylglucosamine (GlcNAc), an amino sugar. The other common amino sugar, glucosamine (GlcN), is also a source of carbon and nitrogen for E. coli, but use of this sugar results in lower growth rates than use of GlcNAc. Both GlcNAc and GlcN are phosphotransferase system (PTS) sugars (38) in E. coli so that their uptake occurs concomitantly with their phosphorylation, which produces intracellular N-acetylglucosamine 6-phosphate (GlcNAc6P) and glucosamine 6-phosphate (GlcN6P). GlcNAc6P is first deacetylated by the nagA gene product, GlcNAc6P deacetylase (NagA; EC 184.108.40.206); this produces GlcN6P, which is then subject to deamination and isomerization by the nagB-encoded GlcN6P deaminase (NagB; EC 188.8.131.52; formerly GlcN6P isomerase), resulting in fructose 6-phosphate, which enters the glycolytic pathway, and ammonia. Thus, the last amino sugar-specific step in the catabolism of both GlcN and GlcNAc depends on the nagB-encoded enzyme (Fig. 1A).
GlcNAc6P has many functions during the metabolism of the amino sugars, both as a metabolite and as a regulator. As well as being the product of GlcNAc transport by the PTS and the substrate of the subsequent enzyme in the degradation pathway (Fig. 1A), it has two key regulatory roles. It is the allosteric activator for GlcN6P deaminase (NagB) (7), and it is also the inducing signal for NagC, the transcriptional repressor of the nag operon (34). The genes for the transport (nagE) and catabolism (nagBA) of GlcNAc are located in the divergent nagE-nagBACD operons (Fig. 1B). Expression of the nagE and nagBA genes is regulated in parallel by NagC binding to two operators, which overlap the promoters, via formation of a DNA repression loop (36). Growth on GlcNAc produces GlcNAc6P, which binds to NagC, displaces it from its operators, and induces expression of the nag genes. GlcN is transported by the rather nonspecific, so-called mannose PTS, EIIABCDMan, which is encoded by manXYZ and is capable of transporting a number of sugars, including glucose and GlcN, and which under normal conditions is the only transporter for GlcN (9, 16, 33, 38) (Fig. 1). Expression of the manXYZ operon is controlled by the Mlc repressor (28). Mlc is primarily involved in controlling utilization of glucose, and the inducing signal for Mlc is generated by growth on glucose and also, to some extent, by growth on other PTS sugars (18, 30, 31).
Growth on GlcN produces intracellular GlcN6P and results in much lower levels of induction of the nagE-nagBA operons than growth on GlcNAc (32). The fact that there is some induction (2- to 4-fold, compared to the 10- to 20-fold for GlcNAc) implies that GlcN6P can be converted to GlcNAc6P by a not-yet-characterized (probably indirect) mechanism. No acetyltransferase that is capable of converting GlcN6P to GlcNAc6P has been identified. Although NagA has been shown to synthesize GlcNAc6P in the presence of high acetate and GlcN6P concentrations in vitro (40), it is unlikely to be the source of GlcNAc6P in vivo, since GlcNAc6P accumulates in a nagA mutant. GlcNAc6P is produced by recycling of peptidoglycan, and NagA appears to have a critical role in the recycling process (27, 41). It is also possible that GlcN6P can act as a secondary inducer of NagC in vivo.
GlcN6P deaminase (NagB) is a homohexameric enzyme whose physiological allosteric activator is GlcNAc6P (7, 20). Like most allosteric enzymes, GlcN6P deaminase has two extreme structural states, one displaying high substrate affinity, which is defined as the R state, and the other displaying low affinity for GlcN6P, the T state. The crystallographic structures of both the R and T forms of NagB have been solved, and the binding sites for GlcNAc6P and GlcN6P have been described (15, 26). The transition from the T state to the R state, which activates the enzyme, can be driven by substrate binding that produces positive cooperativity (homotropic activation) and also by GlcNAc6P binding to the allosteric site (heterotropic activation). The GlcNAc6P binding sites are formed in the subunit interfaces of the hexamer as a consequence of the transition to the R state. The allosteric transition activates the enzyme, increasing its apparent affinity for GlcN6P (Km) without a change in the catalytic constant (kcat) (7). This is called a K system as defined by Monod et al.(22). The affinity for GlcNAc6P is high (Kdis, 0.035 mM), whereas much higher concentrations of GlcN6P (in the millimolar range) are required to produce half-maximum activity of deaminase in vitro in the absence of the allosteric activator (1).
Our initial goal was to try to determine what caused the different growth rates on GlcN and GlcNAc and whether the different growth rates were due to the allosteric regulation of NagB. There are at least three possible rate-limiting steps which could slow growth on GlcN compared to growth on GlcNAc. The following three rate-limiting hypotheses provide nonexclusive explanations for the slower growth on GlcN: (i) the rate of uptake of GlcN is lower than the rate of uptake of GlcNAc; (ii) the amount of the NagB protein, which depends on the level of induction of the nag genes, is smaller with GlcN than with GlcNAc; and (iii) the activity of the NagB enzyme, which depends on the level of allosteric activation of NagB, is lower during growth on GlcN than during growth on GlcNAc. We considered these three hypotheses by testing the effects of (i) an mlc mutation that enhanced the transport of GlcN (via an increase in expression of the manXYZ-encoded transporter), (ii) a nagC mutation that enhanced the level of the nagB-encoded deaminase, and (iii) a nagA mutation which caused the accumulation of GlcNAc6P, the allosteric activator of the deaminase.
The allosteric mechanisms of many allosteric enzymes have been studied in detail by biochemical, biophysical, and crystallographic methods over the years. Allosteric enzymes are often located at the beginning of biochemical pathways, where they are subject to feedback regulation by end products of the pathway (e.g., aspartate transcarbamylase and glutamine synthetase). As first proposed by Monod et al. (21), it is tacitly assumed that the allosteric activation has a function in vivo, adjusting the catalytic activity to the metabolic state of the organism. However, whether the changes in catalytic properties observed in vitro are properly and quantitatively reflected in the physiological properties of the enzyme in vivo has rarely been tested. A notable exception in bacteria is work on glycerol kinase, in which researchers measured the effect of loss of binding of two allosteric effectors, fructose 1,6-bisphosphate and EIIAGlc, on glucose regulation in vivo. For this enzyme it was shown that fructose 1,6-bisphosphate binding was the major factor contributing to glucose control of the enzyme (14).
Several mutants with site-directed mutations in the catalytic or allosteric site of the NagB protein have been constructed and analyzed in vitro. Some of these mutations alter the catalytic constant of the enzyme and modify qualitatively and quantitatively the allosteric properties of the enzyme, in some cases uncoupling the substrate, GlcN6P, induced cooperativity (homotropic effect), and the activation by the allosteric effector, GlcNAc6P (heterotropic effect). The results obtained have allowed a coherent description of the catalytic mechanism and provide some understanding of the allosteric activation mechanism (1, 5, 6, 8, 19, 23, 24).
To better understand the functioning of GlcN6P deaminase in vivo and to determine whether the kinetic data obtained with pure enzymes assayed under standardized pH, ionic strength, and temperature conditions in vitro reflect the behavior of the enzyme in the complex milieu of the cell, we studied in vivo several site-directed mutations in nagB. As deaminase is part of an inducible sugar metabolism operon, it was crucial to have the mutations correctly located on the bacterial chromosome so that their expression was regulated in the same way as the wild-type gene. We took advantage of the RED recombination system (10, 44) to introduce several single-amino-acid mutations into the nagB gene within the nag operon. To assess their effects in vivo, we simply compared their growth rates on the amino sugars GlcN and GlcNAc to those of the wild-type version.
Bacteriological techniques.Strains used in this work are listed in Table 1. P1vir transductions were used to introduce the mlc::tc (28), mlc::Tn10Kan, (11), nagC::tc, and nagA::tc mutations (34). Bacteria were grown in minimal morpholineethanesulfonic acid (MOPS) medium (25) containing a carbon source at a concentration of 0.2% (except glycerol, which was added at a concentration of 0.4%). β-Galactosidase activities of the manX-lacZ fusion were measured as described previously (28).
Construction of strains carrying single nagB point mutations at the chromosomal locus of the nag operon. (i) General strategy.The RED recombination system with PCR fragments described by Yu et al. (44) was used in two steps. In the first step the whole nagB gene in DY329 was replaced by a cat-sacB cassette. The resulting strain was chloramphenicol resistant and sucrose sensitive because of the Bacillus subtilis sacB gene (13). In the second step the nagB::cat-sacB cassette was replaced by the mutated nagB allele, with selection for sucrose resistance and screening for Cms. In some cases it was possible to select directly for Nag+ strains in this second step. Finally, the recombinants were transduced out of the RED strain into MC4100 carrying a ΔnagB::cat replacement, either with selection for Nag+ or by using the adjacent asnB::Tn5 mutation and screening for Cms.
(ii) Construction of DY329 nagB::cat and DY329 nagB::cat-sacB.Approximately 70-nucleotide oligonucleotides having at their 5′ ends 50 nucleotides corresponding to the target gene, nagB, and at their 3′ ends 20 nucleotides corresponding to the cat cassette were synthesized. NagBcm5′ had 50 nucleotides corresponding to amino acids 54 to 70 of the nagB open reading frame joined to the 5′ cat promoter primer, and NagBcm3′ had 50 nucleotides corresponding to amino acid 256 to the nagB stop codon plus 23 nucleotides after the stop codon joined to the 3′ cat primer. NagBsac3′ had the same 3′ nagB sequence joined to a sacB 3′ primer. The nagB::cat fragment (1.03 kb) was amplified by using pACYC184 plasmid DNA as the template and the Pwo enzyme (Roche Diagnostics). The nagB::cat-sacB fragment (3.3 kb) was amplified with chromosomal DNA of strain NC397, which had a cat-sacB insert (a gift from the Don Court lab), by using the Expand High-Fidelity PCR system (Roche Diagnostics). The PCR fragments were purified from agarose gels. The nagB::cat cassette was introduced by electroporation into DY329 after induction of the RED system as described by Yu et al., with selection for Cmr, to obtain LAA1. The nagB::cat-sacB allele was introduced into DY329 carrying the asnB::Tn5 mutation (LAA33), which was adjacent to the nag operon. To ensure that Tn5 (which jumps very frequently during P1 transductions) was adjacent to the nag operon, asnB::Tn5 was introduced into DY329 nagB::cat (LAA1), with selection for Kanr and screening for Cms, to obtain LAA33. Subsequently, the nagB::cat-sacB allele was introduced into LAA33 by electroporation to obtain LAA41 (DY329 asnB::Tn5 nagB::cat-sacB). The sucrose sensitivity produced by the sacB gene was tested on minimal glycerol (0.4%) plates supplemented with nicotinic acid (40 μg/ml), biotin (10 μg/ml), and 4% sucrose at 30°C.
(iii) Replacement of nagB::cat-sacB with nagB point mutations.DNA fragments having point mutations in nagB were amplified from pTZ(NagB) plasmid DNA containing the mutant alleles by using oligonucleotides Nag52 (5′-TCCAGGTTACGCTTAAAGATGCC-3′) and Nag53 (5′-CAGTGAACGTTGTTCGATCTCTGG-3′). The pTZ(NagB) plasmids have a 1.1-kb FspI-ClaI restriction fragment containing nagB inserted into pTZ18R (1). Oligonucleotides Nag52 and Nag53 were chosen because they hybridized near the extremities of the cloned insert. The Nag52-Nag53 fragments, amplified by Pwo polymerase (Roche Diagnostics), were purified from agarose gels and introduced into LAA41 by electroporation, with selection for sucrose resistance or growth on minimal GlcNAc plates at 30°C. Control experiments showed that significant numbers of apparently sucrose-resistant colonies appeared in the absence of any Nag52-Nag53 DNA (10 to 50% of the number of colonies formed with Nag52-Nag53 DNA). Sucrose-resistant colonies from the electroporation plates were transferred onto grids on the same selection plates with and without 10 μg of chloramphenicol per ml to eliminate spontaneous sucrose-resistant strains when the cat-sacB insertion had not been replaced by the nagB mutant allele. Sucrose-resistant, Cms, or Nag+ Cms colonies were purified and kept. The recombinant nagB point mutations were introduced by P1 transduction into LAA20 (MC4100 carrying the nagB::cat mutation), with selection for asnB::Tn5 (or GlcNAc+) and screening for Cms colonies. To introduce nagA::tc and nagC::tc mutations into the strains having point mutations in nagB, P1 grown on LAA44 (= MC4100 nagB::cat, nagA::tc) and LAA43 (nagB::cat, nagC::tc) were used, with selection for Tcr and screening for colonies which were still Cms and so had retained the mutant nagB allele. To verify correct replacement in the chromosome, the Nag52-Nag53 fragment was amplified by PCR from small cultures of the recombinants, and the entire fragment was sequenced (by MWG-Biotech).
Measurement of bacterial growth rates.We compared the growth of MC4100 derivatives carrying the mutated nagB alleles on GlcN, GlcNAc, glucose, and glycerol. Bacteria were grown in minimal MOPS medium at 37°C with vigorous aeration. Precultures were usually grown in the same medium, but the strains having mutations which resulted in a defect in growth on GlcN or GlcNAc were also tested by starting with glycerol-grown precultures. Precultures were diluted 100-fold, and bacterial growth was measured by monitoring the optical density at 650 nm for at least 10 to 12 h or until saturation. Measurements were obtained every 30 min. Doubling times (DT) were calculated by linear regression of points in the exponential phase, generally between A650 of 0.1 and 1.0. Slowly growing bacteria were stored at 4°C overnight and, if necessary, rediluted, and growth was monitored for a second day.
Western blotting.Aliquots of the MC4100 cultures carrying recombinant nagB alleles growing with the different carbon sources were harvested at an A650 of 0.8. Bacterial pellets were resuspended in 10 mM Tris-HCl-1 mM EDTA-10 mM β-mercaptoethanol-10% glycerol and disrupted by sonication. Aliquots (25 μg) were analyzed by sodium dodecyl sulfate-12.5% polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Hybond C) by electroblotting for 1.5 h at 300 mA as described previously (35). The blots were treated with antibodies to NagA and NagB, and the reacting proteins were revealed by treatment with 125I-labeled protein A. The antibodies were not sensitive enough to detect the uninduced chromosomal levels of deaminase and deacetylase. Detection of NagB was particularly difficult because the protein was weakly bound by the membrane and at least 50% passed through the top membrane and could be detected on subsequent membranes (35). These experiments showed that the mutant proteins were present in amounts similar to the wild-type amounts and that their expression was regulated by the nagA and nagC mutations like the expression of the nagB+ protein expressed from the chromosome.
Growth on GlcN compared to growth on GlcNAc.Growth of a wild-type nagB+ strain (MC4100) on GlcNAc resulted in growth rates (μ) comparable to the growth rates on glucose (μ expressed as doublings per hour, 1.1 to 1.2 h−1, which corresponded to DT of about 50 to 55 min), while growth on GlcN was much slower and the growth rates were less than those on glycerol (μ on Gly, 0.8 h−1 [DT, 75 min]; μ on GlcN, 0.6 h−1 [DT, 100 min]) (Table 2 and Fig. 2) (42). In an attempt to assess what limited growth on GlcN, we examined the effects of introduction of three mutations (mlc, nagC, and nagA) on the growth rate of MC4100 with GlcN as the sole source of carbon.
(i) Rate of transport: effect of an mlc mutation.The mlc mutation resulted in a 40% increase in the growth rate, giving a μ of 0.84 h−1 corresponding to a decrease in the DT from about 100 to 70 min (Fig. 2A and Table 2). The mlc mutation increased manX-lacZ expression during growth on GlcN (Table 3). Growth on GlcN or Man resulted in very little induction of Mlc-repressed genes, and there was only a twofold increase in manX-lacZ expression during growth on these sugars compared to growth on glycerol. However, the presence of the mlc mutation during growth on GlcN increased the value nearly twofold (Table 3). This increase in manXYZ expression should be reflected in the rate of GlcN uptake. Previously, it was shown that an mlc mutation resulted in a twofold increase in the rate of uptake of N-acetylmannosamine, another sugar substrate of the ManXYZ transporter (37), demonstrating that for this sugar manXYZ expression limits the rate of sugar uptake. In fact, growth on N-acetylmannosamine was only possible in the presence of the mlc mutation (37), which showed that a twofold effect on expression can have a significant physiological effect. In another example, a twofold change in the phosphofructokinase level was found to have appreciable effects on the growth rate and glycolytic flux (3). We concluded that under normal conditions uptake of GlcN is one factor that limits growth on GlcN. Moreover, we could not exclude the possibility that GlcN uptake was still limiting in the mlc mutant strain.
(ii) Amount of the NagB protein: effect of a nagC mutation.The presence of a nagC mutation resulted in a 60% increase in the growth on GlcN, giving a μ of 0.97 h−1 and a DT of 62 min (Table 2 and Fig. 2). This effect was somewhat stronger than the 40% increase resulting from the mlc mutation. The presence of the nagC mutation resulted in at least a 10-fold increase in nagE-BA expression, and the levels of the NagB and NagA proteins increased similarly, whereas the level of the NagB deaminase during growth of the wild-type strain on GlcN increased only slightly (32, 34, 35) (Fig. 3A). In fact, the level of the NagB protein was even higher in the nagC strain during growth on GlcN than during growth on GlcNAc. This was because growth on GlcNAc generated catabolite repression which reduced the expression of the nagE-BA operons (12, 32). These growth experiments clearly indicated that the amount of the nagB gene product limited growth on GlcN. However, it is interesting that the presence of an mlc mutation further increased the growth rate, to a value nearly equivalent to the growth rate of the wild-type strain on GlcNAc (μ, 1.1 h−1; DT, 55 min) (Table 2 and Fig. 2), showing that increased GlcN uptake in the presence of excess NagB is still advantageous. This is consistent with the notion that increasing all the enzyme levels in a chain of metabolic steps is better than just trying to increase the level of the step that has the highest flux control coefficient (17). Together, these observations strongly suggest that GlcN uptake and enzyme induction are not sufficient for maximum growth of the wild-type strain on GlcN as a sole carbon source.
(iii) Enzymatic activity of NagB: effect of a nagA mutation.The presence of a nagA mutation had an effect on the growth rate on GlcN similar to the effect of the nagC mutation, and as in the case of the nagC mutation, growth was slightly improved by the presence of an mlc mutation. Strains which have a nagA mutation accumulate high (millimolar) concentrations of GlcNAc6P (41, 42; unpublished results), which results in high constitutive expression of the nagE-BA operons via induction of the NagC repressor (34) (Fig. 3A). In addition, the accumulation of GlcNAc6P allows full allosteric activation of the NagB deaminase. A difference in the growth rates of strains having a nagA mutation and strains having a nagC mutation would imply that the accumulated GlcNAc6P contributes to the allosteric activation of deaminase, but no difference was apparent with strains carrying a wild-type nagB allele. Thus, when high levels of the deaminase were present, no effect due to allosteric activation of NagB was detectable. Unfortunately, at the moment we are unable to test the effect of allosteric activation by GlcNAc6P under conditions corresponding to the level of the NagB protein present during growth on GlcN. Increasing the intracellular GlcNAc6P concentration simultaneously derepresses the nag operon and allosterically activates NagB. This is the physiological response when cells grow on GlcNAc; both transcription and enzyme activity are simultaneously increased.
To test whether the allosterically activated form of deaminase, if it is present at the concentrations normally attained during growth on GlcN, allows more rapid growth than the nonactivated form requires a mutant locked in the allosterically activated R form. Such mutants exist (see below), but it is not just their allosteric properties which are changed; their catalytic constants are also significantly lower. Their strongly reduced kcat values mean that the enzymes are not active enough to allow growth unless they are overproduced. However, for some of the mutants described below, the fact that they did grow better on GlcN with a nagA mutation than with a nagC mutation demonstrated that the substrate (homotropic) cooperativity of these mutant forms of the enzyme is not sufficient during growth on GlcN and, at least for these modified forms of the protein, the allosteric activation is functionally significant in vivo.
Finally, it can be noted that the mlc and nagC mutations have no significant effect on growth rates on GlcNAc, implying that during growth of the wild-type strain on this sugar, induction of the nag catabolic genes and transport of the sugar (via NagE) are sufficient. The nagA mutation eliminates growth on GlcNAc. The fact that the nagC and nagA mutations result in similar growth rates on GlcN (or glycerol and glucose [data not shown]) indicates that the intrinsic accumulation of GlcNAc6P in the nagA mutant is not detrimental per se. However, growth of a nagA strain on another carbon source is inhibited when GlcNAc is also added. This is the Nags phenomenon, which is presumably due to accumulation of toxic levels of GlcNAc6P (4, 42). It should also be mentioned that the nagA mutant strains are not very genetically stable; Nagr strains accumulate, and these strains mostly contain noninducible alleles of NagC which keep the nagE-BA genes repressed despite the accumulation of GlcNAc6P (29, 42).
Effects of mutations which affect the allosteric properties of NagB during growth on GlcN. (i) Tyr121 and Tyr254.Spectroscopic studies implicated two tyrosine residues in GlcNAc6P binding, Tyr121 and Tyr254 (2). Mutants with conservative mutations (Tyr121Thr, Tyr121Ser, Tyr121Trp, Tyr254Phe, and Tyr254Trp) were constructed and were found to have modified kinetics and to be affected both in substrate-induced positive cooperativity (homotropic effect) and in the affinity of the allosteric site for GlcNAc6P (1, 23) (Table 4). However, when the crystal structures were obtained, it was clear that neither Tyr121 nor Tyr254 is directly involved in GlcNAc6P binding (15, 26) and that these amino acids must contribute indirectly to GlcNAc6P binding at the allosteric site. Mutants with substitutions at both positions still displayed substrate (homotropic) cooperativity (although it was reduced for mutants with mutations at Tyr254) and were also allosterically activated by GlcNAc6P, but the effect was different. For the wild-type enzyme, GlcNAc6P binding resulted in only an increase in the affinity for GlcN6P (a K-type system). In the tyrosine mutants, activation by GlcNAc6P also resulted in an increase in the apparent catalytic constant and hence the measured Vmax. This is known as mixed K-V allosteric regulation. The GlcNAc6P-induced increases in kcat were 20- and 7-fold for the Tyr121Ser and Tyr254Phe mutants, respectively. Both of these mutants also had lower affinity for GlcNAc6P. The Kdis for GlcNAc6P was 2 orders of magnitude higher for Tyr254Phe, while the Tyr121Ser and Tyr121Trp mutants produced biphasic binding curves so that the first three GlcNAc6P sites were occupied with the same Kdis as the wild-type enzyme and the remaining three sites were occupied with a Kdis nearly 10 times higher (Table 4) (1, 23).
Consistent with these in vitro results with the purified enzymes, when the mutations were transferred to the E. coli chromosome, none of them had any effect on the growth rate on GlcNAc, which allowed full allosteric activation by GlcNAc6P, but all mutations resulted in lower growth rates on GlcN. The growth rates decreased to between 40 and 60% of the nagB+ value (Table 2 and Fig. 2C). Introducing an mlc mutation, to increase the rate of uptake of GlcN, increased the growth rate for all mutants. In particular, for Tyr121Trp and Tyr254Trp, an mlc mutation was sufficient to produce growth rates that were similar to that of the nagB+mlc strain.
As observed for the wild-type nagB+ strain, introduction of a nagC or nagA mutation resulted in dramatic increases in the growth rates (Table 2). This demonstrated that an excess of the defective proteins can compensate for their reduced activity. Introduction of a nagA mutation resulted in decreases in the doubling times similar to the decreases produced by nagC, except in the case of Tyr121Ser and Tyr254Phe, where the nagA mutation resulted in higher growth rates than the nagC mutation (Table 2 and Fig. 2C). This was most obvious for Tyr121Ser, where the nagA mutation resulted in a growth rate of 0.95 h−1 (DT, 63 min), while for the nagC mutant the growth rate was 0.84 h−1 (DT, 74 min). This is consistent with the in vitro defects in the allosteric activation of the mutants. The massive accumulation of GlcNAc6P in the nagA strain forced allosteric activation, even though GlcNAc6P binding was deficient and higher concentrations were required for heterotropic activation in vitro for both mutant enzymes (1, 23). In fact, in the presence of the nagA mutation, all five nagB mutant strains had growth rates comparable to the growth rate of the nagB+nagA strain on GlcN (μ, 0.95 h−1).
Most of the kcat values for the fully activated forms (R forms) of the mutated enzymes in vitro were two- to threefold lower than the value for the wild-type enzyme (Table 4). As strains with these enzymes were capable of normal wild-type growth rates on GlcNAc, this showed that a threefold-lower kcat is still adequate and that the wild-type enzyme, with a kcat of 158 s−1, has unused potential catalytic capacity. As explained above, the Tyr replacements resulted in mixed K-V allosteric activation, and the calculated kcat values for the T forms, in the absence of GlcNAc-6P, for Tyr121Ser and Tyr254Phe were very low (3 and 11 s−1, respectively) (1, 23). This probably accounts for the low growth rates on GlcN in the absence of an adequate supply of the allosteric activator to push the enzyme into the higher-activity R state. In the case of these mutants it is clear that the lack of allosteric activation limited growth on GlcN compared to growth on GlcNAc.
(ii) Mutations in the active site lid: Phe174Ala and Lys160Glu.The active site of the enzyme is covered by a complex lid motif formed by residues 158 to 187. Arg158 and Lys160 contribute to GlcNAc6P binding in the allosteric site, and Arg172 is involved in GlcN6P binding. This motif exhibits high mobility in the crystal structure when the active site is free of ligands, but it has a fixed position when the active site is occupied. The lid plays a critical role in the functional coupling of allosteric and active sites. It changes position during the allosteric transition, thus transmitting the conformational changes produced by GlcNAc6P binding between the allosteric and active sites (15, 39). Phe174 is involved in anchoring the lid to the rest of the molecule, and the Phe174Ala mutation increases the flexibility of the lid (6). The Phe174Ala mutant enzyme is essentially inactive in vitro in the absence of the allosteric activator, GlcNAc6P. It requires the allosteric activator to restructure the active site (6). Consistent with the in vitro data, the Phe174Ala mutant grew slowly on GlcNAc and not at all on GlcN (Table 2). Growth on GlcNAc was improved by a nagC mutation, implying that the amount of the mutant protein is limiting for growth on GlcNAc. For the Phe174Ala mutant, introduction of the nagC mutation resulted in a 70% increase in the growth rate on GlcNAc. However, the growth rate (0.72 h−1; DT, 83 min) was still distinctly lower than that of the wild-type enzyme (1.14 h−1), implying that the catalytic capacity of the Phe174Ala NagB mutant was not sufficient for maximal growth even when the enzyme was overproduced. This is perhaps surprising, since the kcat in vitro for the fully allosterically activated enzyme was the same as the kcat for the wild-type enzyme, 163 s−1, whereas other mutants with lower kcat values in vitro (Tyr121Ser and Tyr254Phe) had normal growth rates on GlcNAc. The level of production of the Phe174Ala NagB protein during growth on the different carbon sources paralleled the level of production of the nagB+ allele (Fig. 3B), showing that the differences in the growth rate were apparently due to the enzyme activity of the mutated protein and not to the amount of protein. Possibly the F174A protein was less stable and the protein accumulated in vivo in an inactive form. However, unstable or misfolded proteins are usually eliminated by protein degradation machinery (43). Alternatively, the in vitro assay conditions may not have reflected the prevailing conditions in the cell, and the mutant enzyme could be particularly sensitive to ionic conditions or pH.
In the absence of a nagC or nagA mutation that increased the amount of the Phe174Ala protein, there was no growth on GlcN. The nagA mutation with Phe174Ala resulted in distinctly higher growth rates than nagC (0.8 h−1 [DT, 75 min] for nagA, compared to 0.53 h−1 [DT, 113 min] for nagC), showing that, as predicted, the accumulation of GlcNAc6P in the nagA strain allosterically activated the enzyme. We also tested to see if GlcN uptake was still limiting under these conditions. An mlc mutation that enhanced GlcN transport had no effect on the Phe174Ala nagA strain but did seem to slightly improve the growth on GlcN of the Phe174Ala nagC strain (Table 2 and Fig. 2B). Increasing GlcN uptake increases the GlcN6P pool; i.e., it increases the substrate concentration for NagB and allows whatever residual homotropic activation that exists in this enzyme to occur and/or facilitates the intracellular conversion of GlcN6P to GlcNAc6P and hence allows some heterotropic activation.
Mutations in another residue in the active site lid were also tested. Lys160 is at the N terminus of the sequence forming the lid and is also part of the allosteric site cleft involved in binding the phospho group of GlcNAc6P. Mutations at Lys160 eliminated homotropic cooperativity and binding of GlcNAc6P so that the enzyme showed hyperbolic kinetics but a lower kcat (S. Lara-González and M. Calcagno, unpublished results) (Table 4). The Lys160Glu mutation resulted in a pattern of growth similar to the Phe174Ala pattern of growth, but there was no apparent stabilization by GlcNAc6P since the growth rate on GlcN was identical for the nagC or nagA version and was also identical to the growth rate of the nagC derivative on GlcNAc. The Lys160Ala mutation, on the other hand, had a more pronounced detrimental effect on growth than Lys160Glu had. There was no growth on either GlcNAc or GlcN, which correlated with the lower kcat of the Lys160Ala version. In the presence of a nagC mutation, the growth rate of the Lys160Ala mutant strain on GlcNAc was unexpectedly much lower than the growth rate on GlcN (0.2 h−1 [DT, 290 min] for GlcNAc and 0.42 h−1 [DT, 142 min] for GlcN). In this respect Lys160Ala behaved like the Asp141Asn and Glu148Gln catalytic site mutations (see below). Slower growth on GlcNAc could be interpreted to mean that GlcNAc6P is an inhibitor for the enzyme, but this does not seem to be the case since the Lys160Ala nagA strain, which should have contained high concentrations of GlcNAc6P, grew as rapidly on GlcN (0.41 h−1 [DT, 146 min]) as the nagC version and grew more rapidly than the Lys160Ala nagC strain on GlcNAc. We do not have an explanation for this behavior (but see the discussion of Asp141Asn and Glu148Gln below).
(iii) Active site mutations: Asp141Asn and Glu148Gln.The enzymatic mechanism of GlcN6P deamination includes a catalytic triad consisting of Asp141, His143, and Glu148 involved in the sugar ring-opening step, followed by a base-catalyzed enolization reaction involving Asp72 (20, 24, 26). Mutants with changes at the crucial residues His143 (e.g., His143Gln) and Asp72 (e.g., Asp72Asn) are essentially inactive (their kcat values are only 0.1 and 0.01% of the wild-type values, respectively) (24), and mutations at these positions were not tested in vivo. However, mutants with substitutions that changed the acid residues of Asp141 and Glu148 to their amides were still appreciably active in vitro, although they showed very marked kinetic changes. In these mutant forms the allosteric activator had become (at concentrations of the substrate GlcN6P above a certain critical but low concentration) an allosteric inhibitor so that binding GlcNAc6P drove the enzyme to a less catalytically active form (8).
When present at the nagB locus, neither mutation allowed growth of E. coli on GlcN or GlcNAc in the absence of a nagC mutation that increased the amount of the mutant proteins (Table 2). Significantly, the growth rates of nagC derivatives of Glu148Gln and Asp141Asn mutants were nearly twofold higher on GlcN (0.52 h−1 [DT, 115 min]) than on GlcNAc (0.31 h−1 [DT, 200 min]). This implies that in vivo, as in vitro, the presence of GlcNAc6P formed by growth on GlcNAc inhibits the growth on GlcNAc compared to the growth on GlcN. According to the same rationale, the presence of a nagA mutation, which results in the accumulation of millimolar amounts of GlcNAc6P, should also reduce growth on GlcN compared to the effect of a nagC mutation. However, the presence of the nagA mutation had only minor effects on the growth rates of Glu148Gln and Asp141Asn mutants on GlcN compared to the presence of the nagC mutation. Possibly the intracellular concentration of GlcN6P was lower during growth on GlcN than during growth on GlcNAc in the nagC strain. The nagC mutation resulted in overexpression of the fully active NagE transporter and NagA deacetylase, leading to higher intracellular levels of GlcN6P during growth on GlcNAc than during growth on GlcN. As allosteric inhibition by GlcNAc6P depends on the concentration of the substrate GlcN6P, this could mean that GlcNAc6P was more inhibitory during growth on GlcNAc, when GlcN6P concentrations were high, than it was in the nagA strain during growth on GlcN, when GlcN uptake was still limiting.
Concluding remarks.At least two factors contribute to the slower growth on GlcN than on GlcNAc: lower induction of the nag genes and limiting GlcN uptake. The major reason appears to be the level of the NagB protein. As measured previously by using nag-lacZ fusions and Western blots (32, 35) and as confirmed here for the wild-type and Phe174Ala mutant strains, GlcN results in much lower levels of induction of the nag genes than GlcNAc (Fig. 3). Either a nagA mutation or a nagC mutation derepresses the nag genes 20-fold so that the levels of the nag operon gene products that are present during growth on GlcN are similar to the levels in a nagA+C+ strain during growth on GlcNAc. Second, the rate of GlcN uptake is also limiting, since a mlc mutation which increased expression of the manXYZ-encoded transporter for GlcN (Table 3) resulted in a nearly 40% increase in the growth rate. For the wild-type enzyme we cannot decide whether a lack of allosteric activation also limits growth on GlcN. Another intriguing question is why in E. coli the metabolism of amino sugars is designed to favor GlcNAc as a carbon source over GlcN.
We tested a series of site-directed mutations in the nagB gene whose effects on the in vitro kinetics made them interesting candidates to study in vivo. The amino acids mutated were picked mostly on the basis of the crystallographic structures, and we chose residues involved in either the allosteric site or the active site. However, in most cases, the amino acid replacements had pleiotropic effects that altered both active site catalysis and allosteric activation. The catalytic constants (which were reflected in the Vmax) were usually decreased, but several mutants were still capable of normal growth on GlcNAc. On the basis of the in vitro kinetic parameters, certain characteristics of the growth pattern could be predicted. For example, mutations that resulted in a defect in homotropic activation (Tyr121Ser) affected only growth on GlcN, and this could be fully compensated for by a nagA mutation, which increased the intracellular GlcNAc6P concentration and produced allosteric activation. Similarly, the Phe174Ala mutant, which was active in vitro only in the presence of GlcNAc6P, grew better on GlcN in the presence of a nagA mutation than in the presence of a nagC mutation, since the accumulation of GlcNAc6P forced allosteric activation. On the other hand, strains having mutations that distorted the active site, so that the allosteric activator behaved as an inhibitor (Asp141Asn and Glu148Gln), grew better on GlcN than on GlcNAc. Although the correlation was not always perfect, these experiments showed that the in vivo growth characteristics of the NagB mutants reflect and can be interpreted in terms of the enzymatic and allosteric properties of the mutant proteins in vitro.
We thank the Don Court lab for gifts of strains and advice on the use of the RED recombination system and Samuel Lara-Gonzáles and Ismael Bustos-Jaimes for providing unpublished results and for useful discussions. L.A.-A. is pleased to acknowledge a leave of absence from the UNAM. We are grateful to the following people for their critical comments on the manuscript: Luis Acerenza, Bernard Badet, Henri Buc, Guy Hervé, Annie Kolb, Rosario Muñoz-Clares, Mathias Springer, and the reviewers of the manuscript.
This work was supported by grants from the CNRS and Université Paris 7-Denis Diderot (to UPR9073) and from CONACYT (grant 41328Q) and PAPIIT-UNAM (grant IN228703) (to M.L.C.).
- Received 19 October 2004.
- Accepted 22 January 2005.
- Copyright © 2005 American Society for Microbiology

subset: commoncrawl


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Hyannis, MA Commercial Realty Advisors has hired a new associate to their team. Commercial real estate agent Michael Belmore has joined the local office.
Belmore began his professional career with the Danish ship-owner Dampskibsselskabet Norden A/S. The company owns large oil vessels (tankers) shipping clean and dirty petroleum products worldwide. He worked his way up from trainee to chartering manager and was transferred to the company's headquarters in Copenhagen, Denmark. His main responsibilities were vessel pricing negotiations, logistics, and complicated contract formation with customers such as Exxon, BP, Chevron, and Shell.
Always having a passion for real estate, Belmore switched gears and became a full-time real estate professional. His focus is commercial real estate and helping businesses and investors achieve their goals through information, guidance, strategies, and resourcefulness.
In 2016 he received a professional degree in Commercial Shipping from the Institute of Chartered Shipbrokers in London.
Belmore currently holds a B.S in International Maritime Business from the Massachusetts Maritime Academy in Buzzards Bay.
Growing up on the Cape, Belmore is excited to be back and help contribute to the local community. He lives in Dennis with his finance Lucia and pup Cali.


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- Ultra-low emission technology of converter gas dry bag dust removal system
- Do you know the boiler flame arrester of Jida Machinery?
- What is the reason for water vapor blocking the bag filter?
- Bag dust collectors are not pneumatic
- What is the post-treatment process of functional dust bag
- The reason why the bag filter has a high resistance during operation
- Pay attention to the air leakage rate when installing the bag filter
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email@example.com E_mail: email@example.com
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Negative pressure suction pump suction type pneumatic conveying equipment
Introduction of negative pressure suction pump
 The negative pressure suction pump and suction pneumatic conveying equipment device system produced by Ruiguang Machinery have unique wear-resistant parts structure and materials to extend the life of suction pump. The vacuum negative pressure generated by the vacuum pump is used as the conveying power. The material is sucked into the conveying pipeline, and the material and gas separator is used to separate the material and gas. After the material is separated, the material is discharged into the receiving bin by electric air lock valve under the action of gravity. Operation, reduce the occurrence of security accidents and reduce the operating costs of the enterprise. Negative pressure pneumatic conveying can be designed according to the needs of users. Its other forming equipment is ship unloader, negative pressure pneumatic conveying-unloading machine, and dock negative pressure suction pump.
Negative pressure pneumatic conveying system (also known as negative pressure air conveying system or negative pressure air conveying system) is the vacuum generated by high pressure centrifugal fan (or Roots vacuum pump), which sucks the bulk powder and granular materials into the designated container with negative pressure air flow. (Silo), vacuum vacuum pressure collection system composed of Ruiguang negative pressure suction pneumatic conveying device, which is especially suitable for short distance, high material temperature and multi-point discharge systems.
The bulk material suction machine is composed of a suction nozzle, a hydraulic suction arm, a separation and discharge device, a roots blower (vacuum pump), and an electronic control system. The materials can be sent directly to the bulk material storage (storage) directly or through the conveying machinery. The pneumatic conveying system for bulk materials consists of negative pressure pneumatic conveying equipment (suction nozzle, hydraulic suction arm, separation device, roots blower or vacuum pump) and positive pressure pneumatic conveying equipment (bin pump, combined pump, air compressor) and electronic control system. composition.
Negative pressure suction pneumatic conveying principle
Negative pressure suction-type pneumatic conveying fluidized bin uses air to make the fly ash particles fully fluidized, so that the fly ash can enter the negative pressure ash suction positive pressure conveying pipeline smoothly. The interior of the negative pressure ash suction fluidization bin is composed of two porous thin steel plates, and a breathable layer is sandwiched between the upper and lower porous plates. The perforated plates inside the entire fluidization bin are arranged at a certain slope (4% -10%). Fly ash enters the combined pump from the separator, and enters the fluidization bin from the combined pump, and the feeding equipment is added to the breathable layer of the feeding opening of the upper casing. The air enters the lower casing from the fluidization bin, and passes through the breathable layer along the fluidization bin to fluidize the fly ash. Feeding into the conveying machinery or entering into the positive pressure conveying equipment, the positive pressure generated by the air compressor or the roots fan passes the feeding pipeline to send the material into the bulk material warehouse (warehouse). The clean air is discharged from the upper part of the separator through the exhaust duct, and is discharged into the atmosphere from the roots of the Roots blower (vacuum pump).
Components of suction-type pneumatic conveying equipment
 The main components of the negative pressure suction-feeding device are separator, suction nozzle (fixed suction nozzle and movable suction nozzle), unloader, vacuum pump, valve and pipe fittings, Control system, etc., suitable for short and medium distance transportation of powdery materials. Because the transportation process is completed in a closed negative pressure pipeline, no leakage and pollution will occur on the transmission line, and it is especially suitable for the transportation of hazardous substances with strict leakage pollution requirements. Conveying system can feed single point or multiple points, the process layout is flexible and convenient.
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 The pressure in the conveying pipe is lower than the ambient pressure, and the dust does not leak outward. It is a better choice for conveying toxic materials. It is often used for ship unloading, vacuum loading, cleaning, etc. application. Suitable for multi-point to one-point conveying. The conveying line is in the state of negative pressure pipeline, and there is no leakage or pollution during the conveying process; the conveying state is uniform and continuous, and the conveying capacity can be adjusted; the air source device adopts dry negative pressure air source, which is simple and reliable; Pulse blowing, compact structure, high separation efficiency; the nozzle device can be specially designed according to different applications and process requirements; the equipment configuration of the conveyor system is simple, the system investment is small; the system maintenance is low, and the maintenance is convenient.
Negative pressure suction classification
1. Negative pressure suction direct loading type;
2. Negative pressure suction mechanical conveying type;
3. Negative pressure mixed pneumatic conveying type.
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BANDUNG-GFTN Indonesia Trade and Networking Coordinator, WWF Nur Maliki Arifiandi, in a release received jabarprov.go.id, Tuesday (15/11) explained Today, November 15, 2015, the first export of Forest Law Enforcement Governance and Trade (FLEGT) licensed wood products from Indonesia to EU countries do. WWF-Indonesia welcomes this momentum which signifies the commitment of Indonesia to combat illegal logging and illegal timber trade.
Indonesia is the first country in the world which has right to issue FLEGT licenses thus Indonesian timber products are free to enter the EU market and Indonesia is one of the largest exporters of tropical wood products ranging from plywood, pulp and paper to furniture and handycrafts. Starting today, a logging company in Europe could place Indonesian timber products into the EU market without going through the completed test.
FLEGT-VPA is an agreement between the Indonesian government and the EU aims to improve governance and promote legal timber trade from Indonesia to the EU market and to support the Wood Legality Verification System (SVLK) meets the requirements of the European Union Timber Regulation (EUTR).
"We welcome the first export of Indonesian forest products to the EU," said Benja V. Mambai PLT CEO, WWF-Indonesia. "This is the fruit of hard work all parties for 10 years in developing Wood Legality Verification System, until the system is becoming an important instrument to ensure Indonesia wood products that is marketed to the European Union and other countries comes from legal sources."
Based on recent data, said Nur Maliki Indonesian wood products which is exported to Europe in 2015 reached USD 882 billion [1]. In addition to the EU, China, Japan and Korea are the main export destinations of Indonesia forestry. (NR)
Meanwhile, Forest Commodity Market Transformation Leader of WWF-Indonesia, Aditya Bayunanda, in his statement describing "FLEGT License will bring a positive impact on the marketing of timber products from Indonesia.
WWF-Indonesia urges the Indonesian authorities and the EU to completely run and monitor the implementation of this system, so that could be the basis to move toward sustainability, ".
"The Indonesian government also has to ensure information access to Non-Governmental Organizations (NGOs) to be able to conduct independent monitoring, including in providing access to relevant data and planning documents. hold such information can make it difficult for NGOs to monitor the implementation of Wood Legality Verification System in field, "he added.
At the moment there are already 2,322 industries in Indonesia (data KLHK, 2016) which has passed Wood Legality Verification System and V-legal certified, and can be directly exporting to various countries. This certificate as proof of Indonesia seriousness to improve forest governance. Implementation of Wood Legality Verification System right can be a solid foundation to implement forest management in Indonesia. (NR)


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COVID-19: FREE SHIPPING Domestic Orders Over $299.99 - Most orders ship out within 2 business days!
Regular price
$ 74.98
New Price
This bolt on battery box for the AG801 small case 8 cell Antigravity battery is the perfect fit for your Honda Shadow bobber build! Made of .125" (1/8") thick steel, this battery box is built for a long life. Two drain holes in the bottom ensure that any moisture that's collected can get out. I know many of you Shadow owners don't have access to a welder, but almost anyone can get their hands on a simple drill and some bolts!
This bolt on battery box is for the Antigravity AG801 8 cell small case batteries, not for OEM batteries.
This bolt on battery box for your Honda Shadow VT750 ACE is ridiculously easy to install! It mounts to the rectangular backbone right below where the rear tank tab is! It can be mounted below the swingarm or above it! There is even a cutout in the swingarm on some model years that the box fits in perfectly so that even under full compression or unloaded, the swingarm won't touch the battery box. This battery box fits the Antigravity Battery Model AG801.
On the spirit models you may need to remove some of the old electronics hangers, these are very thin metal tabs that you wont need anymore.
Check out all the images to see the different ways to mount it!
Includes 2 bolts and retention strap made from thick 3/16" steel!


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Welcome to Eyam Village in Derbyshire, England
Thank you for visiting this website which aims to give you all the information about the plague village of Eyam in Derbyshire, England.
The idea of Eyam being a plague village might seem a morbid one, but whilst the events of the plague were tragic we want to celebrate all that is fantastic, bright and great about this beautiful little village in the English countryside.
Watch this video on YouTube.
Here you will find all the information you need to play your visit to beautiful Eyam, to spend time in the pubs and tea-rooms of the village, and of course to trace the steps of the villagers of this wonderful place back in the time of the notorious bubonic plague outbreak that devastated so many families here.
Enjoy the site and let us know if there's anything we missed


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Special multifunctional unit by atmos company, which serves to control various elements of a house via a mobile phone it has a number of predefined functions that simplify and facilitate its installation and operation. Apprenticeship involves both classroom studies and on-the-job training under the supervision of a certified plumber, called a journeyperson. Psc electrical are.
Added: 2020-05-08 | Comments: 0 | Category: one


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Internacjonalizacja polskich przedsiębiorstw rodzinnych – wybrane wyniki z ogólnokrajowego badania statystycznego
Alicja Hadryś-Nowak , Ewa Więcek-Janka , Adrianna Lewandowska
AbstractThe international expansion of the company is nowadays a necessity. This also applies to family businesses. Researches on internationalization of family busines- ses are ambiguous. Some results indicate that family firms are doing much worse on fo - reign markets because of the family character that makes businesses more conservative and reluctant to risk, including the risk of expansion. Other studies however show that thanks to the involvement of the family, foreign expansion is much easier and that they do not differ from non-family businesses. In many countries which survey the entre - preneurial families, there are no national statistical surveys about family business, not to mention how they operate on foreign markets. Therefore, the aim of this paper is to present the official nationwide statistics regarding the internationalization of Polish family businesses. In the paper the author presents range and the countries of expansion of family businesses, the relationship between internationalization and the size of the family business, the industry in which it operates, gender of business owners and the presence and composition of the board.
|Other language title versions
|Internationalization of Polish Family Firms – Preliminary Results From the First Official Statistics
|Journal series
|Przedsiębiorczość i Zarządzanie, ISSN 1733-2486, (B 14 pkt)
|Issue year
|6, cz. 2
|Publication size in sheets
|Keywords in Polish
|przedsiębiorstwa rodzinne, internacjonalizacja, badanie statystyczne
|Keywords in English
|internationalization, family owned businesses, statistics
|pl polski
|Score (nominal)
|Score source
|= 14.0, 27-03-2020, ArticleFromJournal
|Citation count*
|Additional fields
|Tytuł numeru
|Firmy rodzinne - rozwój teorii i praktyki zarządzania
* presented citation count is obtained through Internet information analysis and it is close to the number calculated by the Publish or Perish system.


text: The RavPower USB dual port 24w wall charger is among the cheapest USB adapters we've seen. Yet it offers workability with USB 3.0 devices and max charging speeds for USB 2.0 and earlier devices. It has well isolated ports, foldable 120 volt plug prongs. Plus, the charger features highly reliable operation, which is perhaps its … Continue reading RAVPower 24w Wall Charger Review, USB, Dual Port


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Hippo Card & Dice Game
Be the first player to give all your rubber rings to Mr Hippo.
This is a brilliantly compact game. A small box containing swimming pool cards which unite to form the board. Ideal for family excursions when you want to bring along something to entertain but weight and size matters.
Hippo is a simple concept that echos other classic games (like Shut the Box) where you have to decide how to play the numbers on the 3 dice that you roll. You can either add all or some of the dice values together, or split them in the most strategically advantageous manner. This means Hippo is a good fun way to practise early maths skills and number bonds.
Luck can play a big role in this game - rolling the number 7 gives you a huge advantage (an extra turn and a 'safe' place for one of your rubber rings). See video for more on how to play.
For 2 - 4 players, aged 6 years +
Boxed Dimensions: 6.5 x 11 x 3 cm
Attention - small parts. Not suitable for children under the age of three years


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Mission Trips Informational Meeting
February 2, 2020
12:15pm – 1:00pm
Location: 1811 Dutch Fork Rd, Irmo, South C 29063Category: Faith Church | Coordinator: Kathy Simpson
Our Lord has called each of us to love and serve Him and others. There are many ways to do that! One way is to serve on a mission trip. The Connecting to Others team is presenting two opportunities this year to do just that! Please pray about being involved, and pray for trip leadership!
WHAT: Spring Break and Summer Mission Trips!
WHERE and WHEN: Nashville, April 13 - 18
Dominican Republic, June 23 - 28
WHO: Men, Women and Youth!
(middle school or older for Nashville; high school who have previously served domestically or older for DR)
WHY: We are called to serve!
HOW: Sign up THIS Sunday if you plan to be involved. There will be sign up sheets in the lobby!


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February 2012: Ford GT versus Ferrari 458 Comparison Test
In February of 2012 I organized a comparison test between an Audi R8 and Ferrari 458. This was for another website I was running at the time and didn't, technically, have anything to do with my Ford GT. Of course, having two more mid-engine sports cars at my disposal made it impossible not to think about how they stacked up against the Ford GT.
Throughout my Ford GT ownership I've also taken every opportunity I could to drive the GT back-to-back against similar cars, and this time was no different. Since getting my GT in 2005 I had only driven two cars that I felt offered a similar level of driving satisfaction. One was the Porsche Cayman RS, the other was the Audi R8 with a V10 engine. But all that changed after driving the Ferrari 458.
I haven't been overly impressed with Ferraris, either from a performance or styling perspective, since the late 1990s. The 360 lacked low-end torque and was too hairy when driven to its extremes. The 430 addressed the torque issue but still lacked the sheer capability I expect from a prancing horse.
The 458 addresses all of these issues. It is an extremely capable machine that honestly stunned me when I drove it aggressively. It was the first car since getting my Ford GT that I knew, without question, was far superior in terms of performance. My confidence level behind the wheel of the 458 was unlike anything I'd experienced perviously, and I happen to think the car is beautiful, too (particularly in white).
That's not to say I'd trade my Ford GT for a Ferrari 458. The GT still has an advantage in terms of raw emotion, styling and heritage. With three pedals and zero electronic nannies it represents the end of an era in sports car design and philosophy. No current or future sports car will offer those traits (automakers can't even build a modern car without stability control). The new Ford GT, which will undoubtedly be more capable than a 458 — or just about any other street car ever made — won't likely have the raw emotion of the 2005-2006 Ford GT.


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Personalise with the couples name and the wedding date. You can also choose from either Pink, Teal, Grey, Navy or Orchid for your cards.
Each Save the Date is a flat A6, postcard style invitation, printed with all of the details on one side. All Save the Dates come with your choice of matching White or Kraft envelopes.
Samples are available for you to see the overall look and quality of our stationery before ordering. All samples are posted within 2-3 days by Royal Mail first class post (the delivery date shown is for full orders of stationery). Samples cannot be personalised, however there is a colour swatch on the back for your reference.
Once your order has been placed we will send over a digital version in JPEG format via a NOTHS enquiry for you to approve of 3-5 days within you ordering. Once these have been approved they will be printed and dispatched to you. Please order 3-4 weeks before the date you need to send them to allow time for proofing, printing and delivery.
Please note that colours may look different on different device screens and we recommend that you order samples prior to ordering to confirm you are happy with the colours. Should you require a different colour to those shown please contact us prior to ordering to confirm suitability, we will of course accommodate your request if we can!
Further pieces in this collection are available here in our NOTHS store, including wedding invitations, RSVP card and Thank You cards.
Made from:
Printed onto Textured card, these A6 flat cards come complete with envelopes.
A6 size flat cards.


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Worried about your small breast size? Just exercising, eating the right food and a few changes in your lifestyle may get you bigger breasts.
These foods will help you get a bigger breast naturally Here we shall recommend some foods which get your hormones moving to get you.
Yes! Foods help you to get bigger breast? Astonishingly, what you consume can create a distinction in increasing your cup size. There are.


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10 Cybersecurity Practices That Create the Best Remote Environment
Top 10 Ways Companies Cook the Books
3 Steps to Building Strong Risk Management
New FAQs Address PPP Loan Forgiveness Issues
IRS to Allow Faxing of Automatic Method Change Requests
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IRS Provides Guidance for April 15 Filing Delay
The IRS officially announced that, because of the ongoing coronavirus pandemic, tax returns due April 15 will not have to be filed until July 15 this year. Notice 2020-18 was released hours after Treasury Secretary Steven Mnuchin had broken the news in a tweet.
The AICPA expressed its thanks to Treasury and the IRS for the filing extension and to its members and the state CPA societies for their outreach to federal legislators and administrators on the issue.
The postponement applies to any "individual, a trust, estate, partnership, association, company or corporation" with a federal income tax return or income tax payment due on April 15 (affected taxpayer). Any affected taxpayer receives an automatic postponement of that deadline until July 15. They do not have to file Form 4868, Application for Automatic Extension of Time to File U.S. Individual Income Tax Return, or Form 7004, Application for Automatic Extension of Time to File Certain Business Income Tax, Information, and Other Returns.
Earlier in the week, the IRS had announced a postponement of federal tax payments due April 15. Under that guidance (Notice 2020-17), C corporations and consolidated groups could postpone up to $10 million in tax payments under this relief. All other taxpayers could postpone up to $1 million in tax payments. Notice 2020-18 removes the limitation on the amount of tax that can be postponed and entirely supersedes Notice 2020-17.
Under Friday's notice, no interest, penalty, or addition to tax for failure to file a federal income tax return or to pay federal income taxes will accrue between April 15, 2020, and July 15, 2020, for any return or payment postponed by the notice.
The IRS is postponing the filing and payment deadlines using its authority under Sec. 7508A, which was triggered on March 13 when President Donald Trump declared the ongoing coronavirus pandemic a national emergency under the Robert T. Stafford Disaster Relief and Emergency Assistance Act, P.L. 100-707.
Friday's notice applies only to federal income tax returns and payments (including self-employment tax payments) due April 15, 2020, for 2019 tax years, and to estimated income tax payments due April 15, 2020, for 2020 tax years. The notice explicitly does not apply to any other type of federal tax or to any federal information returns. For state tax developments related to the coronavirus outbreak, see the AICPA list here.
Because Notice 2020-17 is superseded, the IRS will not be returning phone calls it received concerning that notice.
(Source: AICPA – CPA Letter Daily – Journal of Accountancy – March 23, 2020)


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Acosta had asked a White House official why there was apparently no effort to implement social distancing measures for the audience at the speech. The official's response?
"Everybody is going to catch this thing eventually," the official reportedly said.
It's a staggering comment, for a variety of reasons. The most obvious is that everyone won't catch the novel coronavirus eventually, ideally; the point of developing a vaccine is to keep that from happening. What's more, even if there were no vaccine, there's a big difference between people catching it now and catching it in a year or two when there might be better therapeutic treatments or potentially a cure. It's like shrugging at people getting infections before the invention of penicillin.
But this was also one comment from one official, and it was not something that we could simply ascribe to the administration overall as a deliberate strategy.
Until Monday morning, when The Washington Post reported that something along these lines is gaining acceptance among White House leadership.
"One of President Trump's top medical advisers is urging the White House to embrace a controversial 'herd immunity' strategy to combat the pandemic, which would entail allowing the coronavirus to spread through most of the population to quickly build resistance to the virus, while taking steps to protect those in nursing homes and other vulnerable populations, according to five people familiar with the discussions," The Post's Yasmeen Abutaleb and Josh Dawsey report. "The administration has already begun to implement some policies along these lines, according to current and former officials as well as experts, particularly with regard to testing."
Last week, new guidelines from the Centers for Disease Control and Prevention scaled back the number of tests being completed for the virus, forgoing testing for those who weren't displaying symptoms but had been in contact with someone infected with the virus. One effect of this would be a political benefit for Trump, reducing the number of recorded coronavirus cases. Another effect would be to increase the transmission of the virus, as asymptomatic people can still infect others if not quarantined.
Trump has for some time insisted that the government's time is best spent protecting those in nursing homes and who are otherwise at risk, an approach to the pandemic that allows for more economic activity and — Trump again clearly hopes — fewer negative consequences for his reelection bid.
He's also repeatedly downplayed the risks associated with contracting the virus. At times, he's insisted that 99 percent of infections are "totally harmless," a claim that both misrepresents the deadliness of the virus and ignores the body of evidence showing that some infections yield long-term health effects and potentially permanent damage.
Over the weekend, he sought to bolster the idea that the virus isn't really as deadly as experts say by retweeting a QAnon supporter's claim that a CDC report showed that 94 percent of those identified as dying from covid-19, the disease caused by the virus, had other conditions at the time of their deaths. What the report actually showed was that in most cases, covid-19 killed people who were living with other conditions.
As one expert on cancer put it: "600,000 die of cancer each year. 95% likely have comorbidities. Doesn't mean cancer was not cause of their death."
It's obvious that covid-19 is killing Americans when we consider the number of people who've died relative to prior years. Through the middle of May, data from the New York Times showed 88,000 more deaths in 2020 than in the average number of deaths seen from 2014 to 2019. A report earlier this month estimated that about 200,000 more people had died so far this year than would have been expected from prior years.
In other words, Trump's halfhearted assurances notwithstanding, a shift in the White House's strategy to allow for more widespread infections would likely mean tens or hundreds of thousands more deaths than would be seen with containment measures.
Precisely how many isn't clear. At this point, the country has been averaging more than 900 deaths per day for more than a month. The number of new cases is hovering at about 41,000 per day, on average. And since early July, the percentage of deaths relative to new cases two weeks prior has been between about 1.5 percent and 1.8 percent.
One projection of the death toll from the virus figures that the country will add another 90,000 deaths by Election Day. That assumes a reduction in efforts to contain the virus — which is reportedly what the White House is embracing.
Trump understood the risks of letting the virus spread without containment back in April, when he was defending the administration's decision to endorse a shutdown of economic activity. At a news briefing centered on the virus, Trump was asked about Sweden's decision to embrace a "herd immunity" approach to it, allowing it to spread widely enough that there would be enough people with immunity to slow the spread.
"I think we could have followed that approach," Trump said of Sweden's strategy. "And if we did follow that approach, I think we might have 2 million people dead."
That figure comes from an estimate developed by Imperial College London. When the White House first announced its support of containment measures, it used an upper-end estimate of 2.2 million deaths to describe what could happen if Americans didn't try to contain the virus's spread.
"Sweden," Trump added, "is suffering greatly."
In July, the Times described the country as the world's "cautionary tale," having tried to both continue operating as normal but allowing the virus to spread. The result was a much higher death toll as a function of population than its Scandinavian neighbors without significantly better economic results.
So what changed since April? At the time, there had been only about 21,500 deaths in the country, and the pandemic was seen as an emerging risk. Now, nearly 180,000 people have died, and a steady daily death toll of more than 900 people has become background noise in the national conversation.
It's an example of the saying often incorrectly first attributed to Joseph Stalin, that one death is a tragedy and a million a statistic. In this case, Trump would much rather focus on one death during violent encounters in Portland, Ore., or Kenosha, Wis., than the 12 daily coronavirus deaths that are happening in those states.
One he thinks helps him politically. The other he knows doesn't. So if everyone catches this thing, so be it.


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cutest ever!!!.
I agree with you that you should forget about the girl and move on. She was with you, visits your family with you and then has sex with your brother behind your back? What in the world was she thinking? Clearly you would not want her after this. I doubt your brother wants her now either. The bottom line is move on from her and never and I mean never ever trust your brother again. What a complete slimeball he is. I wish you luck..
I feel like she is trying to make me be the one that tells her im done so she can feel better about it. Should i just keep texting her and bugging her until she tells me to stop?.
I'm pretty simple and easy to get along with. I'm 34, divorced and have a 13 year old son that lives with me on the weekends and during the summer...
Another one for tummyfan.
@arkangel2511- yeah, I uploaded 15 a week ago and they are all still in my queue..
Go over there somewhere..
Are you sure she doesn't want to meet you? Or is that just what your bf is telling you? It sounds like things are still awkward between them, but whether that's him not over her or her not over him (or both) it hard to tell from here. Ask him if he thinks that she's over him, and if he thinks that she isn't over him ask him if he thinks it's a good idea for him to keep seeing her..
Anyway, rant over..
Pretty girl with a very hot body.
You could say the same for women, that they just want to be taken care of financially..
outdoors poolside amputee water wet pigtails pink bikini sideknot tummy bellybutton ruffles smiling teeth sunshine pink nailpolish.
cutoffs hoh key twosome slice duckface.
how has this not been an HP she is perfect.
I met this girl for the first time a year ago and my feelings for her have been growing and growing. I know she likes up to some point. She had a boyfriend for a long time and now he has moved to another country and they are in an open relationship and before they broke up they decided that she will be going there in december to meet(me hating that). I have gone out with her a couple of times, not as formal dates but some hangarounds and chit chats, taning, eating, icecream, downtown..


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Nodines Back Bacon 8 ounce
Click Image for Gallery
Nodine's Back Bacon (8 ounce)Nodine's uses premium boneless pork loins to produce their finest Irish-style back bacon.With their traditional Irish style recipe and brining methods,Nodine's has captured the true flavor and quality of Ireland in the U.S.A.
Nodine's Smokehouse Inc. was started back in 1969 as a small custom smokehouse in Goshen, Connecticut. Over the many years of hard work and dedication, this small family business outgrew the small processing plant located at the farm in Goshen. In 1989, the company moved to a remodeled and modernized facility. At the new production and shipping facility, while using the finest U.S.D.A. inspected ingredients, Nodine's has been able to capture the Old World Gourmet quality and flavor that's missing in most high volume facilities, while consistently maintaining high production.
- Ships expedited in an insulated package to ensure freshness
- Made in the USA
- Using the traditional Irish style recipes and brining methods
- A hearty addition to breakfast or to add that smoky flavor to filet mignon or baked beans
- A favorite among bacon connoisseurs


text: here are a couple ideas i wanted to share. one is a top loading hopper pump and the other is for a bottom mounted detachable magazine fed pump. trigger mechanisms are dowel pins or ball bearings depending on design. they work off the same principal as a quick change tool.


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1,805 Posts
Discussion Starter • #1 •
At the office this morning, I noticed Tito acting a bit strange. I looked over to see what he was doing and he had "made friends" with a big red ant. It was one of the kinds that bite with the mandibles but don't sting, so I just let him play with it. Besides, by the time I saw him, the ant was about dead (as you can tell in the pics). Actually, he found a silverfish just now and he's over there playing w/ it! LOL! Those are the things that eat your clothes. Anyway, he was just too cute while he was playing w/ the ant, so I snapped some close-ups and decided to share.


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Michael Moradzadeh (born December 13, 1979) is the CEO of international law firm Rimon Law P.C. He established the firm in 2008 with COO Yaacov Silberman.
Background and early life
Moradzadeh is a native of Silicon Valley. Technology was part of his life from an early age. He began taking apart computers to increase their capabilities at age 13. He built his first website at age 15, when he started his first online company called EZ Webmall.
Moradzadeh attended University of California, Berkeley where he earned a B.A. with Departmental and General Honors. He graduated in three years and was a member of Phi Beta Kappa. He earned a J.D. from Columbia University, also with Honors. At Columbia, he was the editor of the Columbia Journal of European Law and the Assistant Editor of the Columbia Journal of International Affairs.
At the beginning of his career, Moradzadeh worked with startups Napster and Check Point Software. He began his legal career at the United States Attorney's Office in San Francisco and then as an associate with Ropes & Gray.
Rimon, P.C.
Moradzadeh developed the idea for Rimon Law P.C. with Ropes & Gray transactional associate Yaacov Silberman. They aspired to create a firm with less bureaucracy and pomp. They left Ropes & Gray to start Rimon in 2008. During the initial development of Rimon, Moradzadeh and Silberman lived and worked together in San Francisco's Sunset District.
Moradzadeh and Silberman have presented at Harvard Law School and Stanford Law School on the changing landscape of the legal industry.
Other Work
Moradzadeh is General Counsel of Open Perception, "an internationally renowned group of expert scientists and engineers, working together to solve complicated 3D perception problems."
Personal life
Moradzadeh married his wife Nomi Moradzadeh in 2010 in a traditional Jewish wedding. The couple has three children.
Further reading
Documentaries, videos and podcasts
Related Topics
- 1
Dawson SewellSeeking to explain science and technology through simple writing.
- 2
Julius John VillariscoEvery day is a new learning for me, new opportunity every time. Never limit my self, think outside the box.
- 3
Jude GomilaFounder and CEO of Golden, Entrepreneur. Investor in 200+ companies. Cantabrigian (Gonville and Caius). Interested in the universe.


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All About Airbags
Car shopping for that perfect ride? It's a good idea to take airbags into consideration, too. Airbags and seatbelts have allowed thousands of passengers to walk away from serious accidents with only minor injuries — or less — because of the protection they provide.
Brush up with a few of these facts about airbags, and shop smart for your next car.
Airbag Facts to Know
- Inflated airbags are designed to cushion the head and upper body during a crash.
- Frontal airbags reduce driver fatalities in frontal crashes by 29 percent and fatalities of front-seat passengers age 13 and older by 32 percent, according to the Insurance Institute for Highway Safety.
- Side airbags reduce a car driver's risk of death in driver-side crashes by 37 percent and an SUV driver's risk by 52 percent.
- Rear-window curtain airbags protect people in back seats.
- Front-center airbags keep drivers and front-seat passengers from hitting each other in a crash.
- Inflatable safety belts are aimed at reducing rear-seat injuries.
Airbags and Your Kids
Infants. Never put a rear-facing infant car seat in the front seat. It may be too close to the inflating airbag and its force, which can be transmitted through an infant seat.
Children. Older children should always wear lap and shoulder belts so they're not seriously injured by the force of a deploying airbag. Properly fasten your children in car safety seats or belt-positioning booster seats. Have the lap and shoulder belts corrected for their size.
The back seat is the safest place for children 12 and under.
If a child must ride in the front, move the seat back as far as possible away from the airbag. Be sure the child is restrained properly for his or her size. Keep in mind that your child may still be at risk of injuries from the airbag.
But First, Buckle
Even with airbags, it's still important for everyone in your car to buckle up. Seatbelts keep people in the car and in position so the airbag can do its job — cushion your impact.
Now that you've got the facts, you're all set to peruse the car market, and make sure you get the airbags you want and need before closing the deal. Happy shopping!
Related Topics: On The Road , Car Safety , Safe Driving