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ATP‐sensitive K+ (KATP) channels in the heart are normally closed by high intracellular ATP, but are activated during ischemia to promote cellular survival. These channels are heteromultimers composed of Kir6.2 subunit, an inwardly rectifying K+ channel core, and SUR2A, a regulatory subunit implicated in ligand‐dependent regulation of channel gating. Here, we have shown that the muscle form (M‐LDH), but not heart form (H‐LDH), of lactate dehydrogenase is directly physically associated with the sarcolemmal KATP channel by interacting with the Kir6.2 subunit via its N‐terminus and with the SUR2A subunit via its C‐terminus. The species of LDH bound to the channel regulated the channel activity despite millimolar concentration of intracellular ATP. The presence of M‐LDH in the channel protein complex was required for opening of KATP channels during ischemia and ischemia‐resistant cellular phenotype. We conclude that M‐LDH is an integral part of the sarcolemmal KATP channel protein complex in vivo, where, by virtue of its catalytic activity, it couples the metabolic status of the cell with the KATP channels activity that is essential for cell protection against ischemia.
Sarcolemmal ATP‐sensitive K+ (KATP) channels belong to the group of intracellular ATP sensors, coupling the metabolic status of the cell with the membrane excitability (Noma, 1983). These channels are selectively permeable to K+ ions and are closed by high intracellular ATP levels (Terzic et al., 1995; Ashcroft and Gribble, 1998). It has been suggested that the opening of sarcolemmal KATP channels promotes survival of cardiomyocytes during metabolic stress (Jovanović and Jovanović, 2001a; Light et al., 2001). Ischemia opens sarcolemmal KATP channels before a significant fall in the level of intracellular ATP occurs, suggesting that the intracellular level of ATP is not the only factor that regulates the channel activity (Knopp et al., 1999). Indeed, it is believed that the activity of KATP channels is controlled by a complex interaction of many intracellular factors and signalling pathways. In addition to ATP, the activity of these channels may be regulated by other nucleotides, intracellular pH, lactate, cytoskeleton, protein kinase C (PKC), phosphatidylinositol‐4,5‐bisphosphate and by the operative condition of the channel itself (reviewed by Tanemoto et al., 2001).
Structurally, the cardiac subtype of KATP channels are heteromultimers composed of the Kir6.2 subunit, an inwardly rectifying K+ channel core primarily responsible for K+ permeance, and SUR2A, a regulatory subunit implicated in ligand‐dependent regulation of channel gating (Inagaki et al., 1996). More recently, evidence has been provided that adenylate kinase and creatine kinase, two main regulators of intracellular ATP levels in the heart, are also parts of the cardiac KATP channel protein complex (Carrasco et al., 2001; Crawford et al., 2002). The whole channel protein complex seems to interact with the actin cytoskeleton (Korchev et al., 2000). However, the complexity of channel regulation would further suggest the sarcolemmal KATP channel to be composed of more proteins than those currently recognized.
In this regard, we have employed several strategies to determine whether the sarcolemmal KATP channel protein complex is composed of more proteins than Kir6.2, SUR2A, adenylate kinase and creatine kinase. We have found that the muscle form of lactate dehydrogenase (M‐LDH) is also an integral part of the sarcolemmal KATP channel, where it regulates channel activity. Moreover, the presence of M‐LDH in the channel complex seems to be required for early opening of KATP channels during ischemia and cellular resistance against metabolic stress.
Co‐immunoprecipitation of the cardiac membrane fraction
To immunoprecipitate proteins associated with the cardiac KATP channels, we used anti‐SUR2A antibody (see Ranki et al., 2001; Crawford et al., 2002). Immunoprecipitation revealed putative accessory polypeptides migrated at ∼36 (p36), ∼38 (p38), ∼46 (p46), ∼48 (p48), ∼98 (p98) and ∼150 kDa (p150) (Figure 1). The profile of immunoprecipitated peptides was different when anti‐PKC antibody [nPKCϵ(n‐17); Santa Cruz Biotechnology, Santa Cruz, CA] was used instead of anti‐SUR2A antibody (Figure 1), and anti‐SUR2A immunoprecipitation was blocked with an excess of the corresponding antigenic peptide (Figure 1).
LDH is a part of the sarcolemmal KATP channel complex
Previously, p38, p48 and p150 were identified as Kir6.2, creatine kinase and SUR2A respectively (Crawford et al., 2002). Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF) analysis identified p98, p46 and p36 as β‐myosin, α‐actin (data not shown) and LDH (Figure 2A), respectively. LDH activity was present in anti‐SUR2A immunoprecipitation pellets, whereas no activity was observed when the experimental protocol was applied without the antibody (Figure 2B). Western blotting with anti‐LDH antibody (Abcam, Cambridge, UK; this antibody targets all isoforms of LDH) of both anti‐Kir6.2 and anti‐SUR2A immunoprecipitates revealed signals for LDH (Figure 2C). Conversely, western blotting with anti‐Kir6.2 and anti‐SUR2A antibodies detected Kir6.2 and SUR2A subunits in anti‐LDH immunoprecipitate of cardiac membrane fraction (Figure 2D). Furthermore, double labelling immunofluorescence showed that both Kir6.2 and SUR2A co‐localize with a substantial portion of LDH in cardiomyocytes (Figure 3). This was observed in both rod‐shaped and rounded cells. In contrast, when cardiomyocytes were labelled with antibody against Kv 1.3, much less spatial overlap was noted (Figure 3). To provide even more evidence for physical association between LDH and sarcolemmal KATP channels we performed fluorescence resonance energy transfer (FRET) analysis using antibodies labelled with Alexa 594 (Molecular Probes, Eugene, OR) (anti‐LDH antibody, donor) and Alexa 647 (anti‐Kir6.2 antibody, acceptor) applied on cardiac membrane fraction. The emission spectrum for this sample is shown in Figure 4A, as are the donor and acceptor components. The same data are presented in Figure 4B, except that the magnitude of the extracted acceptor spectrum is calculated from the (ratio)A observed for a sample with only the anti‐Kir6.2 antibody. Comparison of the two panels shows that there is significant energy transfer between the fluorophores attached to the two antibodies. The (ratio)A value is 0.246, one‐third greater than the value of 0.179 determined for the acceptor‐only control. SDS (1%), a disrupter of protein–protein complexes, abolished the observed energy transfer [Figure 4C; (ratio)A decreased from 0.246 in the absence to 0.188 in the presence of SDS, which was not significantly different from the acceptor‐only control (0.192)]. When the same type of experiment was carried out with anti‐IgG antibody (Santa Cruz) in place of anti‐Kir6.2 antibody, no energy transfer was observed [Figure 4D; (ratio)A was 0.177 with acceptor alone and 0.165 with acceptor–donor]. In contrast, the energy transfer between anti‐Kir6.2 (donor) and anti‐SUR2A (acceptor) antibodies was detected [(ratio)A was 0.197 with acceptor alone and 0.213 with acceptor–donor].
M‐LDH, but not the heart form of LDH, is the isoform directly physically associated with the cardiac KATP channel subunits
LDH is a tetramer composed of either M (muscle) and/or H (heart) subunits, which may be combined to form five LDH isozymes (Van Hall, 2000). We have co‐expressed Kir6.2 and/or SUR2A with M‐ and H‐LDH subunits, and examined which of the isozymes associate with the channel subunits. When both H‐ and M‐LDH subunits were co‐expressed with Kir6.2, SUR2A and Kir6.2/SUR2A in A549 cells, cells natively devoid of KATP channels, LDH activity was found in anti‐Kir6.2 [antibody published in Crawford et al. (2002), and it was used for immunoprecipitation cells expressing Kir6.2 alone] and anti‐SUR2A (used for immunoprecipitation cells expressing Kir6.2 alone and Kir6.2/SUR2A) immunoprecipitates of cells expressing subunits both alone and combined (Figure 5A). However, when either H‐ or M‐LDH was co‐expressed with channel subunits, LDH activity was detected in immunoprecipitate from cells transfected with M‐LDH, but not in immunoprecipitate from cells transfected with H‐LDH (Figure 5B and C). Western blotting with the anti‐LDH antibody of anti‐Kir6.2 and anti‐SUR2A immunoprecipitates identified the presence of LDH in cells transfected with Kir6.2, SUR2A, Kir6.2/SUR2A and M‐LDH, but not in those transfected with H‐LDH (Figure 5D and E). To determine whether M‐LDH is the only LDH subunit that associates with the KATP channel subunits in vivo, we subjected anti‐SUR2A immunoprecipitate from cardiac membrane fraction to two‐dimensional (2D) gel analysis using an anti‐LDH antibody. 2D gel analysis revealed a signal corresponding to M‐LDH without any visible tracings of H subunits in immunoprecipitate (Figure 5F).
M‐LDH associates with Kir6.2 and SUR2A subunits via its N‐ and C‐termini, respectively
The results obtained with transfected A549 cells suggested that M‐LDH is associated with the KATP channel protein complex interacting with both Kir6.2 and SUR2A subunits. Based on known LDH structure and primary sequence differences between M‐ and H‐LDH subunits (see Discussion), we hypothesized that M‐LDH interacts with Kir6.2 and SUR2A peptides via terminal ends. To test this hypothesis, we made N‐ and C‐terminus‐truncated forms of M‐LDH (see Materials and methods). When an N‐terminus‐truncated form of M‐LDH was co‐expressed with the channel subunits in A549 cells, the activity was lost only in immunoprecipitate from cells expressing Kir6.2 subunit alone (Figure 6A). Conversely, a C‐terminus truncated form of M‐LDH was absent only in immunoprecipitate of cells expressing the SUR2A subunit alone (Figure 6A). The same pattern of LDH presence was observed when western blotting with anti‐LDH antibody was applied instead of measuring LDH activity (Figure 6B).
LDH substrates regulate sarcolemmal KATP channels activity
The defining attribute of KATP channels is their inhibition by intracellular ATP (Noma, 1983). Upon excision of a membrane patch from guinea pig ventricular cardiomyocyte, opening of KATP channels was inhibited by 1 mM of ATP. Addition of pyruvate plus NADH (20 mM each) on the intracellular face of the excised membrane patch opened KATP channels despite the presence of ATP (1 mM; Figure 7A). On the other hand, the opening of KATP channels with 20 mM of lactate, in the presence of ATP (1 mM), was inhibited by NAD (20 mM; Figure 7B). Furthermore, we measured membrane currents from a guinea pig ventricular myocyte in a whole‐cell configuration. When NADH (20 mM) or pyruvate (20 mM) was present in the patch pipette solution alone, the steady‐state current–voltage relationship (Figure 8A) was in an N shape due to the strong inward rectification of Ik1 channels and absence of active KATP channels (Terzic et al., 1995). However, when pyruvate (20 mM) was combined with NADH (20 mM), the outward K+ current became greater and inward rectification weaker (Figure 8A). The current density was 6.6 ± 0.8 and 7.5 ± 0.8 pA/pF in the presence of NADH and pyruvate alone, respectively, and 11.4 ± 1.2 pA/pF in the presence of NADH plus pyruvate (P = 0.01, n = 6–9; Figure 8A1). Lactate (20 mM)‐induced outward K+ current significantly increased at potentials more positive than −70 mV, and inward rectification of the current–voltage relationship was much weaker (Figure 8B), which is a typical finding when measuring K+ current flows through KATP channels (Terzic et al., 1995). When NAD (20 mM) was also present in the pipette solution with lactate (20 mM), the outward current was decreased and inward rectification was stronger (Figure 8B). The current density was 6.8 ± 1.0 and 18.1 ± 3.2 pA/pF in the presence of NAD and lactate alone, respectively, and 10.2 ± 1.8 pA/pF in the presence of NAD plus lactate (P = 0.019, n = 6–9; Figure 8B1).
Kir6.2/SUR2A/M‐LDH confer resistance to ischemia in A549 cells
At rest, A549 cells, which are natively devoid of KATP channels, have a low cytosolic Ca2+ concentration (30.2 ± 5.7 nM, n = 16; Figure 9A). Exposure to ischemia produced rapid and significant Ca2+ loading (208.5 ± 71.1 nM, n = 16, P = 0.03; Figure 9), which was associated with statistically non‐significant decrease in whole‐cell K+ current as measured by perforated patch–clamp electrophysiology (current density at +80 mV was 21.9 ± 1.9 pA/pF in control and 15.0 ± 2.6 pA/pF in ischemia, n = 6, P = 0.13; Figure 9). Transfection of Kir6.2/SUR2A/M‐LDH almost completely prevented ischemia‐induced Ca2+ loading (resting Ca2+ was 55.6 ± 9.0 nM prior to and 73.3 ± 10.8 nM following ischemia, P = 0.22, n = 9; Figure 9). In transfected cells, ischemia also induced significant increase in whole‐cell K+ membrane current as measured by perforated patch–clamp electrophysiology [in this mode intracellular milieu remains largely undisturbed (Jovanović et al., 2001); current density at +80 mV was 14.7 ± 2.0 pA/pF under control conditions and 28.2 ± 3.1 pA/pF following ischemia, P = 0.007, n = 5, Figure 9].
The opening of recombinant KATP channels as well as intact LDH activity is required for cell protection afforded by Kir6.2/SUR2A/M‐LDH
The resistance of Kir6.2/SUR2A/M‐LDH A549 phenotype to ischemia was significantly reduced when glybenclamide (10 μM), a KATP channel blocker, was present during the experiments (intracellular Ca2+ was 31.2 ± 5.3 nM prior to and 96.4 ± 31.6 nM following ischemia, P = 0.008, n = 9; Figure 10). In addition, glybenclamide (10 μM) abolished the ischemia‐induced increase in whole‐cell membrane K+ current (current density at +80 mV was 23.1 ± 2.9 pA/pF in control and 24.2 ± 3.2 pA/pF in ischemia, n = 5, P = 0.63; Figure 10). When Kir6.2/SUR2A were co‐transfected with the inactive, mutated form of M‐LDH (His193 was mutated into glycine, which greatly reduced the catalytic activity; Clarke et al., 1986) instead of wild‐type enzyme, the protection against ischemia was abolished (intracellular Ca2+ was 37.0 ± 6.5 nM prior to and 193.8 ± 62.7 nM following ischemia, P = 0.026, n = 6; Figure 10), as well as ischemia‐induced increase in whole‐cell membrane K+ current (current density at +80 mV was 20.1 ± 3.1 pA/pF in control and 19.3 ± 2.9 pA/pF in ischemia, n = 5, P = 0.32; Figure 10).
It has recently been suggested that sarcolemmal KATP channel complexes are composed of Kir6.2 and SUR2A subunits as well as two enzymes, regulators of ATP levels, adenylate kinase and creatine kinase (Inagaki et al., 1996; Carrasco et al., 2001; Crawford et al., 2002). In the present study, Coomassie Blue staining of the anti‐SUR2A immunoprecipitate revealed polypeptides that were previously identified as Kir6.2, SUR2A and creatine kinase (Crawford et al., 2002). The presence of adenylate kinase in immunoprecipitate was not visualized, which is plausible since the molecular weight of adenylate kinase would overlap with the primary antibody heavy chain under the conditions applied (Carrasco et al., 2001). MALDI‐TOF analysis identified p46 and p98 as α‐actin and β‐myosin, which is in accord with the cardiac KATP channel protein complex being physically associated with the cytoskeleton (Korchev et al., 2000).
Several lines of evidence, including MALDI‐TOF, LDH assay and western blotting, suggested that a previously unidentified 36 kDa protein found in the immunoprecipitate was LDH. Since immunoprecipitations could have non‐specific contaminants (Harlow and Lane, 1999), we used extreme care to classify LDH as a protein associated with the sarcolemmal KATP channel complex. The following results suggest that the LDH presence in immunoprecipitate is due to the physical association with KATP channel subunits: (i) the appearance of p36 was specific for the anti‐SUR2A antibody and was blocked by the antigenic peptide, suggesting that interaction between the anti‐SUR2A antibody and membrane fraction is required for the presence of LDH in the immunoprecipitate; (ii) there was no cross‐reactivity between the anti‐SUR2A antibody and purified LDH itself, which excludes the possibility that LDH was directly precipitated with the anti‐SUR2A antibody; (iii) LDH was detected in immunoprecipitates obtained with structurally unrelated antibodies against two members of the protein complex, Kir6.2 and SUR2A subunits; and (iv) not only was LDH found in both anti‐SUR2A and anti‐Kir6.2 immunoprecipitates but, vice versa, both SUR2A and Kir6.2 subunits were identified in anti‐LDH immunoprecipitate of cardiac membrane fraction. Immunofluorescence and FRET analysis were also employed to test the conclusions drawn with immunoprecipitation data. Immunofluorescence demonstrated co‐localization of Kir6.2 or SUR2A subunits with LDH in both rod‐shaped and rounded cardiomyocytes, i.e. in both healthy and dead cells, respectively (see S.Jovanović et al., 1998). In healthy cardiomyocytes, it could be argued that LDH might appear to be co‐localized with channel subunits due to ubiquitous intracellular presence of the enzyme. However, since dead cardiac cells release a major fraction of intracellular LDH (Altschuld et al., 1994), this argument does not stand, suggesting that the observed co‐localization is specific for LDH and the channel subunits. This conclusion is supported by finding that much less spatial overlap was observed between Kv 1.3, another K+ channel subunit expressed in the heart (England et al., 1995), and LDH. In addition, FRET analysis detected energy transfer between Kir 6.2 and LDH in cardiac membrane fraction suggesting that these two proteins are in close proximity (see Clegg, 1992). As the Ro for this donor–acceptor pair is 8.4 nm (see the manufacturer's web site: http://www.probes.com), the average distance between the fluorophores must be <17 nm, as neglible FRET is observed at distances greater than twice the Ro. Bearing in mind that this distance includes a sum of anti‐Kir6.2 antibody, Kir6.2 protein, LDH protein and anti‐LDH antibody lengths, it is very unlikely that in pure membrane fraction vigorously cleaned of cytosolic elements Kir6.2 and LDH protein could be so close without being physically associated. This conclusion is supported by the finding that treatment which disrupts protein complexes abolished the energy transfer between Kir6.2 and LDH, while no energy transfer was observed between LDH and IgG antibodies. Taken all together, it reasonable to conclude that LDH is an integral part of the sarcolemmal KATP channel protein complex in vivo.
LDH is a tetramer composed of M (muscle) and/or H (heart) subunits, which may be combined to form five LDH isozymes. In the heart, all LDH isozymes are present; LDH1 (H4) is the predominant form, whereas LDH5 (M4) is present in trace amounts (Van Hall, 2000). When H‐ and M‐LDH subunits in combination and alone were co‐expressed with Kir6.2, SUR2A and Kir6.2/SUR2A in A549 cells, the pattern of LDH presence in anti‐Kir6.2 and anti‐SUR2A immunoprecipitate suggested that M‐LDH, but not H‐LDH, is the isoform that associates with the channel subunits. Since M‐ and H‐LDH subunits are indistinguishable by size but have different isoelectric points (Li, 1990; Read et al., 2001; this study), we applied isoelectric focusing and 2D gel analysis to determine whether M‐LDH is the only LDH subunit that associates with the KATP channel subunits in vivo. This technique revealed a signal corresponding to M‐LDH without any visible tracings of H subunits in immunoprecipitate, confirming the data obtained with the heterologues expression system and suggesting that M‐LDH is indeed a part of the cardiac KATP channel complex in vivo. LDH is composed of 331 amino acids, with the active site localized approximately in the middle of the protein (Li, 1990; Van Hall, 2000). In order to be associated with both Kir6.2 and SUR2A subunits and still retain its activity, interacting parts of M‐LDH with channel subunits probably have to be localized on its terminal sides. In addition, differences in amino acid sequence between M‐LDH (associates with the channel) and H‐LDH (does not associate with the channel) are found primarily in the first 22 and the last 38 residues (Li, 1990), again suggesting that termini are involved in interaction between M‐LDH and Kir6.2/SUR2A. To test this hypothesis, we made N‐ and C‐terminus‐truncated forms of M‐LDH. When an N‐terminus‐truncated form of M‐LDH was co‐expressed with the channel subunits in A549 cells, LDH was lost only in immunoprecipitate from cells expressing Kir6.2 subunit alone, while a C‐terminus‐truncated form of M‐LDH was absent only in immunoprecipitate of cells expressing SUR2A subunit alone. These results suggest that M‐LDH associates with the Kir6.2 subunit via its N‐terminus and with the SUR2A via its C‐terminus. Although LDH is normally a tetramer (Li, 1990; Takatani et al., 2001), the catalytic function of LDH does not depend upon interaction between LDH subunits, and single subunits retain catalytic activity (King and Weber, 1986). This means that the complex of Kir6.2, SUR2A and M‐LDH subunits may be functional even if LDH subunits do not interact with each other. Also, since a single KATP channel is composed of four Kir6.2 and four SUR2A subunits (Shyng and Nichols, 1997), it is possible that four M‐LDH subunits associated with the channel subunits form a three‐dimensional structure that has a spatial relationship similar to those in the traditional enzyme tetramer without necessarily being physically associated.
It is generally accepted that M‐LDH primarily controls the formation of lactate (Van Hall, 2000). Lactate is reported to open sarcolemmal KATP channels in cells despite the presence of millimolar concentrations of ATP (Han et al., 1993). Since LDH catalyses NADH + pyruvate ↔ lactate + NAD reaction, this enzyme has the potential both to open (by lactate production) and to close (by lactate removal) KATP channels. The results obtained, at both single channel and whole‐cell levels, demonstrate that LDH substrates activate or inhibit KATP channels opening in a manner consistent with the hypothesis that LDH is a part of the sarcolemmal KATP channels. The regulation of KATP channel activity was observed despite the continuous intracellular presence of millimolar concentrations of ATP, suggesting that M‐LDH, by virtue of its activity, could open KATP channels at physiological concentrations of ATP. In the heart, KATP channels are opened during ischemia and act in a cardioprotective manner (Knopp et al., 1999; Light et al., 2001). Although Kir6.2/SUR2A reconstitutes basic properties of sarcolemmal KATP channels in cell lines natively devoid of these channels (Inagaki et al., 1996), recombinant KATP channels remain mostly closed throughout the metabolic challenge (Jovanović and Jovanović, 2001c). Yet, when recombinant channels are pharmacologically open host cells are protected against metabolic stress (A.Jovanović et al., 1998; Jovanović et al., 1999). Inability of Kir6.2/SUR2A proteins to create cellular phenotype resistant to metabolic stress may be due to incomplete reconstitution of the channel and impaired metabolic sensing. We tested the hypothesis that M‐LDH, as a part of the sarcolemmal KATP channel complex, may be important for co‐ordinated channel opening and cellular protection. A549 cells responded to ischemia with intracellular Ca2+ loading, which is a major indicator of the cell injury (A.Jovanović et al., 1998), showing that these cells are vulnerable to such an insult. When Kir6.2/SUR2A were co‐expressed with M‐LDH in A549 cells, ischemia‐induced Ca2+ increase was almost abolished and this was associated with significant activation of the outward K+ current. Both increase in K+ current and resistance towards ischemia was inhibited by glybenclamide, an antagonist of KATP channels, suggesting that the opening of recombinant KATP channels was responsible for the observed cellular resistance to ischemia. When Kir6.2/SUR2A was co‐transfected with the inactive, mutated form of M‐LDH, protection against ischemia was abolished, as well as ischemia‐induced increase in whole‐cell membrane K+ current, implying that the presence of M‐LDH with unimpaired activity is required in the cardiac KATP channel protein complex for early opening of KATP channels, clamping the membrane potential and maintaining the intracellular Ca2+ homeostasis.
M‐LDH as a part of the cardiac KATP channel could explain how the channel open during ischemia to protect the cells despite the presence of millimolar levels of ATP. Under anaerobic conditions, pyruvate, NAD and H+ cannot be metabolized further and accumulate (see Kantor et al., 2001), leading to the M‐LDH‐catalysed lactate production within microenvironment surrounding the channel. Lactate opens the channel despite high levels of ATP (Han et al., 1993; this study) and protects the cell against Ca2+ overload and death.
In conclusion, we have demonstrated that M‐LDH is an integral part of the sarcolemmal KATP channel protein complex in vivo, where it couples the metabolic status of the cell with the KATP channels activity which, in turn, is essential for cell protection against ischemia.
Materials and methods
Cardiac cells isolation
Ventricular cardiomyocytes were dissociated from guinea pig hearts as described previously (Ranki et al., 2001). In brief, hearts were retrogradely perfused (at 37°C) with medium 199, followed by Ca2+‐EGTA‐buffered low‐Ca2+ medium (pCa = 7), and finally low‐Ca2+ medium containing pronase E (8 mg/100 ml), proteinase K (1.7 mg/100 ml), bovine serum albumin (BSA; 0.1 g/100 ml, fraction V) and 200 μM CaCl2. Ventricles were cut into fragments in the low‐Ca2+ medium enriched with 200 μM CaCl2. Single cells were isolated by stirring the tissue (at 37°C) in a solution containing pronase E and proteinase K supplemented with collagenase (5 mg/10 ml).
A549 cells, genes, truncations, site‐directed mutagenesis and transfection
A549 cells (ATCC), which are natively devoid of KATP channels, were cultured in a tissue flask (at 5% CO2) containing Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum and 2 mM glutamine. Cells were transfected using 8–24 μl lipofectAMINE (Gibco), with 2–6 μg of total plasmid DNA [full‐length Kir6.2, SUR2A, M‐LDH, H‐LDH, ΔN‐LDH, ΔC‐LDH, gly193‐M‐LDH cDNA and green fluorescent protein (GFP), subcloned into the mammalian expression vector pcDNA3.1+]. Kir6.2 was a gift from Dr S.Seino (Chiba University, Chiba, Japan) and SUR2A was kindly provided by Dr Y.Kurachi (Osaka University, Osaka, Japan). H‐ and M‐LDH were cloned from mouse heart RNA. First strand cDNA was synthesized from 1 μg total RNA using 200 U of MML‐V reverse transcriptase (Promega) and an anchor‐oligo(dT) [5′‐GTCATGGCATGGGATCCTG(T)15; total volume, 20 μl], according to the manufacturer's instructions. The total PCR volume was 25 μl (50 μl for H‐LDH), including 3 μl of the cDNA, 25 pmol (20 pmol for H‐LDH) of each primer and 2.5 U PromegaTag (Promega). PCR was performed in a thermal cycler Model Phenix (Helena Biosciences) or a thermal cycler Mastercycler Gradient (Eppendorf) under the following conditions: the PCR was run with a hot start for 5 min at 95°C (initial melting), followed by 40 cycles of 0.5 min at 94°C, 0.5 min at 55°C and 1.5 min at 72°C (final extension). The primers had the following sequences. H‐LDH‐specific primers: sense, 5′‐GACAAGATGGCAACCCTTAAGGA‐3′; antisense, 5′‐AGTGCAAACATCAAACAAGCCTGG‐3′. M‐LDH‐specific primers: sense, 5′‐ATGGCAACCCTCAAGGACCAG‐3′; antisense, 5′‐GCACAGCTGTGGGGAGTG‐3′. The product obtained (full‐length cDNA), which contain translatable parts of H‐LDH or M‐LDH sequence, was isolated from a 1% agarose gel using Qiagen gel extraction kit, sequenced, ligated into the pcDNA3.1+ mammalian expression vector and used for transfection. Truncation of the first 19 N‐terminal amino acids of M‐LDH was achieved using nested PCR of M‐LDH cDNA and the following primers: sense: 5′‐GAGCAGGCTCCCCAGAACAAGATTACAGTTATG‐3′; antisense, 5′‐GCAGAGCTGTGGGGAGTG‐3′. The total PCR volume was 50 μl, including 3 μl of the cDNA, 20 pmol of each primer and 2.5 U PromegaTag (Promega). PCR was performed in a thermal cycler Mastercycler Gradient (Eppendorf) under the following conditions: the PCR was run with a hot start for 5 min at 94°C (initial melt), followed by for 35 cycles of 10 s at 94°C, 1.5 min at 56°C and 1.5 min at 71°C (final extension). Truncation of 48 C‐terminal amino acids was achieved by PCR using site‐directed mutagenesis to introduce a 3′ stop codon (TAG). The QuikChange site‐directed mutagenesis kit (Stratagene) was used according to the manufacturer's instructions. Mutagenic primers were sense: 5′‐TGATTAAGGGTCTCTATGGAATCAATGTAGATGTCTTCCTCAGTGTCCCATGTATCCTG‐3′; antisense, 5′‐CAGGATACATGGGACACTGAGGAAGACATCTACATTGATTCCATAGAGACCCTTAATCA‐3′. PCR conditions were the same as for N‐terminus truncation.
For site‐directed mutagenesis of His193, the same Stratagene kit as for C‐terminus truncation was used. Mutagenic primers were sense: 5′‐GCTGGGTCCTGGGAGAAGGTGGCGACTCC‐3′; antisense, 5′‐GGA GTCGCCACCTTCTCCCAGGACCCAGC‐3′. The total PCR volume was 50 μl, including 25 ng of non‐mutated M‐LDH inserted into the pcDNA3.1+, 125 ng of each primer, 1 μl of dNTP and 2.5 U DNA polymerase. PCR was performed in a thermal cycler Model Phenix (Helena Biosciences) under the following conditions: the PCR was run with a hot start for 0.5 min at 95°C (initial melting), followed by 16 cycles of 0.5 min at 95°C, 1 min at 55°C and 14 min at 68°C. The parental DNA was destroyed according to the manufacturer's instructions, and the mutated plasmids were further transformed, sequenced and used for experiments. All PCR products in experiments described above were verified by DNA sequencing.
Immunoprecipitation, Coomassie Blue staining and western blot analysis
Sheep anti‐peptide antibodies were raised against synthetic peptides conjugated to a carrier protein, keyhole limpet hemocyanin, and used for immunoprecipitation and western blotting (Crawford et al., 2002). Cardiac membrane fraction was obtained as described previously (Crawford et al., 2002). In brief, guinea pig ventricular tissue was homogenized in buffer I [Tris 10 mM, NaH2PO4 20 mM, EDTA 1 mM, phenylmethylsulfonyl fluoride (PMSF) 0.1 mM, pepstatin 10 μg/ml, leupeptin 10 μg/ml pH 7.8] and incubated for 20 min (at 4°C). The osmolarity was restored with KCl, NaCl and sucrose and the obtained mixture was centrifugated at 500 g. The supernatant was diluted in buffer II (imidazole 30 mM, KCl 120 mM, NaCl 30 mM, NaH2PO4 20 mM, sucrose 250 mM, pepstatin 10 μg/ml, leupeptin 10 μg/ml pH 6.8) and centrifugated at 7000 g, the pellet removed and the supernatant was centrifugated at 30 000 g. The obtained pellet contains membrane fraction. A549 cells or membrane fraction were snap‐frozen and ground to a powder under liquid nitrogen. The powder was resuspended in 10 ml of buffer (20 mM HEPES, 150 mM NaCl, 1% Triton X‐100 pH 7.5) and homogenized. Protein concentration was determined using the method of Bradford. Forty micrograms of the epitope‐specific anti‐SUR2A or anti‐Kir6.2 antibody was pre‐bound to protein G–Sepharose beads and used to immunoprecipitate from 60 μg of protein extract. The pellets of this precipitation were run on two SDS polyacrylamide gels, one for Coomassie Blue stain and one for western blot analysis as described in Crawford et al. (2002). In a separate series of experiments, immunoprecipitation was performed with the anti‐LDH antibody (Abcam) using the same protocol as described above. For the 2D gel analysis, immunoprecipitation was performed using 20 μg of SUR2A specific anti‐peptide antibody to precipitate from 20 μg guinea pig cardiac membrane fraction. The resultant precipitate was run on a Novex (Invitrogen) isoelectric focusing gel encompassing a pH range of 3–10, followed by excision of the lane and further protein separation on the second dimension according to molecular weight. Control samples were purified H‐LDH and H‐LDH from Sigma run using the same protocol (Crawford et al., 2002).
Immunofluorescence and laser confocal microscopy
Cardiomyocytes were fixed with 4% paraformaldehyde for 20 min. After washing with phosphate‐buffered saline (PBS), cells were permeabilized with 0.1% Triton X‐100 in PBS for 15 min, washed and blocked with 5% BSA in PBS for 30 min. Cells were incubated overnight at 4°C with primary antibodies (rabbit anti‐Kir6.2, rabbit anti‐SUR2A, goat anti‐LDH and rabbit anti‐Kv1.3) in PBS and incubated in the dark with the secondary antibodies [anti‐sheep fluorescein (Diagnostic Scotland, Edinburgh, UK) and anti‐rabbit rhodamine (Santa Cruz)] at 1:500 dilution for 1 h. Samples were washed with PBS, mounted on slides using 50% glycerol containing 1% n‐propylgallate and sealed with nail polisher (Marks & Spencer, Dundee, UK). Slides were examined by laser confocal microscopy (LSM‐510; Zeiss, Gottingem, Germany). Images were acquired and analysed using Zeiss Image Examiner Software.
Cardiac membrane fraction was obtained as described in the immunoprecipitation section. Anti‐Kir6.2, anti‐LDH, anti‐SUR2A or anti‐IgG antibodies were labelled with Alexa 594 (donor) or Alexa 647 (acceptor) where appropriate, according to the manufacturers instruction (Molecular Probes). Cardiac membrane fraction was blocked with 1% BSA, incubated with labelled antibodies for 12 h at 4°C, pelleted, and the pellets washed and resuspended with PBS. Fluorescence spectra were recorded at 20°C using an SLM‐Aminco 8100 fluorimeter, in PBS buffer except where otherwise specified. The excitation and emission band passes were 4 and 8 nm, respectively. Scattering of incident light was minimized by setting excitation and emission polarizers crossed at 90°. FRET was measured using the acceptor normalization method (Clegg, 1992), in which an extracted acceptor spectrum FA(ν1,ν′) (excitation at ν′ = 590 nm for Alexa 594, emission at ν1) is normalized to a second spectrum [F(ν1,ν″)] from the same sample excited at a wavelength (ν″ = 655 nm for Alexa 647) at which only the acceptor is excited, with emission at ν2. This gives the acceptor ratio (ratio)A = FA(ν1,ν′)/F(ν,ν″). FRET is considered to be observed if a sample has a (ratio)A value in excess of that recorded for a control sample having only the acceptor fluorophore. Fluorescence intensities were quantified by integrating emission spectra over the interval from 665 to 700 nm. The extracted acceptor spectrum was obtained by using regression to fit the spectrum of a donor‐only control sample to that part of the sample spectrum which has only donor fluorescence (600–620 nm). Subtracting the scaled donor spectrum from the sample spectrum was used to calculate the extracted acceptor spectrum.
MALDI‐TOF analysis
The band of interest was excised from the gel, and the protein trapped within the gel was reduced and alkylated and then digested with trypsin (12.5 ng/μl) overnight. The peptides were extracted, first, by the addition of 25 mM ammonium bicarbonate, followed after a 10 min incubation by an equal volume of acetonitrile, and then by two extractions with 5% formic acid and acetonitrile. The pooled extracts were concentrated to near dryness in vacuo and redissolved in 10% formic acid. Half of the sample was desalted and concentrated using a micro C18 column (0.2 μl ZapTip; Millipore) according to the manufacturer's instructions. The peptides were eluted directly from the tip onto the target in 1.5 μl α‐cyano‐4‐hydroxycinnamic acid (10 mg/ml) in 75% acetonitrile/25% formic acid (10%). Spectra were obtained on a Micromass TofSpec 2E instrument (Micromass, Manchester, UK) equiped with a 337 nm laser and operated in reflectron mode. Three hundred shots were combined and data were calibrated against a mixture of known peptides. Peptide masses were searched against the Swiss‐Prot/TREMBL databases using the ExPASy PeptIdent program and against the NCBI database using the MS‐Fit search program (USCF Mass Spectrometry Facility). The searches were limited to rodent proteins and included an appropriate mass range limit (Shevchenko et al., 1996).
LDH assay
LDH activity was determined in a reagent solution containg 0.2 M Tris–HCl (100 ml), 6.6 M NADH (1 ml), 30 mM sodium pyruvate (10 ml); pH was adjusted to 7.3 at 25°C. LDH activity was measured using a spectrophotometer (Unicam UV2 Spec) set at wavelength 340 nm on pellets (20 μl) dissolved in PBS (total volume was 100 μl) and put in reagent solution (Tris–HCl 1.4 ml, NADH 50 μl, sodium pyruvate 50 μl). Following 5 min incubation of the reagent solution in the spectrophotometer (to achieve temperature equilibration) pellets were added and the absorbance at 340 nm was measured every min until steady‐state is reached (steady‐state was defined as steady reading of absorbance in five consecutive measurements). The reaction velocity was determined by a decrease in absorbance at 340 nm, resulting from the oxidation of NADH indicative of LDH activity.
Patch–clamp electrophysiology
To monitor on‐line behaviour of single channel molecules, the gigaohm seal patch–clamp technique was applied in the inside‐out configuration (Jovanović and Jovanović, 2001b). Cells were superfused with (in mM): KCl 140, MgCl2 1, EGTA 5, HEPES–KOH 5 (pH 7.4). Fire‐polished pipettes, coated with Sylgard (resistance 5–7 MΩ), were filled with (in mM): KCl 140, CaCl2 1, MgCl2 1, HEPES–KOH 5 (pH 7.3). Recordings were made at room temperature (22°C), using a patch–clamp amplifier (Axopatch‐200B). Single‐channel activity was monitored on‐line and stored on a PC. Data were reproduced, low‐pass filtered at 1 KHz (−3 dB) and sampled at 100 μs rate for further analysis with the pClamp8 software. For whole‐cell electrophysiology applied on cardiomyocytes cells were superfused with Tyrode solution (in mM: NaCl 136.5, KCl 5.4, CaCl2 1.8, MgCl2 0.53, glucose 5.5, HEPES–NaOH 5.5; pH 7.4). Pipettes (resistance 3–5 MΩ), were filled with (in mM): KCl 140, MgCl2 1, ATP 3, HEPES–KOH 5 (pH 7.3). The whole‐cell K+ current in untransfected or transfected A549 cells (selection of the transfected cells was based on the GFP fluorescence) was measured using whole‐cell perforated patch–clamp technique with essentially the same pipette solution as above, except ATP was omitted and amphotericin B (240 μg/ml; Sigma) was added (see Jovanović et al., 2001). Also, during ischemia, Tyrode solution was modified as described below (see section Ischemia). For all cells monitored, the membrane potential was normally held at −40 mV and the currents evoked by a series of 400 ms depolarizing and hyperpolarizing current steps (−100 to +80 mV in 20 mV steps) recorded directly to hard disk using an Axopatch‐200B amplifier, Digidata‐1321 interface and pClamp8 software (Axon Instruments, Inc., Forster City, CA). The capacitance compensation was adjusted to null the additional whole‐cell capacitative current. The slow capacitance component measured by this procedure was used as an approximation of the cell surface area and allowed normalization of current amplitude (i.e. current density). Currents were low pass filtered at 2 kHz and sampled at 100‐μs intervals. When the effect of LDH substrates on whole‐cell K+ current was assessed either NADH (20 mM), pyruvate (20 mM), NADH plus pyruvate (20 mM each), NAD (20 mM), lactate (20 mM) or NAD plus pyruvate (20 mM each) was added into the pipette solution (Crawford et al., 2002).
Digital epifluorescent microscopy
A549 cells (transfected cells were selected based on GFP fluorescence) were superfused with Tyrode solution, and loaded with the esterified form of the Ca2+‐sensitive fluorescent probe Fura‐2 (Fura‐2AM, dissolved in dimethyl sulfoxide plus pluronic acid; Molecular Probes). Cells were imaged using a digital epifluorescence imaging system coupled to an inverted microscope (Image Solutions, Standish, UK). A mercury lamp served as a source of light to excite Fura‐2AM at 340 and 380 nm. Fluorescence emitted at 520 nm was captured, after crossing dichroic mirrors, by an intensified charge coupled device camera, and digitized using imaging software. An estimate of the cytosolic Ca2+ concentration, as a function of Fura‐2 fluorescence, was calculated according to the equation: [Ca2+] = (R − Rmin/Rmax − R)Kdβ, where R is the fluorescence ratio recorded from the cell, Rmin and Rmax the minimal and maximal fluorescence ratio, Kd the dissociation constant of the dye (236 nM), and β the ratio of minimum to maximum fluorescence at 380 nm (Ranki et al., 2001).
Untransfected and transfected A549 cells were exposed to ischemia combining hypoxia with modified Tyrode solution (ischemic solution). The ischemic solution (solution otherwise similar to Tyrode solution, contained slightly increased K+ concentration, 16 mM, pH was reduced to 6.5, and glucose was omitted) was continuously bubbled with 100% argon and the exchange of O2 between solution in the chamber and air was prevented by a nitrogen jet. The PO2 under these conditions was ∼20 mmHg. The duration of ischemic perfusion was 10 min (Ranki et al., 2001).
Statistical analysis
Data are presented as mean ± SEM, with n representing the number of patched/imaged cells or examined hearts. Mean values between two groups were compared by the paired or unpaired Student's t‐test or rank tests where appropriate. Mean values between more then two groups were compared by the one‐way or one‐way rank ANOVA. All statistical tests were carried out using using the SigmaStat program (Jandel Scientific). P <0.05 was considered statistically significant.
We thank S.Seino (Chiba University) for the Kir6.2 gene, Y.Kurachi (Osaka University) for the SUR2A gene. We are grateful to C.H.Booting (University of St Andrews) and D.M.J.Lilley (University of Dundee) for MALDI‐TOF and FRET analysis, respectively. This research was supported by grants from the Anonymous Trust, BBSRC, British Heart Foundation, MRC, National Heart Research Fund, TENOVUS and the Wellcome Trust.
- Copyright © 2002 European Molecular Biology Organization


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|MLB Career Stats
|San Diego
|25th in NL
|21st in NL
|9th in NL
|25th in NL
|19th in NL
|16th in NL
|25th in NL
|8th in NL
|5th in NL
|13th in NL
|24th in NL
|15th in NL
|24th in NL
|7th in NL
|4th in NL
|9th in NL
|16th in NL
|13th in NL
|16th in NL
|20th in NL
|6th in NL
|24th in NL
|4th in NL
|10th in NL
|4th in NL
|18th in NL
|9th in NL
|13th in NL
|December 7, 2012
|Salt Lake Bees released RHP Brian Lawrence.
|June 2, 2011
|RHP Brian Lawrence retired.
|April 15, 2011
|Los Angeles Angels signed free agent RHP Brian Lawrence to a minor league contract.
|November 6, 2010
|RHP Brian Lawrence elected free agency.
|April 6, 2010
|Brian Lawrence assigned to New Orleans Zephyrs from Florida Marlins.
|April 1, 2010
|Florida Marlins signed free agent RHP Brian Lawrence to a minor league contract.
|November 9, 2009
|RHP Brian Lawrence elected free agency.
|August 14, 2009
|New Orleans Zephyrs signed free agent RHP Brian Lawrence.
|August 1, 2009
|Portland Beavers released RHP Brian Lawrence.
|June 17, 2009
|Portland Beavers signed free agent RHP Brian Lawrence.
YOU COMPLETE ME: Tossed back to back complete games, allowing one run to the Florida Marlins on August 20 while shutting out the New York Mets on August 25 to become the first Padres pitcher to accomplish the feat since Andy Ashby in 1998... the complete games were the sixth and seventh of his career and the second and third by a Padres pitcher (Valdez; June 5 vs. Milwaukee) in 2004.
I KNOW IT'S SHORT NOTICE BUT...: Worked on three days rest for the first time in his career May 23 at Philadelphia... took the loss after allowing four runs on five hits over 5.0 innings... started again on three days rest September 19 at San Francisco and did not receive a decision despite allowing just two runs (one earned) on four hits over 7.0 innings... four days later made his third and final start on three days rest and took the loss after allowing five runs (four earned) on six hits over 4.0 innings September 23 vs. Los Angeles.
FOR OPENERS: Was the club's Opening Day starter for the second consecutive season... joined Clay Kirby (1972-73), Randy Jones (1975-77), Gaylord Perry (1978-79), Eric Show (1986-87) and Andy Benes (1993-95) as Padres pitchers to have started in consecutive Opening Days.
DOING THE SPLITS: Went 10-5 with a 3.86 ERA (46 ER/107.1 IP) in 18 road starts compared to 5-9 with a 4.42 ERA (47 ER/95.2 IP) in 16 starts at PETCO Park... despite having an ERA over 1.00 run higher, tallied 13 of his 15 wins at night when his ERA was 4.47 compared to a 2-7 mark and a 3.41 (25 ER/66.0 IP) in day games.2003 HIGHLIGHTS: Finished second on the Padres with 10 wins, his second consecutive season with double-digit victories... overall, went 10-15 with a 4.19 ERA (98 ER/210.2 IP)...was more successful at home where he went 6-8 with a 3.18 ERA (45 ER/127.1 IP) than on the road where his ERA was 5.72 (53 ER/83.1 IP)...started off slowly posting a 5-10 mark and 4.45 ERA (64 ER/129.1 IP) during the first half, but regained his 2002 form after the All-Star break to go 5-5 with a 3.76 ERA (34 ER/81.1 IP)...allowed three runs or fewer in 22 of his 33 starts including eight of his final nine outings...boasted a 2.53 ERA (9 ER/32.0 IP) in August and a 2.78 ERA in September (10 ER/32.1 IP). A HORSE IS A HORSE, OF COURSE, OF COURSE: After tossing a career-high 210.2 innings, became the first Padres hurler to surpass 200 innings in consecutive seasons since Andy Ashby in 1998 and 1999. START ME UP: Was the Padres Opening Day starter, drawing the assignment for the first time in his career... did not receive a decision after allowing two earned runs on three hits over 7.0 IP in a 5-2 loss to the San Francisco Giants on March 31. BATMAN: Batted .224 with one home run and five RBI...his home run, the first of his career, was a solo shot off Colorado starter Darren Oliver in the fourth inning of the Padres 6-4 win April 11...the homer came on an 0-2 pitch and was the 49th home run by a Padres hurler...Lawrence was the first Padres pitcher to take one deep since Dennis Tankersley on May 26, 2002 at Milwaukee...additionally he was the first Padres pitcher to hit a home run at Qualcomm Stadium since Joey Hamilton on May 22, 1997 and his shot was the 23rd by a Padres pitcher at the Q...Lawrence became the 16th different Padres pitcher to hit a home run at Qualcomm. DOWN AND DIRTY: After leading the league in 2002, finished third among National League hurlers with 346 groundball outs...trailed only Atlanta's Greg Maddux (375) and Los Angeles' Kevin Brown (374)...also ranked sixth among NL league leaders with 23 double-plays induced. registered an ERA of 3.86 in those six starts. of balls put in play were ground balls. one appearance against the Giants. August 2 vs. Philadelphia. 2002 In his first full season, was the Padres' most dependable and consistent starter...led the team in wins (12), shutouts (2), innings (210.0) and strikeouts...averaged a team-high 6.2 in nings per start...established career highs in vir tu al ly every major category...was 11-6 with a 3.25 ERA after posting a 6-2 win on Au gust 14 vs. New York (NL), but went 1-6 with a 5.87 ERA (25 ER/38.1 IP) there after.
AMONG THE BEST: Ranked among National League leaders in shutouts (tied for 4th with 2), fewest walks per 9 innings pitched (tied for 6th at 2.2), hits allowed (4th with 230) and hit bats men (6th with 11).
BETTER THAN YOU THINK: Finished 12-12, but pitched much better...allowed 2 earned runs or fewer in 16 of 31 starting as signments, including one or none 9 times...allowed 3 runs or less in 5 of 8 no-decisions...suffered from a lack of offensive support, as the Friars plated one run or less in 6 of Brian's 31 starts...registered an ERA of 3.86 in those 6 starts.
WELL GROUNDED: Led the National League with 386 ground-ball outs...tied with Livan Hernandez for the NL lead with 29 GIDPs induced...only Tim Hudson (35) and Jon Garland (32) in the AL had more in all of baseball...among NL pitchers, ranked behind only Mike Hampton (1.41) by recording 1.24 GIDPs per 9 innings pitched...led the NL with a 365/148 ground ball-to-fl y ball ratio (or 2.5/1)...also led the NL as 71.5 percent of balls put in play were ground balls.
THE OLD WEST: Pitched well against division foes, going 6-4 with a 3.28 ERA (30 ER/82.1 IP) against Los Angeles, Colorado, San Francisco and Arizona...strangely, faced the Dodgers 7 times (4 starts), but only made one appearance against the Giants.
PERFECT 10: Recorded his 10th victory in a 5-2 win, August 2 vs. Cincinnati...reached the 10-win plateau quicker than any Padre since 2000, when Matt Clement evened his 2000 record at 10-10 by earning the win on August 2 vs. Philadelphia.
THAT'S BETTER: After going 0-4 at home last season, improved to 6-5 with a 3.57 ERA within the concrete confi nes of Qualcomm Stadium in 2002...posted his fi rst career win at The Q in an 8-0 com plete-game shutout April 8 vs. Arizona.
INNINGS EATER: On September 13 vs. Los Angeles, became the first Padres to eclipse the 200-inning barrier since Matt Clement worked 205.0 innings as a rookie in 1999...despite not taking his final 2 turns in the rotation, missed rank ing among the National League's top 10 in in nings pitched by just 6.0 innings.
FINISH LINE: Tossed 2 complete games: April 8 vs. Arizona and July 17 at Los Angeles...now has 3 career complete games...has one at each of Southern California's 3 big league ballparks (Qualcomm Stadium, Dodger Stadium and Edison International Field).
DON'T SEE THAT TOO OFTEN: Fanned the side on only 9 pitches in the 3rd inning on June 12 at Baltimore...set down Brook Fordyce, Jerry Hairston and Melvin Mora...Brian's feat was the 36th confi rmed 9-pitch, 3-strikeout inning in the his to ry of the game, the 4th of 2002.
OTHER NOTES: Made his 31st and fi nal start of the year on September 13 vs. San Francisco...would have made a team-high 33 starts, but pitched out of the bullpen the fi nal 14 games of the year as a precaution...went 0-1 with a 5.40 ERA in 4 games out of the bullpen (3 ER/5.0 IP)...recorded his first career hold in an 8-4 win September 20 vs. Los Angeles...fanned a ca reer-best 10 twice: June 1 vs. Milwaukee and August 2 vs. Cincinnati...opposing batters hit just .209 with runners in scoring position and 2 outs...gave up only one home run in the first inning all season (Derrek Lee, August 24 vs. Florida)...if the count reached 2 strikes, opposing batters hit just .174...went 6-for-63 (.095) with 2 doubles and 6 RBI at the plate...ended the sea son in a 1-for-39 swoon.2001 SUMMARY: In his major league debut season, finished 5-5 with a 3.45 ERA in 27 games (15 starts) for the Padres...enjoyed 4 separate stints with San Diego, joining the Padres for good on July 31...ranked No. 15 among Baseball America's Top 20 Rookies of 2001...made 12 of his first 14 appearances out of the bullpen before making 13 consecutive starts to close out the season...went 5-5 with a 3.23 ERA (35 ER/97.2 IP) in his 15 starts and 0-0 with a 4.76 ERA (9 ER/17.0 IP) in relief...opponents hit .240 (89-for-371) in his starts and .269 (18-for-67) in his relief outings...the Padres went 6-9 in his 15 starts, 10 of which were quality starts...averaged nearly 6.2 innings per start...upon joining the rotation for good on July 15, Lawrence went 4-5 with a 3.13 ERA (29 ER/83.1 IP) over his final 13 games. UP AND DOWN: Opened the season at Triple-A Portland but was recalled for the first time in his career April 11...optioned back to Triple-A on May 8 after posting no record and a 6.23 ERA (6 ER/8.2 IP) in 6 relief appearances...recalled for the 2nd time on June 6 and made 8 appearances, including his first 2 starts for San Diego, going 1-0 with a 3.57 ERA (9 ER/22.2 IP)...optioned to Portland July 4 but was recalled July 15 to start that night at Anaheim...threw a complete game to beat the Angels 5-1 before again being optioned to Portland the following day...returned for good on July 31, and went 3-5 with a 3.39 ERA (28 ER/74.1 IP) in 12 starts the rest of the way. FIRST TIME FOR EVERYTHING: Made his major league debut April 15 vs. Los Angeles, working 1.2 innings of no-hit, no-run relief...hit one and fanned one...came on in a 4-4 game with the bases loaded and one out in the 6th inning...after Gary Sheffield, the first hitter he faced, hit a sacrifice fly to center, Lawrence fanned Shawn Green to extinguish the threat...set down the side in the 7th with only one man reaching...he hit Paul Lo Duca with 2 outs...earned his first major league win in his first start (after 11 relief appearances), beating the Dodgers 6-2 June 23 in Los Angeles...allowed 2 runs on 4 hits over 7.1 innings, walking one and striking out 7...collected his first career hit and RBI in the game, lacing a 2-out RBI double off Luke Prokopec in the 4th inning to give the Padres a 3-1 lead. GOING THE DISTANCE: Threw his first career complete game in a 5-1 win July 15 at Anaheim...recalled from Triple-A Portland to make the one start, he limited the Angels to a run on 7 hits, walking none and fanning 4 in the 104-pitch gem...returned to Triple-A following the start. MORE GEMS: Beat the Pirates 6-2 August 11 at PNC Park, allowing 2 runs (one earned) on 6 hits over 8.0 innings...walked 2 and fanned 6...August 28 at St. Louis came within one out of his 2nd career complete game in a 5-2 win over Darryl Kile...left with a 5-1 lead and 2 outs in the 9th...was charged with 2 earned runs on 4 hits over 8.2, walking one and striking out 6...in his first start at Coors Field, earned a 3-1 win September 26...limited the Rockies to a first-inning run on 6 hits over 6.0 innings...matched Arizona's Randy Johnson pitch-for-pitch September 2 at The Q, working 8.0 innings of 6-hit shutout ball...walked 4 and fanned 6...neither starter figured in the decision as the Padres prevailed on a leadoff homer by Ryan Klesko off Byung-Hyun Kim in the bottom of the 13th. KING OF THE ROAD: Was 5-1 with a 2.78 ERA (17 ER/55.0 IP) in 11 road games (7 starts) and 0-4 with a 4.07 ERA (27 ER/59.2 IP) in 16 home games (8 starts)...opponents hit .215 (44-for-205) with 4 homers on the road and .270 (63-for-233) with 6 homers at The Q. INSIDE THE NUMBERS: Worked at least 7.0 innings in 7 of his 15 starts, at least 8.0 innings 4 times...gave up 2 or fewer earned runs in 11 of his starts...the Padres managed a total of 10 runs in his 5 losses, including a pair of shutouts...walked 2 or fewer in 12 of his starts, issuing 3 bases on balls once and 4 twice...opponents hit .244 (107-for-438) with 10 homers...lefties batted .281 (55-for-196) with 9 long balls, while righthanders managed only a .215 average (52-for-242) and one homer...opposing base stealers were 7-for-12...posted a 4.47 ERA (29 ER/58.1 IP) in games against N.L. West rivals and a 2.40 ERA (15 ER/56.1 IP) against all other clubs. AT THE PLATE: Batted .115 (3-for-26) with 2 doubles, 3 RBI, 2 sacrifice bunts, a sac fly and 7 strikeouts...delivered an RBI double off Luke Prokopec June 23 at Los Angeles, doubled off Prokopec again June 28 vs. Los Angeles and singled a run home off the Braves' Kevin Millwood August 23 in Atlanta...hit a sacrifice fly off Colorado's John Thomson October 7 at The Q. 2001 AT PORTLAND: Went 1-3 with a 3.80 ERA in 9 games (8 starts) for Triple-A Portland...fanned 42 in 45.0 innings...allowed 2 earned runs or fewer in 5 of his 8 starts...walked 2 or fewer in 6 of 8 starts, issuing a season-high 3 walks only twice...allowed more than 6 hits only once and 3 or fewer 3 times...earned the first and only save of his professional career July 8 vs. Edmonton, working 3.0 innings of 3-hit shutout ball...walked one and fanned 4...opponents hit .239 (42-for-176) with 3 home runs...lefties hit just .188 (16-for-85) with no home runs...fanned a season-high 10 in 8.0 innings July 21 at Nashville. 2000 SUMMARY: Shared Padres Minor League Pitcher of the Year honors with Jacob Peavy on the strength of an 11-6 record and a 2.29 ERA in a career-best 29 starts for Double-A Mobile and Triple-A Las Vegas...pitching above Single-A for the first time, he led the organization in ERA and strikeouts (165) for the 2nd straight year...ranked 3rd among Padres minor leaguers in wins, trailing only Peavy (13) and Mike Bynum (12)...fanned 4.7 hitters for every walk he allowed, striking out 165 and walking 35 in 173.1 innings...averaged 8.6 strikeouts and 1.8 walks per 9 innings...did not walk more than 3 in a start all season...allowed only 147 hits, an average of 7.6 per 9 innings...Southern League and Pacific Coast League opponents hit just .230 (147-for-639) with 12 homers, an average of one long ball every 14.4 innings...threw only one wild pitch in his 29 starts. AT MOBILE: Went 7-6 with a 2.42 ERA in 21 starts for the BayBears...in 126.2 innings, allowed only 99 hits and a .217 opponents average...walked28 and fanned 119...14 of his 21 starts were quality starts...surrendered 3 earned runs or fewer in 19 of 21 starts, limiting the opposition to 2 earned runs 5 times, to one earned run 6 times and to no earned runs 5 times...worked at least 6.0 innings in 14 of 21 starts, at least 7.0 innings in 7...threw a season-high 8.0 innings of 3-hit, 10-strikeout ball June 28 vs. Greenville...never walked more than 3, and walked none or one in 11 of his starts...was 1-4 in his first 8 starts despite a 3.06 ERA (16 ER/47.0 IP)...went 6-2 with a 2.03 ERA (18 ER/79.2 IP) in his next 13 outings before being promoted to Las Vegas...in his last 5 Mobile starts, went 2-0 with a 1.09 ERA (4 ER/33.0 IP)...allowed just one unearned run over 3 starts (20.0 IP) July 3-15. AT LAS VEGAS: Promoted to Triple-A on July 30, Lawrence went 4-0 with a 1.93 ERA in 8 starts to close out the season...allowed one or no earned runs in 6 of the 8 starts...worked 6.0 innings of 2-hit shutout ball in his July 30 Triple-A debut at Tacoma, and was 1-0 with a 0.51 ERA (1 ER/17.2 IP) in his first 3 starts for the Stars...after allowing 4 runs over 6.0 innings in a win at Iowa in his next start on August 14, surrendered only 2 earned runs in his next 3 starts (17.0 IP) for a 1.06 ERA. AT THE PLATE: Batted .205 (8-for-39) with 3 RBI overall...hit .241 (7-for-29) with 2 doubles and an RBI at Mobile and .100 (1-for-10) with a double and 2 RBI at Las Vegas...struck out only 6 times in 39 total at-bats. 1999 Went 12-8 with a 3.39 ERA in 27 starts for Single-A Rancho Cucamonga...led the organization in ERA and with 166 strikeouts, while ranking 2nd in wins...led the California League by issuing only 1.48 walks per 9 innings...ranked 2nd in the league in shutouts (3), strikeouts and innings pitched (175.1), and tied for 3rd in complete games (4)...also placed 4th in ERA and tied for 4th in victories and starts...collected a season-high 10 strikeouts over 7.0 innings in a 3-0 victory May 3 at San Bernardino...faced only 28 batters en route to a 3-hit, 1-0 win May 24 at Visalia...allowed no walks and struck out a season-high 11, retiring 14 consecutive hitters at one point...on June 27, faced only one batter over the minimum in a 2-hit, 2-0 win against Lake Elsinore...struck out 8 and walked none to pick up Cal League Pitcher of the Week honors for June 21-27...carried a perfect game through 5.0 innings but allowed a leadoff single in the 6th...won 5 consecutive starts, posting a 2.43 ERA (9 ER/33.1 IP) June 9-July 2...registered his 2nd 10-strikeout outing in a 10-3 triumph over Lake Elsinore on July 28...beat Stockton 3-0 on August 25 for his 3rd shutout of the season...got a no-decision in his lone playoff start vs. San Bernardino...allowed only 2 runs on 9 hits and 2 walks while striking out 6 in 7.1 innings. 1998 Went 8-3 with a 2.73 ERA in 16 starts for Single-A Idaho Falls and Clinton...his 2.73 ERA was 3rd-best among Padres minor leaguers...registered a 5.6/1 strikeout-to-walk ratio (100 K/18 BB)...tied for the Pioneer League lead with one shutout, despite making only 4 starts for the Braves...also tied for 4th among PL hurlers with 2 complete games...fanned a then-career-high 12 July 11 at West Michigan in his first Clinton start...established a new career high by collecting 13 K's in a 4-1 win August 5 vs. Burlington...pitched a one-hit, complete game to top Michigan 2-1 August 15 in Clinton...walked 3 while allowing his only hit to the 2nd batter he faced in the first inning...it was the 6th straight start in which he allowed 2 earned runs or fewer.


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Why Can't Detroit Have a RoboCop Statue?
They have the money.
Remember the campaign to raise money for a RoboCop statue in Detroit? It worked: $25,000 was brought in via Kickstarter and another $25,000 was chipped in by a private source, which is enough to fund the project. The problem is, Detroit mayor Dave Bing doesn't think it's a good idea: "My own personal opinion is that I don't see where we get a lot of value from that." Organizer Brandon Walley argues that it would draw tourism, like Philly's Rocky Balboa and Milwaukee's Fonzie statues. Also, Peter Weller, the guy who played RoboCop, thinks it's a good idea. And so does Vulture. Come on, Mayor Bing, make it happen! [AP]


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The same man who led the South African cricket team around Switzerland for four days prior to their arrival in England has linked up with the side again ahead of the Test at Lord's, but other than that, it's all systems go as usual for the Proteas. By ANT SIMS.
South Africa might insist that they're not preparing any differently for the third and final Test against England at Lord's, but they did something a little bit extra on Tuesday. Explorer Mike Horn – the same man who led them on their adventures through Switzerland before the start of the tour – joined the team preparations and he will be with the squad for the next three days.
While South Africa's performance at Headingley wasn't quite as convincing as the whipping they handed England at The Oval, Horn linking up with the squad is by no means unplanned or an attempt to recreate the magic from the first Test.
"We always planned on getting him here after our excursion in Switzerland," coach Gary Kirsten said. "He is an inspiration for us, he played a big role in our preparations before the tour and he will no doubt add value to our current team environment."
Horn is no replacement for any sort of technical cricket know-how, however, and the explorer says he is simply with the side to help them on a mental level, to get over a stumbling block they've so often failed to conquer in crunch situations.
"I can't come here and teach the players how to bat or to bowl," said Horn. "I am here to bring something away from the game. They have proven that they are a better team than before. There is a good ambience in the camp and the players are looking forward to performing better as a team. I'm not going to say much, I think my presence will say a lot."
The mental challenge will be a tough test for South Africa, but when it comes to preparing for play, Jacques Kallis says nothing has changed.
"Our focus has been preparing for this Test as if it's just any other Test and we want to put in a big performance. To win a Test series in England is a big deal, it doesn't happen often and not many teams have come here and won," said Kallis.
Kallis is being coy. Winning in England does happen – if you're South Africa. The Proteas win here in 2008 was monumental and they are now on the verge of replicating that feat. There are a few members of the side who tasted that victory. Graeme Smith, Hashim Amla, Kallis, AB de Villiers, Morne Morkel and Dale Steyn are all experienced campaigners who have been through the many highs and lows the South African team has experienced in the last few years and Kallis believes that the side is reaching a maturity it hasn't had before.
"I think our current side is shaping up to be different to any other South African side we've played in. the guys are maturing and we're learning a lot from the way we're doing things," said the all-rounder.
Kallis credits Kirsten for his influence on the side and says the coach has helped the team come together as a unit, helping them morph into the superpower they are slowly becoming.
"We want to play good, aggressive and positive cricket – that's what we're working towards as a team. Kirsten has been a catalyst for how the team has changed and he's very process driven, he's helped us focus on what we can do better as a team," Kallis said. "I think every player in the team knows what their role is and that makes a huge difference to how you approach the game."
South Africa haven't lost an away series since Sri Lanka in 2006 and that record will continue to stand, no matter what the result at Lord's. However, England have been far below their touted best, especially in the first Test where they managed to take just two wickets in the entire match. They came back much stronger in the second Test and English seamer Tim Bresnan reckons there's no need for panic stations just yet.
"I do think our bowling has been slightly below par this series, but I also think that we bowled alright at the Oval, there wasn't much in the pitch. We did much better at Headingley and we bowled well without a great deal of luck. I don't think we need to go another plan just yet," Bresnan said.
With Kevin Pietersen having been dropped, Jonny Bairstow has been recalled to replace him and Bresnan has backed the Yorkshireman to do well, despite his struggles against the West Indies earlier in the summer where the youngster managed just 38 runs in three Tests.
"It's a chance for Bairstow to show the kind of talent he is and personally I believe he can get runs at this level, he just needs a bit confidence. When I've seen him play in county cricket, he just smashes it into the stands and if that's his natural game, then that's what he needs to do," said Bresnan.
Pietersen has had a tremendous series, having scored more runs than any other English player on the tour and has the highest average of all the players in the side. England will have to make do without him, though, and Bresnan reckons the hosts will cope just find without their talisman.
"I think we have just as much chance of winning this Test as we would have had if Kevin Pietersen was playing. We have a squad so deep in talent that it doesn't matter who plays, if we play our best cricket on any given day, we can beat anyone," Bresnan said. DM
Photo: South Africa's captain Graeme Smith (3rd R) passes a rugby ball as his teammates look on during a training session before Thursday's third cricket test match against England at Lord's cricket ground in London August 14, 2012. REUTERS/Philip Brown
The filming of The Beach permanently damaged the ecosystem on the Thai island it was located on.


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<< Back to "Countries, regions, transboundary river basins" page
Almost two-thirds of the countries in the world have rivers flowing into their territories from upstream countries. In an effort to visualize the surface water flowing from one country to the other (note: this does not include flows to the ocean), AQUASTAT has prepared two different chart types:
For both chart types, line thickness represents flow, therefore thicker lines represent greater flow. For the network charts, flows have been divided by 10 for increased readability. Flows are in km3/year, and the scale for each continent is different and automatically adjusted.
Notes on sankey plots: negative flows have been removed. This means that if 10 km3/year flow from country A to B, and 3 km3/year flow from country B to A, then only 7 km3/year are shown flowing from A to B. The other flows that were omitted because they caused cyclicity are:
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Moleskine hack success!
Constantly hunting for my pen whenever I needed to jot something down in my pocket Moleskine was getting a bit old. Since the Moleskine hack community is no small potatoes, I figured someone would have figured out a way to attach the pen to the notebook that required ZERO points in the effort department.
Didn't take long to find one. Duct tape. Pure genius.
Doesn't look half bad, either.
And before anyone asks, yes, that is a knot in the elastic of my Moleskine. It decided to stretch like a lanky contortionist. I wasn't too happy (it's not like I found the notebook in the discount aisle at K-mart), but hell will freeze over before I go to any effort to replace the elastic. The knot does just fine.


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This cemetery is referred to in the book "Vital Records of Bridgewater, Massachusetts to 1850", Published by the New England Historic Genealogical Society, as G.R.7.
This cemetery is referred to in the book "Old Cemeteries of Southeastern Massachusetts", by Charles Thatcher.
This cemetery is referred to in the book "Epitaphs in Old Bridgewater, Massachusetts", by Williams Latham, as "Old Grave-Yard in Bridgewater".
Located near the original Unitarian Church, the land for this cemetery was deeded to the community by John Washburn in 1719. His wife, Rebecca Washburn, was the first person buried in it. The last known burial in the cemetery was in 1867, Lucinda Carver. This cemetery is also the burial location for Cuff Ashport, former slave and Revolutionary War soldier.
When these towns were established, there were three separate and distinct towns in the area known as "Old Bridgewater". Each one has a cemetery titled "Old Graveyard", but, were established in different years - Bridgewater's in 1716, East Bridgewater's in 1685 and West Bridgewater's in 1683.
Sources for this information include: History of Bridgewater by Nahum Mitchell, (pub. 1840, 1897, 1970, 1975); Williams Latham, Epitaphs in Old Bridgewater, Massachusetts. Illustrated with Plans and Views (Bridgewater: Henry T. Pratt, printer, 1882; repr. Bowie, MD: Heritage Books, 1976; Middleborough, MA, 1986). This book was later transcribed by Arthur Richardson and Dale H. Cook. Also, from the Vital records of Bridgewater, MA (to the end of 1850), Volume II, page 6, abbreviations, C.R.1 represents information for this book gleaned from church records of the Unitarian Church.
GPS Coordinates: 41.98887, -70.97286
- Added: 26 Sep 2007
- Find A Grave Cemetery: #2233339
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Dunn Coffee Table with Hourglass Table/Stool
Why this room works
- We love how seamlessly the Dunn coffee table integrates into this space. Dunn has rounded edges so you can position seating anywhere in this room. The glass top is a perfect match, allowing you to see the artfully crafted wood base.
- The Saarinen end table sits evenly with the arm of the chair. Made from white marble, it brightens up the room and is a great companion for the chair, offering easy access to books, beverages and more.
- The Reclaimed Wood stool shows off beautiful, natural wood that echoes the warm wood of the Dunn table. These woods mixed with the light, bright marble of the Saarinen table work together to create an inviting room.
Dunn Coffee Table
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Saarinen End Table
Profile Frames in Black
$59 - $99
Herringbone Pillows
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Case Study Planters with Walnut Base
$149 - $189


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MADISON, Wis. -- The No. 2-ranked University of Michigan softball team used a pair of big innings, including a seven-run fourth, to cruise past Wisconsin, 9-1, in five innings, on Saturday afternoon (April 30) at the Goodman Softball Complex. After missing last weekend with injury, senior RHP Jordan Taylor (Valencia, Calif./Valencia HS) was stellar in her return to the circle, striking out 10 while allowing three hits -- two in the fourth -- and a walk through five innings. Taylor fanned the side in the second and fifth innings en route to her eighth double-digit strikeout performance of the season. She improved to 22-3 on the season. RECAP
Penn State 8, Purdue 5
UNIVERSITY PARK, Pa. - The Nittany Lion softball team overcame a three-run deficit on Saturday at Beard Field to defeat Purdue, 8-5. Penn State used a three-run third inning and a five-run fourth inning rally to cement the victory. Kailyn Johnson (Yorba Linda, Calif.) went 2-for-3 with two RBI and a run scored to lead the Nittany Lions. RECAP
Minnesota 6, Michigan State 3
MINNEAPOLIS - The Minnesota softball team tallied its 28th win of the season on Saturday afternoon, taking down Michigan State 6-3 to lock up a winning record for the 2011 season. After morning showers delayed the start of Saturday's series opener by over two hours, freshman pitcher Sara Moulton picked up her 25th win of the season, moving the freshman into a tie with Angie Recknor (2000) and Steph Klaviter (1996) for second place on the Minnesota single season list. Moulton needs two wins to tie Vicki Swanson (1978) for the school's single season record. RECAP
Indiana 3, Iowa 1
BLOOMINGTON, Ind. - The Indiana Hoosiers (33-15, 13-2 Big Ten) continued their win streak, pushing it to 13 straight games with a 3-1 victory over the Iowa Hawkeyes (26-20, 8-7 Big Ten) at the IU Softball Field. Indiana has now won 14 of 15 at home this season and have won a school-record 12 consecutive Big Ten games. Morgan Melloh (28-14) went the distance in the circle for the Hoosiers, her seventh straight complete game victory. She allowed just two hits in the game and one run while walking three and striking out nine. She improves her Indiana single-season record for strikeouts to 427 in the process and now has 1565 in her career to remain in ninth on the NCAA career list. RECAP
Illinois 5, Northwestern 4
URBANA, Ill. - The University of Illinois Softball squad grabbed its first walk-off home run of the season on Saturday as the Illini earned a 5-4 victory over Northwestern. Down, 4-3, entering the bottom of the seventh, Hollie Pinchback (katy, Texas/Cinco Ranch) drove the first pitch of her at bat over the center field fence with Danielle Zymkowitz (West Hills, Calif./Chaminde) on first base to end the contest. Pinchback also hit a solo shot as part of a three-run third frame for Illinois. RECAP


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This pretty rectangular dresser or vanity box has a lovely glass base and a silverplated lid in an Art Nouveau floral repousse pattern. The lid could use some polishing, and has some areas were the silver has worn off. The glass base is in perfect condition. The piece measures 4" x 2" x 1.5". It would be a sweet addition to your vanity collection. It dates to around the 1920's -1930's.
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It's On Us: Free Shipping. Free Returns.
Product 1/19
General describtion:RELLECIGA Lace Bikini Collection - Lovely lace on the triangle top creates a naturally feminine silhouette. With irresistible Brazilian cut, the bottom trimmed in lace also adds an oh-so-sexy touch. Why not flirt in them?
Features: -Lace horizontally sewed on top-Light removable padding-Adjustable Ties at the neck and back-Lace-waist bottom-1/2 shirring at center back of bottom-80% polyamide and 20% spandex
Advantages in craft:-Color fastness level plus +1 (compare to normal bikinis and lace)-Highly elastic polyamide fabric - high flexibility and elasticity without shirring-Soft Natural Cotton Lining - Comfortable without any irritation-Fine sewing craft


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Meanderings of an Aging Soul, Volume 2
Paperback, 240 Pages
Price: $16.95
Prints in 3-5 business days
In these volumes, with warmth and gentle humor, the author recalls his boyhood on a farm near Lanesboro, Minnesota in the 1940s. Many of his essays in earlier volumes portray a childhood structured by chores, family, school and church obligations. In addition to depicting everyday life, he reflects on may special events (Minnesota State Fair, holidays, WWII "air raid" practice, car trips) and recreation (radios shows, townball league, fishing on the Duschee Creek, roller skating). These charming memoirs capture whimsical details as well as stark realities of Midwestern rural life during that period. Besides giving us a glimpse back into time, on a deeper level William Connelly's writings reveal how his Irish Catholic farm family and small town community shaped the values that would guide him through life. His later writings, including this volume, focus on and characterize how his values have influenced his thinking about more contemporary issues and events. They,... More > too, come alive in his writings.< Less
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Product Details
- 9781105707483
- Copyright
- William L. Connelly (Standard Copyright License)
- Edition
- First
- Publisher
- William L. Connelly
- Published
- April 28, 2012
- Language
- English
- Pages
- 240
- Binding
- Perfect-bound Paperback
- Interior Ink
- Black & white
- Weight
- 1.61 lbs.
- Dimensions (inches)
- 8.5 wide x 11 tall
- Product ID
- 20090601
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Gives a free extra donation! Add this item to your order & enter code 1MANGOTREE before checkout to plant a fruit tree in Africa -- at no additional cost!
Sewn in Kampala, our soft and sporty t-shirt dress is made of 100% organic cotton. Made by seamstresses who found themselves living in poverty when the government closed down their factories -- your purchase offers them new hope for the future.
- 100% organic cotton
- Hand wash cold; line dry; low iron; do not bleach
- Fairly traded from Uganda
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- March 2, 2015
Cute, soft, and versatile (& I agree with the other reviewers comments on the red color) - I really like the organic cotton clothing available through Greater Good.
- November 15, 2014
Nice material and great fit. It's a "soft" red, vibrant but a little more sophisticated. The length is rather short on me (I'm 5'4") but I love it. Packs well.
- August 22, 2014
Lovely styling, both simple & elegant. Very comfortable to wear alone or over slacks. So gratifying to know it's supporting women-made & fairly traded company in Uganda. Would definitely recommend ordering to friends. Great Cause!
- May 29, 2017
The dress is too tight. I am returning it.


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Primetime Preview: The Premiere of Dancing with the StarsMatt Richenthal at . Comments
What should you set your TiVo for tonight? And what should you say Ti-NO to?
Our staff combs through this evening's television listings and helps readers set their viewing schedules:
- 8:00 p.m. It's the eighth season of Dancing with the Stars (ABC); A patient acts just like House (Fox); Summer Glau guest stars on The Big Bang Theory (CBS).
- 8:30 p.m. Laura Prepon guest stars on How I Met Your Mother (CBS).
- 9:00 p.m. Charlie says he loves Chelsea on Two and a Half Men (CBS); Parkman tries not to blow anything up on Heroes (NBC); the President tries not to be killed on 24 (Fox).
- 10:00 p.m. The series debut of Castle (ABC).
Catch the first-ever airing of Castle, tonight on ABC!
Matt Richenthal is the Editor in Chief of TV Fanatic. Follow him on Twitter and on Google+.


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Ad Details
Ad ID: 886
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Sean D. Moore. Swift, the Book, and the Irish Financial Revolution: Satire and Sovereignty in Colonial Ireland. Baltimore: Johns Hopkins University Press, 2010. xi + 268 pp. $65.00 (cloth), ISBN 978-0-8018-9507-4.
Reviewed by Jill M. Bradbury
Published on H-Albion (November, 2010)
Commissioned by Jeffrey R. Wigelsworth (Red Deer College)
Jonathan Swift's Contributions to the Irish Financial Revolution and Anglo-Irish Print Culture
This concise monograph manages the difficult task of interpreting Jonathan Swift's corpus in new contexts, while contributing to existing themes in Swift scholarship. Taking a new economic criticism approach, Sean D. Moore examines Swift's engagement with and contributions to the development of Irish financial institutions. Moore's study profits from existing studies on the English financial revolution, in particular on the representational issues posed by new financial instruments and on the structural overlap between public and private credit. But he adds a new and important dimension by bringing the colonial context of the Irish situation to bear and by extending the homology between paper forms of money and print to the concurrent development of the Irish financial and book markets. While Moore's study makes novel contributions to these different lines of scholarship, it could benefit from further elaboration of certain elements, along with more extensive use of scholarship in economic history and the history of economic thought.
Moore dates the beginnings of the Irish financial revolution to 1716, when a small group of Anglo-Irish landowners loaned the Irish treasury funds for national security. The interest on this first national debt was to be paid in perpetuity from future tax revenues, thus giving investors a strong incentive to resist any attempt by the British Parliament to regulate taxation in Ireland or to restructure the debt. Following George Berkeley, Moore terms this coterie "the Monti" and argues that the political and economic community it formed was the origin of Anglo-Irish national identity. While not a member of the Monti, Swift supported the political and socioeconomic values it represented. Moore views Swift's pamphlets on the national bank, Wood's halfpence, and other financial and commercial issues as successful attempts to mobilize a domestic publishing industry to sway public opinion and policymakers to act in the Monti's interests, which were depicted as those of the Irish nation in general. Moore argues that Anglo-Irish political thought thus originates in this discourse on the national debt, which is at the same time a discourse on a national print culture. Swift's writings on trade and currency, in particular, suggest the potential of an Anglo-Irish publishing industry to counteract British political, economic, and cultural imperialism.
Moore anchors his conjoint reading of the national debt and the national book trade by tracing the motif of weaving in The Battle of the Books, A Tale of a Tub (1704) and A Proposal for the Universal Use of Irish Manufactures (1720). Through this traditional metaphor for writing, Swift attacks the cultural hegemony of British standards of taste in both sartorial and intellectual fashions. This metadiscourse on textiles as a symbol for cultural production appears in several of Swift's tracts on Irish economic issues, as well as in those of other Anglo-Irish writers. Moore also demonstrates how financial issues and print culture were connected in Swift's critique of British colonialism through analysis of scatological imagery, the homology between minting/printing, and the metaphorics of cannibalism. His analysis of the major works thus connects to central issues in recent Swift scholarship, but his argument is most compelling when focused on the shorter pamphlets and unpublished tracts.
Swift, the Book, and the Irish Financial Revolution offers a fresh and engaging reading of familiar material, but some aspects of Moore's argument need further development. Reception issues, in particular, are touched on, but not well explored. More evidence could have been presented that Swift's readers understood his pamphlets to be about both financial issues and the need for a national book trade. The influence of Swift's writings on the actions of both publishers and politicians are similarly gestured at, but not convincingly detailed. The compressed epilogue on the "branding" of Irish identity introduces an entirely new topic and theoretical perspective that would be better unpacked in another study.
Moore's project also displays the tendency of the "literary" new economic criticism to slight economic history and the history of economic thought. His discussion of the Irish financial revolution cites only a few primary texts, while his foundational concept of the fiscal-military imperial state draws largely, if not entirely, from John Brewer's The Sinews of Power (1990). The financial revolution and its contribution to British imperialism has been the subject of extensive research by economic historians, however, and Moore's study would have been strengthened had it incorporated (or even undertaken) more of this work. For example, further background on the composition of the Monti, its self-recognition as such, and the extent to which it did indeed encompass the "Protestant Interest" could have been provided. For scholarship on the history of economic thought, Moore relies on the work of literary critics Clifford Siskin (1998) and James Thompson (1996), rather than studies by economists. This perhaps accounts for Moore's misleading use of "political economy," as in his opening comment that "The first arena of argument chosen for the Monti's publicity campaign was political economy, a discipline of study and genre of print" (p. 6). While some degree of anachronism inheres in historical argument, this usage is particularly unsuited to the period Moore is describing. "Political economy" was first used in print by Antoine de Montchrétien in 1615, but it was not commonly used to refer to the state's harnessing of economic development and policies for political ends until the 1760s and 1770s. It did not come into being as a discipline of study and a genre of print until the nineteenth century. Moore's synoptic use of this term suggests a level of theoretical sophistication and discursive self-awareness that did not exist during the 1720s and 1730s (Moore himself recognizes this at a later point, but handles the intellectual development of economic discourse inconsistently). One could furthermore argue that Swift's works are not representative of the "political economy," such as it was, of the period. Finally, Moore might have benefited from including the economic literature on the social aspects of finance, in addition to the literary studies by Catherine Ingrassia (Authorship, Commerce, and Gender in Early Eighteenth-Century England: A Culture of Paper Credit [1998]), Colin Nicholson (Writing and the Rise of Finance: Capital Satires of the Early Eighteenth Century [1994]), and Sandra Sherman (Finance and Fictionality in the Early Eighteenth Century:Accounting for Defoe [1996]).
A final weakness appears in Moore's use of an orthodox Marxist base/superstructure-material/ideological dichotomy that is at odds with elements of his argument and with the body of literary-financial criticism within which he situates his study. In their studies of the financial revolution, Ingrassia, Nicholson, Sherman and others stress the representational consequences of new credit instruments. Paper money, joint stock company shares, and forms of public credit such as the national debt problematized the assumption of a "real" basis of value and undermined clear distinctions such as material/imaginative or referent/sign. At various points, Moore incorporates this representational "crisis" into his readings of Swift's works, as when he writes, "Property, for Swift, was no longer prior to money; the maintenance of the 'real' that land ownership represented was now codependent with the 'imaginary' realm of discourse and commodity exchange" (p. 156). Yet comments sprinkled throughout his study frame the overall analysis in terms of a distinction between the economic and the ideological: e.g., "The Debt of the Nation thus came to be the material basis for eighteenth-century Anglo-Irish political thought" (p. 4); "Ireland's debt was the economic foundation of the ideology of Anglo-Irish Protestant nationalism" (p. 15); "The manifest gap between the economic base and the ideology of this new imagined community" (p. 136); and "The Anglo-Irish elite had established the economic base of their fledgling state with the founding of the Debt of the Nation in 1716, and that base required a superstructure of ideology to sustain it" (p.149). All of these statements beg the question of how debt, a form of wealth that exists in a textual/fictional medium, could function as a material ground for this base/economic-superstructure/ideology framework.
To sum up, Moore's study is thought-provoking and makes a number of important interventions in both the literary scholarship on the financial revolution and in Swift scholarship more generally. But the execution of the argument is weak in several important respects and readers will find much to argue with in the details.
If there is additional discussion of this review, you may access it through the network, at: https://networks.h-net.org/h-albion.
Jill M. Bradbury. Review of Moore, Sean D., Swift, the Book, and the Irish Financial Revolution: Satire and Sovereignty in Colonial Ireland.
H-Albion, H-Net Reviews.
November, 2010.
URL: http://www.h-net.org/reviews/showrev.php?id=31401
|This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 United States License.


text:
(BaseballSTL) -- The Hall of Fame voters sent another loud message to players of the steroids era.
First-year eligible players Barry Bonds, Roger Clemens, Mike Piazza and Sammy Sosa were all denied entrance into Cooperstown by voters from the Baseball Writers Association of America.
Players must get at least 75 percent of the vote to get in. Bonds got just 32.6 perce, Clemens 37.6 percent, Sosa 12.5 percent and Piazza 57.8 percent. The stats those players put up indicate they are among the very elite players in the history of the game. But the writers aren't buying it. Even if there is no proof of having taken steroids, players in that era are still being denied.
The voting process is flawed. When the Hall of Fame first opened it made sense for the writers to be doing the voting. They were the only people covering all the games. This was before games were televised or broadcast on the radio. The writers were the best judge of Hall of Fame worthiness at the time. Not so anymore. Too many of them want to become the story themselves with their vote, refusing to vote for certain players for reasons sometimes other than what happened on the field. The writers seem to enjoy being the moral compass of baseball. Bob Costas, Vin Scully, Joe Buck, Tim McCarver... none of them get to vote for the Hall of Fame. That makes no sense.
I would have voted in favor of the top players of the steroids era. As miserable a person as Bonds was in his playing days, he was still among the best ever. Same with Clemens. And Piazza and Sosa. I would have voted for all them. I believe the Hall of Fame should group players from the steroids era together, with an asterisk explaining that the numbers these players achieved occurred at a time when many, if not most, major league players were using performance enhancing drugs. Through-out the history of baseball players have tried to gain an unfair advantage with spit balls, thumb tacks in pitcher's gloves, corked bats, greenies, etc.
Steroids helped players cheat. No doubt about that. But cheating, unfortunately, has been a part of the game forever. I believe they should vote in the steroids players whose numbers are Hall of Fame worthy, and let baseball fans decide for themselves just how "immortal" these players are.
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