Kuppan Gokulan

Charged residue changes in the carboxy‐terminus of α‐tropomyosin alter mouse cardiac muscle contractility

Striated muscle tropomyosin (TM) is an essential thin filament protein that is sterically and allosterically involved in calcium‐mediated cardiac contraction. We have previously shown that overexpressing the β‐TM isoform in mouse hearts leads to physiological changes in myocardial relaxation and Ca2+ handling of myofilaments. Two important charge differences in β‐TM compared to α‐TM are the exchange of serine and histidine at positions 229 and 276 with glutamic acid and asparagine, respectively, imparting a more negative charge to β‐TM relative to α‐TM. Our hypothesis is that the net charge at specific sites on TM might be a major determinant of its role in modulating cardiac muscle performance and in regulating Ca2+ sensitivity of the myofilaments. To address this, we generated transgenic (TG) double mutation mouse lines (α‐TM DM) expressing mutated α‐TM at the two residues that differ between α‐ and β‐TM (Ser229Glu + His276Asn). Molecular analyses show 60–88% of the native TM is replaced with α‐TM DM in the different TG lines. Work‐performing heart analyses show that α‐TM DM mouse hearts exhibit decreased rates of pressure development and relaxation (+dP/dt and –dP/dt). Skinned myofibre preparations from the TG hearts indicate a decrease in calcium sensitivity of steady state force. Protein modelling studies show that these two charge alterations in α‐TM cause a change in the surface charges of the molecule. Our results provide the first evidence that charge changes at the carboxy‐terminal of α‐TM alter the functional characteristics of the heart at both the whole organ and myofilament levels.

Salmonella enterica Typhimurium SipA induces CXC-chemokine expression through p38MAPK and JUN pathways

The role of Salmonella typhimurium type III secretion system (T3SS-1)-translocated proteins in chemokines expression and protein phosphorylation was investigated in HeLa cells. Infection of HeLa cells with S. typhimurium activated IL-8 and GRO-alpha expression at higher levels than infection with a S. typhimurium sipAsopABDE2 mutant, confirming that T3SS-1-secreted proteins are required to fully induce chemokine expression in HeLa cells. A S. typhimurium sipAsopABDE2 mutant complemented with sipA or a strain carrying a chromosomal copy of sipA (sopABDE2 mutant) activated chemokines at significantly higher levels than a S. typhimurium sipAsopABDE2 mutant. However, extracellular addition of recombinant SipA failed to induce IL-8 expression. Phosphorylation analyses revealed that S. typhimurium induced a twofold increase in the phosphorylation of B23, CREB1, ERK1, JUN, p38MAPK, and NR1. JUN and p38MAPK were phosphorylated by S. typhimurium carrying a chromosomal copy of sipA (sopABDE2 mutant) while none was more than twofold phosphorylated in cells infected with the S. typhimurium sipAsopABDE2 mutant. Treating cells with JUN and p38MAPK inhibitors significantly decreased IL-8 expression in sopABDE2 mutant infected cells. These data indicate that S. typhimurium SipA induces expression of CXC chemokines through phosphorylation of IL-8-transcription regulatory proteins, JUN and p38MAK.

Crystal structure of Mycobacterium tuberculosis LrpA, a leucine-responsive global regulator associated with starvation response

The bacterial leucine‐responsive regulatory protein (Lrp) is a global transcriptional regulator that controls the expression of many genes during starvation and the transition to stationary phase. The Mycobacterium tuberculosis gene Rv3291c encodes a 150‐amino acid protein (designated here as Mtb LrpA) with homology with Escherichia coli Lrp. The crystal structure of the native form of Mtb LrpA was solved at 2.1 Å. The Mtb LrpA structure shows an N‐terminal DNA‐binding domain with a helix‐turn‐helix (HTH) motif, and a C‐terminal regulatory domain. In comparison to the complex of E. coli AsnC with asparagine, the effector‐binding pocket (including loop 100–106) in LrpA appears to be largely preserved, with hydrophobic substitutions consistent with its specificity for leucine. The effector‐binding pocket is formed at the interface between adjacent dimers, with an opening to the core of the octamer as in AsnC, and an additional substrate‐access channel opening to the outer surface of the octamer. Using electrophoretic mobility shift assays, purified Mtb LrpA protein was shown to form a protein–DNA complex with the lat promoter, demonstrating that the lat operon is a direct target of LrpA. Using computational analysis, a putative motif is identified in this region that is also present upstream of other operons differentially regulated under starvation. This study provides insights into the potential role of LrpA as a global regulator in the transition of M. tuberculosis to a persistent state.

Dimeric β-turn peptidomimetics as ligands for the neurotrophin receptor TrkC

Abstract Twelve dimeric peptidomimetics 1 were prepared via a divergent–convergent strategy. These peptidomimetics incorporated the same amino acids as i+1 and i+2 residues in key β-turns of the neurotrophin NT-3. Cytosensor microphysiometry was used to gauge the effects of the dimers 1 on cells that overexpress the NT-3 receptor, TrkC. Increases in extracellular acidification rates were observed for some monomers 3, but the active dimers gave greater effects.

Mycobacterium tuberculosis acyl carrier protein synthase adopts two different pH-dependent structural conformations.

The crystal structures of acyl carrier protein synthase (AcpS) from Mycobacterium tuberculosis (Mtb) and Corynebacterium ammoniagenes determined at pH 5.3 and pH 6.5, respectively, are reported. Comparison of the Mtb apo-AcpS structure with the recently reported structure of the Mtb AcpS-ADP complex revealed that AcpS adopts two different conformations: the orthorhombic and trigonal space-group structures show structural differences in the α2 helix and in the conformation of the α3-α4 connecting loop, which is in a closed conformation. The apo-AcpS structure shows electron density for the entire model and was obtained at lower pH values (4.4-6.0). In contrast, at a higher pH value (6.5) AcpS undergoes significant conformational changes, resulting in disordered regions that show no electron density in the AcpS model. The solved structures also reveal that C.xa0ammoniagenes AcpS undergoes structural rearrangement in two regions, similar to the recently reported Mtb AcpS-ADP complex structure. In vitro reconstitution experiments show that AcpS has a higher post-translational modification activity between pH 4.4 and 6.0 than at pH values above 6.5, where the activity drops owing to the change in conformation. The results show that apo-AcpS and AcpS-ADP adopt different conformations depending upon the pH conditions of the crystallization solution.

KasA, Another Brick in the Mycobacterial Cell Wall

Mycobacterium tuberculosis , the causative agent of tuberculosis, a disease that has been plaguing humanity for centuries, has a unique cell wall composition believed to be critical for pathogenicity. Luckner etxa0al. (2009) now describe the structure of KasA , an enzyme involved in cell wall biosynthesis.

Vasoactive amine responses in murine cerebrovascular endothelial cells as measured by extracellular acidification rates.

The Cytosensor™ microphysiometer device is capable detecting cellular responses to specific bioactive ligands by measuring the extracellular acidification rate (ECAR). Using microphysiometry, we were able to determine that cerebovascular endothelial cells derived from SJL/J mice were more sensitive to serotonin (maximal ECAR response at 100 nM), whereas BALB/c‐derived cerebrovascular endothelial cells (CVE) were relatively insensitive (maximal ECAR response at 30 μM). Serotonin (5HT)1 and 5HT2 receptor antagonists inhibited the serotonin‐mediated increases in ECAR. The cells responses to histamine in both strains were similar (maximal ECAR required 100 μM of histamine). H1 and H3 receptor subtype antagonists specifically inhibited the histamine responses. Bradykinin also revealed sensitivity differences in that maximal ECAR changes for CVE from SJL/J mice could be observed with 1 μM, as compared to the ECAR responses from BALB/c mice CVE, which required 10 μM. Exposure to bradykinin antagonists revealed that the response was due to the stimulation of B2 receptors. These microphysiometry results reveal that the cerebrovascular endothelial cells of SJL/J mice tend to be more sensitive to vasoactive substances than those of BALB/c mice. J. Neurosci. Res. 60:356–361, 2000

Early changes in metabolism of leukemic cell lines upon induction of apoptosis by cytotoxic drugs.

We evaluated real-time changes in extracellular acidification rates of human U937 and K562 leukemic cells treated with camptothecin or taxol. U937 cells treated with camptothecin or taxol for 30-60 min showed a continuous, irreversible decrease in extracellular acidification rate that was sensitive to amiloride. In contrast, U937 cells exposed to sodium azide showed an immediate, steep decrease in extracellular acidification rate that was reversible upon azide withdrawal. K562 cells required a >20-fold higher dose of camptothecin to promote similar changes in the extracellular acidification rate, with a corresponding resistance in their susceptibility to camptothecin- or taxol-induced apoptosis. The data show that irreversible commitment to apoptosis is associated with rapid metabolic changes that are reflected by decreased extracellular acidification rate and regulated by the Na(+)/H(+) antiporter. Moreover, detection of extracellular acidification rate changes was not restricted to a particular cell type or apoptosis pathway, making this a potentially useful tool to screen compounds for pro-apoptotic activity.

Cellular responses of NG108‐15 and SK‐N‐MC lines to sweet and bitter tastants as measured by extracellular acidification rates

The Cytosensor microphysiometer device (Molecular Devices, Sunnyvale, CA) is capable of detecting small changes in cellular metabolism in response to specific bioactive ligands by measuring the extracellular acidification rate (ECAR). By measuring the ECAR we were able to detect responses of tissue culture cell lines to a variety of sweet‐ and bitter‐tasting compounds. We examined in detail the responses of the NG108‐15 (mouse neuroblastoma × rat glioma hybrid) and SK‐N‐MC (human neuroepithelioma) cell lines. We determined that NG108‐15 cells were consistently very responsive to several potent sweeteners and bitter compounds, such as sodium saccharin, guanidino‐ sweeteners, denatonium benzoate, quinine, and ranitidine. These compounds could evoke changes in cellular metabolism (measured as ECAR) that were rapid in onset, saturable with respect to ligand concentration, and sensitive to several inhibitors of G‐protein‐coupled receptor signaling pathways. In sharp contrast, the neuroepithelioma SK‐N‐MC did not respond to any of the sweet or bitter compounds. Rapid changes in ECAR were easily detectable in both cell lines with the calcium ionophore A23187. Bradykinin elicited changes in the ECAR only in the NG108‐15 cell line, which is known to express the B2 receptor. The changes in ECAR of the NG108‐15 cell line in response to sweet and bitter taste compounds suggest these cells may expresses a receptor(s) specific for small sapid molecules. J. Neurosci. Res. 63:64–71, 2001.