Tropomyosin exon 6b is troponin-specific and required for correct acto-myosin regulation

The specificity of tropomyosin (Tm) exon 6b for interaction with and functioning of troponin (Tn) has been studied using recombinant fibroblast Tm isoforms 5a and 5b. These isoforms differ internally by exons 6a/6b and possess non-muscle exons 1b/9d at the termini, hence they lack the primary TnT(1)-tropomyosin interaction, allowing study of exon 6 exchange in isolation from this. Using kinetic techniques to measure regulation of myosin S1 binding to actin and fluorescently labeled Tm to directly measure Tn binding, we show that binding of Tn to both isoforms is similar (0.1-0.5 microm) and both produce well regulated systems. Calcium has little effect on Tn binding to the actin.Tm complex and both exons produce a 3-fold reduction in the S1 binding rate to actin.Tm.Tn in its absence. This confirms previous results that show exon 6 has little influence on Tn affinity to actin.Tm or its ability to fully inhibit the acto-myosin interaction. Thin filaments reconstituted with Tn and Tm5a or skeletal Tm (containing exon 6b) show nearly identical calcium dependence of acto-myosin regulation. However, Tm5b produces a dramatic increase in calcium sensitivity, shifting the activation mid-point by almost an order of magnitude. This shows that exon 6 sequence and, hence, Tm structure in this region have a significant effect upon the calcium regulation of Tn. This finding supports evidence that familial hypertrophic cardiomyopathy mutations occurring adjacent to this region can effect calcium regulation.     

Tn antigen promotes human colorectal cancer metastasis via H‐Ras mediated epithelial‐mesenchymal transition activation

Tn antigen is a truncated O‐glycan, frequently detected in colorectal cancer (CRC), but its precise role in CRC metastasis is not well addressed. Here we investigated the effects of Core 1 β3Gal‐T specific molecular chaperone (Cosmc) deletion‐mediated Tn antigen exposure on CRC metastasis and its underlying mechanism. We first used CRISPR/Cas9 technology to knockout Cosmc, which is required for normal O‐glycosylation, and thereby obtained Tn‐positive CRC cells. We then investigated the biological consequences of Tn antigen expression in CRC. The results showed that Tn‐positive cells exhibited an enhanced metastatic capability both in vitro and in vivo. A further analysis indicated that Tn antigen expression induced typical activation of epithelial‐mesenchymal transition (EMT). Mechanistically, we found that H‐Ras, which is known to drive EMT, was markedly up‐regulated in Tn‐positive cells, whereas knockdown of H‐Ras suppressed Tn antigen induced activation of EMT. Furthermore, we confirmed that LS174T cells (Tn‐positive) transfected with wild‐type Cosmc, thus expressing no Tn antigen, had down‐regulation of H‐Ras expression and subsequent inhibition of EMT process. In addition, analysis of 438 samples in TCGA cohort demonstrated that Cosmc expression was reversely correlated with H‐Ras, underscoring the significance of Tn antigen‐H‐Ras signalling in CRC patients. These data demonstrated that Cosmc deletion‐mediated Tn antigen exposure promotes CRC metastasis, which is possibly mediated by H‐Ras‐induced EMT activation.     

Structure-function studies of the amino-terminal region of bovine cardiac troponin T

The length and amino acid sequence of the amino-terminal region of troponin T (TnT) is regulated by alternative mRNA processing in both mammals and birds. To study the function of this region, three forms of bovine cardiac TnT were compared: isoforms TnT1 and TnT2, which differ by the presence or absence of residues 15-19 and TnT 39-284. TnT 39-284 was prepared by chemical cleavage of TnT1 at Cys-39. All three forms of TnT successfully reconstituted with troponin I and troponin C, resulting in troponins designated Tn1, Tn2, and TnCN. Three properties of the reconstituted troponins were compared. 1) Tn1 and TnCN had indistinguishable effects on tropomyosin polymerization. Addition of either 8 microM Tn1 or 8 microM TnCN increased the viscosity (eta rel) of 5 microM tropomyosin from 1.0 to 1.63 at 10 degrees C. 2) All of the three troponins conferred Ca2+ dependence to the MgATPase rate of myosin S-1-actin-tropomyosin. In the presence of saturating concentrations of Tn2, Tn1, or TnCN, 50% MgATPase activation occurred at pCa 6.0, 5.9, or 5.75, respectively. 3) The affinity of the Ca2+-specific binding site of reconstituted Tn1 was 50% stronger than the affinity of the same site on TnCN. These results suggest that the amino-terminal region of cardiac TnT is not a completely Ca2+-insensitive domain, but rather modulates the interaction of Ca2+ with troponin and with the thin filament. Furthermore, the effects of TnT on tropomyosin-tropomyosin binding are predominantly due to portions of TnT carboxyl-terminal to residue 38.     

Isolation and characterization of Tn7 transposase gain-of-function mutants: a model for transposase activation

Tn7 transposition has been hypothesized to require a heteromeric transposase formed by two Tn7-encoded proteins, TnsA and TnsB, and accessory proteins that activate the transposase when they are associated with an appropriate target DNA. This study investigates the mechanism of Tn7 transposase activation by isolation and analysis of transposase gain-of-function mutants that are active in the absence of these accessory proteins. This work shows directly that TnsA and TnsB are essential and sufficient components of the Tn7 transposase and also provides insight into the signals that activate the transposase. We also describe a protein-protein interaction between TnsA and TnsC, a regulatory accessory protein, that is likely to be critical for transposase activation.     

Is Bax a mitochondrial mediator in apoptotic death of dopaminergic neurons in Parkinson's disease?

Bax is a proapoptotic member of the Bcl-2 family of proteins. It is believed to exert its action primarily by facilitating the release of cytochrome c from the mitochondrial intermembrane space into the cytosol, leading to caspase activation and cell death. Because alterations in mitochondrial respiratory function, caspase activation and cell death with morphologic features compatible with apoptosis have been observed post mortem in the brain of patients with Parkinson's disease, we tried to clarify the potential role of Bax in this process in an immunohistochemical study on normal and Parkinson's disease post-mortem brain and primary mesencephalic cell cultures treated with MPP(+). We found that Bax is expressed ubiquitously by dopaminergic (DA) neurons in post-mortem brain of normal and Parkinson's disease subjects as well as in vitro. Using an antibody to Bax inserted into the outer mitochondrial membrane as an index of Bax activation, no significant differences were observed between control and Parkinson's disease subjects, regardless of the mesencephalic subregion analysed. However, in Parkinson's disease subjects, the percentage of Bax-positive melanized SNpc neurons containing Lewy bodies, suggestive of DA neuronal suffering, was significantly higher than the overall percentage of Bax-positive neurons among melanized neurons. Furthermore, all melanized SNpc neurons in Parkinson's disease subjects with activated caspase-3 were also immunoreactive for Bax, suggesting that Bax anchored in the outer mitochondrial membrane of melanized SNpc neurons showing signs of neuronal suffering or apoptosis is increased compared with DA neurons that are apparently unaltered. Surprisingly, MPP(+) treatment of tyrosine hydroxylase (TH)-positive neurons in primary mesencephalic cultures did not cause redistribution of Bax, although cytochrome c was released from the mitochondria and nuclear condensation/fragmentation was induced. Taken together, these findings suggest that in the human pathology, Bax may be a cofactor in caspase activation, but our in vitro data fail to indicate a central role for Bax in apoptotic death of DA neurons in an experimental Parkinson's disease paradigm.     

Troponin and titin coordinately regulate length-dependent activation in skinned porcine ventricular muscle

We investigated the molecular mechanism by which troponin (Tn) regulates the Frank-Starling mechanism of the heart. Quasi-complete reconstitution of thin filaments with rabbit fast skeletal Tn (sTn) attenuated length-dependent activation in skinned porcine left ventricular muscle, to a magnitude similar to that observed in rabbit fast skeletal muscle. The rate of force redevelopment increased upon sTn reconstitution at submaximal levels, coupled with an increase in Ca2+ sensitivity of force, suggesting the acceleration of cross-bridge formation and, accordingly, a reduction in the fraction of resting cross-bridges that can potentially produce additional active force. An increase in titin-based passive force, induced by manipulating the prehistory of stretch, enhanced length-dependent activation, in both control and sTn-reconstituted muscles. Furthermore, reconstitution of rabbit fast skeletal muscle with porcine left ventricular Tn enhanced length-dependent activation, accompanied by a decrease in Ca2+ sensitivity of force. These findings demonstrate that Tn plays an important role in the Frank-Starling mechanism of the heart via on-off switching of the thin filament state, in concert with titin-based regulation.     

Interaction of related Tn916-like transposons: analysis of excision events promoted by Tn916 and Tn5386 integrases

In recent work, we described the excision of a large genomic region from Enterococcus faecium D344R in which the sequence from "joint" regions suggested that excision resulted from the interaction of conjugative transposon Tn916 and the related mobile element Tn5386. In the present study, we examined the ability of integrases and integrase-excisase combinations from Tn916 and Tn5386 to promote the excision of constructs consisting of the termini of Tn916, Tn5386, and the VanB mobile element Tn5382. Integrases alone from either Tn916 or Tn5386 promoted the circularization of constructs from the three different transposons, even when the different termini used in the constructs were discordant in their transposon of origin. The termini of Tn916 and Tn5382 found in all joints were consistent with previously identified Tn916 and Tn5382 termini. Substantial variation was seen in the integrase terminus of Tn5386 used to form joints, regardless of the integrase that was responsible for circularization. Variability was observed in joints formed from Tn5386 constructs, in contrast to joints observed with the termini of Tn916 or Tn5382. The coexpression of excisase yielded some variability in the joint regions observed. These data confirm that integrases from some Tn916-like elements can promote circularization with termini derived from heterologous transposons and, as such, could promote excision of large genomic regions flanked by such elements. These findings also raise interesting questions about the sequence specificities of the C terminals of Tn916-like integrases, which bind to the ends and facilitate strand exchange.     

Electromyographic patterns of individuals suffering from lateral tennis elbow.

This study investigated the applicability of using surface electromyography (EMG) as a tool for differentiating between persons suffering from lateral tennis elbow and the healthy age-matched adults. Temporal muscle activation patterns of the tennis elbow group were evaluated to determine if they varied between subject groups and if noted variations might be interpreted as arresting or exacerbating the injury. Sixteen subjects (Healthy Controls, n = 6; Tennis Elbow, n = 10) were tested under simulated tennis playing conditions. All subjects were males (Healthy group (CON) 38.8 +/- 13.1, Injured group (INJ) 40.8 +/- 10.8 yrs). EMG response data, temporal and spatial muscle activities, of the forearm extensors (Ext), the forearm flexors (Flex) and the triceps (Tri) were recorded for each subject during a single test session using all combinations of three different velocities on three different racket head impact locations. Data were collected at a frequency of 1000 Hz. Statistical analysis was performed using a 2 x 3 x 3 (Health status x Impact velocity x Impact location) ANOVA with repeated measures. Results indicated statistically significant differences (p < 0.05) between the CON and INJ subject groups for the response variables associated with forearm extensor muscle activation. During simulated play, the INJ group employed an earlier, longer, and greater activation of Ext than the CON group, such changes may be considered detrimental to the healing process. These results support the use of surface EMG to quantify differences in muscle activation strategies employed by individuals suffering from soft tissue muscle microtrauma injuries and healthy controls.     

Tn5386, a novel Tn916-like mobile element in Enterococcus faecium D344R that interacts with Tn916 to yield a large genomic deletion

We describe Tn5386, a novel ca.-29-kb Tn916-like mobile element discovered to occur in ampicillin-resistant, Tn916-containing Enterococcus faecium D344R. PCR amplification experiments after overnight growth with or without tetracycline revealed "joint" regions of circularized Tn5386 composed of 6-bp sequences linking different transposon termini. In one case (no tetracycline), the termini were consistent with those derived by target site analysis of the integrated element. In the other case, the termini were virtually identical in distance from the integrase binding regions, as seen with Tn916. These data are consistent with a model in which one PCR product results from the action of Tn5386 integrase, whereas the other results from the action of the Tn916 integrase on Tn5386. Spontaneous conversion of D344R to an ampicillin-susceptible phenotype (D344SRF) was associated with a 178-kb deletion extending from the left end of Tn5386 to the left end of Tn916. Examination of the Tn5386 junction after the large deletion event suggests that the deletion resulted from an interaction between the nonintegrase ends of Tn5386 and Tn916. The terminus of Tn5386 identified in this reaction suggested that it may have resulted from the activity of the Tn916 integrase (Int(Tn916)). The "joint" of the circular element resulting from this excision was amplifiable from D344R, the sequence of which revealed a heteroduplex consistent with Int(Tn916)-mediated excision. In contrast, Tn5386 joints amplified from ampicillin-susceptible D344SRF revealed ends consistent with Tn5386 integrase activity, reflecting the absence of Tn916 from this strain. Tn5386 represents a new member of the Tn916 transposon family. Our data suggest that excision of Tn5386 can be catalyzed by the Tn916 integrase and that large genomic deletions may result from the interaction between these heterologous elements.     

Site-specific recombination by mutants of Tn21 resolvase with DNA recognition functions from Tn3 resolvase

The resolvases from the transposons Tn3 and Tn21 are homologous proteins but they possess distinct specificities for the DNA sequence at their respective res sites. The DNA binding domain of resolvase contains an amino acid sequence that can be aligned with the helix-turn-helix motif of other DNA binding proteins. Mutations in the gene for Tn21 resolvase were made by replacing the section of DNA that codes for the helix-turn-helix with synthetic oligonucleotides. Each mutation substituted one amino acid in Tn21 resolvase with either the corresponding residue from Tn3 resolvase or a residue that lacks hydrogen bonding functions. The ability of these proteins to mediate recombination between res sites from either Tn21 or Tn3 was measured in vivo and in vitro. With one exception, where a glutamate residue had been replaced by leucine, the activity of these mutants was similar to that of wild-type Tn21 resolvase. A further mutation was made in which the complete recognition helix of Tn21 resolvase was replaced with that from Tn3 resolvase. This protein retained activity in recombining Tn21 res sites, though at a reduced level relative to wild-type; the reduction can be assigned entirely to weakened binding to this DNA. Neither this mutant nor any other derivative of Tn21 resolvase had any detectable activity for recombination between res sites from Tn3. The exchange of this section of amino acid sequence between the two resolvases is therefore insufficient to alter the DNA sequence specificity for recombination.     

Reconstituted human erythrocyte sugar transporter activity is determined by bilayer lipid head groups

The effects of bilayer lipid head group on human erythrocyte passive sugar transport protein activity were examined by reconstituting the transporter into bilayers of large unilamellar vesicles (LUVs) formed from lipid classes of identical (or similar) acyl chain composition. Two reconstituted transport parameters were measured as a function of temperature. These were Km and turnover number [Tn = Vmax per reconstituted D-glucose-sensitive cytochalasin B binding site (transport molecule)]. Tn for sugar transport was found to be almost entirely a function of the properties of the bulk lipid composition of the reconstituted LUVs. It was found to be independent of both reconstituted transporter density and small amounts (less than or equal to 3%) of endogenous red cell lipids. With the dimyristoylphospholipids, Tn increases at all temperatures in the order phosphatidylcholine less than phosphatidylglycerol less than phosphatidic acid less than phosphatidylserine (at 50 degrees C, Tn for transport in dimyristoylphosphatidylcholine is 100-fold lower than Tn for transport in dimyristoylphosphatidylserine). Similar results are found with egg yolk derived lipids. Only dimyristoyl- and dipalmitoylphosphatidylcholine bilayers are incapable of supporting detectable transport activity at temperatures below the bilayer phase transition, and only the phosphatidylcholines show a clear increase in Tn during the bilayer melt. All other bilayer systems studied (phosphatidic acid, phosphatidylglycerol, phosphatidylserine, and sphingomyelin) support a small or negligible increase in Tn during the bilayer melt, the major change in transport being restricted to altered Km. With the disaturated phosphatidylglycerols (C14-C18), Tn and the activation energy (Ea) for reconstituted transport increase with acyl chain carbon number. Similar results are found with the phosphatidylcholines. Transport in bilayers formed from egg yolk sphingomyelin (a lipid containing a sphingosine rather than a glycerol backbone) is characterized by very high Km and low Tn parameters. Moreover, protein-mediated transport in sphingomyelin bilayers "spikes" during the bilayer phase transition. These and previous findings [Carruthers, A., & Melchior, D.L. (1984) Biochemistry 23, 6901-6911; Connolly, T.J., Carruthers, A., & Melchior, D. L. (1985) Biochemistry 24, 2865-2873] indicate that those bilayer factors influencing reconstituted sugar transporter activity are, in order of importance, lipid head group greater than lipid acyl chain length and saturation/unsaturation greater than lipid backbone greater than bilayer "fluidity".     

Effects of partial extraction of troponin complex upon the tension-pCa relation in rabbit skeletal muscle. Further evidence that tension development involves cooperative effects within the thin filament

Partial extraction of troponin C (TnC) decreases the Ca2+ sensitivity of tension development in mammalian skinned muscle fibers (Moss, R. L., G. G. Giulian, and M. L. Greaser. 1985. Journal of General Physiology. 86:585), which suggests that Ca2+-activated tension development involves molecular cooperativity within the thin filament. This idea has been investigated further in the present study, in which Ca2+-insensitive activation of skinned fibers from rabbit psoas muscles was achieved by removing a small proportion of total troponin (Tn) complexes. Ca2+-activated isometric tension was measured at pCa values (i.e., -log[Ca2+]) between 6.7 and 4.5: (a) in control fiber segments, (b) in the same fibers after partial removal of Tn, and (c) after recombination of Tn. Tn removal was accomplished using contaminant protease activity found in preparations of LC2 from rabbit soleus muscle, and was quantitated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and scanning densitometry. Partial Tn removal resulted in the development of a Ca2+-insensitive active tension, which varied in amount depending on the duration of the extraction, and concomitant decreases in maximal Ca2+-activated tensions. In addition, the tension-pCa relation was shifted to higher pCa values by as much as 0.3 pCa unit after Tn extraction. Readdition of Tn to the fiber segments resulted in the reduction of tension in the relaxing solution to control values and in the return of the tension-pCa relation to its original position. Thus, continuous Ca2+-insensitive activation of randomly spaced functional groups increased the Ca2+ sensitivity of tension development in the remaining functional groups along the thin filament. In addition, the variation in Ca2+-insensitive active tension as a function of Tn content after extraction suggests that only one-third to one-half of the functional groups within a thin filament need to be activated for complete disinhibition of that filament to be achieved.     

Multiple IS10 rearrangements in Escherichia coli

We have investigated the occurrence of multiple transposon-promoted chromosomal rearrangements in Escherichia coli K12 strains containing transposon Tn10. We show that a single Tn10 element, with its two closely spaced insertion sequence (IS10) elements, frequently gives rise to complex rearrangements that can be accounted for as the sum of two "classical" IS10 events. Using a strain containing differentially marked Tn10 elements at widely separated locations, we have investigated the possibility that IS10-promoted rearrangements occur in cell-wide "bursts", as expected if cells could occasionally undergo brief periods when all IS10 transposition events were activated, interspersed with longer periods of relative quiescence. We find no evidence for strong (greater than 60-fold), periodic cell-wide activation under our experimental conditions. The sensitivity of this experiment has been evaluated using an expression for the accumulation of double mutations in populations with heterogeneous, fluctuating mutation rates (see Appendix). We discuss several mechanisms by which two closely linked IS10 elements could undergo coupled double events without cell-wide activation: local activation of small chromosomal regions, periodic bursts of synthesis of cis-acting transposase protein, and/or a propensity for elements that have actually engaged in one rearrangement event to initiate a second successive event immediately thereafter. We favor the last possibility.     

Myofilament calcium sensitivity does not affect cross-bridge activation-relaxation kinetics

We employed single myofibril techniques to test whether the presence of slow skeletal troponin-I (ssTnI) is sufficient to induce increased myofilament calcium sensitivity (EC(50)) and whether modulation of EC(50) affects the dynamics of force development. Studies were performed using rabbit psoas myofibrils activated by rapid solution switch and in which Tn was partially replaced for either recombinant cardiac Tn(cTn) or Tn composed of recombinant cTn-T (cTnT) and cTn-C (cTnC), and recombinant ssTnI (ssTnI-chimera Tn). Tn exchange was performed in rigor solution (0.5 mg/ml Tn; 20 degrees C; 2 h) and confirmed by SDS-PAGE. cTnI exchange induced a decrease in EC(50); ssTnI-chimera Tn exchange induced a further decrease in EC(50) (in microM: endogenous Tn, 1.35 +/- 0.08; cTnI, 1.04 +/- 0.13; ssTnI-chimera Tn, 0.47 +/- 0.03). EC(50) was also decreased by application of 100 microM bepridil (control: 2.04 +/- 0.03 microM; bepridil 1.35 +/- 0.03 microM). Maximum tension was not different between any groups. Despite marked alterations in EC(50), none of the dynamic activation-relaxation parameters were affected under any condition. Our results show that 1) incorporation of ssTnI into the fast skeletal sarcomere is sufficient to induce increased myofilament Ca(2+) sensitivity, and 2) the dynamics of actin-myosin interaction do not correlate with EC(50). This result suggests that intrinsic cross-bridge cycling rate is not altered by the dynamics of thin-filament activation.     

Tn 7 inserts in both orientations at a single chromosomal location and apparently forms cointegrates in Pasteurella multocida

Pasteurella multocida transconjugants isolated after mating with Escherichia coli strains that carry one or the other of two Tn7-containing suicide plasmids, pRKTV5 and pUW964 (pRKTV5::Tn5), were analysed. These plasmids have the ColE1 replication origin and were thus expected to deliver transposons but not be maintained as free replicons in Pasteurella. Five out of six transconjugants selected for acquisition of Tn7 from E. coli (pRKTV5) had simple insertions of the transposon, in either orientation, at a single chromosomal location, while the sixth had pRKTV5 integrated at the same location. By contrast, all of 27 transconjugants selected for acquisition of either Tn7 or Tn5 from E. coli (pUW964) maintained pUW964. Of seven subsequently examined at the molecular level, all had pUW964 (in one case, a deletion derivative) integrated at the same location as the Tn7 insertions obtained with pRKTV5. A copy of Tn7 was present at each boundary between the integrated plasmids (pRKTV5 or pUW964) and the chromosome in each strain. The two copies of Tn7 at either end of an integrated plasmid were either in the same (six cases) or in opposite (two cases) orientations with respect to each other. These seem to be products of replicative transposition by Tn7 but can also derive from conservative mechanisms.     

Definition of three resolvase binding sites at the res loci of Tn21 and Tn1721.

The dual functions of resolvase, site-specific recombination and the regulation of its own expression from tnpR, both require the interaction of this protein with the DNA sequence at res, but the specificity of this interaction differs between groups of Tn3-like elements. In this study, DNA fragments that contained res from Tn21 or Tn1721 were subjected to either cleavage by DNase I or methylation by dimethyl sulphate in the presence of the purified resolvase from Tn21 or Tn1721. These experiments showed that each resolvase bound to the same three sites (I, II and III) within res from Tn1721 and to an equivalent series of three sites on Tn21: the differences in the amino acid sequences of the two proteins did not affect their interaction with either DNA. The DNA sequences at each site had some similarities and, in conjunction with data from the related transposon Tn501, a consensus was established. However, the three sites are functionally distinct: site I (tnpR-distal) spans the recombination cross-over point and sites II and III (tnpR-proximal) overlap the promoter of tnpR. The binding sites on these transposons were compared with those in the gamma delta/Tn3 system: the similarities between the two groups of transposons revealed some general features of resolvase-DNA interactions while the differences in fine structure elucidated the specificity of each resolvase.