Caveolin-3 is adjacent to a group of extradyadic ryanodine receptors

Caveolae are present in almost all cells and concentrate a wide variety of signaling molecules, receptors, transporters, and ion pumps. We have investigated the distribution of the ryanodine receptor, the Na(+)/Ca(2+) exchanger, the predominant Na(+) channel isoform rH1, and the L-type calcium channel, Ca(v)1.2, relative to the muscle-specific caveolin isoform, caveolin-3, in adult rat ventricular myocytes. Three-dimensional immunofluorescence images were deconvolved and analyzed. Caveolin-3 colocalizes with all of these molecules at the surface of the cell, but there is no significant colocalization between caveolin-3 and either the Na(+)/Ca(2+) exchanger or the Na(+) channel in the cell interior. The distribution of the surface colocalization indicates that the caveolae that colocalize with each molecule form distinct populations. This organization indicates that there are multiple populations of caveolae separable by location and occupants. In the interior of the cell, caveolin-3 shows a marked colocalization with a population of ryanodine receptors that are separate from those within the dyad. Because of their location, the signaling molecules contained within these caveolae may have preferred access to the neighboring nondyadic ryanodine receptors.     

The two authentic methionine aminopeptidase genes are differentially expressed in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Background  

Two putative methionine aminopeptidase genes,map(essential) andyflG(non-essential), were identified in the genome sequence ofBacillus subtilis. We investigated whether they can function as methionine aminopeptidases and further explored possible reasons for their essentiality or dispensability inB. subtilis.     

A homology model of restriction endonuclease SfiI in complex with DNA

Background

Restriction enzymes (REases) are commercial reagents commonly used in recombinant DNA technologies. They are attractive models for studying protein-DNA interactions and valuable targets for protein engineering. They are, however, extremely divergent: the amino acid sequence of a typical REase usually shows no detectable similarities to any other proteins, with rare exceptions of other REases that recognize identical or very similar sequences. From structural analyses and bioinformatics studies it has been learned that some REases belong to at least four unrelated and structurally distinct superfamilies of nucleases, PD-DxK, PLD, HNH, and GIY-YIG. Hence, they are extremely hard targets for structure prediction and homology-based inference of sequence-function relationships and the great majority of REases remain structurally and evolutionarily unclassified.

Results

SfiI is a REase which recognizes the interrupted palindromic sequence 5'GGCCNNNN^NGGCC3' and generates 3 nt long 3' overhangs upon cleavage. SfiI is an archetypal Type IIF enzyme, which functions as a tetramer and cleaves two copies of the recognition site in a concerted manner. Its sequence shows no similarity to other proteins and nothing is known about the localization of its active site or residues important for oligomerization. Using the threading approach for protein fold-recognition, we identified a remote relationship between SfiI and BglI, a dimeric Type IIP restriction enzyme from the PD-DxK superfamily of nucleases, which recognizes the 5'GCCNNNN^NGGC3' sequence and whose structure in complex with the substrate DNA is available. We constructed a homology model of SfiI in complex with its target sequence and used it to predict residues important for dimerization, tetramerization, DNA binding and catalysis.

Conclusions

The bioinformatics analysis suggest that SfiI, a Type IIF enzyme, is more closely related to BglI, an "orthodox" Type IIP restriction enzyme, than to any other REase, including other Type IIF REases with known structures, such as NgoMIV. NgoMIV and BglI belong to two different, very remotely related branches of the PD-DxK superfamily: the α-class (EcoRI-like), and the β-class (EcoRV-like), respectively. Thus, our analysis provides evidence that the ability to tetramerize and cut the two DNA sequences in a concerted manner was developed independently at least two times in the evolution of the PD-DxK superfamily of REases. The model of SfiI will also serve as a convenient platform for further experimental analyses.     

Kinetic model of oxidation catalyzed by xanthine oxidase-the final enzyme in degradation of purine nucleosides and nucleotides

A new kinetic model is presented for analysis of experimental data of oxidation process catalyzed by milk xanthine oxidase. The kinetics for two substrates, xanthine and its analog 2-chloroadenine, in a broad pH range (5.8-9.0) are best described by an equation which is a rational function of degree 2:3 and 2:2, respectively.     

Targeted ablation of prostate carcinoma cells through LH receptor using Hecate-CGbeta conjugate: functional characteristic and molecular mechanism of cell death pathway

A Hecate-CGbeta conjugate (lytic peptide and beta-chorionic gonadotropin) selectively destroyed cells possessing LH receptors. This study described functional characteristics of the conjugate and the molecular mechanism of the cell death pathway in prostate cancer cells. Based on in vitro studies, we conclude that the conjugate kills cells possessing luteinizing hormone receptors (LHR) faster than Hecate alone. Competitive studies have shown that blocking of LHR by preincubation with chorionic gonadotropin (100 ng/ml) reduced toxicity of the conjugate in low concentrations. Further studies have also shown that the conjugate in treated cells both did not induce internucleosomal DNA fragmentation and did not induce morphological changes in cells characterized as having apoptotic features. These results proved that cells died by necrosis rather than apoptosis after the conjugate treatment.     

Genomic and proteomic analysis of mammary tumors arising in transgenic mice

Transforming growth factor-beta (TGF-beta) is the prototype of a large family of signaling molecules. TGF-beta signaling profoundly influences tumor development as demonstrated in several engineered mouse models. The present study was designed to identify differences by cDNA microarray and MALDI-TOF MS analyses in mammary carcinomas with and without TGF-beta signaling. The results demonstrate a significant potential for combination of profiling technologies to further understand the molecular mechanisms of breast cancer.     

In vitro contractile activity of porcine myometrium during luteolysis and early pregnancy: effect of oxytocin and progesterone

The present study was undertaken to elucidate the role of OT in myometrial contractility in sows. Spontaneous and OT-stimulated contractions of the inner circular (CM) and outer longitudinal (LM) layers isolated from cyclic (Days 14-16) and early pregnant (Days 14-16) sows were examined in six cyclic and six pregnant sows. In addition, the role of P(4) in the modulation of OT-induced uterine contractions was investigated. The contractile activity of the LM and CM layers was recorded in a tissue chamber filled with Krebs-Ringer solution. Myometrial contractility was expressed as area under the contractility curve (AUC) and frequency of contractions. Myometrial longitudinal and circular muscles exhibited spontaneous contractility in sows during both luteolysis and early-pregnancy. The mean AUC was higher (p<0.05) in the LM layer compared to the CM layer in both cyclic and pregnant animals. In addition, pregnant sows were characterized by higher AUC in both LM and CM layers in comparison to cyclic sows. The CM layer was unresponsive to examined treatments. Oxytocin (1-3x10(-8) and 1-3x10(-7)M) increased the AUC and frequency of contractions of the LM layer in both examined animal groups, being more effective during luteolysis (p<0.001) than early pregnancy (p<0.01). Response of the LM layer to OT appeared to be clearly related to the initial spontaneous level of contractility. This response to OT was inhibited (p<0.05) in the presence of OT antagonist (10(-6)M). Moreover, in pregnant sows, OT-stimulated contractile activity of myometrium was inhibited (p<0.05) by P(4) (10(-5)M). In conclusion, OT receptors present in myometrial cells (especially in the LM layer) are involved in the regulation of contractile activity of porcine myometrium during luteolysis and early-pregnancy. In addition, progesterone appears to be involved in this regulation.     

Insertion of the plant photosystem I subunit G into the thylakoid membrane

Subunit G of photosystem I is a nuclear-encoded protein, predicted to form two transmembrane alpha-helices separated by a loop region. We use in vitro import assays to show that the positively charged loop domain faces the stroma, whilst the N- and C-termini most likely face the lumen. PSI-G constructs in which a His- or Strep-tag is placed at the C-terminus or in the loop region insert with the same topology as wild-type photosystem I subunit G (PSI-G). However, the presence of the tags in the loop make the membrane-inserted protein significantly more sensitive to trypsin, apparently by disrupting the interaction between the loop and the PSI core. Knock-out plants lacking PSI-G were transformed with constructs encoding the C-terminal and loop-tagged PSI-G proteins. Experiments on thylakoids from the transgenic lines show that the C-terminally tagged versions of PSI-G adopt the same topology as wild-type PSI-G, whereas the loop-tagged versions affect the sensitivity of the loop region to trypsin, thus confirming the in vitro observations. Furthermore, purification of PSI complexes from transgenic plants revealed that all the tagged versions of PSI-G are incorporated and retained in the PSI complex, although the C-terminally tagged variants of PSI-G were preferentially retained. This suggests that the loop region of PSI-G is important for proper integration into the PSI core. Our experiments demonstrate that it is possible to produce His- and Strep-tagged PSI in plants, and provide further evidence that the topology of membrane proteins is dictated by the distribution of positive charges, which resist translocation across membranes.     

Fluorescence quenching and kinetic studies of conformational changes induced by DNA and cAMP binding to cAMP receptor protein from Escherichia coli

Cyclic AMP receptor protein (CRP) regulates the expression of more then 100 genes in Escherichia coli. It is known that the allosteric activation of CRP by cAMP involves a long-distance signal transmission from the N-terminal cAMP-binding domain to the C-terminal domain of CRP responsible for the interactions with specific sequences of DNA. In this report we have used a CRP mutant containing a single Trp13 located in the N-terminal domain of the protein. We applied the iodide and acrylamide fluorescence quenching method in order to study how different DNA sequences and cAMP binding induce the conformational changes in the CRP molecule. The results presented provide evidence for the occurrence of a long-distance conformational signal transduction within the protein from the C-terminal DNA-binding domain to the N-terminal domain of CRP. This conformational signal transmission depends on the promoter sequence. We also used the stopped-flow and Forster resonance energy transfer between labeled Cys178 of CRP and fluorescently labeled DNA sequences to study the kinetics of DNA-CRP interactions. The results thus obtained lead to the conclusion that CRP can exist in several conformational states and that their distribution is affected by binding of both the cAMP and of specific DNA sequences.