Antimicrobial Efficacy of Surface-Coated External Fixation Pins

In clinical applications, colonization of metal implants by adhesive and biofilm-forming bacteria not only prolong healing but create additional healthcare costs for implant revision and antimicrobial treatment. An in vitro assay was established investigating the antimicrobial surface activity of external fixation pins intended for use in bone fractures and deformities. Test articles made out of stainless steel and coated with a polymer-containing nanoparticulate silver were compared to non-coated reference controls out of stainless steel, copper and titanium. Staphylococcus epidermidis, known as a predominant cause for implant-related infections was used as test organism. Test pins and bacteria were incubated for a period of 20 h found to be sufficient for initiating biofilm formation. After removing non- and low-adherent bacteria by rinsing, two methods were used to isolate high-adherent (sessile) bacteria from the implant surfaces. Besides shaking the implants in a solution containing small glass beads, a cytobrush technique was used to mechanically harvest viable bacteria. Finally, the amount of detached bacteria was determined by plate counts. Several parameters identified to be critical within the different removal procedures such as the inoculum concentration and the shaking time in the presence of glass beads as well as time of the cytobrush treatment were analysed. The final test scheme resulted in the use of an inoculum of 10⁵ colony forming units (CFU) per millilitre, ten rinsing steps for the removal of low adherent bacteria and 5 min of shaking in the presence of glass beads, detaching the high-adherent bacteria. Due to subjective variations impacting the outcome of the procedure, the cytobrush technique was not favoured and finally rejected. Using the in vitro assay developed, it could be demonstrated that fixation pins coated with silver show a 3 log step reduction in the number of biofilm-forming bacteria compared to a non-coated stainless steel or titanium implant. Pins made out of copper showed the highest antimicrobial efficacy, as the number of detached bacteria was found to be below the detection limit, they served as a positive control within this test.     

Small molecule AKAP-protein kinase A (PKA) interaction disruptors that activate PKA interfere with compartmentalized cAMP signaling in cardiac myocytes

A-kinase anchoring proteins (AKAPs) tether protein kinase A (PKA) and other signaling proteins to defined intracellular sites, thereby establishing compartmentalized cAMP signaling. AKAP-PKA interactions play key roles in various cellular processes, including the regulation of cardiac myocyte contractility. We discovered small molecules, 3,3'-diamino-4,4'-dihydroxydiphenylmethane (FMP-API-1) and its derivatives, which inhibit AKAP-PKA interactions in vitro and in cultured cardiac myocytes. The molecules bind to an allosteric site of regulatory subunits of PKA identifying a hitherto unrecognized region that controls AKAP-PKA interactions. FMP-API-1 also activates PKA. The net effect of FMP-API-1 is a selective interference with compartmentalized cAMP signaling. In cardiac myocytes, FMP-API-1 reveals a novel mechanism involved in terminating β-adrenoreceptor-induced cAMP synthesis. In addition, FMP-API-1 leads to an increase in contractility of cultured rat cardiac myocytes and intact hearts. Thus, FMP-API-1 represents not only a novel means to study compartmentalized cAMP/PKA signaling but, due to its effects on cardiac myocytes and intact hearts, provides the basis for a new concept in the treatment of chronic heart failure.     

The Pseudo Signal Peptide of the Corticotropin-releasing Factor Receptor Type 2a Decreases Receptor Expression and Prevents Gi-mediated Inhibition of Adenylyl Cyclase Activity

The corticotropin-releasing factor receptor type 2a (CRF_2(a)R) belongs to the family of G protein-coupled receptors. The receptor possesses an N-terminal pseudo signal peptide that is unable to mediate targeting of the nascent chain to the endoplasmic reticulum membrane during early receptor biogenesis. The pseudo signal peptide remains uncleaved and consequently forms an additional hydrophobic receptor domain with unknown function that is unique within the large G protein-coupled receptor protein family. Here, we have analyzed the functional significance of this domain in comparison with the conventional signal peptide of the homologous corticotropin-releasing factor receptor type 1 (CRF₁R). We show that the presence of the pseudo signal peptide leads to a very low cell surface receptor expression of the CRF_2(a)R in comparison with the CRF₁R. Moreover, whereas the presence of the pseudo signal peptide did not affect coupling to the G_s protein, G_i-mediated inhibition of adenylyl cyclase activity was abolished. The properties mediated by the pseudo signal peptide were entirely transferable to the CRF₁R in signal peptide exchange experiments. Taken together, our results show that signal peptides do not only influence early protein biogenesis. In the case of the corticotropin-releasing factor receptor subtypes, the use of conventional and pseudo signal peptides have an unexpected influence on signal transduction.     

Pharmacochaperones post-translationally enhance cell surface expression by increasing conformational stability of wild-type and mutant vasopressin V2 receptors

Some membrane-permeable antagonists restore cell surface expression of misfolded receptors retained in the endoplasmic reticulum (ER) and are therefore termed pharmacochaperones. Whether pharmacochaperones increase protein stability, thereby preventing rapid degradation, or assist folding via direct receptor interactions or interfere with quality control components remains elusive. We now show that the cell surface expression and function (binding of the agonist) of the mainly ER-retained wild-type murine vasopressin V2 receptor GFP fusion protein (mV2R.GFP) is restored by the vasopressin receptor antagonists SR49059 and SR121463B with EC50 values similar to their KD values. This effect was preserved when protein synthesis was abolished. In addition, SR121463B rescued eight mutant human V2Rs (hV2Rs, three are responsible for nephrogenic diabetes insipidus) characterized by amino acid exchanges at the C-terminal end of transmembrane helix TM I and TM VII. In contrast, mutants with amino acid exchanges at the interface of TM II and IV were not rescued by either antagonist. The mechanisms involved in successful rescue of cell surface delivery are explained in a three-dimensional homology model of the antagonist-bound hV2R.     

Regulation of the coupling to different G proteins of rat corticotropin-releasing factor receptor type 1 in human embryonic kidney 293 cells

The regulation of G protein activation by the rat corticotropin-releasing factor receptor type 1 (rCRFR1) in human embryonic kidney (HEK)293 (HEK-rCRFR1) cell membranes was studied. Corresponding to a high and low affinity ligand binding site, sauvagine and other peptidic CRFR1 ligands evoked high and low potency responses of G protein activation, differing by 64-fold in their EC(50) values as measured by stimulation of [(35)S]GTPgammaS binding. Contrary to the low potency response, the high potency response was of lower GTPgammaS affinity, pertussis toxin (PTX)-insensitive, and homologously desensitized. Distinct desensitization was also observed in the adenylate cyclase activity, when its high potency stimulation was abolished and the activity became low potently inhibited by sauvagine. From these results and immunoprecipitation of [(35)S]GTPgammaS-bound Galpha(s) and Galpha(i) subunits it is concluded that the high and low potency [(35)S]GTPgammaS binding stimulation reflected coupling to G(s) and G(i) proteins, respectively, only G(s) coupling being homologously desensitized. Immunoprecipitation of [(35)S]GTPgammaS-bound Galpha(q/11) revealed additional coupling to G(q/11), which also was homologously desensitized. Although Galpha(q/11) coupling was PTX-insensitive, half of the sauvagine-stimulated accumulation of inositol phosphates in the cells was PTX-sensitive, suggesting involvement of G(i) in addition to G(q/11)in the stimulation of inositol metabolism. It is concluded that CRFR1 signals through at least two different ways, one leading to G(s)- and G(q/11)-mediated signaling steps and desensitization and another leading to G(i) -mediated signals without being desensitized. Furthermore, the concentrations of the stimulating ligand and GTP and desensitization may be part of a regulatory mechanism determining the actual ratio of the coupling of CRFR1 to different G proteins.     

角质形成细胞生长因子(KGF)在喉粘膜良性、癌前及恶性病变中的mRNA水平分析

利用原位杂交的方法检测KGFmRNA在正常喉粘膜上皮（N）、慢性非特异性炎症（IF）、不典型增生（DYS）及鳞癌（SCC）中的转录水平,探讨KGF在喉粘膜良性及恶性病变中的分布和可能的作用。结果表明,KGFmRNA不仅在间质中的成纤维细胞中表达,少量的炎细胞及血管内皮细胞中亦表达,而且从N、IF、DYS到SCC、KGFmRNA转录水平逐渐增强;上皮细胞及肿瘤性上皮细胞不表达KGFmRNA,KGFmRNA在分化差的SCC周围间质中表达较分化好的SCC周围间质增多。结论：KGF在上皮与间充质细胞的交互作用中发挥着重要的作用,对维持喉粘膜正常结构、代谢及喉癌的发生发展具有重要意义。     

Microtubules are needed for the perinuclear positioning of aquaporin-2 after its endocytic retrieval in renal principal cells

Water reabsorption in the renal collecting duct is regulated by arginine vasopressin (AVP). AVP induces the insertion of the water channel aquaporin-2 (AQP2) into the plasma membrane of principal cells, thereby increasing the osmotic water permeability. The redistribution of AQP2 to the plasma membrane is a cAMP-dependent process and thus a paradigm for cAMP-controlled exocytic processes. Using primary cultured rat inner medullary collecting duct cells, we show that the redistribution of AQP2 to the plasma membrane is accompanied by the reorganization of microtubules and the redistribution of the small GTPase Rab11. In resting cells, AQP2 is colocalized with Rab11 perinuclearly. AVP induced the redistribution of AQP2 to the plasma membrane and of Rab11 to the cell periphery. The redistribution of both proteins was increased when microtubules were depolymerized by nocodazole. In addition, the depolymerization of microtubules prevented the perinuclear positioning of AQP2 and Rab11 in resting cells, which was restored if nocodazole was washed out and microtubules repolymerized. After internalization of AQP2, induced by removal of AVP, forskolin triggered the AQP2 redistribution to the plasma membrane even if microtubules were depolymerized and without the previous positioning of AQP2 in the perinuclear recycling compartment. Collectively, the data indicate that microtubule-dependent transport of AQP2 is predominantly responsible for trafficking and localization of AQP2 inside the cell after its internalization but not for the exocytic transport of the water channel. We also demonstrate that cAMP-signaling regulates the localization of Rab11-positive recycling endosomes in renal principal cells. dynein; Rab11     

The effect of dibenzofuran substitutions in corticotropin-releasing factor

Summary As -turn-inducing motif, 4-(N-Fmoc-2-aminoethyl)-6-dibenzofuranpropionic acid was synthesized and incorporated into the corticotropin-releasing factor. The effect on structural and biological properties was investigated.     

Phylogenetic reconstruction of vertebrate Hox cluster duplications

In vertebrates and the cephalochordate, amphioxus, the closest vertebrate relative, Hox genes are linked in a single cluster. Accompanying the emergence of higher vertebrates, the Hox gene cluster duplicated in either a single step or multiple steps, resulting in the four-cluster state present in teleosts and tetrapods. Mammalian Hox clusters (designated A, B, C, and D) extend over 100 kb and are located on four different chromosomes. Reconstructing the history of the duplications and its relation to vertebrate evolution has been problematic due to the lack of alignable sequence information. In this study, the problem was approached by conducting a statistical analysis of sequences from the fibrillar-type collagens (I, II, III, and IV), genes closely linked to each Hox cluster which likely share the same duplication history as the Hox genes. We find statistical support for the hypothesis that the cluster duplication occurred as multiple distinct events and that the four-cluster situation arose by a three- step sequential process.