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Respiratory chain complex I contains 8-9 iron-sulfur clusters. In several cases, the assignment of these clusters to subunits and binding motifs is still ambiguous. To test the proposed ligation of the tetranuclear iron-sulfur cluster N5 of respiratory chain complex I, we replaced the conserved histidine 129 in the 75-kDa subunit from Yarrowia lipolytica with alanine. In the mutant strain, reduced amounts of fully assembled but destabilized complex I could be detected. Deamino-NADH: ubiquinone oxidoreductase activity was abolished completely by the mutation. However, EPR spectroscopic analysis of mutant complex I exhibited an unchanged cluster N5 signal, excluding histidine 129 as a cluster N5 ligand.
Stable supercomplexes of bacterial respiratory chain complexes III (ubiquinol:cytochrome c oxidoreductase) and IV (cytochrome c oxidase) have been isolated as early as 1985 (Berry, E. A., and Trumpower, B. L. (1985) J. Biol. Chem. 260, 2458-2467). However, these assemblies did not comprise complex I (NADH:ubiquinone oxidoreductase). Using the mild detergent digitonin for solubilization of Paracoccus denitrificans membranes we could isolate NADH oxidase, assembled from complexes I, III, and IV in a 1:4:4 stoichiometry. This is the first chromatographic isolation of a complete “respirasome.” Inactivation of the gene for tightly bound cytochrome c552 did not prevent formation of this supercomplex, indicating that this electron carrier protein is not essential for structurally linking complexes III and IV. Complex I activity was also found in the membranes of mutant strains lacking complexes III or IV. However, no assembled complex I but only dissociated subunits were observed following the same protocols used for electrophoretic separation or chromatographic isolation of the supercomplex from the wild-type strain. This indicates that the P. denitrificans complex I is stabilized by assembly into the NADH oxidase supercomplex. In addition to substrate channeling, structural stabilization of a membrane protein complex thus appears as one of the major functions of respiratory chain supercomplexes.
Quantitative analysis of the cardiac fibroblast transcriptome implications for NO/cGMP signaling
(2004)
Cardiac fibroblasts regulate tissue repair and remodeling in the heart. To quantify transcript levels in these cells we performed a comprehensive gene expression study using serial analysis of gene expression (SAGE). Among 110,169 sequenced tags we could identify 30,507 unique transcripts. A comparison of SAGE data from cardiac fibroblasts with data derived from total mouse heart revealed a number of fibroblast-specific genes. Cardiac fibroblasts expressed a specific collection of collagens, matrix proteins and metalloproteinases, growth factors, and components of signaling pathways. The NO/cGMP signaling pathway was represented by the mRNAs for α1 and β1 subunits of guanylyl cyclase, cGMP-dependent protein kinase type I (cGK I), and, interestingly, the G-kinase-anchoring protein GKAP42. The expression of cGK I was verified by RT-PCR and Western blot. To establish a functional role for cGK I in cardiac fibroblasts we studied its effect on cell proliferation. Selective activation of cGK I with a cGMP analog inhibited the proliferation of serum-stimulated cardiac fibroblasts, which express cGK I, but not higher passage fibroblasts, which contain no detectable cGK I. Currently, our data suggest that cGK I mediates the inhibitory effects of the NO/cGMP pathway on cardiac fibroblast growth. Furthermore the SAGE library of transcripts expressed in cardiac fibroblasts provides a basis for future investigations into the pathological regulatory mechanisms underlying cardiac fibrosis.
The development of image-guided neurosurgery represents a substantial improvement in the microsurgical treatment of tumors, vascular malformations and other intracranial lesions. Despite the wide applicability and many fascinating aspects of image-guided navigation systems, a major drawback of this technology is they use images, mainly MRI pictures, acquired preoperatively, on which the planning of the operative procedure as well as its intraoperative performance is based. As dynamic changes of the intracranial contents regularly occur during the surgical procedure, the surgeon is faced with a continuously changing intraoperative field. Only intraoperatively acquired images will provide the neurosurgeon with the information he needs to perform real intraoperative image-guided surgery. A number of tools have been developed in recent years, like intraoperative ultrasound and dedicated moveable intraoperative CT units. Because of its excellent imaging qualities, combined with the avoidance of ionizing radiation, MRI currently is and definitely will be in the future for the superior imaging method for intraoperative image guidance. In this short overview, the development as well as some of the current and possible future applications of MRI-guided neurosurgery is outlined.
Alix/AIP1 is an adaptor protein involved in regulating the function of receptor and cytoskeleton-associated tyrosine kinases. Here, we investigated its interaction with and regulation by Src. Tyr319 of Alix bound the isolated Src homology-2 (SH2) domain and was necessary for interaction with intact Src. A proline-rich region in the C terminus of Alix bound the Src SH3 domain, but this interaction was dependent on the release of the Src SH2 domain from its Src internal ligand either by interaction with Alix Tyr319 or by mutation of Src Tyr527. Src phosphorylated Alix at a C-terminal region rich in tyrosines, an activity that was stimulated by the presence of the Alix binding partner SETA/CIN85. Phosphorylation of Alix by Src caused it to translocate from the membrane and cytoskeleton to the cytoplasm and reduced its interaction with binding partners SETA/CIN85, epidermal growth factor receptor, and Pyk2. As a consequence of this, Src antagonized the negative regulation of receptor tyrosine kinase internalization and cell adhesion by Alix. We propose a model whereby Src antagonizes the effects of Alix by phosphorylation of its C terminus, leading to the disruption of interactions with target proteins.
Nitric oxide (NO)-sensitive soluble guanylyl cyclase (sGC) is the major cytosolic receptor for NO, catalyzing the conversion of GTP to cGMP. In a search for proteins specifically interacting with human sGC, we have identified the multidomain protein AGAP1, the prototype of an ArfGAP protein with a GTPase-like domain, Ankyrin repeats, and a pleckstrin homology domain. AGAP1 binds through its carboxyl terminal portion to both the α1 and β1 subunits of sGC. We demonstrate that AGAP1 mRNA and protein are co-expressed with sGC in human, murine, and rat cells and tissues and that the two proteins interact in vitro and in vivo. We also show that AGAP1 is prone to tyrosine phosphorylation by Src-like kinases and that tyrosine phosphorylation potently increases the interaction between AGAP1 and sGC, indicating that complex formation is modulated by reversible phosphorylation. Our findings may hint to a potential role of AGAP1 in integrating signals from Arf, NO/cGMP, and tyrosine kinase signaling pathways.
The ubiquitin (Ub) ligase Cbl plays a critical role in attenuation of receptor tyrosine kinase (RTK) signaling by inducing ubiquitination of RTKs and promoting their sorting for endosomal degradation. Herein, we describe the identification of two novel Cbl-interacting proteins, p70 and Clip4 (recently assigned the names Sts-1 and Sts-2, respectively), that inhibit endocytosis of epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor. Sts-1 and Sts-2 contain SH3 domains that interacted with Cbl, Ub-associated domains, which bound directly to mono-Ub or to the EGFR/Ub chimera as well as phosphoglycerate mutase domains that mediated oligomerization of Sts-1/2. Ligand-induced recruitment of Sts-1/Sts-2 into activated EGFR complexes led to inhibition of receptor internalization, reduction in the number of EGFR-containing endocytic vesicles, and subsequent block of receptor degradation followed by prolonged activation of mitogenic signaling pathways. On the other hand, interference with Sts-1/Sts-2 functions diminished ligand-induced receptor degradation, cell proliferation, and oncogenic transformation in cultured fibroblasts. We suggest that Sts-1 and Sts-2 represent a novel class of Ub-binding proteins that regulate RTK endocytosis and control growth factor-induced cellular functions.
Intra-arterial (IA) chemotherapy for curative treatment of head and neck cancer experienced a revival in the last decade. Mainly, it was used in concurrent combination with radiation in organ-preserving settings. The modern method of transfemoral approach for catheterisation, superselective perfusion of the tumour-feeding vessel, and high-dose (150 mg m−2) administration of cisplatin with parallel systemic neutralisation with sodium thiosulphate (9 g m−2) made preoperative usage feasible. The present paper presents the results of a pilot study on a population of 52 patients with resectable stage 1–4 carcinomas of the oral cavity and the oropharynx, who were treated with one cycle of preoperative IA chemotherapy executed as mentioned above and radical surgery. There have been no interventional complications of IA chemotherapy, and acute side effects have been low. One tracheotomy had to be carried out due to swelling. The overall clinical local response has been 69%. There was no interference with surgery, which was carried out 3–4 weeks later. Pathological complete remission was assessed in 25%. The mean observation time was 3 years. A 3-year overall and disease-free survival was 82 and 69%, respectively, and at 5 years 77 and 59%, respectively. Survival results were compared to a treatment-dependent prognosis index for the same population. As a conclusion, it can be stated that IA high-dose chemotherapy with cisplatin and systemic neutralisation in a neoadjuvant setting should be considered a feasible, safe, and effective treatment modality for resectable oral and oropharyngeal cancer. The low toxicity of this local chemotherapy recommends usage especially in stage 1–2 patients. The potential of survival benefit as indicated by the comparison to the prognosis index should be controlled in a randomised study.
Electric stimulation of the auditory nerve via cochlear implants has made the treatment of sensory deafness possible. Advanced signal processing and stimulation paradigms have led to continuously improved results in speech understanding. Consequently, indication criteria have been extended to patients with profound and severe-to-profound hearing loss and limited speech understanding with conventional acoustic amplification.
Outside this group, a considerable number of patients presents with rather wellpreserved, low frequency hearing of 30-60 dB up to 1 kHz, but severe loss in the mid to high frequency range of more than 60-70 dB. Monosyllabic word scores in these patients do not generally exceed 35%, due to missing consonant information. But, even increasing the audibility of these high frequencies by acoustic amplification still has very limited efficiency for discriminating speech, and therefore, these patients obtain only minor benefit from conventional hearing aids. On the other hand, standard cochlear implantation would carry a high risk of causing complete hearing loss. This situation has led to considering a combination of both modes of stimulation for these patients who are on the borderline between hearing aids and cochlear implant.
In our present model, the surviving low frequency region of the cochlea could still be stimulated acoustically-combined with additional electrical stimulation of the impaired mid and high frequency region of the cochlea.
Several questions still have to be answered with regard to combined electric and acoustic stimulation (EAS). The possible interaction of electric and acoustic stimuli on the different levels off the auditory system is a major issue. Animal experiments clearly demonstrate that tuning properties of auditory neurons, in response to acute acoustic stimulation, are essentially preserved in the presence of electric stimulation even at high levels of electric stimulation, and that chronic electric stimulation of tie intact inner ear does not have a significant effect on the compound action potentials (CAP) thresholds or inner ear function.
In a previous report, we were able to show that this combined F.A.S of the auditory system is possible in humans, and that it has a synergistic effect on speech understanding. Further major issues regard the surgical feasibility and reproducibility of cochlear implantation with the preservation of residual hearing.
Encouraged by our findings, a clinical study was initiated on the application of EAS. So far, seven adults have been included in this study. In addition, one child has been implanted outside the study.
Nitric oxide (NO) represents a short lived mediator that pivotally drives keratinocyte movements during cutaneous wound healing. In this study, we have identified p68 DEAD box RNA helicase (p68) from an NO-induced differential keratinocyte cDNA library. Subsequently, we have analyzed regulation of p68 by wound-associated mediators in human and murine keratinocytes. NO, serum, growth factors, and pro-inflammatory cytokines were potent inducers of p68 expression in the cells. p68 was constitutively expressed in the epithelial compartment of murine skin. Upon injury, we found a transient down-regulation of overall p68 protein in wound tissue. However, p68 did not completely disappear during early wound repair, as we found an expression of p68 protein in isolated wound margin tissue 24 h after wounding. Moreover, immunohistochemistry and cell fractionation analysis revealed a restricted localization of p68 in keratinocyte nuclei of the developing epithelium. Accordingly, cultured keratinocytes also showed a nuclear localization of the helicase. Moreover, confocal microscopy revealed a strong localization of p68 protein within the nucleoli of the cells. Functional analyses demonstrated that p68 strongly participated in keratinocyte proliferation and gene expression. Keratinocytes that constitutively overexpressed p68 protein were characterized by a marked increase in serum-induced proliferation and vascular endothelial growth factor expression, whereas down-regulation of endogenous p68 using small interfering RNA markedly attenuated serum-induced proliferation and vascular endothelial growth factor expression. Altogether, our results suggest a tightly controlled expression and nucleolar localization of p68 in keratinocytes in vitro and during skin repair in vivo that functionally contributes to keratinocyte proliferation and gene expression.