Sugar binding induced charge translocation in the melibiose permease from Escherichia coli

Electrogenic events associated with the activity of the melibiose permease (MelB), a transporter from Escherichia coli, were investigated. Proteoliposomes containing purified MelB were adsorbed to a solid supported lipid membrane, activated by a substrate concentration jump, and transient currents were measured. When the transporter was preincubated with Na(+) at saturating concentrations, a charge translocation in the protein upon melibiose binding could still be observed. This result demonstrates that binding of the uncharged substrate melibiose triggers a charge displacement in the protein. Further analysis showed that the charge displacement is neither related to extra Na(+) binding to the transporter, nor to the displacement of already bound Na(+) within the transporter. The electrogenic melibiose binding process is explained by a conformational change with concomitant displacement of charged amino acid side chains and/or a reorientation of helix dipoles. A kinetic model is suggested, in which Na(+) and melibiose binding are distinct electrogenic processes associated with approximately the same charge displacement. These binding reactions are fast in the presence of the respective cosubstrate (k > 50 s(-1)).     

Molecular structure and interaction of recombinant human type XVI collagen

Collagen XVI is a minor component of at least two different extracellular fibrillar networks of specialized regions of skin and cartilage. In skin, collagen XVI is integrated into particular fibrillin-rich microfibrils lacking an amorphous elastin core. In cartilage, collagen XVI is a component of small heterotypic D-banded fibrils, mainly occurring in the territorial matrix of chondrocytes. Here, we present the first direct evidence for the molecular structure and functional properties of these fibril-associated collagens with interrupted triple helices (FACIT). We have expressed recombinantly the full-length alpha1 chain of human collagen XVI in HEK 293 EBNA cells in large quantities using an episomal expression system. Secreted full-length recombinant collagen XVI forms stable disulfide-bonded homotrimers and is rapidly proteolytically processed to distinct fragments at specific protease sequence motifs, one resembling an aggrecanase recognition site. Limited trypsin digestion assays and thermal transition curves imply sequential thermal denaturation of individual triple helical domains of this recombinant collagen, similar to authentic collagen XVI. Molecular images of collagen XVI reveal rod-like molecules which harbor multiple sharp kinks attributing a highly flexible structure presumably introduced by non-collagenous (NC) regions. Terminally located cloverleaf-shaped nodules correspond to the large NC NC11 domain of trimeric collagen XVI. The total length of individual trimeric recombinant collagen XVI molecules constitutes about 240 nm as calculated by atomic force and negative staining electron microscopy. Recombinant collagen XVI interacts with fibrillin-1 and with fibronectin indicating multiple molecular interactions in which this ubiquitously expressed and versatile FACIT-collagen can participate. In vitro generated collagen XVI provides an indispensable tool for future determination of its function during supramolecular assembly of matrix aggregates and its role in maintenance, organization and interaction of fibrillar structures.     

Neurotrophic and neuroprotective effects of the neuregulin glial growth factor-2 on dopaminergic neurons in rat primary midbrain cultures

Glial growth factor-2 (GGF2) and other neuregulin (NRG) isoforms have been shown to play important roles in survival, migration, and differentiation of certain neural and non-neural cells. Because midbrain dopamine (DA) cells express the NRG receptor, ErbB4, the present study examined the potential neurotrophic and/or neuroprotective effects of GGF2 on cultured primary dopaminergic neurons. Embryonic day 14 rat mesencephalic cell cultures were maintained in serum-free medium and treated with GGF2 or vehicle. The number of tyrosine hydroxylase-positive (TH+) neurons and high-affinity [3H]DA uptake were assessed at day in vitro (DIV) 9. Separate midbrain cultures were treated with 100 ng/mL GGF2 on DIV 0 and exposed to the catecholamine-specific neurotoxin 6-hydroxydopamine (6-OHDA) on DIV 4. GGF2 treatment significantly increased DA uptake, the number of TH+ neurons, and neurite outgrowth when compared to the controls in both the serum-free and the 6-OHDA-challenged cultures. Furthermore, three NRG receptors were detected in the midbrain cultures by western blot analysis. Immunostaining for glial fibrillary acidic protein revealed that GGF2 also weakly promoted mesencephalic glial proliferation in the midbrain cultures. These results indicate that GGF2 is neurotrophic and neuroprotective for developing dopaminergic neurons and suggest a role for NRGs in repair of the damaged nigrostriatal system that occurs in Parkinson's disease.     

Insulin receptor substrate of 53 kDa links postsynaptic shank to PSD-95

The insulin receptor substrate of 53 kDa (IRSp53) is a target of the small GTPase cdc42 which is strongly enriched in the postsynaptic density of excitatory synapses. IRSp53 interacts with the postsynaptic shank1 scaffolding molecule in a cdc42 regulated manner. The functional significance of the cdc42/IRSp53 pathway in postsynaptic sites is however, unclear. Here we identify PSD-95 as a second synaptic interaction partner of IRSp53. Interaction is mediated by a C-terminal PDZ binding motif in IRSp53 and the second PDZ domain of PSD-95. In HEK cells, overexpressed IRSp53 induces filopodia and targets PSD-95 into these processes. Immunoprecipitation and immunocytochemistry experiments demonstrate that the interaction occurs at postsynaptic sites in the brain. By virtue of its PDZ-binding and SH3 domains, IRSp53 is capable of inducing the formation of a triple complex (shank1/IRSp53/PSD-95).     

Staurosporine restores GTPgammaS induced block of rapid endocytosis in chromaffin cells

Fast capacitance measurements demonstrated that chromaffin cells retrieve membrane by several kinetically different pathways. Here, we show that rapid endocytosis is blocked and slow endocytosis reduced by intracellular application of GTPgammaS, an activator of G-proteins, but not by the competitive blocker GDPbetaS. The inhibition of rapid endocytosis by GTPgammaS can be restored with GDPbetaS or staurosporine completely. But only staurosporine partially abolishes the reduction of slow endocytosis by GTPgammaS. Besides triggering exocytosis, GTPgammaS elicits large exo- and endocytotic vesicles that contributed significantly to the total membrane traffic, indicating a third pathway of membrane shuttle.     

Munc13-1-mediated vesicle priming contributes to secretory amyloid precursor protein processing

The amyloid precursor protein (APP) gives rise toc beta-amyloid peptides, which are the main constituents of senile plaques in brains of Alzheimer's disease patients. Non-amyloidogenic processing of the APP can be stimulated by phorbol esters (PEs) and by intracellular diacylglycerol (DAG) generation. This led to the hypothesis that classical and novel protein kinase Cs (PKCs), which are activated by DAG/PEs, regulate APP processing. However, in addition to PKCs, there are other DAG/PE receptors present in neurons that may participate in the modulation of APP processing. Munc13-1, a presynaptic protein with an essential role in synaptic vesicle priming, represents such an alternative target of the DAG second messenger pathway. Using Munc13-1 knock-out mice and knock-in mice expressing a Munc13-1(H567K) variant deficient in DAG/PE binding, we determined the relative contributions of PKCs and Munc13-1 to PE-stimulated secretory APP processing. We establish that, in addition to PKC, Munc13-1 significantly contributes to the regulation of secretory APP metabolism.     

Insulin-like growth factors as diagnostic tools in growth hormone deficiency during childhood and adolescence: the KIGS experience

Growth hormone (GH) deficiency in children covers a spectrum of disorders involving an impairment in GH secretion and a clinical syndrome characterized by permanent stunting of growth. Ascertaining impairments in GH secretion directly is complex, especially if GH deficiency (GHD) is isolated and not caused by congenital or acquired pituitary defects or genetic abnormalities. It has been established that the concentrations of GH-dependent peptides, such as insulin-like growth factor I (IGF-I) and IGF-binding protein 3 (IGFBP-3), are low in patients with GHD. Their levels are, however, also influenced by a multitude of factors, such as age, gender, height, liver function, nutritional status and other hormones. In addition, the type of complex formed, e.g. either binary or ternary, may influence the measurements of IGFs and their binding proteins. Therefore, levels of IGF-I and IGFBP-3 are generally lower in short children compared with age-matched norms. The reported diagnostic value of sub-normal basal levels of IGF-I and IGFBP-3 is, in terms of sensitivity and specificity, approximately 70%. Thus, definite proof of GHD can only be achieved by means of GH measurements. As the diagnosis of GHD is somewhat unlikely if IGF testing shows normal values, it is clearly advantageous to schedule these tests as part of the initial diagnostic work-up in short children, as their implementation is not only practical but also inexpensive. The Pfizer International Growth Database (KIGS) analysis of IGF-I (n = 2,750) and IGFBP-3 (n = 1,300) levels in children with idiopathic GHD shows that these two parameters are now firmly embedded in diagnostic strategies around the world.     

In search of the "hair cycle clock": a guided tour

The hair follicle, a unique characteristic of mammals, represents a stem cell-rich, prototypic neuroectodermal-mesodermal interaction system. This factory for pigmented epithelial fibers is unique in that it is the only organ in the mammalian body which, for its entire lifetime, undergoes cyclic transformations from stages of rapid growth (anagen) to apoptosis-driven regression (catagen) and back to anagen, via an interspersed period of relative quiescence (telogen). While it is undisputed that the biological "clock" that drives hair follicle cycling resides in the hair follicle itself, the molecular nature of the underlying oscillator system remains to be clarified. To meet this challenge is of profound general interest, since numerous key problems of modern biology can be studied exemplarily in this versatile model system. It is also clinically important, since the vast majority of patients with hair growth disorders suffers from an undesired alteration of hair follicle cycling. Here, we sketch basic background information and key concepts that one needs to keep in mind when exploring the enigmatic "hair cycle clock"(HCC), and summarize competing models of the HCC. We invite the reader on a very subjective guided tour, which focuses on our own trials, errors, and findings toward the distant goal of unravelling one of the most fascinating mysteries of biology: Why does the hair follicle cycle at all? How does it do it? What are the key players in the underlying molecular controls? Attempting to offer at least some meaningful answers, we share our prejudices and perspectives, and define crucial open questions.