Initiation of lactation and the provision of human milk to preterm infants in German neonatal intensive care units from the mothers' perspective.

BACKGROUND: If infants with a very low birth weight (VLBW) are to be fed exclusively with human milk, it is essential to focus on lactation initiation. The aim of the study is to learn more about the current state of lactation initiation and human milk provision in neonatal intensive care units in Germany from the mothers' perspective. METHODS: Written surveys were conducted with mothers of VLBW infants to learn more about the timing of initiation of lactation, pumping frequency during the first three days postpartum and feeding of the preterm infant during hospitalisation. RESULTS: The data of 437 mothers (response rate: 44.7%) were included in the analyses. Of these, only 7.8% stated that they had initiated lactation immediately after delivery and 38.2% within 6 h. In terms of pumping frequency, 50.1% pumped 7-9 times a day within the first 3 days postpartum; 60.9% reported that their infant received formula feedings during the hospital stay. CONCLUSION: Overall, deficits were still evident with regard to the initiation of lactation in mothers of VLBW infants in Germany, resulting in a large proportion of VLBW infants receiving formula in the hospital. TRIAL REGISTRATION: German Clinical Trial Register: DRKS00017755 .

Diagnostics and therapy of bilateral choanal atresia in association with CHARGE syndrome.

BACKGROUND: Bilateral choanal atresia in patients with CHARGE syndrome becomes symptomatic immediately after birth. A prompt diagnosis, the implementation of sufficient preliminary measures, and the delivery of surgical therapy are crucial. This article is intended to assist in terms of diagnostics and a therapy recommendation. METHODS: We performed a retrospective study using the medical records of all newborns in the University Hospital in Bonn, diagnosed with bilateral choanal atresia and CHARGE syndrome and underwent surgery at the Department of Otorhinolaryngology, Head and Neck Surgery. RESULTS: A total of 21 patients have been treated with a unilateral or bilateral choanal atresia. 14 patients were primarily treated with transnasal endoscopy or underwent transnasal endoscopic surgery as a follow-up intervention (73.68%). Nine patients had a syndromal appearance, which was considered a definite diagnosis in six patients (five with CHARGE syndrome). All five patients with CHARGE syndrome received transnasal endoscopic treatment and a stent was inserted. DISCUSSION: Bilateral choanal atresia can be a life-threatening situation requiring acute measures. The therapeutic trend goes towards transnasal endoscopic resection. Primary intervention should be: minimally invasive, one-stage surgery, functional, and associated with low complication rates. Patency can be increased by saline irrigations, topical corticosteroids, endoscopic controls, and regular dilatation. The insertion of stents is controversially discussed but can be useful in syndromal patients. However, adjuvant therapy with a stent and mitomycin C is increasingly being abandoned. A significantly higher recurrence rate must be expected in association with CHARGE syndrome. Stenting should be considered on an individual basis. Continuous training and support of the parents are obligatory.

Essential cooperation of N-cadherin and neuroligin-1 in the transsynaptic control of vesicle accumulation.

Cell adhesion molecules are key players in transsynaptic communication, precisely coordinating presynaptic differentiation with postsynaptic specialization. At glutamatergic synapses, their retrograde signaling has been proposed to control presynaptic vesicle clustering at active zones. However, how the different types of cell adhesion molecules act together during this decisive step of synapse maturation is largely unexplored. Using a knockout approach, we show that two synaptic adhesion systems, N-cadherin and neuroligin-1, cooperate to control vesicle clustering at nascent synapses. Live cell imaging and fluorescence recovery after photobleaching experiments at individual synaptic boutons revealed a strong impairment of vesicle accumulation in the absence of N-cadherin, whereas the formation of active zones was largely unaffected. Strikingly, also the clustering of synaptic vesicles triggered by neuroligin-1 overexpression required the presence of N-cadherin in cultured neurons. Mechanistically, we found that N-cadherin acts by postsynaptically accumulating neuroligin-1 and activating its function via the scaffolding molecule S-SCAM, leading, in turn, to presynaptic vesicle clustering. A similar cooperation of N-cadherin and neuroligin-1 was observed in immature CA3 pyramidal neurons in an organotypic hippocampal network. Moreover, at mature synapses, N-cadherin was required for the increase in release probability and miniature EPSC frequency induced by expressed neuroligin-1. This cooperation of two cell adhesion systems provides a mechanism for coupling bidirectional synapse maturation mediated by neuroligin-1 to cell type recognition processes mediated by classical cadherins.

The presynaptic cytomatrix of brain synapses.

Synapses are principal sites for communication between neurons via chemical messengers called neurotransmitters. Neurotransmitters are released from presynaptic nerve terminals at the active zone, a restricted area of the cell membrane situated exactly opposite to the postsynaptic neurotransmitter reception apparatus. At the active zone neurotransmitter-containing synaptic vesicles (SVs) dock, fuse, release their content and are recycled in a strictly regulated manner. The cytoskeletal matrix at the active zone (CAZ) is thought to play an essential role in the organization of this SV cycle. Several multi-domain cytoskeleton-associated proteins, including RIM, Bassoon, Piccolo/Aczonin and Munc-13, have been identified, which are specifically localized at the active zone and thus are putative molecular components of the CAZ. This review will summarize our present knowledge about the structure and function of these CAZ-specific proteins. Moreover, we will review our present view of how the exocytotic and endocytic machineries at the site of neurotransmitter release are linked to and organized by the presynaptic cytoskeleton. Finally, we will summarize recent progress that has been made in understanding how active zones are assembled during nervous system development.

Mrnp41 (Rae 1p) associates with microtubules in HeLa cells and in neurons.

Mrnp41 (hRae1p) is an evolutionarily highly conserved protein, which is a potential component of mRNP particles and plays a role in nuclear mRNA export. The protein is mainly localized at the nuclear pore complex, but is also associated with distinct nuclear domains and with a meshwork of numerous small particles in the cytoplasm (Kraemer and Blobel (1997): Proc. Natl. Acad. Sci. USA 91, 1519-1523). We show that the cytoplasmic pattern of mrnp41 is sensitive to treatment with the microtubule (MT)-depolymerizing drug nocodazole which causes disappearance of mrnp41 from the cell periphery and concentration around the nucleus. By immunofluorescence we demonstrate that mrnp41 colocalizes with MT in HeLa cells and displays an MT-like distribution in cultured neurons. Association of mrnp41 with MT is further demonstrated by copurification with MT from pig brain throughout several steps of polymerization and depolymerization. Separation of MT-associated proteins (MAPs) by phosphocellulose (PC) chromatography showed copurification of mrnp41 with MAPs. These data show an association of mrnp41 with MT and, moreover, demonstrate that an intact MT system is necessary for dispersion of mrnp41-containing particles to the cellular periphery. The essential role of mrnp41 in spindle pole separation and cell cycle progression may also be related to its ability to bind to MTs.

Membrane association of presynaptic cytomatrix protein bassoon.

Components of the specialized cytomatrix at active zones of presynaptic nerve terminals are thought to be involved in organizing synaptic events such as immobilisation or translocation of synaptic vesicles and assemblingactive zone components. The 420-kDa non-transmembraneprotein Bassoon is a specific componentof the presynaptic cytomatrix that shares features with both cytoskeleton-associated and peripheral-membrane proteins. Using immunogold electron microscopy we show here that synapse associated Bassoon is distributed in a subregion of active zones. Using a biochemical assay we show that a fraction of Bassoon is membrane associated. Electron microscopy performed on the same biochemical fraction further revealed that Bassoon is associated with vesicular structures. Together these data suggest that at least a fraction of Bassoon is associated with a membraneous compartment in neurons.

The X-ray crystal structure of neuronal Sec1 from squid sheds new light on the role of this protein in exocytosis.

BACKGROUND: Sec1-like molecules have been implicated in a variety of eukaryotic vesicle transport processes including neurotransmitter release by exocytosis. They regulate vesicle transport by binding to a t-SNARE from the syntaxin family. This process is thought to prevent SNARE complex formation, a protein complex required for membrane fusion. Whereas Sec1 molecules are essential for neurotransmitter release and other secretory events, their interaction with syntaxin molecules seems to represent a negative regulatory step in secretion. RESULTS: Here we report the X-ray crystal structure of a neuronal Sec1 homologue from squid, s-Sec1, at 2.4 A resolution. Neuronal s-Sec1 is a modular protein that folds into a V-shaped three-domain assembly. Peptide and mutagenesis studies are discussed with respect to the mechanism of Sec1 regulation. Comparison of the structure of squid s-Sec1 with the previously determined structure of rat neuronal Sec1 (n-Sec1) bound to syntaxin-1a indicates conformational rearrangements in domain III induced by syntaxin binding. CONCLUSIONS: The crystal structure of s-Sec1 provides the molecular scaffold for a number of molecular interactions that have been reported to affect Sec1 function. The structural differences observed between s-Sec1 and the structure of a rat n-Sec1-syntaxin-1a complex suggest that local conformational changes are sufficient to release syntaxin-1a from neuronal Sec1, an active process that is thought to involve additional effector molecule(s).

Crystallization and preliminary X-ray analysis of squid neuronal Sec1.

Sec1 protein family members are involved in the regulation of all intracellular SNARE-mediated (SNARE = soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) vesicle-fusion processes in a step preceding membrane fusion and have been shown to interact with t-SNAREs. To better understand the structural basis and the role of Sec1 in the regulation of the SNARE-complex formation, neuronal Sec1 from the squid Loligo pealei has been expressed and crystallized; this invertebrate protein shows a high sequence homology to the human neuronal Sec1, Munc18a. Here, the production of diffraction-quality native crystals, which belong to space group P3(1)21 and diffract to 3.3 A resolution, is described. In addition, selenomethionyl n-Sec1 crystals in space groups P3(1)21 and P2(1) have been generated. Preliminary analysis of the monoclinic space group indicates that these crystals diffract to a resolution higher than 2.5 A.

Protein interactions implicated in neurotransmitter release.

Biochemical evidence indicates that the exocytotic release of neurotransmitters involves both evolutionary conserved membrane proteins, the SNAREs, as well as ubiquitous cytosolic fusion proteins, NSF and SNAPs. We have analyzed the biochemical properties and the physiological effects of these proteins. Our data suggest models how NSF, SNAPs and SNAREs may function in neurotransmitter exocytosis.

A neuronal Sec1 homolog regulates neurotransmitter release at the squid giant synapse.

Sec1-related proteins are essential for membrane fusion at distinct stages of the constitutive and regulated secretory pathways in eukaryotic cells. Studies of neuronal isoforms of the Sec1 protein family have yielded evidence for both positive and negative regulatory functions of these proteins in neurotransmitter release. Here, we have identified a squid neuronal homolog (s-Sec1) of Sec1 proteins and examined its function in neurotransmitter release at the squid giant synapse. Microinjection of s-Sec1 into the presynaptic terminal of the giant synapse inhibited evoked neurotransmitter release, but this effect was prevented by coinjecting the cytoplasmic domain of squid syntaxin (s-syntaxin), one of the binding partners of s-Sec1. A 24 amino acid peptide fragment of s-Sec1, which inhibited the binding of s-Sec1 to s-syntaxin in vitro, completely blocked release, suggesting an essential function of the s-Sec1/s-syntaxin interaction in transmitter release. Electron microscopy showed that injection of s-Sec1 did not change the spatial distribution of synaptic vesicles at presynaptic release sites ("active zones"), whereas the inhibitory peptide increased the number of docked vesicles. These distinct morphological effects lead us to conclude that Sec1 proteins function at different stages of synaptic vesicle exocytosis, and that an interaction of s-Sec1 with syntaxin-at a stage blocked by the peptide-is necessary for docked vesicles to fuse.

Regulation of neurotransmitter release kinetics by NSF.

NSF (N-ethylmaleimide-sensitive factor) is an adenosine triphosphatase (ATPase) that contributes to a protein complex essential for membrane fusion. The synaptic function of this protein was investigated by injecting, into the giant presynaptic terminal of squid, peptides that inhibit the ATPase activity of NSF stimulated by the soluble NSF attachment protein (SNAP). These peptides reduced the amount and slowed the kinetics of neurotransmitter release as a result of actions that required vesicle turnover and occurred at a step subsequent to vesicle docking. These results define NSF as an essential participant in synaptic vesicle exocytosis that regulates the kinetics of neurotransmitter release and, thereby, the integrative properties of synapses.

Disruption of syntaxin-mediated protein interactions blocks neurotransmitter secretion.

The membrane protein syntaxin participates in several protein-protein interactions that have been implicated in neurotransmitter release. To probe the physiological importance of these interactions, we microinjected into the squid giant presynaptic terminal botulinum toxin C1, which cleaves syntaxin, and the H3 domain of syntaxin, which mediates binding to other proteins. Both reagents inhibited synaptic transmission yet did not affect the number or distribution of synaptic vesicles at the presynaptic active zone. Recombinant H3 domain inhibited the interactions between syntaxin and SNAP-25 that underlie the formation of stable SNARE complexes in vitro. These data support the notion that syntaxin-mediated SNARE complexes are necessary for docked synaptic vesicles to fuse.

SNAP-mediated protein-protein interactions essential for neurotransmitter release.

The constitutive fusion of transport vesicles with intracellular membranes requires soluble proteins called SNAPs. Certain presynaptic proteins implicated in synaptic vesicle exocytosis also bind SNAPs, suggesting that SNAPs participate in the calcium-regulated membrane fusion events mediating neurotransmitter release. Here we show that injection of recombinant SNAPs into the giant synapse of squid enhances transmitter release. Conversely, injection of peptides designed to mimic the sites at which SNAP interacts with its binding partners inhibits transmitter release downstream of synaptic vesicle docking. A SNAP-dependent protein complex must therefore mediate transmitter release, showing that transmitter release shares a common molecular mechanism with constitutive membrane fusion.