Properties of in situ generated gold nanoparticles in the cellular context.

Gold nanostructures that serve as probes for nanospectroscopic analysis of eukaryotic cell cultures can be obtained by the in situ reduction of tetrachloroauric acid (HAuCl4). To understand the formation process of such intracellularly grown particles depending on the incubation medium, the reaction was carried out with 3T3 fibroblast cells in three different incubation media, phosphate buffer, Dulbecco's Modified Eagle Medium (DMEM), and standard cell culture medium (DMEM with fetal calf serum). The size, the optical properties, the biomolecular corona, and the localization of the gold nanoparticles formed in situ vary for the different conditions. The combination of surface-enhanced Raman scattering (SERS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) microscopic mapping and transmission electron microscopy (TEM) provides complementary perspectives on plasmonic nanoparticles and non-plasmonic gold compounds inside the cells. While for the incubation with HAuCl4 in PBS, gold particles provide optical signals from the nucleus, the incubation in standard cell culture medium leads to scavenging of the toxic molecules and the formation of spots of high gold concentration in the cytoplasm without formation of SERS-active particles inside the cells. The biomolecular corona of nanoparticles formed in situ after incubation in buffer and DMEM differs, suggesting that different intracellular molecular species serve for reduction and stabilization. Comparison with data obtained from ready-made gold nanoparticles suggests complementary application of in situ and ex situ generated nanostructures for optical probing.

Treatment efficiency of mini-implant-borne distalization depending on age and second-molar eruption.

OBJECTIVE: The aim of this study was to evaluate the efficiency of molar distalization depending on age and second-molar eruption using the Beneslider. MATERIALS AND METHODS: Treatment of 51 patients (mean age 17.8 ± 9.6 years) was investigated retrospectively by means of pre- and posttreatment cephalograms. Patients were divided into three groups: 14 children with unerupted upper second molars (group 1), 23 adolescents with second molar in place (group 2), and 14 adults (group 3). The distalization forces applied were 2.4 N in group 1 and 5.0 N in groups 2 and 3. Treatment changes were evaluated and examined statistically for significant differences. RESULTS: In all patients a Class I molar relationship was achieved. All mini-implants remained stable during treatment. Mean distalization distance as measured by the displacement of the center of resistance was 3.6 ± 1.9 mm (range 1.2-8.5 mm depending on treatment needs). Since no significant tipping was detected, the type of movement can be described as bodily movement. Mean overall distalization speed was 0.6 ± 0.4 mm per month. There were no statistical differences between the groups. CONCLUSION: We found the Beneslider to be an effective appliance that enables bodily distalization in adequate treatment time. The higher resistance due to erupted second molars can be compensated by the use of higher forces without significantly reducing distalization speed.

Accuracy of torque-limiting devices for mini-implant removal: an in vitro study.

INTRODUCTION: Mini-implants, due to their potential for osseointegration, are exposed to torque levels that may cause them to fracture during removal. Thus, it is advisable to control the torque levels applied during mini-implant removal. MATERIALS AND METHODS: A torque sensor with strain gauges was used to analyze torque-limiting devices for their accuracy in reverse (counterclockwise) operation. Eight devices were tested in this manner, including a group of hand-operated drivers (n=3), a group of battery-operated drivers (n=4), and a mains-operated surgical unit (n=1). Each device was analyzed eight times at each of the various torque levels. Shapiro-Wilk, Kruskal-Wallis H-, and Mann-Whitney U-tests were used to analyze the results. RESULTS: Most of the various devices revealed significant differences upon comparison. The accuracy of torque control offered by the three hand drivers was clinically acceptable. As two of the four battery-operated drivers did not feature torque limitation in reverse mode, they did not prevent high torque levels from occurring. Likewise, some of the maximum torque levels observed in conjunction with the other two battery-operated drivers and the mains-operated surgical unit exceeded considerably the clinically recommended range of 10-25 Ncm. CONCLUSION: Although miniscrews can be removed successfully with hand-operated drivers while limiting torque, we advise against the use of battery-operated drivers or mains-operated surgical units not offering torque limitation in reverse mode.

[Surgical therapy of segmental jejunal, primary intestinal lymphangiectasia]. / Die operative Therapie einer segmentalen, primären intestinalen Lymphangiektasie des Jejunums mit ausgeprägtem Chylaskos.

Primary intestinal lymphangiectasia (PIL) is a protein-losing, exsudative gastroenteropathy causing lymphatic obstruction. Diagnosis depends on clinical examination and histological findings. Conservative treatment modalities include a low-fat diet and enteral nutritional therapy in order to reduce enteric protein loss and to improve fat metabolism. Other treatment options consist of administration of antiplasmin or octreotide to lower lymph flow and secretion. We report on a 58-year-old patient who underwent exploratory laparotomy due to a worsening physical status, recurrent chylaskos and leg oedema under conservative dietary therapy. Intraoperative findings showed a typical PIL of the jejunum about 20 cm distal to the Treitz's ligament. Histological examinations confirmed this diagnosis. One year after segmental small bowel resection (105 cm) with end-to-end anastomosis the patient is healthy, free of symptoms, has gained weight and his serum protein level has increased. Intraabdominal ascites and leg oedema have not reoccurred since.

Multipurpose use of orthodontic mini-implants to achieve different treatment goals.

OBJECTIVES: The goal of the study was to determine whether mini-implants inserted in the palate can be used to achieve more than one treatment goal consecutively or simultaneously in the same patient. MATERIALS AND METHODS: The treatment results of 43 patients were retrospectively assessed. Two implant-supported mechanical systems per patient were applied either consecutively in 19 patients (group A) or simultaneously in 24 patients (group B). Both groups were analyzed and compared by calculating success rates for achievement of the treatment goals, survival of the mini-implants, and quality of anchorage. Durations of treatment were also analyzed for intergroup differences. RESULTS: Except for a single case in group A, the treatment goals were achieved in all patients (success rates 94.7% in group A versus 100% in group B). Anchorage loss was confined to one patient per group (success rates 94.7% in group A and 95.3% in group B). Mini-implant mobility, and hence implant failure, was observed in three implants in group A (survival rate 91.8%) and two implants in group B (survival rate 95.6%). While none of these intergroup differences were statistically significant, the treatment durations in both groups differed widely: those in group B were significantly shorter (10.0 ± 4.2 months) than those in group A (14.4 ± 3.5 months; p = 0.001). CONCLUSION: Mini-implants inserted in the palate for skeletal anchorage can be used to achieve more than one treatment goal in the same patient. Such multipurpose application can succeed consecutively and simultaneously. The latter option can significantly expedite treatments and should, therefore, be preferred when feasible, depending on the nature of coexisting therapeutic indications in a given patient.

Model of wound healing for esophagogastric anastomoses in rats.

BACKGROUND: Anastomotic leakage after esophageal surgery is a significant cause of morbidity and mortality. Postoperative leakage of esophagogastric anastomosis has been reported in 2-30% of surgical patient, resulting in an increased need for reoperation and a high risk of subsequent esophageal stricture formation and fistula. So far, experimental investigations on major factors influencing the healing of esophageal anastomoses, e.g. neovascularization and collagen deposition, have been hindered by the lack of a functional rodent model. METHODS: We developed a novel technique of gastric tube formation followed by end-to-end esophagogastric anastomosis in a rat model. Standardized anastomoses were carried out in 18 Brown-Norway rats and normal esophagogastric healing was studied by measuring anastomotic breaking strength 5 days after surgery. RESULTS: Five animals showed an insufficiency of the esophagogastric anastomosis as determined by anastomotic leakage testing. Normal anastomotic healing was found in 10 animals. The anastomotic breaking strength was 1.93 ± 0.45 N. CONCLUSION: The rat model for performing esophagogastric anastomoses after gastric tube formation may serve as a functional and useful model in future research studies on microvascular and molecular processes of anastomotic healing.

Impact of minimally invasive surgery in the spectrum of current achalasia treatment options.

Minimally invasive Heller myotomy has evolved the "gold standard" procedure for achalasia in the spectrum of current treatment options. The laparoscopic technique has proved superior to the thoracoscopic approach due to improved visualization of the esophagogastric junction. Operative controversies most recently include the length of the myotomy, especially of its fun-dic part, with respect to the balance between postoperative persistent dysphagia and development of gastroesophageal reflux, as well as the type of the added antireflux procedure. Peri-operative mortality should approach 0%, and favorable long-term results can be achieved in > 90%.

[Achalasia in a patient with HIV/HCV coinfection: detection of HCV in the esophageal tissue]. / Achalasie bei HIV/HCV-Koinfektion : HCV-Nachweis im Ösophagusgewebe.

Esophageal involvement in the context of opportunistic infections in human immunodeficiency virus (HIV) positive patients is a frequent phenomenon. However, worldwide esophageal achalasia has been described only twice in HIV-infected patients.We report the case of a 44-year-old Caucasian patient with HIV and Hepatitis C virus (HIV/HCV) coinfection who, within 2.5 years, displayed a progressive symptomatology with dysphagia, retrosternal pain, regurgitation as well as a considerable loss of weight before achalasia was finally diagnosed. Treatment was performed primarily surgically by means of laparoscopic Heller myotomy with an anterior 180° semifundoplication according to Dor.Histopathology of the specimens taken from the lower esophageal sphincter high-pressure zone proved alterations with abundant connective tissue and only scarce parts of the smooth muscular system without inflammatory infiltrations. In addition, the ganglia cells of the myenteric plexus as well as the interstitial cells of Cajal were significantly reduced. Interestingly, specific gene sequences of the hepatitis C virus could be detected in the esophageal tissue specimen. In contrast, analysis of specific HIV-gene sequences in the same tissue revealed a negative result.The possible but previously unknown relationship between esophageal achalasia and coinfection with HIV and HCV, also described as neurotropic viruses, will be discussed.

Impact of bone quality, implant type, and implantation site preparation on insertion torques of mini-implants used for orthodontic anchorage.

Mini-implants are widely used as skeletal anchorage in orthodontics. To reduce implant loss rate, sufficient primary stability is required. This study quantitatively analysed the impact of bone quality and pre-drilling diameter on the insertion torque of five different mini-implants. Twenty pig bone segments were dissected and embedded in resin. The insertion torques of two different mini-implant types (Tomas Pin, Dentaurum, Germany, 8 and 10 mm; and Dual Top, Jeil, Korea, 1.6 mm × 8 and 10 mm plus 2 mm×10 mm) were measured. After preparation of the implant sites using pilot drill diameters 1.0, 1.1, 1.2 and 1.3mm, 30 implants were inserted into each bone segment. Five reference implants were inserted into each segment for comparison. Micro CT evaluated bone compacta thickness. Insertion moments of orthodontic mini-implants, and hence primary stability, varied strongly depending on compacta thickness, implant design, and pre-drilling at the implant site. The Dual Top consistently showed higher primary stability than the Tomas Pin. Insertion moments higher than 230 Nmm resulted in fractures in some cases. Compacta thickness, implant design and preparation of implant site affect the insertion torque of mini-implants for orthodontic anchorage. To avoid fractures and high bone stresses, optimum pre-drilling diameters should be chosen.

[Giant paraesophageal herniation with intermittent prolapse into the thorax of the transverse colon]. / Monströse paraösophageale Zwerchfellhernie mit isolierter Prolabierung des Colon transversum nach intrathorakal.

HISTORY AND ADMISSION FINDINGS: A 74-year-old woman was admitted with a history of recurring dyspnea for several months. During radiological examination of the chest computed tomography demonstrated a giant paraesophageal hernia containing transverse colon with a significant amount of paracolic fat tissue. Physical examination was unremarkable. INVESTIGATIONS: Routine blood tests and abdominal ultrasound were within the normal range. Endoscopy showed a normal upper and lower gastrointestinal tract and barium swallow was normal without any esophageal motor dysfunction. The esophagogastric junction and gastric fundus were below the diaphragm. TREATMENT AND COURSE: Laparoscopy revealed the colonic herniation and mediastinal adhesiolysis, complete resection of the hernia sac and reposition of the intrathoracic migrated transverse colon were undertaken. Hiatal repair was performed by anterior and posterior hiatoplasty and construction of an anterior 180˚ semifundoplication with fundopexy. CONCLUSION: Patients with giant paraesophageal hernias often present with nonspecific cardiac and respiratory symptoms and the condition is often misdiagnosed. If it is demonstrated, a possible abdominal involvement should be looked for. Minimally invasive surgery is feasible and efficacious in this condition and in addition to being better tolerated by the patient provides a far better visualization of the intrathoracic parts of a type IV hiatal hernia to the surgeon.

An adenylyl cyclase signaling pathway predicts direct dopaminergic input to vestibular hair cells.

Adenylyl cyclase (AC) signaling pathways have been identified in a model hair cell preparation from the trout saccule, for which the hair cell is the only intact cell type. The use of degenerate primers targeting cDNA sequence conserved across AC isoforms, and reverse transcription-polymerase chain reaction (RT-PCR), coupled with cloning of amplification products, indicated expression of AC9, AC7 and AC5/6, with cloning efficiencies of 11:5:2. AC9 and AC5/6 are inhibited by Ca(2+), the former in conjunction with calcineurin, and message for calcineurin has also been identified in the trout saccular hair cell layer. AC7 is independent of Ca(2+). Given the lack of detection of calcium/calmodulin-activated isoforms previously suggested to mediate AC activation in the absence of Gαs in mammalian cochlear hair cells, the issue of hair-cell Gαs mRNA expression was re-examined in the teleost vestibular hair cell model. Two full-length coding sequences were obtained for Gαs/olf in the vestibular type II-like hair cells of the trout saccule. Two messages for Gαi have also been detected in the hair cell layer, one with homology to Gαi1 and the second with homology to Gαi3 of higher vertebrates. Both Gαs/olf protein and Gαi1/Gαi3 protein were immunolocalized to stereocilia and to the base of the hair cell, the latter consistent with sites of efferent input. Although a signaling event coupling to Gαs/olf and Gαi1/Gαi3 in the stereocilia is currently unknown, signaling with Gαs/olf, Gαi3, and AC5/6 at the base of the hair cell would be consistent with transduction pathways activated by dopaminergic efferent input. mRNA for dopamine receptors D1A4 and five forms of dopamine D2 were found to be expressed in the teleost saccular hair cell layer, representing information on vestibular hair cell expression not directly available for higher vertebrates. Dopamine D1A receptor would couple to Gαolf and activation of AC5/6. Co-expression with dopamine D2 receptor, which itself couples to Gαi3 and AC5/6, will down-modulate levels of cAMP, thus fine-tuning and gradating the hair-cell response to dopamine D1A. As predicted by the trout saccular hair cell model, evidence has been obtained for the first time that hair cells of mammalian otolithic vestibular end organs (rat/mouse saccule/utricle) express dopamine D1A and D2L receptors, and each receptor co-localizes with AC5/6, with a marked presence of all three proteins in subcuticular regions of type I vestibular hair cells. A putative efferent, presynaptic source of dopamine was identified in tyrosine hydroxylase-positive nerve fibers which passed from underlying connective tissue to the sensory epithelia, ending on type I and type II vestibular hair cells and on afferent calyces.

Pituitary adenylyl cyclase-activating polypeptide (PACAP) and its receptor (PAC1-R) are positioned to modulate afferent signaling in the cochlea.

Pituitary adenylyl cyclase-activating polypeptide (PACAP), via its specific receptor pituitary adenylyl cyclase-activating polypeptide receptor 1 (PAC1-R), is known to have roles in neuromodulation and neuroprotection associated with glutamatergic and cholinergic neurotransmission, which, respectively, are believed to form the primary basis for afferent and efferent signaling in the organ of Corti. Previously, we identified transcripts for PACAP preprotein and multiple splice variants of its receptor, PAC1-R, in microdissected cochlear subfractions. In the present work, neural localizations of PACAP and PAC1-R within the organ of Corti and spiral ganglion were examined, defining sites of PACAP action. Immunolocalization of PACAP and PAC1-R in the organ of Corti and spiral ganglion was compared with immunolocalization of choline acetyltransferase (ChAT) and synaptophysin as efferent neuronal markers, and glutamate receptor 2/3 (GluR2/3) and neurofilament 200 as afferent neuronal markers, for each of the three cochlear turns. Brightfield microscopy giving morphological detail for individual immunolocalizations was followed by immunofluorescence detection of co-localizations. PACAP was found to be co-localized with ChAT in nerve fibers of the intraganglionic spiral bundle and beneath the inner and outer hair cells within the organ of Corti. Further, evidence was obtained that PACAP is expressed in type I afferent axons leaving the spiral ganglion en route to the auditory nerve, potentially serving as a neuromodulator in axonal terminals. In contrast to the efferent localization of PACAP within the organ of Corti, PAC1-R immunoreactivity was co-localized with afferent dendritic neuronal marker GluR2/3 in nerve fibers passing beneath and lateral to the inner hair cell and in fibers at supranuclear and basal sites on outer hair cells. Given the known association of PACAP with catecholaminergic neurotransmission in sympathoadrenal function, we also re-examined the issue of whether the organ of Corti receives adrenergic innervation. We now demonstrate the existence of nerve fibers within the organ of Corti which are immunoreactive for the adrenergic marker dopamine beta-hydroxylase (DBH). DBH immunoreactivity was particularly prominent in nerve fibers both at the base and near the cuticular plate of outer hair cells of the apical turn, extending to the non-sensory Hensen's cell region. Evidence was obtained for limited co-localization of DBH with PAC1-R and PACAP. In the process of this investigation, we obtained evidence that efferent and afferent nerve fibers, in addition to adrenergic nerve fibers, are present at supranuclear sites on outer hair cells and distributed within the non-sensory epithelium of the apical cochlear turn for rat, based upon immunoreactivity for the corresponding neuronal markers. Overall, PACAP is hypothesized to act within the organ of Corti as an efferent neuromodulator of afferent signaling via PAC1-R that is present on type I afferent dendrites, in position to afford protection from excitotoxicity. Additionally, PACAP/PAC1-R may modulate secretion of catecholamines from adrenergic terminals within the organ of Corti.

Pituitary adenylyl cyclase-activating polypeptide (PACAP) and its receptor (PAC1-R) in the cochlea: evidence for specific transcript expression of PAC1-R splice variants in rat microdissected cochlear subfractions.

Pituitary adenylyl cyclase-activating polypeptide (PACAP) is a neuropeptide originally isolated from the hypothalamus, named for its high potency in stimulating adenylyl cyclase in pituitary cells. PACAP acts through the specific receptor PAC1-R to modulate the action of neurotransmitters, and additionally, to regulate cell viability via autocrine/intracrine mechanisms. Evidence has now been obtained that PACAP and multiple splice variants of PAC1-R are expressed in the rat cochlea. mRNA for PACAP precursor protein is found by reverse transcription-polymerase chain reaction (RT-PCR) in microdissected cochlear lateral wall, organ of Corti, and spiral ganglion subfractions. A specific pattern of expression of mRNA for PAC1-R splice variants, which mediate the response to PACAP, has been revealed by RT-PCR and cloning for the cochlear subfractions. Transcript for the short form of PAC1-R is found in all three subfractions. Four additional splice variants -- hop1, hop2, hip, and a novel hop1 splice variant -- are expressed in the lateral wall. For the amino terminus splice region of PAC1-R, a new splice variant has been detected in the organ of Corti, representing a deletion of the first 7 of 21 amino acids detected in the PAC1-R very-short sequence. Overall, from message determinations in cochlear subfractions, there are five PAC1-R splice variants in the lateral wall, two in the organ of Corti and one in the spiral ganglion, indicating multiple possible responses to PACAP and/or mechanisms to modulate the response to PACAP in the cochlea. The variety of PAC1-R splice variants expressed may reflect the diversity in cell function between subfractions that is modulated by PACAP. The neuropeptide and its specific receptor have been immunolocalized in the lateral wall, the source of the largest number of cochlear PAC1-R splice variants. The receptor was targeted by primary antibodies which would elicit immunoreactivity for all splice variants of PAC1-R detected with RT-PCR, and evidence has been obtained with Western blot analysis suggesting that PAC1-R is glycosylated in vivo. Within the lateral wall, PACAP and PAC1-R were immunolocalized primarily to the stria vascularis, with immunoreactivity for both neuropeptide and receptor increasing from the basal to apical cochlear turns. Within the stria, PACAP immunoreactivity was localized to the basolateral extensions of marginal cells, while PAC1-R was clearly associated with tight junctions between the marginal cells close to the endolymphatic compartment. In addition, evidence was obtained that PAC1-R was associated with endothelial cells of the capillaries in the stria vascularis. The large number of splice variants expressed, coupled to the specificity in linkage between PAC1-R splice variants and G-protein-coupled second messenger pathways, could provide a mechanism to closely modulate tight junction integrity in the stria vascularis, impacting the endolymphatic potential.

Molecular identification of an N-type Ca2+ channel in saccular hair cells.

We report new molecular evidence for the presence of an N-type (Ca(v)2.2, alpha1B) voltage-gated Ca(2+) channel in hair cells of the saccular epithelium of the rainbow trout. The Ca(v)2.2 amino-acid sequence shows 68% and 63% identity compared with chick and human Ca(v)2.2, respectively. This channel reveals features that are characteristic of an N-type Ca(2+) channel: an omega-conotoxin GVIA binding domain, G(betagamma) binding regions, and a synaptic protein interaction site. Immunohistochemical studies with a custom antibody show that immunoreactivity for the Ca(v)2.2 is concentrated in the basolateral and apical regions of hair cells. Whereas trout brain and saccular macula express an 11-amino-acid insert in the second G(betagamma) binding domain of the Ca(v)2.2 I-II loop, isolated hair cells appear not to express this variant. We constructed fusion polypeptides representing portions of the I-II loop, beta1 and beta2a auxiliary subunits, the II-III loop, and syntaxin, and examined their intermolecular interactions via immunoprecipitation and surface plasmon resonance. The I-II loop polypeptides bound both beta1 and beta2a subunits with a preference for beta1, and the II-III loop exhibited Ca(2+)-dependent syntaxin binding. We demonstrated syntaxin immunoreactivity near afferent endings in hair cells, at hair-cell apices, and in efferent endings on hair cells, the former two sites consistent with binding of syntaxin to Ca(v)2.2. The present molecular characterization of the Ca(v)2.2 channel provides novel biochemical evidence for an N-type channel in hair cells, and details molecular interactions of this channel that reflect hair-cell function, such as spontaneous activity and vesicular trafficking. The current work, to our knowledge, represents the first demonstration of a putative N-type channel in hair cells as documented by tissue-specific antibody immunoreactivity and hair-cell-specific cDNA sequence.

Cloning and characterization of alpha9 subunits of the nicotinic acetylcholine receptor expressed by saccular hair cells of the rainbow trout (Oncorhynchus mykiss).

alpha9/alpha10 Subunits are thought to constitute the nicotinic acetylcholine receptors mediating cholinergic efferent modulation of vertebrate hair cells. The present report describes the cloning and sequence analysis of subunits of the alpha9-containing receptor of a hair-cell layer from the saccule of the rainbow trout (Oncorhynchus mykiss). A major alpha9 subunit, termed alpha9-I, displayed typical features of a nicotinic alpha subunit, with total coding sequence of 572 amino acids including a 16 amino-acid signal peptide. It possessed an extended cytoplasmic loop between membrane-spanning regions M3 and M4, compared with mammalian homologs. Transcript for alpha9-I was robustly expressed in the saccular hair cell layer and less prominently in trout olfactory mucosa, spleen, pituitary gland, and liver, as determined by reverse transcription-polymerase chain reaction. alpha9-I cDNA was not detected in trout brain, skeletal muscle, retina, and kidney. The alpha9-I nicotinic receptor protein was immunolocalized, with an affinity-purified antibody directed against a trout alpha9-I epitope, to hair-cell and neural sites in the saccular hair-cell layer. Foci were found at basal and basolateral membrane sites on hair cells as well as on afferent nerve. Receptor clustering was observed in hair cells bordering non-sensory epithelium. Since in higher vertebrates the alpha9 is reported to associate with another nicotinic subunit, alpha10, we examined the possibility of expression of additional nicotinic subunits in trout saccular hair cells. Message for another nicotinic subunit, termed alpha9-II, was found to be expressed in the hair cells, although more difficult to amplify than alpha9-I. In contrast to alpha9-I, alpha9-II was expressed in brain, as well as in olfactory mucosa, less prominently in pituitary gland and liver, but not in spleen, skeletal muscle, retina, or kidney. The cloned alpha9-II had a total coding sequence of 550 amino acids, which included a 17-amino-acid signal peptide, and an extended M3-M4 loop. A third nicotinic subunit message, termed alpha9-III, was PCR-amplified from trout olfactory mucosa where it was strongly expressed. However, message for alpha9-III was not detected in hair cells. Message for alpha9-III was moderately expressed in trout brain, retina, and pituitary gland but not in trout spleen, skeletal muscle, liver, and kidney. Thus, alpha9-I and alpha9-II may together contribute to the formation of the hair-cell nicotinic receptor of teleosts, where no ortholog of alpha10 appears to exist. The current work is, to our knowledge, the first description of alpha9 coding sequences directly from a vertebrate hair cell source. Further, the generality of hair cell expression of subunits for the alpha9-containing nicotinic cholinergic receptor has been extended to fishes, suggesting a similar efferent mechanism across all vertebrate octavolateralis sensory systems.

Hyperpolarization-activated, cyclic AMP-gated, HCN1-like cation channel: the primary, full-length HCN isoform expressed in a saccular hair-cell layer.

The expression of transcript for hyperpolarization-activated, cyclic nucleotide-sensitive cation channel (HCN) isoforms underlying hyperpolarization-activated, inward current (I(h)) has been determined for a model hair-cell preparation from the saccule of the rainbow trout, Oncorhynchus mykiss. Based upon identification from homology to known vertebrate HCN cDNA sequence, cloning of PCR products amplified with degenerate primers indicated an expression frequency of 7:2:1 (HCN1:HCN2:HCN4) for the hair-cell sheet compared with 1:1:7 for brain. Full-length sequence has been obtained for the HCN1-like isoform representing the primary HCN transcript expressed in the hair-cell preparation. The channel protein is 938 amino acids in length with 93% amino acid identity for the region extending from the S1-S6 membrane spanning domains through the voltage-pore and cyclic nucleotide-binding domains, compared with HCN1 for rabbit, rat, mouse and human. The N- and C-terminal regions are less homologous, with 39-51% and 43-44% amino acid identities, respectively. Compared with other vertebrate HCN1, the hair-cell HCN1 contains additional consensus phosphorylation sites associated with unique repeats in the carboxy terminus. The HCN1-like transcript has been localized to hair cells of the saccular sensory epithelia by in situ hybridization. Previous electrophysiological studies have identified I(h) as the sole inwardly rectifying ion channel in a specific population of hair cells of the saccule of frogs [J Neurophysiol (1995) 73:1484] and fish [J Physiol (1996) 495:665]. I(h) is an important determinant of the resting membrane potential, and for this population of hair cells, is predicted to maintain the membrane potential within a voltage range allowing the voltage-gated calcium channels to open, permitting "spontaneous" release of transmitter. The molecular properties of the HCN1-like isoform underlying I(h) expressed in the saccular hair cells of the teleost, trout, may consequently impact spontaneous release of transmitter from hair cells of the saccule.

Muscarinic receptor subtypes are differentially distributed in the rat cochlea.

Five different genes encode the muscarinic acetylcholine receptors. The muscarinic receptor subtypes M1, M3, and M5 are typically coupled to activation of the Galpha(q/11)-phosphatidyl inositol pathway, whereas the M2 and M4 subtypes are typically linked to Galpha(i) and adenylyl cyclase inhibition. In order to localize muscarinic receptors in the rat cochlea, we applied polyclonal antibodies for subtypes M1, M2, M3, and M5, and monoclonal antibody for subtype M4 to paraffin sections. In the organ of Corti, outer hair cells exhibited strong immunoreactivity for M3 and weak immunoreactivity for M1. Deiters' cells were strongly immunoreactive to antibodies for the M1 and M2 subtypes, with weak staining observed for M3, and weaker yet for M5. Inner hair cells showed moderate immunoreactivity for the M1 subtype, weaker staining for the M5 subtype, and slight staining for the M3 subtype. Among the spiral ganglion neurons, weak to moderate immunoreactivity was detected for M3 and M5 subtypes and weak staining was observed for the M1 subtype. The efferent fibers of the intraganglionic spiral bundle were positive for M2 and M5. In the lateral wall, weak to moderate staining was detected for M5 in the stria vascularis corresponding in position to the basolateral extensions of marginal cells. Staining for M3 was observed associated with capillaries. Fibrocytes of the spiral ligament exhibited limited but selective subtype immunoreactivity. No immunoreactivity was detected in the cochlea for the M4 subtype. From the present findings we suggest that M3 is the primary muscarinic receptor subtype in outer hair cells mediating a postsynaptic response to the medial olivocochlear cholinergic efferent input. The muscarinic receptor subtypes M1, M3, and M5 appear to subserve the action of cholinergic lateral olivocochlear efferent stimulation on postsynaptic responses in type I afferents. Whether M1, M3, and M5 protein in inner hair cells indicates constitutive or vestigial expression remaining from development is unknown. M2 and M5 muscarinic receptors expressed presynaptically may modulate the efferent signal. Finally, expression by Deiters' cells of several muscarinic subtypes raises the possibility that cholinergic efferents couple to these non-sensory cells through muscarinic receptors.

Differences between two transpalatal arch systems upon first-, second-, and third-order bending activation.

Transpalatal arches are used in passive mode to improve anchor-age and in activated mode to achieve single tooth movement or movement of segments of teeth in first-, second- and third-order. Clinically it seems that the commonly used palatal arches of the Goshgarian type (here in the MIA system) as well as the precision TMA lingual arches of the Burstone system are not equally suitable for all kinds of activations. Using the Orthodontic Measurement and Simulation System (OMSS) the force systems and the efficacy of activated arches of both systems were examined in an experimental study with respect to different malpositions. The following first-, second- and third-order activations were chosen: symmetrical expansion and compression up to 4 mm, symmetrical distal rotation up to 15 degrees, unilateral distal tipping of 15 degrees and symmetrical buccal root torque up to 10 degrees. Attachments of the MIA system (MIA Rotation Lingual Sheaths, 0.072" x 0.036") and of the Burstone system (precision lingual hinge cap, 0.032" x 0.032", unused and a pair after a 4-week introral application period) were measured. Lingual arches made of 0.036" round stainless steel wire (MIA) and 0.032" x 0.032" TMA (Burstone system) were prepared with a height of 18 mm and a width of 30 mm. First-order activation bends (expansion and compression) of the MIA palatal arches caused forces up to 4.4 N compared to 1.8 N of the TMA arches, due to the lower load/deflection rate of the latter. The malpositions were corrected effectively by both systems. Due to the higher stiffness the moments delivered by the MIA palatal arches (39 Nmm) were higher in distal rotation compared to those of the TMA arches (14 Nmm) and the correction was more effective. In second-order activations (tipping) the MIA system delivered no or only small moments because of the curved shape of the attachments. A correction of only 30% was achieved compared to 80% with the Burstone system. In third-order activations, in contrast, the Burstone attachments caused considerable loss of torque. This was obviously due to the strong deformation of the slot by the intraoral loading. If it were possible to improve the dimensional stability of the hinge cap, all corrections carried out with the Burstone TMA system would involve distinctly smaller forces and moments than the MIA system but would still ensure good effectiveness.

Immunohistochemical localization of adenylyl cyclase isoforms in the lateral wall of the rat cochlea.

The enzymatic activity of adenylyl cyclase (AC) is attributable to nine isoforms with individual pharmacology and tissue distribution. Polyclonal antibodies for AC isoforms I-IV, VII and VIII were applied to sections of cochlear lateral wall, a tissue involved in ion transport contributing to the unique ion content of endolymph and electrical potential of scala media. Within the stria vascularis, immunoreactivity primarily to Ca(2+)/calmodulin-independent isoforms II, IV and VII was localized to sites consistent in position to the basolateral extensions of marginal cells. Little immunoreactivity was observed in the stria vascularis for Ca(2+)/calmodulin-dependent isoforms I, III and VIII. Within the spiral ligament, type II and type IV fibrocytes exhibited moderate staining for ACII, IV and VII, less staining for VIII and little for I and III. Immunoreactivity to ACII, IV, VII and VIII was observed in type I fibrocytes. The outer sulcus cells and root processes were highly immunoreactive for isoforms I and VIII, but not for III or the Ca(2+)/calmodulin-independent isoforms. The differential pattern of immunoreactivity in the lateral wall overall appears to reflect subfamily-specific expression with Ca(2+)/calmodulin-independent isoforms expressed in the stria vascularis and Ca(2+)/calmodulin-dependent isoforms expressed in the outer sulcus cells and root processes. cAMP-mediated modulation of ion transport by marginal cells is predicted to exhibit, in the microenvironment of basolateral membrane infoldings, pharmacological characteristics of the AC type II subfamily (II, IV and VII), including activation by protein kinase C (II and VII).

The influence of bracket design on frictional losses in the bracket/arch wire system.

In arch guided tooth movement, the essential role played by bracket configuration with respect to sliding friction has been recognized by the manufacturers, a fact which has had an increasing impact on the design and marketing of new bracket models in recent years. The aim of the present in-vitro study was to investigate the influence of different bracket designs on sliding mechanics. Five differently shaped stainless steel brackets (Discovery: Dentaurum, Damon SL: A-Company, Synergy: Rocky Mountain Orthodontics, Viazis bracket and Omni Arch appliance: GAC) were compared in the 0.022"-slot system. The Orthodontic Measurement and Simulation System (OMSS) was used to quantify the difference between applied force (NiTi coil spring, 1.0 N) and orthodontically effective force and to determine leveling losses occurring during the sliding process in arch guided tooth movement. Simulated canine retraction was performed using continuous arch wires with the dimensions 0.019" x 0.025" (Standard Steel, Unitek) and 0.020" x 0.020" (Ideal Gold, GAC). Comparison of the brackets revealed friction-induced losses ranging from 20 to 70%, with clear-cut advantages resulting from the newly developed bracket types. However, an increased tendency towards leveling losses in terms of distal rotation (maximum 15 degrees) or buccal root torque (maximum 20 degrees) was recorded, especially with those brackets giving the arch wire increased mobility due to their shaping or lack of ligature wire.