Aversive Conditioning of Spatial Position Sharpens Neural Population-Level Tuning in Visual Cortex and Selectively Alters Alpha-Band Activity.

Processing capabilities for many low-level visual features are experientially malleable, aiding sighted organisms in adapting to dynamic environments. Explicit instructions to attend a specific visual field location influence retinotopic visuocortical activity, amplifying responses to stimuli appearing at cued spatial positions. It remains undetermined both how such prioritization affects surrounding nonprioritized locations, and if a given retinotopic spatial position can attain enhanced cortical representation through experience rather than instruction. The current report examined visuocortical response changes as human observers (N = 51, 19 male) learned, through differential classical conditioning, to associate specific screen locations with aversive outcomes. Using dense-array EEG and pupillometry, we tested the preregistered hypotheses of either sharpening or generalization around an aversively associated location following a single conditioning session. Competing hypotheses tested whether mean response changes would take the form of a Gaussian (generalization) or difference-of-Gaussian (sharpening) distribution over spatial positions, peaking at the viewing location paired with a noxious noise. Occipital 15 Hz steady-state visual evoked potential responses were selectively heightened when viewing aversively paired locations and displayed a nonlinear, difference-of-Gaussian profile across neighboring locations, consistent with suppressive surround modulation of nonprioritized positions. Measures of alpha-band (8-12 Hz) activity were differentially altered in anterior versus posterior locations, while pupil diameter exhibited selectively heightened responses to noise-paired locations but did not evince differences across the nonpaired locations. These results indicate that visuocortical spatial representations are sharpened in response to location-specific aversive conditioning, while top-down influences indexed by alpha-power reduction exhibit posterior generalization and anterior sharpening.SIGNIFICANCE STATEMENT It is increasingly recognized that early visual cortex is not a static processor of physical features, but is instead constantly shaped by perceptual experience. It remains unclear, however, to what extent the cortical representation of many fundamental features, including visual field location, is malleable by experience. Using EEG and an aversive classical conditioning paradigm, we observed sharpening of visuocortical responses to stimuli appearing at aversively associated locations along with location-selective facilitation of response systems indexed by pupil diameter and EEG alpha power. These findings highlight the experience-dependent flexibility of retinotopic spatial representations in visual cortex, opening avenues toward novel treatment targets in disorders of attention and spatial cognition.

Effects of affective content and motivational context on neural gain functions during naturalistic scene perception.

Visual scene processing is modulated by semantic, motivational, and emotional factors, in addition to physical scene statistics. An open question is to what extent those factors affect low-level visual processing. One index of low-level visual processing is the contrast response function (CRF), representing the change in neural or psychophysical gain with increasing stimulus contrast. Here we aimed to (a) establish the use of an electrophysiological technique for assessing CRFs with complex emotional scenes and (b) examine the effects of motivational context and emotional content on CRFs elicited by naturalistic stimuli, including faces and complex scenes (humans, animals). Motivational context varied by expectancy of threat (a noxious noise) versus safety. CRFs were measured in 18 participants by means of sweep steady-state visual evoked potentials. Results showed a facilitation in visuocortical sensitivity (contrast gain) under threat, compared with safe conditions, across all stimulus categories. Facial stimuli prompted heightened neural response gain, compared with scenes. Within the scenes, response gain was smaller for scenes high in emotional arousal, compared with low-arousing scenes, consistent with interference effects of emotional content. These findings support the notion that motivational context alters the contrast sensitivity of cortical tissue, differing from changes in response gain (activation) when visual cues themselves carry motivational/affective relevance.

Effects of Experience on Spatial Frequency Tuning in the Visual System: Behavioral, Visuocortical, and Alpha-band Responses.

Using electrophysiology and a classic fear conditioning paradigm, this work examined adaptive visuocortical changes in spatial frequency tuning in a sample of 50 undergraduate students. High-density EEG was recorded while participants viewed 400 total trials of individually presented Gabor patches of 10 different spatial frequencies. Patches were flickered to produce sweep steady-state visual evoked potentials (ssVEPs) at a temporal frequency of 13.33 Hz, with stimulus contrast ramping up from 0% to 41% Michelson over the course of each 2800-msec trial. During the final 200 trials, a selected range of Gabor stimuli (either the lowest or highest spatial frequencies, manipulated between participants) were paired with an aversive 90-dB white noise auditory stimulus. Changes in spatial frequency tuning from before to after conditioning for paired and unpaired gratings were evaluated at the behavioral and electrophysiological level. Specifically, ssVEP amplitude changes were evaluated for lateral inhibition and generalization trends, whereas change in alpha band (8-12 Hz) activity was tested for a generalization trend across spatial frequencies, using permutation-controlled F contrasts. Overall time courses of the sweep ssVEP amplitude envelope and alpha-band power were orthogonal, and ssVEPs proved insensitive to spatial frequency conditioning. Alpha reduction (blocking) was most pronounced when viewing fear-conditioned spatial frequencies, with blocking decreasing along the gradient of spatial frequencies preceding conditioned frequencies, indicating generalization across spatial frequencies. Results suggest that alpha power reduction-conceptually linked to engagement of attention and alertness/arousal mechanisms-to fear-conditioned stimuli operates independently of low-level spatial frequency processing (indexed by ssVEPs) in primary visual cortex.

Evaluation of lower anterior dental changes in patients treated with self-ligating brackets.

The aim of this study was to evaluate and compare changes in linear distance and inclination of lower incisors and canines and intercanine distance after a 30 months orthodontic treatment with self-ligating appliances. Seven patients were treated orthodontically with a Roth prescription passive self-ligating bracket. To perform the measurements and comparisons, CBCT scans were taken before the start of the orthodontic treatment (T1) and after a period of 30 months treatment (T2). The following measurements were performed: (1) the lower incisors and canines inclination in relation to the mandibular plane, (2) intercanine linear distance in millimeters and (3) linear distance in millimeters of the incisal and apical part of lower anterior teeth to a plane (POGM) passing through pogonion point and perpendicular to the mandibular plane. No significant difference were observed between T1 and T2 for canine inclination (p = 0.835), incisors inclination (p = 0.149), canine incisal distance to POGM (p = 0.423) and incisors incisal distance to POGM (p = 0.966), however canine apical distance (p = 0.049) and incisors apical distance (p = 0.002) to POGM was lower at T1 than at T2. The intercanine distance was significantly lower (p = 0.022) at T1 when compared to T2. The use of passive self-ligating brackets in orthodontic treatment to solve 4 mm tooth crowding were able to produce dental arch expansion by bodily tooth movement.

Nucleobindin 1 (NUCB1) is a Golgi-resident marker of neurons.

Nucleobindin 1 (NUCB1; also known as CALNUC or NUC) is a putative DNA- and calcium-binding protein and exhibits significant structural homology with the protein nucleobindin 2 (NUCB2; also known as nesfatin). While NUCB2 has been mapped in detail in the brain and implicated in the hypothalamic control of energy metabolism, no study has to date addressed the presence of NUCB1 in the central nervous system. Here we have explored the expression and distribution of NUCB1 in the rat brain and spinal cord, using RT-PCR, immunofluorescence and in situ hybridization. NUCB1 mRNA and protein was found to be present in all brain regions, extending to the spinal cord and dorsal root ganglia. Double-staining for NUCB1 and NeuN, glial fibrillary acidic protein and myelin basic protein revealed that NUCB1 is exclusively found in neurons, and not in glial or ependymal cells. Notably, NUCB1-immunoreactivity was observed in all neurons examined, making no distinction between previously identified glutamatergic and GABAergic populations, including those that are known not to stain for NeuN. This included the markedly more restricted population of NUCB2-expressing neurons in the brain. The protein was detected in cell somata and proximal dendrites, but not in axons or terminal structures. Further examination of the subcellular distribution of NUCB1 using organelle-specific markers revealed its consistent presence in the Golgi apparatus. These findings identify NUCB1 as a novel pan-neuronal marker. Along with the recent demonstration of broad expression of the protein in endocrine cells, the present results suggest that NUCB1 may play a role in spatiotemporal calcium handling in signaling cells.

[Age-related activation of the muscles during upright standing in patients with haemophilia]. / Altersabhängige Ansteuerung der Muskulatur im Stand bei Patienten mit Hämophilie.

Thehaemophilicarthropathyaffects thefunction of theknee joint muscles. The aim of thisstudywas to investigatethe myoelectrical signal ofknee jointmusclesin different agestages during upright standing. Surface EMG (SEMG) amplitudes of quadriceps, hamstrings and gastrocnemii were measured in 191 patients with severe haemophilia A (n=164) and B (n=27) while standing on an even surface. After an age-based classification of patients into the subgroups H(A): 17-29 (n = 37), H(B): 30-39 (n = 50), HC: 40-49 (n = 61), H(D): 50-70 in years (n = 43) the clinical WFH score for the ankle and knee joint was determined. To normalize the SEMG values amplitude ratios (percentage of cumulated activity) were calculated with respect to the specific limb. With increasing age, the patient showed descriptively a deterioration of the joint situation. The extensors of the knee joint reached significantly higher absolute and percentage levels in the muscle activity with increasing age (p < 0.05). The absolute amplitude values of the Mm. gastrocnemii showed no differences in the age groups while the relative levels were decreased. The present study shows that patients with increasing age and degree of haemophilic arthropathy develop a modified control strategy during upright standing, in the form of a shift from the plantar flexors to the extensors of the knee joint.

Impact of joint status on contraction steadiness of m. quadriceps femoris in people with severe haemophilia.

Impaired contraction steadiness of lower limb muscles affects functional performance and may increase injury risk. We hypothesize that haemophilic arthropathy of the knee and the strength status of quadriceps are relevant factors which compromise a steady contraction. This study addresses the questions if impaired steadiness of the quadriceps is verifiable in people with haemophilia (PWH) and whether a connection between the status of the knee joint and quadriceps strength exists. A total of 157 PWH and 85 controls (C) performed a strength test with a knee extensor device to evaluate their bilateral and unilateral maximal quadriceps strength and steadiness. Isometric steadiness was measured by the coefficient of variation of maximum peak torque (CV-MVIC in %). For classification of the knee joint status the World Federation of Haemophilia (WFH) score was used. Lower steadiness (higher CV values) was found in PWH compared with C during bilateral [PWH vs. C; 0.63 (0.36/1.13) vs. 0.35 (0.15/0.72), median (Q25/Q75) P < 0.001] and unilateral trials [left leg: 0.70 (0.32/1.64) vs. 0.50 (0.23/1.04), P < 0.05; right leg: 0.68 (0.29/1.51) vs. 0.39 (0.18/0.68), P < 0.001]. PWH with a WFH score difference (≥1) between their extremities showed a less steady contraction in the more affected extremity (P < 0.05). More unsteady contractions have also been found in extremities with lower quadriceps strength compared with the contralateral stronger extremities (P < 0.001), whereby the weaker extremities were associated with a worse joint status (P < 0.001). The results of this study verify an impaired ability to realize a steady contraction of quadriceps in PWH and the influence of joint damage and strength on its manifestation.

Reciprocal effects of Interferon-γ and IL-4 on differentiation to osteoclast-like cells by RANKL or LPS.

OBJECTIVE: LPS can induce differentiation to osteoclast-like cells independent of RANKL. In comparison with RANKL, the effects of Th1 and Th2 cytokines on LPS-induced osteoclastogenesis have not been extensively studied. In this study, we investigated the effects of IFN-γ and IL-4 on RANKL- or LPS-induced osteoclastogenesis. MATERIALS AND METHODS: RAW 264.7 cells were induced to differentiate into osteoclast-like cells by RANKL or LPS, in the absence or presence of IFN-γ or IL-4. The number of TRAP-positive, multinucleated (≥ 3 nuclei) cells (MNCs) was counted. mRNA and protein levels of TRAP and cathepsin K were determined by quantitative RT-PCR and Western immunoblot, respectively. Expression of other genes implicated in osteoclast and macrophage differentiation and inflammation was also quantitated and was subsequently assessed in bone marrow-derived macrophages (BMMs). Phagocytic capacity of differentiated RAW264.7 was investigated by the uptake of pHrodo S. aureus bioparticles conjugates. RESULTS: In contrast to the RANKL-treated cell population that gained more macrophage-like properties at the level of gene and protein expression as well as phagocytosis in the presence of IFN-γ or IL-4, the LPS-induced population gained more osteoclast-like properties by the addition of the same factors. CONCLUSION: These data suggest that the adaptive immune system, through either Th1 or Th2 cytokines, is able to modify the differentiation process of osteoclasts in inflammatory situations. Moreover, the study provides an example of different regulation of osteoclast differentiation during physiological and inflammatory conditions.

Measurement of the neutron electric to magnetic form factor ratio at Q2=1.58 GeV2 using the reaction 3He[over →](e[over →],e'n)pp.

A measurement of beam helicity asymmetries in the reaction 3He[over →](e[over →],e'n)pp is performed at the Mainz Microtron in quasielastic kinematics to determine the electric to magnetic form factor ratio of the neutron GEn/GMn at a four-momentum transfer Q2=1.58 GeV2. Longitudinally polarized electrons are scattered on a highly polarized 3He gas target. The scattered electrons are detected with a high-resolution magnetic spectrometer, and the ejected neutrons are detected with a dedicated neutron detector composed of scintillator bars. To reduce systematic errors, data are taken for four different target polarization orientations allowing the determination of GEn/GMn from a double ratio. We find µnGEn/GMn=0.250±0.058(stat)±0.017(syst).

Quadriceps strength, inter-extremity difference (IED) and joint status in adult persons with severe haemophilia in different age stages.

Quadriceps weakness seems to be a hallmark in adult persons with severe haemophilia (PWH). The purpose of this study was to compare PWH and non-haemophilic controls in different age stages with reference to joint status and quadriceps strength. Further aims were to examine the extent of strength-specific inter-extremity-difference (IED) and the prevalence of abnormal IED (AIED). A total of 106 adults with severe haemophilia (H) and 80 controls (C) had undergone an orthopaedic examination for classification of knee and ankle status using the WFH score. Quadriceps strength was evaluated unilaterally as well as bilaterally with a knee extensor device. Each group was divided into four age-related subgroups (HA/CA: 18-29, HB/CB: 30-39, HC/CC: 40-49, HD/CD: 50-70; in years). H presented a worse knee and ankle status than C indicated by higher WFH scores (P < 0.01). Regarding the age-matched subgroups only HB showed higher knee scores than CB (P < 0.05). The ankles were clinically more affected in HB-HD compared with those in age-matched controls (P < 0.05). H showed lower quadriceps strength than C (P < 0.05). In addition, all subgroups of H presented lower strength (HA: 10-17, HB: 19-23, HC: 35-36, HD: 53-61; in%, P < 0.05). IED was higher in H than in C [H: 12.0 (5.3/32.2) vs. C: 7.1 (2.9/10.9); Median (quartiles) in%, P < 0.001] and increased with age in H. We discovered an AIED in 35% of H. These findings highlight the importance for the early implementation of preventive and rehabilitative muscle training programmes in the comprehensive treatment of PWH.

Bone repair using periodontal ligament progenitor cell-seeded constructs.

The success of tissue-engineering therapies is dependent on the ability of scaffolds to guide differentiation of progenitor cells. Here we present a new approach using a biomimetic construct composed of hydroxyapatite modified with an in vitro-derived extracellular matrix (HA-ECM) and seeded with periodontal ligament progenitor cells (PDLCs). The study aimed to investigate the effect of HA-ECM on osteogenic differentiation of PDLCs and in vivo evaluation of the PDLC-seeded HA-ECM constructs using a rat calvarial critical-sized defect model. After flow-cytometric phenotyping of PDLCs for typical mesenchymal stem cell markers, the PDLCs were cultured on HA-ECM or HA alone in osteogenic media and assessed by MTT, alkaline phosphatase (ALP) assays, and real-time qPCR at different time intervals after seeding. New bone formation induced by PDLC-seeded constructs was assessed by histomorphometric analysis at 12 weeks post-operatively. The PDLCs seeded on HA-ECM showed significantly higher ALP activity and up-regulation of bone-related genes. The treatment with PDLC-seeded HA-ECM significantly improved calvarial bone repair, with the highest amount of newly formed bone elicited by cell-seeded constructs cultured for 14 days. Our results highlight the PDLC-seeded HA-ECM constructs as a promising tool for craniofacial bone regeneration.

[Analysis of muscle function with kinetic superficial EMG in children with haemophilia - recognizing subclinical changes, establishing individual therapy, quality control]. / Muskelfunktionsmessung mit kinetischem Oberflächen-EMG bei Kindern mit Hämophilie. Erkennung subklinischer Veränderungen, Etablierung individueller Therapie, Qualitätskontrolle.

UNLABELLED: Electromyography (EMG) measures muscle electricity. It depends on muscle contraction and central motor control. Muscles react very sensitive on external signals (e. g. bleeding), The resulting changes can be shown in EMG. PATIENTS, METHODS: A first study included 51 children and young adults from Costa Rica. They underwent a clinical examination and EMG of the hip, knee and ankle joints. Resting muscle tone, maximal isometric contraction and three typical isotonic movements of the joints were measured. First step of analysis was to characterize typical pathogenic changes in the muscles and to find a corresponding physical therapy to minimize these changes. RESULTS: It showed that EMG is a good marker for muscle condition. It helps to individualize therapy and improve effectivity of physical and physiotherapeutic treatment of the locomotive system of children and young adults with hemophilia. It can help to recognize early subclinical changes and to control the outcome of therapeutic modalities.

TGF-beta/extracellular matrix interactions in dentin matrix: a role in regulating sequestration and protection of bioactivity.

TGF-beta isoforms sequestrated in dentin matrix potentially provide a reservoir of bioactive molecules that may influence cell behavior in the dentin-pulp complex following tissue injury. The association of these growth factors with dentin matrix and the influence of such associations on the bioactivity of growth factors are still unclear. We used surface plasmon resonance technology in the BIAcore 3000 system to investigate the binding of TGF-beta isoforms 1 and 3 to purified decorin, biglycan, and EDTA soluble dentin matrix components. TGF-beta isoforms 1 and 3 were immobilized on sensorchips CM4 through amine coupling. For kinetic studies of protein binding, purified decorin and biglycan, isolated EDTA soluble dentin matrix, and dentin matrix immunodepleted of decorin and/or biglycan were injected over TGF-beta isoforms and allowed to interact. Programmed kinetic analysis software provided sensorgrams for each concentration of proteoglycan or dentin matrix extract injected. Purified decorin and biglycan and dentin matrix extract bound to the TGF-beta isoforms. However, the association with TGF-beta3 was much weaker than that with TGF-beta1. After immunoaffinity depletion of the dentin matrix extract, the level of interaction between the dentin matrix extract and TGF-beta was significantly reduced. These results suggest isoform-specific interactions between decorin/biglycan and TGF-beta isoforms 1 and 3, which may explain why TGF-beta3 is not detected in the dentin matrix despite being expressed at higher levels than TGF-beta1 in odontoblasts. These proteoglycans appear to play a significant role in TGF-beta/extracellular matrix interactions and may be important in the sequestration of these growth factors in the dentin matrix.

[Pathomechanisms of organ failure. Mitochondrial dysfunction in sepsis]. / Pathomechanismen des Organversagens. Zelluläre Sauerstoffverwertungsstörung im Rahmen der Sepsis.

Proinflammatory mediators as well as increased formation of reactive oxygen and nitrogen species impair cellular respiration during sepsis. In particular, the highly reactive peroxynitrite irreversibly damages lipids, proteins and nucleic acids and also inhibits enzyme complexes of the respiratory chain. In this way cellular metabolic functions and subsequently organ functions are also impaired. Repair of DNA by poly(ADP-ribose)polymerase consumes large amounts of nicotinamide adenine dinucleotide (NAD+) which leads to cellular NAD+ depletion further promoting inflammation. This article summarizes central aspects of the pathophysiology of mitochondrial dysfunction during sepsis and gives an overview about newly developed strategies which proved effective in experimental studies and may have a potential clinical application in the future.

Adenosine A1 and A2 receptor agonists reduce endotoxin-induced cellular energy depletion and oedema formation in the lung.

BACKGROUND AND OBJECTIVE: Tissue depletion of adenosine during endotoxaemia has previously been described in the lung. Therapeutic approaches to prevent adenosine depletion and the role of A1 and A2 receptor agonists, however, have not been investigated until now. METHODS: In isolated and ventilated rabbit lungs, it was tested whether pretreatment with adenosine A1 agonist 2-chloro-N6-cyclopentyladenosine (CCPA; 10(-7) mol, n = 6) or A2 receptor agonist 5'-(N-cyclopropyl)-carboxyamido adenosine (CPCA; 10(-7) mol, n = 6) prior to injection of lipopolysaccharide (LPS) (500 pg mL-1) influenced pulmonary artery pressure (PAP), pulmonary energy content and oedema formation as compared with controls, solely infused with LPS (n = 6). Release rates of adenosine and uric acid were determined by high-performance liquid chromatography. Pulmonary tissue concentrations of high-energy phosphates were measured and the adenine nucleotide pool, adenosine 5'-triphosphate (ATP)/adenosine 5'-diphosphate (ADP) ratio and adenylate energy charge of the pulmonary tissue were calculated. RESULTS: Administration of LPS induced increases in PAP within 2 h up to 20.8 +/- 2.9 mmHg (P < 0.01). While pretreatment with the A1 agonist merely decelerated pressure increase (13.8 +/- 1.1 mmHg, P < 0.05), the A2 agonist completely suppressed the pulmonary pressure reaction (9.6 +/- 1.0 mmHg, P < 0.01). Emergence of lung oedema after exclusive injection of LPS up to 12.0 +/- 2.9 g was absent after A1 (0.6 +/- 0.5 g) and A2 (-0.3 +/- 0.2 g) agonists. These observations were paralleled by increased adenosine release rates compared with LPS controls (P < 0.05). Moreover, tissue concentrations of ADP, ATP, guanosine 5'-diphosphate, guanosine 5'-triphosphate, nicotinamide-adenine-dinucleotide and creatine phosphate were significantly reduced after LPS. Consequently, the calculated tissue adenine nucleotide pool and the adenylate energy charge increased after adenosine receptor stimulation (P = 0.001). CONCLUSIONS: Adenosine A1- and A2-receptor agonists reduced LPS-induced vasoconstriction and oedema formation by maintenance of tissue energy content. Thus, adenosine receptor stimulation, in particular of the A2 receptor, might be beneficial during acute lung injury.

Expression of HMGB1 during tooth development.

High mobility group box 1 (HMGB1) is a nuclear and cytosolic protein that can act as a transcription factor, a growth factor, or a cytokine. To elucidate a possible role for HMGB1 in tooth development, we have studied the expression of HMGB1 and its receptor RAGE (receptor for advanced glycation end-products) during the late fetal and early postnatal period of rat by using light- and electron-microscopic immunohistochemistry. Low HMGB1 protein expression was observed during fetal and newborn stages of tooth development. However, from postnatal day 5 (P5) onward, a marked increase occurred in the levels of the protein in most dental cell types. Expression was particularly high in ameloblasts and odontoblasts at regions of ongoing mineralization. Although most HMGB1 immunoreactivity was confined to cell nuclei, it was also present in odontoblast cytoplasm. At P5, ameloblasts and odontoblasts also showed RAGE immunoreactivity, and reverse transcription-polymerase chain reaction demonstrated both HMGB1 and RAGE mRNA in human dental pulp cells in vitro. Immunoblots performed on extracts from bovine dentin demonstrated a principal band at approximately 27 kDa, indicating that HMGB1 participates in tooth mineralization. The expression of both ligand and receptor suggests an autocrine/paracrine HMGB1 signalling axis in odontoblasts.

Amelotin--a Novel Secreted, Ameloblast-specific Protein.

We aimed to analyze the differential gene expression in various murine dental tissues, expecting to find novel factors that are involved in tooth formation. We here describe the identification of a novel ameloblast-specific gene, amelotin (AMTN), by differential display polymerase chain-reaction (DD-PCR) analysis of microdissected ameloblasts, odontoblasts, dental pulp, and alveolar bone cells of 10-day-old mouse incisors. The conceptually translated protein sequence was unique and showed significant homology only with its human orthologue. The amelotin genes from mouse and human displayed a similar exon-intron structure and were expressed from loci on chromosomes 5 and 4, respectively, which have been associated with various forms of amelogenesis imperfecta. Expression of amelotin mRNA was restricted to maturation-stage ameloblasts in developing murine molars and incisors. Amelotin protein was efficiently secreted from transfected cells in culture. Taken together, our findings suggest that amelotin is a novel factor produced by ameloblasts that plays a critical role in the formation of dental enamel.

The hyalectan degrading ADAMTS-1 enzyme is expressed by osteoblasts and up-regulated at regions of new bone formation.

During bone formation, there are numerous pivotal changes in the interrelationships between osteoblasts and molecules of the extracellular matrix (ECM). Consequently, the mechanisms that underlie the temporal and spatial distribution of ECM molecules in bone are of considerable interest in understanding its formation. A subfamily of a disintegrin and metalloproteinase (ADAMs) has been identified, which contain thrombospondin-like motifs (ADAMTS), and can break down several ECM molecules. Using reversed transcribed PCR, we identified ADAMTS-1, -4 and -5 mRNA expression in cultures of rat osteoblasts treated with ascorbic acid, beta-glycerophosphate and dexamethasone, molecules known to drive osteoblast differentiation. Of these, ADAMTS-1 followed most closely the osteogenic marker osteocalcin during in vitro mineralisation. Consequently, we studied, in detail, protein expression of ADAMTS-1 during in vitro osteogenesis together with ADAMTS-1 immunohistochemistry staining of sections from 2- and 10-day-old rat femur. Western analysis of osteoblast proteins showed ADAMTS-1 products that correspond well with both full-length and furin-processed species. In the ECM laid down by osteoblasts, only the mature secreted protein (approximately 90 kDa) and its accumulation during the later stages of osteogenesis in vitro were noticed. Furthermore, immunostaining with an antibody recognising ADAMTS-1 demonstrated strong expression around mineralised nodules and intense focal staining of putative new areas of nodule formation in vitro. Finally, immunohistochemistry of 2- and 10-day-old rat femur localised ADAMTS-1 protein to regions associated with osteogenesis. These data show that ADAMTS-1 protein accumulates in osteoblast ECM during differentiation. Furthermore, the focalised expression of ADAMTS-1 in regions of osteogenesis, both in vitro and in vivo, implicates this multifunctional protein to be involved in mineralised nodule and bone formation.

Nucleobindin--a Ca2+-binding protein present in the cells and mineralized tissues of the tooth.

Nucleobindin, a Ca2+-binding protein, has been previously identified within the nucleus and endoplasmic reticulum, and in association with the Golgi membrane. In addition, nucleobindin has been shown to be a minor constituent of bone extracellular matrix and has been postulated to play a role in mineralization. In the current investigation, we report the expression and localization of nucleobindin within odontoblasts and the dentin matrix. Nucleobindin mRNA transcripts were detected in the tooth, and in situ hybridization analysis substantiated the findings, showing nucleobindin expression within mature odontoblasts and within the cells of surrounding developing alveolar bone. Western blot analysis of tooth protein extracts demonstrated the presence of a 63 kDa protein, which showed immunologic affinity for a rat nucleobindin peptide antibody. The distribution of the protein was shown in mature odontoblasts by using immunohistochemistry. Moreover, immunogold labeling of nucleobindin and subsequent ultrastructural analysis demonstrated a similar pattern of distribution. Nucleobindin was identified within odontoblast cellular compartments: the nucleus, endoplasmic reticulum, and mitochondria. Of interest, nucleobindin localization was observed within the surrounding dentin extracellular matrix, and immunogold labeling was shown to accumulate with tissue development toward the cusp. The study clearly demonstrated the presence of nucleobindin within dental tissues. In consideration of the known functional properties of nucleobindin, it may be postulated that nucleobindin may contribute to the accumulation and transport of Ca2+ ions to the mineralization front prior to hydroxyapatite deposition.