Flow Cytometric Assessment of AKT Signaling in Platelet Activation: An Alternative Diagnostic Tool for Small Volumes of Blood.

INTRODUCTION: The diagnosis of platelet function disorder in children is challenging. Light transmission aggregometry is the gold standard for platelet function disorders. However, large blood volumes are required. Currently, there are no existing tools for the diagnosis of platelet function disorders that use small blood volumes. AKT signaling plays a central role in platelet activation during hemostasis and might be visualized by flow cytometry. METHODS: Platelet-rich plasma obtained by centrifugation of citrated blood from healthy volunteers was activated with arachidonic acid, thrombin receptor activating peptide-6 (TRAP-6), collagen, adenosine diphosphate ADP, collagen-related peptide (CRP), and epinephrine. After platelet activation, the phosphorylation of AKT was assessed by flow cytometer using a Navios cytometer. RESULTS: Healthy volunteers showed a reproducible phosphorylation of AKT upon activation. In comparison to nonactivated platelets, we documented an increase in pAKT expression with all agonists. Especially TRAP-6 and CRP caused considerable increase in percentage of pAKT expression throughout all the tested healthy volunteers. CONCLUSION: An activation of the AKT-signal pathway by different agonists can clearly be detected on the flow cytometer, indicating that the visualization of signaling in platelets by flow cytometry might be an efficient alternative for light transmission aggregometry to test platelet function in children.

Effects of experimental occlusal interference on body posture: an optoelectronic stereophotogrammetric analysis.

In recent years, there has been increasing interest in the relationship between dental occlusion and body posture both among people and in scientific literature. The aim of the present longitudinal study is to investigate the effects of an experimental occlusal interference on body posture by means of a force platform and an optoelectronic stereophotogrammetric analysis. An occlusal interference of a 0- to 2-mm-thick glass composite was prepared to disturb the intercuspal position while not creating interference during lateral or protrusive mandibular excursions. Frontal and sagittal kinematic parameters, dynamic gait measurements and superficial electromyographic (SEMG) activity of head and neck muscles were performed on 12 healthy subjects. Measurements were taken 10 days before the application of the occlusal interference, and then immediately before the application, the day after it, and at a distance of 7 and 14 days under four different exteroceptive conditions. The outcomes of this study show that an occlusal interference does not modify significantly over time static and dynamic parameters of body posture under different exteroceptive conditions. It has a minimal influence only on the frontal kinematic parameters related to mandibular position, and it induces a transient increase of the activity of masticatory muscles. In this study, the experimental occlusal interference did not significantly influence the body posture during a 14-day follow-up period.

Self-assessed bruxism and phobic symptomatology.

AIM: The aim of this observational study was to compare two samples of patients (identified, from a previous survey carried out in 2007, as self-assessed bruxers and not) on the basis of the presence of anxious/phobic symptoms, general and linked to an oral surgery. METHODS: Forty-three bruxers and 207 non-bruxers were identified; among these last ones a sub-sample of 89 subjects was randomly selected as control and analyzed. The instruments for data collecting were two self-administered psychological questionnaires: STAI-Y1, Phobia Scale by Marks-Sheehan, and supplementary items on specific dental fear/phobia. RESULTS: No significant differences were observed for age, gender and occupation data but interestingly bruxers are significantly more represented among widows/divorced and graduated in comparisons with non-bruxers. Alcohol consumers were more frequent in bruxers than in non-bruxers (55.8% and 12.4%, respectively; P=0.0001). Global anxiety (P=0.02), agoraphobia, claustrophobia, pathophobia, social phobia (P<0.05), are more frequent in bruxers as also a suffocation feeling (P=0.02). The severity of behaviours that aim to avoid the same situations that causes phobias is low and similar in the two groups. CONCLUSION: The involuntary habit of clenching is, in our opinion, reported by the patients who control their anxiety/phobias without avoiding behaviours, increasing the muscular activity at a level relevant to bruxism.

Chaperone-like features of bovine serum albumin: a comparison with alpha-crystallin.

The chaperone behaviour of bovine serum albumin was compared with that of alpha-crystallin. The chaperone activity was assessed by measuring: (i) the ability to antagonize protein aggregation induced by heat; (ii) the capability to protect the activity of thermally stressed enzymes and (iii) the effectiveness in assisting the functional recovery of chemically denatured sorbitol dehydrogenase. Despite the lack of structural analogies, both proteins show several functional similarities in preventing inactivation of thermally stressed enzymes and in reactivating chemically denatured sorbitol dehydrogenase. As with alpha-crystallin, the chaperone action of bovine serum albumin appears to be ATP independent. Bovine serum albumin appears significantly less effective than alpha-crystallin only in preventing thermally induced protein aggregation. A possible relationship between chaperone function and structural organization is proposed. Together, our results indicate that bovine serum albumin acts as a molecular chaperone and that, for its particular distribution, can be included in the extracellular chaperone family.

Alpha-crystallin: an ATP-independent complete molecular chaperone toward sorbitol dehydrogenase.

alpha-Crystallin, the major component of the vertebrate lens, is known to interact with proteins undergoing denaturation and to protect them from aggregation phenomena. Bovine lens sorbitol dehydrogenase (SDH) was previously shown to be completely protected by alpha-crystallin from thermally induced aggregation and inactivation. Here we report that alpha-crystallin, in the presence of the SDH pyridine cofactor NAD(H), can exert a remarkable chaperone action by favoring the recovery of the enzyme activity from chemically denaturated SDH up to 77%. Indeed, even in the absence of the cofactor, alpha-crystallin present at a ratio with SDH of 20:1 (w:w) allows a recovery of 35% of the enzyme activity. The effect of ATP in enhancing alpha-crystallin-promoted SDH renaturation appears to be both nonspecific and to not involve hydrolysis phenomena, thus confirming that the chaperone action of alpha-crystallin is not dependent on ATP as energy donor.

Update on burning mouth syndrome: overview and patient management.

Burning Mouth Syndrome (BMS) is a chronic pain syndrome that mainly affects middle-aged/old women with hormonal changes or psychological disorders. This condition is probably of multifactorial origin, often idiopathic, and its etiopathogenesis remains largely enigmatic. The present paper discusses several aspects of BMS, updates current knowledge, and provides guidelines for patient management. There is no consensus on the diagnosis and classification of BMS. The etiopathogenesis seems to be complex and in a large number of patients probably involves interactions among local, systemic, and/or psychogenic factors. In the remaining cases, new interesting associations have recently emerged between BMS and either peripheral nerve damage or dopaminergic system disorders, emphasizing the neuropathic background in BMS. Based on these recent data, we have introduced the concepts of "primary" (idiopathic) and "secondary" (resulting from identified precipitating factors) BMS, since this allows for a more systematic approach to patient management. The latter starts with a differential diagnosis based on the exclusion of both other orofacial chronic pain conditions and painful oral diseases exhibiting muco-sal lesions. However, the occurrence of overlapping/overwhelming oral mucosal pathologies, such as infections, may cause difficulties in the diagnosis ("complicated BMS"). BMS treatment is still unsatisfactory, and there is no definitive cure. As a result, a multidisciplinary approach is required to bring the condition under better control. Importantly, BMS patients should be offered regular follow-up during the symptomatic periods and psychological support for alleviating the psychogenic component of the pain. More research is necessary to confirm the association between BMS and systemic disorders, as well as to investigate possible pathogenic mechanisms involving potential nerve damage. If this goal is to be achieved, a uniform definition of BMS and strict criteria for its classification are mandatory.

Sucralfate: a help during oral management in patients with epidermolysis bullosa.

BACKGROUND: Epidermolysis bullosa (EB) is a group of genetic disorders that lead to blister formation at variable depths in skin and mucosa. Vesicles may arise spontaneously or be caused by friction or trauma. Oral tissue fragility and blistering is common in all EB types. The majority of patients with mild forms of EB are able to receive dental treatment. The prevention of dental caries is most challenging in subjects with severe mucosal involvement, as they are least able to routinely undergo correct preventive procedures. The aim of this study was to evaluate the effectiveness of a sucralfate suspension in reducing both pain and the number of blisters in patients with EB, and to obtain improved oral hygiene and a lower incidence of caries. METHODS: Five patients with dystrophic EB were treated with sucralfate suspension for the prevention and management of oral blisters. Oral blisters were assessed using a quantitative scale, while pain was assessed using visual analogue scale (VAS), and hygiene was evaluated through plaque and gingival indexes. RESULTS: The number of blisters, oral pain, and plaque decreased in all cases. CONCLUSIONS: Oral prophylaxis with sucralfate prevented oral blisters and oral discomfort. The procedure proved to be cost effective and easy to administer. It did not show significant side effects and may be used routinely in patients with EB.

Complete protection by alpha-crystallin of lens sorbitol dehydrogenase undergoing thermal stress.

Sorbitol dehydrogenase (l-iditol:NAD(+) 2-oxidoreductase, E.C. 1.1.1. 14) (SDH) was significantly protected from thermally induced inactivation and aggregation by bovine lens alpha-crystallin. An alpha-crystallin/SDH ratio as low as 1:2 in weight was sufficient to preserve the transparency of the enzyme solution kept for at least 2 h at 55 degrees C. Moreover, an alpha-crystallin/SDH ratio of 5:1 (w/w) was sufficient to preserve the enzyme activity fully at 55 degrees C for at least 40 min. The protection by alpha-crystallin of SDH activity was essentially unaffected by high ionic strength (i.e. 0.5 m NaCl). On the other hand, the transparency of the protein solution was lost at a high salt concentration because of the precipitation of the alpha-crystallin/SDH adduct. Magnesium and calcium ions present at millimolar concentrations antagonized the protective action exerted by alpha-crystallin against the thermally induced inactivation and aggregation of SDH. The lack of protection of alpha-crystallin against the inactivation of SDH induced at 55 degrees C by thiol blocking agents or EDTA together with the additive effect of NADH in stabilizing the enzyme in the presence of alpha-crystallin suggest that functional groups involved in catalysis are freely accessible in SDH while interacting with alpha-crystallin. Two different adducts between alpha-crystallin and SDH were isolated by gel filtration chromatography. One adduct was characterized by a high M(r) of approximately 800,000 and carried exclusively inactive SDH. A second adduct, carrying active SDH, had a size consistent with an interaction of the enzyme with monomers or low M(r) aggregates of alpha-crystallin. Even though it had a reduced efficiency with respect to alpha-crystallin, bovine serum albumin was shown to mimic the chaperone-like activity of alpha-crystallin in protecting SDH from thermal denaturation. These findings suggest that the multimeric structural organization of alpha-crystallin may not be a necessary requirement for the stabilization of the enzyme activity.

Intraoral fluoride releasing device: a new clinical therapy for dentine sensitivity.

BACKGROUND: Dentinal sensitivity (DS) occurs frequently in adult populations in western countries. The purpose of this work was to assess the effectiveness of a new intraoral fluoride releasing device (IFRD) in reducing the level of pain in patients with primary or postsurgical dentine sensitivity. METHODS: A total of 49 individuals were selected for this study, 15 of whom had post-periodontal surgery dentine sensitivity and 34 with primary sensitivity. An IFRD was applied to 39, while 10 received a placebo device. All individuals in the control group suffered from primary sensitivity. The IFRD used in this study consists of sodium fluoride encased in an acrylic polymer which releases fluoride at a rate of approximately 0.04 mg/day. All patients were asked to rinse with cold water (10 degrees C) and to indicate the level of pain on a 0 to 10 visual analog scale, 0 equalling "no pain" and 10 "maximum bearable pain." All subjects were evaluated once a week during 4 months. Statistical analysis of dentine sensitivity was performed as a univariate study, in relation to the main factors: age, gender, and primary sensitivity or postsurgical etiology. RESULTS: Symptoms decreased dramatically in all treated patients. The level of sensitivity did not change during the first week after IFRD application, but decreased significantly within the fourth week and remained absent through the duration of the treatment (P <0.01). Difference in sensitivity with respect to different etiology was significant only after 4 weeks (P= 0.01), while there was no statistical difference with respect to age or gender. CONCLUSIONS: This paper is an initial study to evaluate the effectiveness of the IFRD. The method is fast, painless, inexpensive, and it appears to be suitable as a routine treatment. The presented data support the conclusions at this stage and warrant more comprehensive evaluation.

A new approach against sugar cataract through aldose reductase inhibitors.

Aldose reductase inhibition is one of the therapeutic strategies that has been proposed to prevent or ameliorate long term diabetic complications including retinopathy and sugar cataract. Rats were fed with a galactose rich diet and the aldose reductase inhibitor Tolrestat was topically delivered by ocular instillation. The levels of lens aldose reductase activity, galactitol and the onset of cataract were evaluated during and after treatment with the inhibitor. Topical application of 1-3% Tolrestat (10 microl) four times daily resulted, after 9 days, in a significant decrease in the enzyme activity. Well after interrupting treatment with the drug, the enzyme activity remained impaired and galactose induced cataract was prevented. Our findings may represent the basis for therapeutic plans to prevent sugar cataract by long term cyclic treatments with aldose reductase inhibitors, with reduction in drug doses and side effects.

Stomatognathic function in juvenile rheumatoid arthritis and in developmental open-bite subjects.

Patients affected with juvenile rheumatoid arthritis present clear radiological alterations of the condyles of the temporomandibular joint (TMJ) due to the inflammatory process, with a prevalence ranging from 17 percent to 63 percent. This work is a comparison between a group of subjects with juvenile rheumatoid arthritis presenting signs and symptoms of TMT dysfunction and a group of the same age not suffering from any rheumatic morbidity, but presenting an open bite > 3 mm. The aim is to verify whether the open bite can induce an alteration in mandibular function comparable to that observed in juvenile rheumatoid arthritis. Statistically significant difference is found in the juvenile rheumatoid arthritis group only regarding spontaneous and provoked muscle pain and the lateral opening deviations of the mouth, but mostly the two groups seem alike and present the same stomatognathic pattern.

A retentive system for intra-oral fluoride release during orthodontic treatment.

The aim of this study was to test a particular type of intra-oral fluoride releasing device (IFRD), designed to release 0.04 mg/day of fluoride over a period of 6 months, using customized holders, in patients receiving orthodontic treatment. Discomfort, holder detachment, plaque accumulation near the device, and the presence of gingivitis, bleeding, white spot lesions, and/or decay was recorded in 76 orthodontic patients (53 experimental and 23 controls) before and after wearing the device for 12 months. The system proved to be easy and quick to use, and did not cause discomfort. There were no significant differences between the treated and the control groups for plaque index, bleeding, or the presence of gingivitis. In addition, no carious and/or white spot lesions occurred during the duration of this study in the test group.

Sorbitol dehydrogenase from bovine lens: purification and properties.

Bovine lens sorbitol dehydrogenase (L-iditol:NAD+ 2-oxidoreductase, EC 1.1.1.14) (SDH) was purified to electrophoretic homogeneity (51 U/mg of protein) and characterized for both kinetic and some structural properties. The enzyme proves to be a homotetramer of 156 kDa containing one equivalent of zinc ion per subunit. Metal chelators such as EDTA and 1,10-phenanthroline determine a loss of enzyme activity which can be specifically recovered by addition of either zinc or manganese ions. Inactivation induced not only by metal chelators but also by thiol reagents is effectively prevented by the pyridine cofactor. Bovine lens SDH is active on polyalcohols and keto-sugars with more than three carbon atoms, and also requires special steric constraints for substrate recognition. Of the polyols, xylitol is the most effective substrate (kcat/KM of 8.1 s-1 mM-1), followed by sorbitol (kcat/KM of 1.59 s-1 mM-1); fructose, the most effective carbonyl substrate, displays a kcat/KM of only 0.9 s-1 mM-1. Analysis at the steady state of initial velocities as a function of the concentration of different substrates and cofactors and studies of product inhibition indicate for both fructose reduction and sorbitol oxidation a Theorell and Chance-type kinetic mechanism of action.

Headache and cranio-mandibular disorders during adolescence.

A clinical and epidemiological investigation was performed on 62 adolescents, 21 males and 41 females, with recurring headaches, in order to analyze the connections between the cephalalgia and the signs and symptoms of mandibular dysfunction. The anamnestic and objective data were reviewed with the aid of the Helkimo indices and compared to data on a control group of comparable age who were not headache sufferers. The most statistically significant signs encountered (p < 0.001) were: pain on palpation of masticatory muscles (61%), altered (43%) or reduced (27%) mandibular movement, preauricular tenderness (19%); the high incidence of parafunctions indicated the behavioral substrate. This pilot study reveals a link between the two pathologies and the need for early diagnosis, preventive identification of the subjects at risk and interdisciplinary cooperation.

Specifically targeted modification of human aldose reductase by physiological disulfides.

Aldose reductase is inactivated by physiological disulfides such as GSSG and cystine. To study the mechanism of disulfide-induced enzyme inactivation, we examined the rate and extent of enzyme inactivation using wild-type human aldose reductase and mutants containing cysteine-to-serine substitutions at positions 80 (C80S), 298 (C298S), and 303 (C303S). The wild-type, C80S, and C303S enzymes lost >80% activity following incubation with GSSG, whereas the C298S mutant was not affected. Loss of activity correlated with enzyme thiolation. The binary enzyme-NADP+ complex was less susceptible to enzyme thiolation than the apoenzyme. These results suggest that thiolation of human aldose reductase occurs predominantly at Cys-298. Energy minimization of a hypothetical enzyme complex modified by glutathione at Cys-298 revealed that the glycyl carboxylate of glutathione may participate in a charged interaction with His-110 in a manner strikingly similar to that involving the carboxylate group of the potent aldose reductase inhibitor Zopolrestat. In contrast to what was observed with GSSG and cystine, cystamine inactivated the wild-type enzyme as well as all three cysteine mutants. This suggests that cystamine-induced inactivation of aldose reductase does not involve modification of cysteines exclusively at position 80, 298, or 303.

Simulated disability exercises and their impact on attitudes toward persons with disabilities.

Two separate graduate rehabilitation counselling groups from a mid-south university either did or did not take part in a wheelchair sensitivity training pilot study designed to enhance their understanding as to what life might be like for persons who use a wheelchair. Twenty students were individually accompanied on a designated route across campus by a non-disabled graduate researcher. Eighteen other graduate students formed a control group and did not participate. Both groups completed a contextually different 14-item Likert type questionnaire designed to assess their perceptions of persons with physical disabilities based on either the sensitivity exercise or their experience in the rehabilitation programme. Results from this quasi-experimental study suggested that regardless of whether they participated in the sensitivity training, both groups perceived they would become better counsellors. t-Tests revealed significant differences in the two groups' responses to questions pertaining to daily frustrations experienced by persons with physical disabilities and a pre-occupation with how accessible places are. Other differences noted were that persons with physical disabilities must feel different from being stared at and must have a harder time in society. Implications of the utility and misuses of these one-time experiences are explored.