Fisher's PLSD Research Articles

Importance of the hybrid layer on the bond strength of restorations subjected to cyclic loading

The present study evaluated the long-term microtensile bond strength of collagen-depleted dentin followed by cyclic loading. Bovine flat, midcoronal dentin received one of the following surface treatments: acid-etch or acid-etched + 5% NaOCl for 2 min. The teeth were restored with Single Bond, Scotchbond Multi-purpose, One-Step Plus, or All-Bond 2 adhesive systems. Half of the specimens were randomly assigned to receive 200,000 cycles (50 N force). Teeth were sectioned into 1 x 1 mm thick slices and stored for 24 h, 1 month, 3 months, or 6 months. Specimens were subjected to tensile testing after elapsed storage time. Samples were analyzed by three-way ANOVA and Fisher's PLSD (p < 0.05). The results showed that the deproteinized groups without cyclic loading presented lower bond strength when compared with the control group, but the difference was only statistically significant for Single Bond and All-Bond 2 (p < 0.05). All adhesives presented a decrease in bond strength over time, regardless of the dentin treatment. When cyclic loaded, the decrease in bond strength for the deproteinized group was even greater when compared with the control group (p < 0.05). The results suggest that the hybrid layer is important as a stress-buffering layer when loading is applied, and thus the presence of collagen is essential.

In vitro evaluation of the accuracy of five different electronic apex locators for determining the working length of endodontically retreated teeth

The aim of this study was to evaluate the accuracy of five electronic apex locators (EALs) in determining the working length (WL) of teeth after removal of the root canal obturation materials. A total of 32 extracted straight, single-rooted teeth were used. The actual canal length (AL) was determined and the WL was established by subtracting 0.5 mm from the AL. The root canals were instrumented and divided into two groups. One group (n = 6) served as control, while the other group (n = 26) was the experimental group. In the experimental group, the root canals were obturated using vertically compacted gutta-percha with AH 26 sealer. In both groups, the access cavities were restored with a provisional restoration and stored for 15 days at 37 degrees C and 100% humidity. The root canal obturation material was removed, and the teeth were then mounted in an experimental apparatus. Five EALs were used: Dentaport ZX, ProPex, Foramatron D10, Apex NRG and Apit 7. For the electronic measurement of canal length, a size 25 K-file was used. During measurement, the canal was irrigated with 2.5% sodium hypochlorite. The difference (D) between the AL and the electronically determined length (EDL), AL-EDL, was calculated and recorded for each measurement. Data were analysed by two-way anova and Fisher's PLSD test. In both groups, statistically significant differences were found among the EALs (P < 0.01). In conclusion, the Dentaport ZX, ProPex and Foramatron D10 were more accurate than the other two EALs in determining the WL in teeth after removal of the root canal obturation materials. However, the Apex NRG and Apit 7 were also reliable for determination of the WL in the majority of the cases.

Generalization of Courtship Learning in Drosophila Is Mediated by cis-Vaccenyl Acetate

Reproductive behavior in Drosophila has both stereotyped and plastic components that are driven by age- and sex-specific chemical cues. Males who unsuccessfully court virgin females subsequently avoid females that are of the same age as the trainer. In contrast, males trained with mature mated females associate volatile appetitive and aversive pheromonal cues and learn to suppress courtship of all females. Here we show that the volatile aversive pheromone that leads to generalized learning with mated females is (Z)-11-octadecenyl acetate (cis-vaccenyl acetate, cVA). cVA is a major component of the male cuticular hydrocarbon profile, but it is not found on virgin females. During copulation, cVA is transferred to the female in ejaculate along with sperm and peptides that decrease her sexual receptivity. When males sense cVA (either synthetic or from mated female or male extracts) in the context of female pheromone, they develop a generalized suppression of courtship. The effects of cVA on initial courtship of virgin females can be blocked by expression of tetanus toxin in Or65a, but not Or67d neurons, demonstrating that the aversive effects of this pheromone are mediated by a specific class of olfactory neuron. These findings suggest that transfer of cVA to females during mating may be part of the male's strategy to suppress reproduction by competing males.

Control of Methyl Methacrylate During the Preparation of Orthopedic Bone Cements

The use of methyl methacrylate (MMA) bone cement during orthopedic procedures has been seen as a potential exposure hazard to health care professionals. However, that assessment is based on a number of investigations with problems in experimental design, analysis, and data interpretation. The current investigation quantified differences in MMA vapors produced during the preparation of competing bone cements using various methods of preparation. Unlike previous investigations, this effort employs modern validated sampling and analytical methods, and considers the affect of censored results. Measurements of sufficient quality and number were collected to allow for a statistical treatment of the data. The ability of two controlled preparation techniques to reduce MMA emissions were compared with a traditional open container. The results confirmed that the preparation of bone cement releases MMA vapors into the breathing zone of the preparer. One preparation technique (Stryker Bowl) controlled emissions during mixing and curing and affected a 73% reduction in measured MMA concentrations. In addition to mixing and curing, the second technique (UltraMix System) also controlled the MMA during pouring of the monomer and affected a 90% reduction in MMA concentrations. An ANOVA test of interaction indicates that the reductions are attributable to the preparation technique regardless of the type of cement being used. Both a Fisher's PLSD and Games/Howell post hoc test of the results indicate that the mean differences between the uncontrolled open container and the controlled preparation techniques are significant (p < 0.05).

Effect of a desensitizing agent containing glutaraldehyde and HEMA on bond strength to Er:YAG laser-irradiated dentine

The aim of this study was to investigate whether a desensitizing agent (GLUMA Desensitizer) containing glutaraldehyde and HEMA improved the bond strength and bonding durability of a self-etching primer adhesive to Er:YAG-irradiated dentine. Dentine of 120 human molars was exposed by wet grinding on SiC paper for bond strength testing. Thirty specimens each were allocated to the following treatment groups: (1) control; (2) Er:YAG laser irradiation; (3) Er:YAG laser irradiation followed by application of GLUMA Desensitizer; (4) Er:YAG laser irradiation followed by application of GLUMA Desensitizer and 10s rinsing with water. Composite cylinders were bonded to the dentine surfaces with a self-etching priming adhesive system. Tensile bond strengths (TBS) of 10 specimens of each treatment group were measured after 24-h water storage, 6 months water storage and 12 months water storage, respectively, and the failure modes were analyzed. TBS data were statistically treated by two-way ANOVA and Fisher's PLSD test at a significance level of p<0.05. TBSs for the GLUMA-non rinse and GLUMA-rinse groups were significantly higher than for the laser group at 24 h and 12 months. Specimens from the Er:YAG-irradiated dentine group had significantly lower bond strengths than the control group at each storage time. All control specimens showed cohesive fractures in resin close to the bonding interface whereas the Er:YAG laser-irradiated groups showed both dentine cohesive, resin cohesive and dentine-resin mixed failures. Application of GLUMA Desensitizer to Er:YAG-irradiated dentine increases the bond strength and durability of the self-etching priming adhesive used.

Seasonal Variations of Melatonin Secretion in Young Females under Natural and Artificial Light Conditions in Fukuoka, Japan

The purpose of this study was to examine the seasonal variations of melatonin secretion of subjects and of their surrounding light conditions. Eight Japanese female students (20.1+/-2.6 yrs, Mean+/-SD) living in Fukuoka, Japan, participated in the present study. Saliva samples were collected every 3 hours over the course of a day, and the light intensity during daily life was measured every 1 min for 5 days in the four seasons. Almost all subjects had different melatonin secretory profiles in autumn, with only two subjects showing similar rhythms in all four seasons. The peak values of melatonin secretion calculated by a spline interpolation were higher in autumn than those in other seasons (p<0.001, Fisher's PLSD) and its peak time in this season was significantly delayed compared with those in spring and summer (p<0.05, Fisher's PLSD). The amount of time during daytime exposure to light of >1,000 lux was at least thirty minutes in all the seasons, and there were no significant differences among them. The relationship between peak level of melatonin secretion and amount of time of daytime light exposure to >1,000 lux was significant only in the autumn. During this season, there was a significant positive correlation (r=0.83, p<0.05, n=6), except for two subjects, whose melatonin secretion remained low.

215 APPLICATION OFA MICROFLUIDIC SPERM SORTER TO THE IN VITRO FERTILIZATION OF PORCINE OOCYTES

In porcine IVF, polyspermy is a persistent obstacle to the efficient production of normal embryos in vitro. A microfluidic sperm sorter (MFSS; Strex Inc., Osaka, Japan) has been developed to isolate motile human spermatozoa (from diluted semen by 2 laminar flows in the microchannel). The motile spermatozoa can gradually accumulate in a chamber of the MFSS. In addition, we have succeeded in reducing the incidence of polyspermic penetration by a transient coincubation of oocytes with spermatozoa in the presence of caffeine, followed by culture of the oocytes in the absence of caffeine (2004 Reproduction 128, 789–800). If porcine oocytes could be cocultured with motile spermatozoa in the chamber of the MFSS, the gradual accumulation of motile spermatozoa might further improve the efficiency of production of monospermic fertilized porcine embryos. The current study was undertaken to apply the MFSS technology to porcine IVF. The sperm-rich fraction from 3 Berkshire boars was diluted at 1 � 108 cells mL–1 with modified Modena containing 20% seminal fluid, cooled to 15�C for 4 h, and kept at the same temperature until use within 2 days. Stored, diluted semen with greater than 80% viability was flowed with Tyrode lactate-HEPES-polyvinyl alcohol medium for 5 or 10 min at room temperature. The concentration and viability of spermatozoa mixed in the flowed medium were determined. In the next experiment, a diluted semen sample was flowed with modified TCM-199 containing 5 mm caffeine for 5 min at room temperature. Before flowing, porcine IVM oocytes were put into the chamber of the MFSS, where motile spermatozoa will accumulate with modified TCM-199 containing 5 mm caffeine. After flowing for 5 min, those oocytes were transferred into a 500-µL droplet of caffeine-free modified TCM-199, and culture was continued for 8 h. Statistical analyses were carried out by ANOVA and Fisher's PLSD post hoc test. After flowing for 5 min, the concentration of spermatozoa recollected from the chamber was 5.7 � 105 cells mL–1. The viability of recollected spermatozoa was significantly higher than the original flowed semen. However, when spermatozoa were flowed for 10 min, the viability of recollected spermatozoa was not different from the original flowed semen, whereas the concentration of recollected spermatozoa was 9.7 � 105 cells mL–1. When IVM oocytes were cocultured with spermatozoa gradually accumulated in the chamber of MFSS for 5 min, 35% (22 to 48%) of oocytes were penetrated, and 95% (90 to 100%) of the penetrated oocytes were monospermic. These observations demonstrate that the MFSS can separate penetrable boar spermatozoa with a high viability and sufficient concentration for IVF. Furthermore, the current data suggest the possibility of improving the efficiency of monospermic fertilization of porcine IVM oocytes by a transient coculture with MFSS.

Electromyography Analysis of Shoulder Joint Muscles in Standing with Three Ambulatory Aids

We investigated the muscle activity of the shoulder joint during single-leg standing three types of ambulatory aid, T-shaped cane (T-cane), Quadripod cane (Q-cane) and Lofstrand crutch (L-crutch) in which were used. The left leg of eight healthy volunteers was assumed to be an affected leg. All participants were asked to keep 20% of the weight-bearing load on the ambulatory aid and the electromyography (EMG) was recorded. Results of the analysis of variance followed by Fisher's PLSD post hoc test reveal that EMG values of the medial and posterior part of the deltoid muscle were significantly related to the three types of ambulatory aid, but the anterior part of the deltoid was not. EMG values of the biceps brachii, triceps brachii, and latissimus dorsi were significantly larger in T-cane and Q-cane users than in L-crutch users, and EMG of the pectoralis major was significantly larger in Q-cane users than in L-crutch users. These results permit us to select an appropriate ambulatory aid for patients.

Interruption of CryAB-Amyloid Oligomer Formation by HSP22

An R120G missense mutation in alpha-B-crystallin (CryAB), a small heat-shock protein (HSP), causes a desmin-related cardiomyopathy (DRM) that is characterized by the formation of aggregates containing CryAB and desmin. The mutant CryAB protein leads to the formation of inclusion bodies, which contain amyloid oligomer intermediates (amyloid oligomer) in the cardiomyocytes. To further address the underlying mechanism(s) of amyloid oligomer formation in DRM linked to the CryAB R120G, a recombinant CryAB R120G protein was generated. The purified CryAB R120G protein can form a toxic amyloid oligomer, whereas little immunoreactivity was observed in the wild-type CryAB protein. A native PAGE showed that the oligomerized form was present in the CryAB R120G protein, whereas only a high molecular mass was detected in the wildtype CryAB. The oligomerized CryAB R120G of around 240-480 kDa showed strong positive immunoreactivity against an anti-oligomer antibody. The CryAB R120G amyloid oligomer was unstable and easily lost its conformation by beta-mercaptoethanol and SDS. Recombinant HSP25 or HSP22 proteins can directly interrupt oligomer formation by the CryAB R120G protein, whereas the amyloid oligomer is still present in the mixture of the wild-type CryAB and CryAB R120G proteins. This interruption by HSP25 and HSP22 was confirmed in a cardiomyocyte-based study using an adenoviral transfection system. Blockade of amyloid oligomer formation by HSP25 and HSP22 recovered the ubiquitin proteosomal activity and cellular viability. Blockade of oligomer formation by small HSP may be a new therapeutic strategy for treating DRM as well as other types of amyloid-based degenerative diseases.

213 THE EFFECT OF eCG TREATMENT IN AN ESTRUS SYNCHRONIZATION PROTOCOL ON PREGNANCY RATES AFTER EMBRYO TRANSFER IN HOLSTEIN HEIFERS

Recent studies have shown that the presence of accessory or multiple corpora lutea (CL) and increased progesterone (P4) concentrations reduced early embryonic mortality in cattle. The objective of this study was to evaluate the effect of equine chorionic gonadotropin (eCG) treatment on the number of CL, the P4 concentrations, and pregnancy rates after embryo transfer (ET). Holstein heifers (n = 120) from 7 dairy farms received an intravaginal progesterone-releasing device (CIDR; InterAg, Hamilton, New Zealand) and 2 mg IM of estradiol benzoate (EB; Gynandol®; Sankyo, Tokyo, Japan) at random stages of the estrous cycle. After 7 to 9 days, CIDRs were removed and 15 mg of prostaglandin F2α (PG; Pronalgon®; Pfizer Japan, Nagoya, Japan) were administered, followed by 100 µg IM GnRH (Conceral®; Takeda Pharmaceutical Co., Ltd., Osaka, Japan) 2 days later (Day 0). The heifers were placed at random into 3 groups for eCG treatment. The eCG was not administered in a control group (n = 53); heifers in other 2 groups received 1000 IU eCG (Peamex®; Sankyo, Japan) IM at the time (0 h group, n = 37) or 48 h before (48 h group, n = 30) PG injection/CIDR removal. On Day 7, heifers were examined by ultrasonography (Aloka SSD500; Aloka, Tokyo, Japan) for number of CL; heifers with at least one functional CL received an in vivo-derived frozen–thawed embryo by direct transfer. At the same time, a blood sample was collected to determine P4 concentration. Pregnancy rates were determined on Days 30 and 60 by ultrasonography and rectal palpation, respectively. The data were analyzed by ANOVA and means were compared with Fisher's PLSD. Proportional data were analyzed by the chi-square test. P4 concentrations (mean ± SD) on Day 7 were 1.8 ± 1.0, 5.6 ± 3.3, and 2.2 ± 1.1 ng mL−1 for the control, 48 h, and 0 h groups, respectively (48 h vs. control and 0 h; P &amp;lt; 0.001). The number of CL on Day 7 were 1.1 ± 0.4, 2.5 ± 1.4, and 1.8 ± 0.9 for the control, 48 h, and 0 h groups, respectively (control vs. 48 h and 0 h, and 48 h vs. 0 h; P &amp;lt; 0.01). Pregnancy rates did not differ between 0 and 48 h groups but both were higher than in the control group (Table 1). Results suggest that the estrus synchronization protocol with administration of eCG at the time of CIDR removal or 48 h earlier significantly increased the number of CL and the P4 concentration, and improved pregnancy rates in Holstein heifers after ET. Table 1.Pregnancy rates of Holstein heifers synchronized with CIDR and PG and treated with eCG

Neuroprotective peptides prevent the alcohol-induced alteration in GABA-A3 which plays a role in cleft lip and palate in fetal alcohol syndrome

Prenatal alcohol exposure affects 1/200 live births in US and results in cranio-facial dysmorphology including cleft lip and palate and long-term learning disabilities. The GABA receptor subunit GABA-Aβ3 is critical during embryogenesis for neural tube and palate development. The GABA-Aβ3 knock-out results in cleft palate and neonatal death that were rescued by introduction of the β3-subunit transgene. We have shown that prenatal alcohol exposure downregulated GABA-Aβ3 in the embryo. In a mouse model for fetal alcohol syndrome (FAS), neuroprotective peptides prevented alcohol-induced fetal death, growth restriction and learning deficits. Our objective was to determine if the peptides' prevention of alcohol-induced complications includes GABA-Aβ3. Timed, pregnant C57BL6/J mice were treated on gestational day 8 with either alcohol (0.03 mL/g) or placebo or alcohol+peptides (NAP+SAL 20μg). Embryos were harvested at 24h (E9) and 10 days (E18) after treatment. Using samples from at least 3 litters per time-point, calibrator-normalized relative real time PCR was performed using primers for GABA-Aβ3 with GAPDH standardization. Statistical analysis included ANOVA and Fisher PLSD. Twenty-four hours after treatment, the alcohol-induced decrease in GABA-Aβ3 was not prevented by the pretreatment with the peptides (P=0.8). However, at E18, the alcohol induced GABA-Aβ3 down regulation was prevented by the peptides (P=0.01) with levels higher than controls (P=0.007). Prenatal treatment with neuroprotective peptides prevented the alcohol-induced decline and resulted in a significant elevation in GABA-Aβ3 ten days after alcohol exposure. Since palate formation continues through gestational day 18, the increase in GABA-Aβ3 expression may be an attempt to overcompensate for alcohol's effects and may play a role in the prevention of cleft lip and palate. These findings suggest that NAP and SAL may be beneficial for prevention of cleft palate in FAS and in other pathologic conditions.

Elevated BALF concentrations of α- and β-defensins in patients with pulmonary alveolar proteinosis

Defensins are endogenous antibiotics and regulators of inflammation, immunity and wound repair. Their concentrations are substantially increased in bronchoalveolar lavage fluid (BALF) of patients with infectious lung diseases. alpha-defensin (HAD) levels are also elevated in patients with idiopathic pulmonary fibrosis (IPF) and correlated with the decline in pulmonary function tests, suggesting the association of defensins with the pathogenesis of interstitial lung diseases. The aim of this study was to determine the profile of defensins in interstitial lung diseases. Serum and BALF levels of HAD and beta-defensin 1 and 2 (HBD-1, and -2) were measured by radioimmunoassay in 63 patients with interstitial lung diseases, including idiopathic pulmonary alveolar proteinosis (PAP), IPF, nonspecific interstitial pneumonia (NSIP), cryptogenic organizing pneumonia (COP) and pulmonary sarcoidosis, and in 9 healthy volunteers as controls. Levels of HAD in BALF of patients with PAP were significantly higher than those in controls and patients with COP and sarcoidosis. Serum levels of HAD in all groups were significantly higher than those in controls. Levels of HBD-1 and -2 in BALF of patients with PAP were extremely high in all subjects. Serum levels of HBD-1 were higher in all patient groups, with the exception of those with PAP, and those of HBD-2 were also higher in patients with IPF and sarcoidosis, compared with controls. BALF of PAP patients, but not IPF patients and controls, expressed antimicrobial activity against Pseudomonas aeruginosa and Staphylococcus aureus. Our findings suggest different kinetics of HAD and HBD-1 and -2 in serum and BALF of interstitial lung diseases and that these antimicrobial peptides in the airway lumen may contribute to prevention of bacterial airway infections in PAP.

Evaluation of a new caries detecting dye for primary and permanent carious dentin

This study evaluated the clinical efficacy of a new caries detecting dye using a laser fluorescence device (DIAGNOdent). Primary and permanent teeth with dentin caries were stained with Caries Check (CC), containing 1% acid red in polypropylene glycol (MW=300) or Caries Detector (CD), containing 1% acid red in propylene glycol (MW=76). Primary-CC, primary-CD, permanent-CC and permanent-CD groups were prepared. In the CC groups, stained dentin was completely removed. In the CD groups, pink-stained dentin was retained according to the manufacturers' instructions. Cavities before and after caries removal were measured with the DIAGNOdent. Data were analyzed using ANOVA and Fisher's PLSD multiple comparison test at alpha=0.05. Regression analyses were performed between DIAGNOdent readings and scores obtained from the clinical parameters. For all groups, there were no significant differences in the DIAGNOdent readings before treatment. The DIAGNOdent readings after caries removal were: primary-CC (16.0+/-17.6), primary-CD (9.6+/-5.2), permanent-CC (11.0+/-7.0) and permanent-CD (7.1+/-3.8). Significant differences were identified between the permanent-CC and primary-CD, and permanent-CC and permanent-CD subgroups but not for the primary subgroups. Correlation coefficients between DIAGNOdent readings and clinical parameters were low. When dentin stained with Caries Check was completely removed, the DIAGNOdent readings were higher than those recorded when palely-stained pink dentin was retained with the Caries Detector, with significant difference observed for the permanent teeth. Caries Check may be used clinically to avoid excessive removal of caries-affected or sound dentin in permanent teeth but not in primary teeth.

Pro-inflammatory Cytokine Tumor Necrosis Factor-α Induces Bone Morphogenetic Protein-2 in Chondrocytes via mRNA Stabilization and Transcriptional Up-regulation

In articular chondrocytes, the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) induces the expression of bone morphogenetic protein-2 (BMP-2), a growth factor known to be involved in the induction of cartilage and bone. A study was performed to clarify the mechanism(s) underlying the induction of BMP-2 in chondrogenic ATDC5 cells and primary cultured adult human articular chondrocytes. In ATDC5 cells, the endogenous BMP-2 expression was consistently low throughout the process of chondrogenic differentiation, and TNF-alpha induced BMP-2 expression only after the cells acquired the chondrogenic phenotype. The results of nuclear run-off assay and cycloheximide treatment consistently indicated that ATDC5 cells acquire the capacity to synthesize BMP-2 mRNA in the nuclei during the differentiation process. In an attempt to explain the discrepancy between the active nuclear mRNA synthesis and the observed low expression level in differentiated ATDC5 cells, the stability of BMP-2 mRNA was evaluated, and the cells were found to regulate the expression of BMP-2 at the post-transcriptional level. Human chondrocytes were confirmed to have a similar post-transcriptional regulation. The result of 3'-rapid amplification of cDNA end revealed that both human and mouse BMP-2 mRNAs contain multiple pentameric AUUUA motifs in a conserved manner in the 3'-untranslated regions, and transient transfection experiments demonstrated that TNF-alpha increases the stability of BMP-2 mRNA through the pentameric motifs. Further experiments revealed that TNF-alpha modulates mRNA stability via p38 signal transduction pathway, whereas the cytokine also augmented the expression of BMP-2 through transcriptional up-regulation via the transcriptional factor NF-kappaB.

Uric acid responses to endurance racing and relationships with performance, plasma biochemistry and metabolic alterations

There is limited understanding of the uric acid response to endurance races. To demonstrate uric acid increments and its relationship to diverse biochemical and performance parameters, in horses subjected to a prolonged effort, with and without presentation of metabolic alterations. Blood samples were taken from horses the day before, and 5-10 mins after, successfully finishing a 121 km (Assay 1, n = 24) or 164 km endurance race (Assay 2, n = 17), and from 19 animals eliminated by metabolic disorders during several endurance races (Assay 3). Plasma was obtained and determinations of CK, AST, LDH, AP, uric acid (UA), creatinine (Cr), urea, lactate (La) and plasma proteins (PP) carried out. Sex, age, time in competition, average speed and total recovery time were also recorded. Assays 1 and 2 were arithmetically subdivided into 3 groups each in order to categorise time in competition, average speed and total recovery time. Changes among the groups were evaluated with ANOVA and Fisher's PLSD test. Student's paired t test was used to assess pre- and post exercise differences. A value of P< or =0.05 was considered significantly different. Pearson correlation coefficient was used to assess the relationship between all the variables and UA increases. Average speed of the sampled horses was significantly higher in Assay 1 compared to Assay 2. However, there were no significant differences in plasma biochemistry values between both groups. The fastest horses showed significantly higher UA levels, compared with the slowest (Assays 1 and 2) and medium horses (Assay 1). The animals with alterations in metabolism had significantly higher UA, CK and PP compared with those that adequately concluded the race. There were significant correlations between UA and CK in Assays 1, 2, and 3 and between UA and PP in Assays 1 and 3. UA rises in horses after a prolonged effort, this increase being higher in animals with metabolic commitment, and in the fastest horses. This increase has a direct correlation with CK. UA could be useful in the assessment of metabolic response during endurance exercise.

Periosteal Remodeling at the Femoral Neck in Nonhuman Primates

Periosteal bone turnover is poorly understood. We documented intramembranous periosteal bone turnover in the femoral neck in intact nonhuman primates and an increase in osteoclast numbers at the periosteal surface in sex steroid-deficient animals. Our studies are the first to systematically document periosteal turnover at the femoral neck. Bone size is an important determinant of bone strength, and cellular events at the periosteal surface could alter bone dimensions. We characterized periosteal cellular activity with dynamic histomorphometric studies of nonhuman primate femoral neck and shaft. Femur specimens from 16 intact adult male and female nonhuman primates (Rhesus [Macaca mulatta, n = 9] and Japanese Macaque [Macaca fuscata, n = 7]) were analyzed. Animals were double-labeled with tetracycline, and necropsy was performed 2-7 days after the last dose. We characterized periosteal resorptive activity in an additional group of five intact and four castrate female animals. Multiple group comparisons in intact animals were performed by one-way ANOVA followed by a Fisher PLSD posthoc test. In gonadectomized animals, Fisher's exact test was used for dichotomous and Mann-Whitney U-test for continuous variables. Bone turnover in the periosteum of the femoral neck in intact animals was more rapid than at the femoral shaft but slower than in femoral neck cancellous bone. Similarly, in these intact animals, the eroded surface of cortical bone at the femoral neck periosteal surface was significantly greater than in the cancellous bone compartment (p < 0.0001) or on the femoral shaft (p < 0.0001). Gonadectomized female animals showed an increase in osteoclast number on the periosteal surface compared with intact controls (p < 0.01). We documented intramembranous periosteal bone turnover in the femoral neck by histomorphometric analyses. The tissue level bone formation rate was sufficient to add substantively to femoral neck size over time. Periosteal osteoclastic activity was not the result of the emergence of intracortical tunneling at the bone surface. Sex steroid deficiency produced an increase in osteoclast numbers at the periosteal surface. This is the first systematic documentation of periosteal turnover at the femoral neck.

Application of crosslinkers to dentin collagen enhances the ultimate tensile strength

The stabilization of dentin collagen with biocompatible crosslinking agents may be of clinical importance to improve dentin bond strength. The present study aimed to evaluate the effect of three collagen crosslinking agents on the ultimate tensile strength (UTS) of undemineralized and demineralized dentin. Ten freshly extracted sound molars were sectioned into 0.5 x 0.5 mm2 thick beams. The beams were either demineralized or kept undemineralized. Then, specimens were subdivided into four groups according to treatments--PBS solution (control), 5% glutaraldehyde (GD), 0.5% proanthocyanidin PBS solution (PA), and 0.625% genipin PBS solution (GE). Specimens were kept in their respective solutions for either 4 or 40 h. To assess UTS, specimens were subjected to tensile forces at a crosshead speed of 1 mm/min. Statistical analysis was performed using two-way ANOVA and Fisher's PLSD test (p < 0.05). Statistically significant increases in UTS were observed for demineralized dentin after PA and GE dentin treatment, when compared with those of the control group. Dentin treated with GD showed no statistically significant differences in UTS when compared with that the control. Undemineralized dentin revealed no significant differences as compared to that of the control, regardless of the collagen crosslinkers. The application of two naturally occurring crosslinkers, i.e., PA and GE, to dentin collagen significantly improves UTS, indicating its potential value in restorative dentistry.

Removal of noncollagenous components affects dentin bonding

The structural integrity of fibrillar type I collagen is critical for effective dentin bonding. Since most noncollagenous matrix components in dentin are closely associated with collagen, we hypothesized that they may also contribute to dentin bonding. To test this hypothesis, bovine dentin was acid-etched, treated with chondroitinase ABC (C-ABC), endo-beta-galactosidase (Endo-beta), or trypsin. Controls were prepared in the same manner but without the enzymes. All control and experimental specimens were then bonded with One-Step. Bond strength data were analyzed by one-way ANOVA and Fisher's PLSD test (p < 0.05). When dentin was treated with C-ABC or trypsin, bond strengths significantly decreased for the rewetted groups (p < 0.05). The treatment with Endo-beta showed no effects on bond strengths (p > 0.05). When the treated dentin surfaces were observed under SEM, the C-ABC and trypsin treated groups revealed significant loss of collagen fibril architecture. The results indicate that chondroitin sulfate glycosaminoglycans and trypsin-digestible noncollagenous proteins play roles in maintaining the open dimensions of the collagen fibril scaffold, which is essential for optimal dentin bonding.

Investigation of the dose-dependent neuroprotective effects of agmatine in experimental spinal cord injury: a prospective randomized and placebo–control trial

No definitive treatment for spinal cord injuries (SCIs) exists, and more research is required. The use of agmatine [4-(aminobutyl)-guanidine-NH2-CH2-CH2-CH2-CH2-NH-C(-NH2)(=NH)], a guanidinium compound formed by decarboxylation of L-arginine by arginine decarboxylase, is a neurotransmitter-neuromodulator with both N-methyl-D-aspartate receptor (NMDAR)-antagonizing and nitric oxide synthase (NOS)-inhibiting activities. The purpose of this study was to demonstrate the dose-dependent activity of agmatine, an inducible NOS (iNOS) inhibitor and selective NMDAR antagonist, on biochemical and functional recovery in an experimental rat SCI model. This study involved 40 Wistar albino male rats. The rats were subjected to sleep-awake cycles for 7 days before surgery. In each group, general anesthesia was induced by a 60-mg/kg ketamine injection. For the surgical SCI model, a Yaşargil aneurysm clip was placed in the spinal cord. The study was conducted in the following four main groups: Group I (control group) laminectomy only; Group II, trauma-only group and SCI; Group III, laminectomy, SCI and agmatine 50 mg/kg for 10 days; and Group IV, laminectomy, SCI, and agmatine 100 mg/kg for 10 days. On Day 1, no statistical difference was observed in any group (p < 0.005, analysis of variance [ANOVA] and the Fisher protected least significant difference [PLSD]). On Day 2, no statistical difference was noted among Groups II, III, and IV (p = 0.27, p = 0.42, and p = 0.76, respectively; ANOVA and Fisher PLSD). Beginning on Day 3, recovery in Groups III and IV differed significantly from that in Group II (p < 0.005, ANOVA and Fisher PLSD), and a statistically significant difference between Groups III and IV was observed, which also was present on Days 5, 7, and 10 (p = 0.003, p = 0.0024, and p = 0.0036, respectively; ANOVA and Fisher PLSD). Several observations were noteworthy: motor function scores were reduced significantly in the spinal cord-injured rats compared with the controls (p < 0.005); on Day 1, the agreement of motor function scores in rats in each SCI group indicated that the traumatic event had been replicated equally across all groups (p = 0.59, p = 0.59, and p = 0.28); a statistically significant difference in motor function scores developed on Day 3 between the rats subjected to trauma alone (Group II) and those treated with agmatine (Groups III and IV) (p < 0.005); and no statistically significant intergroup difference in motor function existed at any postinjury interval between the 50- and 100-mg/kg/day agmatine-treated rats (p > 0.005). Agmatine administration following SCI was shown to reduce NO levels significantly. No statistically significant intergroup difference in the reduction of NO levels was found between rats treated with 50- and 100-mg/kg/day doses of agmatine. Administration of a 100-mg/kg/day dose of agmatine reduced the NO levels to those measured in controls. The authors conclude that with additional studies into the role of agmatine, this drug may be helpful in the treatment of patients with SCIs.

Sexual Dimorphism in the Effect of Exercise Stress on Neutrophil ROS Generation in the Rat

Neutrophils rapidly migrate to sites of inflammation and are key players in the inflammatory response in rheumatic diseases (RDs), which have a female predominance, particularly in early events but also in setting up later events. Dysregulation of neutrophil reactive oxygen species (ROS) may be a critical component to the pathophysiology of RDs. Stress, which may produce exacerbations in RDs, created by repeated exposure to sound, produces a sexually dimorphic effect on ROS generation in neutrophils, with neutrophils from females displaying greater ROS generation. Since exercise is used therapeutic ally but can also exacerbate RDs, we determined what effect intense stressful exercise has on immune parameters including neutrophil function. PURPOSE: To evaluate sexual dimorphism in the effect of intense exercise stress on neutrophil ROS generation. METHODS: Male (M, n=11) and female (F, n=12) Sprague-Dawley rats (260–280g) were randomly assigned to either exercise (EX) or control (C) groups. Exercise consisted of a single session of treadmill running at 20 m/min 5% grade for 45 min. Whole blood was collected for analysis of in vitro neutrophil ROS generation before exercise, one hr post-, and 24 hrs post-exercise. ROS data were analyzed using a two-way ANOVA and Fisher's PLSD for post-hoc comparisons. RESULTS: There were no differences between male and female rats in neutrophil ROS before exercise. One hr post-exercise, neutrophils from EX produced more ROS than those from C (M and F, p<0.001) and M-EX produced more ROS than the other groups (p<0.001). Twenty-four hrs post-exercise, neutrophil ROS production from the EX groups was still slightly elevated (p=0.04), however, sexual dimorphism was abolished. CONCLUSIONS: Sexual dimorphism is evident in the effect of intense exercise stress on in vitro neutrophil ROS production. Neutrophils from exercised males produced more ROS than those from females. In RDs, ROS accumulation and inflammation are often associated with increasing disease severity. The presence of glucocorticoid and adrenergic receptors on neutrophils, suggests a role of corticosterone and epinephrine in immune function. Because of interactions of the hypothalamic-pituitary axis and sympathoadrenal axis with inflammation, the adrenal gland, acting through release of corticosterone and epinephrine, may play a role in mediating sex differences in inflammatory responses following exercise stress. Research is in progress to determine the underlying mechanism. Supported by NIH/NIDCR Grant T32-DE07306-10.