Saturated Picric Acid Research Articles

MnS Precipitation Behavior of High-Sulfur Microalloyed Steel Under Sub-rapid Solidification Process

A typical high-sulfur microalloyed steel was investigated by a sub-rapid solidification process for grain refinement of the as-cast microstructure. The size and distribution characteristics of the MnS precipitates were analyzed. The variations in the dendrite morphology and secondary dendrite arm spacing (SDAS) under different cooling rates have been studied, which strongly influence the precipitation behavior of MnS. The 3D-morphology of MnS precipitates was revealed by a novel saturated picric acid deep-etching method. Most MnS precipitates with a length smaller than 5 μm were columnar or equiaxed in the corresponding dendrite zones under sub-rapid solidification conditions at cooling rates of 261 to 2484 K/s. Furthermore, an area scan analysis of the precipitates showed the number of small MnS per square millimeter with lengths lower than 3 μm decrease from 200,537 to 110,067. The percentage of large MnS with a length over 5 μm increased from 2.6 to 6.2 pct as the solidification condition changed from sub-rapid to air cooling. In addition, the size of MnS precipitate was found to depend linearly on the SDAS.

Analysis of Cyanide Content on Yams Using Spectrophotometry Methods

This study aims to determine whether or not cyanide content in various species of yams in the District of Tempel, Sleman, Yogyakarta. The samples were cassava (Manihot esculenta Crantz), water yam (Discorea Alata), intoxicating yam (Dioscorea hispida Dennst), canna lilly (Canna discolor L), and taro (Colocasia esculenta). The qualitative test was performed by cementing the sample with aquadest and 10% tartrate acid and then covering the erlenmeyer that containing the sample with dry filter paper immersed in saturated picric acid and dampened with 8% sodium carbonate and then heating it. Quantitative tests were performed using a spectrum 20 with a wavelength of 590 nm. This quantitative test is based on forming a blue hydrindantin complex at pH 12 when cyanide is reacted with a ninhydrin complex. The results showed that in qualitative test, sample containing cyanide was indicated by the change of color of filter paper from yellow to brownish red. Quantitative results show that cyanide contained in sample as varies levels. The cyanide levels through the various species of yams were 0.1098 mg / 100g for cassava 0.049 mg/100 g for water yam, 0.1394 mg/100g for intoxing yam, 0.0896 mg/100 g for canna lilly and 0.0680 mg / 100g for taro.

Cardioprotective effect of polydatin on ventricular remodeling after myocardial infarction in coronary artery ligation rats.

The purpose of this study was to explore the effect of polydatin on ventricular remodeling after myocardial infarction in coronary artery ligation rats and to elucidate the underlying mechanisms. A rat model of ventricular remodeling after myocardial infarction was established by left coronary artery ligation. Rats with coronary artery ligation were randomly divided into five groups: control, plus 40 mg/kg captopril, plus 25 mg/kg polydatin, plus 50 mg/kg polydatin, and plus 100 mg/kg polydatin. The sham-operated group was used as a negative control. Rats were administered intragastrically with the corresponding drugs or drinking water for seven weeks. At the end of the treatment, the left ventricular weight index and heart weight index were assessed. The cross-sectional size of cardiomyocytes was measured by staining myocardium tissue with hematoxylin and eosin. Collagen content was counted by Sirius red in aqueous saturated picric acid. The concentrations of angiotensin I, angiotensin II, aldosterone, and endothelin 1 in myocardium or serum were determined by radioimmunoassay. Hydroxyproline and nitric oxide concentrations and glutathione peroxidase and catalase activities in serum were measured by ultraviolet spectrophotometry. Our results showed that seven weeks of polydatin treatment resulted in a significantly reduced left ventricular weight index, heart weight index, serum concentrations of hydroxyproline and aldosterone, an increased concentration of nitric oxide as well as enhanced activities of glutathione peroxidase and catalase. Myocardial angiotensin I, angiotensin II, and endothelin 1 levels were also reduced. The cardiomyocyte cross-sectional area and collagen deposition diminished. This study suggests that polydatin may attenuate ventricular remodeling after myocardial infarction in coronary artery ligation rats through restricting the excessive activation of the renin-angiotensin-aldosterone system and inhibiting peroxidation.

The effects of picric acid (2,4,6-trinitrophenol) and a bite-deterrent chemical (denatonium benzoate) on autotomy in rats after peripheral nerve lesion.

Denervation of the hind limb is a technique used to study peripheral nerve regeneration. Autotomy or autophagia is an undesirable response to denervation in such studies. Application of a commercially available lotion used to deter nail biting in humans reduced autotomy in rats after denervation but did not completely prevent it. In this study, this authors evaluated the application of picric acid to prevent autotomy in rats in peripheral nerve experiments. They carried out sciatic nerve transection in 41 adult female Wistar rats and then applied either bite-deterrent lotion (n = 26) or saturated picric acid solution (n = 15) topically to the affected hind limb immediately after surgery and every day for 1 month. Autotomy scores were lower for rats treated with picric acid than for rats treated with bite-deterrent lotion 1 week and 2 weeks after surgery but were not different between the two groups 4 weeks after surgery. The authors conclude that application of picric acid could be used as an alternative strategy to prevent autotomy in peripheral nerve studies.

A New Technique of Electro-Synthesis of Polypyrrole Nanoparticles

We report here a novel two phase electro-oxidation technique of synthesis of submicron size polypyrrole (ppy) from pyrrole (py) onto a steel electrode. Pyrrole forms a hydrogen bonded red colour complex with picric acid which is almost insoluble in water but soluble in di-chloromethane (DCM). Again, water and DCM forms an immiscible solvent pair. We demonstrated that nano sized polypyrrole is deposited on to a steel anode coupled with a Pt cathode by passing a small current of 0.25 mAcm-2 for a period of 600 seconds when electro-oxidation is carried out in aqueous saturated picric acid phase placed over DCM phase containing a solution of pyrrole-picrate complex. TEM picture shows formation of nano sized polymers. This is probably due to slow diffusion of pyrrole- picrate complex from organic phase to aqueous phase and its extremely low concentration at the electrode surface probably control the chain growth kinetics.Key words: Electro-oxidation, Nanosized polypyrrole, Pyrrole-picric acid complex .Introduction Polypyrrole is a promising conducting polymer for wide variety of electronic, opto-electronic and electro-chemical devices including electrode materials of aqueous and non-aqueous batteries. It is easily deposited on to metallic and carbon substrates by electro- polymerization of pyrrole in a suitable organic solvent. Major problems of electro-oxidation of pyrrole in aqueous medium are its poor solubility in water and corrosions of non-noble metal anode in acidic solution. Previously, we had reported electro-polymerization of pyrrole on to steel substrate using aqueous picric acid as dopant. We found polypyrrole films so deposited were brittle. Polymer chain growth kinetics depends on concentration of monomer as well as radical, this can be controlled taking advantage of the fact that both pyrrole and pyrrole-picrate complex are more soluble in DCM but less soluble in water, hence a two phase electro-oxidation as reported here is expected to produce submicron size polymer. To the best of our knowledge, this technique of two phase electro-oxidation of preparation of nanosized polymer has not been reported earlier.Material and MethodChemicalsPyrrole (Fluka), Picric acid (Merck) and Dichloromethane (Merck) were used as obtained. Triple distilled water was prepared using a all glass setup.Pyrrole (1ml) was taken in a 50ml cylindrical beaker to which 1ml saturated aqueous picric acid (picric acid is only slightly soluble in water) was added when reddish globules of Pyrrole-picric acid (py-pic)complex was formed. To this solution 8ml of DCM was added when py-pic complex dissolved forming a yellow solution. Ten ml of saturated picric acid was then poured over this DCM solution of py-pic complex to form a two phase system. A working electrode of thin steel sheet (4cm-2) and a counter Pt electrode of same dimensions were placed in the aqueous phase just over the phase boundary , a current of 0.25 mA cm-2 under galvanostatic condition was passed for 600 seconds when a blue-black deposition of poly-pyrrole was formed on to working electrode .The deposition was taken out with a sharp knife for TEM analysis. For CV of pyrrole-picrate complex in DCM the solution was prepared as stated above using 0.01M KClO3 as electrolyte. All electro-chemical measurements were taken by PAR VarsaStat (II )Potentiostat/Galvanostat. Results and Discussions A simple experimental arrangement was used in this study. It may be mentioned that the electrodes were kept just above the phase boundary in order to avoid large migration distant of the py-pic complex. Pyrrole forms a complex via hydrogen bonding with trinitro-benzene derivatives including picric acid which is soluble in DCM. From CV of py-pic it is known pyrrole oxidation potential is lowered due its hydrogen bonding with –NO2 group of picric acid. We propose py-pic complex will easily deprotonated to form pyrrole anion which will form pyrrole radical upon anodic-oxidation which is prerequisite for polymerization It is reported polypyrrole film depositions on to different substrate material are best obtained under galvanostatic condition at a current density of 25mA cm-2, we have therefore used this optimised condition in our study. With deposition of ppy film, the resistance of steel electrode surface increases as indicated in potential –time deposition curve.Now the major advantage of two phase electro-oxidation in this case is that the ppy-pic complex is soluble in DCM and it slowly diffuse to aqueous phase resulting a low concentration of monomer in the vicinity of the electrode surface which retards the chain growth kinetics of polypyrrole formation. TEM picture shows these polymers are of almost spherical in shape with diameters of 250-300 nm.We concluded that nano size polypyrrole are easily synthesized by a two phase electro-deposition technique using DCM and water as two immiscible solvent pair and picric acid as complexing agent.

Brucella abortus Induces Collagen Deposition and MMP-9 Down-Modulation in Hepatic Stellate Cells via TGF-β1 Production

In patients with active brucellosis, the liver is frequently affected by histopathologic lesions, such as granulomas, inflammatory infiltrations, and parenchymal necrosis. Herein, we examine some potential mechanisms of liver damage in brucellosis. We demonstrate that Brucella abortus infection inhibits matrix metalloproteinase-9 (MMP-9) secretion and induces collagen deposition and tissue inhibitor of matrix metalloproteinase-1 secretion induced by hepatic stellate cells (LX-2). These phenomena depend on transforming growth factor-β1 induction. In contrast, supernatants from B. abortus-infected hepatocytes and monocytes induce MMP-9 secretion and inhibit collagen deposition in hepatic stellate cells. Yet, if LX-2 cells are infected with B. abortus, the capacity of supernatants from B. abortus-infected hepatocytes and monocytes to induce MMP-9 secretion and inhibit collagen deposition is abrogated. These results indicate that depending on the balance between interacting cells and cytokines of the surrounding milieu, the response of LX-2 cells could be turned into an inflammatory or fibrogenic phenotype. Livers from mice infected with B. abortus displayed a fibrogenic phenotype with patches of collagen deposition and transforming growth factor-β1 induction. This study provides potential mechanisms of liver immune response induced by B. abortus-infected hepatic stellate cells. In addition, these results demonstrate that the cross talk of these cells with hepatocytes and macrophages implements a series of interactions that may contribute to explaining some of mechanisms of liver damage observed in human brucellosis.

Low Density Lipoprotein Receptor-related Protein 1 (LRP1) Modulates N-Methyl-d-aspartate (NMDA) Receptor-dependent Intracellular Signaling and NMDA-induced Regulation of Postsynaptic Protein Complexes

The lipoprotein receptor LRP1 is essential in neurons of the central nervous system, as was revealed by the analysis of conditional Lrp1-deficient mouse models. The molecular basis of its neuronal functions, however, is still incompletely understood. Here we show by immunocytochemistry, electron microscopy, and postsynaptic density preparation that LRP1 is located postsynaptically. Basal and NMDA-induced phosphorylation of the transcription factor cAMP-response element-binding protein (CREB) as well as NMDA target gene transcription are reduced in LRP1-deficient neurons. In control neurons, NMDA promotes γ-secretase-dependent release of the LRP1 intracellular domain (LRP1-ICD). However, pull-down and chromatin immunoprecipitation (ChIP) assays showed no direct interaction between the LRP1-ICD and either CREB or target gene promoters. On the other hand, NMDA-induced degradation of the postsynaptic scaffold protein PSD-95 was impaired in the absence of LRP1, whereas its ubiquitination was increased, indicating that LRP1 influences the composition of postsynaptic protein complexes. Accordingly, NMDA-induced internalization of the AMPA receptor subunit GluA1 was impaired in LRP1-deficient neurons. These results show a role of LRP1 in the regulation and turnover of synaptic proteins, which may contribute to the reduced dendritic branching and to the neurological phenotype observed in the absence of LRP1.

Evaluation of glutamate concentration transient in the synaptic cleft of the rat calyx of Held

Establishing the spatiotemporal concentration profile of neurotransmitter following synaptic vesicular release is essential for our understanding of inter-neuronal communication. Such profile is a determinant of synaptic strength, short-term plasticity and inter-synaptic crosstalk. Synaptically released glutamate has been suggested to reach a few millimolar in concentration and last for <1 ms. The synaptic cleft is often conceived as a single concentration compartment, whereas a huge gradient likely exists. Modelling studies have attempted to describe this gradient, but two key parameters, the number of glutamate in a vesicle (N(Glu)) and its diffusion coefficient (D(Glu)) in the extracellular space, remained unresolved. To determine this profile, the rat calyx of Held synapse at postnatal day 12-16 was studied where diffusion of glutamate occurs two-dimensionally and where quantification of AMPA receptor distribution on individual postsynaptic specialization on medial nucleus of the trapezoid body principal cells is possible using SDS-digested freeze-fracture replica labelling. To assess the performance of these receptors as glutamate sensors, a kinetic model of the receptors was constructed from outside-out patch recordings. From here, we simulated synaptic responses and compared them with the EPSC recordings. Combinations of N(Glu) and D(Glu) with an optimum of 7000 and 0.3 μm(2) ms(-1) reproduced the data, suggesting slow diffusion. Further simulations showed that a single vesicle does not saturate the synaptic receptors, and that glutamate spillover does not affect the conductance amplitude at this synapse. Using the estimated profile, we also evaluated how the number of multiple vesicle releases at individual active zones affects the amplitude of postsynaptic signals.

Neurogliaform cells of amygdala: a source of slow phasic inhibition in the basolateral complex

Synaptic inhibition in the amygdala actively participates in processing emotional information. To improve the understanding of interneurons in amygdala networks it is necessary to characterize the GABAergic cell types, their connectivity and physiological roles. We used a mouse line expressing a green fluorescent protein (GFP) under the neuropeptide Y (NPY) promoter. Paired recordings between presynaptic NPY-GFP-expressing (+) cells and postsynaptic principal neurons (PNs) of the basolateral amygdala (BLA) were performed. The NPY-GFP+ neurons displayed small somata and short dendrites embedded in a cloud of highly arborized axon, suggesting a neurogliaform cell (NGFC) type. We discovered that a NPY-GFP+ cell evoked a GABA(A) receptor-mediated slow inhibitory postsynaptic current (IPSC) in a PN and an autaptic IPSC. The slow kinetics of these IPSCs was likely caused by the low concentration and spillover of extracellular GABA. We also report that NGFCs of the BLA fired action potentials phase-locked to hippocampal theta oscillations in anaesthetized rats. When this firing was re-played in NPY+-NGFCs in vitro, it evoked a transient depression of the IPSCs. Presynaptic GABA(B) receptors and functional depletion of synaptic vesicles determined this short-term plasticity. Synaptic contacts made by recorded NGFCs showed close appositions, and rarely identifiable classical synaptic structures. Thus, we report here a novel interneuron type of the amygdala that generates volume transmission of GABA. The peculiar functional mode of NGFCs makes them unique amongst all GABAergic cell types of the amygdala identified so far.

Pressure Overload Induces Early Morphological Changes in the Heart

Cardiac hypertrophy, whether pathological or physiological, induces a variety of additional morphological and physiological changes in the heart, including altered contractility and hemodynamics. Events exacerbating these changes are documented during later stages of hypertrophy (usually termed pathological hypertrophy). Few studies document the morphological and physiological changes during early physiological hypertrophy. We define acute cardiac remodeling events in response to transverse aortic constriction (TAC), including temporal changes in hypertrophy, collagen deposition, capillary density, and the cell populations responsible for these changes. Cardiac hypertrophy induced by TAC in mice was detected 2 days after surgery (as measured by heart weight, myocyte width, and wall thickness) and peaked by day 7. Picrosirius staining revealed increased collagen deposition 7 days after TAC; immunostaining and flow cytometry indicated a concurrent increase in fibroblasts. The findings correlated with angiogenesis in TAC hearts; a decrease in capillary density was observed at day 2, with recovery to sham-surgery levels by day 7. Increased pericyte levels, which were observed 2 days after TAC, may mediate this angiogenic transition. Gene expression suggests a coordinated response in growth, extracellular matrix, and angiogenic factors to mediate the observed morphological changes. Our data demonstrate that morphological changes in response to cardiovascular injury occur rapidly, and the present findings allow correlation of specific events that facilitate these changes.

Inquisition of the Human Intervertebral Disk Histopathology with the Use of Two Histological Staining Methods

IntroductionChronic low back pain (LBP) affects over 10% of the adult population resulting in profound decreases in the quality of life. The intervertebral disk (IVD) is prone to degeneration which may contribute toLBP. To prevent disk degeneration-related LBP, better understanding of disk structure and the degenerative process is needed to understand the underlying mechanisms. Standard Hematoxylin and Eosin (H&E) staining, alone or in combination with other dyes, have been used to assess the normal biology and degeneration of IVDs. Recently, the multichromatic FAST (Alcian blue, Safranin-O, Fast green and Tartrazine) staining method has been developed (Leung V.Y.L. et al., 2009). This method provides additional information unavailable through conventional methods such as differentiation of the structures within the disk and identification of degenerative matrix remodeling through changes in the staining pattern. Although the FAST method has proved helpful in animal models to assess alteration of glycosaminoglycan content, its usefulness to assess human disk histopathology remains unknown.Materials and MethodsHuman disks were obtained (a) surgically from chronic LBP patients with moderate-severe disk degeneration or (b) postmortem from transplant donors. Disks were cut into quadrants, fixed in 4% paraformaldehyde with 14% (v/v) saturated picric acid for 3 days, embedded in OCT and stored at −80°C. Tissue samples were cut on a cryostat and transverse sections of 14 um and 20 um were obtained for H&E and FAST staining, respectively. Slides were coverslipped with DPX and scanned using a Zeiss MIRAX Scan Digital Slide Scanner for further examination.ResultsThe FAST protocol creates high-resolution and polychromatic images of the general disk structure, and reveals the overall organization of the disk. The transition from the nucleus pulposus (NP) to the inner annulus fibrosus (IAF) and the outer annulus fibrosus (OAF) was clearly visible through color boundaries. Signs of degeneration could be observed with the FAST staining method in both IVDs obtained postmortem with no history of LBP and from chronic LBP patients. For example, FAST staining uncovered the loss of a clear boundary between the NP and the IAF that was not visible with H&E. These data suggest that relevant pathological details might be missed if assessing histological changes with the use of H&E only.ConclusionThe combined use of H&E and FAST methods provides a more complete picture of the anatomy and physiology of human IVDs. The FAST profile distinguishes IVD compartments and shows matrix remodeling events within the disk, while H&E provides complementary information regarding the cellular content of the tissue. These two staining methods complement each other to give a clearer differentiation of IVD sub-regions of both normal and degenerated disk architecture.I confirm having declared any potential conflict of interest for all authors listed on this abstractYesDisclosure of InterestNone declaredLeung V.Y.L. et al. Journal of Histochemistry and Cytochemistry 2009;57:249

Inquisition of the Human Intervertebral Disk Histopathology with the Use of Two Histological Staining Methods

Introduction Chronic low back pain (LBP) affects over 10% of the adult population resulting in profound decreases in the quality of life. The intervertebral disk (IVD) is prone to degeneration which may contribute toLBP. To prevent disk degeneration-related LBP, better understanding of disk structure and the degenerative process is needed to understand the underlying mechanisms. Standard Hematoxylin and Eosin (H&amp;E) staining, alone or in combination with other dyes, have been used to assess the normal biology and degeneration of IVDs. Recently, the multichromatic FAST (Alcian blue, Safranin-O, Fast green and Tartrazine) staining method has been developed (Leung V.Y.L. et al., 2009). This method provides additional information unavailable through conventional methods such as differentiation of the structures within the disk and identification of degenerative matrix remodeling through changes in the staining pattern. Although the FAST method has proved helpful in animal models to assess alteration of glycosaminoglycan content, its usefulness to assess human disk histopathology remains unknown. Materials and Methods Human disks were obtained (a) surgically from chronic LBP patients with moderate-severe disk degeneration or (b) postmortem from transplant donors. Disks were cut into quadrants, fixed in 4% paraformaldehyde with 14% (v/v) saturated picric acid for 3 days, embedded in OCT and stored at −80°C. Tissue samples were cut on a cryostat and transverse sections of 14 um and 20 um were obtained for H&amp;E and FAST staining, respectively. Slides were coverslipped with DPX and scanned using a Zeiss MIRAX Scan Digital Slide Scanner for further examination. Results The FAST protocol creates high-resolution and polychromatic images of the general disk structure, and reveals the overall organization of the disk. The transition from the nucleus pulposus (NP) to the inner annulus fibrosus (IAF) and the outer annulus fibrosus (OAF) was clearly visible through color boundaries. Signs of degeneration could be observed with the FAST staining method in both IVDs obtained postmortem with no history of LBP and from chronic LBP patients. For example, FAST staining uncovered the loss of a clear boundary between the NP and the IAF that was not visible with H&amp;E. These data suggest that relevant pathological details might be missed if assessing histological changes with the use of H&amp;E only. Conclusion The combined use of H&amp;E and FAST methods provides a more complete picture of the anatomy and physiology of human IVDs. The FAST profile distinguishes IVD compartments and shows matrix remodeling events within the disk, while H&amp;E provides complementary information regarding the cellular content of the tissue. These two staining methods complement each other to give a clearer differentiation of IVD sub-regions of both normal and degenerated disk architecture. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Leung V.Y.L. et al. Journal of Histochemistry and Cytochemistry 2009;57:249

Intermediate Volume on Computed Tomography Imaging Defines a Fibrotic Compartment that Predicts Glomerular Filtration Rate Decline in Autosomal Dominant Polycystic Kidney Disease Patients

Total kidney and cyst volumes have been used to quantify disease progression in autosomal dominant polycystic kidney disease (ADPKD), but a causal relationship with progression to renal failure has not been demonstrated. Advanced image processing recently allowed to quantify extracystic tissue, and to identify an additional tissue component named "intermediate," appearing hypoenhanced on contrast-enhanced computed tomography (CT). The aim of this study is to provide a histological characterization of intermediate volume, investigate its relation with renal function, and provide preliminary evidence of its role in long-term prediction of functional loss. Three ADPKD patients underwent contrast-enhanced CT scans before nephrectomy. Histological samples of intermediate volume were drawn from the excised kidneys, and stained with hematoxylin and eosin and with saturated picrosirius solution for histological analysis. Intermediate volume showed major structural changes, characterized by tubular dilation and atrophy, microcysts, inflammatory cell infiltrate, vascular sclerosis, and extended peritubular interstitial fibrosis. A significant correlation (r = -0.69, P < 0.001) between relative intermediate volume and baseline renal function was found in 21 ADPKD patients. Long-term prediction of renal functional loss was investigated in an independent cohort of 13 ADPKD patients, followed for 3 to 8 years. Intermediate volume, but not total kidney or cyst volume, significantly correlated with glomerular filtration rate decline (r = -0.79, P < 0.005). These findings suggest that intermediate volume may represent a suitable surrogate marker of ADPKD progression and a novel therapeutic target.

Interaction of Mint2 with TrkA Is Involved in Regulation of Nerve Growth Factor-induced Neurite Outgrowth

TrkA receptor signaling is essential for nerve growth factor (NGF)-induced survival and differentiation of sensory neurons. To identify possible effectors or regulators of TrkA signaling, yeast two-hybrid screening was performed using the intracellular domain of TrkA as bait. We identified muc18-1-interacting protein 2 (Mint2) as a novel TrkA-binding protein and found that the phosphotyrosine binding domain of Mint2 interacted with TrkA in a phosphorylation- and ligand-independent fashion. Coimmunoprecipitation assays showed that endogenous TrkA interacted with Mint2 in rat tissue homogenates, and immunohistochemical evidence revealed that Mint2 and TrkA colocalized in rat dorsal root ganglion neurons. Furthermore, Mint2 overexpression inhibited NGF-induced neurite outgrowth in both PC12 and cultured dorsal root ganglion neurons, whereas inhibition of Mint2 expression by RNA interference facilitated NGF-induced neurite outgrowth. Moreover, Mint2 was found to promote the retention of TrkA in the Golgi apparatus and inhibit its surface sorting. Taken together, our data provide evidence that Mint2 is a novel TrkA-regulating protein that affects NGF-induced neurite outgrowth, possibly through a mechanism involving retention of TrkA in the Golgi apparatus.

The role of calcium/calmodulin‐dependent protein phosphatase (PP2B) isoforms in exocytosis in Paramecium tetraurelia

Paramecium tetraurelia are ciliated protozoa that contain membrane‐docked vesicles called trichocysts. Trichocysts fuse with the membrane in a calcium‐dependent manner and release proteins that form long crystalline spines, which help Paramecium escape predators. A bacteria‐mediated RNA interference (RNAi) protocol was used to determine if members of the calcium/calmodulin‐dependent protein phosphatase family (PP2B) present in Paramecium were involved in trichocyst discharge as suggested by previous work. After treatment, Paramecium cells were selected and exocytosis triggered by the addition of a saturated picric acid solution. After normalization, the extent of exocytosis was compared and indicated that RNAi treatment with one of the isoforms of Paramecium PP2B resulted in a reduced efficiency of exocytosis. The number of cells exhibiting full exocytosis was reduced from 76% in control cells (n=129) to 25% in PP2B treated cells (n=190). Cells exhibiting dramatically reduced exocytosis (&lt;50% full discharge) increased from 2% (control treatment) to 41% (PP2B treatment). Based upon these results, we conclude that PP2B has a role in the regulation of calcium‐dependent exocytosis in Paramecium tetraurelia. Funding for this project was provided by the Wilson Research Awards and the Howard Hughes Medical Institute.

Poster 242: Histologic and Histomorphometric Analysis of Posterior Region of the Human Temporomandibular Disc

Objective. The aim of this study was to analyze histologic and histomorphometric features of the articular disc in groups with and without disc displacement. Study design. A sample of 39 temporomandibular joints TMJs (31 case specimens, 8 control specimens) from 28 patients (mean age 31.2 years) were recruited for this study. The patients were considered to be affected and treated surgically with disc repositioning when presenting painful clinical signs of disc displacement after unsuccessful nonsurgical treatment for at least 6 months. Of the control patients, 4 presented condyle fracture which required opening to be reduced for treatment, and 4 displayed active condyle hyperplasia. The posterior region of the disc was removed and sent for histologic and histomorphometric analysis. Histologic (hematoxylin-eosin) and histomorphometric (picro-Sirius red) analyses were performed. Statistically significant differences between the analyzed groups were accessed through the chi-squared test (P .05). The Mann-Whitney U test was used to observe the differences between mean values when variables did not present normal distribution [Kolmogorov-Smirnov(a) test]. Results. There were no significant differences between the groups in relation to the parameters studied by histologic and histomorphometric analysis (using or not using polarization). Conclusions. To the limits of this study, there were no significant histologic and histomorphometric differences in the articular disc between groups with and without TMJ dysfunction. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;105:e6-e11)

Cox-2 Is Regulated by Toll-Like Receptor-4 (TLR4) Signaling: Role in Proliferation and Apoptosis in the Intestine

We recently showed that mice deficient in Toll-like receptor 4 (TLR4) or its adapter molecule MyD88 have increased signs of colitis compared with wild-type (WT) mice after dextran sodium sulfate (DSS)-induced injury. We wished to test the hypothesis that cyclooxygenase 2 (Cox-2)-derived prostaglandin E2 (PGE2) is important in TLR4-related mucosal repair. Cox-2 expression was analyzed by real-time polymerase chain reaction, immunohistochemistry, Western blotting, and luciferase reporter constructs. Small interfering RNA was used to inhibit expression of MyD88. TLR4-/- or WT mice were given 2.5% DSS for 7 days. Proliferation and apoptosis were assessed using bromodeoxyuridine staining and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assays, respectively. PGE2 was given orally to DSS-treated mice. Intestinal epithelial cell lines up-regulated Cox-2 expression in a TLR4- and MyD88-dependent fashion. Lipopolysaccharide-mediated stimulation of PGE2 production was blocked by a selective Cox-2 inhibitor or small interfering RNA against MyD88. After DSS injury, Cox-2 expression increased only in WT mice. TLR4-/- mice have significantly reduced proliferation and increased apoptosis after DSS injury compared with WT mice. PGE2 supplementation of TLR4-/- mice resulted in improvement in clinical signs of colitis and restoration of proliferation and apoptosis to WT values. The mechanism for improved epithelial repair may be through PGE2-dependent activation of the epidermal growth factor receptor. We describe an important link between TLR4 signaling and Cox-2 expression in the gut. TLR4 and MyD88 signaling are required for optimal proliferation and protection against apoptosis in the injured intestine. Although TLR4 signaling is beneficial in the short term, chronic signaling through TLR4 may lower the threshold for colitis-associated cancer.

The Role of Integrin-linked Kinase in Liver Wound Healing

Liver wound healing is an integrated process in which hepatic stellate cells play a major role. We hypothesized that the cellextracellular signaling protein integrin-linked kinase (ILK) is important in transducing signals from the extracellular matrix to stellate cells and thus plays a critical role in stellate cell activation and fibrogenesis during liver injury. Liver injury and subsequent stellate cell activation led to a 3-fold increase in ILK expression and increased kinase activity. Overexpression of ILK in isolated stellate cells led to enhanced motility and adhesion as well as increases in smooth muscle alpha-actin and type I collagen mRNA expression. The effects of ILK on stellate cell phenotypes were phosphatidylinositol 3-kinase-dependent. Forced expression of ILK in vivo led to increases in type I collagen, smooth muscle alpha-actin, transforming growth factor-beta, and extra domain A (EDA) fibronectin mRNAs (by 3.2-, 3.5-, 2.5-, and 2.2-fold, respectively; n = 8, p < 0.05 for each versus the control), whereas inhibition of ILK in vivo led to significant reductions in these mRNAs. Morphometric analysis revealed that ILK overexpression led to a 31.4% increase in liver collagen content (n = 8, p < 0.05 versus the control); in contrast ILK knockdown in vivo led to a significant reduction in fibrogenesis. We conclude that ILK plays an important pathophysiological role in vivo in liver wound healing.

Improved Methods for Staining Fibrin

Two methods used to demonstrate fibrin are the Fraser-Lendrum and Mallory's phosphotungstic acid-hematoxylin. One problem with the Fraser-Lendrum is that it requires the use of saturated alcoholic picric acid. To prepare this solution, a considerable amount of picric acid is required, which is an explosive. Federal and state regulations prohibit the storage of this chemical in the laboratory unless it is stored under water. We found that saturated aqueous picric acid could be substituted for the saturated alcoholic solution. Other changes were made to simplify the method. Mallory's method for fibrin requires treatment of tissue sections with potassium permanganate and oxalic prior to staining them with phosphotungstic acid-hematoxylin. We found that better staining results are obtained if the tissues are also treated with Zenker's fixative. However, Zenker's fixative contains mercuric chloride, a hazardous chemical that many laboratories have discontinued using. We found that zinc acetate can be substituted for mercuric chloride in preparing Zenker's fixative with excellent staining results when used in the Fraser-Lendrum and Mallory methods. (The J Histotechnol 26:127, 2003)Submitted April 10, 2003; Accepted April 22, 2003

Distribution density of intraepidermal nerve fibers in normal human skin.

A total of 74 specimens was obtained from the normal human skin of patients from 3 to 90 years old. The specimens were roughly classified into 5 groups: 15 for the face group from the face; 15 for the abdomen group from the abdomen; 13 for the back group from the back; 14 for the arm group from the upper arm and forearm; and 17 for the leg group from the thigh and lower leg. They were all fixed in 4% paraformaldehyde and 14% saturated picric acid. Cryostat sections were examined by the immunoperoxidase method and indirect immunofluorescence (IF). Primary antibodies against neurofilament, neuron-specific enolase, protein gene product 9.5 (PGP 9.5) and S-100 protein were used. The most effective method was found to be the combination of IF with PGP 9.5; it visualized the intraepidermal nerve fibers easily and clearly. Of the 74 specimens, 32 (43%) had intraepidermal PGP 9.5-immunoreactive (or nerve) fibers (IPIF), and 42 (57%) did not have any. With reference to the different skin locations, the maximal rate of specimens having IPIF was 57% in the arm group, and the minimum was 23% in the back group. IPIF positive specimens had approximate surface lengths of 6 mm, in which the existence number of the IPIF was 1 to 75. Their distribution density per 1000 epidermal basal cells was highest at 9.63 in the arm group and lowest at 2.89 in the back group. Their thickness was 2.94 +/- 0.83 microns with no significant differences among the five groups. We concluded that intraepidermal nerve fibers may not be distributed evenly in the hairy portions of normal human skin, but they may be present focally. Physiologically, two-point discrimination of itch may be explained by the distribution mode of intraepidermal nerve fibers.