Elastic moduli of faceted aluminum nitride nanotubes measured by contact resonance atomic force microscopy.

A new methodology for determining the radial elastic modulus of a one-dimensional nanostructure laid on a substrate has been developed. The methodology consists of the combination of contact resonance atomic force microscopy (AFM) with finite element analysis, and we illustrate it for the case of faceted AlN nanotubes with triangular cross-sections. By making precision measurements of the resonance frequencies of the AFM cantilever-probe first in air and then in contact with the AlN nanotubes, we determine the contact stiffness at different locations on the nanotubes, i.e. on edges, inner surfaces, and outer facets. From the contact stiffness we have extracted the indentation modulus and found that this modulus depends strongly on the apex angle of the nanotube, varying from 250 to 400 GPa for indentation on the edges of the nanotubes investigated.

Survivin expression in canine spontaneous cutaneous and subcutaneous tumors and its prognostic importance.

AIM: The present study was carried out to know the expression level of survivin, an inhibitor of apoptosis protein with an objective to determine its prognostic importance in cutaneous and subcutaneous tissue tumors of dogs. MATERIALS AND METHODS: Forty cases of canine cutaneous and subcutaneous tissue tumors on histopathological examination revealed various round cell, epithelial, and mesenchymal cell tumors. Survivin gene expression was detected in all tumors tested by TaqMan real-time polymerase chain reaction assay by comparative cycle threshold method. RESULTS: The mean survivin gene expression value of benign tumors was 0.94±0.63 folds and that of malignant tumors was 18.87±5.30 folds. Postsurgical follow up of 30 malignant tumor cases revealed death in 8, recurrence in 7, and neoplastic free alive status in 15 dogs with mean survivin fold difference values of 48.49±12.39, 14.63±6.37, and 5.034±2.27, respectively. The mean survivin gene expression value was significantly higher in malignant (30 cases, 18.87±5.30) compared to benign tumors (10 cases, 0.94±0.63), and it varied between various postsurgical follow-up groups (p<0.05). Survival analysis, using survivin gene expression median cutoff value of 3.74 in 30 malignant tumors, was performed to predict probable survival period in malignant cutaneous and subcutaneous tumors of dogs. CONCLUSIONS: Results of the present study indicated that the expression of survivin in canine cutaneous and subcutaneous tumors has prognostic value, and survivin expression greater than median cutoff value of 3.74 has a poor prognosis.

Activation of phosphatidylinositol 3-kinase and phosphatidylinositol 4-kinase during rat parotid acinar cell proliferation.

We have recently shown that beta-adrenergic agonist, isoproterenol-induced parotid acinar cell proliferation is in part mediated by elevated levels of surface galactosyltransferase which undergoes interaction with the EGF-R. The receptor subsequently undergoes autophosphorylation on the tyrosine residues in a manner similar to its 'receptor-ligand' interaction (Purushotham et al. (1992) Biochem. J. 284, 767-776). In this study, we provide evidence for phosphatidylinositol 3-kinase and 4-kinase as cytoplasmic signalling proteins involved in both the isoproterenol and EGF-stimulated signal transduction upon in vitro and in-vivo stimulation of parotid acinar cells. Total cell lysate activity for the PtdIns 4-kinase was 2- and 3-fold higher than unstimulated control cells, while the PtdIns 3-kinase was 1.4- and 2.8-fold higher following stimulation by isoproterenol or EGF, respectively. Increases of 6- and 2-fold in phosphatidylinositol 3-kinase were observed in anti-phosphotyrosine-antibody-immunoprecipitated cell lysates upon in-vitro growth stimulation with isoproterenol or EGF, respectively. There was an increase in tyrosine phosphorylation of the holoenzyme and association of the p85 subunit of phosphatidylinositol 3-kinase with EGF-R in response to both isoproterenol and EGF treatments. This corresponded with the mobilization of p85 from the cytoplasm to the plasma membrane upon growth stimulation. These results further implicate the phosphoinositide metabolites in the second messenger signalling pathways of isoproterenol-induced rat parotid cell proliferation. The parallel utilization of EGF indicate that the post-transductional mechanisms of isoproterenol-induced acinar cell proliferation are similar to the growth-factor-mediated activation of intracellular signalling pathways for cell growth.

Characterization of the synthesis and expression of the GTA-kinase from transformed and normal rodent cells.

The murine transformed cell line YC-8 and beta-adrenergic receptor agonist (isoproternol) treated rat and mouse parotid gland acinar cells ectopically express cell surface beta 1-4 galactosyltransferase during active proliferation. This activity is dependent upon the expression of the GTA-kinase (p58) in these cells. Using total RNA, cDNA clones for the protein coding region of the kinase were isolated by reverse transcriptase-PCR cloning. DNA sequence analysis failed to show sequence differences with the normal homolog from mouse cells although Southern blot analysis of YC-8, and a second cell line KI81, indicated changes in the restriction enzyme digestion profile relative to murine cell lines which do not express cell surface galactosyltransferase. The rat cDNA clone from isoproterenol-treated salivary glands showed a high degree of protein and nucleic acid sequence homology to the GTA-kinase from both murine and human sources. Northern blot analysis of YC-8 and a control cell line LSTRA revealed the synthesis of a major 3.0 kb mRNA from both cell lines plus the unique expression of a 4.5 kb mRNA in the YC-8 cells. Reverse transcriptase-PCR of LSTRA and YC-8 confirmed the increased steady state levels of the GTA-kinase mRNA in YC-8. In the mouse, induction of cell proliferation by isoproterenol resulted in a 50-fold increase in steady state mRNA levels for the kinase over the low level of expression in quiescent cells. Expression of the rat 3' untranslated region in rat parotid cells in vitro led to an increased rate of DNA synthesis, cell number an ectopic expression of cell surface galactosyltransferase in the sense orientation. Antisense expression or vector alone did not alter growth characteristics of acinar cells. A polyclonal antibody monospecific to a murine amino terminal peptide sequence revealed a uniform distribution of GTA-kinase over the cytoplasm of acinar and duct cells of control mouse parotid glands. However, upon growth stimulation, kinase was detected primarily in a perinuclear and nuclear immunostaining pattern. Western blot analysis confirmed a translocation from a cytoplasmic localization in both LSTRA and quiescent salivary cells to a membrane-associated localization in YC-8 and proliferating salivary cells.

Down-regulation of cellular proto-oncogenes during inhibition of rat parotid acinar cell proliferation.

The role of cell surface galactosyltransferase in mediating isoproterenol-induced parotid gland hypertrophy and hyperplasia was examined in rat parotid gland acinar cells. Introduction of the transferase modifier, alpha-lactalbumin, or galactosyltransferase-associated kinase inhibitor trifluoperazine, into beta-agonist-treated rats prevented acinar cell proliferation as determined by [3H]thymidine incorporation after 96 h of treatment. However, [3H]thymidine incorporation into DNA after 24 h of treatment, with injection of a combination of isoproterenol/alpha-lactalbumin or isoproterenol/trifluoperazine, was similar to injections of isoproterenol alone; suggesting that acinar cells could be stimulated to undergo a single round of DNA synthesis. Northern blot analysis of myc and fos expression followed a similar pattern of down-regulation to control levels after 96 h but not after 24 h. Hybridization with erb B showed little change with proliferation, confirming previous observations on protein levels of the EGF-receptor in acinar cells. Western blot analysis of nuclear protein expression of myc revealed that isoproterenol caused an increase in a 62-kDa protein which was again down-regulated with inhibition of cell proliferation. Analysis of protein levels of Rb110 protein showed no change in protein level in the nucleus with cell proliferation, but did show an associated increase in protein phosphorylation in response to growth stimulation.

Characterization of the synthesis and secretion of transforming growth factor-alpha from salivary glands and saliva.

Whole saliva collected from rat, mouse, and human sources was found to contain high concentrations of transforming growth factor-alpha (TGF alpha) when analyzed by RIA. The concentrations of TGF alpha in unstimulated human saliva (age, 30-45 yr; n = 10; 1.5 +/- 3.1 nM) was reduced with age (age, 55-70 yr; n = 10; 0.4 +/- 0.1 nM), but increased in oral pathologies manifested in xerostomia (age, 57-70; n = 6; 0.8 +/- 0.2 nM) and Paget's disease (age, 58-76; n = 8; 2.0 +/- 0.6 nM). Immunohistochemical localization of TGF alpha in the salivary glands of rats and mice revealed specific immunostaining of the granular ductal cells of the parotid and submandibular glands. Reverse transcription followed by polymerase chain reaction amplification of total RNA from the parotid and submandibular glands of rats and mice demonstrated the presence of TGF alpha mRNA, suggesting endogenous synthesis by the salivary glands. Thus, salivary glands appear to be an exocrine source for a second member of the epidermal growth factor-like growth factor family in the oral cavity.

Hypoglycemia-induced seizures reduce cyclic AMP response element binding protein levels in the rat hippocampus.

Cyclic AMP response element binding protein (CREB) is a transcription factor that has been implicated in the activation of protein synthesis required for long-term memory. Since memory deficits are manifest following seizure, we undertook the present study to investigate the effects of hypoglycemia-induced seizure on CREB-immunoreactive neurons in several brain regions. We induced generalized seizures in male Long Evans rats (n=5) by injecting them with insulin (30 IU/kg, i.p). Animals were recovered by administration of 3 ml of 30% glucose within 5 min of the occurrence of seizure. Control animals (n=3) were injected with saline instead of insulin. All animals were perfused 90 min after recovery and the brains processed for CREB immunohistochemistry. Cell counts were determined for CREB-positive neurons using a computer-assisted program. When compared to control animals there was a 50% decrease (P<0.0001) in CREB-positive neurons in the CA1 region of the experimental animals. In the CA3 and dentate gyrus there was a 36% (P<0.001) and 25% decrease (P<0.001), respectively. Given the importance of hippocampus in memory-related processes and evidence that CREB is critical for memory formation, it is possible that seizures interfere with memory by disrupting CREB-dependent transcription.

Epidermal growth factor activation of rat parotid gland adenylate cyclase and mediation by a GTP-binding regulatory protein.

Injection of rats with a single dose of epidermal growth factor (EGF) or isoproterenol increased parotid gland acinar cell levels of cyclic AMP (cAMP) significantly above control basal concentrations (34, 177 and 11.5 pmol/g tissue/100 g body weight, respectively). Following a chronic regimen of isoproterenol (3 days), EGF, bovine galactosyltransferase (Gal Tase, EC 2.4.1.22) and isoproterenol increased cAMP levels, albeit to a lower level than observed for the single dose (21, 17 and 51 pmol, respectively). Using isolated parotid gland membranes, EGF and bovine galactosyltransferase also stimulated adenylate cyclase (EC 2.7.4.3) activity in a concentration-dependent manner. Introduction of the beta-adrenergic receptor antagonist propranolol blocked isoproterenol-stimulated adenylate cyclase activity and cAMP accumulation, but not that observed with EGF or the transferase treatment. Growth factor-stimulated adenylate cyclase activity required the presence of the guanosine triphosphate (GTP) analogue, guanyl-5'-imidodiphosphate (p[NH]ppG), while cAMP accumulation could additionally be blocked by introducing the GDP analog, guanosine 5'[beta-thio]diphosphate (GDP[S]). The ability of EGF to activate adenylate cyclase was not affected by pretreatment of acinar cell membranes with pertussis toxin, whereas pretreatment with cholera toxin eliminated EGF-stimulated cyclase activity. The experimental results presented here expand to the parotid gland our knowledge of the ability of EGF to stimulate the cAMP second messenger signalling pathway via a G-binding regulatory protein, by a mechanism independent of beta-adrenergic receptor activation.

Detection of insulin and insulin-like growth factors I and II in saliva and potential synthesis in the salivary glands of mice. Effects of type 1 diabetes mellitus.

The salivary glands of mammals synthesize and secrete a number of peptide growth factors that play important roles in cell/tissue homeostasis and embryonic development. Using a radioimmunoassay, insulin, insulin-like growth factor-I (IGF-I) and insulin-like growth factor-II (IGF-II) were detected in saliva from mice. Unlike epidermal growth factor (EGF), there was no sexual dimorphism in the concentrations of the insulin growth factor family. Immunohistochemical localization of IGF-I and IGF-II was confined to the duct cells of both the parotid and the submandibular glands. Reverse transcriptase-polymerase chain reaction amplification of total RNA from parotid and submandibular glands confirmed the presence of all three hormone/growth factor mRNAs in both glands. The levels of insulin and IGF-I were higher in saliva from an animal model for autoimmune type 1 diabetes, the non-obese diabetic (NOD) mouse, than in a second inbred strain, BALB/c. In contrast, the IGF-II levels were decreased relative to the BALB/c strain. With the onset of diabetes in NOD mice, insulin levels declined, while IGF-I and IGF-II levels showed trends toward lower levels of these growth factors when compared with non-diabetic animals. These changes were reflected in the concentrations from parotid and submandibular gland cell lysates.

Saliva and growth factors: the fountain of youth resides in us all.

The predominant focus of research dealing with saliva revolves around the role in the maintenance of oral health through a number of physiological and biological properties of constituent proteins. An ever-expanding literature exists indicating that the salivary glands additionally synthesize, and secrete into saliva, a wide range of growth factors. Animal studies with epidermal growth factor have provided evidence for a role in both oral and systemic health, through the promotion of wound healing rates. Thus, the ability to manipulate their rates of synthesis and absorption from saliva holds the potential to enhance tissue regeneration and homeostasis.

Desipramine induced changes in salivary proteins, cultivable oral microbiota and gingival health in aging female NIA Fischer 344 rats.

Cyclic antidepressants are still a dominating group of psychotherapeutic drugs used in the treatment of depression. One of their major side effect is salivary gland dysfunction (oral dryness, xerostomia), leading in humans to increased oral disease and dysfunction of speech, chewing, swallowing and taste. The purpose of this study was to assess the effects of the long-term administration of the tricyclic antidepressant desipramine and the reversibility of this treatment following a 15 d washout period on specific salivary proteins, composition of oral microbiota, and oral health (gingivitis) of aging female F344 rats. Total salivary proteins showed decreased concentrations with age and desipramine. Similar SDS/PAGE protein profiles appeared in all phases but in different relative amounts with age and treatment. While certain proteins maintained steady levels (lactoferrin) or decreased with age and treatment (amylase), the synthesis of proline-rich proteins, high molecular weight mucin-type glycoproteins, and lysozyme was induced with desipramine and age. The oral microbiota was significantly changed with age and the administration of the antidepressant. The incidence of gingivitis with desipramine was highest in the oldest animals, For the different parameters measured, recovery was delayed with age. These data indicate, that desipramine has profound effects on salivary protein secretion. This may partially explain the changes in microbiota and the increased incidence of gingivitis.

Characterization of an SH2 containing protein tyrosine phosphatase in rat parotid gland acinar cells.

Rat parotid glands were shown to possess protein phosphatase activity capable of catalyzing the dephosphorylation of several model phosphatase substrates, including p-nitrophenyl phosphate, tyrosine phosphorylated myelin basic protein and serine phosphorylated casein. A portion of this activity closely resembled dephosphorylation patterns of known protein tyrosine phosphatases. The reaction showed sensitivity to sodium orthovanadate, proceeded efficiently in the presence of metal chelators and favored acidic pH for optimum activity. Cell lysates from EGF- or isoproterenol-stimulated parotid glands, when immuno-precipitated with anti-Syp antibody, showed the induction of protein tyrosine phosphatase activity significantly higher than the unstimulated controls. The protein of M(r) = 65kDa also had elevated levels of tyrosine phosphorylation following isolation from cells treated to undergo proliferation. Thus parotid gland acinar cells possess protein tyrosine phosphatase activity of the PTPase 1D class associated with inducible cell growth, in addition to other phosphatases.

Regulation of cell-surface galactosyltransferase in isoproterenol-treated mouse parotid glands.

Chronic injection of isoproterenol into mice resulted in hypertrophy and hyperplasia of the parotid gland. As previously described for the rat, cell proliferation was accompanied by an increase in total membrane-associated galactosyltransferase. A plasma membrane localization was determined by fluorescence-activated sorting of intact cells. Co-administration of the galactosyltransferase modifier protein, alpha-lactalbumin, or the calmodulin inhibitor, trifluoperazine, blocked acinar cell proliferation by 96 h post-treatment but not at 24 h. While alpha-lactalbumin appeared to interfere with galactosyltransferase-substrate interactions, trifluoperazine prevented the appearance of cell-surface enzyme in isoproterenol-treated animals.

A biochemical analysis of parotid and submandibular salivary gland function with age after simultaneous stimulation with pilocarpine and isoproterenol in female NIA Fischer 344 rats.

This analysis of physiological, biochemical and molecular changes related to aging was made in 3-, 12- and 24-month-old rats. The salivary gland weight/body weight ratio and the structural membrane proteins did not change with age for either gland, but a significant age-related decline in DNA synthesis for both glands was detected, unrelated to the hormonal responsiveness at the level of the plasma membrane. There was a marked increase in the concentration of soluble proteins in adolescent parotid gland and, for the two older age groups, in submandibular gland. The saliva flow rate was different when expressed as volume per time, as volume per time and g glandular wet weight, and/or kg body weight. The concentration of secreted proteins was not affected by age in either gland. The total amount of proteins secreted over 30 min revealed no age-related perturbation for the parotid gland, but showed a significant age-related increase in submandibular saliva. Sodium dodecyl sulphate-polyacrylamide gel analysis revealed changes in the protein bands between 39 and 50 kDa in the Coomassie blue-stained gels from 12-month-old animals. Amylase showed an initial increase (12 months), followed by a marked decline in its activity in parotid saliva. The glandular supernatant had low residual cellular amylase activity after stimulation. Therefore, secretory impairment with age after pilocarpine-isoproterenol stimulation was excluded. Analysis of total RNA showed a pronounced decrease of amylase mRNA in the parotid gland between 12 and 24 months of age. No amylase mRNA was expressed in any of the submandibular samples. For epidermal growth factor, total saliva showed a decrease with age. It seemed that the submandibular gland followed the same picture with age as the parotid gland, with a specific decline in the biosynthesis of single secretory proteins.

Effects of surgical ovariectomy on rat salivary gland function.

Studies sought to determine whether there are specific changes in salivary gland protein synthesis and secretion in response to hormone deficiency caused by ovariectomy of female rats. After 50 days, the wet weights of the parotid and submandibular glands did not change with hormone loss while that of the sublingual gland increased by 26% when compared to sham-operated controls. Amylase activity in the parotid declined, as did the level of enzyme activity present in saliva. The amount of the acidic proline-rich protein in the parotid was not altered after ovariectomy when compared to control sham-operated animals, using constant quantities of lysate protein. The total of secreted protein per unit volume did not change with ovariectomy. However, sodium dodecylsulphate-polyacrylamide gel electrophoresis of whole saliva showed the loss of a substantial number of proteins, including amylase and the acidic proline-rich proteins, from the experimental group. Epidermal growth factor concentrations were not significantly altered in the submandibular gland, while again showing a decrease in the concentration from saliva in ovariectomized rats.

Effect of EGF on rat parotid gland secretory function.

The present study reports changes in saliva composition from the rat parotid gland in response to single and repeated administration of epidermal growth factor (EGF). Treatment of rats with EGF (10 micrograms/kg, i.p., twice daily for 3 days) caused an increase in amylase activity in saliva collected from cannulated parotid duct, following stimulation of secretion with pilocarpine, with a corresponding decrease in enzyme activity in the gland. Analysis of parotid gland RNA by reverse transcriptase-PCR generated a single predicted amylase-derived cDNA product of 576 bp. The steady-state levels of mRNA for amylase from EGF-treated parotid total RNA showed a 1.8-fold increase compared to untreated controls. A single dose of EGF (15 min following i.p. injection) elicited an activation of both protein kinase A and protein kinase C activities. While the activation of protein kinase A was still maintained under the chronic EGF regimen, the activity levels of protein kinase C showed down-regulation to untreated control values.

Evaluation of phenolic content and antioxidant potency in various parts of Cassia auriculata L.: a traditionally valued plant.

Presence of polyphenolic content in various part of the plant exhibit wide pharmacological activities including antioxidant activity. The present study was designed to evaluate the phenolic contents (total phenols, flavonoid and tannins) and antioxidant properties of ethanolic extracts of flower, leaf, pod, bark and root obtained from Cassia auriculata. Ethanolic extracts of various parts of C. auriculata obtained by sonication extraction techniques are studied for their phenolic contents and DPPH (2,2-diphenyl-1-picrylhydrazine) radical scavenging assay as well as total antioxidant assays using UV visible spectrophotometer. Among the various parts of the plant studied, bark showed significant content of phenolics, flavonoids and tannins followed by the root, leaf, flower and pod. Even bark extract exhibited highest antioxidant capacity in DPPH assay followed by root, leaf, flower and pod with a value of 766.7, 679.3, 644.9, 572.5 and 474.7 mg vitamin C equivalent antioxidant capacity (mg VCEAC)/sample, respectively. In addition, mg VCEAC values obtained from the total antioxidant assay was in the increasing order of bark > root > leaf > flower > pod. Moreover, a strong correlation was also found between phenolic contents and antioxidant values indicating their influence in the found antioxidant activity, hence the bark extract can be employed as an ideal candidate for herbal based pharmaceutical product. Results of the present study also emphasize variation in the chemical composition as well as biological activity ensuring the importance of proper selection of particular part of the plant to evaluate their therapeutic potency.