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.

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.

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.

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.

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.

Reconstituted Cl- pump protein: a novel ion(Cl-)-motive ATPase.

Cl- absorption by the Aplysia californica foregut is effected through an active Cl- transport mechanism located in the basolateral membrane of the epithelial absorptive cells. These basolateral membranes contain both Cl(-)-stimulated ATPase and ATP-dependent Cl- transport activities which can be incorporated into liposomes via reconstitution. Utilizing the proteoliposomal preparation, it was demonstrated that ATP, and its subsequent hydrolysis, Mg2+, Cl-, and a pH optimum of 7.8 were required to generate maximal intraliposomal Cl- accumulation, electrical negativity, and ATPase activity. Additionally, an inwardly-directed valinomycin-induced K+ diffusion potential, making the liposome interior electrically positive, enhanced both ATP-driven Cl- accumulation and electrical potential while an outwardly-directed valinomycin-induced K+ diffusion potential, making the liposome interior electrically negative, decreased both ATP-driven Cl- accumulation and electrical potential compared with proteoliposomes lacking the ionophore. Either orthovanadate or p-chloromercurobenzene sulfonate inhibited both the ATP-dependent intraliposomal Cl- accumulation, intraliposomal negative potential difference, and also Cl(-)-stimulated ATPase activity. Both aspects of Cl- pump transport kinetics and its associated catalytic component kinetics were the first obtained utilizing a reconstituted transporter protein. These results strongly support the hypothesis that Cl(-)-ATPase actively transports Cl- by an electrogenic process.

Inhibition of rat parotid gland growth response induced by chronic isoproterenol following treatment with quinolone antibiotics.

While antibiotics are broadly used in dental and medical therapy, little attention has been directed towards the potential toxic side effects of antibiotics on tissue regeneration. Here we examined the effect of a quinolone antibiotic, pefloxacin (Rhone Poulenc) on rat parotid gland responses to chronic isoproterenol treatment. Groups of rats received injections of isoproterenol to induce glandular growth, saline (controls), pefloxacin, or isoproterenol and pefloxacin in combination. Parotid gland weight decreased significantly after pefloxacin treatment for 7 days as well as inhibiting glandular enlargement provoked by isoproterenol. The same trend was observed for the rates of DNA synthesis, with the incorporation of [3H]-thymidine in isoproterenol/pefloxacin-treated rats reduced to 49% of isoproterenol treatment alone levels. Saline-treated animals were 42% of the rate of [3H]-thymidine incorporation into DNA observed in isoproterenol treated rats. While isoproterenol treatment increased steady-state mRNA levels for fos, jun, myc, src, c-erbB-2, ras and topo II, inclusion of pefloxacin with the isoproterenol regimen blocked these increases. Pefloxacin treatment by itself did not alter proto-oncogene mRNA levels in the parotid gland. Glandular amylase activity was decreased in the pefloxacin treated group, while the combination of isoproterenol with pefloxacin did not decrease glandular amylase levels to the extent of that observed with beta-agonist treatment alone. In acute experiments, pefloxacin significantly decreased the volume of saliva secreted by the parotid gland. These results suggest that quinolone-based antibiotics disturb the secretory function of the parotid gland and can inhibit cell proliferation and regeneration.

An electrogenic chloride pump in a zoological membrane.

Two widely documented mechanisms of chloride transport across animal plasma membranes are anion-coupled antiport and sodium-coupled symport. No direct genetic evidence has yet been provided for primary active chloride transport despite numerous reports of cellular CI(-)-stimulated ATPases coexisting, in the same tissue, with uphill chloride transport that could not be accounted for by the two common chloride transport processes. CI(-)-stimulated ATPases are a common property of practically all animal cells, with the major location being of mitochondrial origin. It also appears that the plasma membranes of animal cells are sites of CI(-)-stimulated ATPase activity. Recent studies of CI(-)-stimulated ATPase activity and chloride transport in the same membrane system, including liposomes, suggest a mediation by the ATPase in net movement of chloride up its electrochemical gradient across animal plasma membranes. Further studies, especially from a molecular biological perspective, are required to confirm a direct transport role to plasma membrane-localized Cl(-)-stimulated ATPases.

Alterations in the level of phosphotyrosine signal transduction constituents in human parotid tumors.

Human parotid tumors were evaluated for the activation of the phosphotyrosine signaling pathway by Western blot, enzyme activity assay, and reverse transcriptase-polymerase chain reaction. Warthin's tumor and mucoepidermoid carcinomas had the greatest level of tyrosine phosphorylated proteins identified in plasma membrane fractions. These tumors, along with pleomorphic adenocarcinoma, showed high levels of membrane expression of the tyrosine kinase receptor, c-erbB-2, and phosphatidylinositol-3-kinase. Expression of the epidermal growth factor receptor was confined to normal tissue. The level of mRNA for c-erb was elevated only in mucoepidermoid carcinomas. Messenger RNA levels for ras were unchanged from control levels in all tumors, while the level of src mRNA was higher in the tumor samples than the normal parotid tissue. The activities of several signal transduction kinases, including protein kinase A and C were elevated in tumor tissue (7.7- to 18.9- and 0.4- to 3.7-fold higher, respectively), relative to surrounding normal tissue. While the level of glandular amylase was reduced (22%-0% of normal levels) in the tumor tissue, epidermal growth factor (EGF) and transforming growth factor-alpha (TGFalpha) content was dramatically higher in the neoplastic tissue (10- to 170-fold and 4.6- to 6.0-fold, respectively). These results suggest that with the presence of elevated levels of EGF, TGFalpha, and the oncoprotein receptor c-erbB-2 in the membrane of parotid tumors, cell proliferation and activation of the phosphotyrosine signal transduction pathway may involve autocrine stimulation through the expression of high levels of growth factor and receptor in the same tissue.

Absorption of epidermal growth factor occurs through the gastrointestinal tract and oral cavity in adult rats.

Introduction of radiolabeled epidermal growth factor (125I-EGF) by gavage or sublingual confinement resulted in a time-dependent uptake and systemic organ dissemination in the adult rat. Intact EGF was recovered primarily from the tongue, parotid, and sublingual/submandibular glands after administration by sublingual lozenge, whereas gastrointestinal administration resulted in 125I-EGF recovery primarily from plasma, stomach, and lung. Recovered radiolabeled EGF retained the ability to bind to the EGF receptor. Sialoadenectomy caused an increase in 125I-EGF in most tissues by both routes of administration. Thus, in the adult rat, at least two pathways exist for the uptake and distribution for salivary gland-derived EGF present in saliva. With further analyses, sublingual absorbance of EGF may therefore provide a potential delivery route for therapeutic use of growth factor, which avoids the hepatic destruction of EGF after oral administration.

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.

Effect of vanadate on amylase secretion and protein tyrosine phosphatase activity in the rat parotid gland.

Treatment of rat parotid acinar cells with sodium orthovanadate (an inhibitor of protein tyrosine phosphatase) caused a dose-dependent inhibition of phosphatase activity as measured by the hydrolysis of para nitrophenylphosphate (pNPP). Inclusion of 50 microM sodium orthovanadate in in vitro gland cultures prevented the amylase secretion from both untreated control and isoproterenol-stimulated parotid acinar cells. Four different tyrosine-phosphorylated proteins with M(r) 40, 70 and 95 kDa, respectively, were identified in secretory granule preparations from rat parotid glands by immunoblot using a monospecific antibody for phosphotyrosine. An increase in the phosphorylation levels of these phosphoproteins was noted in the presence of 50 microM sodium orthovanadate, suggesting that a protein tyrosine phosphatase (PTPase) is involved in parotid gland protein dephosphorylation reactions. Using antibody to Syp (a PTPase belonging to class 1D), a major fraction of subcellular activity was found to be associated with secretory granule membranes. These results suggest the possible involvement of a PTPase (Syp) in parotid gland secretory mechanisms.

A novel Cl(-)-pump: intracellular regulation of transport activity.

Utilizing a purified basolateral plasma membrane vesicle preparation containing Cl(-)-ATPase, it was demonstrated that ATP, and its subsequent hydrolysis, stimulated both intravesicular Cl- accumulation and intravesicular negativity with almost identical kinetics. Mg2+ also stimulated both pump transport characteristics with an optimal concentration of 3 mM. Similarly, the pH optimum for both pump transport expressions was 7.8. Orthovanadate inhibition of both pump transport characteristics was directly related to its concentration. These results suggested that the active Cl- transport mechanism is electrogenic and is driven by a Cl(-)-stimulated ATPase.

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.

The role of phosphotyrosine signaling pathway in a parotid gland proliferation and function.

Tyrosine phosphorylation and the intracellular signaling processes associated with it have been the focus of intense study due to its importance in the regulation of biological processes as diverse as cell proliferation and cell differentiation. While much of what we now understand has been derived from the study of cell lines and tumor cells, the salivary glands provide a model to examine the effects of tyrosine kinases and tyrosine phosphatases in a normal differentiated tissue. This review will focus, therefore, on the role tyrosine kinases and phosphatases play in inducing the transition from stasis to active proliferation and their potential role in mediating secretory function of the salivary glands.

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.

Activation of SH2-containing proteins by insulin in proliferating mouse parotid gland acinar cells.

Chronic treatment of mice with insulin results in hypertrophy and hyperplasia of the parotid and submandibular glands (Wang et al.: 1994, Proc Soc Exp Biol Med 205:353-361). Hyperplasia of the parotid gland is mediated by the elevation of tyrosine phosphorylation of phospholipase C gamma, p21ras-GTPase activating protein (p21ras-GAP) and phosphatidylinositol 3-kinase. These proteins were found to be associated with the insulin receptor substrate-1 most likely through src homology (SH2) domains of these proteins. There was also a transient increase in intracellular cAMP and protein kinase A during the first day of treatment which declined by Day 3 to near control values. Protein kinase C activity, on the other hand, remained elevated for the 3-day injection regimen. Thus, acinar cell proliferation induced by insulin requires activation of many of the same signaling components as other tyrosine kinase possessing growth factor receptors.

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.

Limited modulation of the mitogen-activated protein kinase pathway by cyclic AMP in rat parotid acinar cells.

Treatment of rat parotid acinar cells, in vitro, with agents that elevate intracellular cAMP had only limited impact on the ability of EGF to subsequently stimulate MAP-kinase activity or phosphorylation. A time course of cAMP accumulation following in vivo administration of isoproterenol showed the greatest level of cAMP 15 min following the primary injection. Over a 72 hr injection regimen, agonist-stimulated cAMP levels were gradually reduced to control levels while cAMP-dependent protein kinase A (PKA) activity in immunoprecipitates of Raf-1 or MAP-kinase remained elevated. Raf-1 did not undergo phosphorylation following incubation with the catalytic subunit of PKA, suggesting that in normal rat acinar cells proliferation induced by isoproterenol or EGF involves the p21ras-Raf-MAP-kinase signaling cascade despite the presence of cAMP.

Effect of chronic insulin administration on mouse parotid and submandibular gland function.

Chronic (six-day) injection of insulin (im, 50 microM/animal) into BALB/c mice resulted in changes in secretory function, hypertrophy and hyperplasia of the parotid and submandibular glands. There were no significant changes in the flow rate or concentrations of proline-rich proteins, TGF alpha, or amylase in saliva when measured against constant protein levels. However, amylase enzyme activity and total saliva protein content were reduced when measured against constant saliva volume. In contrast, EGF synthesis and secretion from the submandibular gland was increased. Both the parotid and submandibular gland showed evidence of gland hypertrophy and increased rates of DNA synthesis as indicated by [3H]-thymidine incorporation in response to insulin treatment. Chronic injection of insulin did not effect the level of receptor in the plasma membrane of either gland.