Fisetin ameliorates methotrexate induced liver fibrosis.

OBJECTIVE: Methotrexate (MTX), a widely used chemotherapeutic and immunosuppressive agent, is associated with hepatotoxicity, leading to liver fibrosis and cirrhosis. This study explores the regenerative and reparative effects of fisetin, a flavonoid with known antioxidant and anti-inflammatory properties, on MTX-induced liver fibrosis in a rat model. MATERIALS AND METHODS: Thirty-six male Wistar albino rats were divided into normal, MTX and saline, and MTX and fisetin. Liver injury was induced in the latter two groups using a single intraperitoneal dose of MTX (20 mg/kg). Fisetin (50 mg/kg/day) or saline was administered intraperitoneally for ten days. After sacrifice, liver tissues were subjected to histopathological evaluation and biochemical analyses, including Transforming Growth Factor-ß1 (TGF-beta), sirtuins-1 (SIRT-1), malondialdehyde (MDA), cytokeratin 18, thrombospondin 1, and alanine transaminase (ALT) levels. RESULTS: MTX administration significantly increased liver injury markers, including TGF-beta, MDA, cytokeratin 18, thrombospondin 1, and ALT, while reducing SIRT-1 levels. Fisetin treatment attenuated these effects, demonstrating its potential therapeutic impact. Histopathological analysis confirmed that fisetin mitigated MTX-induced hepatocyte necrosis, fibrosis, and cellular infiltration. CONCLUSIONS: This study proves that fisetin administration can alleviate MTX-induced liver damage in rats. The reduction in oxidative stress, inflammation, and apoptosis, along with the histological improvements, suggests fisetin's potential as a therapeutic agent against MTX-induced hepatotoxicity. Further investigations and clinical studies are warranted to validate these findings and assess fisetin's translational potential in human cases of MTX-induced liver damage.

Meta-analysis of gene expression in human pancreatic islets after in vitro expansion.

Pancreatic islet transplantation as a potential cure for type 1 diabetes (T1D) cannot be scaled up due to a scarcity of human pancreas donors. In vitro expansion of beta-cells from mature human pancreatic islets provides an alternative source of insulin-producing cells. The exact nature of the expanded cells produced by diverse expansion protocols and their potential for differentiation into functional beta-cells remain elusive. We performed a large-scale meta-analysis of gene expression in human pancreatic islet cells, which were processed using three different previously described protocols for expansion and for which redifferentiation was attempted. All three expansion protocols induced dramatic changes in the expression profiles of pancreatic islets; many of these changes are shared among the three protocols. Attempts at redifferentiation of expanded cells induce a limited number of gene expression changes. Nevertheless, these fail to restore a pancreatic islet-like gene expression pattern. Comparison with a collection of public microarray datasets confirmed that expanded cells are highly comparable to mesenchymal stem cells. Genes induced in expanded cells are also enriched for targets of transcription factors important for pluripotency induction. The present data increase our understanding of the active pathways in expanded and redifferentiated islets. Knowledge of the mesenchymal stem cell potential may help development of drug therapeutics to restore beta-cell mass in T1D patients.

[Prosthetic rehabilitation with dental implant in cleft lip and palate: a ten-year retrospective study]. / Réhabilitation implantaire des fentes labiopalatines: étude rétrospective sur dix ans.

INTRODUCTION: Two problems need to be faced during cleft lip and palate surgical treatment to gain a functional and esthetic result: restoration of the maxillary bone defect and the missing lateral incisor tooth, which may be replaced by an implant. The aim of our study was to demonstrate that it is possible to successfully place a dental implant after obtaining adequate bone graft volume. PATIENTS AND METHOD: 12 consecutively treated patients were studied, 7 with unilateral and 5 with bilateral clefts, mean age 21.5 years, with an average follow-up of 5.5 years (range: 1-10 years). Various types of autogenous or alogenous bone grafts were performed to provide adequate bone volume for immediate or secondary implant placement. We studied the number of bone grafts needed to reach this target, and the number of implants placed, focusing on lateral incisor tooth replacement. RESULTS: Enough cleft bone volume for the implant was obtained in 9 out of 12 cases (75%). All endosseous implants placed in bone graft were successfully restored, 8 out of them for lateral incisor. Some surgical cases are described. DISCUSSION: Global improving of cleft lip and palate surgical treatment by implantology is now feasible. Maxillary defect bone grafting, often in several stages, is strictly necessary to place an implant but also to obtain a good cosmetic result in cleft repair. Length, direction, and cervical implant position have to be considered too. Reconstruction of mucosal anatomy also contributes to the end result.

Growth factor-induced signaling of the pancreatic epithelium.

Activated signaling proteins regulate diverse processes, including the differentiation of the pancreatic islet cells during ontogeny. Here we uncover the in vivo phosphorylation status of major growth factor-activated signaling proteins in normal adult mice and during pancreatic islet regeneration. We report elevated phospho-mitogen-activated protein kinase (phospho-MAPK), phospho-c-Jun-NH2-terminal kinase (phospho-JNK), and phospho-p38 MAPK expression during pancreatic regeneration. Immunoblotting experiments demonstrated elevated phosphorylation of p52 Src-homology/collagen (SHC) in the ductal network as well, substantiating the activation of this pathway. Furthermore, protein kinase B (PKB/Akt), a key signaling protein in the anti-apoptotic pathway, was phosphorylated to a greater extent in the ductal network from regenerating pancreas. We observed fibroblast growht factor (FGF)10 and platelet-derived growth factor (PDGF)AA expression in embryonic as well as regenerating adult pancreas. Epidermal growth factor (EGF) and PDGFAA stimulated MAPK and Akt phosphorylation, while FGF10 stimulated MAPK but not Akt phosphorylation in a time-dependent manner in freshly isolated cells from the adult ductal network. These data suggest that a heightened level of expression and stimulation of key signaling proteins underlie the expansion and differentiation processes that support pancreatic ontogeny and regeneration.

Blood mercury levels of dental students and dentists at a dental school.

OBJECTIVE: To determine the blood mercury levels in dental students and clinical teaching staff in a dental school using amalgam as a restorative material. SETTING: A dental school in Ege University, Turkey surveyed during one academic year. SUBJECTS AND METHODS: Cross-sectional study of groups of dental students (n=92) in years I to V, clinical teachers in restorative dentistry (n=16) and controls (n=14). Mercury concentration was estimated in venous blood samples using a cold vapour atomic absorption method at the commencement and end of the academic year. Daily air mercury levels were determined in clinical and teaching areas by measuring the darkening of palladium chloride discs using spectrophotometry. RESULTS: There were statistically significant increases (p<0.001) in plasma mercury concentration between measurements in all groups at the end of the academic year. Red cell mercury levels were also consistently elevated. Although the highest levels of mercury were recorded in persons working with amalgam, increased levels were also found in subjects working in the teaching classrooms but not with amalgam (controls and first year students). CONCLUSION: Increased mercury levels appeared to be due to background exposure from spillage of mercury and amalgam residues on floors. Increased mercury hygiene and regular control of working atmosphere should be implemented to prevent mercury exposure in the dental pre-clinical laboratory.

Insulin activates phospholipase C-gamma1 via a PI-3 kinase dependent mechanism in 3T3-L1 adipocytes.

Previously we have shown that the insulin receptor and phospholipase C-gamma1 physically interact in the 3T3-L1 adipocyte cell line. In this study, we investigated the ability of insulin and PDGF to stimulate PLC-gamma1 enzyme activity as measured by PI-(4,5)P(2) hydrolysis. Both insulin and PDGF caused a rapid (<1 min) increase in PLC activity associated with the respective receptor. PDGF treatment resulted in a higher and more sustained stimulation of PLC-gamma1 activity compared to insulin (0.95 pmol/min/mg vs 0.68 pmol/min/mg). Furthermore, insulin and PDGF promoted increases in total cellular DAG, one of the products of PI-(4,5)P(2) hydrolysis. Insulin-stimulated PLC activity appears to be downstream of PI-3Kinase as the DAG increase was partially blocked by Wortmannin and addition of PI-(3,4,5)P(3) activated PLC-gamma1 in vitro. Inhibition of PLC using U73122 or an inhibitory peptide caused a decrease in insulin-stimulated 2-deoxyglucose transport and GLUT4 translocation that was rescued by the addition of OAG, a cell-permeable synthetic DAG.

Stimulation of MAPK cascades by insulin and osmotic shock: lack of an involvement of p38 mitogen-activated protein kinase in glucose transport in 3T3-L1 adipocytes.

Osmotic shock and insulin stimulate GLUT4 translocation and glucose transport via mechanisms that are for the most part distinct yet convergent. In this article, we investigated the effect of osmotic shock and insulin on the activation of the mitogen-activated protein kinase (MAPK) cascades in differentiated 3T3-L1 adipocytes. The MAPKs are activated by phosphorylation on conserved tyrosine and threonine residues. Both sorbitol and insulin strongly stimulated extracellular regulated kinase (ERK) 1 and 2 phosphorylation (8- and 18-fold, respectively). In contrast, c-jun-NH2-terminal kinase (JNK)/stress-activated protein kinase (SAPK) phosphorylation was stimulated only by sorbitol (sevenfold) and not by insulin. Phosphorylation of p38 MAPK was stimulated strongly by sorbitol (22-fold) but weakly by insulin (2.7-fold). Measurement of intrinsic JNK and p38 MAPK activity confirmed the phosphorylation studies. JNK and p38 MAPK were activated only significantly by sorbitol. The MAPKs are phosphorylated by dual-specificity kinases (mitogen-activated ERK-activating kinase [MEK] or MAPK kinase [MKK]). As expected, sorbitol and insulin both stimulated MEK phosphorylation. MKK4 was phosphorylated only in response to sorbitol, and neither of the stimuli caused phosphorylation of MKK3 or 6. To determine the functional significance of the observed activation of p38 MAPK in response to insulin and osmotic shock, we used three pyridinyl imidazole p38 MAPK inhibitors, SB203580, SB202190, and PD169316. Insulin and osmotic shock-stimulated glucose transport was not inhibited by any inhibitor at concentrations that were shown to block p38 MAPK activity. Furthermore, activation of the p38 MAPK pathway by treatment of cells with anisomycin did not stimulate glucose transport. These results suggest that activation of the p38 MAPK pathway is not involved in the stimulation of glucose transport.

Association of the insulin receptor with phospholipase C-gamma (PLCgamma) in 3T3-L1 adipocytes suggests a role for PLCgamma in metabolic signaling by insulin.

Phospholipase C-gamma (PLCgamma) is the isozyme of PLC phosphorylated by multiple tyrosine kinases including epidermal growth factor, platelet-derived growth factor, nerve growth factor receptors, and nonreceptor tyrosine kinases. In this paper, we present evidence for the association of the insulin receptor (IR) with PLCgamma. Precipitation of the IR with glutathione S-transferase fusion proteins derived from PLCgamma and coimmunoprecipitation of the IR and PLCgamma were observed in 3T3-L1 adipocytes. To determine the functional significance of the interaction of PLCgamma and the IR, we used a specific inhibitor of PLC, U73122, or microinjection of SH2 domain glutathione S-transferase fusion proteins derived from PLCgamma to block insulin-stimulated GLUT4 translocation. We demonstrate inhibition of 2-deoxyglucose uptake in isolated primary rat adipocytes and 3T3-L1 adipocytes pretreated with U73122. Antilipolytic effect of insulin in 3T3-L1 adipocytes is unaffected by U73122. U73122 selectively inhibits mitogen-activated protein kinase, leaving the Akt and p70 S6 kinase pathways unperturbed. We conclude that PLCgamma is an active participant in metabolic and perhaps mitogenic signaling by the insulin receptor in 3T3-L1 adipocytes.

An attempt to predict daily erythrocyte lithium fluctuations.

Erythrocyte lithium concentration, which is a better predictor of brain lithium levels than plasma lithium concentrations, possesses the disadvantage of precise hourly determination following the last intake. The variability in RBC lithium accumulation increases as the extracellular lithium concentration increases. This increase is also time dependent and it would be very useful if the pharmacokinetic rate constant were known. Unfortunately, low lithium levels do not allow measurements within confidence intervals. In this work, we tried to determine, in vitro, the kinetic rate constants in erythrocytes of healthy volunteers. Different high lithium loaded plasma-like media were used for an extrapolation procedure of constants allowing the determination of an erythrocyte load constant namely K0 = 0.0161 +/- 0.0005 h-1 at corresponding plasma lithium concentrations. The abnormalities of lithium transport determined by in vitro procedures would be very useful in understanding the etiology of affective illness. Lithium flux pre-controls corrected with this rate constant would be very helpful in enlarging laboratory time management.

The effect of delayed gastric emptying and absorption on pharmacokinetic parameters of lithium.

Gastrointestinal motility is one of the most important factors than can influence drug absorption from gastrointestinal tract. The aim of the present study was to investigate the effect of delayed gastric emptying and intestinal transit on pharmacokinetic parameters of lithium. Treatment animals were administered an anticholinergic agent (propantheline bromide, 4 mg/kg, p.o.) 10 min before lithium chloride (1.5 mM/kg, p.o.) administration, whereas the control group was administered the same dose of lithium p.o., alone. Plasma lithium levels were measured by flame spectrophotometry and calculated with a computer programme (SIPHAR). Differences detected in AUC, fractionated AUC values, Cmax and tmax suggest that using delayed absorption process, it is possible to prolong by 272% the plateau time of the drug in the therapeutic range and this approach might be an alternative way to prevent some undesirable effects due to peak plasma levels above the maximal therapeutic level. This approach might be more important as an alternative for suitable slow release formulations.

Pharmacokinetics of carbamazepine. Part I: A new bioequivalency parameter based on a relative bioavailability trial.

The relative bioavailability of three carbamazepine generics available in Turkey, were investigated in 5 healthy male volunteers. When issuing a license to any drug, FDA stipulates at most a difference of 20% from the reference drug only in peak concentration and AUC (area under the curve). This condition may cause some problems, as two generics of the same drug can yield the same total amount (AUC) and can be accepted as bioequivalent despite different curves of the two drugs. In this study, to compare drugs from the point of view of bioequivalency, we suggest a new calculation method that takes into account ka (absorption rate constant), ke (elimination rate constant), tmax (time to peak), MRT (mean residence time) and AUC. Should this formula be used in comparison of bioequivalency, all the parameters related to the kinetics of drugs will have been taken into account. The suggested parameter is: [formula: see text] However, amongst three carbamazepine generics-Tegretol, Temporol and Karazepin-the most desirable curve is that of Tegretol, while bioavailability values are respectively F = 0.86, 0.93, 0.85 and AUC = 145, 161, 127. The A parameter values are respectively 49.3, 47.2, 42.9.

Bioequivalency evaluation by comparison of in vitro dissolution and in vivo absorption using reference equations.

Using in vitro dissolution and in vivo absorption of different generics or batches for an appropriate drug and dosage form, a reference equation of form: ka = a+bkd+dkd2 can be proposed. In the case of availability of standardised in vitro-in vivo data for a specific drug, a Level A a correlation of this type containing experimentally determined a, b, d parameters would serve to predict in vivo absorption phase of the drug. The larger number of batches, the stronger will be the predictive power of these parameters.

Effects of dietary protein restriction on the fractional rates of protein synthesis in perfused rat hindlimb.

The effects of dietary protein restriction on protein synthesis were investigated in perfused rat hindlimb. The fractional rate of protein synthesis was measured with [3H]phenylalanine in young adult (7-week-old) rats fed a low protein (5% casein) diet and a protein-free diet for 3 weeks. The low protein diet (LPD) allowed a moderate gain in body weight. The fractional rate of protein synthesis fell to 70% of the control value in LPD group and further fell to less than a half in the protein-free diet (PFD) group. Thus, the protein synthesis rate decreased as the dietary protein content was reduced. The fall of protein synthesis was mainly accompanied by the reduction of RNA activity (mg protein/mg RNA/day) rather than RNA concentration (RNA/protein). The rate of protein breakdown was calculated by subtracting growth rate from protein synthesis rate. The breakdown rate was decreased in LPD group and increased slightly in PFD group. From the low rates of protein synthesis and breakdown, it appears that dietary protein restriction, at least allowing a gain in body weight, makes the turnover rate slow down. The overall changes in protein synthesis obtained in the perfused hindlimb are consistent with the reported results in vivo.

Insulin- and thyroid hormone-independent adaptation of myofibrillar proteolysis to glucocorticoids.

Myofibrillar protein breakdown in skeletal muscle progresses through two distinct phases in response to chronic glucocorticoid administration in the rat, i.e., an early phase lasting 4-5 days, during which proteolysis increases followed by a later phase during which proteolysis decreases. The possible involvement of insulin and the iodothyronines in this phenomenon has now been examined. Diabetic, thyroidectomized, and normal rats were treated with corticosteroid for 10-11 days, and at timed intervals muscle proteolysis was evaluated by measuring the release of 3-methyl-L-histidine (3-MH) and tyrosine from the perfused hindquarter as well as the excretion of 3-MH in the urine. Corticosterone (CTC) administration to normal rats increased plasma insulin, whereas plasma 3,5,3'-triiodothyronine responded with an early rise followed by a fall after 4-5 days. However, the biphasic response of myofibrillar proteolysis to chronic glucocorticoid treatment was not abolished in CTC-treated diabetic or thyroidectomized rats. CTC treatment increased release of tyrosine by perfused muscle of diabetic rats but, unlike 3-MH release, did not diminish later. Thus the adaptation of myofibrillar proteolysis to chronic glucocorticoid treatment appears to be independent of insulin and thyroid hormones. However, insulin may play a role in curtailing glucocorticoid-induced breakdown of nonmyofibrillar proteins.

Sensitivity of myofibrillar proteins to glucocorticoid-induced muscle proteolysis.

Previous studies have reported that prolonged administration of pharmacological doses of glucocorticoids in young rats results in a rise in urinary 3-methyl-L-histidine (3-MH) excretion followed by a fall to initial levels by 8 days. To determine whether this response reflects events in skeletal muscle, protein breakdown in this tissue was evaluated using the perfused hindquarter preparation with rats treated with corticosterone (10 mg X 100 g-1 X day-1) for 2, 4, or 8 days. Myofibrillar and total cell proteolysis were evaluated by measuring the release of 3-MH and tyrosine, respectively, after inhibition of protein synthesis with cycloheximide. Corticosterone treatment resulted in an early increase (1-4 days) followed by a fall (4-8 days) in 3-MH excretion. 3-MH release by the perfused hindquarter of treated rats responded in a similar manner, in that its release increased at days 2 and 4 and decreased to control levels by day 8. On the other hand, corticosterone treatment did not affect the release of tyrosine by the perfused hindquarter. Corticosterone treatment diminished protein synthesis in muscle by 30-50% (P less than 0.01), which unlike 3-MH release by perfused muscle persisted throughout the treatment period. The data indicate that glucocorticoids specifically augment the breakdown of myofibrillar proteins in skeletal muscle. This response is attenuated with prolonged treatment and is unrelated to a loss of metabolic effectiveness of the steroid. Also our findings suggest that the breakdown of myofibrillar and nonmyofibrillar proteins might be regulated independently.

Synergism of triiodothyronine and corticosterone on muscle protein breakdown.

The concerted effect of triiodothyronine (T3) and corticosterone on muscle protein synthesis and breakdown was studied. Thyroidectomized young male rats were treated with T3 (1.5 microgram/100 g body weight per day), corticosterone (10 mg/100 g body weight per day) and both T3 and corticosterone for 4 days. On the 3rd day of the experiment urine was collected to measure N tau-methylhistidine excretion as an index of muscle protein breakdown. On the last day of the experiment, the rates of protein synthesis in skeletal muscles were measured by the large-dose [3H]phenylalanine method. N tau-Methylhistidine excretion was slightly increased by T3 treatment and it was increased about 3-times by corticosterone treatment. When both T3 and corticosterone were administered, it was increased about 6-fold. The rate of muscle protein breakdown calculated from the difference between the rate of protein synthesis and the growth rate was consistent with these findings. The rate of muscle protein synthesis was increased by T3, and it was decreased by corticosterone. The rate was the same as that of the thyroidectomized control group when the animals were given T3 and corticosterone, showing that T3 restrained the inhibiting effect of corticosterone on muscle protein synthesis. The results indicate that a physiological level of T3 enhances the catabolic action of pharmacological doses of glucocorticoids on muscle protein breakdown.