Photoaffinity heterobifunctional cross-linking reagents based on N-(azidobenzoyl)tyrosines

New heterobifunctional cross-linking reagents that possessed a photoactive terminus, an electrophilic terminus, and a linking arm between the two termini that had a radiolabeled, enzymatically cleavable bond were synthesized. In a model study, succinimidyl N-[N'-(4-azidobenzoyl)tyrosyl]-beta-alanate (16A) was coupled to n-butylamine (a Lys surrogate), iodinated, and cleaved with chymotrypsin in the presence of tyrosylamide to afford the desired adduct (N-(N'-(4-azidobenzoyl)-3-iodotyrosyl)tyrosinamide, thereby demonstrating the feasibility of the enzymatic cleavage. In a biochemical study, succinimidyl N-[N'-(3-azido-5-nitrobenzoyl)tyrosyl]-beta-alanate (16C) was coupled to Lys-75 of calmodulin (CaM), and the radioiodinated monoadduct was successfully photo-cross-linked, in a calcium-dependent manner, to the human erythrocyte plasma membrane Ca2+,Mg2(+)-ATPase and to a synthetic fragment (M13) containing the CaM-binding region of myosin light-chain kinase. In the latter case, densitometry readings indicated 20% cross-linking efficiency.     

Cytosolic and non-cytosolic carbonic anhydrase enzymes from bovine leukocytes.

In this study, carbonic anhydrase (CA) enzyme has been purified and separately characterized according to bound form in 4 steps as outer peripheral, cytosolic, inner peripheral, and integral from bovine leukocyte. Affinity chromatography has also been used for purification of the enzyme in four steps. CA has been found for each step. Measurment of enzyme activity has been done by CO2 hydratase activity and esterase activity methods. Optimum pH and optimum temperature have been defined for each step of purified enzyme. The behaviors of CA with specific inhibitors, such as KSCN and NaN3 have been investigated. In each step, molecular weight and purity have been determined by gel filtration and SDS-PAGE electrophoresis. In addition, enzyme K(M) and Vmax values have been determined with the method of Lineweaver-Burk.     

Effects of estrous cycle stage and transport temperature of ovaries on in vitro maturation of canine oocytes

Unlike other domestic animals, in vitro maturation (IVM) of canine oocytes still has limited success. The present study investigated the effects of estrous cycle stage and transport temperature of ovaries on in vitro maturation of canine oocytes. The donor bitches were categorized into three groups based on stage of estrus cycle: follicular (proestrus or estrous), luteal (diestrus) and anestrus. One ovary of each pair collected from 39 mature bitches was transported in Phosphate Buffer Saline (PBS) at 4 °C while the other was transported at 37 °C. A total of 1138 Grade I COCs obtained from all ovaries were grouped and matured in modified synthetic oviduct fluid (mSOF) supplemented with follicle stimulating hormone (FSH), luteinizing hormone (LH), essential and non-essential amino acids at 38.5 °C in a humidified 5% CO₂, 5% O₂, and 90% N₂ atmosphere for 72 h. The nuclear maturation rates were evaluated by aceto–orcein staining.Oocytes harvested from follicular and luteal ovaries have a significantly higher maturation rates (MI + MII) than the oocytes from anestrual ovaries in the 37 °C group (p < 0.05). However, oocytes harvested from anestrual ovaries transported at 4 °C had the highest maturation (MI + MII) rate, and the difference between anestrual and luteal ovary groups was significant (p < 0.05). The oocytes from anestrual ovaries transported at 4 °C have significantly higher maturation rates than those transported at 37 °C (p < 0.0001). However, the transport temperature (37 or 4 °C) did not significantly affect the maturation (MI + MII) rates of oocytes harvested from the luteal (p = 0.61) and follicular (p = 0.48) stage ovaries.It can be concluded from this study that (1) both transport temperature and transport temperature × estrus cycle stage interaction effected the maturation rates, while estrus cycle stage alone did not, and (2) transporting canine ovaries at 4 °C can improve in vitro maturation rates in oocytes harvested from anestrous ovaries.     

Chitosan film containing fucoidan as a wound dressing for dermal burn healing: Preparation and in vitro/in vivo evaluation

The aim of this study was to develop chitosan film containing fucoidan and to investigate its suitability for the treatment of dermal burns on rabbits. Porous films, thickness between 29.7 and 269.0 μm, were obtained by the solvent dropping method. Water vapor permeability (3.3–16.6/0.1 g), the swelling (0.67–1.77 g/g), tensile strength (7.1–45.8 N), and bioadhesion (0.076–1.771 mJ/cm²) of the films were determined. The thinnest films were obtained with the lowest chitosan concentration (P<.05). The water absorption capacity of the films sharply increased with the freeze-drying technique. The film having the thickness of 29.7 μm showed the highest amount of moisture permeability (16.6 g/0.1 g). Higher chitosan concentration significantly increased tensile strength of the films (P<.05). Using higher concentration of lactic acid made films more elastic and applicable, and these films were selected for in vivo studies. Seven adult male New Zealand white rabbits were used for the evaluation of the films on superficial dermal burns. Biopsy samples were taken at 7, 14, and 21 days after wounding, and each wound site was examined macroscopically and histopathologically. After 7 days treatment, fibroplasia and scar were observed on wounds treated with fucoidan-chitosan film. The best regenerated dermal papillary formation, best reepithelization, and the fastest closure of wounds were found in the fucoidan-chitosan film treatment group after 14 days compared with other treatment and control groups. It can be concluded that fucoidan-chitosan films might be a potential treatment system for dermal burns and that changing formulation variables can modulate the characterizations of the films. Published: May 24, 2007     

Role of the nitric oxide pathway in ischemia-reperfusion injury in isolated perfused guinea pig lungs

We examined the role of the nitric oxide (NO) pathway on ischemia-reperfusion injury via the use of isolated perfused guinea pig lungs. We administered both L-Arginine and N-nitro-L-arginine methyl ester (L-NAME) to the lungs in or after 3 h of ischemia. We observed pulmonary artery pressures as well as tissue and perfusate malondialdehyde (MDA) and glutathione (GSH) levels. We observed that L-NAME significantly increased both tissue and perfusate GSH levels and pulmonary artery pressures, but it decreased both tissue and perfusate MDA levels. On the other hand, L-arginine significantly decreased pulmonary artery pressure and both tissue and perfusate glutathione levels, but it increased both tissue and perfusate MDA levels. Electron microscopic evaluation supported our findings by indicating the preservation of lamellar bodies of type II pneumocytes. We concluded that L-NAME administration during reperfusion improves lung recovery from ischemic injury.     

Zoledronic acid is synergic with vinblastine to induce apoptosis in a multidrug resistance protein-1 dependent way: an in vitro study

We have explored the action of zoledronic acid, which has an apoptotic effect and is used as an agent for treating skeletal metastases and osteoporosis, in the presence of vinblastine, and whether this effect is associated with MRP-1 (multidrug resistance protein-1) expression. HEK (human embryonic kidney) 293 cells were transfected to form the multidrug resistant cell line designated 293MRP (MRP-1 expressing HEK293 cells). Both lines were treated with varying concentrations of vinblastine and zoledronic acid. Apoptosis was determined by the TUNEL (deoxyuridine triphosphate nick end-labeling) method. The type of treatment, MRP-1 expression status, and the type of treatment with respect to MRP-1 expression status significantly affected (P < 0.001) the degree of apoptosis. The largest increase in cytotoxicity was noted in HEK293 cells, when 100 micromol zoledronic acid was added to 4 microg/ml vinblastine (an increment of 80.3%, P < 0.001). This preliminary work shows that zoledronic acid acts synergistically with vinblastine to induce apoptosis in an MRP-1 dependent way.     

The therapeutic effect of silibinin against 5-fluorouracil-induced ovarian toxicity in rats

5-Fluorouracil (5-FU) is a fluoropyrimidine group antineoplastic drug with antimetabolite properties and ovotoxicity is one of the most important side effects. Silibinin (SLB) is a natural compound that is used worldwide and stands out with its antioxidant and anti-inflammatory properties. The aim of this study was to evaluate the therapeutic effect of SLB in 5-FU-induced ovototoxicity using biochemical and histological analysis. This study was carried out in five main groups containing six rats in each group: control, SLB (5 mg/kg), 5-FU (100 mg/kg), 5-FU + SLB (2.5 mg/kg), and 5-FU + SLB (5 mg/kg). The levels of ovarian malondialdehyde (MDA), total oxidant status (TOS), total antioxidant status (TAS), superoxide dismutase (SOD), catalase (CAT), 8-hydroxy-2′-deoxyguanosine (8-OHdG), tumor necrosis factor-alpha (TNF-α), myeloperoxidase (MPO), and caspase-3 were determined using spectrophotometric methods. Hematoxylin and eosin staining method was employed for histopathological examination. MDA, TOS, 8-OHdG, TNF-α, MPO, and caspase-3 levels in 5-FU group were significantly increased compared with the control group, while the levels of TAS, SOD, and CAT were decreased (p < 0.05). SLB treatments statistically significantly restored this damage in a dose-dependent manner (p < 0.05). Although vascular congestion, edema, hemorrhage, follicular degeneration, and leukocyte infiltration were significantly higher in the 5-FU group compared with the control group, SLB treatments also statistically significantly restored these damages (p < 0.05). In conclusion, SLB has a therapeutic effect on the ovarian damage induced by 5-FU via decreasing the levels of oxidative stress, inflammation, and apoptosis. It may be helpful to consider the usefulness of SLB as an adjuvant therapy to counteract the side effects of chemotherapy.