Hydrocortisone stimulates fibronectin synthesis in cultured fibroblasts

The effect of hydrocortisone on fibronectin synthesis was investigated in cultured skin fibroblasis. Confluent cells were treated with hydrocortisone (10^?7 M to 10^?5 M) for 2 days and labeled with [³H]proline for 24 h. Fibronectin levels in both the culture medium and the cell layer were studied by gelatin-Sepharose affinity chromatography and SDS-polyacrylamide gel electrophoresis. In control cultures of human fetal skin fibroblasts, fibronectin constituted 8% of the total labeled proteins in the medium. The proportion of fibronectin increased to 13.1% at 10^?7 M hydrocortisone, 15.5% at 10^?6 M and to 19.4% at 10^?5 M. The proportion of fibronectin associated with the cell layer remained at 2-3% of total [³H]prolne-labeled proteins and did not increase with hydrocortisone exposure. The stimulating effect of hydrocortisone on medium fibronectin was also demonstrated in cultured human newborn foreskin fibroblasts and in rabbit skin fibroblasts.     

Drug in Adhesive Patch of Zolmitriptan: Formulation and In vitro /In vivo Correlation

The objective of the present study was to develop transdermal patch for zolmitriptan, determine its in vivo absorption using the rabbit skin. Solvent evaporation technique prepared zolmitriptan patch was settled in two-chamber diffusion cell combined with excised rabbit abdomen skin for permeation study. A sufficient cumulative penetration amount of zolmitriptan (258.5 ± 26.9 μg/cm² in 24 h) was achieved by the formulation of 4% zolmitriptan, 10% Azone, and adhesive of DURO-TAK® 87–4098. Pharmacokinetic parameters were determined via i.v. and transdermal administrations using animal model of rabbit. The results revealed that the absolute bioavailability was about 63%. Zolmitriptan could be detected with drug level of 88 ± 51 ng/mL after transdermal administration of 15 min. The in vivo absorption curve obtained by deconvolution approach using WinNonlin® program was correlated well with the in vitro permeation curve, the correlation coefficient R is 0.84, and the result indicated that in vitro skin permeation experiments were useful to predict the in vivo performance. In addition, little skin irritation was found in the irritation study. As a conclusion, the optimized zolmitriptan transdermal patches could effectively deliver adequate drug into systemic circulation in short time without producing any irritation phenomenon and worth to be developed.KEY WORDS: chemical enhancer, drug-in-adhesive patch, in vitro/in vivo correlation, pharmacokinetic, zolmitriptan     

Immunoprecipitation of a male-specific polypeptide after in vitro translation of testicular poly(A)+ RNA

Summary Poly(A)⁺ RNAs from male and female gonads of 20-day-old rats were translated in vitro using rabbit reticulocyte lysates. The resulting polypeptides were incubated with specific anti-male (anti-H-Y) antisera raisedin female rats by intrasplenic immunization with syngeneic male skin. Using a second antibody, a male-specific polypeptide of molecular weight approximately 18,000 was immunoprecipitated. This male-specific polypeptide was not cotranslationally modified in vitro and appeared to be identical to serological H-Y antigen.     

Synthesis of diphtheria toxin in E. coli cell-free lysate

An $\text{E. coli}$ cell-free lysate was used to translate $\text{C. diphtheriae}$ RNA from nontoxinogenic C₇(?), C₇ infected with β tox⁺ corynebacteriophage, and $\text{C. diphtheriae}$ strain PW8. De novo synthesis of toxin was detected by immune precipitation with antitoxin, ADP-ribosylation of mammalian elongation factor 2 and rabbit skin test. The results indicated that toxin is produced in the $\text{E. coli}$ protein synthesizing system primed with RNA from cells infected with tox⁺ bacteriophage and is absent in systems primed with RNA from C₇(?) cells.     

Effect of Backing Films on the Transdermal Delivery of Donepezil from Patches

The aim of this work was to investigate the effect of backing films on transdermal delivery of donepezil (DP) from patches. Three backing films, Cotran^TM 9700, Cotran^TM 9701, and Cotran^TM 9726 were chosen as backing layers to prepare transdermal patches containing DP. The transdermal penetration and release amount of DP from each patch were evaluated by rabbit abdominal skin in vitro. The partitioning experiments and attentuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy were performed to confirm the existence of interaction between backing films and DP. Results showed that the cumulative release amount of DP from patches with different backing films had the same order of cumulative amount penetrated, i.e. Cotran^TM 9701 < Cotran^TM 9700 < Cotran^TM 9726, which demonstrated that the permeation of DP was mainly limited by release behavior. Partitioning experiments and ATR-FTIR study indicated that Cotran^TM 9700 and Cotran^TM 9701 had interaction with DP by H bond formation which decreased the release of drug from the patches. By contrast, Cotran^TM 9726 could provide the highest flux of skin permeation of DP, because such interaction between them was not found. Moreover, the parameters of backing films were found to have relation to skin hydration, thus affecting the penetration behavior of DP from patches. In conclusion, the effect of backing films on the flux of DP permeation could be attributed to both the interaction of backing films and the changes of skin hydration. Backing films could be a key factor in formulation screening of DP patches.KEY WORDS: ATR-FTIR, backing films, donepezil, release, transdermal patches     

Phosphorylation of myosin light chain by protease activated kinase I

Protease activated kinase I from rabbit reticulocytes has been shown to phosphorylate the P-light chain of myosin light chains isolated from rabbit skeletal muscle. The enzyme is not activated by Ca²⁺ and calmodulin or phospholipids. Protease activated kinase I is not inhibited by trifluoperazine at concentrations up to 200 μM or by the antibody to the Ca²⁺, calmodulin-dependent myosin light chain kinase from rabbit skeletal muscle. Two-dimensional peptide mapping of chymotryptic digests of myosin P-light chain show the site phosphorylated by the protease activated kinase is different from that phosphorylated by the Ca²⁺, calmodulin-dependent myosin light chain kinase.     

Subinhibitory concentrations of organic ammonium salts: The effect on biological properties ofSalmonella typhimurium

The effect of subinhibitory concentrations (subMICs) of new organic ammonium salts of four homologous series of alkylammonium bromides (32 compounds) was determined with respect to the induction of lysogenic strain prophage, influence of permeability reactions in a rabbit skin test and cytotoxic changes of monolayers of Vero cells. The culture filtrates were prepared by 1-d cultivation ofSalmonella typhimurium in a synthetic culture medium under conditions of intensive aeration at 37°C after addition of subinhibitory concentrations of organic ammonium salts. The results showed that substances of the homologous series of 2-(10-undecenoyloxy)ethyl-alkyldimethylammonium bromides were characterized by a prophage-inducing effect in lysogenic strain cells. The induction of prophage raised with rising concentrations of subMICs of the substances, and its titer in the culture filtrates was mostly 4.10⁶ PFU/mL. SubstancesC3, C9 andC12 of the same homologous series had the strongest effect on the permeability reaction in rabbit skin in 1/2 MICs. One-half MICs of four substances (B14, C3, C12, C14) and 1/4 MICs of substanceA16 influenced cytotoxic changes on Vero cells, the other substances were ineffective.     

Chemical composition and immunological properties of glycoproteins of Sporothrix species

Glycoproteins of 11Sporothrix species were purified from their respective culture filtrates by use of DEAE-Sephadex A-50 and QAE-Sephadex A-25 column chromatography and investigated for their chemical and immunological properties. On the basis of sugar composition, the glycoproteins of the 11Sporothrix species could be divided into two groups, i.e., rhamnose containing (i.e., Rha⁺), and non rhamnose containing (i.e., Rha^–) groups. The species in the former group wereS. curviconia, S. inflata, S. schenckii andS. schenckii var. luriei, and those in the latter group wereS. cyanescens, S. foliorum, S. fungorum, S. ghanensis, S. imectorum, S. luteoalba andS. ramosissima. The glycoproteins of four of the (Rha⁺) species were relatively similar in elution patterns of DEAE-Sephadex A-50 chromatograms, sugar and amino acid compositions, serological reactivity with rabbit andS. schenckii serum and rabbit antiKlebsiella pneumoniae K47 serum, and cutaneous delayed hypersensitivity. In the case of the (Rha^–) species, the glycoproteins of five species cross-reacted with rabbit antiS. schenckii serum and all, but theS. cyanescens, glycoprotein were reactive to some degree in skin tests in sporotrichotic patients. These results strongly suggest that the chemical and immunological properties of these glycoproteins correspond with the morphological observations amongSporothrix species.     

Experimental infection of wild‐caught European rabbits (Oryctolagus cuniculus) with Sarcoptes scabiei from a naturally infected wild rabbit

Scabies was recently reported for the first time in the European wild rabbit, Oryctolagus cuniculus (Lagomorpha: Leporidae). We experimentally exposed 10 seronegative wild‐caught rabbits to skin from a mangy wild rabbit. Serological, physiological, parasitological and histopathological changes were recorded. Three rabbits developed antibodies at 2–5 weeks post‐infection (w.p.i.), two of which then developed lesions at 7 w.p.i. One of these had a small area of alopecia on the hind limb that healed naturally within 1 week; the other developed more extensive lesions restricted to the hind limbs (as typically observed in wild rabbits) that lasted until the rabbit died (12.5 w.p.i.). The third rabbit died of trauma 5 w.p.i. before developing any lesions. Antibodies in the healed rabbit disappeared from serum at 8 w.p.i., whereas antibody levels in the sick rabbit increased until its death. Disseminated intravascular coagulation and hepatic necrosis, probably arising from a concomitant infection with rabbit haemorrhagic disease virus, were the likely final cause of death in this rabbit. The mangy rabbit that served as a donor died of a multifocal fibrinosuppurative pneumonia that may have been secondary to the skin bacterial pyoderma.     

Amiloride is a cholinergic antagonist in the rabbit pancreas

The effect of amiloride on fluid and protein secretion in the isolated rabbit pancreas and on amylase secretion in rabbit pancreatic acini has been studied. Amiloride (1 mM) has no effect on the pancreatic fluid secretion either in a normal incubation medium (143 mM Na⁺), or in a medium containing only 25 mM Na⁺. The carbachol-induced enzyme secretion is inhibited by amiloride in both systems, whereas the enzyme secretion induced by the C-terminal octapeptide of cholecystokinin (PzO) is not affected. Amiloride also inhibits the carbachol-induced ⁴⁵Ca efflux from rabbit pancreatic acini, but again not that induced by PzO. The amiloride concentrations for half-maximal inhibition of carbachol-induced amylase secretion and ⁴⁵Ca efflux are 40 and 80 μM, respectively. Amiloride also competitively inhibits the specific binding of [³H]quinuclidinyl benzylate ([³H]QNB) to rabbit pancreatic acini, suggesting that the amiloride effect is due to competition on the level of the muscarinic acetylcholine receptor.     

Eradication of rabbits from landscape scale exclosures: pipedream or possibility?

Summary High densities of the European Rabbit (Oryctolagus cuniculus) were eradicated from 60 km² of the Arid Recovery Reserve between 1996 and 2001. Eradication was possible due to an initial knockdown caused by rabbit haemorrhagic disease, followed by effective exclusion fencing, broadscale poison baiting, targeted shooting, warren destruction and trapping on rabbit burrows and buckheaps. The efficacy of different broadscale control and intensive rabbit eradication techniques was subsequently estimated in a 26 km² expansion to the Reserve from 2002 to 2006. Non‐target implications of these control techniques were also assessed where possible. An estimated 8000 rabbits were removed in total from both areas and results suggest that rabbit eradication is possible at a landscape scale. Strategies for eliminating rabbits from confined areas are suggested.     

Cartilage-derived factor (CDF) : II. Somatomedin-like action on cultured chondrocytes

Previously, we showed that fetal bovine cartilage contains a polypeptide that stimulates the incorporation of [³⁵S]sulfate into proteoglycans synthesized by rat and rabbit costal chondrocytes in culture. In this paper, we report that the cartilage-derived factor (CDF) increases not only [³⁵S]sulfate incorporation but also [³H]thymidine incorporation into rabbit chondrocytes in monolayer culture. The dose-response curve of CDF stimulation of DNA synthesis was similar in profile to that of CDF stimulation of proteoglycan synthesis. In addition, CDF markedly enhanced [³H]uridine incorporation into rabbit chondrocytes and significantly enhanced [³H]serine incorporation into total protein. These findings indicate that fetal bovine cartilage contains a factor that shows somatomedin-like activity in monolayer cultures of rabbit chondrocytes.     

Control of hyperglycaemia in diabetic rabbits by a combination of implants

Insulin demand varies with meal intake and physical activity. In this study the feasibility of using two implants to meet varying insulin demands was tested in rabbits with alloxan-induced diabetes. One group of severely diabetic rabbits was maintained on a basal dose released by a 50-mg implant made of a compressed admixture of 15% insulin in palmitic acid. The other group of mildly diabetic rabbits required no basal dose implant, but displayed a transient hyperglycaemia as well upon challenge. The supplemental dose was provided by another silicone implant with reservoirs containing 6 mg of compressed insulin. Serous fluid entered the 100μ l internal volume of the silicone implant slowly through an orifice, and dissolved some of the solid insulin. When required, sideways compression of this second implant over the abdominal skin fold of the rabbit delivered the supplemental dose. Typically, a severely diabetic rabbit on a basal dose implant exhibited a transient hyperglycaemia after drinking sweetened water, which raised the blood glucose from 5.4 ± 1.3 mmol l⁻¹ to 14.0 ± 0.5 mmol l⁻¹ for 3 to 4.5 h. In the three test runs, the supplemental bolus of insulin from the silicone implant interrupted the expected rise in blood glucose at 6.1 ± 2.2 mmol l⁻¹ within 1 to 2 h, which then decreased to 3.0 ± 0.2 mmol l⁻¹ for 4 to 5 h before returning to the basal level. A mildly diabetic rabbit showed a blood glucose level of 10.5 ± 1.9 mmol l⁻¹ without the basal dose implant. Its expected transient hyperglycaemia rise to 13.1 ± 0.3 mmol l⁻¹ could also be prevented by the supplemental insulin dose from the silicone implant, and kept at 2.5 ± 0.3 mmol l⁻¹ for 1 to 1.5 h, before returning to the mildly diabetic level in 8 to 9 h. The results demonstrated the feasibility of using an erodible implant to provide a basal insulin dose which could be supplemented by a second implant for better control of transient hyperglycaemia episodes.     

Characterization of a voltage-dependent L-type calcium channel from rabbit and turtle brain

The binding of [³H]nitrendipine to membrane preparation from turtle and rabbit brain was studied. A single population of [³H]nitrendipine binding sites was detected in both species. [³H]nitrendipine bound with high affinity to brain membrane from both rabbit and turtle, revealing a significant population of binding sites (K _d values of 0.55±0.05 nM and 0.56±0.04 nM and B_max values of 122±11 and 275±18 fmol/mg of protein, respectively). Displacement studies showed a similar order of potency of various unlabeled ligands against [³H]nitrendipine both in rabbit or in turtle: nitrendipine > nifedipine ≥ nicardipine ≫ verapamil ≥ diltiazem. Our results show that a two fold increment of [³H]nitrendipine binding sites exists in the turtle brain respect to the rabbit.     

Amino-acid Sequence Homology in the Muscle Aldolases from Sturgeons of Different Species

STUDIES on the primary structure of aldolases isolated from ox, pig and rabbit muscle show that the amino-acid sequence of fructose 1,6-diphosphate aldolase [EC 4.1.2.13] has been highly conserved throughout mammalian evolution¹. But comparison of the primary structure of the enzyme from these species with that from the muscle of a single North Sea sturgeon, presumably Acipenser sturio, indicated that although the proteins were homologous, a number of amino-acid replacements occurred between sturgeon aldolase and the aldolases of the phylogenetically distant mammalian species¹. As a knowledge of the nature and number of amino-acid replacements between homologous proteins caft provide information both about the functional role of individual residues and about evolution, further comparative studies of rabbit and sturgeon aldolases were undertaken and an account of the sequence homology around the active-site-lysine residue of aldolases from rabbit muscle, rabbit liver and the muscle of the river sturgeon of Eastern Canada, Acipenser fulvescens, has been given^2,3.     

Design and Evaluation of a Novel Felbinac Transdermal Patch: Combining Ion-Pair and Chemical Enhancer Strategy

The aim of this study was to design a novel felbinac (FEL) patch with significantly higher (P?<?0.05) skin permeation amount than the commercial product SELTOUCH® using ion-pair and chemical enhancer strategy, overcoming the disadvantage of the large application area of SELTOUCH®. Six complexes of FEL with organic amines diethylamine (DEA), triethylamine (TEA), N-(2′-hydroxy-ethanol)-piperdine (HEPP), monoethanolamine (MEtA), diethanolamine (DEtA), and triethanolamine (TEtA) were prepared by ion-pair interaction, and their formation were confirmed by differential scanning calorimetry (DSC), powder X-ray diffraction (pXRD), infared spectroscopy (IR), and proton nuclear magnetic resonance spectroscopy (¹H-NMR). Subsequently, the effect of ion-pair complexes and chemical enhancers were investigated through in vitro and in vivo experiments using rabbit abdominal skin. Results showed that FEL-TEA was the most potential candidate both in isopropyl palmitate (IPP) solution and transdermal patches. Combining use of 10% N-dodecylazepan-2-one (Azone), the optimized FEL-TEA patch achieved a flux of 18.29?±?2.59 μg/cm²/h, which was twice the amount of the product SELTOUCH® (J?=?9.18?±?1.26 μg/cm²/h). Similarly, the area under the concentration curve from time 0 to time t (AUC_0-t ) in FEL-TEA patch group (15.94?±?3.58 h.μg/mL) was also twice as that in SELTOUCH® group (7.31?±?1.16 h.μg/mL). Furthermore, the in vitro skin permeation results of FEL-TEA patch was found to have a good correlation with the in vivo absorption results in rabbit. These findings indicated that a combination of ion-pair and chemical enhancer strategy could be useful in developing a novel transdermal patch of FEL.     

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches¹. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible¹. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization¹. The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart². It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling³. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart^4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy^8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view^10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia^14-16.     

Prostaglandin Biosynthesis from Endogenous Precursors in Rabbit Kidney

THIS report describes the biosynthesis of the naturally occurring renal prostaglandins E₂ (PGE₂) and F_2α (PGF_2α)^1,2 by homogenates and slices of rabbit renal medulla, from endogenous precursors. I have confirmed that rabbit renal cortex contains little prostaglandin and cannot synthesize them from endogenous lipids³. Hamberg has reported that arachidonic acid, which is converted to PGE₂ and PGF_2α by enzymes present in ram seminal vesicles⁴, can be efficiently converted to PGE₂ and PGF_2α by homogenates of rabbit renal medulla³. I have now confirmed that arachidonic acid, added to such medullary homogenates, can increase the quantities of prostaglandins synthesized. There was no evidence that the major prostaglandin biosynthesized, PGE2, was further metabolized to inactive products.