Effects of intraplantar injections of nociceptin and its N-terminal fragments on nociceptive and desensitized responses induced by capsaicin in mice

Injection of capsaicin into the hindpaw has been employed as a model of chemogenic nociception in mice. Intraplantar injection of nociceptin (30–240 pmol) produced a significant and dose-dependent antinociceptive activity in the capsaicin test. The nociceptin N-terminal fragments, (1–11) and (1–13), were also active with a potency higher than nociceptin and comparable to nociceptin, respectively. Intraplantar injection of the nociceptin (1–7) fragment had no effect on capsaicin-induced nociception. Antinociception induced by nociceptin or nociceptin (1–13) was reversed significantly by intraplantar co-injection of [Nphe¹]nociceptin (1–13)NH₂, an orphan opioid receptor-like 1 (ORL1) receptor antagonist, whereas local injection of the antagonist did not interfere with the action of nociceptin (1–11). Nociceptin (1–11) was approximately 2.0-fold more potent than naturally occurring peptide nociceptin, and 10-fold more active than intraplantar morphine. Nociceptive licking/biting response to intraplantar injection of capsaicin was desensitized by repeated injections of capsaicin at the interval of 15 min. Desensitization induced by capsaicin was attenuated significantly by co-injection of nociceptin at much lower doses than antinociceptive ED₅₀ for nociceptin. Capsaicin desensitization was also decreased by co-injection of nociceptin (1–11) and (1–13) to a similar extent. The present results indicate that not only nociceptin but also the N-terminal fragment (1–13) possesses a local peripheral antinociceptive action, which may be mediated by peripheral ORL1 receptors. In addition, the difference of the effective doses suggests that the antinociceptive action and inhibition of capsaicin-induced desenitization by nociceptin, nociceptin (1–11) and (1–13), may involve distinct mechanisms at the level of the peripheral nerve terminal.     

Cryopreserved Morulae can be used to Efficiently Generate Germline-transmitting Chimeras by Blastocyst Injection

The production of chimeric mice is a complex process, requiring the careful coordination of tissue culture cell growth, production of a large number (30–75) of competent blastocysts and the availability of appropriately timed pseudo pregnant female mice. Failure at any of these steps can impinge upon the rapid production of chimeras. One potential improvement for the efficient generation of chimeric mice would be the utilization of cryopreserved embryos suitable for injection. C57Bl/6 morulae were frozen using a standard 2-step protocol with ethylene glycol as the cryopreservation agent. We determined that cryopreserved morulae could thaw, culture to blastocyst stage in KSOM media and survive injection at rates equivalent to control embryos. Cryopreserved morulae were also equivalent to controls at all later stages in the process of production of chimeric mice, including birth rate, percentage chimerism of resulting animals and ability to produce germline progeny. Hence, cryopreservation of morulae for blastocyst injection is a suitable option to enhance the efficiency of chimeric mouse generation.     

Numerical simulation of selective freezing of target biological tissues following injection of solutions with specific thermal properties

Recently, we proposed a method for controlling the extent of freezing during cryosurgery by percutaneously injecting some solutions with particular thermal properties into the target tissues. In order to better understand the mechanism of the enhancement of freezing by these injections, a new numerical algorithm was developed to simulate the corresponding heat transfer process that is involved. The three-dimensional phase change processes in biological tissues subjected to cryoprobe freezing, with or without injection, were compared numerically. Two specific cases were investigated to illustrate the selective freezing method: the injection of solutions with high thermal conductivity; the injection of solutions with low latent heat. It was found that the localized injection of such solutions could significantly enhance the freezing effect and decrease the lowest temperature in the target tissues. The result also suggests that the injection of these solutions may be a feasible and flexible way to control the size of the ice ball and its direction of growth during cryosurgery, which will help to optimize the treatment process.     

Electro-transfer of small interfering RNA ameliorated arthritis in rats

RNA interference provides the powerful means of sequence-specific gene silencing. Particularly, small interfering RNA (siRNA) duplexes may be potentially useful for therapeutic molecular targeting of human diseases, although novel delivery systems should be devised to achieve efficient and organ-specific transduction of siRNA. In the present study, we demonstrated that electro-transfer of a siRNA-polyamine complex made efficient and specific gene knockdown possible in the articular synovium. Targeted suppression of the tumor necrosis factor-alpha gene through this procedure significantly ameliorated collagen-induced arthritis in rats. Our results suggest the potential feasibility of therapeutic intervention with RNA medicines for treatment of rheumatoid and other locomotor diseases.     

Determination of the refractive index increment (dn/dc) of molecule and macromolecule solutions by surface plasmon resonance

An automated method for dn/dc determination using a surface plasmon resonance instrument in tandem with a flow injection gradient system (FIG-SPR) is proposed. dn/dc determinations of small molecule and biomolecule, surfactant, polymer, and biopolymer solutions with precision around 1-2% and good accuracy were performed using the new method. dn/dc measurements were also carried out manually on a conventional SPR equipment with similar accuracy and precision. The FIG-SPR instrument is inexpensive and could be easily coupled to commercially available SPR and liquid chromatography instruments to obtain several properties of the solutions, which are based on measurements of refractive index.     

Study and application of flow injection spectrofluorimetry with a fluorescent probe of 2-(2-pyridil)-benzothiazoline for superoxide anion radicals

This paper presents an automatic spectrofluorimetric method (flow injection spectrofluorimetry) using a novel fluorescent probe named H. Py. Bzt (2-(2-pyridil)-benzothiazoline) for determining superoxide dismutase (SOD) activity. The fluorescent probe was synthesized in house and fully characterized by elemental analysis and by infrared and (1)H nuclear magnetic resonance spectra. It could specially identify and trap O(2)(*-) and was oxidized by O(2)(*-) to form a strong fluorescence product. Based on this reaction, the flow injection spectrofluorimetric method was proposed and successfully used to determine SOD activity. The proposed method has a better selectivity in the determination of reactive oxygen species because the probe can be oxidized only by O(2)(*-) excluding H(2)O(2). As a kind of simple, rapid, precise, sensitive and automatic technique, it was applied to measurement of SOD activity in scallion, garlic, and onion with satisfactory results.     

Determination of the DNA-binding characteristics of ethidium bromide, proflavine, and cisplatin by flow injection analysis: usefulness in studies on antitumor drugs

Flow injection analysis was used to study the reactions occurring between DNA and certain compounds that bind to its double helix, deforming this and even breaking it, such that some of them (e.g., cisplatin) are endowed with antitumoral activity. Use of this technique in the merging zones and stopped-flow modes afforded data on the binding parameters and the kinetic characteristics of the process. The first compound studied was ethidium bromide (EtdBr), used as a fluorescent marker because its fluorescence is enhanced when it binds to DNA. The DNA-EtdBr binding parameters, the apparent intrinsic binding constant (0.31+/-0.02 microM(-1)), and the maximum number of binding sites per nucleotide (0.327+/-0.009) were determined. The modification introduced in these parameters by the presence of proflavine (Prf), a classic competitive inhibitor of the binding of EtdBr to the DNA double helix, was also studied, determining the value of the intrinsic binding constant of Prf (K(Prf) = 0.119+/-9x10(-3) microM(-1)). Finally, we determined the binding parameters between DNA and EtdBr in the presence of the antitumor agent cisplatin, a noncompetitive inhibitor of such binding. This provided information about the binding mechanism as well as the duration and activity of the binding of the compound in its pharmacological use.     

The dynamic distribution of fluoro-gold and its interrelation with neural nitric oxide synthase following intracerebroventricular injection into rat brain

We mapped the dynamic distribution of fluoro-gold (FG) within rat brain following intracerebroventricular (icv) injection into the lateral ventricle and observed its interrelation with neural nitric oxide synthase (nNOS) using FG fluorescent microphotography combined with nNOS immunohistochemistry. We also detected the amount of icv administered FG entering the peripheral circulation using a fluorescence microplate assay. The degree of periventricular penetration of FG was significantly increased over time. At 2 min after icv injection, FG primarily labeled the choroid plexus in the lateral and third ventricles, with limited penetration into the ependyma and the subependyma of the same ventricles. Some FG/nNOS-double labeled cerebrospinal fluid-contacting neurons were observed in these ventricles as well. At 15 and 30 min, FG penetrated mainly into forebrain ventricular organs and parenchymal structures. Many FG/nNOS double labeled neurons were found at each of these sites. In addition, at 30 min intense FG labeling was found in the hypophysis, while limited periventricular penetration of FG was detected in the hindbrain circumventricular areas. In the peripheral circulation, a low concentration of FG was detected 2 min after icv injection. The concentration increased slowly, peaked at 20 min, then gradually decreased until the end of the experiment at 30 min. These findings indicate that dynamic penetration of icv administrated agents into the periventricular tissues and peripheral circulation should be considered when designing icv experiments.     

Visual marker and Agrobacterium-delivered recombinase enable the manipulation of the plastid genome in greenhouse-grown tobacco plants

Successful manipulation of the plastid genome (ptDNA) has been carried out so far only in tissue-culture cells, a limitation that prevents plastid transformation being applied in major agronomic crops. Our objective is to develop a tissue-culture independent protocol that enables manipulation of plastid genomes directly in plants to yield genetically stable seed progeny. We report that in planta excision of a plastid aurea bar gene (bar(au) ) is detectable in greenhouse-grown plants by restoration of the green pigmentation in tobacco leaves. The P1 phage Cre or PhiC31 phage Int site-specific recombinase was delivered on the Agrobacterium T-DNA injected at the axillary bud site, resulting in the excision of the target-site flanked marker gene. Differentiation of new apical meristems was forced by decapitating the plants above the injection site. The new shoot apex that differentiated at the injection site contained bar(au)-free plastids in 30-40% of the injected plants, of which 7% transmitted the bar(au)-free plastids to the seed progeny. The success of obtaining seed with bar(au)-free plastids depended on repeatedly forcing shoot development from axillary buds, a process that was guided by the size and position of green sectors in the leaves. The success of in planta plastid marker excision proved that manipulation of the plastid genomes is feasible within an intact plant. Extension of the protocol to in planta plastid transformation depends on the development of new protocols for the delivery of transforming DNA encoding visual markers.