Antinociceptive effect of the novel compound OT-7100 in a diabetic neuropathy model.

We previously reported that OT-7100 (5-n-butyl-7-(3,4,5-trimethoxybenzoylamino)pyrazolo[1,5-alpha]pyrimidine) had antinociceptive potency in various animal models. To further characterize this compound, the present study examined the effects of OT-7100 on mechanical hyperalgesia and motor nerve conduction velocity in streptozotocin-induced diabetic rats. OT-7100 significantly increased the nociceptive threshold in the diabetic rat in a dose-dependent manner. Gabapentin (anticonvulsant agent) and insulin strongly increased the nociceptive threshold but gabapentin increased it above normal levels. An aldose reductase inhibitor slightly increased the nociceptive threshold at a high dose. We also measured glucose levels and motor nerve conduction velocity in OT-7100-treated rats. Insulin decreased glucose levels but OT-7100 had no effect on glucose levels or on motor nerve conduction velocity. These results suggest that OT-7100 alleviates hyperalgesia in a diabetic neuropathy model in a different manner from gabapentin or aldose reductase inhibitor and may be a new treatment for the pain associated with peripheral nerve injury.

FR901469, a novel antifungal antibiotic from an unidentified fungus No.11243. II. In vitro and in vivo activities.

FR901469 is a water-soluble macrocyclic lipopeptidolactone (C71H116N14O23) that has inhibitory activity against 1,3-beta-glucan synthase and exhibits in vitro and in vivo antifungal activity against both Candida albicans and Aspergillus fumigatus. The MICs of FR901469 against Candida albicans FP633 and Aspergillus fumigatus FP1305 in a micro-broth dilution test were 0.63 and 0.16 microg/ml, respectively. FR901469 showed excellent efficacy by subcutaneous injection against both Candida albicans and Aspergillus fumigatus in a murine systemic infection mode, with ED50s of 0.32 and 0.2 mg/kg, respectively. This compound also showed potent anti-Pneumocystis activity in the nude mice model with experimental Pneumocystis pneumonia. The hemolytic activity of FR901469 towards mouse red blood cells, is about 30-fold weaker than that of amphotericin B.

Transport of magnesium by two isoforms of the Na+-Ca2+ exchanger expressed in CCL39 fibroblasts.

Cytoplasmic concentrations of Ca2+ ([Ca2+]i) and Mg2+ ([Mg2+]i) were measured with fluorescent indicators in CCL39 cells, a cell line established from Chinese hamster lung fibroblasts, transfected with complementary deoxyribonucleic acid (cDNA) of the Na+-Ca2+ exchanger isolated either from canine heart (NCX1) or from rat brain (NCX3). Raising extracellular [Mg2+] to 10 mM increased Mg2+ influx and the resultant change in [Mg2+]i (delta[Mg2+]i) was monitored with furaptra under Ca2+-free conditions. In control (vector-transfected) cells, delta[Mg2+]i at 45 min was similar with or without extracellular Na+ (130 mM or 0 mM) and when [Na+]i was raised by 1 mM ouabain treatment. delta[Mg2+]i in NCX1-transfected cells was attenuated significantly in the presence of 130 mM Na+, but became comparable to (or slightly larger than) that in control cells on either removal of extracellular Na+ or treatment with 1 mM ouabain. Cells expressing NCX3 showed an intermediate dependence of delta[Mg2+]i on Na+, probably reflecting a lower degree of expression of the exchanger protein. Extracellular Na+-dependent changes in [Ca2+]i (measured with fura-2 in the presence of extracellular Ca2+ and 10 microM ionomycin, a Ca2+ ionophore) were minimal in control cells, marked in the NCX1-transfected cells and intermediate in the NCX3-transfected cells. These results suggest that the Na+-Ca2+ exchanger (either NCX1 or NCX3) can transport Mg2+ and may play a role in the extrusion of magnesium from cells.

Effect of testosterone on the number of NADPH diaphorase-stained nerve fibers in the rat corpus cavernosum and dorsal nerve.

OBJECTIVES: To elucidate the effect of testosterone on penile nerve supply. METHODS: Three groups of 10 rats each were assessed; two groups were castrated and the third underwent a sham operation (control). After castration, one group received subcutaneous injection of testosterone while the others received sesame oil. At 8 weeks, the rats underwent a functional analysis. The evaluation included a subcutaneous injection with apomorphine to study centrally mediated erection, and cavernous nerve electrostimulation and papaverine injection to study peripherally mediated erection. At death, a penile midshaft specimen was taken for nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase staining. RESULTS: In the apomorphine study, castrated rats had no erections, but the erectile function of those receiving testosterone was restored to the level of the controls. The mean numbers of NADPH-diaphorase-stained nerve fibers in the copora cavernosa and both dorsal nerves of castrated rats, at 165.8 +/- 20.0 and 271.3 +/- 21.1, respectively, were significantly lower than those of the controls, at 271.7 +/- 14.6 and 471.2 +/- 27.6, respectively. Those of the testosterone replacement group, at 290.7 +/- 10.1 and 500.7 +/- 23.9, respectively, recovered to the control level. The intracavernosal pressure decreased significantly in the absence of testosterone, both after electrostimulation and intracavernosal papaverine injection, and recovered to the control level after testosterone replacement. CONCLUSIONS: Our results indicate that testosterone acts on the nervous system to mediate erection. When it is absent, there may be downregulation of both the production and activity of nitric oxide (NO), thereby decreasing the response to peripheral stimulation via the NO pathway. Testosterone replacement may upregulate NO activity to the control level.

Delayed testosterone replacement restores nitric oxide synthase-containing nerve fibres and the erectile response in rat penis.

OBJECTIVE: To elucidate the effect of testosterone on penile innervation. Materials and methods Three groups of six rats each were assessed; two groups (1 and 2) were castrated and the third (group 3) underwent a sham operation (control). Eight weeks after castration, group 2 received a subcutaneous injection with testosterone. At 8 weeks, the rats in group 1 and 3 underwent a final functional analysis while those in group 2 did so at 12 weeks. The evaluation included a subcutaneous injection with apomorphine to study centrally mediated erection, and cavernosal nerve electrostimulation and papaverine injection to study peripherally mediated erection. At death a penile mid-shaft specimen was taken for NADPH-diaphorase staining. RESULTS: In the apomorphine study, castration resulted in significantly fewer yawns and erections than in the control, and those in group 2 significantly better central erectile function than in the controls. The mean (SEM) number of nitric oxide synthase (NOS)-containing nerve fibres in the corpora cavernosa and both dorsal nerves of castrated rats, at 46.2 (9.1) and 203 (32.1), respectively, were significantly lower than in rats in group 2, at 84.1 (11.2) and 300.6 (17.1), and than in the controls, at 88.6 (10.9) and 306.3 (22.9), respectively. The intracavernosal pressure decreased significantly in the absence of testosterone, both after electrostimulation and intracavernosal papaverine injection. However, there was no difference between the control and group 2 rats in either the number of NOS-containing nerve fibres or in the peripheral erectile functional study. CONCLUSIONS: Testosterone acts on the nervous system to mediate erection; when it is absent there may be down-regulation of both the production and activity of NO, thereby decreasing the response to peripheral stimulation via the NO pathway. The restoration of erectile function seen in rats in group 2 supports this phenomenon. Delayed testosterone replacement has no detrimental effect on the restoration of the erectile mechanism after castration.

Synthetic mutants of Clostridium pasteurianum ferredoxin: open iron sites and testing carboxylate coordination.

The entire polypeptide chains for two new Clostridium pasteurianum ferredoxin (Fd) mutants were prepared with the following site-specific substitutions: Cys11Asp and Cys11 alpha-aminobutyric acid (Cys11 alpha-Aba), the latter being a non-naturally occurring amino acid. Standard t-Boc procedures were used for the synthesis and the peptides. The two apoproteins were reconstituted to the 2[4Fe-4S] holoprotein and their spectroscopic, redox and thermal properties were compared with those of native C.pasteurianum Fds. The fully reconstituted Cys11Asp and Cys11 alpha-Aba mutants were initially found to have both clusters intact, i.e. they were 2[4Fe-4S] ferredoxins. The unconventional ligands of Asp and alpha-Aba led to holo-Fds that were not very stable and easily released an iron to form the [3Fe-4S] cluster, presumably through oxidation. The Cys11 alpha-Aba mutant was somewhat more thermally stable than Cys11Asp. In contrast, while both mutants were less stable than the native protein upon exposure to oxygen, the Cys11 alpha-Aba mutant was less stable than Cys11Asp. The Cys11Gly mutant was also prepared, but all attempts, despite repeated and varied experimental conditions, at reconstitution to the Cys11Gly holo 2[4Fe-4S] Fd were unsuccessful, probably because a Gly-Gly sequence is known to break structure. This work, when compared with molecular biological site-specific mutagenesis, shows some of the advantages of chemical/in vitro reconstitution: certain mutants which cannot be detected as holoproteins by site-specific mutagenesis can be formed after all in vitro. Nonetheless, it seems apparent that altering any of the Cys coordination sites of the Fd clusters results in fundamentally more unstable ferredoxins.

Characterization of the immunoregulatory action of saikosaponin-d.

The immunoregulatory action of saikosaponin-d (SSd), which was isolated from the root of Bupleurum falcatum L. and has a steroid-like structure, was examined on splenic T lymphocytes of C57BL/6 mice. SSd displayed a definite action in vitro to bidirectionally control the growth response of T lymphocytes stimulated by concanavalin A, anti-CD3 monoclonal antibody, and calcium ionophore A23187 plus phorbol 12-myristate 13-acetate. Low concentrations (1-3 micrograms/ml) of SSd upregulated the responses to suboptimum stimuli of agonists, particularly during the relatively late stage of the responses, whereas it downregulated the responses to supraoptimal stimuli. Under appropriate experimental conditions, SSd promoted interleukin-2 (IL-2) production and IL-2 receptor expression. It also accelerated c-fos gene transcription, but it did not modulate the level of tyrosine phosphorylation of cellular proteins. We concluded from these results that SSd uniquely modulates T lymphocyte function and that at least one target of the action of SSd is located at or before the step of c-fos gene transcription and after T-cell receptor/CD3-mediated protein tyrosine kinase activation.

Effects of DS-4574, a new orally active antiallergic agent, in experimental allergic and inflammatory models.

The anti-allergic and anti-inflammatory activities of DS-4574, which possesses leukotriene antagonism and inhibits the release of immunologically stimulated mediators such as histamine and leukotrienes, were evaluated in several animal models. DS-4574 had dose-dependent inhibitory effects on IgE-mediated passive cutaneous anaphylaxis and the passive Arthus reaction in rats and the phase I response of Forssman antibody-induced bronchoconstriction. In contrast, this compound had no effect on the phase II response of Forssman antibody-induced bronchoconstriction in guinea pigs, the reverse cutaneous anaphylaxis in rats, complement-dependent hemolysis of sheep erythrocytes and the delayed-type hypersensitivity induced by methylated bovine serum albumin in mice. The results obtained in a double sensitization with two IgE antibodies suggested that DS-4574, as well as disodium cromoglycate, did not impair antigen-antibody combination but prevents the release of chemical mediators such as histamine. DS-4574 also had a weak inhibitory activity on carrageenin paw edema in rats, arachidonic acid ear edema in mice and adjuvant arthritis in rats. In addition, this compound inhibited increased vascular permeability in rat skin induced by leukotriene D4 and platelet activating factor-induced pleurisy in rats in a dose-dependent manner. These results indicate that DS-4574 inhibited type III allergic reactions and some inflammatory reactions. Therefore, DS-4574 could be useful in the treatment of allergic diseases such as asthma.

[Effects of cerebral cortical lesions on the ipsilateral thalamus].

An injury to the central nervous system causes a focal logical disturbance, and further may affect the blood flow, metabolism, and function of other brain regions. Recent studies using PET or SPECT have demonstrated that impairment of regional hemodynamics or metabolism in cerebrovascular disease involves not only the site of the lesion itself but also more remote areas. Although depression of the metabolism of the ipsilateral thalamus in patients with cerebral cortical lesions has been shown by PET study, the pathophysiological implications of this remain unclear. The functional and morphological effects of cortical infarcts on the ipsilateral thalamus were studied by assessment of cerebral blood flow using 123I-IMP SPECT and by determining atrophic changes on CT or MRI. Nine out of 17 patients with cortical infarcts showed hypoperfusion of the ipsilateral thalamus, especially patients with larger infarcts involving the frontal or parietal cortex. Thalamic hypoperfusion persisted from early after the insult to several months or even years later. In addition, atrophy of the ipsilateral thalamus was not uncommon following larger cortical infarcts. This tended to be evident about 1 year after the infarct and progressed over several years. Furthermore, atrophic changes in the thalamus was often demonstrated in such patients as hypoperfusion in the later stages. Thus, cortical lesions had functional and morphological effects on the ipsilateral thalamus ranging from early hypoperfusion to later irreversible atrophic changes.(ABSTRACT TRUNCATED AT 250 WORDS)

Myocardial infarct size-limiting effect of ischemic preconditioning was not attenuated by oxygen free-radical scavengers in the rabbit.

BACKGROUND: The limiting effect of ischemic preconditioning on infarct size has been reported in canine hearts, which contain considerable amounts of xanthine oxidase, a free radical-producing enzyme. Furthermore, a recent study suggested that free radicals generated during preconditioning may contribute to the cardioprotective effect of preconditioning. The present study examined 1) whether preconditioning limits infarct size in rabbits, which, like humans, lack myocardial xanthine oxidase and 2) whether the cardioprotective effect of PC is mediated by free radicals. METHODS AND RESULTS: A branch of the circumflex coronary artery in rabbits was occluded for 30 minutes and then reperfused for 72 hours. Myocardial infarct size and area at risk were determined by histology and fluorescent particles, respectively. Five groups were studied: an untreated control group, a preconditioned group (PC group), a high-dose superoxide dismutase (SOD)-treated preconditioned group (high-dose SOD-PC group), a low-dose SOD-treated preconditioned group (low-dose SOD-PC group), and a SOD-plus-catalase-treated preconditioned group (SOD/CAT-PC group). Preconditioning was performed with four episodes of 5 minutes of ischemia and 5 minutes of reperfusion. The free radical scavengers (30,000 units/kg SOD for high-dose SOD-PC group, 15,000 units/kg SOD for low-dose SOD-PC group, and 30,000 units/kg SOD plus 55,000 units/kg catalase for SOD/CAT-PC group) were infused intravenously over 60 minutes starting 20 minutes before preconditioning. Infarct size as the percentage of area at risk was 45.1 +/- 3.5% (mean +/- SEM) in the control group (n = 11), 13.3 +/- 3.0% in the PC group (n = 12), 9.7 +/- 1.8% in the high-dose SOD-PC group (n = 8), 11.9 +/- 2.2% in the low-dose SOD-PC group (n = 6), and 9.6 +/- 2.3% in the SOD/CAT-PC group (n = 6) (p less than 0.05 versus control for the last four values). The differences in infarct size as the percent of area at risk among the PC, high-dose SOD-PC, low-dose SOD-PC, and SOD/CAT-PC groups were not significant. CONCLUSION: Ischemic preconditioning delays ischemic myocardial necrosis regardless of myocardial xanthine oxidase content. Free radicals are unlikely to have a major role in the mechanism of the preconditioning in rabbits.

Histopathologic effects of ultrasonically induced hyperthermia in intraocular malignant melanoma.

Four cases of human intraocular malignant melanoma were treated with ultrasonically induced hyperthermia immediately before enucleation. Tumors were treated in two regimens: 30 minutes at 43 degrees to 45 degrees C and 5 minutes at greater than 50 degrees C. Temperatures were estimated from applied power levels, based on empirical data and mathematical models. Histopathologic changes observed in human tumors were compared with changes seen in malignant melanoma xenografts in athymic nude mice which were treated with ultrasonically induced hyperthermia for 30 minutes at 42 degrees to 46 degrees C. The effects of treatment were similar to changes seen in the animal model treated under analogous conditions: increased intercellular spacing, cytoplasmic vacuole formation, clumping of chromatin, breaks in cell membranes, and swelling and collapse of cells. Perivascular and peripheral zones sometimes showed decreased damage levels. The high temperature (greater than 50 degrees C) technique is presently being used as a means of "sterilizing" tumors before planned enucleation. The moderate temperature (43 degrees-45 degrees C) technique has been used in combination with radiotherapy to treat tumors when vision can be salvaged.

Scleral hyperplasia induced by heat.

We evaluated low-level heating as a stimulus to induce hyperplasia of normal rabbit sclera. Heat treatments were administered by placing an etched-element heater on bare sclera. Contact thermotherapy with a conductive device provided a favorable dose distribution for local scleral heating. A purely conductive heat source was selected to minimize intraocular heat penetration and to determine whether the scleral reaction was a primary thermal effect. Histologic examination of treated specimens showed thickening of normal rabbit sclera with preservation of the underlying normal ocular structures. Electron microscopic examination of treated sclera showed newly formed collagen fibrils adjacent to sclerocytes. These fibroblasts were activated, as manifested by well-developed rough-surfaced endoplasmic reticulum and hypertrophic Golgi complexes.