Novel protein kinase C-beta isoform selective inhibitor JTT-010 ameliorates both hyper- and hypoalgesia in streptozotocin- induced diabetic rats.

AIM: Activation of protein kinase C (PKC) is thought to play an important role in the pathogenesis of diabetic microvascular complications. PKC-beta is elevated in hyperglycaemic conditions, both in vivo and in vitro. In this study, pharmacological effects of a novel PKC-beta isoform selective inhibitor, JTT-010 ((2R)-3-(2-aminomethyl-2,3-dihydro-1H-3a-azacyclopenta(a)inden-8-yl)-4-phenylaminopyrrole-2,5-dione monomethanesulphonate), on diabetic neuropathy were examined. METHODS: PKC inhibitory activity of JTT-010 was evaluated with an enzyme assay. For the in vivo study, streptozotocin (STZ)-induced diabetic rats were treated with JTT-010 for 12 weeks and tail/sciatic nerve conduction velocity (NCV) evaluated. Hyper/hypoalgesia was evaluated using tail-flick and formalin tests. RESULTS: JTT-010 inhibited PKC-betaI and -betaII with IC50 values of 4.0 and 2.3 nm respectively. For other PKC isoforms, IC50 values were 54 nm or greater. In STZ-induced diabetic rats showing a reduction in tail/sciatic nerve conduction velocities, JTT-010 (0.3-3 mg/kg) ameliorated the reduction of these velocities. In a formalin test, STZ-induced diabetic rats had hyperalgesia in the first phase. JTT-010 reduced nociceptive response at doses of 0.1 mg/kg or higher. Furthermore, STZ-induced diabetic rats showed hypoalgesia in the second phase of the formalin test and tail-flick test. JTT-010 also ameliorates these symptoms at doses of 0.1 mg/kg or higher. CONCLUSIONS: These observations suggest that PKC-beta contributes not only to diabetic hyperalgesia, but also to hypoalgesia and also contributes to defects in NCV. PKC-beta inhibitor, JTT-010, may be beneficial in suppressing the development of diabetic nerve dysfunction, including hyperalgesia and hypoalgesia.

Association of protein-tyrosine phosphatase PTP-BAS with the transcription-factor-inhibitory protein IkappaBalpha through interaction between the PDZ1 domain and ankyrin repeats.

PTP-BAS is a membrane-associated protein tyrosine phosphatase containing a band-4.1 homology region and five PDZ (PSD-95 Dlg ZO-1) [discs-large homology region ('DHR')/Gly-Leu-Gly-Phe ('GLGF')] domains. The second and fourth PDZ domains were reported to associate with Fas/CD95. By using the first PDZ domain as a bait in yeast two-hybrid screening, we have identified IkappaBalpha as a binding protein. IkappaBalpha associated with PDZ1 through the stretch of the N-terminal three ankyrin repeats. The association was also confirmed in HeLa cells by co-immunoprecipitation experiments. Inhibition of PTP-BAS by expression of dominant-negative PTP-BAS mutant resulted in tyrosine-phosphorylation of IkappaBalpha. Tyrosine-phosphorylation of IkappaBalpha is a key event in activation of nuclear factor (NF)-kappaB during reoxygenation. PTP-BAS may thus play a regulatory role in activation of NF-kappaB under high oxidative stress.

Effects of lidocaine and mexiletine on defibrillation energy requirements in animals treated with flecainide.

We previously reported that mexiletine alone did not increase the ventricular defibrillation threshold (DFT). However it has been stressed that the interaction of antiarrhythmic drugs may be one of the cause of sudden death in patients taking flecainide or encainide. In the present study, the effect of lidocaine and mexiletine combined with flecainide on DFT was investigated. Experiments were performed on 27 mongrel dogs. Flecainide 2 mg kg(-1) was administered i.v. as a loading dose and repeated for maintenance. Lidocaine (n = 10) or mexiletine (n = 10), was cumulatively administered 15 min later. Saline was administered in control group (n = 7). In these groups, fibrillation/defibrillation trials were repeated. The flecainide concentration ranged from 0.84 to 1.02 microg ml(-1). The lidocaine and mexiletine concentrations increased up to 12.54 and 7.47 microg ml(-1), respectively. DFT15, 15 min after the administration of flecainide, increased from 2.0 to 3.2 J in lidocaine group (mean +/- S.E.M.; P < 0.05), from 2.1 to 3.7 J in mexiletine group (mean +/- S.E.M.; P < 0.05). DFT increased with lidocaine concentrations of 3.42 microg ml(-1) or higher, and mexiletine of 3.60 microg ml(-1) or higher (P < 0.05). In conclusion, both lidocaine and mexiletine elevated the DFT in the dog treated with flecainide, especially with lidocaine in a therapeutic concentration.

Secondary lymphoid-tissue chemokine is a functional ligand for the CC chemokine receptor CCR7.

Secondary Lymphoid-tissue Chemokine (SLC) is a recently identified CC chemokine that is constitutively expressed in various lymphoid tissues and is a potent and specific chemoattractant for lymphocytes. The SLC gene and the gene encoding another lymphocyte-specific CC chemokine, EBI1-ligand chemokine (ELC), form a mini-cluster at human chromosome 9p13. Here, we show that SLC is a high affinity functional ligand for chemokine receptor 7 (CCR7) that is expressed on T and B lymphocytes and a known receptor for ELC. SLC induced a vigorous calcium mobilization in murine L1.2 cells stably expressing human CCR7. SLC tagged with the secreted form of alkaline phosphatase (SLC-SEAP) showed specific binding to CCR7 that was fully competed by SLC with an IC50 of 0.5 nM. SLC also induced a vigorous chemotactic response in CCR7-expressing L1.2 cells with a typical bell-shaped dose-response curve and a maximal migration at 10 nM. When assessed using CCR7-transfected L1.2 cells, SLC and ELC were essentially equivalent in terms of cross desensitization in calcium mobilization via CCR7, cross-competition in binding to CCR7, and induction of chemotaxis via CCR7. SLC and ELC were also shown to fully share receptors expressed on cultured normal T cells known to express CCR7. Notably, however, SLC was somehow less efficient in cross-desensitization against ELC in calcium mobilization and in cross-competition with ELC for binding when assessed using cultured normal T cells. Thus, SLC and ELC, even though sharing only 32% amino acid identity, constitute a genetically and functionally highly related subgroup of CC chemokines.

Clinical course of newly developed or progressive patchy chorioretinal atrophy in pathological myopia.

Regional chorioretinal atrophy in the posterior fundus (patchy chorioretinal atrophy) in pathological myopia impairs vision severely when it covers the macula. The aim of this study was to assess the course of development and progression of patchy chorioretinal atrophy in pathological myopia. The location and progression of patchy chorioretinal atrophy that was either newly developed or had progressed during the follow-up period (mean 5.25 years) were analyzed. A total of 41 lesions of patchy atrophy were newly developed in 30 eyes of 25 patients. These lesions were more likely to occur in marginal regions of a posterior staphyloma but frequency per unit area was highest in the macula. There were 138 lesions of patchy chorioretinal atrophy that progressed in 75 eyes of 53 patients. Sixty percent of the lesions of patchy chorioretinal atrophy in marginal regions of a posterior staphyloma spread toward the center. Seventy percent of the lesions of patchy chorioretinal atrophy in the macula spread in all directions. Fluorescein angiography of newly developed patchy chorioretinal atrophy showed hyperfluorescence in 50% and hypofluorescence in 27%. Fluorescein angiography of progressive lesions of patchy chorioretinal atrophy showed hypofluorescence in 69%. Fluorescein angiography of some progressive areas of patchy chorioretinal atrophy, which showed a change from hyperfluorescence to hypofluorescence within several years, suggested that damage to the retinal pigment epithelium preceded the progression of the patchy chorioretinal atrophy. In conclusion, the patchy chorioretinal atrophy is most likely to occur in the macula and to enlarge in all directions. And it is suggested that the patchy chorioretinal atrophy which shows hyperfluorescence by fluorescein angiography should be kept under observation because our data suggest that this finding indicates progression in the future.

Development of a colon delivery capsule and the pharmacological activity of recombinant human granulocyte colony-stimulating factor (rhG-CSF) in beagle dogs.

A peroral dosage form was examined to deliver recombinant human granulocyte colony-stimulating factor (rhG-CSF) to the colon in beagle dogs. A new gelatin capsule with its inside surface coated with ethylcellulose was prepared for this purpose. RhG-CSF was dissolved with propylene glycol and was filled in the capsule. Several kinds of ethylcellulose-gelatin capsules with an ethylcellulose layer of thickness 46 to 221 mm were used. The capsule was filled with propylene glycol solution containing fluorescein as an absorption marker, castor oil derivative and citric acid. The hardness of the capsule was tested after the gelatin layer was dissolved using a hardness tester and was dependent on the thickness of the ethylcellulose layer of the capsule. The time, Tmax, at which plasma fluorescein level reaches its maximum following oral administration of ethylcellulose capsules was used as a parameter for the in-vivo disintegration time of the ethylcellulose capsule into the colon. Capsules of thickness 84 mm with a Tmax of 4-6 h were filled with rhG-CSF solution containing fluorescein and were administered to dogs. After administration, blood samples were collected for 96 h and the blood total leucocyte (BTL) counts were measured as a pharmacological index of rhG-CSF. The maximum BTL count appeared at 10 h then gradually decreased and returned to its normal level at 48 h. These results suggest the usefulness of ethylcellulose capsules for the delivery of rhG-CSF to the colon and the possibility of a new oral rhG-CSF dosage form has been elucidated.

Molecular cloning of a novel protein-tyrosine phosphatase containing a membrane-binding domain and GLGF repeats.

A full-length cDNA encoding a novel cytosolic protein-tyrosine phosphatase (PTP), PTP-BAS, was cloned from human basophils. Due to in-frame deletions in the coding region, PTP-BAS exists in three isoforms: 7,455 bp (2,485 aa) for type 1, 7,398 bp (2,466 aa) for type 2 and 6,882 bp (2,294 aa) for type 3. All three isoforms contain a single PTP catalytic domain at the carboxyl termini as well as two distinct structural sequences. Amino terminal sequences of 300 amino acids are homologous to membrane-binding domains of cytoskeleton-associated proteins. Three 90 amino acid internal repetitive sequences are homologous to the GLGF repeats found in guanylate kinase proteins. PTP-BAS was expressed in various human tissues, especially highly in the kidney and lung. Interestingly, the BAS mRNA level in the fetal brain was remarkably high.

Conjunctival malignant melanoma with xeroderma pigmentosum.

A 10-year-old male patient with xeroderma pigmentosum had a recurrent, pigmented, conjunctival tumor. Conjunctival malignant melanoma was diagnosed from the histopathological examination of the resected biopsy specimens. To our knowledge, this case of conjunctival malignant melanoma in a patient with xeroderma pigmentosum may be the second such report in the literature. We believe that malignant melanoma must be considered with squamous cell carcinoma in the differential diagnosis of conjunctival tumors in patients with xeroderma pigmentosum. Fontana-Masson and S-100 staining techniques helped diagnose this conjunctival malignant melanoma.

Determination of the sequence coding for the beta subunit of the human high-affinity IgE receptor.

The cDNA encoding the beta subunit of the human high-affinity IgE receptor was cloned by a combination of various polymerase chain reactions (PCR). A major portion of the beta cDNA was amplified using primers homologous within the sequences of rat and mouse. The 3' unknown sequence was preferentially amplified using the RNA template-specific PCR and the improved two-step PCR. The 5' unknown sequence was specifically amplified by our newly developed PCR walking. Random heptanucleotides tagged with a unique sequence at the 5' end were used as the walking primer. Finally, the entire coding region was amplified and sequenced. The two extracellular loops of the human beta subunit were the least homologous to those of rat and mouse.

[Pharmacokinetic and clinical studies of imipenem/cilastatin sodium in the field of obstetrics and gynecology].

Imipenem/cilastatin sodium (MK-0787/MK-0791) was studied for its penetration into the adnexa uteri and uterine tissue, as well as for its clinical efficacy in the treatment of patients with obstetric and gynecologic infections. The following results were obtained. When 500 mg/500 mg of MK-0787/MK-0791 was administered by an intravenous drip infusion, peak levels of MK-0787 in tissues of adnexa uteri and uterus ranged from 14.6 micrograms/g to 25.8 micrograms/g, Tmax ranged from 0.55 hour to 0.98 hour, and the AUC ranged from 25.6 micrograms X hr/g to 45.2 micrograms X hr/g. Thus, the penetration of the drug into these tissues was good. Clinical efficacy of MK-0787 was evaluated in 30 patients in the field of obstetrics and gynecology. The clinical efficacy was excellent or good in all patients. Bacteriological effects of MK-0787/MK-0791 were very good, and 90% of the organisms detected before the treatment were eradicated. The antimicrobial activity of MK-0787 was tested against pathogens isolated before, during and after the treatment. Mean MIC80 values of MK-0787 were 0.39-0.78 micrograms/ml against all Gram-positive bacteria, 0.20-0.39 micrograms/ml against all Gram-negative bacteria, and less than or equal to 0.10-0.20 micrograms/ml against all anaerobic bacteria. The antimicrobial activity of MK-0787 appeared very good. No side effects or abnormal laboratory findings were observed except a slight elevation of S-GPT in 1 patient.

[Preclinical and clinical studies on aztreonam in the field of obstetrics and gynecology].

Penetration of aztreonam (AZT) into the uterus and the adnexal tissues and usefulness and safety of AZT for obstetric and gynecologic infections were studied with the following results. By one shot intravenous injection of AZT 1 g, the uterus and the adnexal tissues showed favorable penetration with Cmax 27.0-48.5 micrograms/g, AUC 29.4-84.9 micrograms X hr/g and Tmax 0.10-0.44 hours. MIC50, MIC80 and MIC90 of AZT for Gram-negative bacteria measured prior to administration were very low being 0.10 micrograms/ml, 0.20 micrograms/ml and 1.56 micrograms/ml, respectively. Clinical effect of AZT for 30 infection cases in obstetrics and gynecology was evaluated according to an overall efficacy criteria resulting in "good" for all the cases. With regard to microbiological effect, 90.9% of the pathogens isolated prior to the administration were eliminated by AZT. During and after the administration of AZT, side effect due seemingly to AZT was not observed in subjective and objective symptoms and laboratory values.

[Clinical trial of cefpiramide in the gynecological field].

Cefpiramide (CPM) was administrated intramuscularly to 27 cases of gynecological infections to evaluate its clinical efficacy and safety and the following results wee obtained. CPM was effective to all the cases of gynecological infections, and excellent was seen in 14 cases and good was seen in 13 cases. CPM eliminated 75% of clinical isolates. Laboratory tests were performed to blood samples and functions of liver and kidney before and after CPM treatment. Elevation of GPT was observed in 2 cases but required no treatment.

[Fundamental and clinical studies on cefpimizole in the field of obstetrics and gynecology].

Cefpimizole (AC-1370) was studied for its transference into adnexa uteri and uterine tissues as well as for its effects and safety on gynecological infections. The results obtained are as follows: Peak levels of AC-1370 were obtained in the antecubital vein and uterine artery at 10 minutes, in the tissues of adnexa uteri and uteri about 30 minutes after one shot intravenous injection of AC-1370 1 g, and relatively high concentrations were maintained for several hours. In the treatment of 30 cases of gynecological infections, the clinical efficacy of AC-1370 was assessed as effective in all cases. As for the bacteriological effects of AC-1370, 77.6% of isolated organisms were eradicated and 90% of all cases were effective. Side effects and abnormal laboratory findings due to AC-1370 were not observed during and after administration.

Corticoidogenic effect of acetylcholine in bovine adrenocortical cells.

The effect of acetylcholine (ACh) on corticoidogenesis in primary cultured bovine adrenocortical cells was examined. One hour exposure to 10(-3) M ACh resulted in a stimulative effect on corticoidogenesis in the freshly isolated cells, and the effect of ACh grew intense during primary culture and reached the maximum on day 2. ACh showed the effect at a higher concentration than 10(-6) M. Thus the primary 2-day cultured cells were used. The corticoidogenic effect of ACh was inhibited by atropine but not by hexamethonium. The effect of ACh was dose dependent, and the extracellular Ca++ was obligatory in inducing the effect. These results suggest that the corticoidogenic effect of ACh may be due to an increase in Ca++-influx via muscarinic receptor in adrenocortical cells.

[Clinical studies on sulbactam/cefoperazone in the field of obstetrics and gynecology].

Efficacy and safety of sulbactam/cefoperazone (SBT/CPZ) was studied on gynecological infections. The results obtained are as follows: In the treatment of 31 cases of gynecological infections, the clinical efficacy of SBT/CPZ was assessed as excellent in 9 cases and effective in 22 cases. As for the bacteriological effects of SBT/CPZ, clinically isolated organisms were completely (100%) eradicated. In comparison with MICs of CPZ, SBT/CPZ was found to show a combined effect on Gram-negative and Gram-positive organisms in the order mentioned, but this effect was not observed against anaerobes. The combined effect of SBT/CPZ on beta-lactamase producing bacteria was also investigated in the same manner. As a result, SBT/CPZ was found to exert a combined effect on beta-lactamase strains of S. aureus, S. epidermidis, E. coli, B. catarrhalis and B. fragilis. The laboratory tests performed before and after administration of SBT/CPZ revealed rise in GOT and GPT values in 1 case, GPT values in 2 cases and eosinophil in 1 case. However, these rises were all mild and required no particular measures.

Effect of adrenaline on steroidogenesis in primary cultured bovine adrenocortical cells.

In primary 2-day cultured bovine adrenocortical cells, adrenaline stimulated the steroidogenesis, while the effect of adrenaline did not appear in the freshly isolated cells. Thus the primary 2-day cultured cells were used to study the effect of adrenaline on steroidogenesis. Adrenaline showed the steroidogenesis-stimulating effect at concentrations higher than 10(-9) M, and the maximum effect was obtained between 10(-6) M and 10(-5) M in the primary 2-day cultured cells. The maximum effect of adrenaline was 50-70% of that of adrenocorticotropic hormone (ACTH). Noradrenaline, isoproterenol and phenylephrine also stimulated the steroidogenesis. However, the order of the potency was isoproterenol much greater than adrenaline = noradrenaline much much greater than phenylephrine. Propranolol and alprenolol inhibited the effect of adrenaline, but phenoxybenzamine and phentolamine did not inhibit the effect. Moreover, adrenaline stimulated the cyclic AMP production dose-dependently at concentrations higher than 10(-8) M. These results suggest that there are steroidogenesis-linked adrenergic receptors in primary 2-day cultured bovine adrenocortical cell membrane and that the steroidogenesis-stimulating effect of adrenaline occurs through the beta-adrenergic receptor.

Possible involvement of Ca2+-calmodulin system in cyclic AMP action in cholesterol ester hydrolytic response to ACTH in bovine adrenocortical cells.

In order to elucidate the relationship between cyclic AMP and the Ca2+-calmodulin system in the steroidogenic response to adrenocorticotropic hormone (ACTH), the effects of calmodulin inhibitors (trifluoperazine and W-7) on cortisol production and cellular cholesterol ester hydrolysis induced by ACTH or dibutyryl cyclic AMP in bovine adrenocortical cells were examined in the absence of extracellular Ca2+. These calmodulin inhibitors inhibited not only the cortisol production and the cholesterol ester hydrolysis induced by ACTH in the absence of extracellular Ca2+, but also inhibited the dibutyryl cyclic AMP-induced cortisol production and the cholesterol ester hydrolysis in the absence of extracellular Ca2+. These results suggested the possibility that cyclic AMP action was mediated by the Ca2+-calmodulin system in the activation process of cellular cholesterol ester hydrolysis in the steroidogenic response to ACTH.

[Clinical study of cefotetan in obstetrics and gynecology].

A new cephamycin antibiotic, cefotetan, was administered intramuscularly to 45 patients with female genital infections including 2 cases with abscess of Bartholin's gland, 14 cases with endometritis, 23 cases with adnexitis and 6 cases with pelvic peritonitis. The daily doses of the drug were 1 to 3 g, with 2 g daily being the most frequent regimen. The treatment was given twice daily in most patients. All cases responded to the drug, and marked response was seen in 22 cases and moderate response in 23. The eradication rate for causative organisms was 64.3%. In 16 cases of S. faecalis, it was rather low at 37.5%. Neither side effects nor abnormalities in clinical laboratory findings attributable to the drug were seen.

A possible regulatory role of Ca++-calmodulin system in cellular cholesterol ester hydrolysis in the steroidogenic response to ACTH in bovine adrenocortical cells.

In order to corroborate the regulatory role of Ca++-calmodulin system in the steroidogenic response to adrenocorticotropic hormone (ACTH), the effects of calmodulin inhibitors (chlorpromazine, trifluoperazine, and W-7) on cortisol production and cellular cholesterol ester hydrolysis induced by ACTH in bovine adrenocortical cells were examined. Three calmodulin inhibitors diminished not only the cholesterol ester hydrolysis and cortisol production induced by ACTH in the presence of Ca++, but also inhibited the Ca++-induced hydrolysis and cortisol production in the absence of ACTH. Neither cortisol production in crude mitochondrial fraction nor the ACTH-induced Ca++-influx was affected by chlorpromazine. These results indicate that Ca++f-calmodulin system plays a significant regulatory role in the supply of free cholesterol to the adrenal mitochondria in the steroidogenic response to ACTH.