Antitumor effect of Nocardia rubra cell wall skeleton on syngeneically transplanted P388 tumors.

The antitumor activity of the immunomodulator, Nocardia rubra cell wall skeleton (N-CWS), was investigated using syngeneically transplanted P388 leukemia cells in a solid form. The s.c. growth of P388 tumors in DBA/2 mice was significantly suppressed by systemically administered N-CWS, and the effect was dose dependent. The antitumor effect of N-CWS was partially but significantly abrogated in splenectomized mice but not in T-cell or natural killer cell-deficient mice. Although spleen cells from mice treated with 1600 micrograms N-CWS contained no cytolytic activity, they exerted a significant cytostatic effect on P388 cell growth both in vitro and in vivo. Splenic cytostatic activity did not reside in T- or natural killer cells, but in plastic adherent cell population, macrophages. The response to N-CWS immunotherapy appeared to be associated with the number of macrophages infiltrating into the tumor lesions, and this was confirmed by histological analysis showing that P388 tumors from N-CWS-treated mice were intensively and dominantly infiltrated by macrophages. Furthermore, these were shown to be strongly tumor necrosis factor-positive by immunohistochemical analysis. These findings indicate that macrophages are the main effector cells playing a critical role in the suppression of P388 tumor growth in DBA/2 mice, and that tumor necrosis factor produced by these cells may be involved in the macrophage-mediated cytostatic effect induced by N-CWS. The fact that N-CWS suppressed the growth of weakly immunogenic P388 cells in syngeneic DBA/2 mice even when it was systemically injected would support the clinical potential of this agent.

Induction of a tumor necrosis factor-like activity by Nocardia rubra cell wall skeleton.

Nocardia rubra cell wall skeleton (N-CWS) stimulated adherent cells harvested from the peritoneal cavities of thioglycollate-treated mice to produce cytotoxic activity. Depletion of macrophages from the adherent cells by 2-chloroadenosine or silica abrogated the production of this cytotoxic activity, whereas treatment of the adherent cells with anti-Thy-1.2 antibody and complement did not. This suggested that macrophages were the producer cells of the activity. Cytotoxic activity became detectable as early as 2 h after N-CWS treatment and reached peak activity at 9 h, then declined to a lower level, indicating rapid onset without persistent effects. N-CWS-induced cytotoxic factors have a fairly narrow temperature range, pH optimum for storage, and are sensitive to pronase and trypsin. By using column chromatography, N-CWS-induced cytotoxic factors were compared in detail with tumor necrosis serum obtained from Bacillus Calmette-Guérin endotoxin-treated mice. Both toxins were found to be nearly identical with respect to their behavior in ion-exchange, gel filtration, and concanavalin A affinity columns. N-CWS also induced human peripheral blood lymphocytes to release cytotoxic activity. Monocytes predominantly participated in production of this activity as confirmed by treatment with monoclonal antibody and complement. The cytotoxic activity was completely neutralized by anti-human tumor necrosis factor antiserum, but not by anti-human lymphotoxin antiserum. The fact that human peripheral blood lymphocytes release tumor necrosis-like factors after stimulation with N-CWS might account for the antitumor effects of this agent.

Cloning and structural analysis of genomic DNA for human renal dipeptidase.

A genomic DNA for human renal dipeptidase was isolated from a human genomic library using probes for human renal dipeptidase cDNA. The human renal dipeptidase gene, containing ten exons and nine introns, had a total length of approx. 6 kbp. The DNA sequence of these exons was slightly different from that of the human renal dipeptidase cDNA reported by Adachi et al. [1]. From the results of a comparison of the deduced amino acid sequence of each exon with various mammalian renal dipeptidases, the fourth exon was found to be highly conserved (90%).

Purification and molecular cloning of mouse renal dipeptidase.

Mouse renal dipeptidase (mouseRDP, EC 3.4.13.11) was purified from the membrane fraction of kidney. The molecular mass of the enzyme was 115 kDa by size-exclusion HPLC and SDS-PAGE under non-reduced conditions and 58 kDa by SDS-PAGE under reduced conditions. The mouseRDP cDNA fragment was amplified from mouse kidney total RNA by reverse transcription-polymerase offin reaction (RT-PCR). The mouseRDP cDNA was isolated from a kidney cDNA library using the probe. The primary structure of mouseRDP deduced from the cDNA showed a high homology with renal dipeptidase from various mammals, except for the amino-terminal and carboxy-terminal domains. Recombinant mouseRDP obtained from transfected mouse L929 cells containing the expression plasmids has the same Km value and molecular mass as native mouse renal dipeptidase. From Northern blotting analysis, expression of the mouseRDP gene was recognized in both kidney and liver.

Prolongation of skin allograft survival in rats by a novel immunosuppressive agent, FK506.

FK506, an immunosuppresant, was isolated from Streptomyces tsukubaensis. Intramuscular administration of FK506 (0.32 mg/kg or more) 5 days a week for two weeks after grafting prolonged the acceptance time of F344 skin allograft to WKA rats. Similar results were obtained with cyclosporine at 32 mg/kg or more, but other immunosuppressives (i.e., prednisolone, azathioprine, and bredinin) gave only a marginal prolongation. The prolonging effect of FK506 was obtained in various donor-recipient combinations across a major or minor histocompatibility barrier. The agent also prolonged the acceptance time of mouse skin xenografts to rats. Furthermore, maintenance doses of 3.2 or 0.32 mg/kg twice a week after an initial 14-day treatment with the agent at 3.2 mg/kg gave graft survival as long as the treatment was continued for more than 120 days. Our findings show that FK506 has a potent immunosuppressive effect in rats and suggest that the agent merits further investigation.

Widespread activation of the brainstem preceding the recruiting rhythm in human epilepsies.

The excitability change of the brainstem was investigated before and during the conspicuous epileptic discharge in six patients with generalized convulsive seizures. The discharge consisted of a short duration of recruiting rhythm, which was considered equivalent to the seizure discharge on electroencephalogram. The excitability of the brainstem was measured with the parameters (amplitude and area) of component waves (wave-III and -V) of brainstem auditory evoked potentials. The theoretical background of the analysis is that brainstem auditory evoked potentials are 'far-field' potentials, by which they convey the information on the activity change of the brainstem even during the paroxysmal discharge within the cortex. The excitability of both the ventral (parameters of wave-III) and the dorsal brainstem (parameters of wave-V) exhibited a synchronized change (activation-inactivation). They were enhanced from -2.4+/-0.4 s, reaching the maxima before the onset of the seizure discharge, and decayed corresponding to the emergence of the recruiting rhythm. The results suggest the possibility that the widespread (ventral and dorsal) and synchronized activation of the brainstem triggers the seizure discharge in human generalized epilepsy. During the widespread activation of the brainstem, both the thalamus and the cortex probably undergo a suppressed inhibitory state through the cholinergic activation, precipitating the seizure discharge.

Structure and expression of cDNA for D-amino acid oxidase active against cephalosporin C from Fusarium solani.

D-Amino acid oxidase (DAO) was extracted and purified from cultured mycelia of Fusarium solani M-0718 (FERM P-2688). The enzyme was able to oxidatively deaminate cephalosporin C to 7-beta-(5-carboxy-5-oxopentanamido)cephalosporanic acid. Ninety-eight amino acid residues of the F. solani DAO were determined by sequence analysis of 9 peptides derived from Acromobacter protease I digests of the protein. Complementary DNAs encoding F. solani DAO were isolated from the F. solani cDNA library by hybridization with synthetic oligonucleotide probes corresponding to the partial amino acid sequences. Analysis of the nucleotide sequences of the clones revealed a 1,186-nucleotide sequence with a 5'-terminal untranslated region of 41 nucleotides, an open reading frame of 1,083 nucleotides that encoded 361 amino acids, and a 3'-terminal untranslated region of 62 nucleotides. The amino acid sequence of F. solani DAO had 25% homology to that of porcine kidney DAO [EC 1.4.3.3] and 37% homology to that of Trigonopsis variabilis DAO. The constructed plasmid overproduced F. solani DAO in Escherichia coli. The recombinant DAO had almost the same molecular activity as the native DAO against cephalosporin C.

Brainstem triggers absence seizures in human generalized epilepsy.

Simultaneous analysis of brainstem auditory evoked potentials (BAEPs) with reference to electroencephalography (EEG) was designed to examine the brainstem function corresponding to the EEG event. With this method, we investigated the brainstem function pre- and during the paroxysmal discharge in human absence seizures classified as primary generalized epilepsy (PGE). Two types of functional change in the lower brainstem were revealed as parameters of wave-III components (amplitude and area) of BAEPs without significant change in the upper brainstem. One was long-range biphasic fluctuation (acceleration followed by abrupt deceleration with the maximum -6.4+/-3.2 s before the seizure onset), and the other was rhythmic oscillation with 3 Hz. The latter, synchronized with the cortical spike-and-wave complex, imposed on the descending slope of the former. One important point is that both preceded the onset of cortical paroxysmal discharge. The results reappraise the classical hypothesis of "centrencephalic system" on seizure generating mechanism in human PGE. The results prove the primary triggering role of the lower brainstem that is independent of sleep-related synchronizations. The method is applicable to other types of EEG event for the investigation of brainstem involvement.

Dual control of the brainstem on the spindle oscillation in humans.

In human subjects, the excitability change of the brainstem was investigated over the course of the spindle oscillation. The investigation was carried out by a sequential analysis of brainstem auditory evoked potentials (BAEPs) with reference to one sequence of spindle oscillation. The method was based on the characteristics of BAEPs, i.e. far-field evoked potential. The brainstem revealed two types of excitability change: one in the lower ventral brainstem (wave-III components), and the other in the upper dorsal brainstem (wave-V components). The excitability in the dorsal brainstem showed an oscillation with one cycle period of about 1.5 s, whereas in the ventral brainstem, the excitability showed a long-range biphasic (decaying-growing) fluctuation. Both excitability changes in the brainstem preceded the spindle oscillation, and the phase was reversed during the emerging period of spindle oscillation. The results suggest a primary triggering mechanism of the brainstem for the spindle oscillation, which is independent of preceding cortical drives (K-complexes) upon the thalamus. The difference of the excitability change between the spindle oscillation and the paroxysmal discharge (spike-and-wave complex) was also discussed.

Brainstem activates paroxysmal discharge in human generalized epilepsy.

In nine patients with generalized epilepsy of convulsive seizures, the excitability change of the brainstem was evaluated over the course of the interictal paroxysmal discharge (poly spike-and-wave complex, poly SWC). The evaluation was carried out by a sequential analysis of brainstem auditory evoked potentials (BAEPs) before and during one sequence of poly SWC. The characteristics of BAEPs, i.e. far-field evoked potentials, allowed the evaluation of the excitability change in the brainstem, which was not influenced by the cortical activity. The excitability in the ventral brainstem, measured with the parameters of wave-III, showed a biphasic fluctuation (deceleration--acceleration) before the onset of poly SWC (minima at -0.7+/-0.4 s). On the other hand, the excitability in the dorsal brainstem, measured with the parameters of wave-V, showed no significant difference over the course of poly SWC. The results suggest that the biphasic excitability change in the ventral brainstem is conveyed to the cortex through the ascending activating system. The excitability acceleration preceded by deceleration in the ventral brainstem probably synchronizes the cortical activity profoundly enough to produce poly SWC through the activation of intralaminar thalamic neurons.

Vasorelaxant mechanism of the new vasodilator, FK409.

To define the vasorelaxation mechanism of FK409, we examined the effect of the compound on vascular tension and cyclic nucleotide levels in isolated rat thoracic aorta contracted with norepinephrine, and on activities of guanylate cyclase and cyclic GMP phosphodiesterase prepared from rat or rabbit thoracic aorta. FK409 (1 x 10(-9) to 1 x 10(-6) M), like nitroglycerin (1 x 10(-9) to 1 x 10(-6) M), produced a potent vasorelaxant effect associated with an increase in cyclic GMP content of the tissue. There was no change in cyclic AMP levels. The vasorelaxant effect of FK409 was independent of the integrity of the endothelium, and was unaffected by L-NG-monomethylarginine (0.1 mM) or oxyhemoglobin (1 microM). On the other hand, FK409 (3.2 x 10(-7) M) activated soluble guanylate cyclase, and the activating effect was completely inhibited by oxyhemoglobin (10 nM). Cyclic GMP phosphodiesterase was unaffected by FK409 (1 x 10(-7) to 1 x 10(-5) M). Furthermore, in rat aortic soluble fraction FK409 (3 mM) was found to liberate nitric oxide (NO) which was evaluated spectrophotometrically after diazotization of sulfanilic acid and coupling with N-(1-naphthyl)-ethylenediamine. The liberation occurred even in the absence of L-cysteine (5 mM), in contrast to the case with nitroglycerin (3 mM). These results suggest that the vasorelaxant effect of FK409 is associated with an increase in intracellular cyclic GMP, and that the cyclic GMP accumulation is due to activation of soluble guanylate cyclase. The enzyme activation is probably due to NO released from the compound molecule in the vascular smooth muscle cells.

Nilvadipine as a neuroprotective calcium entry blocker in a rat model of global cerebral ischemia. A comparative study with nicardipine hydrochloride.

The effects of two dihydropyridine type calcium entry blockers, nilvadipine and nicardipine hydrochloride (nicardipine), on the liberation of free fatty acids (FFAs) were investigated using an experimental model of global cerebral ischemia in rats, and were compared with their pharmacokinetic properties. Nilvadipine, but not nicardipine, at a dose of 100 micrograms/kg i.v., significantly attenuated the liberation of FFAs, particularly docosahexaenoic and arachidonic acid. Furthermore, the brain concentration of nilvadipine was higher than that of nicardipine after equivalent dosing. The results of the present study demonstrate that pharmacokinetic differences between these two calcium entry blockers might explain the difference in their pharmacological efficacy.

Midday exposure to bright light changes the circadian organization of plasma melatonin rhythm in humans.

Effects of bright light exposure at midday were examined on plasma melatonin rhythm in humans under controlled living conditions. Bright light of 5000 1x was provided from the ceiling at midday (1100-1700 h) for 3 consecutive days and the circadian rhythm in plasma melatonin was determined from the fourth to fifth day. The control study was performed in the same subjects who spend four days under dim light conditions (less than 200 1x). The subjects were allowed to sleep from 2400 to 0800 h. The onset phase, but not the end phase, of plasma melatonin rhythm was significantly phase-advanced by bright light exposure. Furthermore, the area under the curve of nocturnal melatonin rise was significantly larger under bright light exposure than under dim light. These findings indicate that midday exposure to bright light for 3 consecutive days changes the circadian organization of plasma melatonin rhythm in humans.

Vitamin B12 enhances the phase-response of circadian melatonin rhythm to a single bright light exposure in humans.

Eight young males were subjected to a single blind cross-over test to see the effects of vitamin B12 (methylcobalamin; VB12) on the phase-response of the circadian melatonin rhythm to a single bright light exposure. VB12 (0.5 mg/day) or vehicle was injected intravenously at 1230 h for 11 days, which was followed by oral administration (2 mg x 3/day) for 7 days. A serial blood sampling was performed under dim light condition (less than 200 lx) and plasma melatonin rhythm was determined before and after a single bright light exposure (2500 lx for 3 h) at 0700 h. The melatonin rhythm before the light exposure showed a smaller amplitude in the VB12 trial than in the placebo. The light exposure phase-advanced the melatonin rhythm significantly in the VB12 trail, but not in the placebo. These findings indicate that VB12 enhances the light-induced phase-shift in the human circadian rhythm.

Platelet activating factor (PAF) involvement in endotoxin-induced thrombocytopenia in rabbits: studies with FR-900452, a specific inhibitor of PAF.

PAF (1 ug/kg) injected intravenously (i.v.) into anesthetized rabbits resulted in marked loss of circulating platelets and leukocytes. Administration of FR-900452 1-methyl-3-(1-(5-methylthiomethyl-6-oxo-3-(2-oxo-3-cyclopenten-1-y lidene)- 2-piperazinyl) ethyl)-2-indolinone, a specific PAF inhibitor, at a dose of 10 mg/kg i.v. with 10 min prior to the PAF injection significantly prevented both changes. On the other hand, PAF has been considered as a mediator of endotoxin shock. Therefore, in order to determine whether endogenous PAF contributes to the occurrence of thrombocytopenia or leukopenia in endotoxin shock, we assessed the effect of FR-900452 on the thrombocytopenia and the leukopenia following bolus i.v. injection of E.coli endotoxin (0.03 mg/kg) in rabbits. As a result, pretreatment with the compound (10 mg/kg, i.v.) significantly reduced the thrombocytopenia at 60 and 180 min after the endotoxin injection. In contrast, FR-900452 did not reduced the leukopenia at any time of after endotoxin. These results indicate that PAF might be involved in the occurrence of thrombocytopenia in rabbit endotoxemia and the contribution of PAF to the leukopenia is much less extent than that to the thrombocytopenia.

Estradiol-17beta measurement in women receiving conjugated estrogens. Dissociation between two commercial methods.

To investigate problems associated with measurement of estradiol-17beta (E2) in hormone replacement therapy (HRT), two commercial immunometric methods (Coat-A-Count E2 and Immulyze E2) were used to assay E2 concentrations and the two results were expressed as E2 ratios. Samples were obtained from 97 Japanese women receiving HRT and 168 unmedicated women. The largest differences between methods (P < 0.001) occurred in patients receiving oral conjugated estrogen (CE), while the best concordance was found in unmedicated women; like these controls, patients receiving oral estriol or transdermal E2 showed no significant difference between methods. Defining an E2 ratio > or = 2.0 as an abnormal discordance, the mean E2 ratio and the frequency of abnormal discordance in the CE group were 2.15 +/- 1.18 and 43.6%, respectively. No abnormal discordance for E2 occurred in other groups. In serial serum samples from the control group, no significant difference was seen between the mean E2 ratio at first measurement and those at a subsequent measurement. Similarly, no significant difference in the ratio was seen when two serial samples from CE patients were compared. However, E2 ratios after prescription of CE were significantly higher than before treatment in all patients. In conclusion, although measurement of E2 is important in patients receiving HRT, validity of the test methods must be carefully weighed for patients receiving CE.

Multi-center study of seasonal affective disorders in Japan. A preliminary report.

A multi-center study on seasonal affective disorder (SAD) was conducted from the autumn of 1988 to the spring of 1989 with the cooperation of 16 facilities in Japan. Forty-six SAD patients were identified among 1104 respondents to our advertisements in mass media, or patients seen at the outpatient clinics. Essentially similar findings to other previous reports were obtained in terms of onset age of the first episode, duration of episode, high proportion of depression in first-degree relatives and atypical vegetative symptoms. However, a nearly equal sex ratio, together with a high proportion of unipolar depression, is characteristic of the present study. Increased appetite and carbohydrate craving were predominant only in female patients, whereas hypersomnia was prominent in both sexes. Effective response to light therapy was found in 17 SAD patients. However, a controlled study on a large number of patients is required to allow final conclusions on the efficacy of light therapy in Japanese SAD patients.

Atypical depressive symptoms possibly predict responsiveness to phototherapy in seasonal affective disorder.

Phototherapy was administered to 24 depressed patients with seasonal affective disorder (SAD), of which 62%, 24%, and 14%, respectively, showed improvements of greater than or equal to 50%, 25-50%, and less than 25% based on the Hamilton rating scale for depression for SAD (HAMSAD). No patients showed aggravation or side effects. Although the improvement rate in HAMSAD correlated significantly with the pretreatment severity of atypical symptoms of depression, it did not correlate with that of typical symptoms. This suggests that phototherapy is a useful treatment in SAD and that responsiveness to phototherapy in SAD can possibly be predicted by the atypical depressive symptoms before treatment.

Gene structural analysis and expression of human renal dipeptidase.

Human renal dipeptidase cDNA and genomic DNA were isolated from human kidney cDNA and genomic libraries, respectively. The human renal dipeptidase gene has a total length of approximately 6 kb and consists of ten exons and nine introns. The exons and cDNA each encode the 411 amino acid residues of the precursor protein, including 16 amino acid residues of signal sequence and a hydrophobic carboxyl terminal sequence for the attachment of a phosphatidylinositol glycan. Although the cDNA was slightly different from the cDNA reported by Adachi et al. (1990), the differences observed suggest, by comparison with human genomic DNA, that it may not represent an allelic variant but a cloning artifact. The recombinant human renal dipeptidase was produced on the surface of transfected L929 cells and had the same character as native renal dipeptidase. Northern blotting hybridization analysis showed that renal dipeptidase mRNA is only transcribed in kidney.

Studies on a new antibiotic FR-900336 taxonomy, isolation and characterization.

A new lipophilic antibiotic, FR-900336 was isolated from a culture of Streptomyces sioyaensis subsp. tanegashimaensis . FR-900336 is light yellow and has a molecular formula C30H30NO13C1 . The characterization by IR, UV, 1H NMR and 13C NMR spectra makes a quinone structure very probable. FR-900336 is active against Gram-positive bacteria and fungi.