Potassium currents inhibition by gambierol analogs prevents human T lymphocyte activation.

Gambierol is a marine polycyclic ether toxin, produced along with ciguatoxin congeners by the dinoflagellate Gambierdiscus toxicus. We have recently reported that two truncated skeletal analogs of gambierol comprising the EFGH- and BCDEFGH-rings of the parent compound showed similar potency to gambierol on voltage-gated potassium channels (Kv) inhibition in neurons. Gambierol and its truncated analogs share the main crucial elements for biological activity, which are the C28=C29 double bond within the H-ring and the unsaturated side chain. Since Kv channels are critical for the regulation of calcium signaling, proliferation, secretion and migration in human T lymphocytes, we evaluated the activity of both the tetracyclic and heptacyclic analogs of gambierol on potassium currents in resting T lymphocyte and their effects on interleukin-2 (IL-2) release and gene expression in activated T lymphocytes. The results presented in this work clearly demonstrate that both truncated analogs of gambierol inhibit Kv channels present in resting T lymphocytes (Kv1.3) and prevented lymphocyte activation by concanavalin A. The main effects of the heptacyclic and tetracyclic analogs of gambierol in human T cells are: (1) inhibition of potassium channels in resting and concanavalin-activated T cells in the nanomolar range, (2) inhibition of IL-2 release from concanavalin-activated T cells and (3) negatively affect the expression of genes involved in cell proliferation and immune response observed in concanavalin-activated lymphocytes. These results together with the lack of toxicity in this cellular model, indicates that both analogs of gambierol have additional potential for the development of therapeutic tools in autoimmune diseases.

Comparative cytotoxicity of gambierol versus other marine neurotoxins.

Many microalgae produce compounds that exhibit potent biological activities. Ingestion of marine organisms contaminated with those toxins results in seafood poisonings. In many cases, the lack of toxic material turns out to be an obstacle to make the toxicological investigations needed. In this study, we evaluate the cytotoxicity of several marine toxins on neuroblastoma cells, focusing on gambierol and its effect on cytosolic calcium levels. In addition, we compared the effects of this toxin with ciguatoxin, brevetoxin, and gymnocin-A, with which gambierol shares a similar ladder-like backbone, as well as with polycavernoside A analogue 5, a glycosidic macrolide toxin. For this purpose, different fluorescent dyes were used: Fura-2 to monitor variations in cytosolic calcium levels, Alamar Blue to detect cytotoxicity, and Oregon Green 514 Phalloidin to quantify and visualize modifications in the actin cytoskeleton. Data showed that, while gambierol and ciguatoxin were successful in producing a calcium influx in neuroblastoma cells, gymnocin-A was unable to modify this parameter. Nevertheless, none of the toxins induced morphological changes or alterations in the actin assembly. Although polycavernoside A analogue 5 evoked a sharp reduction of the cellular metabolism of neuroblastoma cells, gambierol scarcely reduced it, and ciguatoxin, brevetoxin, and gymnocin-A failed to produce any signs of cytotoxicity. According to this, sharing a similar polycyclic ether backbone is not enough to produce the same effects on neuroblastoma cells; therefore, more studies should be carried out with these toxins, whose effects may be being underestimated.

Calcium oscillations induced by gambierol in cerebellar granule cells.

Gambierol is a marine polyether ladder toxin derived from the dinoflagellate Gambierdiscus toxicus. To date, gambierol has been reported to act either as a partial agonist or as an antagonist of sodium channels or as a blocker of voltage-dependent potassium channels. In this work, we examined the cellular effect of gambierol on cytosolic calcium concentration, membrane potential and sodium and potassium membrane currents in primary cultures of cerebellar granule cells. We found that at concentrations ranging from 0.1 to 30 microM, gambierol-evoked [Ca(2+)]c oscillations that were dependent on the presence of extracellular calcium, irreversible and highly synchronous. Gambierol-evoked [Ca(2+)]c oscillations were completely eliminated by the NMDA receptor antagonist APV and by riluzole and delayed by CNQX. In addition, the K(+) channel blocker 4-aminopyridine (4-AP)-evoked cytosolic calcium oscillations in this neuronal system that were blocked by APV and delayed in the presence of CNQX. Electrophysiological recordings indicated that gambierol caused membrane potential oscillations, decreased inward sodium current amplitude and decreased also outward IA and IK current amplitude. The results presented here point to a common mechanism of action for gambierol and 4-AP and indicate that gambierol-induced oscillations in cerebellar neurons are most likely secondary to a blocking action of the toxin on voltage-dependent potassium channels and hyperpolarization of sodium current activation.

Synthetic studies toward gambierol. Convergent synthesis of the octacyclic polyether core.

[structure: see text]. A convergent synthetic route to the octacyclic polyether core of gambierol, a marine polycyclic ether toxin, has been developed. The synthesis involves construction of two fragments representing the ABC and EFGH ring systems followed by their coupling via a B-alkyl Suzuki reaction.

[Analysis of old surgical operation records of the Drs. Ishin Fuwa who graduated from Hanaoka School of Medicine].

Ninety-five surgical records prepared by Dr. Ishin Rensai Fuwa from 1826 to 1859, and by his son, Dr. Ishin Kyousai Fuwa, from 1860 to 1871, were analyzed. These two surgeons lived in Fuwaishiki Village in the Mino area and performed many surgical operations under general anesthesia more than a century ago. The former was one of the trainees of Dr. Seishu Hanaoka who had performed the first operation under general anesthesia by Mafutsusan in 1804. Forty-eight patients were from the Mino area, and thirty-one from the Owari area; the others were from the Ise, Mikawa and Oomi areas. Seventy-three of 95 operations were for breast cancer, four for neck tumor, four for facial tumor, three for cleft palate and so on. The ages of breast cancer patients ranged from 15 to 68 years (mean 44 years), and 27% of 73 patients had an operation for breast cancer with removal of axillary nodes. Even at that time, informed consent for surgery seemed to have been obtained when a cancer was at the advanced stage and recurrence was suspected. These records clarify the medical situation, especially with regard to surgery, in one district of the Mino area at the end of the Edo era before Western medicine had been introduced to Japan.

Lysosomal glycogen accumulation in rat liver and its in vivo kinetics after a single intraperitoneal injection of acarbose, an alpha-glucosidase inhibitor.

A single intraperitoneal injection of acarbose (400 mg/kg) into rats caused lysosomal accumulation of glycogen in the liver, mimicking the cytological characteristics of human glycogen storage disease type II (Pompe's disease). The animal model is therefore useful for studying the pathogenesis of the disease. In the present study, we applied this model to examine the lysosomal hydrolytic pathway of glycogen in vivo. To quantify the lysosomal glycogen, the lysosome-rich fraction was rapidly prepared from liver homogenate by agglutination in the presence of Ca2+. Then the fraction was treated with alpha-amylase in isotonic medium to remove cytosolic glycogen, followed by transfer to hypotonic conditions in the presence of Triton X-100 to destroy total glycogen. The amount of lysosomal glycogen was calculated from the difference between the glycogen levels measured before and after the treatment under hypotonic conditions, and then it was corrected based on measurements of the intactness (%) of lysosomes and the recovery (%) of the lysosomal marker enzyme (beta NAGase). We observed no measurable lysosomal glycogen in normal liver by this method, and this was confirmed by electron microscopy. After administration of acarbose, the lysosomal glycogen level increased to 2.5 mg/g liver within 2 days, and then decreased gradually at a rate of 0.4 mg/day/g. The accumulation of glycogen in the lysosomes at an initial velocity of 1.5 mg/day/g liver may be considered as the amount of glycogen that would normally be degraded by acid alpha-glucosidase. Therefore, assuming that the liver breaks down about 40 mg glycogen/day/g, we estimated that about 3% of the glycogen would be hydrolyzed by the lysosomal pathway.

Some properties of large and small granules of waxy barley (Hordeum vulgare L.) endosperm starch.

Large and small starch granules were isolated and characterized from mature barley kernels with waxy endosperms. The large granules of any given waxy cultivar contained more amylose than the small granules of the same cultivar. It was also found that large granules contained a greater amount of long amylopectin B chains and had a lower fraction III:fraction II ratio, one of the structural characteristics of amylopectin, than the small granules in the same cultivar. Small granules showed a wider range of gelatinization and smaller heat of gelatinization by differential scanning calorimetry.

The influence of hardly digestive starch granules on sucrase and isomaltase in small intestinal mucosa of rats.

The effects of potato-starch granules (PSG) on sucrase and isomaltase activities in the small intestinal mucosa of rats were examined by gel filtration on a Sephacryl S-300 column. Young and adult rats were administered the diet containing 50% of either PSG or pregelatinized potato starch (PPS) as a control for 7 days. Mucosal homogenates were prepared from the small intestine and separated into their component disaccharidases on the column. The sucrase activity, increased by feeding young rats on the PSG diet, was detected in the sucrase-isomaltase complex by changing the activity ratio of the two enzymes. In adult rats, however, the complex seemed to be mature and the increased sucrase moiety was newly combined with a free isomaltase moiety and comprised a portion of the complex. These changes were observed not only in soluble disaccharidases but also in membrane-bound ones. Furthermore, there was a remarkable change of sucrase activity in the distal portion of the small intestine rather than in the proximal.

Effects of leucine/isoleucine ratio in amino acid mixture-diets simulating normal and high-lysine maize proteins on growth, nitrogen balance, and tryptophan-niacin metabolism in rats.

To re-evaluate nutritive values of opaque-2 (o2) and brittle-2 opaque-2 (bt2o2) maize protein and to re-estimate Gopalan's hypothesis [Lancet, i, 954-957 (1960)] that pathogenesis of pellagra might be related with intake of excess leucine and with chronic consumption of maize or jowar which contains relative high leucine, rats were fed on synthetic diets composed of amino acid mixtures simulating the protein of normal and high-lysine maize, o2 and bt2o2 maize. In order to investigate the effect of intake of excess leucine, leucine was supplemented to o2 and bt2o2 diet at the level of 0.43 and 0.73%, respectively, to adjust the ratio of leucine to isoleucine to that of normal maize protein. Judging from body weight gains and carcass nitrogen of weanling rats fed on these diets, the protein quality (amino acid composition) of bt2o2 maize was 30% superior nutritionally to that of o2 maize, and body composition of bt2o2 diet group were similar to that of casein diet group. Leucine supplementation did not affect these values except for a significant decrease in plasma valine levels. In young adult rats fed on leucine supplemented o2 diet, excretion of urinary nitrogen increased significantly compared with o2 diet alone, suggesting that a slight amino acid imbalance took place. But leucine supplementation altered neither N1-methylnicotinamide level in urine nor total niacin levels in the liver and the brain. These results suggest that supplementation of leucine to a high lysine maize diet did not affect tryptophan and niacin metabolism in rats under these conditions.

Some properties of starches of opaque-2, sugary-2 opaque-2, and waxy opaque-2 mutants of two broad-based synthetic cultivars of maize.

Starches of the opaque-2, sugary-2, opaque-2, and waxy opaque-2 endosperm mutants of two broad-based synthetic cultivars (Temp HA and Temp HB) of maize adapted to the more temperate areas of the world are, in general, similar to the respective starches of inbred maize background with regard to amylose percentage and the distribution of linear alpha-D-(1 leads to 4) linked unit-chains of amylopectin, starch-granule susceptibility to amylases, and properties examined by X-ray diffractometry and photopastegraphy. Changes in starch content and morphology of starch granules by scanning electron microscopy in dissected endosperms of the two broad-based synthetic maize cultivars following germination 0, 2, 4 and 6 days after planting were also investigated.

Some properties of starches of grain amaranths and several millets.

Starch granules were prepared from seeds of Amaranthus hypochondriacus L., A. caudatus L., proso millet, Japanese barnyard millet and foxtail millet. Amylose contents and the distribution of alpha-1,4 linked chain of amylopectin were determined by gel filtration of isoamylase-debranched starches. Some physical and chemical properties of the starches were also examined by scanning electron microscopy, X-ray diffractometry, photopastegraphy, and differential scanning calorimetry, together with starch-granule susceptibility to amylases. The existence of both normal and waxy types in the same species of a grain amaranth, A. hypochondriacus L., was confirmed. A. caudatas starches were identified to consist of mainly typical amylopectin and 5-7% amylose. The starches have some unique properties, namely, high starch-granule susceptibility values to amylases as well as those of A. hypochondriacus and unique pasting properties.

Comparative susceptibility of starch granules of double- and triple-mutants containing amylose-extender, waxy, sugary-1, sugary-2 and dull genes of maize inbred OH43 (Zea mays L.) to amylase.

Starch granules were prepared from 14 double- and 26 triple-mutants containing amylose-extender (ae), 14 double- and 18 triple-mutants containing waxy (wx), 15 double- and 20 triple-mutants containing sugary-1 (su1), 13 double- and 23 triple-mutants containing sugary-2 (su2), and 14 double- and 19 triple-mutants containing dull (du) of maize inbred Oh43 (Zea mays L.). The relative susceptibilities of these starch granules to fungal glucoamylase were determined and the starch granules were examined by scanning electron microscopy. A commercial normal maize starch was used as a control. Starch granules of the double- and triple-mutants containing su1 and su2 were digested two to eight times faster than normal. The ae gene reduced susceptibility and seems to be epistatic to su1 and su2. Starch granules of the double- and triple-mutants containing wx were digested about two times faster than normal and those containing shrunken-2 (sh2) were digested 1.2 to eight times faster than normal. Starch granules of triple-mutant combinations with opaque-2 (o2) showed digestion properties which were comparable to those of their respective monopaque double-mutant counterpart.

Comparative susceptibility to amylases of starch granules of several single endosperm mutants representative of floury-opaque, starch-deficient, and modified starch types and their double-mutant combinations with opaque-2 in four inbred lines of maize.

Starch granules were prepared from kernels of eight single endosperm mutants, brittle-1, (bt1), brittle-2 (bt2), floury-1, floury-2, soft starch, opaque-1 (o1), shrunken-2 (sh2), and sugary-2 (su2), and their double-mutant combinations with opaque-2 (o2) of four inbred lines of maize (Zea mays L.), B37, C103, Oh43 and W64A. We compared the susceptibility of various starch granules to Rhizopus glucoamylase and pancreatin. Starch granules of the su2 and su2o2 mutants were digested by amylases much faster than those of the normal counterparts. Starch granules of the bt1, bt2, o1 and sh2 mutants tended to be digested by amylases faster than those of normal maize. Starch granules of double-mutant combinations with the o2 gene were, in general, digested to an extent very comparable to their respective non-opaque single mutant counterparts in each of their four inbred backgrounds. We followed the relative digestion of starch granules by using scanning electron microscopy. Starch granules of endosperm mutants susceptible to amylases showed numerous pin holes on the surface layer and the pores penetrated into the inner layers of the granules during the attack by amylases. In some of the granules the inner portion, which appeared terraced or step-shaped, could be seen. This may be indicative of layered internal structures of the granules.

Effects of dietary protein and calcium levels on renal glucose-6-phosphatase activity of intact and thyroparathyroidectomized rats.

In order to determine the relationship of parathyroid hormone and levels of dietary protein and calcium with the activity of renal glucose-6-phosphatase (G6Pase), effects of two levels of dietary protein, namely, 25 and 75%, on the enzyme activity were compared at three levels of dietary calcium, namely, 0.06, 0.63, and 1.83%, with the use of intact and thyroparathyroidectomized (TPTX) rats. In intact rats, 0.06% dietary calcium caused an increase in renal G6Pase activity in rats fed the high carbohydrate diet, and dietary calcium in excess (1.83%) caused the enzyme activity to decrease. Similar responses in the activity of renal G6Pase to the variation of dietary calcium levels were seen in rats fed the high protein diet, but significant differences were not obtained. In TPTX rats fed the high carbohydrate diet, the activity of renal G6Pase was significantly decreased compared with that of intact rats. When TPTX rats were fed the high protein diet, however, no significant decrease in the enzyme activity was observed. Free access to aqueous 0.1% CaC1(2) solution by TPTX rats tended to restore the activity of renal G6Pase and serum calcium concentrations depressed by thyroparathyroidectomy. In addition, a significant correlation was observed between the total activity of renal G6Pase and serum calcium concentrations. Hypothyroidism produced by oral administration of propylthiouracil (0.05% of diets) did not affect the enzyme activity in the kidneys of rats fed the high carbohydrate and the high protein diets. The results suggest that the activity of renal G6Pase of rats fed the high protein diet might be less susceptible both to dietary calcium levels and to parathyroid function than that of rats fed the high carbohydrate diet.