Ras-related C3 botulinum toxin substrate 1 (RAC1) regulates glucose-stimulated insulin secretion via modulation of F-actin.

AIMS/HYPOTHESIS: The small G-protein ras-related C3 botulinum toxin substrate 1 (RAC1) plays various roles in mammalian cells, such as in the regulation of cytoskeletal organisation, cell adhesion, migration and morphological changes. The present study examines the effects of RAC1 ablation on pancreatic beta cell function. METHODS: Isolated islets from pancreatic beta cell-specific Rac1-knockout (betaRac1(-/-)) mice and RAC1 knockdown INS-1 insulinoma cells treated with small interfering RNA were used to investigate insulin secretion and cytoskeletal organisation in pancreatic beta cells. RESULTS: BetaRac1(-/-) mice showed decreased glucose-stimulated insulin secretion, while there were no apparent differences in islet morphology. Isolated islets from the mice had blunted insulin secretion in response to high glucose levels. In RAC1 knockdown INS-1 cells, insulin secretion was also decreased in response to high glucose levels, consistent with the phenotype of betaRac1(-/-) mice. Even under high glucose levels, RAC1 knockdown INS-1 cells remained intact with F-actin, which inhibits the recruitment of the insulin granules, resulting in an inhibition of insulin secretion. CONCLUSIONS/INTERPRETATION: In RAC1-deficient pancreatic beta cells, F-actin acts as a barrier for insulin granules and reduces glucose-stimulated insulin secretion.

Incidence of chromatoblastomas in aging goldfish (Carassius auratus).

The incidence of chromatoblastomas in aging goldfish, Carassius auratus, was observed for 2 successive years. Tumors first appeared in fish at 5 years of age, and the tumor frequency increased with increasing age of the fish. The cumulative incidence rate of chromatoblastomas and the mean number of tumors per fish were estimated for each age. The mean number of tumors per fish fitted a straight line with a slope of approximately 3 on a double logarithmic graph against age. Thus tumor incidence seemed proportional to about the third power of age, and tumor appearance seemed dependent on some mutational events.

Effect of high energy electrons on H⁻ production and destruction in a high current DC negative ion source for cyclotron.

Recently, a filament driven multi-cusp negative ion source has been developed for proton cyclotrons in medical applications. In this study, numerical modeling of the filament arc-discharge source plasma has been done with kinetic modeling of electrons in the ion source plasmas by the multi-cusp arc-discharge code and zero dimensional rate equations for hydrogen molecules and negative ions. In this paper, main focus is placed on the effects of the arc-discharge power on the electron energy distribution function and the resultant H(-) production. The modelling results reasonably explains the dependence of the H(-) extraction current on the arc-discharge power in the experiments.

High current DC negative ion source for cyclotron.

A filament driven multi-cusp negative ion source has been developed for proton cyclotrons in medical applications. In Cs-free operation, continuous H(-) beam of 10 mA and D(-) beam of 3.3 mA were obtained stably at an arc-discharge power of 3 kW and 2.4 kW, respectively. In Cs-seeded operation, H(-) beam current reached 22 mA at a lower arc power of 2.6 kW with less co-extracted electron current. The optimum gas flow rate, which gives the highest H(-) current, was 15 sccm in the Cs-free operation, while it decreased to 4 sccm in the Cs-seeded operation. The relationship between H(-) production and the design/operating parameters has been also investigated by a numerical study with KEIO-MARC code, which gives a reasonable explanation to the experimental results of the H(-) current dependence on the arc power.

Movement of beta-irradiated epidermal basal cells to the spinous-granular layers in the absence of cell division.

Guinea-pig epidermis was irradiated with 3000 rad of beta rays 1 hr after two injections of [3-H]thymidine 5 hr apart (labeled cells in S phase and G2 phase) or 18 hr after injection (labeled early G1 cells). In nonirradiated epidermis labeled basal cells divided within 24 hr with daughter cells remaining in the basal layer, and approximately 50% of the labeled cells moved into the spinal layer by the 3rd day. Cell division in nonirradiated epidermis diluted the number of silver grains/nucleus, and lightly labeled cells were found in the granular layer by day 7. Beta irradiation inhibited cell division but it did not slow the rate of transit (ca 8 days) of irradiated labeled cells from basal to granular layer, some of these remaining heavily labeled. Although cell division may play some role in upward movement of basal cells in normal epidermis detachment of a basal cell from the basement membrane and its transit to the granular layer is unimpaired in the absence of cell division. These findings suggest that some radioresistant metabolic function(s), not cell division, is responsible for upward movement of basal cells.

Cancer chemopreventive activity of euglobal-G1 from leaves of Eucalyptus grandis.

In the course of our continuing search for novel cancer chemopreventive agents from natural sources, several kinds of Eucalyptus plants were screened. Consequently, the phlorogrucinol-monoterpene derivative, euglobal-G1 (EG-1), was obtained from the leaves of Eucalyptus grandis as an active constituent. EG-1 exhibited the remarkable inhibitory effect on two-stage carcinogenesis test of mouse skin tumors induced by 7, 12-dimethylbenz[a]anthracene (DMBA) as an initiator and fumonisin-B1, which has been known as one of mycotoxins produced by Fusarium monifliforme, as a promoter. Further, EG-1 exhibited potent anti-tumor-promoting activity on two-stage carcinogenesis test of mouse pulmonary tumor using 4-nitroquinoline-N-oxide (4-NQO) as an initiator and glycerol as a promoter.

Desmoplakin I and II in acantholytic dermatoses: preservation in pemphigus vulgaris and pemphigus erythematosus and dissolution in Hailey-Hailey's disease and Darier's disease.

Desmoplakin I and II are important components of the attachment plaque of the desmosome which mediates cell to cell adhesion, in epithelial cells. In this study we used well-characterized antibody against desmoplakin I and II immunohistochemically and immunoelectron microscopically on two cases of pemphigus vulgaris and one case of pemphigus erythematosus and two cases each of Hailey-Hailey's disease and Darier's disease. In the normal human epidermis the desmosomes were demonstrated in a dotted pattern along cell periphery. In pemphigus vulgaris and pemphigus erythematosus acantholytic cells and the perilesional cells exhibited normal dotted pattern along the cell periphery. In Hailey-Hailey's disease and Darier's disease, the dotted pattern is lost in acantholysed and perilesional areas and anti-desmoplakin I + II positive proteins were observed diffusely in the cytoplasm. Immunoelectron microscopical findings correspond to these light microscopical observations. It is concluded that in autoimmune acantholytic disease such as pemphigus vulgaris and pemphigus erythematosus, desmoplakins are intact even in acantholytic cells, whereas in genodermatoses such as vulgaris and pemphigus erythematosus, desmoplakins are intact even in acantholytic cells, whereas in genodermatoses such as Hailey-Hailey's disease and Darier's disease primary or secondary abnormalities abnormalities of desmosomes may be involved in their pathogenesis.

Vertebral malformations in medaka (teleost fish) after exposure to tritiated water in the embryonic stage.

This study was designed to investigate radiation-induced vertebral malformations in medaka embryos irradiated with beta particles from tritium. Embryos of two inbred strains (HO4 and HO5) of medaka, Oryzias latipes, were exposed either to different concentrations of tritiated water (9.25-37 MBq/ml) or to 137Cs gamma rays (dose rates of 0.44-1.89 Gy/day) continuously from morula to hatching. The newly hatched fry were removed from the radiation field and kept under usual conditions for 1 month. Young fish were fixed in 10% buffered formalin, cleared in 1% KOH, stained with alizarin red S, and kept in glycerine. There was almost no difference in the response to radiation between medaka strains. No marked reduction of hatching rate was observed after chronic irradiation with beta particles and gamma rays, but a considerable reduction in survival of fry was detected in irradiated groups within 1 month after hatching. From observation of whole-mounted skeleton specimens, the following vertebral malformations were found in irradiated groups: fusion of two or more vertebrae, incomplete formation of vertebrae, and lack of vertebral process. The incidence of vertebral malformations increased significantly in both groups irradiated with tritium beta particles and 137Cs gamma rays. A similarity in the incidences was also observed between beta-particle- and gamma-irradiated groups. The RBE of beta particles relative to gamma rays was estimated to be 1 based on the dose-response relationships observed.

Effects of tritiated water on germ cells in medaka embryos.

Embryos of medaka, Oryzias latipes, were exposed to tritiated water and 137Cs gamma rays continuously from the one-cell stage until hatching (10 days at 26 degrees C). Germ cells in the gonads of newly hatched fry were counted in histological sections and compared with controls. The accumulated dose for 50% survival of germ cells was 195 rad for tritium beta rays and 350 rad for 137Cs gamma rays. Female progeny were produced using Yamamoto's method. The 50% survival doses for female germ cells treated in a manner similar to that described above were 140 rad for beta rays and 305 rad for gamma rays. When embryos of medaka were irradiated with gamma rays below an accumulated dose of 475 rad or treated with tritiated water at a concentration of 0.2 mCi/ml or lower, the dose response of the germ cells showed an exponential relationship. It appeared that there was no threshold or significant dose-rate effect for either beta or gamma rays on germ cell survival, and that tritium beta rays were more effective than 137Cs gamma rays in germ cell killing.

Erysubins C-F, four isoflavonoids from Erythrina suberosa var. glabrescences.

Four isoflavonoids, erysubins C-F, together with ten known compounds were isolated from the roots of Erythrina suberosa var. glabrescences, and their structures were elucidated on the basis of spectroscopic evidence. Erysubin C is an unusual pterocarpan derivative with a formyl group.

X-ray-induced dicentric yields in lymphocytes of the teleost, Umbra limi.

A microculture technique was applied to the study of lymphocytes of Umbra limi, which have a low number of large meta- and submetacentric chromosomes (2n = 22). On the 5th day (90h) and later after initiation of culture at 20 degrees C, some cultures provided well spread metaphase chromosomes for analyses. After initiation, cultures were irradiated with 50, 100, 150 and 200 R of 200 kVp X-rays. The cultures were harvested on the 5th day, at which time all arrested metaphase chromosomes were in their first division. The dicentric yields induced in X-irradiated Umbra lymphocytes were observed to be significantly (P = 0.05) lower than those in human lymphocytes. The resulting dose-response relationship for dicentric yield was described by the quadratic equation Y = aD + bD2. The advantage of this method lies in the fact that small amounts (0.1 ml) of blood can be repeatedly withdrawn from the fish after a minimal interval of 2 weeks. The microculture technique with Umbra lymphocytes is thought to have a wide range of applications in studies of the potential genotoxic effects of clastogenic agents in the aquatic environment.

Analysis of initial fouling process in coastal environment: effects of settlement, attachment, and metamorphosis promoters.

Effects of lumichrome, L-tryptophan, and curcumin on fouling organisms were examined on panels immersed in a near-shore aquatic environment. These products showed effective action in aquariums in preliminary screening tests for promoting different steps of fouling. Lumichrome showed metamorphosis-inducing activity for ascidian larvae (Halocynthia), L-tryptophan was a settlement-inducer for larvae of barnacles (Balanus), and curcumin showed attachment-promoting activity on the blue mussel (Mytilus). In order to establish that these tests are helpful in screening actual fouling or antifouling compounds, we examined the action of these three compounds in a coastal environment by following the first steps of biofouling, that is, by studying the quantity of chlorophyll, the number of bacterial cells, and the larvae settled. These experiments on the seashore indicated that these compounds did not act as promoters for the target organisms; however, they did show promoting effects on some nontarget organisms.

Cytogenetic adaptive response of cultured fish cells to low doses of X-rays.

The adaptive response was examining chromosomal aberrations and micronucleus in cultured fish cells, ULF-23 (mudminnow) and CAF-31 (gold fish). When cultured fish cells were first irradiated with small doses of X-rays, they became less sensitive to subsequent exposures to high doses. The effective adaptive dose was 4.8 cGy-9.5 cGy. Adaptive doses given cells in the G1 phase were more effective than when given in the S phase. The adaptive response was maximal at 5 hours and disappeared at 10 hours after the adaptive dose. The expression of the response was inhibited by treatment with 3-aminobenzamide, as reported for mammalian cells, and with arabinofuranoside cytosine, an inhibitor of DNA polymerase alpha. Caffeine, an inhibitor of post-replicational repair, had no effect on the response.

Purification and properties of beta-N-acetylglucosaminidase from Vibrio alginolyticus H-8.

Beta-N-acetylglucosaminidase [EC 3.2.1.30] from Vibrio alginolyticus H-8 was purified by column chromatography on DEAE-Sepharose FF, phenyl-Sepharose HP, Superdex 200HR, and Mono Q. The molecular weight of the enzyme was estimated by SDS-gel electrophoresis (SDS-PAGE) to be 75 kDa. The pI was 4.6. The activity was inhibited by Ag+, Hg2+, iodoacetate, p-chloromercuribenzoate, and N-ethylmaleimide.

Cloning and sequencing of a chitinase gene from Vibrio alginolyticus H-8.

A gene from Vibrio alginolyticus H-8, encoding chitinase, designated as chitinase B, was cloned by the shot-gun method using pUC118 and sequenced. The open reading frame consisted of 846 amino acids including a signal peptide. The molecular mass of the enzyme estimated based on the amino acid sequence data was 90 kDa. The N-terminal amino acid sequence of the enzyme was different from that of chitinase C1 which we had previously reported. This cloned chitinase B was considered one out of four chitinases (A, B, D, and E) which had been newly isolated from the culture broth and cell extract of V. alginolyticus H-8. The gene contained a chitin-binding domain and typical conserved regions of chitinases reported previously. The deduced amino acid sequence of the cloned chitinase B showed high sequence homology with the chitinase from V. parahaemolyticus (84% identity) and the chitinase from V. anguillarum (76.6%), but low sequence homology with the chitinase from V. harveyi (24.4%), and the chitodextrinase from V. furnissii (23.9%). Chitinase E found in cell extract is considered an intracellular chitinase which is different from chitodextrinases.

Cloning and sequencing of the deacetylase gene from Vibrio alginolyticus H-8.

A gene encoding deacetylase DA1 that is specific for N, N'-diacetylchitobiose was cloned using the shot-gun method with pUC118 and sequenced. The open reading frame encoded a protein of 427 amino acids including the signal peptide. The molecular mass of the mature enzyme estimated from the amino acid sequence data was 44.7 kDa, which is approximately similar to that, estimated by SDS-PAGE (48.0 kDa), of the purified enzyme reported previously. The N-terminal amino acid sequence deduced from the cloned deacetylase gene showed partial sequence homology with the Nod B protein from Rhizobium sp. (37% identity) and chitin deacetylase from Mucor rouxii (28%). It contained a domain, which showed homology with a chitin-binding domain of chitinase A from Bacillus circulans (39%).

Sixty cases of partial or total toe transfer for repair of finger losses.

Our experience with partial or total toe transfers for the reconstruction of finger deficits is presented. Sixty-one toes were transferred to reconstruct finger deficits in 60 patients. The transfers from the big toe consisted of 3 vascularized whole-nail grafts, 4 onychocutaneous flaps which included the nail and a skin flap from the toetip, 16 thin osteo-onychocutaneous flaps, 4 wrap-around flaps, and 3 combined wrap-around and dorsalis pedis flaps. The transfers from the second toe consisted of 6 trimmed toetips including the nail, 16 second toes, 1 combined second toe and a dorsalis pedis flap, and 1 second toe and third toe. The other transfers mainly consisted of other flaps, including a hemipulp flap and a first web space flap. Regarding the transfers from the big toe, vascularized nail grafts and onychocutaneous flaps were found to be most suitable for the treatment of total nail loss, thin osteo-onychocutaneous flaps for distal phalangeal loss of the thumb, wrap-around flaps with a vascularized iliac bone graft for thumb loss above the metacarpal joint, and the combined wrap-around and dorsalis pedis flaps for a total thumb deficit. Regarding the transfers from the second toe, the trimmed toetips including the nail were most suitable for claw nail deformities, the second toe was most suitable for finger loss except for the thumb with the proximal interphalangeal joint, and the combined second toe and dorsalis pedis flap was most applicable for a total thumb deficit including thenar skin loss.

Free combined thin wrap-around flap with a second toe proximal interphalangeal joint transfer for reconstruction of the thumb.

The combined thin wrap-around flap from the big toe and the proximal interphalangeal joint of the second toe is characterized by (1) a single vascularized joint, which is used to preserve the second toe with a free iliac bone graft, (2) a thin wrap-around flap, which allows the pulpal fatty tissue on the remaining bone of the big toe to be retained and accept a skin graft, (3) a wrap-around flap with a partial distal phalangeal bone, and (4) a microplate for firm fixation at the proximal bone union and early joint motion. The advantages of this method are (1) the cosmetic appearance is excellent with use of the thin wrap-around flap; (2) there is joint motion in the reconstructed thumb with strong pinch and vice pinch; (3) the vascularized joint with a microplate allows for early postoperative motion; (4) bone grafting from another donor site is unnecessary; (5) bone growth is possible in children with open epiphyses; and (6) the big and second toes are preserved with minimal donor-site morbidity. This method is indicated for thumb losses at a level distal to the metacarpophalangeal joint or at the level of the proximal phalanx.

Flow-through anterior thigh flaps for one-stage reconstruction of soft-tissue defects and revascularization of ischemic extremities.

"Flow-through" anterior thigh flaps are used to reconstruct defects accompanied by major vessel damage in the extremities. These flaps have long flow-through pedicle vessels (which are the descending branch of the lateral circumflex femoral system) that can be interposed into the vascular gaps in the extremities and are composed anatomically of the rectus femoris muscle. The defects of eight patients were repaired successfully with flow-through anterior thigh flaps, which were from 10 to 30 cm in length and 7 to 15 cm in width. The advantages of these flaps are (1) the damaged main vessels (< 20 cm in length) can be reconstructed simultaneously with large skin defects (up to 30 cm in length and 15 cm in width), (2) double arterial inflow by means of both ends of the pedicle artery ensures safe blood circulation of the flap, (3) the lateral circumflex femoral system, the pedicle of the anterior thigh flaps, can be used for chimeric composite flaps for the reconstruction of composite defects with major vessel loss in the extremities, (4) two concomitant veins can be used as a drainage system in extremities with severe edema, and (5) multistage flap transfers are possible to repair recurring defects in the extremities with a single major vessel using additional transection and pedicle interposition.

[Two cases of pulmonary dirofilariasis in Nagasaki Prefecture].

We reported two cases of pulmonary dirofilariasis in Nagasaki Prefecture. Each case was admitted to our hospitals for evaluation of an abnormal shadow on chest X-ray film taken for examination. We strongly suspected lung cancer before operation. It was resected in order to confirm the pathological diagnosis. There was a Dirofilaria immitis in the vessels surrounded by the necrotic tissues. The case of pulmonary dirofilariasis tends to increase in Japan, but the positive rate of dogs with Dirofilaria immitis tends to decrease, because of decreasing breeding places of mosquitoes by the spread of the public sewage system in Nagasaki City.