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Biomedical subjects

H Bando

Publications and source records attributed to H Bando.

At least 19 recordsLinked to original sources

Somatostatin release as measured by in vivo microdialysis: circadian variation and effect of prolonged food deprivation.

In vivo microdialysis was used to determine SRIF release from the hypothalamus in unanesthetized male rats over a period of 24 h and in rats deprived of food for 72 h, in relation to changes in plasma GH levels. Before the experiment, a microdialysis probe was inserted into the anterior pituitary gland of the rats with an indwelling right atrial cannula. Dialysates and blood samples were collected serially, after normal feeding or 72-h deprivation of food. Normal rats implanted with the microdialysis probe showed an episodical pattern of GH secretion at intervals of 3 h. SRIF was secreted in a pulsatile fashion in the dark period in a similar manner to the light period. Mean SRIF pulse amplitude and mean SRIF level were significantly increased in the dark period. There was no significant correlation between the SRIF and GH pulses in the light period. SRIF levels in dialysates obtained from fed rats and food-deprived rats showed a pulsatile pattern. Food deprivation resulted in significant increases in mean SRIF level and mean SRIF pulse amplitude. These results suggest that the existence of circadian rhythm in SRIF release and the increase in SRIF release play an important role in suppressing GH secretion during prolonged food deprivation.

Animals

Characterization of a cry2A gene cloned from an isolate of Bacillus thuringiensis serovar sotto.

A cry2A-type gene, designated as cry2(SKW), was cloned from Bacillus thuringiensis serovar sotto SKW01-10.2-06, and some unique features of the gene were revealed. The cry2(SKW) gene encoded a polypeptide of 635 residues with a predicted molecular mass of 71,137 Da. Cry2(SKW) had 95.4% identity with Cry2Aa in amino acid sequence and was two residues longer than Cry2Aa. Two open reading frames (ORFs), designated as orf1 and orf2, were present upstream of the cry2(SKW) and showed high homology with the corresponding ORFs in the cry2Aa operon. The Orf2 from SKW01-10.2-06 contained a region of repeated sequences. However, unlike Cry2Aa, Cry2(SKW) formed the cuboidal crystalline inclusions when the cry2(SKW) gene was expressed in an acrystalliferous B. thuringiensis strain in the absence of the upstream ORFs. Furthermore, Cry2(SKW) was less toxic to a lepidopteran species, Bombyx mori, than Cry2Aa in spite of high homology between the two proteins.

Amino Acid Sequence

Detection of replicative intermediate with closed terminus of Bombyx densonucleosis virus.

Two kinds of Bombyx densonucleosis virus (BmDNV), BmDNV-1 and 2, have been isolated from sericultural farms in Japan or China. These viruses are classified into the family Parvoviridae because of the small spherical virus particle containing a single-stranded linear DNA genome. Recent studies on the genome structure of these viruses suggested that BmDNV-2 was a new type of virus with unique replication mechanism, though that of BmDNV-1 was similar to parvoviruses. However, details about the replication mechanism of BmDNVs have not been reported so far. Here, in order to elucidate the difference on replication mechanism between BmDNVs and parvoviruses, we analyzed the structure of the replicative intermediate (RI) of BmDNV DNAs by PCR using specific primers designed for detection of RI with closed terminal structure (RI-CT) which is expected to be formed by replication with self-priming mechanism. PCR using the DNA from the cells infected with BmDNV-1 could detect the expected DNA fragment, showing the existence of RI-CT. On the other hand, no fragment could be amplified from the virion DNA of the BmDNVs and the DNA extracted from BmDNV-2-infected cells, respectively. These observations strongly suggested that the BmDNV-1 replicates with the "self-priming and hairpin-transfer" mechanism similar to the human parvoviruses, while BmDNV-2 does not.

Animals

Effects of skin metabolism on percutaneous penetration of lipophilic drugs.

Effects of skin metabolism on percutaneous penetration of drugs with high lipophilicity were studied in vitro using rat skin pretreated with and without an esterase inhibitor, diisopropyl phosphorofluoridate [also known as diisopropyl fluorophosphate (DFP)]. Without DFP, about 96% of the total penetrated amount appeared as metabolized p-hydroxybenzoic acid in the receptor fluid after application of butylparaben, whereas about 30% penetrated as intact form after application of propylparaben. On the other hand, metabolized p-hydroxybenzoic acid was not defected in the receptor fluid under pretreatment with DFP. DFP significantly decreased (p < 0.05) the total amount that penetrated after application of butylparaben, but it did not significantly affect that of propylparaben. The results indicate that skin metabolism directly affects total amount that penetrated in the case of highly lipophilic drugs, and it was found that the higher metabolic rate to hydrophilic drugs is, the greater the amount that penetrated the skin would be. Thus, when optimal prodrugs are designed for the purpose of enhancing percutaneous penetration, we propose that the bioconversion rate to parent drugs as well as the lipophilicity of prodrugs becomes an important consideration.

Animals

In vivo evaluation of acyclovir prodrug penetration and metabolism through rat skin using a diffusion/bioconversion model.

PURPOSE: In order to evaluate the in vivo penetration of prodrugs which undergo metabolism in skin, we analyzed the in vivo penetration profiles of acyclovir prodrugs based on a two-layer skin diffusion model in consideration of metabolic process. METHODS: Acyclovir prodrugs (e.g., valerate, isovalerate and pivarate) were used as model prodrugs and the amounts excreted in urine were measured after percutaneous application. In vivo penetration profiles were then estimated by employing a deconvolution method and the penetration of acyclovir prodrugs was analyzed using a diffusion model. Subsequently, diffusion, partitioning and metabolic parameters were compared under in vitro and in vivo conditions. RESULTS: Although total penetration amounts at the end of the experiment were similar for the three prodrugs, the ratio of intact prodrug to total penetration amount differed significantly. Moreover, the excretion and absorption profiles were also very different for each prodrug. Enzymatic hydrolysis rate constants calculated under in vivo conditions were considerably larger than those obtained in the skin homogenate and in vitro penetration experiments. CONCLUSIONS: The present skin diffusion/bioconversion model combined with computer analysis enables us to comprehensively account for diffusion, partitioning and metabolism during in vivo percutaneous absorption. Nevertheless, different enzymatic hydrolysis rate constants obtained under both in vivo and in vitro conditions demonstrate the difficulty of obtaining accurate values for in vivo enzymatic activity from related in vitro experiments.

Acyclovir

Cloning of novel enterotoxin genes from Bacillus cereus and Bacillus thuringiensis.

A novel enterotoxin gene was cloned from Bacillus cereus FM1, and its nucleotide sequence was determined. Previously, a 45-kDa protein causing characteristic enterotoxin symptoms in higher animals had been isolated (K. Shinagawa, p. 181-193, in A. E. Pohland et al., ed., Microbial Toxins in Foods and Feeds, 1990) from the same B. cereus strain, but no report of cloning of the enterotoxin gene has been published. In the present study, a specific antibody to the purified enterotoxin was produced and used to screen the genomic library of B. cereus FM1 made with the lambda gt11 vector. An immunologically positive clone was found to contain the full protein-coding region and some 5' and 3' flanking regions. The deduced amino acid sequence of the cloned gene indicated that the protein is rich in beta structures and contains some unusual sequences, such as consecutive Asn residues. In order to clone enterotoxin genes from Bacillus thuringiensis, two PCR primers were synthesized based on the nucleotide sequence of the B. cereus gene. These primers were designed to amplify the full protein-coding region. PCR conducted with DNA preparations from the B. thuringiensis subsp. sotto and B. thuringiensis subsp. israelensis strains successfully amplified a segment of DNA with a size almost identical to that of the protein-coding region of the B. cereus enterotoxin. Nucleotide sequences of the amplified DNA segments showed that these B. thuringiensis strains contain an enterotoxin gene very similar to that of B. cereus. Further PCR screening of additional B. thuringiensis strains with four primer pairs in one reaction revealed that some additional B. thuringiensis strains contain enterotoxin-like genes.

Amino Acid Sequence

Effects of alkaloids from Aconitum yesoense var. macroyesoense on cutaneous blood flow in mice.

Nine alkaloid constituents in the root of Aconitum yesoense var. macroyesoense, as well as three acetylated derivatives, were examined for their peripheral vaso-activities by measuring laser-flowmetrically the cutaneous blood flow in the hind foot of mice after intravenous administration. The major constitutive delcosine (1), 14-acetyldelcosine (2) and lucidusculine (3), respectively, had little or very mild vaso-activity. Kobusine (4) and pseudokobusine (5) and three minor constituents, luciculine (6), 1-acetylluciculine (7) and dehydroluciculine (8), together exhibited a rapid increase in blood flow reaching a peak with a magnitude almost equal to that produced by hydralazine, when administered intravenously at the same dosage level of 20 mg/kg. Among them, 4 was characterized by successive reversal of the increase to a decrease in blood flow, while 7 produced a flow with a more delayed peak time. Dehydrolucidusculine (9) exhibited a transient decrease in blood flow prior to occurrence of the increase, as did papaverine. Consequently, it is assumed that the alkaloids, especially those of the C20-diterpenoid type, in the root of this Aconitum plant have peripherally vaso-dilating activities to varying degrees in mice, probably due to their direct action on the cutaneous microvasculature in a similar fashion to that shown by hydralazine. The laser blood flowmetric method would be useful as an in vivo means of qualitative as well as quantitative screening of chemically modified derivatives of peripherally vasoactive agents in mice.

Alkaloids

Insecticidal activity of the protein encoded by the cryV gene of Bacillus thuringiensis kurstaki INA-02.

A new host specificity was discovered with the insecticidal protein encoded by the cryV gene. The cryV gene was cloned from the Bacillus thuringiensis kurstaki INA-02 strain, which was selected among a number of B. thuringiensis isolates because of its high activity against Spodoptera litura. Analyses by polymerase chain reaction (PCR) revealed that INA-02 contained the cryIA(a) and cryV genes. Since no Spodoptera activity was observed with B. thuringiensis sotto, which contained only cryIA(a), insecticidal activity of the protein encoded by the cryV gene was investigated with several insect species including S. litura. For bioassay, the cryV gene was highly expressed in an acrystalliferous B. thuringiensis strain, BT51. The CryV protein from BT51 was assayed against larvae of three lepidopteran species, Bombyx mori, S. litura, and Plutella xylostella. The protein was highly active against S. litura and P. xylostella, suggestive that the protein contributes to the unique activity of INA-02.

Animals

Theoretical design of prodrug-enhancer combination based on a skin diffusion model: prediction of permeation of acyclovir prodrugs treated with 1-geranylazacycloheptan-2-one.

PURPOSE: A theoretical design of percutaneous penetration enhancement in which prodrug derivation and enhancer application are combined is proposed based on the skin diffusion model and it is experimentally verified. METHODS: Employing acyclovir as a model drug, the hypothesis was tested by synthesis of its prodrugs and evaluation of their in vitro permeation in the rat skin, with or without a penetration enhancer, 1-geranylazacycloheptan-2-one(GACH). RESULTS: Among five acyclovir prodrugs, those with higher lipophilicities (propionate, butyrate, valerate, and hexanoate prodrugs) showed greater skin penetration than those of hydrophilic prodrugs (acetate), when administered in combination with GACH. Furthermore, the observed enhancement ratios were in good agreement with those predicted by theoretical consideration. CONCLUSIONS: Thus, skin permeation of prodrugs applied with an enhancer can be predicted and optimized by model analysis.

Acyclovir

[A laser flow meter method for evaluating the peripheral blood flow in mice].

We have developed a method using a laser flow meter for investigating the effect of compounds on peripheral blood flow. The differences of blood flow in several portions and areas of the probe of a laser flow meter in the mouse and the effect of solvents for dissolving compounds on the blood flow were observed. The results indicated that the invisible area of blood vessels at the back or hind paw of the mouse was the most suitable placement position for the probe. In these placement positions, none of the tested solvents, 0.9% physiological saline solution, dimethylsulfoxide and 50% ethanol, had any significant effect on the peripheral blood flow. Next, using this method, we determined how the peripheral blood flow is affected by several drugs active on the circulatory system. By comparison between the present results from mice and previously reported data from other animals, it was shown that the effects of these cardiovascular agents on the peripheral blood flow in mouse skin showed similar tendencies to those in the previous reports using larger animals and sites other than the skin. However, a few of the drugs used in this experiment showed characteristic action on the peripheral blood flow in mice. Consequently, it was demonstrated that this method using the laser flow meter was useful as a simple first screening method to evaluate the effect of a compound on the peripheral blood flow.

Animals

Effects of thyroidectomy on thyrotropin-releasing hormone (TRH) and somatotropin release-inhibiting factor (SRIF) patterns in intrahypophysial microdialysates in rats.

The effects of thyroidectomy on patterns of TRH and somatotropin release-inhibiting factor (SRIF) release from the hypothalamus were investigated by using a microdialysis technique. Thyroidectomized and sham-operated rats underwent placement of a guide cannula into the anterior pituitary gland to obtain dialysates, or implantation of an intravenous cannula into the right atrium for blood sampling. Seven days postoperatively dialysates were collected at a flow rate of 2 microliters/min every 1 h. TRH concentrations in dialysates from thyroidectomized rats (0.43 +/- 0.2 pg/h) were significantly higher than those from control rats (0.17 +/- 0.02 pg/h). In contrast, SRIF concentrations in dialysates from thyroidectomized rats (2.45 +/- 0.05 pg/h) were significantly lower than those from control rats (3.80 +/- 0.22 pg/h). In addition, plasma TSH concentrations in thyroidectomized rats (24.8 +/- 0.5 ng/ml) were increased compared with those in control rats (2.5 +/- 0.1 ng/ml), and plasma GH concentrations were decreased from 68.6 +/- 6.4 ng/ml in control rats to 21.2 +/- 0.6 ng/ml in thyroidectomized rats. These findings indicate that TRH and SRIF releases from the hypothalamus are detectable by microdialysis method, and directly show the increase in TRH secretion and the decrease in SRIF secretion from hypothalamus in the hypothyroid state.

Animals

A case report of an elderly patient with acromegaly.

We encountered a 91-year-old patient with acromegalic features. The serum levels of growth hormone (GH) and insulin-like growth factor-I (IGF-I) were increased to 23.3 ng/ml and to 268 ng/ml, respectively. Both thyrotropin-releasing hormone and luteinizing hormone-releasing hormone tests demonstrated a 2-3 fold increase in the serum GH level. Magnetic resonance imaging disclosed a pituitary mass in the enlarged sella. The patient was diagnosed as having acromegaly due to overproduction of GH from a pituitary tumor. She manifested cardiac hypertrophy with severe aortic stenosis and mild hypertension, but without diabetes mellitus. After the administration of octreotide subcutaneously at a dose of 25 to 50 micrograms daily for 20 days, the serum GH level increased transiently but decreased rapidly to approximately half the initial level, and suppression of the GH level persisted thereafter for over 2.5 months. This patient seems to be the oldest patient with acromegaly among those reported in Japan.

Acromegaly

Double pituitary adenoma--two case reports.

A 43-year-old male and a 39-year-old male presented with multiple pituitary adenomas with two distinct histological types. The first patient who had multiple endocrine neoplasia type 1 had developed acromegaly due to a growth hormone-releasing hormone (GHRH)-producing pancreatic tumor. Both plasma GHRH and growth hormone (GH) levels decreased to normal after resection of the pancreatic tumor. However, the plasma GH level gradually increased again and magnetic resonance imaging revealed pituitary adenoma formation. Histological examination revealed two different histological types of pituitary adenoma: GH cell adenoma and null cell adenoma. The second patient, with no such genetic condition, had a non-functioning pituitary adenoma. Histological examination revealed two different histological types of silent GH cell adenoma and silent gonadotroph adenoma. Careful histological examination is required to exclude the possibility of multiple pituitary adenomas.

Adenoma

Thyrotropin-releasing hormone release in normal and hyperthyroid rats as measured by microdialysis.

To measure the thyrotropin-releasing hormone (TRH) release from the hypothalamus in euthyroid and hyperthyroid states, we investigated the changes in TRH levels in the anterior pituitary of conscious male rats using an in vivo microdialysis technique. In the euthyroid rats (n = 18), TRH levels in the extracellular dialysates of the anterior pituitary varied from 1.0 to 101.0 pg/ml in a pulsatile fashion: 15.9 +/- 13.9 (mean +/- SD) pg/ml with 5.8 +/- 1.5 pulses/24 h. In the hyperthyroid rats (n = 5) who received L-thyroxine at 10 micrograms/100 g body weight for 7 days, TRH levels in the dialysates during 6 h was 3.6 +/- 1.7 pg/ml, and significantly lower (P < 0.05) than those of the control rats (15.5 +/- 14.1 pg/ml); the pulse frequency was unchanged. These findings demonstrated for the first time the existence of pulsatile TRH release from the hypothalamus, and showed that thyroid hormone inhibits TRH release by the reduction of pulse amplitude, but not of pulse frequency.

Animals

Analysis of the genetic information of a DNA segment of a new virus from silkworm.

In 1983, a parvo-like virus (Yamanashi isolate) was newly isolated from silkworm. However, unlike parvovirus, two DNA molecules (VD1 and 2) were always extracted from purified virions. To investigate the structure and organization of the virus genomes, we determined the complete nucleotide sequence of VD2. The sequence consisted of 6031 nucleotides (nts) and contained a large open reading frame (ORF1) with 3513 nts. A smaller open reading frame (ORF2) with 702 nts was found in the complementary sequence. Computer analysis revealed that both ORFs did not code for the major structural proteins (VP1, 2, 3, and 4). These results suggest that VD2 has not enough information to produce progeny virions by itself. Further, the structural importance of the terminal sequence (CTS) common to both VD1 and VD2 was also predicted by a computer analysis.

Amino Acid Sequence