Biomedical subjects

M Mita

Publications and source records attributed to M Mita.

At least 37 records · Page 2Linked to original sources

PubMed1997

Cloning, sequencing, and expression of Arthrobacter protophormiae endo-beta-N-acetylglucosaminidase in Escherichia coli.

The gene encoding endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) was cloned, and its nucleotide sequence was determined. A single open reading frame consisting of 1935 base pairs and encoding a polypeptide composed of signal peptides of 24 amino acids and a mature protein of 621 amino acids was found. The primary structure of Endo-A exhibited significant homology with FO1F.10 gene product from Caenorhabditis elegans and weak homology with peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase from Flavobacterium meningosepticum and chitinase from Streptomyces olivaceoviridis. However, the enzyme had no significant homology with any previously reported endo-beta-N-acetylglucosaminidases. Transformed Escherichia coli cells carrying the 4.5-kb fragment expressed Endo-A activity. This enzyme activity was detected in the medium as well as in the periplasmic space of cells under the control of the Endo-A gene promoter. The recombinant Endo-A was efficiently isolated from the periplasmic space of the cells. N-terminal sequence analysis revealed that native and recombinant Endo-A have the same N-terminus. Recombinant and native Endo-A also showed very similar optimum pH profiles and transglycosylation activity.

Amino Acid Sequence↗

PubMed1997

Inhibition of muscarinic receptor-operated Ca2+ sensitization by short-term desensitization of alpha-toxin-permeabilized smooth muscle cells from guinea pig stomach.

1. Isolated single smooth muscle cells from the guinea pig stomach were permeabilized with Staphylococcus aureus alpha-toxin. 2. The permeabilized single cells showed a shortening in response to Ca2+ in an all-or-none manner. Moreover, the addition of acetylcholine (ACh) or guanosine 5'-triphosphate (GTP) resulted in a decrease in concentration of Ca2+ required to trigger a threshold response, suggesting that Ca2+ sensitization is induced by the stimulation of muscarinic acetylcholine receptors (mAChRs) or GTP-binding protein(s). 3. Short-term desensitization was induced by incubating the permeabilized cells with 100 microM ACh for 10 min. 4. In desensitized cells, the concentration of Ca2+ required to trigger a threshold response in the presence of ACh was increased, however, the cell shortening in response to Ca2+ in the absence of ACh and GTP-induced Ca2+ sensitization was not affected by short-term desensitization. 5. These results suggest that the receptor-operated augmentation of Ca2+ sensitivity is inhibited by short-term desensitization and that the development of short-term desensitization is due to an uncoupling of mAChR/GTP-binding protein(s).

Acetylcholine↗

PubMed1997

Probable participation of phospholipase A2 reaction in the process of fertilization-induced activation of sea urchin eggs.

In sea urchin eggs activated by sperm, A23187 or melittin, BPB (4-bromophenacyl bromide, a phospholipase A2 inhibitor) blocked fertilization envelope formation and transient CN(-)-insensitive respiration in a concentration-dependent manner. BPB had virtually no effect on the increase in [Ca2+]i (cytosolic Ca2+ level), the activity of phosphorylase a and the rate of protein synthesis, as well as acid production and augmentation of CN(-)-sensitive respiration. BPB also inhibited fertilization envelope formation and augmentation of CN(-)-insensitive respiration induced by melittin. Melittin, known to be an activator of phospholipase A2, induced the envelope formation, acid production, augmentation of CN(-)-insensitive and sensitive respiration, but did not cause any increase in [Ca2+]i, the phosphorylase a activity and the rate of protein synthesis. An activation of phospholipase A2 induced by Ca2+ or melittin seems to result in cortical vesicle discharge and production of fatty acids, which are to be utilized in CN(-)-insensitive lipid peroxidase reactions. Activation of other examined cell functions in eggs activated by sperm or A23187, probably results from Ca(2+)-triggered sequential reactions other than Ca(2+)-caused activation of phospholipase A2.

Acetophenones↗

PubMed1996

Cyclic AMP-dependent protein kinase in ovarian follicle cells of starfish Asterina pectinifera.

Adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) in ovarian follicle cells of the starfish Asterina pectinifera was studied. Protein kinase activity in follicle cell homogenate was activated by cAMP in a dose-dependent manner, and Ka was obtained with 10(-7) M cAMP. The PKA activity required Mg2+ at concentrations between 2 and 10 mM. On Sephacryl S-300 column chromatography of partially purified PKA, Mr of the holoenzyme was estimated to be about 180,000. [2,3-3H]cAMP binding activity also suggested a regulatory subunit of Mr about 50,000. DE-52 column chromatography of the cell extract resolved the enzyme activity into two peaks, which eluted between 0.05 and 0.1 M NaCl (type I), and between 0.15 and 0.25 M NaCl (type II). The type I enzyme was the predominant form of PKA in starfish follicle cells. In a cell-free system, a 70 kDa protein was phosphorylated during incubation with [gamma-32P]ATP in the presence of cAMP. These results suggest that PKA stimulates the phosphorylation of a 70 kDa protein following an increase in the level of cAMP.

Animals↗

PubMed1996

[Myelodysplastic syndrome with multiple chromosome aberrations in a patient with Werner's syndrome].

An unusual case of Werner's syndrome with myelodysplastic syndrome (MDS) in a 63-year-old male is reported. He was transferred to our hospital for evaluation of pancytopenia in August 1994. He had surgery for cataracts at age 47. On admission, his weight was 39 kg and his height was 148 cm. He looked pale, had thin limbs, a bird-like face, loss of hair, a hoarse voice, and atrophic skin with ulcers. A complete blood count showed: hemoglobin 8.7 g/dl; platelet count 1.5 x 10(4)/microliters; and white blood cell count 2,900/microliters with 3% blasts. A bone marrow aspiration revealed hypercellularity with 11.8% blasts and trilineage myelodysplasia. A diagnosis of RAEB was made according to the FAB classification. Cytogenetic analysis of the bone marrow cells revealed multiple aberrations such as 44, XY, -3, -5, add (4) (q?31), add(6) (p2?), del(7) (q22), add(10) (q24), del(12) (q?), add(14) (q32), -15, -16, -17, + mar1, +mar2, +mar3. He died of pneumonia on day 11. Although approximately 700 cases (about 300 cases in Japan) of Werner's syndrome have been reported, the occurrence of hematopoietic malignancies in Werner's syndrome is rare, and the literature includes 8 cases of leukemia and 4 cases of MDS.

Bone Marrow Cells↗

PubMed1995

Endogenous substrate for energy metabolism and ultrastructural correlates in spermatozoa of the sea urchin Diadema setosum.

Energy metabolism in spermatozoa of the sea urchin Diadema setosum of the order Diadematoida was examined. The spermatozoa contained not only several kinds of phospholipids and cholesterol, but also triglyceride (TG). Glycogen and glucose were present at extremely low levels. Following dilution of dry sperm and incubation in seawater, the TG content decreased rapidly. Other lipids, however, remained at constant levels, except for a slight increase in the level of free fatty acid. High lipase activity was demonstrated in the spermatozoa. 14C-labeled fatty acid was oxidized to 14CO2. Ultrastructural study also showed that lipid globules were present at the bottom of the midpiece. After incubation in seawater, morphological changes in the lipid globules were observed and some vacuoles appeared. Thus, the results obtained strongly suggest that D. setosum spermatozoa obtain energy through oxidation of fatty acid from TG stored in the lipid globules at the midpieces.

Animals↗

PubMed1995

All-or-none augmentation of Ca2+ sensitivity in alpha-toxin-permeabilized single smooth muscle cells from guinea-pig taenia caecum.

1. Isolated smooth muscle cells from guinea-pig taenia caecum were permeabilized by use of Staphylococcus aureus alpha-toxin, and the sarcoplasmic reticulum Ca2+ store was depleted by exposure to 0.1 microM A23187. 2. Shortening of alpha-toxin-permeabilized single smooth muscle cells was induced by increasing free Ca2+ but was not induced by 0.2 microM free Ca2+. 3. Shortening of the permeabilized cells was caused by application of acetylcholine (ACh) with free Ca2+ concentration held at 0.2 microM. Permeabilized smooth muscle cells responded to 0.3 microM or 1 microM ACh with 0.2 microM Ca2+ with maximal shortening. The concentration-response relationship to ACh had a very steep slope and the cell shortening appeared to be an all-or-none response rather than a graded response, as was the shortening of intact cells to ACh. 4. The shortening of permeabilized cells was also induced by application of guanosine 5'-triphosphate (GTP) with 0.2 microM free Ca2+, showing an all-or-none response. The threshold concentration of GTP that induced an all-or-none response was between 10 microM and 30 microM. 5. These results suggest that Ca2+ sensitivity is augmented by stimulation of the muscarinic receptor or GTP-binding protein(s) in an all-or-none manner. It seems probable that this contributes to the all-or-none response to ACh in intact smooth muscle cells.

Acetylcholine↗

PubMed1994

Analysis of lung surfactant in the metamorphosing bullfrog (Rana catesbeiana).

In bullfrog (Rana catesbeiana) tadpoles, the lung begins to function at an advanced stage of metamorphosis. As a preliminary step for investigation of the mechanisms involved in lung maturation, pulmonary surfactant was prepared from tadpoles at advanced stages of metamorphosis and its biochemical properties were analyzed. Surfactant phospholipid analysis revealed that the major constituent was phosphatidylcholine (PC), as examined in the animals at late climax (stage 24). Other detectable phospholipids were phosphatidylethanolamine, phosphatidylserine, sphingomyelin, and phosphatidylglycerol, a marker lipid in mammalian surfactant. As in mammals, PC in the surfactant was rich in saturated fatty acids, about 50% of fatty acid moieties being palmitic acid. The content of surfactant PC in the lung increased moderately around mid-climax and markedly at the end of climax. The effect of antiserum against bullfrog prolactin (PRL) on the pulmonary surfactant was studied in climactic tadpoles. The content of surfactant PC in the lung of the antiserum-treated larvae was lower than that in the lung of the normal rabbit serum-injected larvae, whereas the content of PC in the whole lung did not differ between the antiserum-treated and control groups. The results suggest that synthesis of surfactant in the amphibian lung is enhanced as metamorphosis progresses and that PRL is involved in lung maturation.

Animals↗

PubMed1994

G-proteins and adenylyl cyclase in ovarian granulosa cells of amago salmon (Oncorhynchus rhodurus).

The involvement of guanine nucleotide-binding regulatory proteins (G-proteins) and adenylyl cyclase in the gonadotropin stimulation of cAMP was investigated using crude membrane fractions from granulosa cells of amago salmon (Oncorhynchus rhodurus) postvitellogenic ovarian follicles. Although cholera toxin-catalyzed ADP ribosylation occurred in 45- and 50-kDa proteins, only the former was recognized by an antibody against the alpha-subunit of Gs. With pertussis toxin, only the 41-kDa protein was ADP-ribosylated. This 41-kDa protein was recognized by an antibody against the alpha-subunit of Gi. Partially purified chum salmon gonadotropin (SGA) stimulated adenylyl cyclase activity in crude membrane preparations of granulosa cells only in the presence of pertussis toxin in the incubation medium. Adenosine inhibited adenylyl cyclase in the presence of GTP and pertussis toxin reversed it. Unlike SGA, forskolin, which acts upon adenylyl cyclase without G-protein interaction, markedly stimulated adenylyl cyclase activity in the absence of pertussis toxin. These results provide evidence that both stimulatory (Gs) and inhibitory (Gi) regulation by adenylyl cyclase operates in the granulosa cells of amago salmon postvitellogenic ovarian follicles. It is possible that, although a stimulatory receptor interacts with Gs, its activity is influenced by the functional state of Gi.

Adenosine↗

PubMed1994

Energy metabolism of spermatozoa of the sand dollar Clypeaster japonicus: the endogenous substrate and ultrastructural correlates.

Energy metabolism in spermatozoa of the sand dollar-sea urchin Clypeaster japonicus was examined. The spermatozoa contained triglyceride and cholesterol ester besides several kinds of phospholipids and cholesterol. Glycogen and glucose were present at extremely low levels. Following incubation of spermatozoa in seawater, the triglyceride content decreased rapidly. Other lipids, however, remained at constant levels. High lipase activity was demonstrated in the spermatozoa. Also, [1-14C]oleic acid was oxidized to 14CO2. Ultrastructural study showed that lipid globules were present at the bottom of the midpiece. After incubation in seawater, morphological changes in the lipid globules were observed and vacuoles of various sizes appeared near the lipid globules. Thus, it is concluded that C. japonicus spermatozoa obtain energy through oxidation of fatty acid from triglyceride stored in the lipid globules at the midpieces.

Animals↗

PubMed1994

[Desensitization of the m3-muscarinic acetylcholine-receptor in the smooth muscle and the intracellular signal transduction].

Desensitization of the m3-muscarinic acetylcholine-receptor in the smooth muscle of the digestive tract is discussed together with the changes in intracellular signal transduction. Isolated single cells that show an all-or-none contractile response to acetylcholine were desensitized by treatment with 0.1 mM acetylcholine for 10 min, resulting in an increase in the threshold concentration of acetylcholine for contraction, but without changing any of the binding characteristics. Permeabilized cells showed that the desensitization is via uncoupling between the receptor and G-protein. Secretory cells (rat basophil leukemia-2H3 cells) transfected with human m3-receptor showed desensitization when treated with 0.1 mM carbachol for 30 min. The coupling between the receptor and G-protein was not impaired, but some unknown Ca(2+)-independent mechanism may be involved. Smooth muscle tissue was tested for its time-course of desensitization, and a novel transient resensitization was found at 1 min of incubation with 0.1 mM carbachol. This resensitization, and the desensitization prior to it, were accompanied with changes in binding affinity. However, the affinity was not changed, in parallel with desensitization afterwards, but the positive feedback loop of Ca(2+)-influx caused by alkalization via receptor-stimulation was suppressed. After a 30-min treatment, a Ca(2+)-independent mechanism caused the uncoupling and affinity decrease. Treatment for 3 hr increased the number of binding sites without recovery of the response. The desensitizing process is very diverse to achieve selectivity, but its purpose is in unity.

Animals↗

PubMed1993

Prevention of carcinogenicity of anticancer drugs by metallothionein induction.

We examined the efficacy of metallothionein induction in the prevention of the carcinogenic action of cis-platinum and melphalan administered repeatedly to mice over a relatively long period. The increased pulmonary metallothionein induced by bismuth or zinc compounds during the period of chemotherapy with cis-platinum or melphalan protected the mice from carcinogenesis of these drugs in the lung. These results suggested the efficacy of metallothionein inducers in suppression of carcinogenicity considered as a secondary effect of anticancer agents in cancer chemotherapy.

Animals↗

PubMed1993

Lipid globules at the midpieces of Glyptocidaris crenularis spermatozoa and their relation to energy metabolism.

Spermatozoa of the sea urchin, Glyptocidaris crenularis, use endogenous triglycerides (TG), not phosphatidylcholine, to produce energy for swimming. This study was undertaken to examine ultrastructurally the location of TG available for utilization in energy metabolism in these spermatozoa. Each spermatozoon contained several lipid globules at the bottom of the midpiece. Following incubation with sea water, both the number and size of the lipid globules decreased significantly. The total volume of lipid globules in each spermatozoon was roughly halved after 1 h of incubation. Similarly, about half of the TG was metabolized during the same incubation period. Oxygen consumption by spermatozoa during the incubation indicated the oxidation of fatty acid derived from TG. Thus it appears that G. crenularis spermatozoa obtain energy through oxidation of fatty acid from TG stored in the lipid globules within their midpieces.

Animals↗

PubMed1993

All-or-none shortening of isolated single smooth muscle cells from different organs to acetylcholine.

1. Single smooth muscle cells from the guinea-pig taenia caecum and the fundus of guinea-pig stomach were prepared by collagenase digestion under different, mild conditions. 2. Most of the cells either from taenia caecum or from the fundus of stomach responded repeatedly, showing an all-or-none response to acetylcholine (ACh). 3. The threshold concentrations of ACh were different for the cells of the two tissues. Although individual cells showed an all-or-none response to ACh, the average responses of all the cells were graded, like that of whole tissues. 4. Thus, isolated single smooth muscle cells from different tissues and under different conditions responded to ACh in an all-or-none manner such as the twitch observed in skeletal muscle. 5. These results suggest that the isolation of cells reveals the fundamental characteristics of smooth muscle cells as excitable.

Acetylcholine↗

PubMed1993

[All-or-none response of isolated smooth muscle cells from guinea pig taenia caecum to acetylcholine].

Most isolated smooth muscle cells from guinea pig taenia caecum responded repeatedly, showing an all-or-none response to acetylcholine (ACh). However, the average responses of all the cells were graded owing to the difference in the threshold concentration of ACh, like that of whole tissue. The sensitivity of the muscarinic receptor on individual cells was the same as that of whole tissue and ACh bound to the receptor concentration-dependently. The shortening of the cells in response to ACh depended upon the influx of extracellular Ca2+ through the Ca channel. 45Ca2+ influx stimulated by ACh was very sharp and highly correlated with the shortening of the cells. The shortening of alpha-toxin-permeabilized smooth muscle cells was induced by increasing free Ca2+. The concentration-response relationship to free Ca2+ had a very steep slope, and the shortening appeared to be an all-or-none response rather than a graded response. In conclusion, it is suggested that isolated smooth muscle cells show an all-or-none response as a result of a slight increase in the intracellular free Ca2+ level over the threshold concentration when early signalling coupled to ACh-receptor stimulation reaches the threshold to evoke Ca2+ influx and Ca2+ release.

Acetylcholine↗

PubMed1992

Involvement of cyclic adenosine 3',5'-monophosphate in methylation during 1-methyladenine production by starfish ovarian follicle cells.

Resumption of meiosis in starfish oocytes is induced by 1-methyladenine (1-MeAde) produced by ovarian follicle cells under the influence of a gonad-stimulating substance (GSS). With respect to 1-MeAde production, the effect of GSS on follicle cells results in the receptor-mediated formation of cyclic AMP (cAMP). It has also been reported that methylation is involved in 1-MeAde production by GSS. This study was undertaken to determine whether cAMP is the agent responsible for mediating methylation in 1-MeAde biosynthesis by isolated follicle cells of the starfish Asterina pectinifera. Methionine and selenomethionine enhanced 1-MeAde production by GSS in follicle cells. These stimulatory effects were dependent on the GSS concentration. Production of 1-MeAde by GSS was inhibited by ethionine and selenoethionine, competitive inhibitors of methionine. Like GSS, 1-MeAde production induced by concanavalin A, trypsin, and 3-isobutyl-1-methylxanthine (IBMX), which stimulated cAMP accumulation in follicle cells, was influenced by methionine and its related compounds. In contrast, although 1-methyladenosine (1-MeAde-R) induced 1-MeAde production by follicle cells without increasing cAMP levels, methionine and its related compounds had no effect. Use of [methyl-14C]methionine showed that a radiolabel was incorporated into 1-MeAde during incubation with GSS and IBMX, but not with 1-MeAde-R. These results strongly suggest that cAMP plays an important role in the process of methylation during 1-MeAde biosynthesis induced by GSS.

1-Methyl-3-isobutylxanthine↗

PubMed1992

Characterization of the heredity of kininogen deficiency in brown Norway Katholiek strain rats.

Brown Norway rat strain has been studied for mode of inheritance of its congenital deficiency in plasma high molecular weight (HMW)-kininogen and low molecular weight (LMW)-kininogen, and low plasma level of prekallikrein. We examined the genetics of the deficiency by performing a mixed breeding experiment between B/N-Katholiek (B/N-Ka, deficient) and B/N-Kitasato (B/N-Ki, normal) strains. Incidence of the deficiency was judged by the plasma level of HMW-kininogen. Plasma level of HMW-kininogen was around 50% of the normal level in all F1 generations of the hybrid between male B/N-Ka and female B/N-Ki (Exp. 1), and between female B/N-Ka and male B/N-Ki (Exp. 2). Incidence of deficiency (plasma HMW-kininogen level less than 5%) in Exp. 1 was 23.8% in male F2 and 20.0% in female F2 generations. By Exp. 2 also the incidence was 25.0% in male and 30.0% in female F2 generations. There was no significant difference of the incidence between the two experiments or sexes. These results indicate the inheritance of the kininogen-deficiency to be Mendelian autosomal recessive, the same as for the reported cases of human kininogen deficiency. Gel filtration study suggests that prekallikrein in the B/N-Ka plasma may be free form, while that in the B/N-Ki plasma may form complex with HMW-kininogen.

Animals↗

PubMed1992

Fatty chains of alkenylacyl, alkylacyl and diacyl phospholipids in sea urchin spermatozoa.

1. Alkenylacyl, alkylacyl and diacyl phospholipids were analyzed in the spermatozoa of the sea urchin, Hemicentrotus pulcherrimus. 2. Choline phosphoglycerides (CPG) contained alkylacyl component (19%) in addition to the diacyl component (81%), and alkenylacyl analog was present in a trace amount. The ethanolamine phosphoglycerides (EPG) contained alkenylacyl (51%), alkylacyl (2%) and diacyl (47%) components and the serine phosphoglycerides (SPG), alkylacyl (9%) and diacyl (91%) derivatives. 3. Analysis by gas-liquid chromatography indicated that the fatty chain at the 1-position in alkenylacyl, alkylacyl and diacyl compounds of CPG, EPG and SPG was mainly composed of saturated and monoenoic types (16:0, 18:0, 18:1 and 20:1). In contrast, considerable amounts of polyunsaturated types (20:4 and 20:5) were noted at the 2-position.

Animals↗