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Biomedical subjects

K Kusano

Publications and source records attributed to K Kusano.

At least 55 records · Page 3Linked to original sources
PubMed1995

Endogenous bone-resorbing factors in estrogen deficiency: cooperative effects of IL-1 and IL-6.

Estrogen deficiency causes a marked bone loss by stimulating osteoclastic bone resorption. To explore the endogenous bone-resorbing factors involved in estrogen deficiency, we examined the bone-resorbing activity present in the supernatant fraction of mouse bone marrow collected from ovariectomized (OVX) mice. Adding bone marrow supernatants at 20-80% to organ cultures of mouse long bones dose-dependently stimulated bone resorption. The endogenous bone-resorbing activity present in bone marrow supernatants from OVX mice was much higher than that from sham-operated mice 2-4 weeks after surgery, and it was significantly diminished by indomethacin in vitro. Anti-IL-1 alpha antibody completely neutralized the bone-resorbing activity present in bone marrow supernatants from OVX mice. Antibodies against IL-1 beta, IL-6, and IL-6 receptors also neutralized it, but partially. The concentration of IL-1 alpha measured by ELISA was much higher in bone marrow supernatants than in sera, but it was not appreciably changed before or after OVX. The concentration of IL-1 beta in bone marrow supernatants from OVX mice was less than the detection limit. OVX stimulated IL-1 activity in bone marrow supernatants measured by means of the proliferation of thymocytes. However, the level of IL-1 alpha present in bone marrow supernatants from OVX mice was insufficient to stimulate bone resorption. Compared with the serum concentration, bone marrow supernatants contained a much higher level of IL-6 as well, and it was further increased by OVX. However, IL-6 alone present in bone marrow supernatants from OVX mice again did not stimulate bone resorption.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Evidence for conservative (two-progeny) DNA double-strand break repair.

The double-strand break repair models for homologous recombination propose that a double-strand break in a duplex DNA segment is repaired by gene conversion copying a homologous DNA segment. This is a type of conservative recombination, or two-progeny recombination, which generates two duplex DNA segments from two duplex DNA segments. Transformation with a plasmid carrying a double-strand gap and an intact homologous DNA segment resulted in products expected from such conservative (two-progeny) repair in Escherichia coli cells with active E. coli RecE pathway (recBC sbcA) or with active bacteriophage lambda Red pathway. Apparently conservative double-strand break repair, however, might result from successive events of nonconservative recombination, or one-progeny recombination, which generates only one recombinant duplex DNA segment from two segments, involving multiple plasmid molecules. Contribution of such intermolecular recombination was evaluated by transformation with a mixture of two isogenic parental plasmids marked with a restriction site polymorphism. Most of the gap repair products were from intramolecular and, therefore, conservative (two-progeny) reaction under the conditions chosen. Most were conservative even in the absence of RecA protein. The double-strand gap repair reaction was not affected by inversion of the unidirectional replication origin on the plasmid. These results demonstrate the presence of the conservative (two-progeny) double-strand break repair mechanism. These experiments do not rule out the occurrence of nonconservative (one-progeny) recombination since we set up experimental conditions that should favor detection of conservative (two-progeny) recombination.

Bacteriophage lambda↗
PubMed1995

Effects of cofilin on actin filamentous structures in cultured muscle cells. Intracellular regulation of cofilin action.

The previous investigation (Abe et al. (1989) J. Biochem. 106, 696-702) suggested that cofilin is deeply involved in the regulation of actin assembly in developing skeletal muscle. In this study, to examine further the function of cofilin in living myogenic cells in culture, recombinant cofilin having extra Cys residues at the N terminus was produced in Escherichia coli and was labeled with tetramethylrhodamine-iodoacetamide (IATMR). When the cofilin labeled with IATMR (IATMR-cofilin) was introduced into myogenic cells, actin filaments in the cytoplasm or nascent myofibrils were promptly disrupted, and many cytoplasmic rods which contained both IATMR-cofilin and actin were generated. Sarcomeric myofibrillar structures were not disrupted but tropomyosin was dissociated from the structures by the exogenous cofilin, and the IATMR-cofilin became localized in I-band regions. 24 hours after the injection, however, the actin-cofilin rods disappeared completely and the IATMR-cofilin became diffused in the cytoplasm as endogenous cofilin. Concomitantly, actin filaments were recovered and tropomyosin was re-associated with sarcomeric I-bands. At this point, the IATMR-cofilin in the cells still retained the functional activity to form intranuclear actin-cofilin rods in response to stimulation by DMSO just as endogenous cofilin. FITC-labeled actin introduced into myogenic cells at first failed to assemble into filamentous structures in the presence of the exogenous cofilin, but was gradually incorporated into myofibrils with time. The drastic effects of the exogenous cofilin on actin assembly were suppressed by phosphatidylinositol 4,5-bisphosphate (PIP2). These results indicate that the exogenous cofilin is active and alters actin dynamics remarkably in muscle cells, but its activity in the cytoplasm gradually becomes regulated by the action of some factors including PIP2-binding.

Actin Depolymerizing Factors↗
PubMed1995

[Symmetrical thalamic lesions due to perinatal brain damage].

There have been many reports concerning cases with basal ganglionic and thalamic lesions due to neonatal hypoxic-ischemic encephalopathy (HIE), which are considered as a clinicopathological syndrome. But the clinical symptoms and extension of the brain damage varied from case to case. We previously reported seven cases with bilateral basal ganglionic and thalamic lesions (BBTL) due to neonatal HIE. The affected lesions were posterior putamen, lateral thalami, hippocampus and perirolandic cortex. Now, we have experienced two cases who presented symmetrical thalamic lesions (STL) with involvement of the brainstem and periventricular white matter due to neonatal HIE. Of these lesions of the basal ganglia and thalamus, STL comprised calcification and atrophy of the bilateral thalami, and BBTL comprised cystic lesions of the bilateral putamen and thalami. The cerebral lesion in the cases of BBTL were observed in the perirolandic cortex, which is a watershed area of the main vascular territories. While the cerebral lesion in the cases of STL were found in the periventricular white matter, which is a watershed area in premature babies. The brainstem was more severely injured in the case of STL than BBTL. The distribution of the affected lesions suggested that the cases with STL had a different clinicopathological syndrome from those with BBTL. We discussed the developmental mechanism of the cases.

Basal Ganglia Diseases↗
PubMed1995

[Pattern of the brain damages and its developmental mechanisms in monozygotic twins].

Neurologic morbidity is observed more frequently in twins than in singletons. Low birthweight and premature birth are observed more frequently in twins but cannot always explain the cause of cerebral palsy, although circumstances related to twinning may cause brain damage. We attempted to select the patients whose brain damages might be caused by circumstances related to twinning and discussed the developmental mechanisms of their brain damages. We divided the cases into 3 groups. One comprised the monozygotic (MZ) twins whose co-twins had died. The second comprised the MZ twins with the complication of the twin-twin transfusion syndrome or discordant twins. The third comprised the MZ twins with concordant co-twins. We postulated that group I and II had complication of the disturbance of fetal circulation, but group III did not. Hydranencephaly (group I) and polymicrogyria (group II) might develop when the disruption occurs at an intermediate stage of morphogenesis (before the 6th month of gestation). In the cases with cerebral infarction (group I), the distribution of the lesions were related to the main vascular territories of the mature form, might be caused by occlusion of blood vessels associated with intrauterine disseminated intravascular coagulation. In the cases of MCE (group I), multiple cavities are present mainly in the white matter, the gray matter having a tendency to be spared. As the distribution of the cavities is not related to the main vascular territories, it was less likely that the lesions was produced by occluded vessels. The death of one fetus might cause severe blood deprivation, leading to hypoxic-ischemic brain insults in the surviving fetus.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain↗
PubMed1995

[A case of spastic paraparesis, mental retardation and thinning of corpus callosum].

There are several reports of the patients who have peculiar clinical presentation, that is, spastic paraparesis, mental disturbance and thinning of corpus callosum. We presented a sporadic case with similar symptoms. He was born by normal vaginal delivery without problems from consanguineous parents. His development had delayed since his infantile period. He started to have spasticity of the legs at the age 13 years and it worsened with advancing age. On examination at age 19, he was mentally slow, his full-scale IQ being 35. The tone in the arms was slightly increased and marked in the legs. The finger to nose test caused mild dysmetria. Rest of the neurological examinations were normal. MRI demonstrated characteristic thinning of corpus callosum, dominant in anterior portion. In addition, it showed wide cavum vergae and cavum septi pellucidi, and abnormal high intensity in periventricular white matter. The findings of MRI were possibly suggestive of the static developmental disturbance of brain.

Adult↗
PubMed1994

Thymine starvation-induced structural changes in Escherichia coli DNA. Detection by pulsed field gel electrophoresis and evidence for involvement of homologous recombination.

Effect of thymine starvation on Escherichia coli DNA was investigated by using pulsed field gel electrophoresis combined with cell lysis in agarose gel. Post-lysis treatment with restriction enzymes generating relatively large fragments (NheI, SpeI or XbaI) revealed peculiar electrophoretic profiles specific for thymine-starved cells. Thus, a substantial portion of the DNA remained in the origin of electrophoresis (non-migrating DNA), and the amounts of the migrating fragments correspondingly decreased in an inverse relation to the map distance between the origin of replication (oriC) and each fragment. The formation of non-migrating DNA seems to depend upon the presence of replicated portions of the chromosome (sister duplexes), as judged by the effect of a preincubation at the non-permissive temperature in a dnaA(Ts) mutant. Electron microscopy showed that the non-migrating fraction of DNA was enriched with such structures as single-stranded tails or gaps and branchings with single-stranded arms. It was also found that the appearance of non-migrating DNA was highly dependent on the functional recA gene and moderately on certain RecF-family genes. These results strongly suggest that homologous recombination between sister duplexes is involved in the formation of the peculiar structures found in non-migrating DNA. A possible causal relationship between the formation of non-migrating DNA and viability loss (thymineless death) is also discussed.

Biotransformation↗
PubMed1994

DNA double-strand break repair: genetic determinants of flanking crossing-over.

Whether or not homologous interaction of two DNA molecules results in crossing-over of the flanking sequences is an important decision in view of genome organization. Several homologous recombination models, including the double-strand break repair models, explain this decision as choice between two alternative modes of resolution of Holliday-type intermediates. We have demonstrated that a double-strand gap can be repaired through gene conversion copying a homologous duplex, as predicted by the double-strand break repair models, in the RecE pathway of Escherichia coli. This gap repair is often accompanied by crossing-over of the flanking sequences. Mutations in ruvC and recG, whose products interact with Holliday structures in vitro, do not block double-strand gap repair or its association with flanking crossing-over. However, two mutations in the recJ gene, which encodes a single-strand 5'-->3' exonuclease, severely decrease association of flanking crossing-over. Two mutations in the recQ gene, which encodes a helicase, moderately decrease association of flanking crossing-over by themselves and suppress the severe effect of a recJ mutation. Similar relationships of recJ and recQ mutations are observed in cell survival after ultraviolet light irradiation, gamma-ray irradiation, and H2O2 treatment. We discuss how cooperation of the recQ gene product and the recJ gene product brings about double-strand break repair accompanied by flanking crossing-over. We also discuss how this reaction is related to repair of chromosome damages.

Adenosine Triphosphatases↗
PubMed1994

Involvement of RecE exonuclease and RecT annealing protein in DNA double-strand break repair by homologous recombination.

We demonstrated that a double-stranded (ds) gap in DNA is repaired by a gene conversion mechanism in an Escherichia coli recBC sbcA23 strain, as predicted by the ds break repair models for homologous recombination. The sbcA mutation is known to induce several gene products encoded on the Rac prophage present in most strains of E. coli K-12. These include exonuclease VIII (Exo VIII), a 5' to 3' exonuclease working from the end of a duplex DNA, and RecT, an annealing protein. We found that a rac- strain (lacking the Rac prophage) cannot support this repair. A plasmid carrying part of the Rac prophage supported highly efficient ds gap repair activity in a rac- strain, but two ExoVIII+ recT- plasmids did not. The recE159 mutation that blocks ds gap repair was found to be recT+, since these ExoVIII+ recT- plasmids complemented the recE159 mutation in repair of ultraviolet light damage. From these observations, we conclude that both ExoVIII and RecT are essential for ds gap repair. We discuss their possible roles in the ds break repair reaction.

Bacterial Proteins↗
PubMed1994

Monozygous twin with polymicrogyria and normal co-twin.

Monozygous twinning has been associated with brain lesions such as holoprosencephaly, hydranencephaly, multicystic encephalomalacia, and porencephaly. Polymicrogyria is rare but has been reported in autopsied cases. We present a monozygotic twin with complicating twin-twin transfusion syndrome, the donor exhibiting normal psychomotor development and the recipient psychomotor delay associated with the brain anomaly, polymicrogyria. The brain anomaly might be due to blood pressure instability or a circulatory disturbance resulting from increased coagulability due to polycythemia.

Cerebral Cortex↗
PubMed1994

Estrogen deficiency stimulates B lymphopoiesis in mouse bone marrow.

We have found that an estrogen deficiency causes a marked increase in bone marrow cells. To examine the effect of estrogen on hemopoiesis, we characterized the increased population of bone marrow cells after ovariectomy (OVX). In OVX mice, the percentage of myeloid cells and granulocytes was decreased, whereas that of B220-positive B lymphocytes was selectively increased 2-4 wk after surgery. The total number of myeloid cells and granulocytes did not change appreciably, but that of B220-positive cells was greatly increased by OVX. When OVX mice were treated with estrogen, the increased B lymphopoiesis returned to normal. B220-positive cells were classified into two subpopulations, B220low and B220high. The majority of the B220low cells were negative for the IgM mu chain, whereas most of the B220high cells were mu-positive. OVX selectively increased the precursors of B lymphocytes identified by B220low. mu-negative phenotype, suggesting that an estrogen deficiency stimulates accumulation of B lymphocyte precursors. When bone marrow-derived stromal cells (ST2) were pretreated with estrogen then co-cultured with bone marrow cells in the presence of estrogen, the stromal cell-dependent B lymphopoiesis was greatly inhibited. The present study suggests that estrogen plays an important role in the regulation of B lymphocyte development in mouse bone marrow.

Animals↗
PubMed1994

Development of radioimmunoassay for the novel platelet activating factor receptor antagonist, E6123, and its application to pharmacokinetics in laboratory animals.

A direct radioimmunoassay for the determination of E6123, a novel antagonist of platelet activating factor (PAF) receptor, was developed in order to study the pharmacokinetics at low dose. This procedure used [3H]E6123 as the radioligand and an antiserum obtained from rabbits immunized with the hapten covalently bound to bovine serum albumin. M1B, one of the main metabolites of E6123, exhibited cross-reactivity with antisera. But this metabolite had no effect on measurements of E6123, because the amount of M1B in plasma radioactivity after administration of [14C]E6123 to dogs and monkeys was low. The sensitivity limit of this assay was 25 pg/ml of plasma when 0.1 ml of plasma was used and the assay showed good accuracy and high precision. The validity of the radioimmunoassay was demonstrated by comparative analysis of a number of samples after oral and intravenous administration (1.0 mg/kg) by HPLC-UV method (r = 0.972-0.984, slope = 1.0314-1.2143). The pharmacokinetics of E6123 was studied at a dose of 30 micrograms/kg. After intravenous administration, the plasma concentration-time curves in all species fitted a two-compartment model and the terminal half-lives in guinea pigs, dogs and monkeys (both poor and extensive metabolizers) were 4.77, 1.71, 5.34 and 1.07 h, respectively. After oral administration, the maximum plasma concentrations were obtained within 0.83-3.00 h and the half-life for each animal was almost the same as that after intravenous administration. The mean bioavailabilities of E6123 in guinea pigs, dogs and monkeys (poor and extensive metabolizers) were 106.9, 45.7, 59.1 and 22.8%, respectively.

Animals↗
PubMed1994

Prophylactic efficacy of milbemycin oxime against multiple infection of dogs with Dirofilaria immitis.

In order to examine the prophylactic effects of milbemycin oxime (MO) against Dirofilaria immitis infection, experiments were carried out under multiple infection with D. immitis. Ten filaria-free beagles of age 4 to 8 months were each inoculated with a total number of 480 larvae 12 times at intervals of 15 days over a period of 6 months, and MO was given monthly for the 6 months at a dose of 0.25 mg/kg. The infection rate in the medicated group of dogs was nil, this suggesting complete protection of the infection, while in the non-medicated control group it ranged from 6.5 to 14.8% (mean, 11.4%).

Animals↗
PubMed1994

[Epilepsy in patient with structural autosomal abnormality].

Few cases have been reported on the structural autosomal abnormality (SAA) focusing on epilepsy excluding those of Down syndrome and Klinefelter syndrome. We investigated patients who had SAA with special reference to epilepsy. Various types of epilepsy were observed in its severity in our cases as well as previously reported cases. There was no correlation between the degree of mental retardation, motor dysfunction, brain damage on CT scan, and severity of epilepsy. Some cases had brain dysplasia, such as agenesis of corpus callosum, pachygyria, and mega cisterna magna. No correlation was found between these brain dysplasia and severity of epilepsy. It is important for a pediatrician to find a common epileptic syndrome or EEG abnormality in a SAA. An observation of symptoms in patients with the same chromosomal deletion or duplication will lead to identification of responsible gene for an epileptic symptom.

Adolescent↗
PubMed1994

[Clinical consideration of patients with neonatal bilateral basal ganglia-thalamic lesion due to hypoxic ischemic encephalopathy].

We describe herein the clinical symptoms, clinical course and results of investigation of 7 patients with bilateral basal ganglia-thalamic lesions (BBTL). All patients had spastic quadriplegia with rigidity. They were unable to sit and turn over. They could follow objects, turn head towards a sound and recognize parents to some degree. They were all evaluated as having the most severe degree of disability (Oshima's classification 1). They all had dysphagia and 2 patients had a episode of bradycardia and hypothermia, which might be evidences of brain stem disorders. Muscle hypertonia, vomiting, hematemesis and obstructive respiration, which were the major complications for the patients, worsened with age. High percentage of histories of birth asphyxia and poor feeding in the neonatal period suggested that perinatal brain insults might be one of the important factors for developing BBTL. It seemed to be difficult to explain that such diffuse brain injuries in our cases were caused by only the insults during parturition. Brain insults during parturition as well as prenatal factors probably participate in developing BBTL. Although the cerebrum of the patients seem to be relatively preserved in the images of head CT-scan, MRI of the patients revealed diffuse brain lesions. All of five patients tested had an abnormal auditory brain stem response (ABR). These investigations demonstrated that patients with BBTL have diffuse brain damage including brain stem. Further observation is needed to verify the mechanisms of development and the time of onset of BBTL.

Adolescent↗
PubMed1993

Targeted recombination with single-stranded DNA vectors in mammalian cells.

We studied the ability of single-stranded DNA (ssDNA) to participate in targeted recombination in mammalian cells. A 5' end-deleted adenine phosphoribosyltransferase (aprt) gene was subcloned into M13 vector, and the resulting ssDNA and its double-stranded DNA (dsDNA) were transfected to APRT-Chinese hamster ovary cells with a deleted aprt gene. APRT+ recombinants with the ssDNA was obtained at a frequency of 3 x 10(-7) per survivor, which was almost equal to that with the double-stranded equivalent. Analysis of the genome in recombinant clones produced by ssDNA revealed that 12 of 14 clones resulted from correction of the deletion in the aprt locus. On the other hand, the locus of the remaining 2 was not corrected; instead, the 5' deletion of the vector was corrected by end extension, followed by integration into random sites of the genome. To exclude the possibility that input ssDNA was converted into its duplex form before participating in a recombination reaction, we compared the frequency of extrachromosomal recombination between noncomplementary ssDNAs, and between one ssDNA and one dsDNA, of two phage vectors. The frequency with the ssDNAs was 0.4 x 10(-5), being 10-fold lower than that observed with the ssDNA and the dsDNA, suggesting that as little as 10% of the transfected ssDNA was converted into duplex forms before the recombination event, hence 90% remained unchanged as single-stranded molecules. Nevertheless, the above finding that ssDNA was as efficient as dsDNA in targeted recombination suggests that ssDNA itself is able to participate directly in targeted recombination reactions in mammalian cells.

Adenine Phosphoribosyltransferase↗