[Organ lesions and progress in diagnosis of collagen diseases. 4) Lesions of the nervous system].
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Autoimmune Diseases↗
Biomedical subjects
H Kondo
Publications and source records attributed to H Kondo.
Electrochemical analysis of DNA amplified by the polymerase chain reaction with a ferrocenylated oligonucleotide.
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Chromatography, High Pressure Liquid↗
p47 is a cofactor for p97-mediated membrane fusion.
At least two distinct ATPases, NSF and p97, are known to be involved in the heterotypic fusion of transport vesicles with their target membranes and the homotypic fusion of membrane compartments. The NSF-mediated fusion pathway is the best characterized, many of the components having been identified and their functions analysed. In contrast, none of the accessory proteins for the p97-mediated fusion pathway has been identified. Now we have identified the first such component, a protein of relative molecular mass 47,000 (p47), which forms a tight, stoichiometric complex with cytosolic p97 (one trimer of p47 per hexamer of p97). It is essential for the p97-mediated regrowth of Golgi cisternae from mitotic Golgi fragments, a process restricted to animal cells. As a homologue of p47 exists in budding yeast, this indicates that it might also be involved in other membrane fusion reactions catalysed by p97, such as karyogamy.
Adenosine Triphosphatases↗
Cloning and nucleotide sequence of Bacillus stearothermophilus pyruvate carboxylase.
A gene for prokaryotic pyruvate carboxylase (PC) was cloned from Bacillus stearothermophilus. It has an open reading frame of 3441 base pairs which can code for a protein of 128,353 Da. Not only the molecular size and domain organization but also the deduced amino acid sequence of B. stearothermophilus PC are similar to those of eukaryotic PCs.
Amino Acid Sequence↗
Cloning of a novel human diacylglycerol kinase (DGKtheta) containing three cysteine-rich domains, a proline-rich region, and a pleckstrin homology domain with an overlapping Ras-associating domain.
Diacylglycerol kinase (DGK) attenuates levels of second messenger diacylglycerol in cells and produces another (putative) messenger, phosphatidic acid. We have previously purified a 110-kDa DGK from rat brain (Kato, M., and Takenawa, T. (1990) J. Biol. Chem. 265, 794-800). Here we report the cDNA cloning from human brain and retina cDNA libraries. The cDNA encodes a novel DGK isotype, termed DGKtheta, of 941 amino acids with an apparent molecular mass of 110 kDa. DGKtheta contains a C-terminal putative catalytic domain, which is present in all eukaryotic DGKs. In contrast to other DGK isotypes, DGKtheta contains three cysteine-rich domains instead of two. The third cysteine-rich domain is most homologous to the second one in other DGK isotypes. This particular sequence homology extends C-terminally beyond the typical cysteine/histidine core structure and is DGK-specific. DGKtheta furthermore contains various domains for protein-protein interaction, such as a proline- and glycine-rich domain with a putative SH3 domain-binding site and a pleckstrin homology domain with an overlapping Ras-associating domain. DGKtheta is expressed in the brain and, to a lesser extent, in the small intestine, duodenum, and liver. In situ hybridization of DGKtheta mRNA in adult rat brain reveals high expression in the cerebellar cortex and hippocampus. DGKtheta activity in COS cell lysates is optimal toward diacylglycerols containing an unsaturated fatty acid at the sn-2 position.
Amino Acid Sequence↗
Gene cloning and characterization of CDP-diacylglycerol synthase from rat brain.
A cDNA encoded a 462-amino acid protein, which showed CDP-diacylglycerol synthase (CDS) activity was cloned for the first time as the vertebrate enzyme molecule from rat brain cDNA library. The deduced molecular mass of this rat CDS was 53 kDa, and putative primary structure included several possible membrane- spanning regions. At the amino acid sequence level, rat CDS shared 55.5%, 31. 7%, and 20.9% identity with already known Drosophila, Saccharomyces cerevisiae, and Escherichia coli CDS, respectively. This rat CDS preferred 1-stearoyl-2-arachidonoyl phosphatidic acid as a substrate, and its activity was strongly inhibited by phosphatidylglycerol 4, 5-bisphosphate. By immunoblotting analysis of COS cells overexpressed with the epitope-tagged for rat CDS, a 60-kDa band was detected. By epitope-tag immunocytochemistry, the CDS protein was mainly localized in close association with the membrane of the endoplasmic reticulum of the transfected cells. The intense mRNA expression of CDS was localized in the cerebellar Purkinje cells, the pineal body, and the inner segment of photoreceptor cells. Additionally, very intense expression was detected in postmitotic spermatocytes and spermatids.
Amino Acid Sequence↗
Glial tau-positive structures lack the sequence encoded by exon 3 of the tau protein gene.
Tau protein comprises six distinct isoforms defined by the presence or absence of sequences encoded by alternatively spliced exon 2, 3 and 10. We have investigated immunohistochemically the expression of exon 3-derived fragment (E-3) of tau protein in brains of patients with Alzheimer's disease (AD) and other neurodegenerative diseases in which the abnormal accumulation of tau protein takes place. In AD, a subset of neurofibrillary tangles, neuropil threads and dystrophic neurites in senile plaques were stained positively with an anti-E-3 antibody. In sharp contrast, glial tau-positive structures, such as astrocytic plaques and oligodendroglial coiled bodies, were negative for E-3 in all cases examined in this study. This is the first report to discriminate tau-positive inclusions in glial cells from those in neurons at the molecular level.
Aged↗
Ca2+ overloading causes the negative inotropic effect of doxorubicin in myocytes isolated from guinea-pig hearts.
We reported previously that conditions shown to increase Ca2+ loading augment the negative inotropic effect of doxorubicin: To examine if the negative inotropic effect is caused by Ca2+ overloading doxorubicin-induced changes in diastolic and systolic Ca2+ concentrations and twitch contractions were studied under altered Ca2+ loading. Intracellular Ca2+ concentrations ([Ca2+]i) were monitored in fura-2-loaded myocytes isolated from the ventricular muscle of guinea-pig hearts. Twitch contractions were estimated from the shortening of myocytes. In myocytes incubated at 34 degrees C a medium containing 1.2 mM CaCl2 (standard conditions), doxorubicin (100 microM) caused a significant decrease in diastolic length, and an increase in the amplitude of contraction. The positive inotropic effect of doxorubicin was followed by a negative effect. Concomitant with these changes in myocyte contractions, a monophasic increase in diastolic Ca2+ and an increase and a subsequent decrease in the amplitude of Ca2+ transients (peak [Ca2+]i minus diastolic [Ca2+]i) were observed. When the Ca2+ load of myocytes was increased by an incubation at a low temperature (25 degrees C) or in the presence of high Ca2+ in the incubation medium (2.4 mM CaCl2), diastolic [Ca2+]i was elevated. Doxorubicin further increased diastolic [Ca2+]i. Under these conditions, the doxorubicin-induced increase in the twitch contraction lasted a shorter period of time and the subsequent decrease in contraction was significantly enhanced. The peak [Ca2+]i initially increased slightly and then decreased. Thus, the decrease in the amplitude of Ca2+ transients, as well as myocyte contraction was greater compared to the corresponding values observed under the standard conditions. These changes in the contraction and Ca2+ transient developed with the same time course. The effects of the low temperature incubation were antagonized by verapamil. These results indicate that the negative inotropic effect of doxorubicin results from a decrease in the amplitude of Ca2+ transients caused by an increased diastolic [Ca2+]i and a decreased peak [Ca2+]i. These changes are likely to be caused by myocardial Ca2+ overload.
Animals↗
Neuropathology of sporadic amyotrophic lateral sclerosis of long duration.
We performed post-mortem examinations of three patients with progressive neurogenic amyotrophy of long duration. One patient had been clinically diagnosed as having sporadic amyotrophic lateral sclerosis (ALS) and two had been diagnosed with progressive spinal muscular atrophy (PSMA). The disease durations were 10, 17 and 20 years, respectively, and all of the patients died of respiratory failure with no artificial respiratory support. In all of the patients, both the upper and lower motor neuron systems were affected; degeneration of the former was definite, but was milder than that usually encountered in sporadic ALS patients, and the histopathology of the latter was identical to that of sporadic ALS. Light microscopy revealed Bunina bodies, which are characteristic of sporadic ALS, in the remaining anterior horn cells of each patient. In addition, ubiquitin-positive skein-like inclusions were also identified, immunohistochemically, in the remaining anterior horn cells of each patient. Neuron counts indicated that the number of neurons was preserved in Clarke's column in these patients, but was significantly reduced in the intermediolateral nucleus, compared with control subjects. Based on these findings, we think that these three patients, with long disease durations, were affected by essentially the same underlying disease process as that of sporadic, classical ALS. Moreover, we question the neuropathological occurrence of sporadic ALS without involvement of the upper motor neuron system, namely, pure PSMA or lower motor neuron disease.
Aged↗
Novel regulation of delta-aminolevulinate synthase in the rat harderian gland.
The mode of expression of delta-aminolevulinate synthase (ALAS), as well as that of mRNAs for other heme pathway enzymes, was examined in the rat Harderian gland. Northern blot and in situ hybridization analyses demonstrated that the non-specific ALAS (ALAS-N) mRNA is highly expressed in this tissue, whereas the erythroid-specific ALAS (ALAS-E) mRNA is not. Immunoblot analysis of ALAS also confirmed this finding at the protein level. ALAS-N mRNA was maximally induced in the Harderian gland and was not increased further by treatment of animals with 2-allyl-2-isopropylacetamide (AIA). The levels of mRNAs for other heme pathway enzymes, i.e., delta-aminolevulinate dehydratase, porphobilinogen deaminase, uroporphyrinogen decarboxylase, and coproporphyrinogen oxidase, also were increased markedly in the Harderian gland and not influenced by AIA treatment. The level of ferrochelatase (FeC) mRNA in the gland was, however, lower than that in the liver. The gland contained an extremely high level of protoporphyrin, while heme was undetectable. Microsomal heme oxygenase-1 (HO-1) mRNA levels were significantly higher in the Harderian gland than in the liver. When isolated glands were incubated with hemin in vitro in organ cultures, the level of HO-1 mRNA was increased, whereas the ALAS-N mRNA level was not. These findings indicate that markedly elevated levels of protoporphyrin and extremely low levels of heme in the Harderian gland are the results of both decreased expression of FeC and markedly increased expression of ALAS-N and HO-1. The constitutive expression of the ALAS-N gene in the Harderian gland suggests a novel transcriptional control mechanism of this gene.
5-Aminolevulinate Synthetase↗
Detection of K-ras gene mutations at codon 12 in the pancreatic juice of patients with intraductal papillary mucinous tumors of the pancreas.
BACKGROUND: The authors previously found specific mutations of the K-ras gene at codon 12 in the pancreatic juice of 67% of patients (6 of 9) with pancreatic ductal carcinoma, and the detection of these mutations was useful for diagnosis. This study was performed to detect and evaluate K-ras mutations in pancreatic juice from patients with intraductal papillary mucinous tumor of the pancreas, which is considered a low grade malignancy. The results were interpreted from the viewpoint of clinical significance. METHODS: K-ras mutations were examined using seminested polymerase chain reaction analysis combined with restriction enzyme digestion, followed by nonradioisotopic single strand DNA conformation polymorphism. RESULTS: Twelve of thirteen cases (92%) of intraductal papillary mucinous tumor of the pancreas, confirmed histologically (9 adenomas and 4 carcinomas), and 26 of 43 cases (60%) of ductal carcinoma showed specific K-ras gene mutations in the pancreatic juice. Furthermore, 4 of 22 patients (18%) with chronic pancreatitis, followed for more than 1 year without a sign of pancreatic tumor, showed K-ras mutations. In contrast, no mutations of the K-ras gene were detected in the pancreatic juice from 28 normal controls. CONCLUSIONS: K-ras mutations were found in the pancreatic juice of all but one patient with intraductal papillary mucinous tumor of the pancreas, but they were not useful for distinguishing carcinoma from adenoma. The authors concluded that K-ras mutations are not a specific marker for pancreatic neoplasms because similar mutations were detected in the pancreatic juice from patients with chronic pancreatitis. At the present time, the detection of K-ras mutations in pancreatic juice should be used clinically as an adjunct diagnostic modality for pancreatic diseases.
Adenocarcinoma, Mucinous↗
Inhibition of UDP-glucuronosyltransferase activity by fatty acyl-CoA. Kinetic studies and structure-activity relationship.
We previously identified and purified UDP-glucuronosyltransferase (UGT) isoforms as targets of protein acylation from rat liver microsomes (Yamashita et al., Biochem J 312: 301-308, 1995). The acylation of UGT isoforms occurred upon incubation with acyl-CoA without another protein acyltransferase, suggesting that it was autoacylation. The study revealed the interaction of UGT isoforms with acyl-CoA. In the present study, the effects of fatty acyl-CoA on UGT activities were examined thoroughly, using a rat liver microsomal and purified enzyme fractions. The UGT activities of both fractions were inhibited by acyl-CoA in a concentration-dependent manner. The effect of acyl-CoA was observed on the activities toward various substrates, suggesting that the effect shows the wide spectrum of the isoforms of UGT. To assess the mechanism underlying the inhibition of UGT activity by acyl-CoA, the relationship of the inhibition, acyl-CoA binding to the proteins, and changes in the tertiary structure of the enzyme were examined. The kinetics of these phenomena were related closely with each other. Furthermore, the inhibition of UGT activity was specified for acyl-CoA, though a structurally related compound, acyl-3-dephosphoCoA, had no inhibitory effect. The results suggested that the specific binding of acyl-CoA to UGT isoforms induced conformational changes of the enzymes and resultant inhibition of UGT activity.
Acyl Coenzyme A↗
Changes in gene expression for skin-type fatty acid binding protein in hypoglossal motor neurons following nerve crush.
The changeability in the gene expression for five species of fatty acid binding protein (FABP) was investigated in the crushed hypoglossal nucleus by in situ hybridization histochemistry. Increased gene expression for skin-type fatty acid binding protein (S-FABP) was evident in the affected hypoglossal neurons on the 3rd day after nerve crush, and it lasted until the postoperative day 14. On the other hand, no significant gene expression for heart-, liver-, intestinal- or brain-type FABPs was detected in the hypoglossal neurons of normal control or in these neurons for 3 weeks after the nerve crush. These findings suggest that skin-type FABP may selectively contribute to some important roles in morphological and biochemical changes of neuronal cells associated with the nerve regeneration.
Animals↗
Studies on selectin blockers. 4. Structure-function relationships of sulfated sialyl Lewis X hexasaccharide ceramides toward E-, P-, and L-selectin binding.
In order to clarify the real ligand structure of L-selectin proposed by Rosen et al., we first synthesized 6-sulfated sLe(x) hexasaccharide ceramide 1, 6'-sulfated sLe(x) hexasaccharide ceramide 2, and 6,6'-disulfated sLe(x) hexasaccharide ceramide 3 and examined their binding avidities for L-selectin. As a result, we found that the 6'-sulfated sLe(x) hexasaccharide ceramides 1-3 have similar binding avidities to L-selectin and their binding to L-selectin appeared somewhat stronger than that of sLe(x). For P-selectin, the sulfated sLe(x) derivatives 1-3 showed a similar avidity to sLe(x). On the other hand, 6-sulfated sLe(x) 2 was recognized to E-selectin and the binding avidity was apparently weak as compared to that of sLe(x) hexasaccharide ceramide. Surprisingly, 6'-sulfated and 6,6'-disulfated sLe(x)s 1 and 3 did not bind to E-selectin at all. We constructed the E-selectin-sLe(x) complex model and investigated the binding mode. Namely, the galactose 6'-position was directed toward the negatively charged residues, Glu80 and Asp100. Our results with E-selectin indicate that the replacement of 6'-OH position from anionic charged group to cationic charged one, e.g., amino groups, could have a marked affect on E-selectin recognition. These results could provide useful information for the drug design of selectin blockers.
Animals↗
Selectin-ligand interactions revealed by molecular dynamics simulation in solution.
Through a computer modeling and simulation technique, we investigated the binding mode of a complex of E-selectin-GSC-150, which is a novel selectin blocker. GSC-150 is the 3'-sulfated Lewis X derivative with a long, branched alkyl chain. Initial attempts to construct a model for E-selectin-GSC-150 complex were performed based on a previously reported model of E-selectin-sialyl Lewis X (sLex) complex [Kogan, T.P.; Revelle, B.M.; Tapp, S.; Scott, D.; Beck, P.J.J. Biol. Chem. 1995, 270, 14047-14055]. In our model, the carbohydrate portion of GSC-150 interacted with the protein in a similar manner as that of sLex reported previously. Interestingly, each of the branched alkyl chains extended on the surface of E-selectin and interacted with two different hydrophobic portions. One of these hydrophobic portions consists of Tyr44, Pro46, and Tyr48. Another portion forms a shallow cavity, and it consists of Ala9, Leu114, and the alkyl moieties of the side chains of Lys111, Lys112, and Lys113. A subsequent 200-ps molecular dynamics simulation in solution revealed that the interactions involved in the sugar portion of the ligand were relatively weak, whereas the hydrophobic interactions involved in the branched alkyl chains were fairly stable in solution. These results suggest that the branched alkyl chain serves as an "anchor" for the tight binding of GSC-150 on the surface of E-Selectin. This is the first attempt to evaluate the dynamics of E-Selectin-ligand interactions in solution, and it sheds light on the nature of ligand recognition by selectins.
Carbohydrate Sequence↗
Attenuation of monosodium urate crystal-induced arthritis in rabbits by a neutralizing antibody against interleukin-8.
Accumulating evidence implicates interleukin-8 (IL-8) as an essential mediator in neutrophil-mediated acute inflammation. Neutrophils have also been shown to have a crucial role in the pathogenesis of acute gouty arthritis. Thus, we investigate the pathophysiological role of IL-8 in an experimental model of acute gout, monosodium urate (MSU) crystal-induced arthritis in rabbits. The injection of MSU crystals into knee joints caused a marked swelling of joints. Concomitantly, the infiltration ofleukocytes, mostly neutrophils, was observed in synovial membrane and synovial fluids. The injection of MSU crystals also induced an elevation in synovial fluid IL-8 levels preceding neutrophil infiltration into synovial fluids, without an accompanying increase in plasma IL-8 levels. Immunoreactive IL-8 protein was detected in synovial lining cells at 12-24 h after the injection. IL-8 protein was also observed in infiltrated leukocytes in synovium as early as 3-24 h after the injection. Finally, the intraarticular injection of a neutralizing anti-IL-8 antibody significantly attenuated the crystal-induced joint swelling that occurred at 12 h, and neutrophil infiltration into arthritic joints at 12 and 24 h after the induction. These results provide evidence on the pathogenic roles of locally produced IL-8 in MSU crystal-induced gouty arthritis.
Animals↗
Rapid induction of neutrophil apoptosis by sulfasalazine: implications of reactive oxygen species in the apoptotic process.
Accumulating evidence indicates that neutrophils are crucially involved in the pathogenesis of inflammatory bowel diseases and rheumatoid arthritis. We therefore investigated the effect of sulfasalazine (SSZ), which is widely used in the treatment of these diseases, on neutrophil apoptosis in vitro. The apoptosis of neutrophils was determined by morphology, a DNA histogram of propidium iodide-stained nuclei, and DNA fragmentation. SSZ rapidly accelerated the rate of spontaneous neutrophil apoptosis within clinically relevant concentrations. This effect is unique to neutrophils because other types of leukocytes and a number of leukocyte cell lines are resistant to SSZ. Neutrophil apoptosis caused by SSZ was abrogated by a tyrosine kinase inhibitor, a protein kinase A inhibitor, and antioxidants. The subsequent results provided pharmacological evidence that the phosphorylation of tyrosine kinase and protein kinase A and generation of reactive oxygen species are involved in SSZ-mediated neutrophil apoptosis. These data suggest that SSZ-induced neutrophil apoptosis may account, in part, for the clinical benefits of SSZ on inflammatory bowel diseases and rheumatoid arthritis.
Anti-Inflammatory Agents↗
Crystallization and preliminary X-ray studies of two serine proteinase inhibitors, BGIA and BGIT, from the seeds of bitter gourd.
Two serine proteinase inhibitors from seeds of the bitter gourd, BGIA (bitter gourd inhibitor against acidic amino acid-specific proteinase of Streptomyces griseus) and BGIT (bitter gourd trypsin inhibitor), were crystallized for X-ray structure determination. Crystals of BGIA belong to the monoclinic space group C2 with cell dimensions of a = 54.0 A, b = 23.7 A, c = 47.9 A, and beta = 105.4 degrees, and diffracted X-ray up to 1.5 A resolution. Crystals of BGIT belong to the triclinic space group P1 with cell dimensions of a = 22.8 A, b = 23.5 A, c = 28.4 A, alpha = 93.1 degrees, beta = 99.6 degrees, and gamma = 101.0 degrees, giving X-ray diffraction of over 1.2 A resolution. Intensity data of BGIA and BGIT crystals were collected using synchrotron radiation up to 1.7 and 1.4 A, respectively.
Crystallization↗