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M Kitagawa

Publications and source records attributed to M Kitagawa.

At least 19 recordsLinked to original sources
PubMed1995

Differences in substrate specificity between Cdk2-cyclin A and Cdk2-cyclin E in vitro.

Cyclin-dependent kinase 2 (Cdk2), when bound to either cyclin A or cyclin E, recognizes the Ser/Thr-Pro-X-basic amino acid (motif A) as a phosphorylation site. In this study, we designed several peptides based on motif A and examined the substrate specificity of Cdk2-cyclin A and Cdk2-cyclin E using these peptides. Peptides containing a proline residue in the sequence Pro-X-Thr-Pro-X-basic amino acid (motif B) had higher affinity for both Cdk2 complexes than peptides containing motif A. Furthermore, differences in substrate affinity between the two Cdk2 complexes were caused by a proline residue adjacent to or three positions before the threonine residue. Similarly, the presence of different basic amino acids in motif B also had different effects on affinity for each complex. We demonstrate the possibility that the substrate specificity of Cdk2 bound to cyclin might be regulated by the species of cyclin.

Amino Acid Sequence↗
PubMed1995

Purification and characterization of a fatty acid-activated protein kinase (PKN) from rat testis.

PKN, a novel protein kinase with a catalytic domain homologous to that of the protein kinase C (PKC) family and unique N-terminal leucine-zipper-like sequences, was identified by molecular cloning from a human hippocampus cDNA library [Mukai and Ono (1994) Biochem. Biophys. Res. Commun. 199, 897-904]. Recently we partially purified recombinant PKN from COS7 cells transfected with the cDNA construct encoding human PKN, and demonstrated that the recombinant PKN was activated by unsaturated fatty acids and limited proteolysis [Mukai, Kitagawa, Shibata et al. (1994) Biochem. Biophys. Res. Commun. 204, 348-356]. The present work has focused on the further purification and characterization of PKN from native rat tissue. Immunochemical measurement revealed that PKN was found in every tissue, and was especially abundant in testis, spleen and brain; subcellular fractionation of rat brain showed that half of the PKN was localized in the soluble cytosolic fraction. PKN was purified approx. 8000-fold to apparent homogeneity from the cytosolic fraction of rat testis by DEAE-cellulose chromatography, ammonium sulphate fractionation and chromatography on butyl-Sepharose, heparin-Sepharose, Mono Q and protamine-CH-Sepharose. The enzyme migrates as a band of apparent molecular mass 120 kDa. Using serine-containing peptides based on the pseudosubstrate sequence of PKC-delta as phosphate acceptors, the kinase activity was stimulated several-fold by 40 microM unsaturated fatty acids or by detergents such as 0.04% sodium deoxycholate and 0.004% SDS. In the absence of modifiers, protamine sulphate, myelin basic protein and synthetic peptides based on the pseudosubstrate site of PKCs or ribosomal S6 protein were good substrates for phosphorylation by the kinase. In the presence of 40 microM arachidonic acid the kinase activity of PKN for these phosphate acceptors was increased 2-18-fold. The autophosphorylation activity of purified PKN was partially inhibited by pretreatment with alkaline phosphatase. These properties appear to distinguish PKN from many protein kinases isolated previously.

Amino Acid Sequence↗
PubMed1995

Cell-free transmission of Fv-4 resistance gene product controlling Friend leukemia virus-induced leukemogenesis: a unique mechanism for interference with viral infection.

Fv-4 is a mouse gene that dominantly confers resistance to infection by ecotropic murine leukemia virus (MuLV). We previously demonstrated that mixed radiation bone marrow chimeras containing Fv-4r-bearing BALB/c-Fv-4Wr (C4W) bone marrow and Fv-4r-bearing C3H/He (C3H) bone marrow grafted into C3H recipient mice (C4W+C3H-->C3H) were resistant to Friend leukemia virus (FLV)-induced leukemogenesis, even when they contained as high as 70% C3H-derived cells. This indicates that FLV-sensitive C3H-derived cells are rendered refractory to infection and/or transformation with FLV when they coexist in mice with Fv-4r-bearing cells. To investigate the mechanism of Fv-4 resistance to FLV-induced leukemogenesis, we first examined the expression of Fv-4r env antigen in the peripheral blood mononuclear cells (PBMC) of these chimeras. The Fv-4r env antigen was present not only on C4W-derived cells, but also on Fv-4r-bearing C3H-derived cells in C4W+C3H-->C3H mixed bone marrow chimeras. The Fv-4r env antigen that binds to the cells surface of C3H cells was found in sera from normal C4W mice, C4W-->C3H chimeras, and C4W+C3H-->C3H mixed chimeras. The serum Fv-4r env antigen binds to ecotropic MuLV receptors, shown by specific binding to transfectant mink cells expressing ecotropic MuLV receptor, but not to parental mink cells. To determine whether the binding of Fv-4r env antigen to the putative MuLV receptors would block FLV infection, C3H thymocytes or spleen cells that had been preincubated with C4W serum were mixed with FLV and the subsequent production of MuLV specific antigens was examined. C3H thymocytes or spleen cells treated with C4W serum became refractory to binding by FLV. These results provide evidence that the Fv-4r env antigen is released from C4W-derived cells in vivo and binds to cells expressing surface receptors for ecotropic MuLV, thereby protecting them from infection with FLV. The implication of these findings for gene therapy of retrovirus-induced disease such as acquired immune deficiency syndrome (AIDS) is discussed.

Animals↗
PubMed1995

The interactions of E2F with pRB and with p107 are regulated via the phosphorylation of pRB and p107 by a cyclin-dependent kinase.

It has been postulated that the product (pRB) of the retinoblastoma gene dissociates from the E2F-pRB complex upon phosphorylation by cyclin-dependent kinase(s) (cdk). However, there is no direct evident for the regulation of formation of the E2F-pRB complex via phosphorylation by purified cdk. Therefore, we investigated the regulation of formation of this complex by phosphorylation using pRB and purified cyclin A-cdk2, cyclin E-cdk2 or cyclin D1-cdk4. Purified pRB was incubated with nuclear extracts prepared from pRB-defective cells and then subjected to gel mobility shift assays. We confirmed that unphosphorylated pRB associated with various types of E2F but pRB has been phosphorylated by cyclin A-cdk2 did not. We found that E2F-pRB complexes were disrupted as a consequence of phosphorylation by cyclin A-cdk2, and the levels of the free forms of E2Fs increased. We also found that not only the E2F-pRB complexes but also the E2F-p107 complexes were disrupted upon phosphorylation by cyclin A-cdk2. Furthermore, E2F-pRB complexes were disrupted through phosphorylation by cyclin D1-cdk4 and cyclin E-cdk2, as well as by cyclin A-cdk2. These results clearly demonstrate that the phosphorylation of pRB and p107 by cdks regulates the formation of complexes between E2F and pRB or p107.

Base Sequence↗
PubMed1995

Xenopus PKN: cloning and sequencing of the cDNA and identification of conserved domains.

cDNA clone encoding Xenopus laevis PKN has been isolated from Xenopus kidney library. Sequencing of this clone has revealed a single open reading frame encoding a protein of 901 amino acids. Immunoprecipitate from cytoplasmic fraction of COS7 cells transfected with this cDNA construct using antiserum against bacterially expressed Xenopus PKN revealed arachidonic acid-dependent autophosphorylation activity. Comparison of the closely related sequences of human and rat PKN with a protein from evolutionarily distant Xenopus, revealed several highly invariant domains in the NH2-terminal regulatory regions, suggesting that they participate in binding interaction with arachidonic acid.

Amino Acid Sequence↗
PubMed1995

A variant form of cyclin-dependent kinase 2 (Cdk2) in a malignantly transformed rat thyroid (FRTL-Tc) cell line.

Cyclin-dependent kinase 2 (Cdk2) controls the transition from the G1 to the S phase in the mammalian cell cycle. We found by immunoblotting that anti-Cdk2 antibodies recognize three Cdk2 proteins (of 33, 34 and 39 kDa) in FRTL-5 and FRTL-Tc cells (malignantly transformed FRTL cells). Although 33 kDa protein is a phosphorylated form of 34 kDa protein previously reported, the nature of 39 kDa protein is unknown. In order to determine the nature of this protein, we screened a FRTL-5 cDNA library. Two cDNA clones of the rat homologue (rat Cdk2-alpha and -beta) of human Cdk2 were isolated. The open reading frame of rat Cdk2-alpha cDNA encoded a protein with 428 amino acids and has a high degree of conservation with human Cdk2. The calculated molecular weight of Cdk2-alpha protein is 33892 Da. The rat Cdk2-beta cDNA was identical to Cdk2-alpha cDNA except that it had extra 144 bp; this coincided with insertion of 48 amino acids into Cdk2-alpha protein between Met 196 and Val 197. The calculated molecular weight of Cdk2-beta protein is 39087 Da. Northern blot analysis indicated that the sizes of rat Cdk2-alpha and -beta mRNAs are approximately 2.5 kb and 3.0 kb, respectively. Partial proteolytic mapping showed that Cdk2-beta gene product is 39 kDa Cdk2 in the immunoblotting. We also found that Cdk2-beta protein binds to cyclin A and suc1 proteins. During G1-S phase in FRTL-Tc cells, Cdk2-alpha protein level is constant, but is gradually phosphorylated. In contrast, the level of Cdk2-beta protein increases through the S phase and decreases at the early G2 phase. These results suggest that a variant form of Cdk2 protein might be required for entry into the S phase of the cell cycle in FRTL-Tc cells.

Amino Acid Sequence↗
PubMed1995

Possible involvement of the transcription factor ISGF3 gamma in virus-induced expression of the IFN-beta gene.

Two virus-inducible transcription factors, IRF-1 and IRF-2 have been identified as an activator and a repressor, respectively, of the type I interferon (IFN) genes. Recent studies with mice carrying null mutations for the IRF-1 or IRF-2 alleles have revealed the existence of IRF-1-dependent and -independent pathways mediating IFN-beta gene induction. Here we report that the expression of an IRF family member ISGF3 gamma is induced upon viral infection in IRF-1-/-, IRF-2-/- embryonic fibroblasts. Furthermore, ISGF3 gamma can bind to a virus-inducible promoter element in the IFN-beta gene. These results suggest that ISGF3 gamma or complex containing ISGF3 gamma is involved in the IRF-1-independent pathway mediating IFN-beta gene regulation.

Amino Acid Sequence↗
PubMed1995

Phosphorylation of E2F-1 by cyclin A-cdk2.

Transcription factor E2F-1 has a putative consensus sequence for phosphorylation by cyclin dependent kinase (Ser-Pro-X-Lys/Arg). Therefore, we studied the phosphorylation of E2F-1 in vivo and in vitro and its biological functions. E2F-1 was prepared by immunoprecipitation with anti-E2F-1 antibody from IMR32 lysates and was effectively phosphorylated by human cyclin A-cdk2 which was expressed in insect cells using baculovirus system. GST-E2F-1 was phosphorylated by cyclin A-cdk2 more efficiently than by cyclin E-cdk2. Cyclin D1-cdk4 phosphorylated pRB but scarcely phosphorylated GST-E2F-1 or H1 histone. The 60 kd protein precipitated with anti-E2F-1 antibody was phosphorylated in vivo. Phospho-peptide mapping indicated that its cleavage profile was identical with that of E2F-1 phosphorylated by cyclin A-cdk2 in vitro. This 60 kd protein, which is likely to be E2F-1, was not phosphorylated during the G0 and early G1 phase. Phosphorylation of E2F-1 began from the S phase while phosphorylation of pRB started nearly at G1/S. The in vivo phosphorylation of E2F-1 was inhibited by butyrolactone I, a cyclin-dependent kinase inhibitor (Kitagawa et al., 1993, Oncogene, 8, 2425-2432). The binding of E2F-1 to E2 promoter was found to be reduced by phosphorylation of E2F-1 by cyclin A-cdk2, suggesting that phosphorylation of E2F-1 may induce shut off of gene expression at the transcriptional level. These results suggest that E2F-1 is phosphorylated by cyclin A-cdk2 in the S phase in vivo as well as in vitro and that its phosphorylation by cyclin A-cdk2 may modulate its activity.

Baculoviridae↗
PubMed1995

Targeted disruption of the neurotrophin-3 gene with lacZ induces loss of trkC-positive neurons in sensory ganglia but not in spinal cords.

We have replaced the NT-3 gene with Escherichia coli-derived lacZ gene by means of homologous recombination in embryonic stem cells and thus produced null mutant mice. Mice homozygous for this mutation developed to birth, but most of them could not suck well and died within 2 days after birth. The surviving homozygous mutant mice displayed movement disorder similar to ataxia. The expression of lacZ was widely distributed in the target tissues of peripheral nerves, spinal motor neurons, lumbar dorsal root ganglia and trigeminal ganglia during the prenatal periods. A neuroanatomical examination revealed that there was marked cell reduction present in trigeminal and lumbar dorsal root ganglia in the developing homozygous mutant mice. In these tissues, the expression of trkC, a high-affinity receptor for NT-3, was markedly reduced. In contrast, we did not find any morphological abnormalities, significant cell loss or decreased levels of trkC expression in the motor neurons present in the ventral horn of the spinal cord. These results indicate that the absence of the NT-3 gene leads to a defect in the sensory nervous system, but it may be complemented by other neurotrophins in the motor nervous system during the development.

Animals↗
PubMed1995

Clinicopathological study of primary malignant tumors of the lung: an analysis of 993 tumors resected at the Kanazawa University Hospital between 1979-1993.

A retrospective study was conducted of 993 malignant tumors of the lung in 977 patients. The male-female sex ratio was 2.6:1. The age distribution of the males did not differ from that of the females and the overall mean age was 64.3 +/- 9.5 years (range, 13-85 years). Forty-three percent of the patients were in their seventh decade. The location was peripheral in 81% of the tumors and central in 19%. Of the central tumors, 85% were squamous cell (epidermoid) carcinoma (SCC), while 58% of the peripheral tumors were adenocarcinoma (ADENO). In males, 49% of all tumors were SCC and 36% ADENO, while in females 76% were ADENO. The incidence of positive lymph node metastasis increased in parallel with the pT status in both central and peripheral carcinomas. However, it was noted that, of peripheral lung carcinomas, no SCC with a diameter of 15 mm or less had lymph node metastasis, whereas 18.9% of the ADENO of this size were positive for lymph node metastasis. These results indicate that lymph node metastasis can occur even in small-sized peripheral ADENO less than 15 mm. Thus, early detection and surgery together with mediastinal lymph node dissection are necessary even for small-sized tumors to improve the prognosis in patients with peripheral ADENO of the lung.

Adolescent↗
PubMed1995

Distribution pattern of the basement membrane components is one of the significant prognostic correlates in peripheral lung adenocarcinomas.

Clinicopathological variables that might have an effect on prognosis were analyzed in 98 samples of resectable peripheral lung adenocarcinoma. Pathological stage (stage I v stage II, III, or IV; P < .001), degree of central fibrosis (grade 1 or 2 v grade 3 or 4; P < .01), and histological grade (well differentiated v moderately or poorly differentiated; P < .05) were shown to be prognostic factors, whereas any other variable, including patient age at diagnosis, sex, cigarette smoking habits, and tumor histological subtype and cytological type, showed no meaningful correlation with patient length of survival. The distribution pattern of the basement membranes (BMs) was significantly associated with pathological stage (intact v disrupted or absent; P < .001). It was noteworthy that in stage I samples a significant relationship between the distribution pattern of the BMs and patient length of survival was found (intact v disrupted or absent; P < .001), although such a relationship could not be detected when analyzed in samples from the other pathological stages. The distribution pattern of the BMs also was correlated with the presence or absence of lymph node metastasis but not with its extension. There were no meaningful relationships between the distribution pattern of the BMs and any other clinicopathological variables, including degree of central fibrosis, histological grade, etc. The distribution pattern of the BMs in peripheral lung adenocarcinomas might be useful for postoperative therapeutic strategy and could serve as an important prognostic indicator for stage I lesions.

Adenocarcinoma↗
PubMed1995

Pancreatic stone protein and lactoferrin in human pancreatic juice in chronic pancreatitis.

Lactoferrin and pancreatic stone protein (PSP) are thought to be closely related to pancreatic stone formation in chronic pancreatitis. However, the results reported so far have not been conclusive. To reevaluate the pathological importance of PSP in chronic pancreatitis, compared to lactoferrin, levels of PSP were determined by applying an immunoassay specific to PSP to pure pancreatic juice taken from a total of 52 patients. The patients consisted of 16 controls, 19 chronic pancreatitis patients (13 noncalcified and 6 calcified), and 17 probable cases of pancreatitis. The monoclonal antibody PSP antagonist used in the study recognizes both forms of the protein, PSP S1 and S2-5, with equal effectiveness. No significant reduction of PSP was observed in either calcified (mean +/- SEM, 111 +/- 30 micrograms/mg and 24 +/- 3 micrograms/mg protein) or noncalcified (305 +/- 133 and 97 +/- 47) chronic pancreatitis patients compared with controls (85 +/- 23 and 34 +/- 16). PSP levels did not decrease, at least not in the complete forms of the protein found in chronic pancreatitis. PSP antibody and assay results indicated that a reduction of PSP S2-5 alone could not be ruled out in chronic pancreatitis either.

Adult↗
PubMed1995

Activation of trypsinogen in experimental models of acute pancreatitis in rats.

Trypsinogen activation peptide (TAP) concentration and alpha 2-macroglobulin-trypsin complex (alpha 2M-T) activity were measured in two experimental models of acute pancreatitis in rats to evaluate the significance of activation of trypsinogen in acute pancreatitis. TAP concentration and alpha 2M-T activity in serum rose significantly in trypsin-taurocholate-induced hemorrhagic acute pancreatitis, while in cerulein-induced edematous acute pancreatitis they did not rise in spite of a similar increase in immunoreactive trypsin. When rats in trypsin-taurocholate-induced pancreatitis were treated by protease inhibitor (FUT-175; nafamostat mesilate; FUT group), alpha 2M-T activity in serum was significantly lower than that in nontreated controls (mean +/- SEM, 20.8 +/- 1.43 U/L in the FUT group vs 79.1 +/- 24.5 in controls; p < 0.01). The survival rate at 24 h was significantly improved in the FUT group compared with the controls (70 vs 43%; p < 0.05). The increase in TAP concentration in the FUT group was similar to that in controls. The TAP concentration in pancreatic tissue at 24 h was significantly (p < 0.01) lower in the survival group (7.8 +/- 0.8 ng/ml) than in the lethal group (25.9 +/- 3.7 ng/ml). Activation of trypsinogen and its subsequent enzyme activity play an important role in the evolution of severe acute pancreatitis.

Acute Disease↗
PubMed1995

Morphometric studies of renal lesions in Itai-itai disease: chronic cadmium nephropathy.

Renal cortex in 15 cases with Itai-itai disease was morphometrically examined, i.e. the cross-size of tubuli in the renal cortex was measured using a color image analyzer (Olympus CIA-102) and the ratio of preserved urinary tubuli occupying renal cortex was calculated by means of the point-counting method. The former and the latter decreased gradually together as atrophy of kidney advanced. However, their decrease was stopped at about 60 g of kidney weight and it was inferred to be the limit of histopathologic changes of the kidneys with Itai-itai disease. Comparing the cross-size of the tubuli of the outer cortex area [= S(o)] with one of the inner cortex area [= S(i)], changes were more marked in S(o). This revealed that the outer cortex area was more sensitive to Cd intoxication and it was more affected by systemic atherosclerosis than the inner cortex area. It was concluded that when atrophy of kidneys as seen with Itai-itai disease was extremely progressing, decreases in the number and volume of tubuli in the cortex usually occurred at the same time and in a regular manner.

Aged↗
PubMed1995

Cadherin 11 expression marks the mesenchymal phenotype: towards new functions for cadherins?

Cadherin-11 (cad-11) belongs to the cell adhesion type II cadherin family, which seems to have different functions from the classic cadherin family. This study shows the overall pattern of cad-11 gene expression during rat embryonic development, from the pregastrula to very late embryonic stage. Cad-11 is the first cadherin found to be highly expressed in the dispersed and migrating mesenchymal cells that originate from the neuroectodermal neural crest cells and from the pre-chordal and paraxial mesoderm. A burst of cad-11 expression appears during the epithelial to mesenchymal transition, as observed by sclerotome formation. Cad-11 mRNAs were present in all mesenchymal cells throughout the embryo, regardless of their embryonic origin. A proximo-distal and antero-posterior gradient of cad-11 expression is seen in the limb buds, genitalia, and tail. As development proceeds, while all epithelium are negative, cad-11 is present in all mesenchymal cells involved in various morphogenetic events, such as the mesenchyme condensations during chondrogenesis and in the formation of sclera, cornea, naris, palate and meninges. Cad-11 was strongly expressed in mesenchyme during lung or kidney branching morphogenesis or the many epithelium to mesenchyme inductions that operate in the nasal septum, skin, vibrissae, teeth and various glands. High levels of cad-11 transcripts were also found in the dispersed cells of the hyaloid plexus in the vitreous body and in the invading mesenchyme within the trabeculae of the outflow tract of the heart. Cad-11 is thus specific to the mesenchymal phenotype whatever the stage of embryonic development.

Amino Acid Sequence↗
PubMed1995

Gualamycin, a novel acaricide produced by Streptomyces sp. NK11687. II. Structural elucidation.

A novel acaricide, gualamycin was isolated from the culture broth of Streptomyces sp. NK11687. The structure of gualamycin was determined to be (2R,3S,4S)-2-O-[4-O-(2-amino-2-deoxy-beta-D-gulopyranosyl)-alpha-D - galactopyranosyl]-2,3,4-trihydroxy-4-[(2S,3S,4S,5S)-3,4-dihydroxy-5-hydr oxy - methylpyrrolidin-2-yl] butanoic acid by FAB-MS, 1H and 13C NMR, COSY, HMQC, HMBC, IR, X-ray crystallographic analyses and synthetic studies.

Disaccharides↗