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Biomedical subjects

K Kohno

Publications and source records attributed to K Kohno.

At least 19 recordsLinked to original sources

Histone-like proteins are required for cell growth and constraint of supercoils in DNA.

The gene hns of Escherichia coli K-12, which encodes the histone-like protein H-NS, was inactivated by insertion of a DNA (gene hph) encoding hygromycin phosphotransferase. The growth rates of two mutants lacking either one or the other of the histone-like proteins, HU and IHF, were not affected by introduction of the hns mutation. However, cells depleted of HU, IHF and H-NS simultaneously, could not be constructed by P1 phage-mediated transduction. These results, together with our previous finding that cells deficient in both HU and IHF are viable at 30-37 degrees C [Kano and Imamoto, Gene 89 (1990) 133-137] showed that E. coli cells deficient in any two of these three histone-like proteins are viable at 30-37 degrees C, and suggested that simultaneous deficiency of all three of the proteins is lethal. There were no detectable differences in the levels of superhelicity of the reporter plasmids isolated from cells deficient in either IHF or H-NS, or from wild-type cells, but about 15% decrease in negative superhelicity was detected for the reporter plasmid isolated from cells lacking HU and lacking both HU and H-NS. However, the cryptic bgl operon, whose expression was reported to be regulated through topological changes of cellular DNA, could not be activated in cells depleted of HU or IHF. The bgl operon was expressed in cells depleted of both HU and H-NS as well as in cells depleted of H-NS.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Outer Membrane Proteins

Different sensitivities of human esophageal cancer cells to multiple anti-cancer agents and related mechanisms.

Mechanisms responsible for drug resistance in human esophageal cancer cell lines were investigated. Three cell lines established from human esophageal carcinoma (TE-1, SH-1, and TH) showed different sensitivities to vindesine, vincristine, cisplatin (CDDP), etoposide (VP-16), and pepleomycin. Both SH-1 and TH cell lines were twofold to sevenfold more resistant to pepleomycin, vindesine, and vincristine than TE-1 was. SH-1 showed twofold more resistance to CDDP than either TE-1 or TH did, and TH and TE-1 showed a 3-fold or 1.5-fold more resistance, respectively, to VP-16 than SH-1 did. The accumulation of tritiated vincristine in SH-1 and TH was approximately 50% that in TE-1. Two multidrug resistance reversal agents, cepharanthine and a synthetic dihydropyridine analogue (NK-252; Nikken Chemicals, Saitama, Japan), potentiated the cytocidal actions of vindesine against SH-1, TH, and TE-1 cells, with no apparent expression of P-glycoprotein in the three cell lines. The glutathione S-transferase pi gene was expressed in all three cell lines. DNA topoisomerase II levels were lowest in TE-1, followed by SH-1 and TH, although the accumulation of tritiated VP-16 was less in both TH and SH-1 than in TE-1. Differential sensitivities to anti-cancer drugs appear to be mediated through pleiotropic mechanisms.

ATP Binding Cassette Transporter, Subfamily B, Mem

Amino acid substitution in the C-terminal arm domain of HU-2 results in an enhanced affinity for DNA.

Three mutants of the Escherichia coli hupA gene, encoding the HU-2 protein, were constructed by synthetic oligodeoxyribonucleotide-directed, site-specific mutagenesis on M13mp18 vectors. The resulting HupAN10, HupAN11 and HupAN12 proteins contained Thr59-->Lys, Gln64-->Lys and Asn53-->Arg substitutions, respectively. These amino acid (aa) changes increased the positive charge of the N-terminal half of the two-strand, antiparallel beta-ribbon of the arm structure, which is believed to be a domain for DNA binding. The three mutant proteins bound to DNA more tightly than wild-type HU-2, and their affinities for DNA increased in the order of HupAN10, HupAN11, HupAN12. The mutant proteins showed a slightly increased HU activity for supporting Mu phage development. A mutant HU-2 protein with increased basicity, but with an altered aa sequence in the arm region due to a frameshift mutation, was also constructed. This mutant protein showed a reduced affinity to DNA and was unable to support Mu growth, suggesting that a unique aa sequence of the arm domain, rather than mere basicity of this domain, is required for efficient binding to DNA.

Amino Acid Sequence

Activation of human multidrug resistance-1 gene promoter in response to heat shock stress.

The multidrug resistance (MDR1) gene encodes a P-glycoprotein, which catalyzes the energy-dependent efflux of anticancer agents. Various environmental stresses including heat shock can induce the expression of endogenous MDR1 genes. In order to study the regulatory mechanisms of MDR1 gene expression, we have established human cancer KB cell lines which could stably integrate bacterial chloramphenicol acetyltransferase (CAT) gene driven by various lengths of the MDR1 promoter. Kst-6 has an integrated plasmid, pMDRCAT1, containing the human MDR1 promoter of -2 kilobases. The MDR1 gene promoter contains a typical heat shock element (HSE) motif located -152 bp to -178 bp from the initiation site. Heat shock at 45 degrees C for 90 min significantly induced CAT activity in Kst-6 cells. Northern blot analysis showed a 4-5 fold increase in CAT mRNA levels in Kst-6 cells. Deletion analysis of the MDR1 promoter demonstrated that the induction of CAT activity was observed in Kxh-14 cells containing a HSE-deleted MDR1 promoter construct, pMDRCAT7. However, further deletion analysis showed that heat shock could not induce CAT activity in Khp-1 cells containing -76 approximately +121 base sequence of the promoter, suggesting that a new heat shock responsible element was located at between -136 and -76. Gel shift assay showed that the heat shock factor (HSF) could bind to the HSE motif located at -152 bp to -178 bp in the MDR1 promoter. We also found that one distinct DNA-protein complex formed specifically within the MDR1 promoter region -99 to -66 was not significantly increased, but relatively more stabilized under mild denaturing condition in the nuclear extract of heat-shocked cells. In our present assay system, activation of the MDR1 promoter in response to heat shock appears to be mediated through both a new heat shock responsive element and MDR1 specific transcription factor.

ATP Binding Cassette Transporter, Subfamily B, Mem

Purification and characterization of BiP/Kar2 protein from Saccharomyces cerevisiae.

Using specific anti-BiP/Kar2 antibody as the probe, we have developed an efficient purification method of BiP/Kar2 protein from the total cell extract of Saccharomyces cerevisiae. Overproduction of BiP/Kar2 protein was achieved by the cloning of the KAR2 gene on multicopy plasmids and the treatment of cells harboring the cloned KAR2 gene with tunicamycin. Freeze-thaw treatment, hydroxyapatite high pressure liquid chromatography, and ATP-agarose column chromatography of crude extract yielded homogeneous BiP/Kar2 protein (including less than 0.2% of degradative derivative) with a 430-fold purification and 28% recovery. Edman degradation of purified BiP/Kar2 suggests that the mature protein corresponds to a processed product with the removal of a 42-amino acid presequence. It is active as a homodimer and exhibits ATPase activity with a specific activity of 2 pmol/min/micrograms of protein. Protease susceptibility indicated that the ADP form of BiP/Kar2 is more resistant than the ATP form to the chymotrypsin digestion and that BiP/Kar2 required the presence of ATP to avoid the irreversible denaturation. Synthesis of BiP/Kar2 was induced by the inducible expression of an aberrant heterologous protein, yeast killer prepro-signal mouse alpha-amylase fusion protein.

Adenosine Triphosphatases

A model system for tumor angiogenesis: involvement of transforming growth factor-alpha in tube formation of human microvascular endothelial cells induced by esophageal cancer cells.

Tumor growth is dependent on angiogenesis, which is thought to be mediated through growth factors, such as transforming growth factor-alpha (TGF-alpha) and -beta (TGF-beta), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF), produced by tumor cells. We have developed a model system for tumor angiogenesis in vitro: tube formation of human omentum microvascular endothelial (HOME) cells in type I collagen gels when these cells are co-cultured with tumor cells. Exogenously added TGF-alpha induced tube formation of HOME cells in collagen gel. In contrast, TGF-beta inhibited the TGF-alpha-induced tube formation of endothelial cells. We investigated whether tube formation could be induced in HOME cells in collagen gel when the HOME cells were co-cultured with three esophageal cancer cell lines, TE1, TE2, and TE5. TE1 and TE2 cells expressed both TGF-alpha and TGF-beta mRNA, but the level of TGF-alpha mRNA in TE2 was found to be much lower than in TE1 cells. TE5 did not express either TGF-alpha or TGF-beta. The tube formation of HOME cell was induced when they were co-cultured with TE1 cells, while both TE2 and TE5 cell lines induced tube formation at much lower rates than TE1. TE1-induced tube formation of HOME cells was specifically blocked by co-administration of anti-TGF-alpha-antibody, but not by anti-bFGF-antibody. The present study suggests that, in our model system, esophageal tumor angiogenesis is partly controlled by TGF-alpha, possibly through a paracrine pathway.

Blotting, Northern

Induction of low density lipoprotein receptor and a transcription factor SP-1 by tumor necrosis factor in human microvascular endothelial cells.

We have previously reported that tumor necrosis factor-alpha (TNF-alpha) enhances expression of interleukin-6, collagenase, plasminogen activator inhibitor-1, and basic fibroblast growth factor genes in human omental microvascular endothelial (HOME) cells in culture. In this study, we found that treatment of HOME cells with TNF-alpha or interleukin-1 (IL-1) caused enhanced expression of low density lipoprotein (LDL) receptor. A few-fold increase in both LDL binding activity and the receptor mRNA levels was observed when HOME cells were treated with either TNF-alpha or IL-1. Northern blot analysis showed that cellular expression of LDL receptor gene was significantly increased 12-24 h after exposure to TNF-alpha. No significant changes in the life-span of LDL receptor mRNA were observed in untreated and TNF-alpha-treated cells. Scatchard analysis showed an increased receptor number for LDL in TNF-alpha-treated cells. Parallel to increased LDL binding activity, internalization and degradation of LDL were also increased in HOME cells treated with TNF-alpha or IL-1. TNF-alpha-induced enhancement of LDL receptor gene expression was not observed when cycloheximide was present. Cellular mRNA level of SP-1 gene was increased about 3-4-fold at 12 h after treatment with TNF-alpha. Nuclear run-on assays showed increased transcription of LDL receptor gene as well as SP-1 gene by TNF-alpha. Gel retardation assay with the SP-1 consensus fragment showed that SP-1 binding activity was increased about 4-5-fold 12-24 h after treatment with TNF-alpha. NF-kB binding activity was also dramatically increased, but there is no NF-kB motif on the promoter for LDL receptor gene. The induction of LDL receptor by TNF might be mediated through a transcription factor, SP-1.

Base Sequence

The human multidrug resistance 1 promoter has an element that responds to serum starvation.

We have previously demonstrated in transient expression assay systems that a human multidrug resistance 1 (MDR1) promoter can be directly activated by cytotoxic anticancer agents. In this study, we examined whether the MDR1 promoter could be regulated in response to growth arrest induced by serum starvation. We have established human and rodent cell lines which stably expressed the chloramphenicol acetyltransferase (CAT) gene driven by various lengths of the MDR1, the viral thymidine kinase (TK) and the simian virus 40 (SV40) promoters. Serum starvation caused enhanced expression of CAT gene with MDR1 promoter, but not with two viral gene promoters in human cancer KB cells. Hydroxyurea activated the MDR1 promoter, but not TK and SV40 promoters. By contrast, the DNA topoisomerase II inhibitor, etoposide, equally activated the MDR1, TK and SV 40 promoters. Increased CAT gene expression by serum starvation was also specifically observed in stable transfectants of human adrenal SW-13 cell lines, but not in stable transfectants of mouse fibroblast NIH3T3 and adrenal Y-1 cell lines when the human MDR1 promoter-CAT was introduced. Etoposide, however, effectively induced CAT activity in both human and rodent cells. Assays with deletion constructs of the MDR1 promoter showed that serum starvation activated the MDR1 promoter carrying -258 approximately +121 base sequence of the promoter, but not -198 approximately +121 of the promoter. These results suggest that the expression of the MDR1 gene induced by serum starvation is regulated at the transcriptional level in a promoter sequence-specific manner in human cells.

Animals

A 22 bp cis-acting element is necessary and sufficient for the induction of the yeast KAR2 (BiP) gene by unfolded proteins.

The KAR2 gene of Saccharomyces cerevisiae codes for an essential chaperone protein (BiP) that is localized in the lumen of the endoplasmic reticulum (ER). The high basal rate of transcription of KAR2 is increased transiently by heat shock: prolonged induction occurs when unfolded proteins accumulate in the ER. Three cis-acting elements in the KAR2 promoter control expression of KAR2: (i) a GC-rich region that contributes to the high level of constitutive expression, (ii) a functional heat shock element (HSE) and (iii) an element (UPR) that is involved in the induction of BiP mRNA by unfolded proteins. By analyzing internal deletion mutants of the KAR2 promoter, we demonstrate here that these three elements regulate transcription of KAR2 independently. Furthermore, the 22 bp UPR element causes a heterologous (CYC1) promoter to respond to the presence of unfolded proteins in the ER. Extracts of both stressed and unstressed yeast cells contain proteins that bind specifically to synthetic HSE and UPR elements and retard their migration through gels. Binding proteins specific for the UPR element can be fractionated by ammonium sulfate precipitation. Two of the proteins UPRF-1 and UPRF-2 (which is apparently a proteolytic degradation product of UPRF-1) bind inefficiently to mutant versions of the UPR that are unable to confer responsiveness to unfolded proteins to the (CYC1) promoter. UPRF-1 therefore displays the properties expected of a transcription factor that is involved in the sustained response of the KAR2 promoter to unfolded proteins in the ER. These experiments show that yeast cells can activate a transcription factor that stimulates expression of a nuclear gene in response to the accumulation of unfolded proteins in another cellular compartment.

Base Sequence

Decreased response to epidermal growth factor during cellular senescence in cultured human microvascular endothelial cells.

We have previously demonstrated that epidermal growth factor (EGF) induces cell migration, tissue-type plasminogen activator synthesis, as well as tubular formation in microvascular endothelial cells from human omental tissue. In this study, we compared the responsiveness to EGF of late passaged (senescent) human omental microvascular endothelial (HOME) cells with that of early passaged (young) HOME cells. We have employed HOME cells derived from surgically resected omental samples from 14 patients. EGF-stimulated cell migration significantly more in the young cells than in the senescent cells during serial cultivation (aging) in vitro. Scatchard analysis demonstrated that the number for both high and low affinity receptors for EGF in HOME cells was decreased dramatically during serial cultivation. The expression of EGF receptor mRNA was also decreased in the senescent HOME cells. Treatment of HOME cells with EGF significantly increased cellular mRNA levels of tissue-type plasminogen activator, and two protooncogenes, c-fos and c-myc, in young HOME cells, but not in senescent HOME cells. Thus HOME cells aged in vitro show a decreased responsiveness to EGF, resulting in decreased migration of human endothelial cells. The serial cultivation of human endothelial cells in vitro may downregulate EGF receptor and decrease responsiveness to exogenous EGF, a potent angiogenic factor.

Blotting, Northern

Morphology of sympathetic preganglionic neurons innervating the superior cervical ganglion in the chicken: an immunohistochemical study using retrograde labeling of cholera toxin subunit B.

Preganglionic sympathetic neurons (SPNs) in the chicken were demonstrated immunohistochemically using cholera toxin subunit B (CTb) as a retrograde tracer. After injection of CTb-solution into the superior cervical ganglion, labeled SPNs were mainly found in the ipsilateral sympathetic preganglionic column of Terni (the column of Terni), with only a few in the intermediate zone. They were observed from the caudal half of the 15th cervical segment to the rostral tip of the 3rd thoracic segment. Cell somata of SPNs were loosely packed within the column of Terni, where they had an elliptic shape with the long axis oriented rostrocaudally. In the horizontal plane three kinds of dendrites could be discriminated on the basis of their orientation. Longitudinally oriented dendrites emanated from the rostral and the caudal poles of the SPNs. Medially oriented dendrites were observed to cross the midline and enter the contralateral column of Terni, where they further branched to form a loose dendritic plexus; some extended beyond the lateral limit of the contralateral column of Terni to reach the intermediate zone. Laterally oriented dendrites formed periodically arranged dendritic bundles projecting into the intermediate zone. The present findings provide a detailed account of the dendritic organization of SPNs in the chicken, and suggest that avian SPNs share certain structural features in common with mammalian SPNs.

Animals

Antibodies against 70-kD heat shock cognate protein inhibit mediated nuclear import of karyophilic proteins.

Previously, we found that anti-DDDED antibodies strongly inhibited in vivo nuclear transport of nuclear proteins and that these antibodies recognized a protein of 69 kD (p69) from rat liver nuclear envelopes that showed specific binding activities to the nuclear location sequences (NLSs) of nucleoplasmin and SV-40 large T-antigen. Here we identified this protein as the 70-kD heat shock cognate protein (hsc70) based on its mass, isoelectric point, cellular localization, and partial amino acid sequences. Competition studies indicated that the recombinant hsc70 expressed in Escherichia coli binds to transport competent SV-40 T-antigen NLS more strongly than to the point mutated transport incompetent mutant NLS. To investigate the possible involvement of hsc70 in nuclear transport, we examined the effect of anti-hsc70 rabbit antibodies on the nuclear accumulation of karyophilic proteins. When injected into the cytoplasm of tissue culture cells, anti-hsc70 strongly inhibited the nuclear import of nucleoplasmin, SV-40 T-antigen NLS bearing BSA and histone H1. In contrast, anti-hsc70 IgG did not prevent the diffusion of lysozyme or 17.4-kD FITC-dextran into the nuclei. After injection of these antibodies, cells continued RNA synthesis and were viable. These results indicate that hsc70 interacts with NLS-containing proteins in the cytoplasm before their nuclear import.

Amino Acid Sequence

DNA topoisomerase-targeting antitumor agents and drug resistance.

A review of the chemotherapeutic agents which have been developed by targeting DNA topoisomerase I and II is presented. Camptothecins as topoisomerase I-targeting agents and newly developed topoisomerase II-targeting agents with unique properties are expected to be promising anticancer agents in the near future. An important issue is how cellular sensitivity to these agents is controlled. One approach is to establish and characterize drug-resistant human cancer cell lines, which would provide powerful tools to understand their intracellular target sites and also the mechanisms for acquirement of drug resistance to topoisomerase inhibitors. Drug resistance to topoisomerase-targeting agents appears to be closely correlated with two events, namely decreased expression and point mutation of topoisomerase genes.

Antineoplastic Agents

A pathologic and bacteriologic study on otitis media in swine.

A pathologic and bacteriologic study on otitis media in swine was performed on 237 swine, ranging in age from 1 day to 1 year. These 237 swine from eight selected farms were slaughtered due to unfavorable prognosis associated with clinical signs of illness. One hundred sixty-three (68.8%) of the 237 swine were found to be affected with otitis, though only a few swine showed clinical signs characteristic of the disease. One hundred fifty-one (63.7%), 53 (22.4%), and 39 (16.5%) had lesions in the middle, external, and internal ear, respectively. Of the 151 cases with otitis media, bilateral and unilateral infection accounted for 114 (75.5%) and 37 (24.5%), respectively. Only 5/53 swine with otitis externa and 6/39 swine with otitis interna failed to show otitis media. All swine with infection in the tympanic cavity had inflammatory lesions in the auditory tube. Examination of the swine grouped by age revealed that, regardless of age, incidence of otitis media was 50-70%. In swine younger than 1 month old, otitis media tended to center on the auditory tube, where a mild infiltration of neutrophils into the mucous membrane was frequent. Thereafter, otitis media increased in degree and extent with age. Forty (26.5%) of the 151 swine affected with otitis media were accompanied by a substantial amount of inflammatory exudate in the tympanic cavity and bulla. Purulent exudate was found in 20/40 (50%) swine. Severe otitis media prevailed in swine between 1 and 4 months of age, showing inspissation of a suppurative exudate in the tympanic cavity and lysis of the underlying osseous wall of the tympanic bulla with subsequent fibrosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Actinomyces

Intra- and extracellular localization of hyaluronic acid and proteoglycan constituents (chondroitin sulfate, keratan sulfate, and protein core) in articular cartilage of rabbit tibia.

To demonstrate the intra- and extracellular localization of hyaluronic acid (HA) in articular cartilage of the rabbit tibia, biotinylated HA binding region, which specifically binds to the HA molecule, was applied to the tissue. In comparison with the localization of HA, that of chondroitin sulfate (CS), keratan sulfate (KS), and the protein core (PC) of the proteoglycan was examined by immunohistochemistry. Strong positive staining for HA was detected in chondrocytes located in the transition between the superficial and middle zones of the tissue. Pre-treatment with chondroitinase ABC, keratanase II, or trypsin enhanced the stainability for HA in peri- and intercellular matrices. Immunohistochemistry with or without enzymatic pre-treatment demonstrated that immunoreactivity for CS, KS, and PC was distinctly discerned in chondrocytes and in the extracellular matrix located in the middle and deep zones. In particular, the immunoreactivity for KS and PC was augmented by pre-treatment with chondroitinase ABC not only in chondrocytes but in the extracellular matrix located in the middle and deep zones. Microbiochemical analysis corresponded well with histochemical and immunohistochemical results. These results suggest that HA is abundantly synthesized and secreted in chondrocytes located in the transition between the superficial and middle zones.

Aggrecans

Localization of hyaluronic acid, chondroitin sulfate, and CD44 in rabbit cornea.

To demonstrate the localization of hyaluronic acid (HA) in rabbit cornea, the biotinylated HA-binding region, which specifically binds to the HA molecule, was applied to the tissue. Localization of chondroitin sulfate (CS) and CD44, a possible cell surface receptor for HA, were also examined by immunohistochemistry. The stainability of HA changed depending on the fixatives used. Reaction products for HA were distinctly detected in epithelial cells and stromal keratocytes, but faintly in the extracellular matrix of the stroma when unfixed cryosections were applied. No positive reaction was found in the endothelium, except that the positive deposits formed a continuous layer on the apical surface of the endothelium. Electron microscopy using samples fixed with 2% paraformaldehyde revealed gold particles indicating HA labeling the intercellular space of the epithelium and stromal extracellular matrix. No intracellular deposition was detected in epithelial cells, whereas the gold labeling was seen in vacuolar structures of stromal keratocytes. Immunodeposits for CS were intensely localized in the epithelium and stroma, and weakly in the endothelium. Immunoreactivity for CD44 was found in the epithelial, endothelial and stromal cells. In particular, immuno-deposits for CD44 were detected in basal parts of epithelial cells, while they were localized in the apical surface of endothelial cells. These results suggest that HA is synthesized in and secreted from epithelial and stromal cells of rabbit cornea, while the localization of HA in the apical surface of the endothelium is closely associated with that of CD44. Moreover, the presence of CS in corneal tissue may play a role in its transparency, as has been suggested for keratan sulfate and dermatan sulfate.

Animals