Search PubMed⌕ Search

Biomedical subjects

A Kihara

Publications and source records attributed to A Kihara.

At least 37 records · Page 2Linked to original sources
PubMed1996

FtsH (HflB) is an ATP-dependent protease selectively acting on SecY and some other membrane proteins.

The FtsH protein is a membrane-bound ATPase of Escherichia coli that was proposed to be involved in membrane protein assembly as well as degradation of some unstable proteins. SecY, a subunit of protein translocase, is FtsH dependently degraded in vivo when it fails to associate with its partner (the SecE protein). We constructed a series of mutants in which mutations were introduced into conserved residues in the two ATP binding consensus sequences or the zinc binding sequence of FtsH. We purified wild-type and mutant FtsH proteins by making use of a polyhistidine tag attached to their carboxyl termini. Complementation analysis and ATPase activity assays in vitro indicated that, of the two sets of ATP binding sequence motifs, the one located C-terminally (A1) is essential for ATPase activity and in vivo functioning of FtsH. Wild-type FtsH protein degraded purified SecY in an ATP hydrolysis-dependent manner in vitro. Mutant proteins without ATPase activity were inactive in proteolysis. A zinc binding motif mutant showed a decreased proteolytic activity. SecY and FtsH were cross-linkable with each other in the membrane, provided that FtsH had an ATPase-inactivating mutation. These results demonstrate that FtsH binds to and degrades SecY, its A1 motif and the zinc binding motif being important for the proteolytic activity. FtsH-dependent proteolysis was also demonstrated for SecY in crude membrane extracts, whereas a majority of other membrane proteins were not degraded, indicating that FtsH has high selectivity in protein degradation.

ATP-Dependent Proteases↗
PubMed1996

A protease complex in the Escherichia coli plasma membrane: HflKC (HflA) forms a complex with FtsH (HflB), regulating its proteolytic activity against SecY.

Escherichia coli FtsH (HflB), a membrane-bound ATPase is required for proteolytic degradation of uncomplexed forms of the protein translocase SecY subunit. We have now isolated SecY-stabilizing mutations that cause an amino acid substitution in the HflK-HflC membrane protein complex. Although HflKC protein was believed to have a proteolytic activity against lambda cII protein, deletion of hflK-hflC did not stabilize SecY. Instead, the mutant alleles were partially dominant and overexpression of ftsH suppressed the mutational effects, suggesting that the mutant proteins antagonized the degradation of SecY. These results raise the possibility that even the wild-type HflKC protein acts to antagonize FtsH. Consistent with this notion, the hflkC null mutation accelerated degradation of the SecY24 protein. Furthermore cross-linking, co-immunoprecipitation, histidine-tagging and gel filtration experiments all indicated that FtsH and HflKC form a complex in vivo and in vitro. Finally, purified HflKC protein inhibited the SecY-degrading activity of purified FtsH protein in vitro. These results indicate that the proteolytic activity of FtsH is modulated negatively by its association with HflKC.

ATP-Dependent Proteases↗
PubMed1995

Intracellular stability of alpha fragments of beta-galactosidase: effects of amino-terminally fused polypeptides.

Intracellular stability of alpha fragments of beta-galactosidase in Escherichia coli has been studied by pulse-chase/immunoprecipitation experiments. An alpha fragment encoded by the pUC118 vector was relatively stable with an estimated half-life of about 12 min at 37 degrees C, whereas another vector, pSTV28, encoded a less stable alpha fragment that had a different carboxy-terminal sequence. Stability of the fragment was found to be affected markedly by amino-terminal attachment of other sequences. An amino-terminal fusion of a sequence derived from cytoplasmic domain 4 of the SecY protein shortened the half-life of the alpha fragment to less than 1 min. In contrast, an amino-terminal sequence from the NusG protein had no apparent effect on the stability of the fragment. In a fusion protein in which the intact SecY protein was fused to the alpha fragment, stabilization of the SecY part by overproduction of the partner SecE protein resulted in an increased alpha complementation activity of beta-galactosidase. These results indicate that stability of alpha fragment can be dictated by the stability of the fused protein. The alpha fragment of beta-galactosidase, which is unique in that it is largely unstructured but can be "active" in alpha complementation, may be used as an in vivo indicator of stability of proteins attached to it.

Amino Acid Sequence↗
PubMed1995

FtsH is required for proteolytic elimination of uncomplexed forms of SecY, an essential protein translocase subunit.

When secY is overexpressed over secE or secE is underexpressed, a fraction of SecY protein is rapidly degraded in vivo. This proteolysis was unaffected in previously described protease-defective mutants examined. We found, however, that some mutations in ftsH, encoding a membrane protein that belongs to the AAA (ATPase associated with a variety of cellular activities) family, stabilized oversynthesized SecY. This stabilization was due to a loss of FtsH function, and overproduction of the wild-type FtsH protein accelerated the degradation. The ftsH mutations also suppressed, by alleviating proteolysis of an altered form of SecY, the temperature sensitivity of the secY24 mutation, which alters SecY such that its interaction with SecE is weakened and it is destabilized at 42 degrees C. We were able to isolate a number of additional mutants with decreased ftsH expression or with an altered form of FtsH using selection/screening based on suppression of secY24 and stabilization of oversynthesized SecY. These results indicate that FtsH is required for degradation of SecY. Overproduction of SecY in the ftsH mutant cells proved to deleteriously affect cell growth and protein export, suggesting that elimination of uncomplexed SecY is important for optimum protein translocation and for the integrity of the membrane. The primary role of FtsH is discussed in light of the quite pleiotropic mutational effects, which now include stabilization of uncomplexed SecY.

ATP-Dependent Proteases↗
PubMed1995

Product of a new gene, syd, functionally interacts with SecY when overproduced in Escherichia coli.

A mutant form of SecY, SecY-d1, was previously suggested to sequester a component(s) of the protein translocator complex. Its synthesis from a plasmid leads to interference with protein export in Escherichia coli. SecE is a target of this sequestration, and its overproduction cancels the export interference. We now report that overexpression of another gene, termed syd, also suppresses secY-d1. The nucleotide sequence of syd predicted that it encodes a protein of 181 amino acid residues, which has been identified by overproduction, purification, and determination of the amino-terminal sequence. Cell fractionation experiments suggested that Syd is loosely associated with the cytoplasmic surface of the cytoplasmic membrane. SecY may be involved in the membrane association of Syd since the association is saturable, the extent of which depends on the overproduction of SecY. SecY is rapidly degraded in vivo unless its primary partner, SecE, is sufficiently available. Overproduction of Syd was found to stabilize oversynthesized SecY. However, Syd cannot stabilize the SecY-d1 form of SecY. Thus, in the presence of both secY+ and secY-d1, Syd increases the effective SecY+/SecY-d1 ratio in the cell and cancels the dominant interference by the latter. We also found that overproduction of Syd dramatically inhibits protein export in the secY24 mutant cell in which SecY-SecE interaction has been weakened. These results indicate that Syd, especially when it is overproduced, has abilities to interact with SecY. Possible significance of such interactions is discussed in conjunction with the apparent lack of phenotypic consequences of genetic disruption of syd.

ATP-Binding Cassette Transporters↗
PubMed1995

Cytotoxic activity of chimeric toxins containing the epidermal growth factor-like domain of heregulins fused to PE38KDEL, a truncated recombinant form of Pseudomonas exotoxin.

The EGF-like domains of heregulin alpha, beta 1, beta 2, and beta 3 were fused to a truncated form of Pseudomonas exotoxin (PE38KDEL), which contains a modified carboxyl-terminal sequence, KDEL, that increases that toxin activity. The resulting chimeric toxins were produced in Escherichia coli, purified to near homogeneity, and shown to be cytotoxic to target cells with very high activity on HTB20, N-87 MCF-7, and HepG2 cells; high activity on A431 and MDA-MB468 cells; and low activity toward SK-OV3, L929, and KB cells. The fact that cytotoxicity did not correlate with the levels of erbB2 expression indicated that another receptor in the erb family might be involved. Accordingly, cytotoxicity assays were performed on NIH/3T3 cell lines transfected with EGFR, ErbB2, ErbB3, or ErbB4. The results indicate that the heregulin toxins target ErbB4 or possibly ErbB3 but not ErbB2.

3T3 Cells↗
PubMed1994

Small chimeric toxins containing only transforming growth factor alpha and domain III of Pseudomonas exotoxin with good antitumor activity in mice.

Chimeric toxins composed of transforming growth factor alpha (TGF alpha) fused to mutant forms of Pseudomonas exotoxin (PE) bind to the epidermal growth factor receptor and kill cells bearing epidermal growth factor receptors. Initially, the binding domain (Ia; amino acids 1-252) of PE was deleted and replaced with TGF alpha to make TGF alpha-PE40 in which TGF alpha is fused to domains II, Ib, and III of PE (amino acids 253-613). That drug is currently undergoing clinical study for the intravesical therapy of bladder cancer. To generate smaller molecules that would have increased tumor penetration, several deletion mutants were constructed. In one of these, TGF alpha was inserted near the carboxyl terminus of PE, and residues in domains II and Ib of PE (amino acids 253-279 and 365-380) were deleted so that the chimeric toxin did not need to be cleaved by an intracellular protease to be activated (Theuer et al., J. Biol. Chem., 267: 16872-16877, 1992). We have now constructed chimeric toxins which contain only domain III, yet still exhibit high cytotoxic activity on epidermal growth factor receptor-containing cells and produce substantial tumor regressions in mice bearing a human xenograft. The high cytotoxic activity of these severely truncated toxins provides new insights on the proposed functions of domains II and III of PE.

Adenosine Diphosphate Ribose↗
PubMed1992

Alanine scanning mutagenesis identifies surface amino acids on domain II of Pseudomonas exotoxin required for cytotoxicity, proper folding, and secretion into periplasm.

Pseudomonas exotoxin A (PE) is a single polypeptide chain that contains 613 amino acids and is arranged into three major structural domains. Domain Ia is responsible for cell recognition, domain II for translocation of PE across the membrane, and domain III for ADP-ribosylation of elongation factor 2. Recombinant PE can be produced in Escherichia coli and is efficiently secreted into the periplasm when an OmpA signal sequence is present. To investigate the role of the amino acids located on the surface of domain II in the action of the toxin against mammalian cells, we substituted alanine for each of the 27 surface amino acids present in domain II. Surprisingly, all 27 mutant proteins had some alteration in cytotoxicity when tested on human A431 or MCF7 cells or mouse L929 cells. Native PE has a compact structure and therefore is relatively protease resistant and very little ADP-ribosylation activity is detected in the absence of the denaturing agents like urea and dithiothreitol. Several of the mutations resulted in altered protease sensitivity of the toxin. Seven of the mutant molecules exhibited ADP-ribosylation activity without urea and dithiothreitol, indicating they are partially unfolded. Out of these seven mutants, six had increased cytotoxic activity on at least one of the target cell lines and the other retained its native cytotoxic potency.

ADP Ribose Transferases↗
PubMed1991

Effect of the calcium antagonist nicardipine hydrochloride on glucose tolerance and insulin secretion.

The calcium antagonist nicardipine was administered to 42 patients at a dose of 60 or 120 mg/day for an average of 7.8 weeks to determine its effects on glucose tolerance and insulin secretion. Glucose tolerance and immunoreactive insulin levels were essentially unchanged by nicardipine regardless of the preexisting level of glucose tolerance. Mean systolic and diastolic blood pressures decreased in all patients. Concentrations of triglycerides decreased and high-density lipoprotein cholesterol levels increased significantly (p less than 0.05) in association with nicardipine administration. Therefore it is concluded that, regardless of the preexisting level of glucose tolerance, nicardipine exerted hypotensive effects without significant adverse effects on glucose tolerance or insulin secretion.

Adult↗
PubMed1989

Quantitative study on the interface between bone tissue and Blade-vent implants using the image processing system.

Undecalcified histopathologic sections of two titanium blade-vent implants (which had survived for five years in a dog) were made, and image analyses of the bone-implant interface were determined. One implant was inserted conventionally in the right mandible, and the other was inserted shallower than usual in the left mandible, so that the shoulder was located at the same level as the residual bone ridge. Using an image analysis technique, we observed, on the deeper implant, a 0-56% rate of bony contact around the implant and a 71-84% rate of bone-vent occupancy in the vents. With the shallower implant, a 0-46% rate of bony contact and a 26-60% rate of bone-vent occupancy were observed. When the two implants were compared, the deeper implant showed relatively higher bony contact and bone-vent occupancy than its less-submerged control in the five-year study.

Animals↗
PubMed1989

Improved method using a bubble-free adhesion technique for the preparation of semi-serial undecalcified histologic sections containing dental implants.

The preparation technique, with the minimum of artifacts for the semi-serial undecalcified histologic sections containing dental implants, is presented in this study. The sections enabled finer light-microscopic observations to be made. A formalin-fixed tissue block containing a dental implant was dehydrated in ethanol and acetone, and then embedded in polyester resin under 76 cm Hg reduced pressure. The embedded block was trimmed by a cutter and ground by abrasive paper. In a 1.5 Kg f/cm2 pressurized chamber, its polished surface was bonded to a methacrylate slide by means of ethylcyanoacrylate used in an adhesion loading device. This meant that no bubbles could arise in the interface between the slide and the block. The slide-block was then attached to an adsorptive specimen-holder of a hard-tissue cutting machine and cut to a thickness of approximately 50 microns, with use of a diamond blade. The slide-section was ground to 15-40 microns with wet-type abrasive paper and film on a polishing table. Etching with weak acid and surface staining with toluidine blue and methylene blue/basic fuchsin/light green were performed on the section.

Acrylic Resins↗
PubMed1988

Differences between the bony interfaces of titanium and hydroxyapatite-alumina plasma-sprayed titanium blade implants.

Histopathologic study was done to reveal the difference of bony interface in the mandibular bone between titanium blade implants and titanium implants coated with the mixture of hydroxyapatite-alumina by the plasma-spray technique, using the improved undecalcified histologic technique and the image analyzing system. In 4 adult shepherd dogs, the implantation was performed in the molar region of the mandible 2 months after tooth extraction. The coated implants and the non-coated ones were inserted respectively in the mandibles. They were sacrificed on the 5th, 7th, 14th and 28th day postoperatively. In 5-day implant after insertion, granulation tissues or fibrous connective tissues were observed around both implants in bone tissues. In 7-day implant, osteoid tissues regenerated around the apex of both implants, but attached a little to them. In 14-day implant, osteoid tissues or woven bone regenerated around both implants and attached more to the coated implant (approximately 13.7%) than the non-coated one (approximately 6.0%). In 28-day implant, regenerative woven or lamellar bone attached much more to the coated implant (approximately 63.4%) than the non-coated one (approximately 8.8%). Bony interface increased remarkably in the coated implants, chronologically. The results showed that the plasma-sprayed hydroxyapatite-alumina coating was effective for the initial fixation of endosseous dental implants.

Aluminum Oxide↗
PubMed1987

Clinico-pathological study on denture stomatitis.

The clinical and pathological study was performed in order to determine the histopathological and cytoimmunological characteristics of denture stomatitis. All specimens were biopsy materials from seventeen patients with denture stomatitis. Normal palatal mucosae from ten patients served as the control. In addition to the usual staining methods, naphtol AS-D chloroacetate esterase stain and peroxidase-antiperoxidase method were used to detect mast cells and plasma cells. Denture stomatitis could be divided into atrophic and hyperplastic types. The former showed a smooth and atrophic mucosa. The latter showed a large number of exophytic projections which were composed of marked acanthosis and submucosal fibrosis, and was further subdivided into granular and papillary subtype according to the size of projections. In the present study, there were six cases of the atrophic type, and eleven cases of the hyperplastic type (consisting of seven granular and four papillary subtypes). The hyperplastic type was more frequently observed in patients with partial dentures compared with complete dentures and was associated frequently with ill fitting of the denture base as well as agglutination of denture plaque. Cytoimmunological study revealed that there was a pronounced increase of plasma cells, especially IgG- and IgA-producing cells, and a moderate increase of lymphocytes as well as mast cells in both types of denture stomatitis. Mast cells were always noted in the area with marked plasma cell infiltration, suggesting an intimate relation between both cells. These findings suggest that the immunological reactions play some role in the pathogenesis of denture stomatitis.

Adolescent↗