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Biomedical subjects

K Kohama

Publications and source records attributed to K Kohama.

At least 19 recordsLinked to original sources
PubMed2002

TGF-beta-3 promotes scarless repair of cleft lip in mouse fetuses.

TGF-beta3 mediates epithelial-mesenchymal transformation during normal fusion of lip and palate, but how TGF-beta3 functions during cleft lip repair remains unexplored. We hypothesize that TGF-beta3 promotes fetal cleft lip repair and fusion by increasing the availability of mesenchymal cells. In this investigation, we demonstrated that cleft lips in mouse fetuses were repaired by fetal surgery, producing scarless fusion. At the site of the operation, we first observed an infusion of platelets expressing TGF-beta3, followed by increased expression of cyclin D1 and tenascin-C, and coupled with increased mesenchymal cell proliferation. In an ex vivo serumless culture system, cleft lip explants fused in the presence of exogenous TGF-beta3. Cultured lips also showed up-regulation in cyclin D1 and tenascin-C expression. These findings suggest that microsurgical repair of cleft lip in the fetus that produced scarless fusion is mediated by TGF-beta3 regulation of mesenchymal cell proliferation and migration at the site of repair.

Animals↗
PubMed2001

Involvement of gicerin in the extension of microvilli.

Gicerin is a cell adhesion molecule belonging to the immunoglobulin superfamily. To study the functional differences between l- and s-gicerin, we first examined the distribution of endogenous gicerin in B16 cells and found that l-gicerin was densely localized in microvilli. To clarify the relationship between gicerin and the microvilli, we established independent stable cell lines expressing l- and s-gicerin in L cells and found that l-gicerin localized to the microvilli. Scanning electron microscopic analysis revealed that the microvilli of l-gicerin-transfected cells were longer than those of s-gicerin and control transfectants. This suggested that l-gicerin might participate in the elongation of the microvilli. When cells were double-stained with antibodies to gicerin and moesin, a microvilli-specific protein, the staining of l-gicerin corresponded to that of moesin in the elongated microvilli. Moesin was coprecipitated with glutathione S-transferase-fusion proteins of the l-gicerin cytoplasmic domain but not with the s-gicerin cytoplasmic domain. To determine the region involved in the extension of microvilli, we generated transfectants of two truncated forms of l-gicerin cytoplasmic domain, and we found that only the transfectants of the longer mutant had the longer microvilli, while the shorter mutant exhibited short microvilli. These results suggested that l-gicerin-specific amino acid residues, especially amino acids 16-39, within the cytoplasmic domain of l-gicerin might be involved in the extension of microvilli.

Actins↗
PubMed2001

Propofol activates vanilloid receptor channels expressed in human embryonic kidney 293 cells.

Propofol (2,6-diisopropylphenol) is an intravenous anesthetic agent structurally unrelated to any other intravenous anesthetics. We examined the effect of propofol on a rat vanilloid receptor that was expressed in the human embryonic kidney (HEK) 293 cells by using calcium imaging method. Propofol caused a concentration-dependent increase in [Ca(2+)](i) in the HEK293 cells with the receptor. These responses were inhibited by removing extracellular calcium ions. The propofol-evoked increase in [Ca(2+)](i) in the HEK293 cells with the receptor was partially inhibited by capsazepine, a competitive antagonist of capsaicin. We conclude that propofol acts as an agonist for the receptor.

Anesthetics, Intravenous↗
PubMed2001

A highly sensitive method for measurement of myosin ATPase activity by reversed-phase high-performance liquid chromatography.

A new method for measurement of myosin ATPase activity has been developed utilizing reversed-phase high-performance liquid chromatography (HPLC), which detects as low as 0.05 nmol of ADP hydrolyzed from ATP. After termination of the ATPase reaction by addition of perchloric acid, the hydrolysate ADP and substrate ATP were separated by reversed-phase HPLC. The absorbance of ADP was monitored at 259 nm, and the amount of ADP was quantified from its peak area on the chromatogram by use of the NIH Image computer software. Our method showed linearity over a wide range from 0.05 to 10 nmol of ADP per 20 microl with a coefficient of determination (r(2)) of 0.99. Myosin ATPase activities determined by the HPLC method were almost identical to those determined by the malachite green method, a widely used spectrophotometric method with range of detection from 1 to 8 nmol of phosphate. Because our method requires only a small volume of reaction solution, it will be a powerful tool for measuring ATPase activity of motor proteins, which are difficult to obtain in large amount.

Adenosine Diphosphate↗
PubMed2001

Myosin light chain kinase as a multifunctional regulatory protein of smooth muscle contraction.

Myosin light chain kinase (MLCK) is a regulatory protein for smooth muscle contraction, which acts by phosphorylating 20-kDa myosin light chain (MLC20) to activate the myosin ATPase activity. Although this mode of action is well-established, there are numerous reports of smooth muscle contraction that is not associated with MLC20 phosphorylation. The kinase activity for the phosphorylation is localized at the central part of MLCK, which is also furnished with actin-binding activity at its N terminal and myosin-binding activity at its C terminal. This article overviews as to how such multifunctional properties of MLCK modify the actin-myosin interaction and presents our observations that the phosphorylation is not obligatory in induction of smooth muscle contraction.

Actins↗
PubMed2001

[High throughput pharmacology for drug discovery].

High Throughput Screening (HTS) now plays an important role in the discovery of new lead compounds for novel therapeutic targets. The advantage of HTS over the conventional method, now termed as Low Throughput Screening (LTS), is that valuable compounds can be selected rapidly from a large number of samples with minimal human involvement. In spite of the growing awareness of HTS, the importance of the LTS in the drug discovery and development is still not changed. Advances in pharmacogenomics will also provide us many pharmacological targets, and thus increase the number of compounds that should be assayed by HTS and LTS. In this review, we will first describe the outline of HTS. We will next describe new approaches to develop and brush up the LTS: 1) screening method of drugs acting on ion channels by voltage-sensitive fluorescent dye, 2) functional assay method using reconstituted smooth muscle fiber, and 3) organ culture method as a useful model of vascular proliferative disease. These approaches, which work cooperatively with HTS, will contribute greatly to the development of new drugs.

Animals↗
PubMed2001

[Targeting of myosin light chain kinase in vascular smooth muscle cells, and its implication for drug discovery].

We constructed a plasmid vector to have a 1.4-kb insert of myosin light chain kinase (MLCK) cDNA in an antisense direction to express antisense mRNA. The construct was then transfected to SM3, a cell line from vascular smooth muscle cells (VSMCs), producing a few stable transformants. We explained the methods in detail. The down-regulation of MLCK expression in the transfectants was confirmed by both Northern and Western blots. The control showed chemotactic motility to the platelet derived growth factor. However, the transfectants did not show chemotactic motility, indicating the essential role of MLCK in the motility. It was discussed that down-regulation of MLCK expression could be utilized to discover the drug for arteriosclerosis which prevents the proliferating VSMCs from forming arterial plaques.

Amino Acid Sequence↗
PubMed2001

[Drugs that modulate the cellular function of calcium ion].

Cytoplasmic Ca(2+) concentration is kept low level of about 10(-7)M. When Ca(2+) concentration increases by specific stimuli via opening of channel proteins, Ca(2+) binds to Ca(2+)-binding proteins, activating specific proteins, results in responding the stimuli. In this report, we summarize the effect of drugs, of which targets are the proteins above, including Ca(2+)-pump, ion exchanger, ion channels, calmodulin and calmodulin-binding proteins, and other Ca(2+) binding proteins.

English Abstract↗
PubMed2000

How can Ca2+ selectively activate recoverin in the presence of Mg2+? Surface plasmon resonance and FT-IR spectroscopic studies.

We investigated the relationship between metal ion selective conformational changes of recoverin and its metal-bound coordination structures. Recoverin is a 23 kDa heterogeneously myristoylated Ca(2+)-binding protein that inhibits rhodopsin kinase. Upon accommodating two Ca(2+) ions, recoverin extrudes a myristoyl group and associates with the lipid bilayer membrane, which was monitored by the surface plasmon resonance (SPR) technique. Large changes in SPR signals were observed for Sr(2+), Ba(2+), Cd(2+), and Mn(2+) as well as Ca(2+), indicating that upon binding to these ions, recoverin underwent a large conformational change to extrude the myristoyl group, and thereby interacted with lipid membranes. In contrast, no SPR signal was induced by Mg(2+), confirming that even though it accommodates two Mg(2+) ions, recoverin does not induce the large conformational change. To investigate the coordination structures of metal-bound Ca(2+) binding sites, FT-IR studies were performed. The EF-hands, Ca(2+)-binding regions each comprising 12 residues, arrange to coordinate Ca(2+) with seven oxygen ligands, two of which are provided by a conserved bidentate Glu at the 12th relative position in the EF-hand. FT-IR analysis confirmed that Sr(2+), Ba(2+), Cd(2+), and Mn(2+) were coordinated to COO(-) of Glu by a bidentate state as well as Ca(2+), while coordination of COO(-) with Mg(2+) was a pseudobridging state with six-coordinate geometry. These SPR and FT-IR results taken together reveal that metal ions with seven-coordinate geometry in the EF-hands induce a large conformational change in recoverin so that it extrudes the myristoyl group, while metal ions with six-coordinate geometry in the EF-hands such as Mg(2+) remain the myristoyl group sequestered in recoverin.

Animals↗
PubMed2000

Propofol potentiates ATP-activated currents of recombinant P2X(4) receptor channels expressed in human embryonic kidney 293 cells.

We examined the effects of a general anesthetic 2, 6-diisopropylphenol (propofol) on ATP- and alpha,beta-methylene ATP (alphabetameATP)-activated currents in the human embryonic kidney 293 (HEK 293) cells expressing recombinant P2X receptor channels, using the whole-cell patch-clamp method. Propofol at clinical relevant concentrations ( approximately 56 microM) potentiated the current responses through the P2X(4) receptor in a dose-dependent manner, whereas propofol did not affect the responses through the P2X(2) receptor or through the heterologous complex of the P2X(2) and P2X(3) (P2X(2+3)) receptor. These results suggest that activation of P2X(4) subtype in the brain and the motor neurons of the spinal anterior horn might be involved in the excitatory effect by propofol such as convulsion and unexpected movements.

Adenosine Triphosphate↗
PubMed2000

Calcium binding properties of recombinant calcium binding protein 40, a major calcium binding protein of lower eukaryote Physarum polycephalum.

Calcium binding protein 40 (CBP40) is a Ca(2+)-binding protein abundant in the plasmodia of Physarum polycephalum. CBP40 consists four EF-hand domains in the COOH-terminal half and a putative alpha-helix domain in the NH(2)-terminal half. We expressed recombinant proteins of CBP40 in Escherichia coli to investigate its Ca(2+)-binding properties. Recombinant proteins of CBP40 bound 4 mol of Ca(2+) with much higher affinity (pCa(1/2) = 6.5) than that of calmodulin. When residues 1-196 of the alpha-helix domain were deleted, the affinity for Ca(2+) decreased to pCa(1/2) = 4.6. A chimeric calmodulin was generated by conjugating the alpha-helix domain of CBP40 with calmodulin. The affinity of Ca(2+) for the chimeric calmodulin was higher than that for calmodulin, suggesting that the alpha-helix domain is responsible for the high affinity of CBP40 for Ca(2+). CBP40 forms large aggregates reversibly in a Ca(2+)-dependent manner. A mutant protein with a deletion of NH(2)-terminal 32 residues, however, could not aggregate, indicating the importance of these residues for the aggregation. The aggregation occurs above micromolar levels of Ca(2+) concentration, so it may only occur when CBP40 is secreted out of the plasmodial cells.

Animals↗
PubMed2000

The molecular structure of the fastest myosin from green algae, Chara.

Chara myosin in green algae, Chara corallina, is the fastest myosin of all those observed so far. To shed light on the molecular mechanism of this fast sliding, we determined the primary structure of Chara myosin heavy chain (hc). It has a motor domain, six IQ motifs for calmodulin binding, a coiled-coil structure to dimerize, and a globular tail. Chara myosin hc is very similar to some plant myosins and has been predicted to belong to the class XI. Short loop 1 and loop 2 may account for the characteristics of mechanochemical properties of Chara myosin.

Amino Acid Sequence↗
PubMed2000

Assembly of smooth muscle myosin by the 38k protein, a homologue of a subunit of pre-mRNA splicing factor-2.

Smooth muscle myosin in the dephosphorylated state does not form filaments in vitro. However, thick filaments, which are composed of myosin and myosin-binding protein(s), persist in smooth muscle cells, even if myosin is subjected to the phosphorylation- dephosphorylation cycle. The characterization of telokin as a myosin-assembling protein successfully explained the discrepancy. However, smooth muscle cells that are devoid of telokin have been observed. We expected to find another ubiquitous protein with a similar role, and attempted to purify it from chicken gizzard. The 38k protein bound to both phosphorylated and dephosphorylated myosin to a similar extent. The effect of the myosin-binding activity was to assemble dephosphorylated myosin into filaments, although it had no effect on the phosphorylated myosin. The 38k protein bound to myosin with both COOH-terminal 20 and NH(2)-terminal 28 residues of the 38k protein being essential for myosin binding. The amino acid sequence of the 38k protein was not homologous to telokin, but to human p32, which was originally found in nuclei as a subunit of pre-mRNA splicing factor-2. Western blotting showed that the protein was expressed in various smooth muscles. Immunofluorescence microscopy with cultured smooth muscle cells revealed colocalization of the 38k protein with myosin and with other cytoskeletal elements. The absence of nuclear immunostaining was discussed in relation to smooth muscle differentiation.

Amino Acid Sequence↗
PubMed2000

Stable transfectants of smooth muscle cell line lacking the expression of myosin light chain kinase and their characterization with respect to the actomyosin system.

We constructed a plasmid vector having a 1.4-kilobase pair insert of myosin light chain kinase (MLCK) cDNA in an antisense direction to express antisense mRNA. The construct was then transfected to SM3, a cell line from vascular smooth muscle cells, producing a few stable transfectants. The down-regulation of MLCK expression in the transfectants was confirmed by both Northern and Western blots. The control SM3 showed chemotaxic motility to platelet-derived growth factor-BB, which was supported by lamellipodia. However, the transfectants showed neither chemotaxic motility nor developed lamellipodia, indicating the essential role of MLCK in the motility. The specificity for the targeting was assessed by a few tests including the rescue experiment. Despite this importance of MLCK, platelet-derived growth factor-BB failed to induce MLC20 phosphorylation in not only the transfectants but also in SM3. The mode in which MLCK was involved in the development of membrane ruffling is discussed with special reference to the novel property of MLCK that stimulates the ATPase activity of smooth muscle myosin without phosphorylating its light chain (Ye, L.-H., Kishi, H., Nakamura, A., Okagaki, T., Tanaka, T., Oiwa, K., and Kohama, K. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 6666-6671).

Actomyosin↗
PubMed2000

Inhibitory effects of ML-9, wortmannin, and Y-27632 on the chemotaxis of vascular smooth muscle cells in response to platelet-derived growth factor-BB.

The chemotactic migration toward platelet-derived growth factor-BB of SM3, a cell line established from rabbit aorta smooth muscle, was examined by the Boyden chamber method. Myosin light-chain (MLC) kinase inhibitors ML-9 and wortmannin, and the Rho kinase inhibitor Y-27632 effectively reduced the migration. However, neither membrane ruffling nor the phosphorylation of MLC was inhibited concomitantly. The reduction is discussed with reference to a novel property of MLC kinase, which stimulates myosin ATPase activity without phosphorylating MLC [Ye et al. (1999) Proc. Natl. Acad. Sci. USA 96, 6666-6671].

Amides↗
PubMed2000

Retinal fascin: functional nature, subcellular distribution, and chromosomal localization.

PURPOSE: To investigate the functional properties, subcellular localization, and chromosomal location of retinal fascin. METHODS: Recombinant retinal fascin protein was prepared by using a baculovirus-insect expression system. Actin-binding and -bundling assays were performed with chick actin purified from skeletal muscle. Western blot analysis and immunohistochemistry were performed with a polyclonal antibody raised against bovine retinal fascin. A human retinal cDNA library was screened with an expressed sequence tag cDNA fragment. Chromosomal location was determined with fluorescent in situ hybridization. RESULTS: The actin-binding and actin-bundling activities of retinal fascin were demonstrated by high- and low-speed centrifugation assays. Formation of filamentous (F)-actin bundles by retinal fascin in vitro was also morphologically confirmed by fluorescence microscopy and electron microscopy. Immunohistochemical analysis revealed that retinal fascin protein was localized specifically in the outer and inner segments of the photoreceptor cells in the retina. Two splicing variants of human retinal fascin cDNA were also located. One clone encoded 492 amino acids, and the other encoded 516 amino acids. The gene encoding retinal fascin was localized to human chromosome 17, region q24 -25. CONCLUSIONS: These results suggest that retinal fascin may play a role in formation of unique morphologic structures of the photoreceptor cells and is a candidate gene for retinal degenerative disorders.

Actins↗
PubMed1999

Inhibitory effect of phosphorylated myosin light chain kinase on the ATP-dependent actin-myosin interaction.

Myosin light chain kinase (MLCK) phosphorylates the regulatory light chain of myosin in the presence of Ca(2+) and calmodulin (Ca(2+)-CaM) so that myosin can interact with actin filaments. MLCK has another activity that is not attributable to this kinase activity, i.e., it inhibits the ATP-dependent movement of actin filaments on a myosin-coated glass surface. MLCK itself can be phosphorylated at site A and site B with a few kinases. The phosphorylation at site A reduces kinase activity. However, we have no knowledge as to how phosphorylation of MLCK affects the inhibitory activity of MLCK. When MLCK was phosphorylated at site B, it exerted an inhibitory effect on the movement in much lower concentrations. When Ca(2+)-CaM or ML-9 was present, the inhibition was reduced. The reduction was less when the movement was arrested by the MLCK phosphorylated at site B. This observation was explained by the increase in the affinity of MLCK to myosin upon the phosphorylation at site B.

Actin Cytoskeleton↗