Masahito Kawata

Small GTP-Binding Proteins

Small GTP-binding proteins (G proteins) exist in eukaryotes from yeast to human and constitute a superfamily consisting of more than 100 members. This superfamily is structurally classified into at least five families: the Ras, Rho, Rab, Sar1/Arf, and Ran families. They regulate a wide variety of cell functions as biological timers (biotimers) that initiate and terminate specific cell functions and determine the periods of time for the continuation of the specific cell functions. They furthermore play key roles in not only temporal but also spatial determination of specific cell functions. The Ras family regulates gene expression, the Rho family regulates cytoskeletal reorganization and gene expression, the Rab and Sar1/Arf families regulate vesicle trafficking, and the Ran family regulates nucleocytoplasmic transport and microtubule organization. Many upstream regulators and downstream effectors of small G proteins have been isolated, and their modes of activation and action have gradually been elucidated. Cascades and cross-talks of small G proteins have also been clarified. In this review, functions of small G proteins and their modes of activation and action are described.

A geranylgeranyltransferase for rhoA p21 distinct from the farnesyltransferase for ras p21A

We have clarified that rhoA p21 purified from bovine aortic smooth muscle is geranylgeranylated at the cysteine residue in the C-terminal CAAX motif (A is an aliphatic amino acid and X is any amino acid). In this paper, a geranylgeranyltransferase for rhoA p21 (rhoA p21 GGT) was partially purified from bovine brain cytosol. This enzyme transferred a geranylgeranyl moiety from geranylgeranyl pyrophosphate to rhoA p21 having the CAAX motif (rhoA p21-CAAX) but not to rhoA p21 lacking the AAX portion. rhoA p21 GGT was separated from the previously reported farnesyltransferase for ras p21s (ras p21 FT) by column chromatographies and did not geranylgeranylate or farnesylate c-Ha-ras p21-CAAX. ras p21 FT did not geranylgeranylate or farnesylate rhoA p21-CAAX. These results indicate that rhoA p21 GGT distinct from ras p21 FT is present in bovine brain cytosol.

Identification of a platelet Mr 22,000 GTP-binding protein as the novel smg-21 gene product having the same putative effector domain as the ras gene products

We have recently purified a Mr 22,000 GTP-binding protein (G protein) to near homogeneity from human platelet membranes and characterized it (Ohmori, T., Kikuchi, A., Yamamoto, K., Kim, S. and Takai, Y. (1989) J. Biol. Chem. in press). This platelet G protein was present most abundantly among several G proteins in platelets and showed a Mr of about 22,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This platelet G protein showed kinetic and physical properties very similar to those of the novel smg-21 gene product, having the same putative effector domain as the ras gene products, which we have recently purified to near homogeneity from bovine brain membranes and characterized (Kawata, M., Matsui, Y., Kondo, J., Hishida, T., Teranishi, Y. and Takai, Y. (1988) J. Biol. Chem. in press). Moreover, the peptide map of the platelet G protein was identical with that of the smg-21 gene product and the partial amino acid sequence of the platelet G protein was identical with that of the smg-21 gene product. These results indicate that this human platelet G protein is the smg-21 gene product.

Molecular cloning of smg p21B and identification of smg p21 purified from bovine brain and human platelets as smg p21B

We have previously purified smg p21 from bovine brain membranes and isolated its cDNA from a bovine brain cDNA library. In the present studies, we have performed extensive screening of the bovine brain cDNA library with the cloned smg p21 cDNA as a probe and isolated another cDNA encoding a protein highly homologous to smg p21. The proteins encoded by the previously and newly isolated cDNAs are designated as smg p21A and -B, respectively. Since the partial amino acid sequences determined previously from the smg p21 purified from bovine brain were identical with the common amino acid sequences between smg p21A and -B, we have further sequenced smg p21 and identified it as smg p21B. We have also further sequenced the smg p21 purified from human platelet membranes and identified it as smg p21B. Amino acid sequence analysis indicates that smg p21A is identical with the rap1A and Krev-1 proteins and smg p21B is identical with the rap1B protein.

Identification of a major GTP-binding protein in bovine aortic smooth muscle membranes as smg p21, a GTP-binding protein having the same effector domain as ras p21s.

At least two GTP-binding proteins (G proteins) with Mr values of about 20,000 were extracted from bovine aortic smooth muscle membranes by sodium cholate. The most abundant G protein (22K G) was purified to near homogeneity by successive column chromatographies of Ultrogel AcA-44, phenyl-Sepharose CL-4B, hydroxyapatite and Mono Q HR5/5. 22K G showed kinetic and physical properties very similar to those of smg p21, a G protein recently isolated from bovine brain and human platelet membranes, having the same effector domain as ras p21s. Moreover, 22K G was recognized specifically by the anti-smg p21 antibody. These results indicate that the major G protein in bovine aortic smooth muscle membranes is smg p21.

A case of coronary lesion with lotus root appearance treated by percutaneous coronary intervention with intravascular ultrasound guidance

A 66-year-old man underwent percutaneous coronary intervention (PCI). Coronary angiography showed a diffuse lesion with lotus root appearance and severe stenosis in the left anterior descending artery (LAD). Multiple channels were observed by intravascular ultrasound (IVUS). Different channels were connected to the first diagonal branch, the first septal branch and LAD lumen separately. To prevent obstruction of side branches, we made connections to the branches from the main channel of LAD with tapered-tip guide wire, followed by balloon dilatation and stenting without side branch obstruction. IVUS findings were helpful for the PCI with a lotus root appearance lesion.

Percutaneous interventional techniques to remove embolized silicone port catheters from heart and great vessels

Rupture of a silicon port catheter is a relatively rare complication and sometimes it is difficult to remove it. We experienced three cases of retrieval of silicone port catheters migrating into cardiac ventricle or pulmonary artery. Several devices such as a snare wire, an ablation catheter, and a basket catheter in combination with interventional guiding catheter were applied to retrieve them. These interventional techniques are applicable for retrieval of embolized vascular access port system and other catheter fragments.

Binding of ras p21 to bands 4.2 and 6 of human erythrocyte membranes

The direct binding protein(s) of ras p21 was (were) investigated in inside‐out vesicles of human erythrocyte ghosts using the pure v‐Kirsten (Ki)‐ras p21 synthesized in E. coli. The bound ras p21 was detected immunochemicallly using an anti‐v‐Ki‐ras p21 monoclonal antibody. ras p21 bound to vesicles. Prior digestion of the vesicles with trypsin reduced this binding significantly. When ras p21 was laid over vesicle proteins immobilized on a nitrocellulose sheet by transfer from the gel of SDS‐polyacrylamide gel electrophoresis, ras p21 bound to bands 4.2 and 6. ras p21 binding to these proteins was reduced by prior incubation of ras p21 with the purified band 4.2 or 6 protein. These results indicate that v‐Ki‐ras p21 can bind directly to bands 4.2 and 6 of human erythrocyte membranes as far as tested in an in vitro cell‐free system.

Primary pulmonary valve papillary fibroelastoma

Papillary fibroelastoma (PFE) is a rare and benign cardiac tumor typically found on the valvular endocardium. In most cases, PFE is identified incidentally on echocardiography or during cardiac surgery. The patient was a 73-year-old man who had been treated for hepatocellular carcinoma for 5 years. On echocardiography, a 2.5-cm diameter mass was detected in the pulmonary trunk just above the pulmonary valve. Through a transpulmonary arterial approach with cardiopulmonary bypass, the mass identified on the commissure of the right and posterior pulmonary cusp was surgically excised together with the attached endocardium. Despite the benign histology of PFE, lethal embolic events such as stroke, myocardial infarction, and pulmonary embolism are reported in some cases. To prevent such complications, tumor identification and surgical excision are essential.

Familial Carney complex with biatrial cardiac myxoma

We report a case of Carney complex (CNC) with biatrial cardiac myxoma. The patient had left and right atrial myxomas which were resected in a surgery. She showed bilateral adrenal tumors and multiple mammary tumors. She had pigmentation on her lower lip. Previously, her daughter was also diagnosed with CNC with cardiac myxoma. Both of them showed mutations in the PRKAR1A gene. <Learning objective: Carney complex is a syndrome with skin pigmentation, myxomas, and endocrine abnormalities. It is an autosomal dominant disease and shows PRKAR1A gene mutation. We experienced a rare case of familial Carney complex with biatrial cardiac myoxomas found by echocardiography and treated surgically.>