Epsin binds to the EH domain of POB1 and regulates receptor-mediated endocytosis.

POB1 has been identified as a RalBP1-binding protein and has the Eps15 homology (EH) domain. The EH domain-containing proteins have been suggested to be involved in clathrin-dependent endocytosis. To clarify the function of POB1, we purified a protein which binds to the EH domain of POB1 from bovine brain cytosol and identified it as Epsin, which is known to bind to the EH domain of Eps15. Epsin has three Asn-Pro-Phe (NPF) motifs in the C-terminal region, which are known to form the core sequence for the binding to the EH domain. The EH domain of POB1 interacted directly with the region containing the NPF motifs of Epsin. Expression of Epsin in CHO-IR cells inhibited internalization of insulin although it affected neither insulin-binding nor autophosphorylation activities of the insulin receptor. Taken together with the observations that Epsin is involved in internalization of the receptors for epidermal growth factor and transferrin, these results suggest that Epsin is a binding partner of POB1 and their binding regulates receptor-mediated endocytosis.

Katanin, a microtubule-severing protein, is a novel AAA ATPase that targets to the centrosome using a WD40-containing subunit.

Microtubule disassembly at centrosomes is involved in mitotic spindle function. The microtubule-severing protein katanin, a heterodimer of 60 and 80 kDa subunits, was previously purified and shown to localize to centrosomes in vivo. Here we report the sequences and activities of the katanin subunits. p60 is a new member of the AAA family of ATPases, and we show that expressed p60 has microtubule-stimulated ATPase and microtubule-severing activities in the absence of p80. p80 is a novel protein containing WD40 repeats, which are frequently involved in protein-protein interactions. The p80 WD40 domain does not participate in p60 dimerization, but localizes to centrosomes in transfected mammalian cells. These results indicate katanin's activities are segregated into a subunit (p60) that possesses enzymatic activity and a subunit (p80) that targets the enzyme to the centrosome.

Effect of bucillamine on the rat trinitrobenzene sulfonic acid induced model of colitis.

Salicylazosulphapyridine and corticosteroids are the only remedies for inflammatory bowel disease currently in clinical use. They do not, however, necessarily bring about satisfactory therapeutic benefits, so that new agents are needed. In this study, we evaluated the effect of bucillamine, a new antirheumatic agent, in experimental rat colitis induced by trinitrobenzene sulfonic acid enema. Wistar rats were given vehicle alone (n = 16) or treated with 50 (n = 20) or 150 (n = 20) mg/kg of bucillamine daily for three weeks after induction of colitis. Conventional histological sections of the colon stained by haematoxylin and eosin were prepared and observed under a light microscope to determine colonic damage scores. The determinations were significantly lower in the group treated with 50 mg/kg of bucillamine and tended to be lower in the group treated with 150 mg/kg of bucillamine than in the untreated group, which implied that the experimentally induced colonic inflammatory changes and ulcerations were alleviated by bucillamine. Blood tests showed no abnormal values at the end of the treatment. The present observations suggest that bucillamine should be developed further as a possible therapy for inflammatory bowel disease.

Rho-associated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho.

The small GTP binding protein Rho is implicated in cytoskeletal responses to extracellular signals such as lysophosphatidic acid to form stress fibers and focal contacts. Here we have purified a Rho-interacting protein with a molecular mass of approximately 164 kDa (p164) from bovine brain. This protein bound to GTPgammaS (a non-hydrolyzable GTP analog).RhoA but not to GDP.RhoA or GTPgammaS.RhoA with a mutation in the effector domain (RhoAA37).p164 had a kinase activity which was specifically stimulated by GTPgammaS.RhoA. We obtained the cDNA encoding p164 on the basis of its partial amino acid sequences and named it Rho-associated kinase (Rho-kinase). Rho-kinase has a catalytic domain in the N-terminal portion, a coiled coil domain in the middle portion and a zinc finger-like motif in the C-terminal portion. The catalytic domain shares 72% sequence homology with that of myotonic dystrophy kinase and the coiled coil domain contains a Rho-interacting interface. When COS7 cells were cotransfected with Rho-kinase and activated RhoA, some Rho-kinase was recruited to membranes. Thus it is likely that Rho-kinase is a putative target serine/threonine kinase for Rho and serves as a mediator of the Rho-dependent signaling pathway.

The small GTP-binding protein Rho binds to and activates a 160 kDa Ser/Thr protein kinase homologous to myotonic dystrophy kinase.

The small GTP-binding protein Rho functions as a molecular switch in the formation of focal adhesions and stress fibers, cytokinesis and transcriptional activation. The biochemical mechanism underlying these actions remains unknown. Using a ligand overlay assay, we purified a 160 kDa platelet protein that bound specifically to GTP-bound Rho. This protein, p160, underwent autophosphorylation at its serine and threonine residues and showed the kinase activity to exogenous substrates. Both activities were enhanced by the addition of GTP-bound Rho. A cDNA encoding p160 coded for a 1354 amino acid protein. This protein has a Ser/Thr kinase domain in its N-terminus, followed by a coiled-coil structure approximately 600 amino acids long, and a cysteine-rich zinc finger-like motif and a pleckstrin homology region in the C-terminus. The N-terminus region including a kinase domain and a part of coiled-coil structure showed strong homology to myotonic dystrophy kinase over 500 residues. When co-expressed with RhoA in COS cells, p160 was co-precipitated with the expressed Rho and its kinase activity was activated, indicating that p160 can associate physically and functionally with Rho both in vitro and in vivo.

Effect of granulocyte colony-stimulating factor on hematopoietic recovery after peripheral blood progenitor cell transplantation.

To examine the effect of granulocyte colony-stimulating factor (G-CSF) on hematopoietic recovery after high-dose chemotherapy and peripheral blood progenitor cell transplantation (PBPCT), 20 patients with hematologic malignancies were divided into two groups. One group was given G-CSF at a daily dose of 50 micrograms/m2 subcutaneously, the other received no G-CSF. Neutrophil recovery was accelerated in the G-CSF treated patients and exceeded 0.5 x 10(9)/l at a median of 10 days post-PBPCT compared with 14 days in the control group (p < 0.01). This reduction led to a decrease in antibiotic use and a trend toward fewer febrile days in the G-CSF treated group.

Autoimmune thrombocytopenia following peripheral blood stem cell autografting.

A 22-year-old woman diagnosed as AML (M3) received myeloablative chemotherapy followed by autologous peripheral stem cell transplantation (PBSCT). Rapid hematopoietic reconstitution occurred. By day 10, the neutrophil count was > 0.5 x 10(9)/l and the platelet count > 50 x 10(9)/l. The platelet count was 145 x 10(9)/l on day 20. Purpura developed on the anterior chest and legs on day 50, at which time the platelet count fell to 17 x 10(9)/l. The BM was hypocellular with an increase in megakaryocytes. Platelet-associated IgG (PAIgG) was 88.1 ng/10(7) platelets (normal range 9-25 ng/10(7)); a diagnosis of idiopathic thrombocytopenic purpura (ITP) was made. Prednisolone administration led to an increase in the platelet count and a decrease in PAIgG. Analysis of lymphocyte subsets revealed an increased number of CD3+ gamma/delta T cells. It is postulated that the thrombocytopenia in this case was due to an autoimmune mechanism such as ITP.

Collection of peripheral blood stem cells mobilized by high-dose Ara-C plus VP-16 or aclarubicin followed by recombinant human granulocyte-colony stimulating factor.

We developed an effective method for harvesting large numbers of peripheral blood stem cells (PBSC) for use in autotransplantation. Twenty patients with hematological malignancies were treated with high doses of Ara-C (12 g/m2) and VP-16/aclarubicin followed by administration of rhG-CSF (50 micrograms/m2). The optimal time for starting PBSC collection was determined by monitoring the CD34-positive stem cells in blood using immunomagnetic beads. PBSC were collected with a CS-3000 blood cell separator. A total blood volume between 7000 and 9000 ml was processed in each apheresis. Under these conditions, a total of 64 apheresis procedures was performed in the 20 patients. The mean numbers of mononuclear cells and of CFU-GM harvested per apheresis were 4.1 x 10(8)/kg and 110 x 10(4)/kg, respectively. A number of CFU-GM sufficient for engraftment (> 30 x 10(4)/kg) could be harvested by a single apheresis in 15 of the 20 patients. So far, 11 patients have been transplanted with PBSC and obtained rapid hematopoietic recovery. The median time to recover neutrophils more than 0.5 x 10(9)/l was 10 days, and that for platelets 50 x 10(9)/l was 11 days. This method for harvesting large numbers of PBSC allows safer autotransplantation in patients with chemoradiosensitive tumors, and is applicable to older patients.

Predictive factors for the response of ulcerative colitis patients during the acute-phase treatment.

Twenty-six consecutive admissions of 24 patients with severe ulcerative colitis (UC) hospitalized in our Department at some time between January 1983 and December 1988 were studied to identify factors useful in the prediction of response to medical treatment in the acute inflammatory phase of this disease. Results of laboratory tests (white blood cells, red blood cells, platelet count, hemoglobin, erythrocyte sedimentation rate, total protein, albumin, alpha 2-microglobulin, cholinesterase, total cholesterol, and triglycerides) and of endoscopic findings (extent of disease, progress of the lesions, sparing of the rectum, and presence of geographic ulcers, longitudinal ulcers, and polypoid mucosal tags) were analyzed for any relationship with the effect of medical treatment during the acute phase. The effect of treatment was evaluated in terms of days it took for a severe condition to improve to an intermediate one defined by Truelove and Witts' categories for UC severity. C-Reactive protein, nutritive condition (total protein, albumin, and cholinesterase), extent of the lesions, and existence of polypoid mucosal tags provide predictive factors useful in the management of UC during the acute phase.

[Neonatal hyperbilirubinemia of inadequately breast-fed infants and the effect of formula supplementation].

One hundred and fifty full-term breast-fed infants were analysed for their oral intake in the first 6 days. Fifty infants with less than 330 ml/kg/6 days breast feeding were defined as inadequately breast-fed infants. Twenty inadequately breast-fed infants without formula supplementation had significantly lower total fluid intake, lower total calorie intake, more body weight loss and significantly higher rates of hyperbilirubinemia and requirement of phototherapy when compared with the control group of 100 infants with more than 330 ml/kg/6 days breast feeding. On the other hand, 30 inadequately breast-fed infants with formula supplementation had significantly higher total fluid intake, higher total calorie intake, lower body weight loss and requirement of phototherapy than those without formula supplementation. From these data, we suggest that (1) Inadequate feeding may be a factor responsible for the higher prevalence of early neonatal jaundice in breast-fed infants reported in the literature. (2) Lower fluid intake, lower calorie intake and greater body weight loss may be associated with the higher incidence of hyperbilirubinemia and requirement of phototherapy. (3) Formula supplementation may be helpful in decreasing the risk of hyperbilirubinemia in inadequately breast-fed infants.