Glial cell line-derived neurotrophic factor/neurturin-induced differentiation and its enhancement by retinoic acid in primary human neuroblastomas expressing c-Ret, GFR alpha-1, and GFR alpha-2.

Neuroblastomas often undergo spontaneous differentiation and/or regression in vivo, which is at least partly regulated by the signals through neurotrophins and their receptors. Recently, glial cell line-derived neurotrophic factor (GDNF) and a second family member, neurturin (NTN), have been found to mediate their signals by binding to a heterotetrameric complex of c-Ret tyrosine kinase receptors and glycosylphosphatidylinositol-linked proteins, GFR alpha-1 (GDNFR-alpha) or GFR alpha-2 (TrnR2/GDNFR-beta/NTNR-alpha/RETL2). Here, we studied the effect of GDNF and NTN on human neuroblastomas in the short-term primary culture system, as well as the expression of c-Ret, GFR alpha-1, GFR alpha-2, GDNF, and NTN. GDNF (1-100 ng/ml) induced morphological differentiation in 34 of 38 primary neuroblastomas and an accompanying increase in c-Fos induction. These effects were markedly enhanced by treatment with 5 microM all-trans-retinoic acid. Although GDNF alone induced a rather weak differentiation independent of the disease stages, the enhancement of neurite outgrowth induced by treatment with both GDNF and all-trans-retinoic acid was significantly correlated with younger age (less than 1 year; P = 0.0039), non-stage 4 diseases (P = 0.0023), a single copy of N-myc (P = 0.027), and high levels of TRK-A expression (P = 0.0062). To examine the expression levels of GFR alpha-1, we cloned a short form of the human GFR alpha-1 gene with a 15-bp deletion by screening a human adult substantia nigra cDNA library. Many primary neuroblastomas expressed c-Ret, GFR alpha-1, and GFR alpha-2 as well as their ligands, GDNF and NTN, suggesting the presence of a paracrine or autocrine signaling system within the tumor tissue. The effect of NTN on primary culture cells of neuroblastoma was similar to that of GDNF. These imply that the GDNF(NTN)/c-Ret/GFR alpha-1(GFR alpha-2) signaling may have an important role in regulating the growth, differentiation, and cell death of neuroblastomas.

Enhanced expression of N-myc messenger RNA in neuroblastomas found by mass screening.

A substantial fraction of neuroblastomas found by mass screening have been suggested to regress spontaneously because of the high incidence of infantile neuroblastomas in the screening population. In this study, 70 neuroblastomas were analyzed for expression of proto-oncogenes related to neuronal differentiation to clarify the biological significance of proto-oncogene expression in the screening-positive and -negative tumors. The tumors consisted of 39 neuroblastomas found by screening (group 1), 16 non-N-myc-amplified neuroblastomas found by clinical symptom(s) (group 2), and 15 N-myc-amplified neuroblastomas found by clinical symptom(s) (group 3). The expression of c-src, trk A, and N-myc in tumor tissues was analyzed by quantitative RNA PCR. Neuronal c-srcN2 expression varied significantly in the following order: group 1 > group 2 > group 3. The level of expression of trk A was markedly reduced in group 3 but did not differ in groups 1 and 2. Most tumors in group 3 overexpressed N-myc. However, N-myc expression in group 1 was significantly higher than that in group 2. Thus, the characteristics of proto-oncogene expression in screening-positive tumors included enhanced expression of c-srcN2 and N-myc mRNA, regardless of nonamplification of N-myc. Our results suggest that the role of N-myc differs in neuroblastomas detected by screening and in N-myc-amplified tumors.

X-ray crystal structure and catalytic properties of Thr252Ile mutant of cytochrome P450cam: roles of Thr252 and water in the active center.

The structure-function relationship in cytochrome P450cam monooxygenase was studied by employing its active site mutant Thr252Ile. X-ray crystallographic analyses of the ferric d-camphor-bound form of the mutant revealed that the mutation caused a structural change in the active site giving an enlarged oxygen-binding pocket that did not contain any hydrophilic group such as the OH group of Thr and H(2)O. The enzyme showed a low monooxygenase activity of ca. 1/10 of the activity of the wild-type enzyme. Kinetic analyses of each catalytic step revealed that the rate of proton-coupled reduction of the oxygenated intermediate of the enzyme, a ternary complex of dioxygen and d-camphor with the ferrous enzyme, decreased to about 1/30 of that of the wild-type enzyme, while the rates of other catalytic steps including the reduction of the ferric d-camphor-bound form by reduced putidaredoxin did not change significantly. These results indicated that a hydrophilic group(s) such as water and/or hydroxyl group in the active site is prerequisite to a proton supply for the reduction of the oxygenated intermediate, thereby giving support for the operation of a proton transfer network composed of Thr252, Asp251, and two other amino acids and water proposed by previous investigators.

Identification of a new cellular protein that can interact specifically with DAN.

Recently, we demonstrated that the DAN gene product contains a growth- and/or a tumor-suppressive activity in vitro. In the present work, using a yeast two-hybrid system, we searched for cellular proteins that can associate with the DAN gene product. A cDNA clone, termed DA41, was initially isolated from an adult rat lung cDNA library. The DA41 gene was expressed in all adult tissues examined, however, the levels of expression varied significantly among the different tissues. Like the DAN gene product, the DA41 protein was similarly restricted to the cytoplasm. Sequence analysis revealed that DA41 cDNA is 2,167 nucleotides in length and contains a single open reading frame (ORF) of 582 amino acids (61,945 daltons). A homology search revealed that the DA41 gene product shares no structural similarity with those filed in the data base. In a synchronous 3Y1 cell culture, DA41 mRNA was expressed at a low level in quiescent cells; however, its level was significantly increased between the G1 and S phases of the cell cycle. On the other hand, the expression level of DAN mRNA did not change throughout the cell cycle progression. These results suggest that the DAN-DA41 complex might play a crucial role in the regulation of the cell cycle progression.

GDNF-induced neurite formation was stimulated by protein kinase inhibitors and suppressed by Ras inhibitors.

The effects of various inhibitors on the glial cell line-derived neurotrophic factor (GDNF)-induced neurite formation in TGW human neuroblastoma cells were investigated. Treatment of cells with Ser/Thr protein kinase inhibitors such as staurosporine, H-7, H-8 and HA-1004, induced neurite formation without GDNF. On the other hand, tyrosine kinase inhibitors such as erbstatin, genistein and herbimycin A did not produce neurites per se, but effectively enhanced the GDNF-induced neurite formation. A phosphatase inhibitor, okadaic acid, and Ras inhibitors such as oxanosine, damnacanthal and conophylline strongly suppressed the effect of GDNF. These results suggest that a tyrosine protein kinase has a suppressive role in the neurite formation induced by GDNF and that Ras is necessary for the signaling initiated by GDNF.

Nonoperative management of blunt pancreatic injury in childhood.

PURPOSE: Nonoperative management for blunt pancreatic injury in children was performed between 1977 and 1998. The efficiency and safety of nonoperative management was examined. METHODS: Pancreatic injury was diagnosed in 20 children. The surgical indication was determined by hemodynamic instability and the management of associated injuries. Children without surgical indications were treated initially by nonoperative management. RESULTS: Nineteen of 20 children were treated initially nonoperatively, and 18 of the 19 survived. Surgical exploration was performed in only 1 child with perforation of the duodenum and bile duct. One child died of complications of total parenteral nutrition. Ultrasound scan and computed tomography scan showed pancreatic contusion in 9, laceration in 6, and injury of the main pancreatic duct (MPD) in 5. Pseudocysts were detected in 10 (5 laceration and 5 MPD injury). Pseudocysts smaller than 10 cm disappeared after nonoperative management, and those larger than 10 cm required operative management. Rupture of pseudocysts occurred in 2 children by rotating the upper torso. CONCLUSIONS: Nonoperative management of pancreatic injuries is effective in children, although careful management is required to avoid complications. Pseudocysts smaller than 10 cm were treated successfully by nonoperative management, and those larger than 10 cm required surgical management.

Patient information exchange guideline MERIT-9 using medical markup language MML.

To realize clinical data exchange between healthcare providers, there must be many standards in many layers. Terms and codes should be standardized, syntax to wrap the data must be mutually parsable, then transfer protocol or exchange media should be agreed. Among many standards for the syntax, HL7 and DICOM are most successful. However, everything could not be handled by HL7 solely. DICOM is good for radiology images, but, other clinical images are already handled by other "lighter" data formats like JPEG, TIFF. So, it is not realistic to use only one standard for every area of clinical information. For description of medical records, especially for narrative information, we created SGML DTD for medical information, called MML (Medical Markup Language). It is already implemented in more than 10 healthcare providers in Japan. As it is a hierarchical description of information, it is easily used as a basis of object request brokering. It is again not realistic to use MML solely for clinical information in various level of detail. Therefore, we proposed a guide-line for use of available medical standards to facilitate clinical information exchange between healthcare providers. It is called MERIT-9 (MEdical Records, Images, Texts,--Information eXchange). A typical use is HL7 files, DICOM files, referred from an MML file in a patient record, as external entities. Both MML and MERIT-9 are research projects of Japanese Ministry of Health and Welfare, and the purpose is to facilitate clinical data exchanges. They are becoming to be used in technical specifications for new hospital information systems in Japan.

[Morphometric analysis of normal glomerular epithelial cells in rat and human].

It has been postulated that morphological changes of podocytes might be related to glomerular sclerotic lesions in experimental models and patients with glomerular diseases. To estimate the absolute number of podocytes in mammalian normal glomerulus, we analyzed normal glomeruli in four rats and six humans. In PAS stained light microscopic sections, at least 25 midsections of open glomeruli were photographed. Stereologic estimation was performed to obtain the following values: absolute values of glomerular volume (V), glomerular surface area (S), podocyte and intraglomerular cell number per glomerulus (P and IGC), glomerular surface area covered by one podocyte (S/P) and glomerular volume occupied by one intraglomerular cell (V/IGC). The glomerular volume, glomerular surface area and podocyte and intraglomerular cell numbers per glomerulus of human were significantly increased compared with those of the rat (V: 2.70 +/- 0.86 > 0.89 +/- 0.19, S: 4.84 +/- 1.26 > 1.88 +/- 0.26, P: 407.7 +/- 88.2 > 153.8 +/- 84.0, p < 0.01 vs rat). On the other hand, there were no significant differences in glomerular surface area covered by one podocyte and glomerular volume occupied by one intraglomerular cell between the humans and rats (S/P: 1.25 +/- 0.20, 1.29 +/- 0.05, V/IGC: 2,471 +/- 487, 2,227 +/- 201, p < 0.01 vs rat). These data were almost the same as previously reported values. It appears that these values can be considered as standards for rats and humans in morphometric analysis of the glomerulus.

Two adult cases of IgM-associated mesangial proliferative glomerulonephritis.

Two adult patients with mesangial proliferative glomerulonephritis with diffuse IgM deposition in the glomeruli are reported. Case 1 was a 25-year-old female with nephrotic syndrome who showed complete remission after treatment with prednisolone (PSL). Case 2 was a 46-year-old male with asymptomatic proteinuria who showed incomplete remission (0.5-1.0 g/24 hr) of urinary protein without any medication. In light microscopy, these patients revealed minimal or slight proliferation of glomerular mesangial cells without glomerular sclerosis and crescent formation. Deposition of IgM and C3 was observed in the glomerular mesangial areas and capillary walls by immunofluorescence. Electron-dense deposits were observed in the glomerular mesangial areas in these patients. Mesangial proliferative glomerulonephritis associated with diffuse IgM deposition in the glomeruli appears to have a benign clinical course. It has also been suggested that this disease has variant clinical courses since we recently experienced two other patients with mesangial proliferative glomerulonephritis with focal IgM deposits who showed renal tubular dysfunction or chronic renal failure.

Prediction of postnatal outcomes in congenital diaphragmatic hernia using MRI signal intensity of the fetal lung.

OBJECTIVE: Prognostic prediction in prenatally diagnosed congenital diaphragmatic hernia (CDH) is needed. The aim of the study was to evaluate magnetic resonance imaging (MRI) signal intensity of the fetal lung as a predictor of prognosis in CDH. STUDY DESIGN: The subjects consisted of 12 fetuses with prenatally diagnosed CDH, who were treated soon after the birth in our institution. They all underwent MRI at 29 to 37 weeks of gestation. The ratio of the lung signal intensity to the spinal fluid signal intensity (L/SF) was calculated using region-of-interest analysis of T2-weighted images. The relationship between L/SF and clinical data was then examined. RESULT: L/SF were significantly larger in survivors compared with deaths (0.815 vs 0.614, P<0.05). In survivors, L/SF significantly correlated with duration of tracheal intubation (rs=-0.938, P<0.01). CONCLUSION: L/SF is a unique factor to predict the survival prognosis and likely to quantify the degree of pulmonary hypoplasia in CDH.

Plk1 regulates liver tumor cell death by phosphorylation of TAp63.

We previously found that Plk1 inhibited the p53/p73 activity through its direct phosphorylation. In this study, we investigated the functional role of Plk1 in modulating the p53 family member TAp63, resulting in the control of apoptotic cell death in liver tumor cells. Immunoprecipitation and in vitro pull-down assay showed that p63 binds to the kinase domain of Plk1 through its DNA-binding region. in vitro kinase assay indicated that p63 is phosphorylated by Plk1 at Ser-52 of the transactivating (TA) domain. Plk1 decreased the protein stability of TAp63 by its phosphorylation and suppressed TAp63-induced cell death. Furthermore, Plk1 knockdown in p53-mutated liver tumor cells transactivated p53 family downstream effectors, PUMA, p21(Cip1/WAF1) and 14-3-3sigma, and induced apoptotic cell death. Double knockdown of Plk1/p63 attenuated Plk1 knockdown-induced apoptotic cell death and transactivation. Intriguingly, both Plk1 and p63 are highly expressed in the side population (SP) fraction of liver tumor cells compared to non-SP fraction cells, suggesting the significance of Plk1/TAp63 in the control of cell death in tumor-initiating SP fraction cells. Thus, Plk1 controls TAp63 by its phosphorylation and regulates apoptotic cell death in liver tumor cells. Plk1/TAp63 may be a suitable candidate as a molecular target of liver tumor treatments.

Coiled-coil domain containing 85B suppresses the beta-catenin activity in a p53-dependent manner.

Aberrant accumulation of beta-catenin is closely related to carcinogenesis. Mutations in the p53 gene are reported to induce the aberrant accumulation of beta-catenin in the absence of dysfunction in the glycogen synthase kinase 3beta (GSK3beta)-mediated degradation pathway, but the mechanism remains incompletely understood. Here, we show that human coiled-coil domain containing 85B (CCDC85B) is induced by p53 and regulates beta-catenin activity via interaction with the T-cell factor 4 in the nucleus. Moreover, CCDC85B enhances the degradation of beta-catenin and suppresses tumor cell growth. In conclusion, we revealed that CCDC85B-induced degradation of beta-catenin is independent of GSK3beta and other p53-inducible products, Siah-1L, suggesting that CCDC85B constitutes the one of the frameworks of p53-induced multiple regulatory pathways for beta-catenin activity.

BodyMap: a human and mouse gene expression database.

BodyMap is a human and mouse gene expression database that has been maintained since 1993. It is based on site-directed 3'-ESTs collected from non-biased cDNA libraries constructed at Osaka University and contains >270 000 sequences from 60 human and 38 mouse tissues. The site-directed nature of the sequence tags allows unequivocal grouping of tags representing the same transcript and provides abundance information for each transcript in different parts of the body. Our collection of ESTs was compared periodically with other public databases for cross referencing. The histological resolution of source tissues and unique cloning strategy that minimized cloning bias enabled BodyMap to support three unique mRNA based experiments in silico. First, the recurrence information for clones in each library provides a rough estimate of the mRNA composition of each source tissue. Second, a user can search the entire data set with nucleotide sequences or keywords to assess expression patterns of particular genes. Third, and most important, BodyMap allows a user to select genes that have a desired expression pattern in humans and mice. BodyMap is accessible through the WWW at http://bodymap.ims.u-tokyo.ac.jp

Podocyte injury predicts prognosis in patients with iga nephropathy using a small amount of renal biopsy tissue.

To predict the progression in patients with IgA nephropathy, we analyzed glomerular lesions except for sclerosis, adhesion and/or crescents in 34 patients with this disease by morphometric analysis. Levels of urinary protein excretion (UP), creatinine clearance (Ccr), serum creatinine (sCr) and mean blood pressure (MBP) at the time of renal biopsy were used as the clinical parameters. The slope of 1/sCr was also used as a prognostic parameter. Renal specimens were obtained by echo-guided biopsy. In PAS-stained light microscopic renal sections, three mid sections of open glomeruli were selected and photographed. Stereologic estimation was performed as follows: absolute values of glomerular volume (V(G)), glomerular surface area (S(G)), podocyte and nonpodocyte cell number per glomerulus (N(G(pod)) and N(G(Non-pod))), glomerular surface area covered by one podocyte S(G)/N(G(pod))) and glomerular volume occupied by one nonpodocyte cell (V(G)/N(G(Non-pod))). There was a significant correlation between the levels of UP and the change of podocyte injury parameters (N(G(pod)) and S(G)/N(G(pod))) or N(G(Non-pod)). N(G(pod)) was negatively but S(G)/N(G(pod)) and N(G(Non-pod)) were positively correlated with UP. S(G)/N(G(pod)) or N(G(Non-pod)) was correlated with MBP. N(G(pod)), S(G)/N(G(pod)), N(G(Non-pod)), UP or MBP was significantly correlated with the slope of 1/sCr. High specificity was observed for N(G(pod)), S(G)/N(G(pod)) and MBP. High sensitivity was also observed for N(G(Non-pod)) and UP. It appears that podocyte injury might provide additional prognostic information in patients with IgA nephropathy.

Successful surgical treatment of two cases of congenital chylous ascites.

The authors report on 2 patients with congenital chylous ascites who underwent successful lymphatic duct ligation after a laparoscopic lymphoid dye test. Fetal ascites had been detected in both cases, and both babies were born with marked abdominal swelling. Given that conservative treatment by medium-chain triglyceride (MCT) milk and total parenteral nutrition (TPN) was ineffective, the authors elected to perform lymphatic duct ligation on the 95th postnatal day in the former case and on the 27th postnatal day in the latter case. Lipophilic dye was administered preoperatively both through oral and subcutaneous routes, and the peritoneal cavity was explored using laparoscopy. This laparoscopic lymphoid dye test precisely identified the area of chylous leakage, and the authors were able to repair the malformed lymphatic duct directly at laparotomy. Both postoperative courses have been favorable with no recurrence of symptoms. The lymphatic duct ligation should be considered in cases resistant to conservative treatment for over a month. The present laparoscopic lymphoid dye test is a novel and useful procedure that allows surgeons to identify the exact location of chylous leakage, and thus successfully ligate the lymphatic duct.

Intralobar bronchopulmonary sequestration evaluated by contrast-enhanced three-dimensional MR angiography.

Bronchopulmonary sequestration (PS) is characterized by non-functioning lung tissue fed from one or several aberrant systemic arteries. The condition is diagnosed by visualizing the feeding arteries using non-invasive CT, MRI, colour Doppler sonography or conventional angiography. We present a 5-year-old boy in whom intralobar sequestration was diagnosed using contrast-enhanced 3D MR angiography, which visualised fine blood vessels in the thoraco-abdominal region without arterial puncture. This technique is useful for diagnosing PS.

Putidaredoxin-cytochrome p450cam interaction. Spin state of the heme iron modulates putidaredoxin structure.

During the monooxygenase reaction catalyzed by cytochrome P450cam (P450cam), a ternary complex of P450cam, reduced putidaredoxin, and d-camphor is formed as an obligatory reaction intermediate. When ligands such as CO, NO, and O2 bind to the heme iron of P450cam in the intermediate complex, the EPR spectrum of reduced putidaredoxin with a characteristic signal at 346 millitesla at 77 K changed into a spectrum having a new signal at 348 millitesla. The experiment with O2 was carried out by employing a mutant P450cam with Asp251 --> Asn or Gly where the rate of electron transfer from putidaredoxin to oxyferrous P450cam is considerably reduced. Such a ligand-induced EPR spectral change of putidaredoxin was also shown in situ in Pseudomonas putida. Mutations introduced into the neighborhood of the iron-sulfur cluster of putidaredoxin revealed that a Ser44 --> Gly mutation mimicked the ligand-induced spectral change of putidaredoxin. Arg109 and Arg112, which are in the putative putidaredoxin binding site of P450cam, were essential for the spectral changes of putidaredoxin in the complex. These results indicate that a change in the P450cam active site that is the consequence of an altered spin state is transmitted to putidaredoxin within the ternary complex and produces a conformational change of the 2Fe-2S active center.

Follow-up study on urinary type IV collagen in patients with early stage diabetic nephropathy.

Type IV collagen is a major component released from the glomerular and tubular basement membranes. To investigate the alteration of renal type IV collagen turnover in early stage diabetic nephropathy, urinary type IV collagen was measured by a highly sensitive one-step sandwich enzyme immunoassay (EIA). Urinary samples were obtained from 94 diabetic patients without overt proteinuria. Among those patients, 61 were normoalbuminuric and 33 patients were in the microalbuminuric group. Levels of urinary type IV collagen were serially examined at the start of this study and again one year later. The levels of urinary type IV collagen in patients in the microalbuminuric group were significantly higher than those in the normoalbuminuric group (P < 0.01). There was a significant correlation between the concentration of urinary albumin and urinary type IV collagen in both groups (P < 0.05). Twenty-eight patients (45.3%) in the normoalbuminuric group who showed an abnormal elevation of urinary type IV collagen in comparison to the reference range of normal healthy adults (normal range; less than 3.5 microg/g x Cr). Seven (25%) out of these 28 normoalbuminuric patients with increased urinary type IV collagen progressed to the microalbuminuric group one year later. The levels of urinary type IV collagen in such patients were significantly increased. In the 21 patients who stayed within the normoalbuminuric group, the urinary type IV collagen levels were significantly decreased one year later. It appears that the levels of urinary type IV collagen might reflect ongoing alteration of the extracellular matrix (ECM) turnover and might define more specifically the early stage diabetic nephropathy than the detection of microalbuminuria. It is concluded that the serial measurement of urinary type IV collagen can be a useful marker for detecting renal injury in diabetes.

Expression of MRP and cMOAT in childhood neuroblastomas and malignant liver tumors and its relevance to clinical behavior.

Advanced neuroblastoma and malignant liver tumor are representative childhood cancers for which combined chemotherapy including cisplatin and doxorubicin is routinely performed. The prognosis of patients with tumors which develop multiple drug resistance (MDR) is unfavorable. To elucidate the role of multidrug resistance-associated protein (MRP) and canalicular multispecific organic anion transporter (cMOAT) in the clinical behavior of the tumors, we examined 42 neuroblastomas and 10 malignant liver tumors for the expressions of MRP and cMOAT by quantitative RNA-polymerase chain reaction (PCR). The amplification and expression of N-myc oncogene in the neuroblastomas were also investigated. We found a close association between MRP and N-myc expression in each neuroblastoma sample but no significant relationship between MRP expression and the patients' outcome. The forced expression of N-myc failed to enhance the expression of MRP in N-myc transfected neuroblastoma cell lines. cMOAT was rarely expressed in the neuroblastomas, but was frequently expressed in the malignant liver tumors. The expression of MRP and cMOAT in the childhood liver tumors was more common and higher, especially in advanced cases with a poor outcome, than that observed in normal liver or in 9 hepatocellular carcinomas from adult patients. The enhanced expression of these genes might be characteristic of childhood malignant liver tumors and related to their clinical chemoresistance.

Developing NLP Tools for Genome Informatics: An Information Extraction Perspective.

Huge quantities of on-line medical texts such as Medline are available, and we would hope to extract useful information from these resources, as much as possible, hopefully in an automatic way, with the aid of computer technologies. Especially, recent advances in Natural Language Processing (NLP) techniques raise new challenges and opportunities for tackling genome-related on-line text; combining NLP techniques with genome informatics extends beyond the traditional realms of either technology to a variety of emerging applications. In this paper, we explain some of our current efforts for developing various NLP-based tools for tackling genome-related on-line documents for information extraction task.