Identification and characterization of a 500-kb homozygously deleted region at 1p36.2-p36.3 in a neuroblastoma cell line.

Loss of heterozygosity of the distal region of chromosome 1p where tumor suppressor gene(s) might harbor is frequently observed in many human cancers including neuroblastoma (NBL) with MYCN amplification and poor prognosis. We have identified for the first time a homozygously deleted region at the marker D1S244 within the smallest region of overlap at 1p36.2-p36.3 in two NBL cell lines, NB-1 and NB-C201 (MASS-NB-SCH1), although our genotyping has suggested the possibility that both lines are derived from the same origin. The 800-kb PAC contig covering the entire region of homozygous deletion was made and partially sequenced (about 60%). The estimated length of the deleted region was 500 kb. We have, thus far, identified six genes within the region which include three known genes (DFF45, PGD, and CORT) as well as three other genes which have been reported during processing our present project for the last 3(1/2) years (HDNB1/UFD2, KIAA0591F/KIF1B-beta, and PEX14). They include the genes related to apoptosis, glucose metabolism, ubiquitin-proteasome pathway, a neuronal microtubule-associated motor molecule and biogenesis of peroxisome. At least three genes (HDNB1/UFD2, KIAA0591F/KIF1B-beta, and PEX14) were differentially expressed at high levels in favorable and at low levels in unfavorable subsets of primary neuroblastoma. Since the 1p distal region is reported to be imprinted, those differentially expressed genes could be the new members of the candidate NBL suppressor, although RT-PCR-SSCP analysis has demonstrated infrequent mutation of the genes so far identified. Full-sequencing and gene prediction for the region of homozygous deletion would elucidate more detailed structure of this region and might lead to discovery of additional candidate genes. Oncogene (2000) 19, 4302 - 4307

Proteolytic cleavage sites of band 3 protein in alkali-treated membranes: fidelity of hydropathy prediction for band 3 protein.

To assess the fidelity of hydropathy prediction for band 3 protein, we determined the cleavage sites of the protein and the portions of the protein tightly bound to the membrane lipid bilayer by means of in situ proteolytic digestion. For the removal of all anticipated hydrophilic connector loops from membranes, we had to denature the band 3 protein molecule in situ by alkali treatment. When the alkali-treated membranes were digested with trypsin, chymotrypsin, and pepsin, the majority of the anticipated transmembrane portions remained in the membrane fraction. However, five anticipated transmembrane portions were released into the supernatant fraction. Thus, the first, second, third, sixth and tenth anticipated transmembrane portions, in accordance with the hydropathy prediction, were released into the supernatant with the proteolytic digestion method. This indicates that these anticipated transmembrane portions are not bound with the boundary lipids although the hydrophobicity of these portions is comparable to that of the portions experimentally remaining in the membrane fraction. It is conceivable that the membrane peptide portions of band 3 protein could be classified into at least two categories, i.e. one bound to the boundary lipids and the other free from the boundary lipids. Approximately 90% of the transmembrane domain of the band 3 protein are recovered in either the supernatant fraction or the membrane fraction. The fidelity of hydropathy prediction for polytopic membrane proteins and the nature of the membrane embedded peptide portions are discussed.

Secondary structures of synthetic peptides corresponding to the first membrane-contact portion of normal band 3 and its deletion mutant (Southeast Asian ovalocytosis).

The conformations of synthetic peptides corresponding to the first membrane-contact portion from Tyr390 to Lys430 of band 3 (band 3-1a) and the counterpart portion of South-East Asian ovalocytosis (SAO) band 3 (band 3-1b) in lipid bilayers were examined by means of circular dichroism (CD), Fourier transform infrared (FTIR) spectroscopy as well as a proteolytic digestion method. The CD and FTIR studies showed that band 3-1a and band 3-1b in a membrane lipid bilayer cannot assume an alpha-helix rich structure but instead assume a beta-structure rich conformation. The proteolytic digestion experiments demonstrated that the cleavage sites of Tyr392 and Phe423 were common to both the model and erythrocyte membranes. Taken together with our previous work, which indicated that the first membrane-contact portion was the portion embedded in the erythrocyte membrane without tight lipid-peptide interactions [Hamasaki et al. (1997) J. Biochem. 122, 577-585], we imply herein that the first membrane-contact portion of band 3 by itself can not assume the ordinary alpha-helix conformation in the membrane lipid bilayers. A proteinase-resistant portion, from Ser402 to Phe423, was observed when liposomes containing band 3-1a were digested with proteinase K, while no proteinase-resistant core portion was found in the case of band 3-1b (DeltaAla400-Ala408). This suggests the crucial role of the deleted portion, from Ala400 to Ala408, in the interaction of the first membrane-contact portion of band 3 with a membrane lipid bilayer.

Failure of dacron arterial prostheses caused by structural defects.

We have observed unusual aneurysmal complications associated with structural defects of Dacron fabrics in three patients. The first patient had beadshape aneurysms along the course of the bilateral axillo-femoral prosthetic bypasses 4 or 5 years after surgery. Round defects or longitudinal rents in the lower third of the prostheses (Vascular-D knitted Dacron graft of high porosity, USCI) were the source of the aneurysmal complications. In the second patient, the transverse diameter of the aorto-femoral Dacron graft (Vascular-D, USCI) was increased to twice that of the original graft with formation of an anastomotic false aneurysm in the groin 3 1/2 years after surgery. The defective prostheses in these two cases were successfully replaced by Cooley double velour knitted Dacron grafts. The third patient developed an arterio-ureteral fistula originating from a round defect of the aorto-femoral prosthesis (Tetoron that is woven Dacron graft with a low porosity, Nakao-Filter, Japan) 8 years postoperatively and died from massive hematuria and subsequent disseminated intravascular coagulopathy. Microscopic examination of the removed prostheses revealed a complete loss of the conformation of the weave with fragmentation and disruption of the graft fibers. Therefore, patients with Dacron arterial prostheses should be followed up regularly and with great care.

Occluded persistent sciatic artery.

The case of a patient with ischemic symptoms due to occlusion of a persistent left sciatic artery is presented. Femoral arteriography revealed a tapering hypoplastic superficial femoral artery terminating as small branches in the distal thigh and a hyperplastic profunda femoris artery. Also, translumbar aortography demonstrated an abrupt occlusion of a hyperplastic left hypogastric artery just distal to the inferior gluteal artery. The popliteal artery was found to be patent and perfused by collaterals. A bypass graft using a composite E-PTFE prosthesis and an autogenous vein graft was successfully performed.

Optimum apportionment of presentation time in visual display.

With regard to the better display of visual stimuli, one of the key parameters to match with human short-term memory is the presentation time of the information. We have studied the optimum-apportionment modes of the presentation time of seven Roman alphabet letters sequentially presented to five young males by means of our time-continuous model of short-term memory. These modes are thought to allow for the easiest retention by the short-term memory. The average optimum-apportionment mode, as an average of individual scores of the optimum-apportionment mode, was also obtained. With free-recall experiments using the Roman characters, four constants for the model were determined for the subjects. Then, the theoretical optimum-apportionment modes were calculated using a computer simulation for each subject. With slight modifications of these results, eight types of apportionment modes were obtained. The most efficient mode for the recall of the Roman characters in the free-recall experiment was determined as the optimum-apportionment mode of the individual.

Database access method for autonomous distributed total hospital information system and its object-oriented design.

ADITHIS (Autonomous Distributed Total Hospital Information System) is an information system providing a distributed computing environment for a hospital. The access method to its database enables users to access any patient data stored in individual departmental databases. Its functional design based on the object-oriented approach and its characteristics are described.

A new concept in polytopic membrane proteins following from the study of band 3 protein.

In the present communication, we introduce a novel concept in multispanning polytopic membrane proteins revealed by the study of the band 3 protein. The transmembrane domain of such proteins can be divided into three categories, that is, hydrophilic loops connecting transmembrane peptides (category 1), portions embedded by peptide-peptide interactions (category 2), and portions embedded by peptide-lipid interactions (category 3). Category 2 peptides of polytopic membrane proteins were found to stably reside in the lipid bilayer without peptide-lipid interactions that had been thought to be essential for transmembrane segments. Category 3 peptides are equivalent to single-spanning segments of bitopic membrane proteins. Three different experiments, namely proteolytic digestion, chemical modification of the band 3 protein, and cell free transcription and translation, were used to categorize the transmembrane peptides.

Hunting the subset-specific genes of neuroblastoma: expression profiling and differential screening of the full-length-enriched oligo-capping cDNA libraries.

BACKGROUND: Neuroblastoma (NBL) has a distinct nature in different prognostic subgroups. PROCEDURE: To understand the molecular mechanism of NBL's genesis and biology as well as that of the neural crest development, we constructed full-length-enriched cDNA libraries by an oligo-capping method from two different subsets of primary NBL, one with favorable biology and the other with MYCN amplification. RESULTS: Sequencing analysis of these libraries revealed that the expression profile was markedly different between both subsets. To identify the genes differentially expressed between the subsets, semi-quantitative RT-PCR analyses are proceeding. CONCLUSION: So far, 54 transcripts have been found to be expressed at high levels in favorable NBLs, and significantly at low levels in unfavorable NBLs.

A cytoplasmic RNA vector derived from nontransmissible Sendai virus with efficient gene transfer and expression.

We have recovered a virion from defective cDNA of Sendai virus (SeV) that is capable of self-replication but incapable of transmissible-virion production. This virion delivers and expresses foreign genes in infected cells, and this is the first report of a gene expression vector derived from a defective viral genome of the Paramyxoviridae. First, functional ribonucleoprotein complexes (RNPs) were recovered from SeV cloned cDNA defective in the F (envelope fusion protein) gene, in the presence of plasmids expressing nucleocapsid protein and viral RNA polymerase. Then the RNPs were transfected to the cells inducibly expressing F protein. Virion-like particles thus obtained had a titer of 0.5 x 10(8) to 1. 0 x 10(8) cell infectious units/ml and contained F-defective RNA genome. This defective vector amplified specifically in an F-expressing packaging cell line in a trypsin-dependent manner but did not spread to F-nonexpressing cells. This vector infected and expressed an enhanced green fluorescent protein reporter gene in various types of animal and human cells, including nondividing cells, with high efficiency. These results suggest that this vector has great potential for use in human gene therapy and vaccine delivery systems.

Identification of the homozygously deleted region at chromosome 1p36.2 in human neuroblastoma.

BACKGROUND: We have identified for the first time a homozygously deleted region within the smallest region of overlap at 1p36.2-3 in two neuroblastoma cell lines. PROCEDURE: The 800-kb PAC contig covering the entire homozygously deleted region was made and sequenced. To date, approximately 70% of sequencing has been accomplished, and the estimated length of the deleted region was 500 kb. RESULTS: Currently, we have found six genes within the region, which include three known genes as well as three other genes that have been reported during processing of our present project for the last 3(1/2) years. We report here the results of expression and mutation analyses of those genes. CONCLUSIONS: Full sequencing for the region of homozygous deletion as well as further analyses of the genes mapped within the region may reveal whether or not there is a neuroblastoma suppressor gene as proposed by the Knudson's two-hit hypothesis.