PubMed Health⌕ Search

Biomedical subjects

L Ji

Publications and source records attributed to L Ji.

At least 19 recordsLinked to original sources
PubMed1997

Calmodulin-dependent protein kinase II mediates signal transduction in apoptosis.

The present studies describe a new function for calmodulin-dependent protein kinase II (CaM-KII) in signal transduction leading to apoptosis. Both tumor necrosis factor alpha (TNF) and UV light rapidly stimulated Ca2+-independent activity of CaM-KII in the monocytic leukemia, U937. Two mechanistically different inhibitors of CaM-KII blocked activation of CaM-KII and prevented DNA fragmentation and death. Activation of CaM-KII during apoptosis and inhibition of DNA fragmentation by the two CaM-KII inhibitors were reproduced in several other lines including KGla, HL-60, and YAC-1. However, K562, which is relatively resistant to apoptosis induced by either TNF or UV light, did not activate CaM-KII in response to these stimuli. A variant derived from U937 that is resistant to TNF- or UV light-induced apoptosis also lacked a CaM-KII response. Activation of Cam-KII was blocked by two protease inhibitors, VAD-fmk and TPCK, but not by other inhibitors of serine proteases. Both inhibitors of CaM-KII and the protease inhibitors blocked activation of AP24, a serine protease originally isolated from apoptotic cells that induces DNA fragmentation in nuclei. Our evidence supports a model in which proteolytic activity functions upstream of CaM-KII. This kinase then leads to activation of AP24, which transmits signals to the nucleus to initiate DNA fragmentation.

Apoptosis↗
PubMed1997

DNA measurement of overlapping cell nuclei in thick tissue sections.

The paper describes an improved image analysis procedure for measuring the DNA content of cell nuclei in thick sections of liver tissue by absorption densitometry. Whereas previous methods only permitted the analysis of isolated nuclei, the new technique enables both isolated and overlapping nuclei to be measured. A 3D segmentation procedure determines whether each object is an isolated nucleus or a pair of overlapping nuclei; in the latter case the combined optical density is redistributed to the individual nuclei. A selection procedure ensures that only complete nuclei are measured. The method has been tested on specially-prepared Feulgen-stained 20micro sections of normal liver tissue. The overall distribution of the nuclear DNA measurements shows well-defined diploid and tetraploid peaks, with coefficient of variations of less than 10%. Similar distributions were obtained from both the isolated nuclei and overlapped nuclei sub-populations.

Cell Nucleus↗
PubMed1997

New susceptibility locus for NIDDM is localized to human chromosome 20q.

To test the hypothesis that a gene (or genes) in the "MODY1 region" of the long arm of chromosome 20 contributes to the development of NIDDM, we conducted linkage studies in 29 extended Caucasian families in which many members were affected with NIDDM. A total of 498 individuals, including 159 NIDDM patients with an average age at diagnosis of 47 years, were genotyped for eight highly polymorphic microsatellite markers spanning a 31-cM region on chromosome 20q12-13.1. Using affected sib-pair analysis, we obtained evidence suggesting linkage between NIDDM and markers D20S119, D20S178, and D20S197 (allele sharing identical-by-descent [IBD], 0.56 for all three; P = 0.005, P = 0.009, and P = 0.004, respectively). Multipoint nonparametric linkage (NPL) analysis also showed evidence for linkage of NIDDM with the same three markers. The evidence for linkage was much stronger (allele sharing IBD by affected sibpairs, 0.64 [P < 0.0001]; maximum NPL score, 3.3 [P = 0.009]) in the 14 families whose average age at diagnosis of NIDDM was above the median (47 years) for all families. In these 14 families, one particular allele of the microsatellite D20S197 was transmitted from heterozygous parents to NIDDM offspring more frequently than expected (P < 0.01). This indicates that the marker allele and the disease allele are in linkage disequilibrium, implying that they are in close proximity. Consequently, the recently identified MODY1 gene (hepatocyte nuclear factor 4) is an unlikely candidate gene for NIDDM in our families, since it is located about 8 cM centromeric of D20S197. In conclusion, we have identified a new region on chromosome 20q that contains one or more NIDDM genes distinct from the recently identified MODY1 gene.

Adult↗
PubMed1996

CREB proteins function as positive regulators of the translocated bcl-2 allele in t(14;18) lymphomas.

The translocated and normal bcl-2 alleles in the DHL-4 cell line with the t(14;18) translocation were separated by pulsed field electrophoresis. An in vivo footprint over a cAMP response element (CRE) in the bcl-2 5'-flanking sequence was identified on the translocated allele. Electrophoretic mobility shift assays with the bcl-2 CRE demonstrated complexes with mobilities identical to those with a consensus CRE. UV cross-linking experiments revealed that proteins with molecular masses of 34, 43, and 67 kDa bound to the bcl-2 CRE site. Electrophoretic mobility shift assay with an antibody specific to the phosphorylated cAMP response-binding protein (CREB) demonstrated that phosphorylated CREB was present in DHL-4 cells. Treatment with phorbol 12-myristate 13-acetate (PMA) led to an increase in both the amount of phosphorylated CREB and the bcl-2 promoter activity. The response to PMA was dependent on an intact CRE site. The activity of the bcl-2 promoter was increased 20-fold in a construct with the immunoglobulin heavy chain enhancers, and mutation of the CRE site abolished most of the induction. The addition of PMA increased the activity of the bcl-2-immunoglobulin enhancer construct by 3.5-fold. Access to the CRE site is blocked in the silent normal bcl-2 allele, while CREB proteins bind to the site on the translocated allele. We conclude that the CRE site functions as a positive regulatory site for the translocated bcl-2 allele in t(14;18) lymphomas.

Alleles↗
PubMed1995

Tumor necrosis factor alpha increases tyrosine phosphorylation of a 23-kDa nuclear protein in U937 cells through ceramide signaling pathway.

In many tumor cell lines, tumor necrosis factor alpha (TNF alpha) causes profound alterations in nucleus including chromatin condensation and DNA fragmentation. However the mechanism is largely unknown. Here we demonstrate that TNF alpha treatment of U937 cells increased tyrosine phosphorylation of a 23-kDa nuclear protein (P23) maximally by 13-fold, which occurred after 2.5 h treatment concomitantly with occurrence of DNA fragmentation. Tyrosine kinase inhibitor, herbimycin A, inhibited either tyrosine phosphorylation of P23 or DNA fragmentation, suggesting that the P23 phosphoprotein may be involved in the TNF alpha-induced changes in nucleus. Increase of intracellular Cer level by hydrolysis of cell surface SM with bacterial sphingomyelinase or addition of either C2-Cer or natural Cer also resulted in increase of P23 tyrosine phosphorylation, which was detectable at 1.5 h treatment and reached maximum after 2 h treatment, indicating that P23 phosphoprotein may be a downstream effector of Cer signaling pathway. Our data provide further evidence that Cer signaling pathway may mediate signal transduction of TNF alpha.

Benzoquinones↗
PubMed1995

Inhibition of tumor necrosis factor alpha- and ceramide-induced internucleosomal DNA fragmentation by herbimycin A in U937 cells.

In many tumor cell lines, tumor necrosis factor alpha (TNF alpha) causes apoptosis with characteristic internucleosomal DNA fragmentation. However, the mechanism is largely unknown. Here we examined the involvement of protein tyrosine kinases by using their inhibitors. Among various tyrosine kinase inhibitors tested, only herbimycin A was found to inhibit internucleosomal DNA fragmentation but not apoptotic morphological changes and cell death induced by TNF alpha in U937 cells. Herbimycin A was able to block DNA fragmentation when it was added to the cell culture as late as 1.5 h after TNF alpha treatment. These results demonstrate that herbimycin A selectively inhibits a later event involved in the process of apoptois that results in internucleosomal DNA fragmentation. Sphingomyelinase and ceramide (Cer) induced internucleosomal DNA fragmentation was also inhibited by herbimycin A, supporting the hypothesis that Cer may be a novel second messenger mediating the cytotoxic effect of TNF alpha.

Apoptosis↗
PubMed1995

The transcription factor, Nm23H2, binds to and activates the translocated c-myc allele in Burkitt's lymphoma.

We have identified an in vivo footprint over the PuF site on the translocated c-myc allele in Burkitt's lymphoma cells. The PuF site on the silent normal c-myc allele was unoccupied. We demonstrated by electrophoretic mobility shift assay, electrophoretic mobility shift assay with antibody, UV cross-linking followed by SDS-gel electrophoresis, and Western analysis that Nm23H2 in B cell nuclear extracts bound to the c-myc PuF site. Transfection experiments with c-myc promoter constructs in both DHL-9 and Raji cells revealed that the PuF site functioned as a positive regulatory element in B cells with a drop in activity with mutation of this site. Access to this site is blocked in the normal silent c-myc allele; these data suggest that the Nm23H2 protein is involved in deregulation of the translocated c-myc allele in Burkitt's lymphoma cells.

Alleles↗
PubMed1995

Induction of apoptotic DNA fragmentation and cell death by natural ceramide.

We have found that a solvent mixture of ethanol and dodecane was able to disperse natural ceramide (Cer) into aqueous solution. In U937 cells, addition of natural Cer to medium caused a characteristic pattern of DNA fragmentation, which is indistinguishable from that caused by TNF alpha. The effective concentration of Cer is as low as 25 nM. The present study provides direct evidence that natural Cer functions as a second messenger mediating TNF alpha-induced DNA fragmentation. The use of this solvent to deliver hydrophobic natural Cer to cells will contribute to the elucidation of the biological function of Cer.

Apoptosis↗
PubMed1995

The hydrolysis of cell surface glycosphingolipids by endoglycoceramidase reduces epidermal growth factor receptor phosphorylation in A431 cells.

This paper presents a new method to evaluate the biological significance of glycosphingolipids (GSLs) using a GSL-specific enzyme, endoglycoceramidase (EGCase), by which GSL-sugar chains are removed from the cell surface of living cells. In this report, the effects of EGCase on epidermal growth factor (EGF)-dependent tyrosine-specific EGF receptor (EGFR) phosphorylation of A431 cells are described. After treatment of A431 cells with EGCase II (20 mU/ml) in the presence of the activator for 12 h, all acidic GSLs tested were reduced to approximately 70% of control, but no hydrolysis occurred on Gal alpha 1,4Gal beta 1,4Glc beta 1,1Cer (Gb3Cer) and GalNAc beta 1,3Gal alpha 1,4Gal beta 1,4Glc beta 1,1Cer (Gb4Cer). In plasma membrane fractions of A431 cells, the reduction of gangliosides by EGCase II was found to be much faster than that of intact cells and reached 54.8% reduction of total gangliosides after 2 h incubation with the enzyme. EGF-dependent phosphorylation of EGFR of A431 cells was inhibited by the reduction of cell surface GSLs by EGCase when either intact A431 cells or their plasma membrane fractions were used, while EGF binding to their receptors was not changed. Neither hydrolysis of cell surface GSLs nor reduction of EGFR phosphorylation occurred when A431 cells were incubated with the activator or EGCase alone. The exogenous addition of ceramides or sphingosines, and treatment of cell membranes with sphingomyelinase, had no effect on the EGFR phosphorylation of purified membrane fractions, while the inhibitory effect of EGCase II on EGFR phosphorylation was restored by the addition of GM3-sugar chains, but not by lactose or sialic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1995

Trinucleotide repeats at the rad locus. Allele distributions in NIDDM and mapping to a 3-cM region on chromosome 16q.

A 10-allele polymorphism was identified in rad (ras associated with diabetes), a gene that is overexpressed in non-insulin-dependent diabetes mellitus (NIDDM) muscle. The polymorphism, designated RAD1, consists of a variable number of trinucleotide repeats (GTT and ATT) located in the poly(A) region of an intronic Alu sequence. Based on the number of GTT and ATT repetitions, the alleles can be grouped into four classes (I-IV). RAD1 allele frequencies were determined in 210 NIDDM patients and 133 nondiabetic control subjects, all Caucasians. One allele (number 8, class III) accounted for > 80% of the chromosomes in both groups. However, an excess of minor alleles, all belonging to class I, II, or IV, was observed among NIDDM chromosomes (P < 0.025), suggesting a possible association between RAD1 and NIDDM predisposition. To promote further studies to test the hypothesis that genetic variability at the rad locus contributes to NIDDM, we mapped rad on the human genome. Using the fluorescence in situ chromosomal hybridization technique, rad was unequivocally assigned to chromosomal band 16q22. In families that were informative for RAD1, the rad locus was mapped within a 3-cM region defined by the markers D16S265, D16S186, and D16S397 (logarithm of odds scores = 10.08, 10.9, and 10.84 at recombination fractions of 0.024, 0.001, and 0.03, respectively). The high degree of heterozygosity of these markers will allow large-scale family studies to be performed to test the presence of linkage between rad and NIDDM.

Alleles↗
PubMed1994

Fully automatic chromosome segmentation.

This paper is concerned with automatic segmentation of high resolution digitized metaphases. This includes automatic detection and rejection of interphase nuclei, stain debris, and other "noise"; automatic detection and segmentation of touching and overlapping chromosome clusters; and automatic rejection of cells which are evaluated as being incomplete, or incorrectly segmented, or where the cell is otherwise unsuitable for further analysis. In this paper, a rule-based approach is described which treats the cell as a whole rather than as a series of individual chromosomes or clusters. The rules adapt classification and segmentation parameters for each cell. Initially, different sets of parameters are chosen according to the staining method of the cells, and the goal of the segmentation. A chromosome number predictor is used to guide the adaptation of the parameters and to estimate the performance. The adaptation is iterative, and the self-adjustment will stop when either a satisfactory result is achieved or if the cell is rejected. The method was implemented on both a Sun workstation and a Cytoscan, a commercial machine for chromosome analysis. Seven hundred and thirteen cells from real data have been tested. A success rate of 90-95% has been achieved. The procedure has been implemented in an automatic aberration scoring system for routine use.

Chromosome Aberrations↗
PubMed1994

Automatic fluorescence metaphase finder speeds translocation scoring in FISH painted chromosomes.

A fluorescence metaphase finder was constructed with commercially available hardware and a standard Unix workstation. Its accuracy was measured in terms of the number of false positive and false negative detected metaphases on a variety of different slide preparations. The metaphase finder was used in a translocation scoring experiment in which metaphase preparations of human peripheral blood lymphocytes were hybridized with whole chromosome probes to chromosomes #1, #2, and #4. The automatic finder presented metaphases to the cytogeneticist, centered in the eyepieces at x63. The cytogeneticist's scores of analyzable metaphases and of painted chromosomes involved in rearrangements were recorded. The time for the analysis was recorded and compared to the time to analyze a similar number of cells in a purely visual experiment in which the cytogeneticist scanned for cells and analyzed them, both at x63. The results showed that, neglecting the machine time spent scanning unattended, the amount of time required for the analysis was reduced by a factor of three. Furthermore, in this experiment the metaphase finder found more scorable metaphases than the cytogeneticist found by visual scanning. Machine-assisted scoring had additional, less quantifiable, benefits; notably that digital images of metaphases sometimes assisted the analysis of chromosome rearrangements, that cells could be revisited easily, and that the analysis was much less fatiguing.

Cytogenetics↗
PubMed1994

Cloning and sequence analysis of a cDNA encoding ferric leghemoglobin reductase from soybean nodules.

A cDNA encoding soybean (Glycine max [L.] Merr) ferric leghemoglobin reductase (FLbR), an enzyme that is postulated to play an important role in maintaining leghemoglobin in its functional ferrous state, has been cloned and characterized. A group of highly degenerate oligonucleotides deduced from the N-terminal amino acid sequence of FLbR was used to prime the polymerase chain reaction (PCR) on soybean nodule mRNA and cDNA. A full-length clone of FLbR cDNA was isolated by screening a lambda gt11 soybean nodule cDNA library using the specific PCR-amplified FLbR cDNA fragment as a probe. The cDNA contained about 1.8 kb and had a coding sequence for 523 amino acids with a predicted molecular mass of 55,729 D, which included a putative 30-residue signal peptide and a 493-residue mature protein. Computer-aided analysis of the deduced FLbR amino acid sequence showed considerable homology (varied from 20-50% with enzymes and species) to dihydrolipoamide dehydrogenase (EC 1.8.1.4), glutathione reductase (EC 1.6.4.2), mercuric reductase (EC 1.16.1.1), and trypanothione reductase (EC 1.6.4.8) in a superfamily of pyridine nucleotide-disulfide oxidoreductases from various organisms. Northern blot analysis using FLbR cDNA as a probe showed that the FLbR gene was expressed in soybean nodules, leaves, roots, and stems, with a greater level of expression in nodules and leaves than in roots and stems. Southern blot analysis of the genomic DNA showed the presence of two homologous FLbR genes in the soybean genome.

Amino Acid Sequence↗
PubMed1994

NF-kappa B sites function as positive regulators of expression of the translocated c-myc allele in Burkitt's lymphoma.

An in vivo footprint over a potential NF-kappa B site in the first exon of the c-myc gene has been identified on the translocated allele in the Ramos Burkitt's lymphoma cell line. The potential NF-kappa B site in the 5' flanking sequence of c-myc was found to be occupied on the translocated allele in the Raji Burkitt's cell line. Electrophoretic mobility shift assays with each of these sequences demonstrated complexes with mobilities identical to those of the NF-kappa B site from the kappa light-chain gene. A supershift was obtained with anti-p50 antibody with the exon site. The upstream-site shift complex disappeared with the addition of anti-p50 antibody. Binding of NF-kappa B proteins to the c-myc exon and upstream sites was demonstrated by induction of binding upon differentiation of pre-B 70Z/3 cells to B cells. UV cross-linking experiments revealed that a protein with a molecular mass of 50 kDa bound to the exon and upstream sites. Transfection experiments with Raji cells demonstrated that both sites functioned as positive regulatory regions, with a drop in activity level when either site was mutated. Access to these sites is blocked in the silent normal c-myc allele in Burkitt's lymphoma cells, while Rel family proteins bind to these sites in the translocated allele. We conclude that the two NF-kappa B sites function as positive regulatory regions for the translocated c-myc gene in Burkitt's lymphoma.

B-Lymphocytes↗
PubMed1994

[Estimation of age from epiphyseal union degrees of the sternal end of the clavicle].

The sternal ends of right clavicles of Japanese aged 13-31 years (n = 54) autopsied during 1982-1992 were observed for determining degree of epiphyseal union to establish a criterion for age estimation. As the degree of union 5 stages (McKern and Stewart, 1957) were applied to samples: Stage 0; without any union between the trunk and the epiphyseal cap, stage 1; between stage 0 and stage 2, stage 2; half united, stage 3; between stage 2 and stage 4, and stage 4; completely united. As results, in females union appears to proceed faster than in males. There were cases in which union reached stage 4 in younger ages. As a criterion for age estimation it is proposed at present that in both sex, when union is in stage 0, age is certainly estimated to be less than 22 years old and in stage 4, more than 19 years old. Comparing the present data with the investigation about American males by McKern and Stewart (1957), in Japanese males union proceeds more fast than them. The authors realized that the correct criteria should be used in age estimation on a sample belonging to the same race and to the times. The clavicles in skeletal remains tend to be preserved outdoors and are suitable for estimating age from adolescence to about 30 years old. Therefore the present criterion would be very useful in districts such as Hokkaido where skeletal remains are commonly found.

Adolescent↗