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Biomedical subjects

J Lu

Publications and source records attributed to J Lu.

At least 469 records · Page 26Linked to original sources
PubMed1996

Characterization of a high mobility group 1/2 homolog in yeast.

A 35-kDa polypeptide belonging to the high mobility group family of proteins was purified from the yeast Saccharomyces cerevisiae on the basis of its association with a DNA helicase activity. Amino acid sequence alignment suggests that this protein, Hmo1p, is related to the HMG1/2 class of chromatin-associated proteins. Consistent with this prediction, the Hmo1 protein immunolocalizes to the nucleus, binds single-stranded DNA, and unwinds DNA in the presence of eukaryotic DNA topoisomerase I. While the purified protein has no DNA helicase activity on its own, immunoprecipitation experiments confirm that Hmo1p associates with a 5' to 3' DNA helicase activity in nuclear extracts. The in vivo role of the protein was investigated by constructing an hmo1 deletion mutant. This strain has a severe growth defect, reduced plasmid stability, and chromatin that is hypersensitive to micrococcal nuclease digestion. Taken together, the data indicate that HMO1 is likely to be the homolog of HMG1/2 in higher cells and that it plays an important role in genome maintenance.

Amino Acid Sequence↗
PubMed1996

Complete 1H and 13C resonance assignments of a 21-amino acid glycopeptide prepared from human serum transferrin.

A 21-amino acid glycopeptide (Gp21) was isolated and purified in multi-milligram yields from commercially available human serum transferrin (HSTF) by a combination of tryptic digestion, Con A affinity chromatography, and reverse phase HPLC. The peptide chain of Gp21 contains a single N-glycosylation site to which a diantennary oligosaccharide is attached. The amino acid sequence and the glycan primary structure of Gp21 have been verified by peptide sequencing, electrospray mass spectrometry, and one-dimensional 1H NMR spectroscopy. Different glycoforms were found for the glycan of Gp21 derived from two different batches of commercial HSTF. These glycoforms differ from one another in the number of NeuAc residues (ranging from 0 to 2) and/or the number of Gal residues (ranging from 1 to 2). As for the monogalacto species, in the two-dimensional nuclear Overhauser effect (NOE) spectrum of Gp21, interglycosidic NOEs were observed between Man4 in the alpha (1-->3) branch and the terminal GlcNAc beta (1-->2) residue. No interglycosidic NOE was observed between Man4' in the alpha (1-->6) branch and the terminal GlcNAc residue. These observations indicate that the terminal GlcNAc residue in the minor glycoforms of Gp21 is exclusively located in the alpha (1-->3) branch of the Gp21 glycan. The occurrence of such a carbohydrate structure in HSTF has not been reported before. The 1H and 13C NMR spectra of Gp21 have been completely assigned by two-dimensional homonuclear and heteronuclear spectroscopy. The close similarity of the 1H and 13C chemical shift values for the Gp21 glycan with the respective values for the peptide-free diantennary oligosaccharide (Wieruszeski et al., Glycoconjugate J., 6 (1989) 183-194) indicates that the 1H and 13C chemical shifts of the diantennary oligosaccharide are not perturbed by the presence of the Gp21 peptide fragment. The complete 1H and 13C resonance assignments and the full characterization of the primary structure of Gp21 will permit us to study the conformation and dynamics of the N-linked diantennary oligosaccharides while covalently attached to a polypeptide fragment.

Carbohydrate Sequence↗
PubMed1996

Bcl-xS enhances adenoviral vector-induced apoptosis in neuroblastoma cells.

bcl-x is a member of the bcl-2 family of genes and by alternative splicing gives rise to two distinct mRNAs: bcl-xL and bcl-xS. We have previously investigated the expression of Bcl-x in neuroblastoma (NB) cell lines and have shown that Bcl-xL is expressed and functions to inhibit chemotherapy-induced apoptosis. However, none of the NB cell lines expressed Bcl-xS. The aim of the present study was to determine the effects of Bcl-xS expression on the viability of NB cells. A panel of NB cell lines (CHP-382, GOTO, SHEP-1, SHSY-5Y, and GI-CA-N) were infected with either a bcl-xS adenovirus (pAdRSV-bcl-xS) or a control virus (pAdRSV-lac-z). NB cells showed loss of viability with both viruses, although the bcl-xS virus was most toxic. Importantly, infection with the bcl-xS adenovirus resulted in rapid loss of cell viability, DNA fragmentation, and morphological features of apoptosis even in NB cells transfected to overexpress Bcl-2 and Bcl-xL. These findings suggest that deregulated expression of Bcl-xS using an adenovirus may provide a novel mechanism for initiating cell death in tumors that express Bcl-2 or Bcl-xL.

Adenoviridae↗
PubMed1996

Anatomic considerations for posterior approach to the sacroiliac joint.

STUDY DESIGN: This anatomic study describes a new intraosseous, posterior approach to the sacroiliac joint. OBJECTIVES: To define a transosseous approach to the sacroiliac joint in which a triangular bony window is raised on the posterosuperior aspect of the ilium that provides improved access to the sacroiliac joint for posterior fusion. SUMMARY OF BACKGROUND DATA: A posterior approach to the sacroiliac joint has been widely used for debridement of infectious diseases and for fusion. Most conventional approaches to the sacroiliac joint are interosseous, and there is a relative lack of information on transiliac approaches. METHODS: The projection of the sacroiliac joint on the outer table of the ilium and the thickness of the posterior ilium forming part of the sacroiliac joint were determined in 15 cadaveric pelves. A right angle, triangular bony window was raised from the posterior ilium to investigate the suitability of a transiliac approach in performing sacroiliac debridement and arthrodesis. A horizontal reference line 3-3.5 cm in length was drawn between a point 1 cm anterosuperior to the posteroinferior iliac spine and a point 1.5 cm superior to the superior border of the greater sciatic notch. A vertical reference line was extended superiorly for 2-2.5 cm perpendicular to and beginning at the anterior end of the horizontal reference line. The oblique arm of the right triangle was created by joining the superior end of the vertical reference line to the posterior end of the horizontal line. RESULTS: Thirty percent to fifty percent of the articular surface of the iliac bone was removed with this triangular segment of bone, and a corresponding area of the sacral articular surface was visualized directly. It was possible to remove the rest of the articular cartilage with angled curettes in all specimens. CONCLUSIONS: This approach facilities improved access to the sacroiliac joint for debridement and arthrodesis with minimal soft tissue dissection and iliac bone resection.

Adult↗
PubMed1996

Germline BRCA2 gene mutations in patients with apparently sporadic pancreatic carcinomas.

Germline mutations in BRCA2 predispose carriers to the development of breast, ovarian, and a variety of other cancers. The original localization of the BRCA2 gene was aided by its homozygous deletion in a pancreatic carcinoma; indeed, an excess of pancreatic carcinoma has been seen in some BRCA2 cancer families. To determine the involvement of BRCA2 in pancreatic carcinomas, we screened for BRCA2 alterations in an unselected panel of 41 adenocarcinomas of the pancreas (30 pancreatic adenocarcinoma xenografts and 11 pancreatic cancer cell lines). Of the 15 (27%) that had allelic loss at the BRCA2 locus, 4 (9.8%) had abnormalities in the second allele upon screening of the entire BRCA2 gene by in vitro synthesized protein assay. Three of the four mutations were considered germline in origin (7.3% overall; two were confirmed in normal tissue, and one was the 6174delT mutation from the pancreatic cancer cell line CAPAN-1, for which normal tissue was unavailable). The identification of two 6174delT mutations in this series prompted us to evaluate the prevalence of this mutation in an overlapping consecutive series of 245 patients who underwent pancreatoduodenectomy for adenocarcinoma of the pancreas. Sequence analysis of this limited region of the gene identified two additional mutations: (a) one additional germline 6174delT mutation (2 of 245, 0.8% overall); and (b) a second nearby germline 6158insT mutation. One of the patients with a germline mutation had a single relative with breast cancer, and another had a single relative with prostate cancer. None had a family history of pancreatic cancer. The incidence of germline BRCA2 mutations in apparently sporadic pancreatic cancer may be at least as high as in breast or ovarian cancer. Our results suggest that some familial risks for carcinoma will be evident only through a population-based application of gene screening techniques because a low disease penetrance of the germline mutations in some families often evades clinical suspicion.

Adenocarcinoma↗
PubMed1996

Localisation of the C1q binding site within C1q receptor/calreticulin.

C1q receptor (C1qR/collectin receptor) is located on many cell types. Binding of C1q to these cells elicits numerous responses. Protein sequencing has shown that C1qR is almost identical to calreticulin (CaR), an abundant multifunctional protein. Radioiodinated C1qR and CaR bind to C1q with identical characteristics. Three recombinant C1qR/CaR domains (N-, C-terminal domains and central P-domain) were expressed using the Thiofusion system, and used to study the interaction with C1q. Both the N- and P-domains were implicated in C1q binding. A region, termed the S-domain, spanning the N and P intersection was expressed, and showed concentration-dependent binding to C1q, demonstrating that the C1q binding site lies within this region.

Amino Acid Sequence↗
PubMed1996

Improvements on the purification of mannan-binding lectin and demonstration of its Ca(2+)-independent association with a C1s-like serine protease.

Mannan-binding lectin (MBL), previously called 'mannan-binding protein' or MBP, is a plasma C-type lectin which, upon binding to carbohydrate structures on micro-organisms, activates the classical pathway of complement. Purification of MBL relies on its Ca(2+)-dependent affinity for carbohydrate, but existing methods are susceptible to contamination by anti-carbohydrate antibodies. In the present study a sequential-sugar-elution method has been developed which can achieve a preparation of virtually pure MBL and its associated serine protease (MBL-associated serine protease, MASP) by two steps of affinity chromatography. In further separation of MASP from MBL, it was found that activated MASP was associated with MBL independent of Ca2+. Since MBL was found to bind to underivatized Sepharose 4B, the MBL-MASP complex was purified using Sepharose 4B and protease inhibitors were included to purify the complex with MASP in its proenzyme form. Analysis of thus-purified MBL-MASP complex by gel filtration on a Sephacryl S-300 column at pH 7.8 showed that the proenzyme MASP was also associated with MBL independently of Ca2+, but that the complex could be disrupted at a low pH (5.0). Therefore the mechanism of MBL-MASP-mediated complement activation appears to be significantly different from the C1-mediated classical pathway.

Calcium↗
PubMed1996

A hierarchy of SSB protomers in replication protein A.

Replication Protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein (SSB) found in all eukaryotic cells. RPA is known to be required for many of the same reactions catalyzed by the homotetrameric SSB of bacteria, but its origin, subunit functions, and mechanism of binding remain a mystery. Here we show that the three subunits of yeast RPA contain a total of four domains with weak sequence similarity to the Escherichia coli SSB protomer. We refer to these four regions as potential ssDNA-binding domains (SBDs). The p69 subunit, which is known to bind ssDNA on its own, contains two SBDs that together confer stable binding to ssDNA. The p36 and p13 subunits each contain a single SBD that does not bind stably, but corresponds to the minimal region required for viability in yeast. Photocross-linking of recombinant protein to ssDNA indicates that an SBD consists of approximately 120 amino acids with two centrally located aromatic residues. Mutation of these aromatic residues inactivates ssDNA binding and is a lethal event in three of the four domains. Finally, we present evidence that the p36 subunit binds ssDNA, as part of the RPA complex, in a salt-dependent reaction similar to the wrapping of ssDNA about E. coli SSB. The results are consistent with the notion that RPA arose by duplication of an ancestral SSB gene and that tetrameric ssDNA-binding domains and higher order binding are essential features of cellular SSBs.

Amino Acid Sequence↗
PubMed1996

Vulnerability of great medullary artery.

STUDY DESIGN: The present study describes anatomic observations on great medullary artery and intercostal arteries pertinent to thoracolumbar spinal surgery. OBJECTIVES: This study reveals the vulnerable course of the great medullary artery and its relationship to the lateral or posterolateral approach to thoracic spine. SUMMARY OF BACKGROUND DATA: There are no previous anatomic data on the length of the great medullary artery, its intradural course, its relationship with the anterior spinal artery, and the distance between two adjacent intercostal arteries. METHODS: The location of the intercostal arteries was defined, and the distance between two adjacent arteries was measured at a point on the lateral surface of the vertebra midway between its anteroposterior diameter. The intradural length of the great medullary artery and the angle it formed with the anterior spinal artery at the point of anastomosis were also measured. RESULTS: The mean intradural length of the great medullary artery was 3.6 cm (range, 1.7-8.1 cm), and it passed over 1-3 disc spaces before joining the anterior spinal artery at a mean angle of 20.1 degrees (range, 12-28 degrees). The average distance between two adjacent intercostal arteries from T6 to L2 was 3.6 cm (range, 2.8-4.0 cm), which provides a safe window through which a herniated thoracic disc may be approached if surgery is indicated. CONCLUSIONS: The acute angle between the great medullary artery and anterior spinal artery indicates that these two arteries are in close proximity for considerable length and are liable to be compressed together with the intervening vascular collaterals by a space-occupying lesion, such as disc herniation or a fractured fragment. The longer the intradural course of the great medullary artery, the more vulnerable it is to compression by disc herniation or fracture. The intercostal and lumbar arteries are located at the midportion of the lateral aspect of the vertebral bodies rather than at the level of intervertebral discs. Discectomy or decompression of the anterior thoracic canal may be accomplished through a lateral or posterolateral extracavitary approach between two intercostal or lumbar arteries.

Aorta, Thoracic↗
PubMed1996

An anatomic study of the thickness of the occipital bone. Implications for occipitocervical instrumentation.

STUDY DESIGN: The authors measured the thickness and quality of occipital bone regions to determine screw placement during occipitocervical fusion and described the projection of the posterior dural venous sinuses. OBJECTIVE: This study provides anatomic data relevant to areas of screw placement into the occiput during occipitocervical fixation. SUMMARY OF BACKGROUND DATA: Few reports exist regarding the morphometrics of the occipital bone and intracranial structures relevant to occipitocervical fusion. METHOD: The thickness of the posterior inferior occipital bone was measured relative to a 10 x 5 cm grid. Sections were evaluated grossly and histologically. The projections of the posterior dural venous sinuses were determined by direct measurements. RESULTS: The maximum thickness of the occipital bone, which ranged from 11.5 to 15.1 mm in males and from 9.7 to 12.0 mm in females, was at the level of the external occipital protuberance. The occipital bone was thicker than 8 mm in an area extending laterally from the external occipital protuberance for 23 mm and consisted of dense cortical bone with little or no diploic bone. The projection of most of the torcula on the external surface of the occipital bone was located superior to the center of the external occipital protuberance (mean, 12.6 mm superior and 4.7 mm inferior to external occipital protuberance), whereas that of the transverse sinus was distributed more evenly above and below the external occipital protuberance (mean, 7.3 mm superior and 6.5 mm inferior). CONCLUSIONS: Screws that are 8-mm long may be inserted in the region of the superior nuchal line (Level 0) extending 2 cm laterally from the center of the external occipital protuberance, 1 cm from the midline at a level 1 cm inferior to the external occipital protuberance (Level 1), and 0.5 cm from the midline at a level 2 cm inferior to the external occipital protuberance (Level 2). The major dural venous sinuses are situated immediately beneath the thickest regions of the occiput and are at risk of penetrative injury during screw placement.

Bone Plates↗
PubMed1996

Identification of a second transmembrane protein tyrosine phosphatase, IA-2beta, as an autoantigen in insulin-dependent diabetes mellitus: precursor of the 37-kDa tryptic fragment.

A novel cDNA, IA-2beta, was isolated from a mouse neonatal brain library. The predicted protein sequence revealed an extracellular domain, a transmembrane region, and an intracellular domain. The intracellular domain is 376 amino acids long and 74% identical to the intracellular domain of IA-2, a major autoantigen in insulin-dependent diabetes mellitus (IDDM). A partial sequence of the extracellular domain of IA-2beta indicates that it differs substantially (only 26% identical) from that of IA-2. Both molecules are expressed in islets and brain tissue. Forty-six percent (23 of 50) of the IDDM sera but none of the sera from normal controls (0 of 50) immunoprecipitated the intracellular domain of IA-2beta. Competitive inhibition experiments showed that IDDM sera have autoantibodies that recognize both common and distinct determinants on IA-2 and IA-2beta. Many IDDM sera are known to immunoprecipitate 37-kDa and 40-kDa tryptic fragments from islet cells, but the identity of the precursor protein(s) has remained elusive. The current study shows that treatment of recombinant IA-2beta and IA-2 with trypsin yields a 37-kDa fragment and a 40-kDa fragment, respectively, and that these fragments can be immunoprecipitated with diabetic sera. Absorption of diabetic sera with unlabeled recombinant IA-2 or IA-2beta, prior to incubation with radiolabeled 37-kDa and 40-kDa tryptic fragments derived from insulinoma or glucagonoma cells, blocks the immunoprecipitation of both of these radiolabeled tryptic fragments. We conclude that IA-2beta and IA-2 are the precursors of the 37-kDa and 40-kDa islet cell autoantigens, respectively, and that both IA-2 and IA-2beta are major autoantigens in IDDM.

Amino Acid Sequence↗
PubMed1996

A filter elution assay for the simultaneous detection of DNA double and single strand breaks.

This paper reports a gravity-flow filter elution assay that permits the simultaneous detection of DNA double strand breaks (DSBs, as neutral elutable DNA) and DNA single strand breaks (SSBs, as alkaline elutable DNA). This assay is based on a modification of a conventional gravity-flow alkaline elution procedure. The elution fraction resulting from cell lysis before the application of the alkaline elution buffer provides an estimate of DNA DSBs, while the fraction eluted by the alkaline buffer (pH 12.3) measures the extent of DNA SSBs. Data show that, in addition to its good detection sensitivity for SSBs, this method provides a sensitive measure of DNA DSBs. As an illustration of its specificity, this method unambiguously distinguished DNA SSBs induced by X-irradiation of mouse leukemia L1210 cells from DNA DSBs occurring in cells undergoing nucleosomal DNA fragmentation and apoptosis. The neutral elution fraction is a good predictor of loss of cell viability induced either by selenite at pharmacological levels or by growth factor depletion. The filter elution method reported is more specific and informative than a nick translation assay for the detection of DNA strand breaks.

Animals↗
PubMed1996

Natural killer cell precursors in the CD44neg/dim T-cell receptor population of mouse bone marrow.

Natural killer (NK) cells develop from the nonadherent cell component of NK long-term bone marrow (BM) cultures (NK-LTBMC). Because these nonadherent cells are depleted of mature NK cells and T cells, but appear to enriched for NK precursors, they were used as a starting population to begin to define the NK precursors that function in NK-LTBMC. As the stromal cell component of NK-LTBMC has been shown to support interleukin (IL)-2-induced, CD44 dependent, NK cell development from nonadherent NK precursors, NK-LTBMC stroma was used in a limiting dilution assay (LDA) to quantitate the precursors. NK-LTBMC in 96-well plates were irradiated (20 Gy) to kill hematopoietic cells (including the NK precursors), seeded with limiting dilutions of the cells to be quantitated, cultured with 500 U/mL IL-2 for 13 days and assayed for development of NK activity by adding 51Cr-labeled YAC-1 cells to the wells and evaluating the release of 51Cr after 4 hours. Flow cytometric analysis, sorting, and quantitation of the nonadherent cell component of NK-LTBMC showed that NK precursors were concentrated in the CD44neg/dim subset that comprised 10% of the "lymphoid" gated cells. When the CD44neg/dim subset was sorted from BM of mice treated with 5-fluorouracil (5-FU) day before (-1FUBM), there were about 30% T cells, but no NK-1.1+ cells. When the T cells were removed by sorting and the CD44neg/dim, alphabeta, gammadelta T-cell receptorneg (TCR-) subpopulation was seeded onto irradiated stroma with IL-2, they proliferated, developed NK activity, became NK-1.1+ and CD44bright and remained alphabeta, gammadelta TCR-. The frequency of NK precursors in this population as estimated from the LDA was about 1/500.

Animals↗
PubMed1996

Scavenging effects of phenylpropanoid glycosides from Pedicularis on superoxide anion and hydroxyl radical by the spin trapping method(95)02255-4.

The scavenging activities of six phenylpropanoid glycosides, i.e. leucosceptoside A and martynoside, isolated from Pedicularis alashanica Maxim, verbascoside, and pediculariosides A, M and N isolated from Pedicularis striata Pall ssp. arachnoidea Franch Tsoong, on superoxide anion and hydroxyl radicals have been studied by the spin trapping method. The results demonstrated that the number of phenolic hydroxyl groups in the structures of the phenylpropanoid glycosides is related to their scavenging activities. The scavenging effects of the phenylpropanoid glycosides possessing two o-dihydroxyl groups were stronger than the effects of the compounds possessing two o-hydroxy-methoxy groups.

Cyclic N-Oxides↗
PubMed1996

Electrophoretically uniform fluorescent dyes for automated DNA sequencing.

A class of dyes, BODIPY fluorophores, has been identified for automated DNA sequencing that has improved spectral characteristics compared with conventional fluorescein and rhodamine dyes. Single and double BODIPY dye primers were characterized in commercially available DNA sequencers and showed uniform electrophoretic mobilities and high fluorescence intensities. The improved physical properties of BODIPY dye primers were demonstrated by direct base-calling from the unprocessed fluorescent signals and improved heterozygote analyses of mixed-base populations. The high sensitivity of BODIPY dye primers requires at least 33 percent less reagent consumed per reaction than conventional dye primers, which should affect the costs of large genome-sequencing efforts.

Autoanalysis↗
PubMed1996

Human ficolin: cDNA cloning, demonstration of peripheral blood leucocytes as the major site of synthesis and assignment of the gene to chromosome 9.

Pig ficolins and a number of other proteins contain sequences that are homologous to the C-terminal halves of fibrinogen beta- and gamma-chains. To clone the cDNA for human ficolin, two degenerate oligonucleotide primers were synthesized, based on two stretches of protein sequence that were highly conserved among those proteins, and used for PCR with cDNA from a human uterus lambda gt11 library as a template. A PCR product with a predicted size of 300 bp was obtained and this was used to screen a uterus cDNA library. Of the positive clones isolated, two (U1 and U2), containing inserts of 1.7 and 1.1 kb respectively, were found to encode human ficolin. The cDNA-derived amino acid sequence of human ficolin has approx. 75% identity with, and a similar domain organization to, the two pig ficolin sequences, which are characterized by the presence of a leader peptide, a short N-terminal segment followed by a collagen-like region and then by a C-terminal fibrinogen-like domain. The 1.1 kb insert of clone U2 was used in Northern-blot analysis, and a very strong signal for a 1.4 kb mRNA species was detected in mRNA from human peripheral blood leucocytes. This showed that, despite the initial characterization of pig ficolin as a putative receptor on uterine cells for transforming growth factor beta 1, blood leucocytes are probably the major site of human ficolin synthesis. Much weaker signals of the same size were also detected in spleen, lung and thymus and may be due to the presence of tissue macrophages or trapped blood in these tissues. An mRNA species of approx. 1.3 kb in human liver also weakly hybridized to the U2 probe, indicating the presence of a sequence that was distinct from, but related to, ficolin. The gene for human ficolin has been mapped to chromosome 9.

Amino Acid Sequence↗