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J K Wu

Publications and source records attributed to J K Wu.

15 recordsLinked to original sources
PubMed1992

Transfection of mouse cytotoxic T lymphocyte with an antisense granzyme A vector reduces lytic activity.

Murine CTL have seven serine proteases, known as granzymes, in their lytic granules. Despite considerable effort, convincing evidence that these enzymes play an obligatory role in the lytic process has not been presented. To investigate the function of one of these proteases, granzyme A (GA), we utilized an antisense expression vector to lower the level of the enzyme in the cells. An expression vector containing antisense cDNA for GA and the gene for hygromycin B resistance was constructed and electroporated into the murine CTL line, AR1. Transfectants were selected based on resistance to hygromycin B, and a number of stable lines were developed. One of the antisense lines had greatly reduced levels of GA mRNA, when compared to the parental cells or to control lines transfected with the vector lacking the antisense DNA. The message levels for two other CTL granule proteins, granzyme B and perforin, were unaffected by the antisense vector. The amount of GA, as measured by enzymatic activity, was 3- to 10-fold lower in the transfectant. Most significantly, this line also consistently showed 50 to 70% lower ability to lyse nucleated target cells and to degrade their DNA. Furthermore, it exhibited 90 to 95% lower lytic activity to anti-CD3-coated SRBC. Conjugate formation with target cells, however, was normal. These data provide strong evidence that GA plays an important role in the cytolytic cycle, and that the quantity of enzyme is a limiting factor in these cytolytic cells.

Animals↗
PubMed1992

High pressure gel-permeation assay for the proteolysis of human aggrecan by human stromelysin-1: kinetic constants for aggrecan hydrolysis.

The adaptation of an analytical procedure for aggrecan based upon gel-permeation chromatography to an FPLC-based protocol has significantly sped up the analysis. The faster assay has permitted determination of the kinetic constants for digestion of human aggrecan by human stromelysin-1. Monomeric aggrecan appeared to be hydrolyzed by stromelysin-1 to multiple forms with lower molecular weight. The disappearance of high-molecular-weight aggrecan was first-order, showing Km much larger than 2 microM and kc/Km = 4000 M-1 s-1 at pH 7.5. The disappearance of high-molecular-weight aggrecan upon hydrolysis by stromelysin-1 at pH 5.5 was also first-order, with kc/Km = 10,700 M-1 s-1. The disappearance of high-molecular-weight aggrecan at pH 7.5 was first-order for digestion by human leukocyte elastase with kc/Km = 230,000 M-1 s-1, by human cathepsin G with kc/Km = 4200 M-1 S-1, and by human plasma plasmin with kc/Km = 2800 M-1 s-1, all with Km much larger than 2 microM.

Aggrecans↗
PubMed1992

Molecular cloning and cell-free expression of mouse antithrombin III.

The homology between antithrombin III (AT-III) of mouse, of man, and that of other species was investigated. Preliminary experiments showed that mouse AT-III inhibited human alpha-thrombin efficiently (second order rate constant [K2nd] 5.8 x 10(3) M-1 s-1) as compared to human AT-III (K2nd 6.7 x 10(3) M-1), but was not recognized on immunoblots by antibodies that recognized both human and rabbit AT-III. In order to compare AT-III from different species at the molecular level, a cDNA clone for murine AT-III was isolated from a lambda ZAP mouse liver cDNA library on the basis of hybridization to a rabbit AT-III cDNA probe. The 1509 bp murine AT-III cDNA consists of a 1398 bp open reading frame, preceded by a 15 bp 5' untranslated region, followed by a 75 bp 3' untranslated region. The deduced primary protein structure consists of a 32 amino acid signal sequence, with a mature portion of 433 residues. Mature murine AT-III is 89% identical to its human counterpart, 86% identical to bovine AT-III, and 82% identical to that of the rabbit. Constructs lacking the nucleotides encoding the signal sequence were engineered and expressed in a cell-free system. The resulting 47 kDa non-glycosylated translation product was capable of being cleaved by human alpha-thrombin, of forming SDS-stable complexes with the protease, and of binding to immobilized heparin. Isolation of the murine AT-III cDNA will make feasible molecularly defined experiments with murine AT-III in the mouse system.

Amino Acid Sequence↗
PubMed1992

Loss of heterozygosity on the short arm of chromosome 17 in human astrocytomas.

Using restriction fragment length polymorphism (RFLP) analysis, we demonstrated in 4 of 20 patients with astrocytomas loss of heterozygosity on the short arm of chromosome 17 (17p), in the telomeric segment distal to DNA marker pEW301 (locus D17S58). The loss of heterozygosity may uncover a mutation in a tumour suppressor gene and thus lead to or permit tumour formation. The p53 tumour suppressor gene, which is localized at 17p13, is a likely candidate for the tumour suppressor gene involved. Of the 4 patients with loss of heterozygosity on 17p, one patient had a grade I astrocytoma, another patient had a grade II astrocytoma and 2 patients had glioblastoma multiforme. Since the loss of heterozygosity on 17p was detected in low-grade as well as in high-grade astrocytomas, it is possible that p53 suppressor gene loss may be an early genetic event in the multistep process of astrocytoma formation.

Adult↗
PubMed1992

Cerebellar infarction in the young.

BACKGROUND AND PURPOSE: Ischemic cerebrovascular disease in children and young adults usually affects the anterior circulation. SUMMARY OF REPORT: We describe two cases of cerebellar infarction in the territory of vertebral artery supply, associated with physical exertion, in a young adult and in a child. Review of 31 previous cases of cerebellar infarction occurring in the first 2 decades of life demonstrated a mostly obscure causation; where a likely cause was found, trauma was most frequent. In 12 of the 31 patients, a vertebral artery (usually the left) was occluded. Patients were sometimes predisposed to such occlusions by subluxation between the first and second cervical vertebrae, allowing abnormal neck movements that can cause arterial injury and thromboembolism. Some of these cerebellar infarcts, like those of our patients, have followed physical exertion. CONCLUSIONS: Cerebellar infarction can be life-threatening, but half of the patients, including ours, have had complete or near-complete recovery.

Adult↗
PubMed1992

Surgical reversal of a subacute complete unilateral visual loss from an ovarian metastasis to the pituitary gland.

A 56-year-old woman with ovarian papillary cystadenocarcinoma that metastasized to the pituitary gland sought treatment with a 7-day history of total loss of vision in one eye. Ten days after transsphenoidal tumor resection, the patient's vision suddenly returned to baseline. This unusual case indicates that surgical decompression of the optic nerve and chiasm can completely salvage vision, even after prolonged total visual loss.

Blindness↗
PubMed1992

Aneurysm recurrence following endovascular balloon occlusion.

Endovascular balloon occlusion is an alternative treatment for surgically unclippable cerebral aneurysms. The results of aneurysm occlusion with either a silicone or a latex balloon in a common carotid artery bifurcation aneurysm model are compared to determine which type of balloon was least likely to result in aneurysm recurrence. Five rabbits each underwent endovascular balloon occlusion with either a silicone or a latex balloon, with seven rabbits serving as controls. At 3 months postocclusion, nine of the 10 balloon-treated aneurysms had recurred. The recurrent aneurysm tended to be larger in animals treated with silicone than with latex balloons. A dense fibrotic response was present around the collar of the latex balloons, but no significant fibrotic response was found in the silicone balloon group. This study suggests that with currently available balloons, the initial complete angiographic obliteration of an aneurysm following balloon occlusion should not be interpreted as a cure and that periodic follow-up angiography should be performed.

Aneurysm↗
PubMed1992

Instrumented laxity test for the evaluation of posterior cruciate ligament reconstructed knee.

By making anterior and posterior drawer tests using "Laxity tester" is an objective way to evaluate the resting position of tibia plateau and knee laxity. This study followed up 16 cases of PCL reconstructed or pull-out repaired knees for an average of two years and seven months. A force of 20 1b was applied to evaluate knee laxity at 30 degree and 90 degree knee flexion. From paired t-test, the stability of reconstructed knees was not statistically different from that of control contralateral normal knees. The functional scores measured by the Gillquist scoring system for injured knees after reconstruction were satisfactory. Therefore the restabilized PCL deficient knees by the modified Clancy method or pull-out repair yielded good evaluation scores. The data of contralateral normal knee laxity obtained in this study was used as reference in pre-operative and post-operative evaluation of PCL injured knee.

Female↗
PubMed1991

Human cytotoxic lymphocyte granzyme B. Its purification from granules and the characterization of substrate and inhibitor specificity.

Granzyme B has been purified to homogeneity from the granules of a human cytolytic lymphocyte line, Q31, in an enzymatically active form by a three-step procedure. Q31 granzyme B hydrolyzed Na-t-butyloxycarbonyl-L-alanyl-L-alanyl-L-aspartyl (Boc-Ala-Ala-Asp) thiobenzyl ester with a kcat of 11 +/- 5 mol/s/mol enzyme and catalytic efficiency kcat/Km of 76,000 +/- 44,000 M-1 s-1. The hydrolysis of Boc-Ala-Ala-Asp thiobenzyl ester by crude Q31 Percoll fractions paralleled the tryptase activity for granule-containing fractions, which showed that granzyme B was associated with granules. When chromatographed on Sephacryl S-300, Q31 granzyme B eluted in two broad bands corresponding to dimer and monomer, both of which electrophoresed at 35 kDa in reducing NaDodSo4 polyacrylamide, and both of which showed a lag phase in assays. The lag phase in assays could be extended with 0.03 mM pepstatin. Upon elution from ion-exchange chromatography Q31 granzyme B electrophoresed at 32 kDa in reducing NaDodSO4 polyacrylamide and did not have a lag phase in assays. The amino-terminal sequence of the 32-kDa Q31 granzyme B was identical to four other human cytotoxic T-lymphocyte granzymes B in 18 of 18 positions sequenced. Purified Q31 granzyme B had a preference for substrates with Glu or Asp as the residue amino-terminal to the scissile bond; little or no activity was noted with oligopeptide substrates for trypsin-like, chymotrypsin-like, and elastase-like proteases. Human plasma alpha 1-protease inhibitor, human plasma alpha 2-protease macroglobulin, soybean and lima-bean trypsin inhibitors, bovine aprotinin, phosphoramidon, and chymostatin inhibited Q31 granzyme B. The inhibition by alpha 1-protease inhibitor was rapid enough to be of physiological significance.

Amino Acid Sequence↗
PubMed1991

The enzymatic activity of human cytotoxic T-lymphocyte granzyme A and cytolysis mediated by cytotoxic T-lymphocytes are potently inhibited by a synthetic antiprotease, FUT-175.

The synthetic antiprotease, FUT-175 (6-amidino-2-naphthyl-4-guanidinobenzoate), was found to be an extraordinarily potent and rapid inhibitor of human Q31 cytotoxic T-lymphocyte granzyme A. The granzyme A was inhibited in a time-dependent manner with kobs/i = 430,000 +/- 80,000 M-1 s-1. Four other FUT-175 analogs were also found to be potent, rapid Q31 granzyme A inhibitors. All five compounds inhibited Q31 cytotoxic T-lymphocyte-mediated cytolysis of human JY lymphoma cells, but at concentrations far in excess of those needed for granzyme A inhibition. The data presented suggest that postmarketing surveillance of FUT-175 should include a review of possible immunosuppressive side-effects, such as increased susceptibility to viral infections and to neoplastic transformations.

Benzamidines↗
PubMed1990

Differential expression of ros oncogene in primary human astrocytomas and astrocytoma cell lines.

Overexpression of oncogenes has been associated with the pathogenesis of some human cancers. The ros oncogene, which encodes a putative receptor with tyrosine kinase activity, has been recently shown to be specifically expressed in high levels in human astrocytoma and glioblastoma cell lines. Using transcription mapping analysis, we surveyed 25 primary astrocytomas of all histological grades, including glioblastomas, and failed to demonstrate elevated expression of ros in these tumors. This difference between the cell lines and the primary tumors may be due to dilution of the ros-positive clones by larger populations of ros-negative cells in the primary tumors or to induction of ros oncogene when the tumors are adapted to tissue culture.

Astrocytoma↗
PubMed1990

Myelopathy presenting decades after surgery for congenital cervical cutaneous lesions.

We report on two patients in whom cervical myelopathy developed decades after they had undergone surgery for congenital cervical cutaneous lesions. Preoperative magnetic resonance imaging demonstrated dorsal tethering and cavitation of the cervical cord in the area of the previous surgery and was helpful in decision making regarding surgical exploration and in planning for it. We stress the importance of long-term follow-up by both clinical examination and magnetic resonance imaging in children who have been operated on for cervical cutaneous masses that may have central connections.

Adult↗
PubMed1988

Human cytotoxic lymphocyte tryptase. Its purification from granules and the characterization of inhibitor and substrate specificity.

A trypsin-like enzyme (tryptase) has been purified to homogeneity from the granules of a human cytolytic lymphocyte (CTL) line, Q31, by a three-step procedure. By including 0.3% (v/v) Triton X-100 and 1 mg/ml heparin in purification buffers, near total yields of tryptase activity were obtained during the purification. The enzyme, referred to as Q31 tryptase, migrated in polyacrylamide gels with sodium dodecyl sulfate at a position corresponding to 28 kDa with and to 45 kDa without 2-mercaptoethanol. It had an amino-terminal sequence identical to a previously reported human CTL tryptase at 20 of 22 positions identified. It hydrolyzed N alpha-carbobenzyloxy-L-lysyl-thiobenzyl ester (BLT), and this BLT esterase activity was most efficient at slightly alkaline pH and was relatively more active near neutral pH than mouse CTL tryptase. Human alpha 1-protease inhibitor, human antithrombin III, phenylmethanesulfonyl fluoride, and p-aminobenzamidine inhibited the Q31 tryptase. The inhibition by human antithrombin III was rapid enough to be of physiological significance. A survey of oligopeptide p-nitroanilides found that the best substrate for human Q31 tryptase is H-D-(epsilon-carbobenzyloxy)Lys-L-Pro-L-Arg-p-nitroanilide. The Q31 tryptase appears to have broad specificity for amino acid residues at P2 and P3, i.e. at 2 and 3 residues amino-terminal to the scissile bond.

Cytoplasmic Granules↗