Absence of anticardiolipin antibody in non-treated and heat-inactivated normal human sera.
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Antibodies, Anticardiolipin↗
Biomedical subjects
T Kinoshita
Publications and source records attributed to T Kinoshita.
trans-5-prostaglandin E2 stimulates plasminogen activation by tissue-type plasminogen activator.
The effect of trans-5-prostaglandin E2 (trans-PGE2) on fibrinolysis was examined in vitro using synthetic chromogenic substrate S-2251. trans-PGE2 was found to enhance plasminogen (PLG) activation mediated by tissue-type plasminogen activator (tPA). The enhancing effect was dependent on the concentration of trans-PGE2. cis-PGE2 and the other PGs (PGE1 and PGI2) did not show such an effect as trans-PGE2, despite to the fact that their structures are similar to that of trans-PGE2. trans-Configuration around the double bond at the 5-position seems to be important in the enhancement of the fibrinolytic activity.
Amino Acid Sequence↗
Developmental changes in hepatocyte growth factor mRNA and its receptor in rat liver, kidney and lung.
Hepatocyte growth factor (HGF) is a mesenchymal-derived factor which induces mitosis, cell movement and morphogenesis of tissue-like structure. We analyzed changes in HGF mRNA and its receptor, the c-met proto-oncogene product, in the liver, kidney and lung during late fetal and postnatal development in rats. In the liver, the HGF-mRNA level was very low during late gestation and in neonates, it increased remarkably and reached a maximum two weeks postnatally, to be followed by a decrease to 33% of the maximum. HGF mRNA in the kidney and lung was either undetectable or very low during late gestation and the neonatal period and increased markedly to reach a maximum, respectively, 3-4 weeks postnatally. HGF-mRNA level in the adult rat lung was fivefold higher than that in the liver and kidney. The number of HGF receptors on plasma membranes of these tissues was low in neonates but there was a rapid increase after birth and a maximum was reached within three weeks. The number of HGF receptors/ng plasma membrane protein at the maximal level was highest in the liver and lowest in the lung. c-met/HGF-receptor mRNA in the liver was also low during late-gestation or in early neonatal periods and increased postnatally. Since HGF-mRNA and HGF-receptor levels changed differently in liver, kidney and lung, the expression of HGF and its receptor may be independently regulated in each organ. However, in these organs, HGF mRNA and the HGF receptor increased within a few weeks of birth, HGF may play roles in organ growth, organ maturation and the maintenance of tissue homeostasis during the postnatal period, presumably through its potential to act as mitogen, motogen and morphogen.
Aging↗
Quantification of gene expression over a wide range by the polymerase chain reaction.
We investigated the usefulness of the polymerase chain reaction (PCR) method for the relative quantification of gene expression using a simultaneously amplified sequence of beta-actin mRNA as an internal control for the target sequence of tax/rex mRNA of human T-cell leukemia virus type I. The PCR product of the internal control was reduced by delaying the addition of the primers for its sequence. The photostimulated luminescence of the bands was measured with a laser image analyzer, and the values were plotted against the cycle number. The cycle differences between the logarithmic phase of the curves for the target sequence and for beta-actin (delta cycle) showed a linear correlation with the initial concentration of the sample. This method is highly sensitive for evaluating gene expression over a wide range.
Actins↗
Mapping of epitopes, glycosylation sites, and complement regulatory domains in human decay accelerating factor.
Decay accelerating factor (DAF, CD55) is a glycophospholipid-anchored membrane protein that protects cells from complement-mediated damage by inhibiting the formation and accelerating the decay of C3/C5 convertases. DAF deletion mutants lacking each of the four short consensus repeats (SCR) or the serine/threonine-rich region (S/T) were created by site-directed mutagenesis. These deletion mutants were expressed by stable transfection in Chinese hamster ovary cells for the purpose of mapping important structural and functional sites in DAF. The epitopes on DAF for 16 murine mAb were mapped by immunoprecipitation studies as follows: SCR1, 6; SCR2, 3; SCR3, 3; SCR4, 3; S/T, 1. Testing of 13 mAb showed complete blocking of DAF function only by 1C6 and 1H4, both directed at SCR3. The single N-linked glycosylation site was confirmed at a location between SCR1 and SCR2, and the multiple O-linked oligosaccharides were localized to the S/T region. Functional activity of DAF mutants was assessed by the ability of these transfected constructs to protect Chinese hamster ovary cells from cytotoxicity induced by rabbit antibody plus human complement. Removal of SCR1 had no effect on DAF function, but individual deletion of SCR2, SCR3, or SCR4 totally abolished DAF function. Surprisingly, deletion of the S/T region totally abrogated DAF function, but this could be restored by a fusion construct placing the four SCR domains of DAF onto the HLA-B44 molecule, implying that the O-glycosylated S/T region serves as an important but nonspecific spacer projecting the DAF functional domains above the plasma membrane. Overall, the creation of DAF deletion mutants has elucidated important structure-function relations in the DAF molecule.
Animals↗
Primary structure and expression of a gamete lytic enzyme in Chlamydomonas reinhardtii: similarity of functional domains to matrix metalloproteases.
A gamete lytic enzyme (GLE) of Chlamydomonas reinhardtii is a zinc metalloprotease and mediates digestion of the cell walls of the two mating-type gametes during mating as a necessary prelude to cell fusion. The nucleotide sequence analysis of a cDNA revealed that GLE is synthesized in a preproenzyme form, a 638-amino acid polypeptide (Mr, 69,824) with a 28-amino acid signal peptide, a 155-amino acid propolypeptide, and a 455-amino acid mature polypeptide (Mr, 49,633). A potential site for autocatalytic activation was contained within the propolypeptide and a zinc binding site found within the mature polypeptide; both sites were highly homologous to those in mammalian collagenase. A putative calcium binding site was present in the near C-terminal region of the mature GLE. Both propolypeptide and mature polypeptide had potential sites for asparagine-linked glycosylation, and the Arg-(Pro)3 and Arg-(Pro)2 motifs, which are known to exist in hydroxyproline-rich glycoproteins of the Chlamydomonas cell wall. Northern blot analysis revealed that steady-state levels of the 2.4-kilobase GLE mRNA increased during growth and mitotic cell division in the vegetative cell cycle and also increased markedly during gametogenesis under nitrogen-starved conditions.
Amino Acid Sequence↗
Mutations of p53 gene and their relation to disease progression in B-cell lymphoma.
The alteration of p53 tumor suppressor gene was studied in 48 patients with B-cell lymphoma. A sequential combined technique of polymerase chain reaction-mediated single-strand conformational polymorphism (PCR-SSCP) or reverse transcription (RT)-PCR-SSCP and direct sequencing were used as a simple and sensitive approach to analyze nucleotide changes. By these methods, we identified 8 missense point mutations and 2 codon deletions in 9 of the 48 patients. These mutations were located in or close to the evolutionally highly conserved regions of the p53 gene. Eight of nine patients having p53 gene alterations were in advanced clinical stage (IV). It is the first report of p53 gene mutations in follicular and diffuse lymphoma. These observations suggest that the p53 gene alteration may play an important role in lymphomagenesis and/or disease progression in some types of B-cell lymphoma.
Base Sequence↗
Identification and characterization of "injurin," an inducer of expression of the gene for hepatocyte growth factor.
The marked and rapid increase of hepatocyte growth factor (HGF) mRNA in the intact lung of rats after partial hepatectomy or unilateral nephrectomy suggests the existence of a humoral factor mediating a signal of injury to distal organs and may induce the expression of HGF gene in these organs. We have now identified a proteinous factor in the sera of rats with injury of liver or kidney that increases HGF mRNA in the intact lung. When the serum of rats with liver insult caused by partial hepatectomy or ischemic treatment was injected i.p. into normal noninjured rats, it induced a marked HGF mRNA expression in the lung of the recipient rats. The addition of serum from rats with various hepatic or renal injuries to MRC-5 human embryonic lung fibroblasts in culture also led to the induction of HGF mRNA expression, so that the production of HGF by MRC-5 cells after treatment with the sera was remarkably increased in the culture medium. However, serum from the normal intact rat induced no HGF production and no HGF mRNA in the lung in vivo and lung fibroblasts in vitro. This factor, which increases HGF production, was purified greater than 200-fold from sera of CCl4-treated rats. The factor proved to be an acid- and heat-stable protein with an apparent molecular mass of 10-20 kDa in SDS/PAGE. Its activity markedly increased within 3-6 hr in the plasma of rats after various treatments that injured the liver or kidney. These results suggest that the factor specifically appears in the blood of rats with organ injury and may be involved in organ regeneration through the potential to increase the synthesis of HGF. Since the factor seems to mediate various organ injuries, we named it "injurin."
Animals↗
Covalent binding of C3b to C4b within the classical complement pathway C5 convertase. Determination of amino acid residues involved in ester linkage formation.
C5 convertase of the classical complement pathway is a protein complex consisting of C4b, C2a, and C3b. Within this complex C3b binds to C4b via an ester linkage. We now present evidence that the covalent C3b-binding site on human C4b is Ser at position 1217 of C4. We also show that formation of the covalently linked C4b.C3b complex occurs in the mouse complement system and that the C3b-binding site on mouse C4b is Ser at position 1213 which is homologous to Ser-1217 of human C4. Therefore, covalent binding of C3b to a single specific site on C4b within the classical pathway C5 convertase is likely a common phenomenon in the mammalian complement system. Specific noncovalent association of metastable C3b with C4b would occur first, leading to reaction of the thioester with a specific hydroxy group. This is supported by two lines of experimental evidence, one which shows that a mutant C4 that does not make a covalent linkage with C3b is still capable of forming C5 convertase and a second in which the C4b.C3b complex has been demonstrated by cross-linking erythrocytes bearing this C5 convertase.
Amino Acid Sequence↗
Role of TraT protein, an anticomplementary protein produced in Escherichia coli by R100 factor, in serum resistance.
Escherichia coli K12 strain W3110/SM bearing a plasmid containing the traT gene (traT+ strain) was more resistant to the bactericidal activity of guinea pig serum than the same strain bearing this plasmid without the traT gene (traT- strain). A murine mAb was generated against synthetic TraT peptide (86-99). This antibody reacted only with denatured TraT protein, but it was used for monitoring TraT protein by immunoblotting during purification of the protein. Six mAb were then generated against partially purified traT protein from the solubilized membrane fraction of the traT+ strain. These mAb reacted with the native protein even on living cells, and their F(ab) fragments were found to suppress the inhibitory effect of the TraT protein on the bactericidal activity of serum. TraT protein was purified from solubilized membranes of the traT+ strain by ion exchange and gel filtration chromatographies. The purified TraT protein inhibited the lysis of sensitized erythrocytes by serum complement. Its inhibitory action was mainly on the C6 step. It strongly inhibited the reaction of C6 with EAC14b2a3b and excess C5, C7, C8, and C9. TraT protein also inhibited the reaction of C7-deficient human serum with guinea pig erythrocytes when it was activated by cobra venom factor. It did not inhibit the reaction of preformed C5b6 complexes. However, TraT did not have any effect on the cleavage of 125I[C5] to 125I[C5b] in similar conditions. It also partially inhibited the reaction steps of C4, C5, and factor B and limited guinea pig complement serum in 0.1% gelatin veronal buffered saline, pH 7.4, containing 10 mM EDTA with their respective preceding intermediate cells. It had no effect on either the binding of C3 to EAC14b2a or the cleavage of C3b by factors H and I. TraT protein probably inhibits the formation of C5b6 complex or causes structural alteration of the complex to a nonfunctional form.
Amino Acid Sequence↗
Bactericidal activity of C9-deficient human serum.
Escherichia coli B/SM, strain 1-1, was killed dose dependently by human hereditary C9-deficient serum (C9DHS), which was shown to contain no C9 Ag by an ELISA method. On the other hand, human hereditary C7-deficient serum did not kill the bacteria under similar conditions. The bactericidal activity of C9DHS was inhibited by rabbit anti-C5 antibody but not by murine anti-C9 mAb. The anti-C9 antibody decreased the bactericidal activity of normal human serum (NHS) to the level of that with C9DHS. Sheep anti-human lysozyme antibody did not affect the bactericidal activity of C9DHS or NHS even when added at more than twice the concentration required to block the serum lysozyme activity on Micrococcus luteus. After treatment with C9DHS and washing, surviving Escherichia coli were killed by C9, but not by lysozyme, transferrin, or both. Other strains of E. coli (K12 W3110, C600, and NIHJ) and Salmonella typhimurium (strain NCTC 74), all maintained in the laboratory, were also killed by C9DHS. However, pathogenic strains recently isolated from patients with traveler's diarrhea and some strains of S. typhimurium were resistant to both C9DHS and NHS, at least at the serum concentration tested. A concentration of 0.1 M Tris did not increase the susceptibility of serum-resistant strains of bacteria to C9DHS, but made one strain of S. typhimurium tested susceptible to NHS, but not to C9DHS. These results clearly showed that C9DHS kills bacteria that are sensitive to NHS through activation of C up to the step of C8 in the same way that C9-deficient C serum lyzed sensitized erythrocytes.
Blood Bactericidal Activity↗
Distinct receptor and regulatory properties of recombinant mouse complement receptor 1 (CR1) and Crry, the two genetic homologues of human CR1.
The relationship between the characterized mouse regulators of complement activation (RCA) genes and the 190-kD mouse complement receptor 1 (MCR1), 155-kD mouse complement receptor 2 (MCR2), and mouse p65 is unclear. One mouse RCA gene, designated MCR2 (or Cr2), encodes alternatively spliced 21 and 15 short consensus repeat (SCR)-containing transcripts that crosshybridize with cDNAs of both human CR2 and CR1, or CR2 alone, respectively. A five SCR-containing transcript derived from a second unique gene, designated Crry, also crosshybridizes with human CR1. We have previously shown that the 155-kD MCR2 is encoded by the 15 SCR-containing transcript. To analyze the protein products of the other transcripts, which are considered the genetic homologues of human CR1, we have expressed the 21 and the 5 SCR-containing cDNAs in the human K562 erythroleukemia cell line. We demonstrate that cells expressing the 21 SCR transcript express the 190-kD MCR1 protein. These cells react with five unique rat anti-MCR1 monoclonal antibodies, including the 8C12 antibody considered to be monospecific for MCR1. In addition, these cells efficiently form rosettes with mouse C3b-bearing sheep erythrocytes. In contrast, cells expressing the five SCR-containing Crry transcript are strongly recognized by an anti-human CR1 antibody that also defines the mouse p65 protein. Using a functional assay that measures the surface deposition of C3 activated via the classical complement pathway, we show that Crry/p65-expressing cells have a markedly decreased amount of C3 deposited on them as compared with control cells expressing the antisense construct or cells expressing MCR1 or MCR2. This suggests that Crry has intrinsic complement regulatory activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Preoperative assessment of lymph node metastases in patients with gastric cancer: evaluation of the Maruyama computer program.
The probability of survival of patients with gastric cancer depends upon depth of wall penetration by the primary tumour and metastatic lymph node burden. Radical lymphadenectomy may lead to prolonged survival but with increased morbidity. A computer program from Maruyama, National Cancer Centre, Tokyo enables evaluation of individual survival time and infiltration of lymph nodes. This analysis was applied to a German population. Computer-aided predictions were determined retrospectively using the prognostic factors of sex, age, Borrmann classification, histology, depth of wall penetration, location and diameter of the tumour. Data were collected from 222 patients at the Technical University of Munich (median age 66 years, sex ratio (M:F) 2:1), who had been operated on (72 per cent total gastrectomy, 28 per cent subtotal gastrectomy) for gastric cancer. The predicted results were compared with the postoperative pathological findings. The prediction of node metastases was highly accurate (lymph nodes 13-16, 96 per cent; 7-12, 89 per cent; 1-6, 82 per cent). These computer predictions may provide perioperative information of therapeutic value.
Aged↗
Structural analysis of non-volatile compounds by liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry: thermal isomerization of benzylpenicillin in a Plasmaspray interface.
Isomerization and degradation of benzylpenicillin ((2S,5R,6R)-3,3-dimethyl-7-oxo-6-(2-phenylacetamido)-4-thia-1- azabicyclo[3.2.0]heptane-2-carboxylic acid) were studied using a combination of Plasmaspray (PSP) liquid chromatography/tandem mass spectrometry (LC/MS/MS) and liquid secondary ion tandem mass spectrometry (LSI MS/MS). Benzylpenicillin was isomerized to benzylpenicillenic acid (3-mercapto-N-[[5-oxo-2-(phenylmethyl)-4(5H)- oxazolylidene]methyl]valine) in the PSP interface/ion source. The isomerization was inferred from the probe temperature dependence of PSP LC tandem mass spectra and discrepancies in the daughter ions between PSP LC and LSI tandem mass spectra. High temperature at the PSP interface was responsible for the isomerization, since the difference between PSP LC and LSI tandem mass spectra became smaller as the probe temperature was lowered. It was also found that benzylpenicillin was decomposed to benzylpenilloic acid (5,5-dimethyl-2-[(phenylacetamido)methyl]thiazolidine-4-carboxylic acid), N-(phenylacetyl)glycine, N-(phenylacetyl)glycinal and 3-mercaptovaline in the PSP interface/ion source. The degradation products formed in the interface/ion source were identical to those formed in acidic solution. The results show that degradation of penicillins can be investigated by PSP LC/MS and PSP LC/MS/MS.
Chromatography, Liquid↗
Influence of surgical stress on bactericidal activity of neutrophils and complications of infection in patients with esophageal cancer.
The association between surgical stress-related depression in bactericidal activities of neutrophils and the occurrence of postoperative infections was investigated. Bactericidal activities of neutrophils were measured in 19 patients undergoing esophagectomy, 15 gastrectomy, and 16 cholecystectomy. Five patients had complications of infection following esophagectomy. In 45 patients with no postoperative infections, intracellular killing index (KI) and superoxide anion production (SOP) levels decreased on postoperative day 1 while myeloperoxidase (MPO) activity increased on days 1-3. In 5 patients with esophageal cancer and postoperative infections, decreases in KI and SOP were less prominent, as compared to findings in 14 esophageal cancer patients without such problems but the MPO activity decreased on days 1-3. This evidence suggests that postoperative septic complications are not directly associated with surgical stress-related transient depression of bactericidal activities immediately after surgery but rather with neutrophil-mediated tissue injuries based on degranulation.
Adenocarcinoma↗
Impaired neutrophil bactericidal activity correlates with the infection occurring after surgery for esophageal cancer.
We examined whether or not preoperative impaired bactericidal activities of polymorphonuclear neutrophils (PMN) are associated with infections following surgery for esophageal cancer. Intracellular killing (KI), superoxide anion-producing capacity (SOP), and myeloperoxidase (MPO) activity were measured in 22 patients with esophageal cancer, 27 with gastric cancer, and 13 age-matched controls. The average level of KI was significantly depressed in patients with esophageal cancer or with gastric cancer, to a similar extent, compared to findings in controls, but SOP was not. In esophageal cancer patients, the SOP level was significantly higher in those with postoperative septic complications than in those without such problems, whereas the KI level was depressed to a similar extent in both. Therefore, a depression of KI with elevation of SOP of PMN may serve to predict complications of infection following surgery in patients with esophageal cancer.
Adult↗
Factors contributing to deficiencies in cell-mediated immunity in esophageal cancer patients.
Based on the previous data which indicated a preoperative decrease in cell-mediated immunity (CMI) is associated with the occurrence of infectious complications following surgery on patients with esophageal cancer, we examined possible factors contributing to a decrease in CMI levels. A multiple linear regression analysis was made on data from 76 patients with esophageal cancer and 53 with gastric cancer as the control. In patients with esophageal cancer, both protein-calorie malnutrition (PCM) and age factor contributed to a decrease in CMI, although the contribution of the latter was weak while the stage of the cancer and the grade of dysphagia showed no such contribution. The PCM and stage of the cancer were contributing factors in patients with gastric cancer. Thus, these results indicate that PCM and old age, and not the presence of malignant tumors, play a significant role in deficiency in CMI in patients with esophageal cancer.
Age Factors↗
Increases in immunoglobulin and complement in patients with esophageal or gastric cancer.
Based on data providing evidence that the enhancement of serum IgG and IgA is associated with the occurrence of infectious complications following surgery in patients with esophageal cancer, we examined the possible factors contributing to alterations in the serum IgG, IgA, IgM, C3, C4, and CH50 levels. A multiple linear regression analysis was made on data obtained from 71 patients with esophageal cancer and 57 with gastric cancer. In the patients with esophageal cancer, age and protein-calorie malnutrition (PCM) were related to the elevation of IgG levels while the stage of cancer was linked to that of IgA. The sex and IgM levels were also seen to be related. Age and the stage of cancer were associated with reductions in C3, C4, and CH50 levels, although in the patients with gastric cancer, the stage of cancer and elevations of these complement levels were related. Thus, age, PCM, and tumor malignancy are all factors related to the enhancement of IgG or IgA in patients with esophageal cancer.
Adenocarcinoma↗