Biomedical subjects

C C Liu

Publications and source records attributed to C C Liu.

At least 217 records · Page 12Linked to original sources

PubMed1990

Cytotoxic mechanisms of murine lymphokine-activated killer cells: functional and biochemical characterization of homogeneous populations of spleen LAK cells.

A highly purified population of murine lymphokine-activated killer (LAK) cells was obtained by selecting plastic-adherent splenocytes after incubation in high doses of recombinant IL-2. The population obtained was shown to be more than 95% positive for the cell marker asialo-GM1, and negative for both Lyt-1 (CD5) and Lyt-2 (CD8). The cells presented typical large granular lymphocyte morphology, and killed NK-susceptible target cells in an exclusively calcium-dependent fashion. A target cell DNA fragmentation activity of LAK cells could be detected even before target cell death. The presence of Hanukkah Factor/granzyme A/serine esterase 1, CTLA-1/granzyme B/serine esterase 2, and pore-forming protein (PFP/perforin) in these LAK cells was demonstrated by Northern blot analysis, suggesting that these markers are not exclusively associated with cytotoxic T lymphocytes. On immunoblots, antibodies specific for a lymphocyte PFP/perforin reacted with a 70-kDa protein of LAK cells. PFP/perforin was localized by immunofluorescence to the cell granules. A 50-kDa protein antigenically related to the macrophage cytokine tumor necrosis factor (TNF) was detected by immunoblotting and localized by immunofluorescence to both the cell granules and the cytosol. No RNA for TNF, however, could be detected using TNF-specific probes, suggesting that LAK cells may contain a cytotoxic factor which is related to, but distinct from, TNF. The work presented here demonstrates that cytotoxic mediators identified in cell lines are also present in primary cell cultures.

Animals↗

PubMed1990

Anti-idiotypic antibodies derived against C8, C9 and perforin bind homologous restriction factor.

A pore-forming protein (PFP/perforin/cytolysin), stored in the cytoplasmic granules of cytolytic lymphocytes, lyses a variety of target cells but not the cytolytic lymphocytes. In the complement (C) system, a C8-binding protein (C8bp) or homologous restriction factor (HRF) has been described that protects cells against lysis mediated by homologous C. C8bp/HRF is known to bind to C8 and C9 and has also been suggested to protect lymphocytes against perforin-mediated lysis. Here, using an anti-idiotypic antibody approach, several polyclonal antisera were raised against IgGs that are specific for mouse perforin, and human C8 and C9. These anti-idiotypic antisera were shown to react against an overlapping epitope(s) on C8bp/HRF as indicated by the following evidence: (i) all three types of antisera reacted against partially purified C8bp/HRF and against a 65 kDa protein band in cell lysates; reactivity was only observed against disulfide-reduced antigens; (ii) the three antibodies react with a protein band in normal erythrocytes (E) but not with type III E of patients with paroxysmal nocturnal hemoglobinuria or with a mutant B lymphoblastoid cell line, both of which cell types are known to be deficient in C8bp/HRF; and (iii) the three antibodies compete with each other for binding to C8bp/HRF. Type III E and the C8bp/HRF-deficient mutant lymphoblastoid cell line, however, are as susceptible to perforin-mediated lysis as type I E and wild-type lymphoblastoid cell line, respectively, indicating that C8bp/HRF does not play a role in protecting cells against perforin-mediated lysis. These paradoxical findings suggest that perforin may share with C8 and C9 the same domain(s) that bind to C8bp/HRF and yet, unlike C8 and C9, perforin is not inactivated by this type of putative interaction. Since C8 and C9 are now readily available, the anti-idiotypic approach described here provides a convenient protocol for production of antisera specific for C8bp/HRF.

Animals↗

PubMed1990

Induction of perforin and serine esterases in a murine cytotoxic T lymphocyte clone.

The expression of perforin and serine esterase (SE) activities and genes was examined in a murine cytotoxic T lymphocyte line (R8i) that does not require exogenous IL-2 for proliferation. Although perforin (hemolytic) activity was detected in unstimulated R8i, it was induced 2- to 14-fold in the presence of IL-2, IL-3, IL-4, and IL-6, and to a lesser degree (less than 4-fold) by TNF and IFN-gamma. A transient induction was also observed at the mRNA level. Peak perforin protein and mRNA levels were reached within 24 h and started to decline 48 h after stimulation. A trypsinlike SE activity which cleaves the chromogenic substrate N, alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester was also induced 2- to 4-fold in the presence of the various IL tested. At the mRNA level, the message for SE SE1/granzyme A/Hanukah factor was absent from R8i whereas SE2/granzyme B/CTLA-1 increased by greater than 3-fold in the presence of IL-2, IL-3, IL-4, and IL-6 and occurred with the same kinetics and pattern as perforin. The induction response occurred without any enhancement of cell proliferation, suggesting that the cytokines tested may provide a direct differentiation signal to CTL. The induction response was abrogated effectively by inhibitors of protein (cycloheximide or emetine) and RNA (actinomycin D) syntheses. These findings suggest that the various IL may provide both a growth signal and a differentiation signal to CTL, resulting in the direct activation of perforin and SE genes.

Animals↗

PubMed1990

Human parathyroid hormone-(1-34) prevents bone loss and augments bone formation in sexually mature ovariectomized rats.

A group of 3-month-old Sprague-Dawley rats were sham operated or ovariectomized and given daily injections of human PTH-(1-34) (8 or 16 micrograms per 100 g body weight) for 5 weeks. At the termination of the study histomorphometric techniques were used to examine changes in cortical and cancellous bone in the diaphysis and proximal metaphysis of the tibia. Ovariectomy resulted in a 50% decrease in cancellous bone that was accompanied by a 41 and 120% increase in osteoclasts and osteoblasts, respectively. In contrast, in the ovariectomized animals treated with PTH, the metaphyseal cancellous bone increased by over 300% to a level in excess of that present in the sham-operated control animals. The increase in cancellous bone induced by PTH was associated with an over 70% increase in osteoblasts and tetracycline-labeled area and an unexpected decrease in trabecular osteoclasts. In the tibial diaphysis PTH also decreased endosteal osteoclasts and at the same time increased osteoblast size and number as well as endosteal and periosteal bone formation; ovariectomy increased only periosteal bone formation. Our findings demonstrate that intermittent administration of PTH prevents ovariectomy-induced bone loss and augments cancellous and cortical bone formation in sexually mature ovariectomized rats. Although the basis of the bone anabolic action of PTH remains elusive, our data indicate that it may involve the uncoupling of bone formation and resorption such that the latter is inhibited as bone formation is enhanced. Our findings are also compatible with the view that intermittent administration of PTH increases bone mass, in part by stimulating the proliferation and differentiation of osteoblast progenitors while inhibiting osteoclast proliferation.

Animals↗

PubMed1990

Expression of mRNAs for pore-forming protein and two serine esterases in murine primary and cloned effector lymphocytes.

The cDNAs encoding several proteins present in the granules of cytolytic effector lymphocytes have now been cloned. These include the cytolytic pore-forming protein (PFP) or perforin, and at least six serine esterases (SE), also called granzymes. The cDNA probes for PFP, SE-1, and SE-2 are used here to study the expression of these proteins in murine primary effector lymphocytes. Among the stimuli effective in inducing the expression of PFP, SE-1, and SE-2 were recombinant interleukin-2, the lectin concanavalin A in the presence of phorbol esters, and allogeneic cells in mixed lymphocyte cultures. Some correlation was seen between the levels of PFP and SE mRNAs and cytotoxicity measured in a standard 51Cr release assay. We also examined a panel of 13 cloned cytotoxic T lymphocyte (CTL) lines and found that mRNAs for PFP and SE-2 were expressed in all CTL lines, including some that were previously considered not to produce PFP. Twelve of the 13 CTL lines also proved to possess the mRNA for SE-1. One thymoma cell line, TIMI.4, did not express mRNA for PFP, although it expressed mRNA for SE-1 and SE-2.

Animals↗

PubMed1990

Limited Multiplication of Symbiotic Cyanobacteria of Azolla spp. on Artificial Media.

We examined various media and conditions to isolate symbiotic cyanobacteria from the leaf cavities of Azolla spp. Cyanobacteria survived and multiplied to a limited extent on a medium with fructose, Casamino Acids, yeast extract, and NaNO(3) under 1% O(2). These cyanobacteria were antigenically identical to the endosymbionts.

Journal Article↗

PubMed1990

How lymphocytes kill.

Cytotoxic T lymphocytes and natural killer cells are potent killers of target cells. These lymphocytes have large cytoplasmic granules containing cytotoxic peptides and other factors. Several of these molecules have been isolated and their functions elucidated. These molecules may be directly involved in the killing of virus-infected and transformed cells as well as in the development of cell-mediated autoimmune disorders.

Animals↗

PubMed1990

[Acute hemodynamic effects of upper airway obstruction in normal dogs].

Eight normal dogs were anesthetized to assess the effects of an upper airway obstruction (UAO) on arterial blood gases (ABG) and hemodynamic (HD) parameters. Each dog was fitted with an arterial line, a Swan-Ganz catheter and an endotracheal tube. The HD parameters including heart rate, systolic blood pressure, central venous pressure (CVP), cardiac index (CI), pulmonary capillary wedge pressure (PCWP), systemic vascular resistance index (SVRI), pulmonary vascular resistance index (PVRI), left ventricle stroke work index (LVSWI) and right ventricle stroke work index (RVSWI), were monitored. The baseline ABG and HD parameters were taken prior to endotracheal tube clamping, and then checked at 0.3, 1.5, 3.0, 4.0 and 5.0 minutes. The clamp was subsequently removed to allow spontaneous breathing, and then another set of measurements were taken at 5, 15 and 30 minutes, respectively. The above procedures were then repeated a second time. The results showed that UAO can produce the following: (1) decreases in the CI, PaO2 and pH; (2) increases in the mean systemic and pulmonary arterial pressure, PCWP, SVRI, PVRI, LVSWI, RVSWI, and PaCO2; (3) significant and acute changes in PaO2 and pH, with the most significant changes occurring within 90 seconds after clamping and then reaching a plateau; and (4) repeated UAO increases in SVRI and PVRI. In conclusion, upper airway obstructions may possibly induce serious ABG and HD changes in humans, as it does in normal dogs.

Airway Obstruction↗

PubMed1990

Comparison of lower concentrations of lidocaine to suppress bucking before extubation during recovery of general anesthesia.

Thirty females, aged 25-50 years old, scheduled for abdominal total hysterectomy were randomly divided to two groups. For patients in group I, 2 mL 1% lidocaine solution was injected through the catheter on the tube to desensitize the trachea while surgeon started to close the peritoneum. In group II, 2 mL 2% lidocaine was used. The peak cuff pressures generated in the awakening group I patients were 49.0 +/- 2.5 cmH2 O (mean +/- SD) which shows greater than that in group II (27.9 +/- 7.2 cmH2 O), p less than 0.01. Bucking before awakening was also evaluated clinically. One hundred % (15/15) in group I patients bucked compared with 13.3% (2/15) in group II patients (p less than 0.01). Gag reflex was preserved in all patients and none suffered from aspiration postoperatively. Intratracheal administration of two mL 2% lidocaine through the catheter of modified endotracheal tube showed significant effect for suppressing bucking during recovery of general anesthesia.

Adult↗

PubMed1990

Red cell distribution width in the detection of iron deficiency in maintenance hemodialysis patients.

Iron deficiency is a frequent complication in maintenance hemodialysis patients. Red cell distribution width (RDW) was recently used as an indicator in early detection of iron deficiency. One hundred and thirty nine patients maintained on hemodialysis were studied for the usefulness of RDW in detection of iron deficiency. Serum ferritin less than 30 ug/dl was defined as iron deficiency in these patients. Another 69 patients with iron deficiency not entering hemodialysis were chosen as a control group. The sensitivity of RDW elevation to detect iron deficiency in chronic hemodialysis patients was only 36.7% which was much lower than that of the control group (62.3%). However, it was still more sensitive than microcytosis in the detection of iron deficiency in chronic hemodialysis patients because none of them showed microcytosis. The specificity and positive predictive value of RDW elevation in respect to iron deficiency were 62.4% and 9.6% respectively. This study demonstrated that the use of RDW in the detection of iron deficiency in chronic hemodialysis patients was superior to mean corpuscular volume but still limited.

Erythrocyte Count↗

PubMed1989

In vivo conversion of gamma-aminobutyric acid and 1,4-butanediol to gamma-hydroxybutyric acid in rat brain. Studies using stable isotopes.

The formation of 4-[1,4-13C]hydroxybutyric acid ([13C]gamma-hydroxybutyric acid; [13C]GHB) in rat brain was studied following intracerebroventricular (i.c.v.) administration of either 4-[1,4-13C]aminobutyric acid ([13C]GABA or 1,4-[1,4-13C]butanediol ([13C]1,4-BD) to awake, freely moving animals. GHB and [13C]GHB were measured with a gas chromatographic mass spectrometric (GC/MS) technique designed to detect the lactone derivative of GHB with the acid or lactone being determined by conditions of tissue extraction. [13C]GHB was detected following i.c.v. administration of [13C]GABA with a turnover rate of 2.04 nmol/g tissue/hr and [13C]1,4-BD with a turnover rate of 1.4 nmol/g/hr. The formation of [13C]GHB from [13C]GABA was blocked by an inhibitor of GABA-transaminase, but this drug had no effect on the formation of [13C]GHB from [13C]1,4-BD. The latter pathway was also unaffected by alcohol dehydrogenase inhibitors, compounds which block this pathway in the periphery. Further, in the course of these experiments, naturally occurring endogenous gamma-butyrolactone (GBL) was detected in rat brain in a concentration of 200 pmol/g tissue weight, but lactonization in vivo of [13C]GHB formed from either labeled GABA or 1,4-BD was not demonstrated. These data confirm two separate pathways of synthesis for GHB in brain, demonstrate the presence of GBL in brain, and illustrate the utility of a new GC/MS technique for analysis of GHB and for GBL which does not involve extensive derivatization.

Animals↗

PubMed1989

Purification of a ubiquitin protein peptidase from yeast with efficient in vitro assays.

In eukaryotic cells ubiquitin is synthesized as a polyubiquitin protein or as a protein fused at the carboxyl terminus to other polypeptides. An enzyme activity, ubiquitin protein peptidase, has been proposed to process these precursors by cleaving the peptide bond between adjoining ubiquitin molecules or between ubiquitin and the fused peptides. Using the cleavage of a 35S-labeled yeast ubiquitin protein fused to a synthetic 38-residue peptide obtained by in vivo metabolic labeling in Escherichia coli in an expression system based on the interaction of bacteriophage T7 RNA polymerase and its promoter, it is possible to detect a processing activity in soluble yeast extract. The specificity of the cleavage suggests this activity could be the in vivo processing activity for various ubiquitin precursor proteins in yeast cells. A similarly labeled ubiquitin protein fused to one cysteine residue was also utilized to detect an activity capable of removing a single cysteine residue from ubiquitin in a soluble extract. Employing assays based on the cleavage of labeled ubiquitin protein fusions, a ubiquitin protein peptidase activity from Saccharomyces cerevisiae was purified about 15,000-fold to yield a protein mixture consisting of only a few protein species. The major protein band which comigrated with the activities in in vitro assays has an apparent molecular weight of 29,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two other protein species, about 20,000 and 10,000 in molecular weight, also comigrated with the in vitro activities throughout the purification procedure. Though our most purified protein fraction was shown to cleave various artificial ubiquitin protein fusions under our experimental conditions, it cannot cleave a ubiquitin dimer protein, suggesting the existence of functionally distinct ubiquitin protein peptidases. Our experimental protocol for preparing various labeled ubiquitin protein precursors provides a means to explore various processing enzymes existing in cells. The same protocol may also be adapted to prepare substrates for the study of other specific protein processing enzymes.

Amino Acid Sequence↗

PubMed1989

Perforin and serine esterase gene expression in stimulated human T cells. Kinetics, mitogen requirements, and effects of cyclosporin A.

A pore-forming protein (PFP; perforin) and various serine esterases (SE) have been identified in the cytoplasmic granules of CTL and NK cells. Perforin and several SE have recently been cloned. Northern blotting analysis was performed here using cDNA probes specific for human perforin and two SE (SE 1/HS and SE 2/GB) to monitor the levels of specific mRNAs in mitogen-stimulated primary human T cells. These mRNAs were rapidly induced by IL-2 with optimal responses at 300 U/ml. After IL-2 treatment, mRNAs for perforin, SE 1, and SE 2 peaked at 12-24 h and decreased after 48 h. The three mRNAs were also induced in T cells treated with a combination of PMA plus lectin, OKT3 mAb, or plastic-adherent accessory cells. However, the induction induced by PMA/mitogen followed a slower kinetics, peaking at 48 h. In general, we found that SE 1 mRNA was more readily induced by IL-2, while SE 2 responded better to PMA/mitogen. Similar patterns of mRNA expression were observed for both unprimed T cells and PHA-primed T blasts. After stimulation with IL-2 and PMA/mitogen, the T8+ subset was shown to be the main producer of perforin, SE 1, and SE 2. Low levels of all three mRNAs, however, were also detected in the T4+ subset. The induction of all three mRNAs by either IL-2 or PMA/mitogen was partially blocked by the immunosuppressive drug cyclosporin A (CsA), but not by the biologically inactive analogue cyclosporin H. Together, these results point to some similarities and differences with upregulation of granule mediator mRNAs relative to lymphokine mRNAs. Both sets of genes require two signals for their induction by mitogens. In contrast to lymphokines, there is a strong response of granule mRNAs to IL-2, and the induction of these transcripts is only partially blocked by CsA.

Cyclosporins↗

PubMed1989

Heterogeneity of granules of murine cytolytic T lymphocytes. Isolation of a homogeneous population of dense granules.

A separation procedure based on Percoll gradient centrifugation is described here that allows enrichment for two main populations of granules, with average densities of 1.05 and 1.1 g/ml. The heavy-density granules, which comprise 5-20% of the total amount of membrane-lytic activity, are morphologically and biochemically more homogeneous than the light-density granules, which are contaminated with other subcellular organelles. A trypsin-like serine esterase activity, used in the past as a granule marker, was mainly found in the free cytosol and in the light-density fractions, while a small portion of this activity was associated with the heavy-density fractions. This enzyme marker is therefore not exclusively associated with granules and should not be used as the only granule marker.

Animals↗

PubMed1989

Purified perforin induces target cell lysis but not DNA fragmentation.

Rapid and extensive target cell DNA fragmentation is a unique characteristic of CTL-mediated killing. We studied the role of the granule pore-forming protein (PFP/perforin/cytolysin) of CTL in mediating lysis and DNA fragmentation of target cells. Perforin was isolated from murine CTL by sequential application of perforin-enriched granule fractions to four chromatographic columns: DEAE-Sepharose, Q-Sepharose, Polyanion SI, and Superose 12. Purified perforin was eluted as a single band of 70 kD in SDS-PAGE. While purified perforin produced potent lysis of a variety of target cells tested, it did not induce any measurable amount of DNA fragmentation. In parallel experiments, intact CTL produced marked DNA fragmentation of the same target cell populations. Our results suggest that perforin alone is not responsible for the DNA fragmentation observed during CTL-mediated killing and that other, as yet unknown, mediators or mechanisms are likely to be involved in the induction of target cell nuclear damage.

Animals↗

PubMed1989

Resistance of cytolytic lymphocytes to perforin-mediated killing. Induction of resistance correlates with increase in cytotoxicity.

CTL and NK cells cultured in vitro are known to produce a cytolytic pore-forming protein (PFP, perforin) localized in their cytoplasmic granules. Using purified perforin, we showed here that both cloned CTL and primary killer cell populations, including allospecific CTL, NK/lymphokine-activated killer cells, and MHC-non-restricted CTL, were more resistant to perforin-mediated killing than other lymphocyte populations and cell types. Similar results were obtained with both murine and human cytolytic lymphocyte populations. Resistance of killer cells to perforin correlated in general with their cytolytic capability. Thus, cells that have acquired competence to kill after stimulation with Con A, IL-2, or leukocyte-conditioned medium, were also the more resistant cells. IL-2-independent CTL lines and hybridomas derived in our laboratories could be triggered to become cytotoxic and perforin resistant by short-term stimulation with various cytokines, indicating that the acquisition of resistance to perforin-mediated lysis was independent of cell proliferation. Activation of one IL-2-independent CTL line with IL-2 also resulted in enhanced production of perforin and in enhanced serine esterase activity. The acquisition of cell resistance to perforin by these IL-2-independent cell lines after activation with stimulatory reagents was independent of protein and RNA neosynthesis: emetine, cycloheximide, and actinomycin D, while effectively blocking the incorporation of [35S]methionine into cell proteins, did not affect the induced increase in perforin resistance.

Animals↗

PubMed1989

The structure of the mouse lymphocyte pore-forming protein perforin.

Purified murine lymphocyte pore-forming protein (PFP or perforin) was partially sequenced. Oligonucleotides synthesized on the basis of this sequence information were used to screen a murine cytotoxic T lymphocyte (CTL) cDNA library. Seven clones were obtained, two of which were sequenced, providing full-length sequence information on PFP. Murine PFP (534 a.a.) is 68% identical to human PFP. Hydropathic analysis revealed a predominantly hydrophilic protein with some hydrophobic domains, including a region (a.a. 191-251) that could contain putative membrane-spanning domains. PFP is approx. 20% identical to human C7, C8 and C9 within a region encompassing 270 a.a., confirming previous immunological cross-reactivity studies. Northern blot analysis showed that expression of PFP but not of a serine esterase transcript is enhanced in a CTL line by antigen receptor-stimulation. Southern blot analysis of mouse genomic DNA indicated that PFP is encoded as a single-copy gene with the coding region contained within 10 kilobases of genomic DNA.

Amino Acid Sequence↗

PubMed1989

The lymphocyte pore-forming protein perforin is associated with granules by a pH-dependent mechanism.

A pore-forming protein (PFP, perforin or cytolysin) has been found in the cytoplasmic granules of cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. Extraction of granules with high-salt buffers or by freezing-and-thawing results in the release of perforin, which occurs only when the buffer pH is above 7.0. While high-salt extraction and freezing-and-thawing of granules at low pH (below 7.0) do not result in perforin release, these treatments render granules susceptible to a subsequent incubation with low-salt buffers (pH 7-8) that then solubilizes perforin completely. Granules may thus have been made leaky by high-salt extraction or freezing-and-thawing that may occur regardless of the buffer pH, while dissociation of perforin from granules may be exquisitely pH-sensitive. Freezing-and-thawing intact CTL and NK cells in physiological buffers with pH in the range of 7-8 (but not below 7) also causes release of perforin activity to the cell supernatant, thus providing a simple procedure by which perforin activity can be quantitated in small cell samples. Our results suggest that during lymphocyte-mediated killing, the extracellularly released perforin may rapidly dissociate from granules as a result of pH change and, in the process, become cytolytically active.

Buffers↗