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C C Liu

Publications and source records attributed to C C Liu.

At least 235 records · Page 13Linked to original sources
PubMed1989

Identification and characterization of a membrane-bound cytotoxin of murine cytolytic lymphocytes that is related to tumor necrosis factor/cachectin.

Cytotoxic T lymphocytes (CTLs) kill their targets by a contact-dependent mechanism. We investigated the possibility that the CTL membranes themselves could exert direct cytotoxic activity. Murine CTLs that had been fixed with paraformaldehyde retained a slow cytotoxic activity toward various target cells that are also sensitive to another cytokine, tumor necrosis factor (TNF)/cachectin. This cytotoxic activity was neutralized by antibodies specific for TNF. Membrane fractions obtained from CTLs were cytotoxic to TNF-sensitive targets but not to several TNF-resistant cell lines. Immunoblot analysis revealed a membrane protein band of 50-60 kDa from CTLs that reacts with anti-TNF antibodies. The surface localization of this cytokine was further ascertained by flow cytometry, indirect immunofluorescence, and immunoelectron microscopy studies using TNF-specific antibodies. Radioiodination of CTL surface proteins followed by immunoprecipitation with anti-TNF antibodies confirmed the presence of a TNF-related cytokine in the plasma membranes of CTLs that migrated with an apparent molecular mass of 50-60 kDa under disulfide-reducing conditions. This cytokine can be removed from membranes by treatment with detergents but not with high-salt buffers, suggesting that it may be an integral membrane protein.

Animals↗
PubMed1989

Purification and Characterization of Pectinmethylesterase from Ficus awkeotsang Makino Achenes.

Pectinmethylesterase from the pericarp of jelly fig (Ficus awkeotsang) achenes was extracted and purified to a specific activity of 289 micromole proton produced per minute per milligram protein. Pectinmethylesterase, a major protein with high specific activity in the crude extract, was monomeric with a molecular weight of 38,000. The enzyme preparation was stable in distilled water at 4 degrees C for at least 6 months, and at 60 degrees C for at least 10 minutes. This enzyme functioned optimally at pH 6.5 to 7.5 when the assay mixture contained no NaCl or at low NaCl concentration. The pH optimum shifted to lower pH as the NaCl concentration was increased. The K(m) value for pectin was 0.75 milligram per milliliter pectin, corresponding to a V(max) value of 310 micromoles per minute per milligram protein. Inhibition studies with antibodies indicated that jelly fig achene pectinmethylesterase and the two other pectinmethylesterases from orange and tomato were similar in their active site conformation; however, the surface determinants may be very different because no precipitation between anti-jelly fig pectinmethylesterase immune serum and the pectin methylesterase from orange and tomato could be observed in the double immunodiffusion analysis. Specific antisera raised against jelly fig achene pectinmethylesterase in a Western blot experiment also showed low similarity between jelly fig pectinmethylesterase with that from orange and tomato. This observation was also supported by the very low isoelectric point (pH 3.5) of jelly fig pectinmethylesterase, compared with high isoelectric points reported for most of the pectinmethylesterases. Amino acid composition and N-terminal sequence have been obtained. High homology of the N-terminal amino acid residues between jelly fig and tomato pectinmethylesterase (O Markovic, H Jornvall [1986] Eur J Biochem 158: 455-462) was observed. Pectinmethylesterase activity causes the release of protons from the deesterification of pectin such that a low pH environment is created, and this may be related to the cell growth. Pectinmethylesterase is not needed for jelly fig seed germination, however the gel formed from pectin and pectinmethylesterase may insure a water source for the germinating jelly fig seeds.

Journal Article↗
PubMed1989

The aged rat model of ovarian hormone deficiency bone loss.

Three studies were carried out. First, the effects of aging on the maturation of the female skeleton were assessed. Second, the hypothesis that has linked ovarian hormone deficiency bone loss to hypercalcemic suppression of the parathyroids leading to a decrease in 1,25-dihydroxyvitamin D synthesis and gut absorption of calcium was examined. Third, the effects of ovariectomy and a combination of ovarian hormone deficiency and low dietary calcium on bone and the calcium-regulating hormones were evaluated. After 6 months, ovariectomy and a low calcium diet independently decreased the density of the ilium, the femur, and the fourth lumbar vertebra as well as the calcium content of the latter two. The effects of the two treatment regimens were additive and more marked in the vertebral bone. Ovariectomy lowered serum calcitonin only in animals fed a normal diet and had no effect on serum PTH and vitamin D metabolites, while a low calcium diet caused a significant increase in serum 1,25-dihydroxyvitamin D. In both dietary regimens ovariectomy resulted in about a 30% decrease in intestinal calcium absorption. A low calcium diet increased morphometric indices of bone formation and bone resorption as did ovariectomy, with resorption exceeding formation. The discussion of our findings led to the conclusion that the aged rat model of ovarian hormone deficiency bone loss qualifies for serious consideration as a practical convenient cost-effective animal model for exploring aspects of the pathogenesis and treatment of postmenopausal bone loss.

Aging↗
PubMed1989

Thyrotropin-releasing hormone gene expression in normal thyroid parafollicular cells.

The rat TRH gene encodes a 255-amino-acid precursor polypeptide, preproTRH, containing five copies of TRH and seven non-TRH peptides. Expression of this gene is well documented in the central nervous system, particularly in the hypothalamus. Thyroids also contain TRH immunoreactivity, but it is unknown whether this immunoreactivity results from expression of the TRH gene or from other genes encoding TRH-like products. Since the CA77 neoplastic parafollicular cell line expresses the TRH gene, we investigated whether TRH gene expression also occurs in normal thyroid parafollicular cells. Northern analysis of total thyroid RNA with a preproTRH-specific RNA probe identified a single hybridizing band the same size as authentic TRH mRNA found in hypothalamus and CA77 cells. Gel filtration analysis of thyroid extracts identified the same 7-kilodalton and 3-kilodalton species of immunoreactive preproTRH53-74 previously identified in hypothalamus and CA77 cells. Immunoreactive preproTRH115-151, not previously identified, was found in all three tissues. Part of this immunoreactivity comigrated with the synthetic preproTRH115-151 standard on gel filtration and reversed-phase HPLC. PreproTRH53-74 was localized to thyroid parafollicular cells by immunostaining. These findings demonstrate authentic TRH gene expression by normal rat thyroid parafollicular cells and establish the CA77 cell line as the only model system of a normal TRH-producing tissue. In addition to expanding the range of neuroendocrine peptides known to be produced by parafollicular cells, these results also suggest a potential paracrine regulatory role for TRH gene products within the thyroid.

Animals↗
PubMed1989

Parathyroid hormone potentiates the effect of insulin-like growth factor-I on bone formation.

Insulin-like growth factor-I and parathyroid hormone are both known regulators of bone formation. In this study, human recombinant IGF-I and bovine PTH (1-34) and their combination were studied for their effects in vitro on the proliferation of embryonic chick osteoblast-like cells (osteoblasts) and in vivo on bone formation in normal rats. Osteoblasts from 17-day-old chick embryos were cultured in serum-free BGJb medium containing 0.1% bovine albumin. After 2 days, IGF-I and/or PTH were added. Twenty-four hours later [3H]thymidine incorporation into trichloroacetic acid precipitable material was quantified as an index of cell proliferation. This has previously been shown to reflect actual cell division. IGF-I at doses ranging from 0.85 to 13.6 nmol/l caused a dose-dependent increase in [3H]thymidine incorporation into osteoblasts. PTH alone (10 to 1000 pmol/l) had no significant effect. However, when combined with IGF-I, PTH potentiated the mitogenic effect of IGF-I and achieved statistical significance at 30 and 100 pmol/l (p less than 0.05). This potentiation was also studied in vivo. The right hind-limbs of rats weighing 150 g were infused intra-arterially by an osmotic minipump with graded doses of IGF-I (0.1 to 0.4 nmol/day) and/or PTH (0.27 nmol/day) for 7 days. The rate of trabecular bone apposition (formation) was measured by double tetracycline labelling and compared with the contralateral uninfused limb which acted as the control. Histomorphometric data revealed that neither IGF-I nor PTH alone had a significant effect on trabecular bone apposition rate compared with control limbs.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Structural and metabolic integrity of sheep external intercostal muscle during extended culture.

The objectives of this study were to examine the structural and metabolic integrity of isolated sheep external intercostal muscle bundles following variable lengths of preincubation (0 to 192 h). Samples of intact external intercostal muscle (10 to 15 g), with tendons attached, were prepared from growing wethers and maintained at their resting lengths during preincubation for 0 to 192 h. Protein synthesis (PS), protein degradation (PD), acetate oxidation and ultrastructural integrity of muscle samples were examined at 0 to 192 h, 0 to 96 h, 0 to 48 h and 0 to 96 h following isolation, respectively. Additionally, the effects of variable fetal calf serum (FCS) concentrations (0 to 20%; w/v) on PS and PD and acetate oxidation were examined. Rate of PS increased as preincubation time increased to 192 h; however, most of this increase was due to the proliferation of fibroblasts on the surface of the muscle sample. Addition of cytosine arabinoside to the incubation media prevented the fibroblast-dependent increase in PS; however, it did not entirely prevent the preincubation time-dependent increase in PS. Rate of PD increased greatly upon preincubation. The nitrogen balance of incubated muscles was negative at all times examined. Acetate oxidation was maintained through 12 h of preincubation and thereafter declined. Relatively normal myofibrillar structure was maintained through 48 h of preincubation; however, loss of mitochondrial integrity and dissolution of Z-disks at 48 h and at 96 h of preincubation were evident. Isolated tissues were able to respond to FCS concentration in medium following 48 h of preincubation.

Animals↗
PubMed1989

In vivo calvarial bone cell responses to dietary perturbations and the implications for mineral homeostasis.

Calvariae from small animals have been an important source for in vitro studies of bone. However, few in vivo studies have been undertaken on quantitative cell changes in calvariae. In the present study of mineral perturbations, rats were first deprived of calcium. After 18 days endosteal osteoclasts and nuclei/osteoclast in the parietal bone had increased 120% (P less than 0.001) and 26% (P less than 0.001), respectively, the marrow space had increased 141% (P less than 0.001), and the bone area experienced a 49% decrease (P less than 0.001). This thinning and weakening of the calvaria was accompanied by a compensatory increase in the number of endosteal osteoblasts (297%, P less than 0.001). These rats were then replenished with calcium, and after 14 days the number of endosteal osteoclasts had decreased to 86% (P less than 0.001) below the control and the endosteal surface was almost completely covered by osteoblasts (866% above the control, P less than 0.001). Bone area was increased by 51% (P less than 0.01). Similarly, in calcium-deficient rats in the tibial diaphysis at the fibular junction, the number of endosteal osteoclasts and the medullary space increased 1606% (P less than 0.001) and 63% (P less than 0.001), respectively, which were accompanied by a 32% decrease (P less than 0.001) in cortical bone area. After calcium replenishment, most endosteal osteoclasts in the tibial diaphysis disappeared from the endosteal surface and were replaced by osteoblasts (increased 487%, P less than 0.001). These results indicate that changes in bone cell activity in response to calcium deficiency are similar in calvariae and long bones, and that mobilization of calcium from the calvaria during calcium deficiency occurs at the expense of the protective action of the calvaria. Therefore, long bones as well as membranous bones are apparently important for the maintenance of mineral homeostasis.

Animals↗
PubMed1988

Resistance of cytolytic lymphocytes to perforin-mediated killing. Lack of correlation with complement-associated homologous species restriction.

CTL and NK cells resist self-mediated killing and lysis by their own pore-forming protein (PFP; perforin). Perforin, like C, lyses RBC. Efficient C-mediated lysis of RBC occurs when both C and RBC are from different species (homologous species restriction). A protective surface protein (C8-binding protein, homologous restriction factor) has been reported to mediate both homologous species restriction in C-dependent cytolysis and protection of some target cells against perforin-induced lysis. We show here that perforin, unlike C, lyses target cells across a variety of species, including the homologous one, while the same target cell populations resist the attack by homologous C. Perforin-containing extracts of CTL and LAK/NK cells from three species (rat, mouse, and human) and purified mouse perforin were tested against RBC from 10 different species, several nucleated target cell lines, and one primary cell population (thymocytes). While resisting lysis by homologous C, most of these cell types were lysed effectively by perforin without any homologous restriction pattern. CTL and NK cells, like other nucleated targets, are resistant to lysis by homologous but not heterologous C; however, these cell types are resistant to both homologous and heterologous perforin. Together, our results suggest that the protective mechanisms associated with C- and perforin-mediated lysis are distinct.

Animals↗
PubMed1988

A semi-automated microassay for complement activity.

A simple, automated microassay for the serum complement-dependent hemolytic activity is described here. In contrast to the traditional titration hemolysis assay, the new method depends on a single experimental step using a fixed volume of serum specimen and sheep erythrocytes. The assay is based on the change in light scattering properties of erythrocytes upon hemolysis. It relies on the spectrophotometric reading of microtiter well samples at 700 nm by using a microplate reader. The measured absorbance correlates proportionally with the extent of hemolysis. A good correlation between the results obtained using this technique and those obtained by the traditional CH50 titration method is observed. This simple procedure can be applied to the rapid, semi-quantitative diagnostic screening of complement activities of a large number of serum specimens.

Animals↗
PubMed1988

Cloned cytotoxic T lymphocytes as target cells. II. Polarity of lysis revisited.

The original polarity of lysis experiments suggested that CTL are themselves sensitive to whatever mechanism it is that CTL use to lyse their targets. This concept has placed certain limitations on possible mechanisms of lysis by CTL. Recently, we found in studies with cloned CTL as targets that cloned CTL are in fact highly resistant to lysis by other CTL, as well as to their cytotoxic granule proteins. We show here that although cloned CTL are extremely resistant to lysis by primary and cloned CTL, they are readily inactivated functionally by all primary CTL and by at least one CTL clone. Moreover, cloned CTL are also functionally inactivated by cytotoxic granule proteins. The activation of CTL, which we call inhibitin, is Ca2+ insensitive and distinct from hemolytic activity, and is, thus, unlikely to be perforin. These experiments suggest a possible alternative interpretation of the original polarity of lysis experiments.

Animals↗
PubMed1988

Perforin-dependent and -independent pathways of cytotoxicity mediated by lymphocytes.

There is little doubt at the present time that both perforin-dependent and -independent pathways are important in mediating the cytotoxicity associated with lymphocytes. The cell distribution of perforin, initially thought to include both CTL and NK cells, now must be viewed with caution because all previous biochemical studies on CTL have been conducted with cell lines propagated in long-term cultures in the presence of T cell growth factors (IL-2 and perhaps some still undefined factors). Under these conditions, CTL are known to assume a broader, NK-like specificity in target cell killing and may thus differ significantly from primary CTL generated in the body. Accordingly, perforin does not seem to be present in primary CTL activated directly through mixed lymphocyte reactions. It remains to be shown how primary CTL lyse target cells in vivo. Initial studies conducted in several laboratories have already provided some clues. It now seems that even in cultured, perforin-containing CTL, the perforin pathway is not an obligatory mechanism required for target cell killing. Other pathways, possibly involving TNF/lymphotoxin-like molecules, may play a direct role in this type of cytotoxicity. Other still unidentified factors now also need to be sought, including membrane polypeptides that may develop cytotoxicity directly upon cell contact and binding. Although from the studies reviewed here it is clear now that perforin has a more limited role in cell killing than originally proposed, it is still intriguing that it should share structural and functional homologies with complement proteins, drawing paradoxical analogies between two systems (the cellular and the humoral immune systems) which have evolved to become specialized to carry out separate immunological tasks. The cloning of the genes for perforin and for all the C proteins that comprise the MAC should reveal important information on how these genes originated and then diverged during evolution. The cellular distribution of other granule products, such as serine esterases, also must be viewed with caution. A serine esterase activity was initially thought to be CTL-specific. This information stimulated an intensive research activity in many laboratories that resulted in both the purification of a serine esterase family and the cloning of several serine esterase transcripts. It is becoming clear from recent evidence that this group of enzymes is not truly CTL-specific and therefore would not be expected to develop any function rendered absolutely necessary for cytolysis.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

Molecular mechanisms of lymphocyte-mediated killing.

Lymphocytes kill tumor cells and other target cells by a contact-dependent mechanism. Killer lymphocytes acquire cytoplasmic granules upon lymphokine stimulation and these granules in turn contain potent cytotoxic mediators, some of which have been characterized recently. These include (i) a pore-forming protein (perforin) of 70 kDa that polymerizes into transmembrane tubules in the presence of calcium and that is structurally related to the terminal components (C5b-6, C7, C8 and C9) of the complement cascade, (ii) a cytokine related to tumor necrosis factor and lymphotoxin that causes DNA fragmentation in target cells, and (iii) a family of serine esterases presently without any known function. The relevance of these mediators in lymphocyte-mediated killing is currently being investigated. The identification and characterization of additional putative mediators of cytotoxicity in the future will undoubtedly provide us with a better understanding of the molecular basis of cell-mediated killing.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1987

A calcium- and perforin-independent pathway of killing mediated by murine cytolytic lymphocytes.

Cytotoxic T lymphocytes have been thought to lyse cellular targets in the past by a calcium-dependent pathway. This notion was recently supported by the identification and purification of a pore-forming protein (perforin) from the granules of these cell types. Here, we show that perforin is absent from a number of cell lines that nevertheless display vigorous cytolytic activity toward target cells. The cytotoxic activity of eight murine CTL lines is completely or partially retained in the absence of calcium. The calcium-independent lytic activity is associated with two subcellular fraction peaks isolated by Percoll gradient centrifugation, e.g., a heavy density band migrating with granule markers and a lighter band corresponding to free cytosolic material. These results suggest a complex picture of lymphocyte-mediated killing involving probably multiple mechanisms and mediators that may operate in concert or independently in the delivery of the lethal hit.

Animals↗
PubMed1987

Identification, isolation, and characterization of a novel cytotoxin in murine cytolytic lymphocytes.

Murine cytotoxic T lymphocytes contain, in addition to the cytotoxic pore-forming protein perforin, another cytolytic factor localized in both cytoplasm and granules. Like perforin, this CTL cytotoxin lyses a variety of tumor cells; unlike perforin, it is stable in the presence of calcium, requires several hours to induce maximal lytic activity, and is antigenically related to the previously described tumor necrosis factor (TNF) and lymphotoxin (LT). However, it differs from TNF and LT in a number of biochemical and functional properties. TNF- and LT-specific cDNA probes did not hybridize with any CTL-specific message, indicating that the CTL cytotoxin is distinct from those two factors. It has an apparent Mr of 50 and 70 kd under reducing and nonreducing conditions, respectively, is secreted by secretagogue-stimulated CTLs, and causes DNA fragmentation in several targets, a phenomenon previously attributed to target cell damage by CTLs. These results suggest that killing by lymphocytes may encompass multiple mechanisms and polypeptides.

Animals↗
PubMed1987

Resistance of cloned cytotoxic T lymphocytes to cell-mediated cytotoxicity.

Cloned CTLs show an unusually high resistance to lysis by effector CTLs. Several cloned CTL lines in our laboratories are absolutely refractory to lysis by other cloned CTLs, either (a) directly, (b) in the presence of lectin, or (c) by PMA-induced CTLs. They can be lysed to some extent by primary CTL, although they are less than 5% as sensitive as target cells normally used to assay primary CTL lytic activity. Lysis of cloned CTLs by primary CTL effector cells is not enhanced by the presence of lectin, and cloned T cells are also highly resistant to lysis by primary lymphokine-activated killer cells. Cloned CTLs are highly resistant to lysis by isolated CTL granules that contain the membranolytic pore-forming protein (PFP or perforin), while non-CTL targets are highly susceptible to granule-mediated killing, indicating that cloned CTLs resist lysis not only at the intact effector cell level but also when soluble effector proteins are used. This resistance mechanism may explain how CTLs kill but spare themselves from being killed during the cytolytic event.

Animals↗
PubMed1987

Production of granulocyte-stimulating and bone cell-modulating activities from a neutrophilia hypercalcemia-inducing murine mammary cancer cell line.

We have previously shown that a transplantable murine tumor (CE mammary carcinoma) causes marked neutrophilia and excessive bone resorption in vivo. In order to understand the humoral mechanism of these tumor-induced phenomena, we successfully established a cell line (CE 816) and subsequently cloned CE mammary carcinoma cells in serum-free culture conditions. Cultured CE tumor cells continued to induce neutrophilia and hypercalcemia when they were transplanted back into mice. Conditioned medium (CM) prepared from the CE 816 cell line and control non-neutrophilia-inducing tumor cells were evaluated for stimulation of neutrophilic colony formation, embryonic bone cell proliferation, and bone resorption in vitro assays. Both the CE 816 and control tumor CM demonstrated colony-stimulating activity, but the CE 816 CM stimulated more neutrophilic colonies than the control tumor at all experimental conditions examined. The CE 816 CM demonstrated bone-resorbing activity but not control tumor CM. Both types of CM stimulated proliferation of embryonic bone cells. Production of colony-stimulating and bone-mitogenic activities was directly related to the tumor cell growth in vitro. CM prepared from four clones of CE tumor cells demonstrated both colony-stimulating and bone cell-mitogenic activities. These studies provided evidence that CE mammary carcinoma cells produce factors affecting granulopoiesis and bone cells in vitro, and these activities are clonal in origin.

Animals↗