Expression of telomerase inhibits hydroxyl radical-induced apoptosis in normal telomerase negative human lung fibroblasts.

In tumor cells telomerase activity is associated with resistance to apoptosis and the introduction of the human telomerase reverse transcriptase (hTERT) subunit into normal human cells is associated with life span extension of the cells. To determine the role of telomerase in regulating apoptosis, telomerase negative human embryo lung fibroblasts were transfected with the hTERT gene. Unlike the control fibroblasts, the telomerase-expressing cells had elongated telomeres and were resistant to apoptosis induced by hydroxyl radicals. The results indicate that expression of telomerase and, thus, the maintenance of telomere length in normal human somatic cells caused resistance to not only cellular senescence but also apoptosis. Moreover, we found that hydroxyl radical-induced apoptosis in telomerase-expressing and control fibroblasts was caspase-3 independent. These findings have revealed a new type of interrelation between telomerase and caspase-3, which may indicate that in this case the expressed telomerase may inhibit apoptosis at a site not related to the caspase-3 cascade.

Intrinsic fluorescence study of the interaction of human apolipoprotein H with phospholipid vesicles.

Apolipoprotein H (ApoH) is a plasma glycoprotein with its in vivo physiological and pathogenic roles being closely related to its interaction with negatively charged membranes. In this paper, the interaction of ApoH with phospholipid vesicles was characterized by (i) detecting the wavelength shift of the fluorescence spectrum of ApoH and (ii) measuring the fluorescence quenching extent of ApoH by the membrane resident quencher 1-palmitoyl-2-stearoyl-(5-doxyl)-sn-glycero-3-phosphocholine (DPC). The observed blue shift upon addition of DMPG vesicles indicated that the tryptophan residues of ApoH moved from a polar to a nonpolar environment. The insertion ability of ApoH into PG-containing vesicles did not depend on the PG content in a stoichiometric way as did the blue shift, indicating that the negatively charged DMPG does not serve as a specific binding site but rather provides a suitable microenvironment for ApoH interaction. The finding that the detachment effect of cations on the blue shift is remarkably different from that on the quenching extent suggests that ApoH is capable of existing in two different conformations when membrane-bound.

The insertion of human apolipoprotein H into phospholipid membranes: a monolayer study.

Apolipoprotein H (ApoH) is a plasma glycoprotein isolated from human serum. The interactions of ApoH with lipid membrane were reported to be essential for its physiological and pathogenic roles. In this paper we studied the ability of ApoH to insert into phospholipid membranes using the monolayer approach. The results show that ApoH is surface active and can insert into the lipid monolayers. The insertion ability of ApoH is stronger when a higher content of negatively charged lipids is present in the membrane. The acidic-pH and low-ionic-strength conditions will also enhance ApoH insertion, but these factors may not have much influence on the final insertion ability of ApoH, suggesting that, in the mechanism of ApoH insertion, not only electrostatic forces, but also hydrophobic interactions, are evidently involved. Modification by heat inactivation and reduction/alkylation does not change the critical insertion pressure (pic) of ApoH, suggesting a stable domain, maybe a linear sequence motif, but not the native three-dimensional structure of ApoH, is responsible for its insertion. The extent to which insertion of ApoH into phospholipid membranes may facilitate the 'immune cleaning' of plasma liposomes is discussed.

Fluorescence characterization of type I collagen from normal and silicotic rats and its quenching dynamics induced by hypocrellin B.

In this paper, we studied the quenching mechanism of intrinsic fluorescence of type I collagen by a new type photosensitizer and fluorescence quencher, hypocrellin B (HB). It was indicated that type I collagen can emit Tyr-intrinsic fluorescence with the excitation wavelength of Tyr (lambda(ex) = 269 nm). Its fluorescence decay conform to the triexponential rule of the fluorescence lifetime. The intrinsic fluorescence of type I collagen can be effectively quenched by HB through a process of charge and energy transference, which is involved in the collisional quenching, the dipolar inducement, and the formation of exciplex between HB and excited fluorophores of collagen. The fluorescence quenching would be weakened by higher ionic environments. The fluorescence emission and its quenching rate of abnormal silicotic collagen show falling trends, implying its much weakened potential of charge and energy transference, and its lessen bioelectric activities. In conclusion, the bioelectric properties of collagen depends on the perfect order of its molecular structure and orderly intramolecular and intermolecular interactions, which is important in its performing normal physiological functions. It is also demonstrated that the fluorescence quenching technique, using HB as a quencher, is truly an effectively method for biomolecular studies.

D1-like dopaminergic activation of phosphoinositide hydrolysis is independent of D1A dopamine receptors: evidence from D1A knockout mice.

Accumulated evidence suggests that dopamine and dopamine D1 agonists can activate phospholipase C in both brain and peripheral tissue. The receptor that mediates the hydrolysis of phosphoinositides has not been identified. The cloned dopamine D1A receptor that is generally thought to be linked to adenylyl cyclase, has also been proposed to couple to phospholipase C. However, a number of studies have suggested that this signaling pathway is mediated via a distinct D1-like dopamine receptor. We tested whether the D1A site plays a role in stimulating phosphoinositide hydrolysis by using the dopamine D1A-deficient mutant mice as a test model. Results show that although D1 dopamine receptor-mediated product on of cAMP is completely absent in membranes of D1A-deficient mice, D1 receptor-mediated accumulation of inositol phosphate is identical in tissues of mutant and wild-type animals. Furthermore, the coupling of [3H]SCH23390 binding sites in striatal or frontal cortex membranes to G alpha s is markedly reduced, although coupling of [3H]SCH23390 binding sites to G alpha q was unaltered in tissue taken from D1A mutant mice compared with control animals. These results clearly demonstrate that dopaminergic stimulation of inositol phosphate formation is mediated by a D1 dopamine receptor subtype that is distinct from the D1A receptor that activates adenylyl cyclase.

A novel bifunctional fusion enzyme catalyzing ethylene synthesis via 1-aminocyclopropane1-carboxylic acid.

A C terminus truncated soybean 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (466 aa) was fused to an N terminus truncated tomato ACC oxidase (312 aa) to create a 778-amino acid fusion polypeptide. This ACC synthase-ACC oxidase fusion enzyme (ACSO) was expressed in a heterologous prokaryotic Escherichia coli system, which is capable of converting endogenous S-adenosyl-L-methionine (AdoMet) to ethylene. The molecular weight of the fusion enzyme, ACSO, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 90 +/- 3 kDa. Gel filtration analysis indicates that the native ACSO is oligomeric and is capable of converting exogenously supplied AdoMet to ethylene. The ethylene production rate of ACSO fusion enzyme was determined to be 6.0 nmol h-1 mg-1 under our assaying conditions using the partially purified enzyme extract. In the enzyme reaction mixture, an increase in ethylene production catalyzed by the bifunctional ACSO was accompanied by a decrease in ACC accumulation. Similarly, in E. coli cells, the level of ACC, produced as an intermediate during the sequential reactions from AdoMet to ethylene, was also found to arise earlier than that of ethylene. Because ACSO could produce ethylene from the ubiquitous AdoMet in living cell and the method commonly used to measure gaseous ethylene is simple, fast, and sensitive, we anticipate this bifunctional fusion enzyme to be useful as a reporter and for research in molecular biology, developmental biology, fermentation, and genetic engineering.

Studies on the inhibitory effect of tetrandrine on silica-induced fibrogenesis in vitro.

Two cell culture systems composed of rat peritoneal macrophages and 2BS fibroblasts were used to study the inhibitory action of tetrandrine on collagen and glycosaminoglycan synthesis. The activation of silica-treated macrophage supernatants stimulated collagen synthesis by 2BS fibroblast cells. Tetrandrine had an inhibitory effect on the biosynthesis of collagen and glycosaminoglycans. The increase in the content of hydroxyproline, representing collagen, was verified by comparing it with that of DNA.

Studies on the effect of tetrandrine on microtubules. I. Biochemical observation and electron microscopy.

Microtubules were purified by using two cycles of assembly and disassembly processes on fresh brain homogenates from 30 guinea pigs. The yield was about 60 mg. The effect of tetrandrine on tubulin was determined by spectrophotometric analysis and electron microscopy. In addition, we used the indirect immunofluorescent method including tubulin antibody to locate the presence of microtubules in 3T3 cells by fluorescence microscopy. The effects of colchicine and P204 were studied for comparison at the same time. The results showed that colchicine can effectively depolymerize microtubules, while tetrandrine showed aggregation, and in a different manner. The shape and structure of microtubules were definitely destroyed by colchicine, but were not affected by P204 which protected against the destructive effect of tetrandrine. This result indicated the safety of using a combination of P204 and tetrandrine in the treatment of silicosis.

Studies on the effect of tetrandrine on microtubules. II. Observation with immunofluorescence techniques.

Microtubules were purified by using two cycles of assembly and disassembly processes on fresh brain homogenates from 30 guinea pigs. The yield was about 60 mg. The effect of tetrandrine on tubulin was determined by spectrophotometric analysis and electron microscopy. In addition, we used the indirect immunofluorescent method including tubulin antibody to locate the presence of microtubules in 3T3 cells by fluorescence microscopy. The effects of colchicine and P204 were studied for comparison at the same time. The results showed that colchicine can effectively depolymerize microtubules, while tetrandrine showed aggregation, and in a different manner. The shape and structure of microtubules were definitely destroyed by colchicine, but were not affected by P204 which protected against the destructive effect of tetrandrine. This result indicated the safety of using a combination of P204 and tetrandrine in the treatment of silicosis.