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Biomedical subjects

S Zhuang

Publications and source records attributed to S Zhuang.

32 records · Page 2Linked to original sources
PubMed1999

p53 overexpression in synovial tissue from patients with early and longstanding rheumatoid arthritis compared with patients with reactive arthritis and osteoarthritis.

OBJECTIVE: The p53 tumor suppressor gene is overexpressed in synovial tissue (ST) from patients with longstanding rheumatoid arthritis (RA), and may contain somatic mutations. The aim of this study was to determine p53 expression in ST from RA patients in different stages of the disease, compared with disease controls. METHODS: ST biopsy specimens were obtained from the knee joints of 31 RA patients in varying disease phases, 8 patients with reactive arthritis (ReA), 10 patients with inflammatory osteoarthritis (OA), and 6 control patients (4 with meniscus pathology, 2 with vascular insufficiency). ST was also obtained from the clinically uninvolved knee joints of 9 RA patients. Expression of p53 was determined by immunohistology with DO1 monoclonal antibody (mAb) in all patients and by Western blot analysis with DO7 mAb in a subgroup of the patients. RESULTS: The p53 protein was detected by immunohistology in 10 of the 13 patients with early RA (duration <6 months) and in 12 of the 14 patients with longstanding RA (duration >5 years). The p53 protein was also demonstrated in clinically uninvolved knee joints. Western blots revealed immunoreactive p53 in ST extracts from all RA patients. Expression of p53 was about twice as high in ST from patients with longstanding RA as in early RA samples, but the difference did not reach statistical significance. Small amounts of p53 were also detected in ST from ReA and OA patients, although the expression in RA synovium was significantly higher. Immunohistologic analysis of normal ST gave negative results for p53. CONCLUSION: This study shows that p53 overexpression is specific for RA, compared with OA and ReA. This phenomenon is probably secondary to increased production of wild-type p53 protein in response to DNA damage and secondary to somatic mutations caused by the genotoxic local environment in inflamed ST. Of interest, p53 overexpression can also be found in the earliest stages of RA and in clinically uninvolved joints.

Aged↗
PubMed1999

Intra-articular IL-4 gene therapy in arthritis: anti-inflammatory effect and enhanced th2activity.

Gene therapy has been explored as a potential method for treating chronic inflammatory diseases such as rheumatoid arthritis. To determine the efficacy of intra-articular IL-4 gene therapy in an animal model of arthritis using a retroviral vector, a retrovirus encoding rat IL-4 (DA-IL-4) was engineered, purified and concentrated to high titer (>/=109 CFU/ml). Infectivity and expression levels were demonstrated in vitro using cultured fibroblast-like synoviocytes. Efficacy was evaluated in the rat adjuvant arthritis model. DA-IL-4 or DA-beta-gal retrovirus was injected into the intra-articular joint space of the right ankle on day 12 after immunization. Three days after joint injection, the injected paw contained increased levels of IL-4 compared with control or with the contralateral uninjected paw, demonstrating successful transgene expression. Surprisingly, 8 days after treatment IL-4 levels continued to increase in the injected and contralateral paw compared with DA-beta-gal-treated animals. Serum IL-4 levels were also elevated in DA-IL-4-treated rats. RT-PCR studies demonstrated that the transgene was expressed in the injected ankle but not in the contralateral joint. IL-4 gene therapy resulted in a significant reduction in paw swelling and decreased radiographic evidence of bone destruction. This is the first demonstration of successful intra-articular retroviral gene treatment using a therapeutic gene. In addition to its anti-inflammatory effect, this study supports the potential application of intra-articular gene therapy as a method for enhancing systemic Th2 function.

Animals↗
PubMed1998

Effects of long-term exposure to delta9-THC on expression of cannabinoid receptor (CB1) mRNA in different rat brain regions.

The time course of changes across 21 days of continuous exposure to Delta9-tetrahydrocannabinol (Delta9-THC) was assessed for the level of cannabinoid receptor (CB1) mRNA expression in three different rat brain regions: cerebellum, hippocampus and corpus striatum. Expression levels of CB1 mRNA were determined using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) following a protocol which included a gene standard, 28S ribonucleic acid protein (rRNA), for normalization of levels of RNA in the three different brain regions. The levels of CB1 mRNA were assessed in four different rats at each of seven time points (6 h, and 1, 2, 3, 7, 14 and 21 days) during a 21-day Delta9-THC one dose day-1 (10 mg kg-1) treatment regimen. In the cerebellum and hippocampus, CB1 mRNA levels were increased above vehicle control animals at 7 and 14 days of treatment. In the striatum the levels of CB1 transcripts were severely reduced from days 2-14. CB1 message expression in all three brain areas returned to vehicle control levels by day 21 of Delta9-THC treatment, a time at which behavioral tolerance has been previously reported. An additional measure, receptor stimulated GTPgammaS binding, performed over the same time period revealed differential desensitization within the 3 brain areas as a function of chronic exposure to Delta9-THC. Hippocampus was the earliest to desensitize decreasing to 35% of control by treatment day 7, followed by a decrease in the cerebellum to that same level on day 14 of treatment. The striatum showed only half that degree of desensitization (65%) over the entire 21-day treatment period. Comparisons suggests that CB1 message may be regulated by different effector systems in each of the three areas during chronic Delta9-THC exposure.

Animals↗
PubMed1998

Activation of protein kinase C is required for protection of cells against apoptosis induced by singlet oxygen.

We evaluated the role of protein kinase C (PKC) in the regulation of apoptosis triggered by singlet oxygen. Activation of PKC by short-term 12-O-tetradecanoyl phorbol 13-acetate (TPA) treatment inhibited apoptosis, whereas inhibition of PKC with several inhibitors potentiated this process. The antiapoptotic effect of TPA was accompanied by phosphorylation of extracelluar signal-regulated kinase 1/2 (ERK1/2). Pretreatment of cells with MEK inhibitor, PD98059, inhibited TPA-induced phosphorylation of ERK1/2 and the cytoprotective ability of TPA. These results suggest that activation of PKC in HL-60 cells confers protection against apoptosis induced by singlet oxygen and that ERK1/2 mediates antiapoptotic signaling of PKC.

Apoptosis↗
PubMed1998

Transcatheter endomyocardial laser revascularization: a feasibility test.

By means of a special catheter system, a total of 76 endomyocardial laser channels were percutaneously produced in a controllable manner at selected sites in 6 beating canine hearts. Acute patency of channels (length = 4-11 mm, diameter = 0.5-1.2 mm) was documented angioscopically and histologically. This minimally invasive method might be useful for revascularising certain patients with ischemic heart disease without resorting to open-chest surgery.

Angioscopy↗
PubMed1998

[Studies on optimal conditions for inulinase production by Kluyveromyces sp. Y-85].

The application of response surface method to optimal medium-screeing for inulinase production by Kluyveromyces sp. Y-85 has been investigated. The experiments of condition control for the enzyme production was carried out in a 15 L fermenter. A large scale fermentation (1000 L fermenter) was performed five batches, and average inulinase activity of 68.9 u/ml achieved.

Culture Media↗
PubMed1997

Calmodulin binds to caldesmon in an antiparallel manner.

Two of the five tryptophan residues (W659 and W692) in chicken gizzard smooth muscle caldesmon (CaD) are located within the calmodulin (CaM) binding sites in the C-terminal region of the molecule. When these Trp residues are replaced with Gly in either recombinant fragments or synthetic peptides of CaD, the affinity for CaM is decreased by at least 10-fold, suggesting that both of these residues are important for the interaction of CaD with CaM. To gain information about the topography of the CaM-CaD complex, we have carried out fluorescence titrations of CaM with Tb3+ as a substitute for Ca2+ in the presence of wild-type or mutated CaD variants. By exciting Trp residues of CaD fragments or peptides while monitoring the enhanced luminescence of CaM-bound Tb3+ ions via resonance energy transfer, we were able to estimate the relative proximity between the bound metal ions in the two domains of CaM and the Trp residues of CaD. Our results suggest that in the CaM-CaD complex the metal-binding sites III and IV in the C-terminal domain of CaM are very close to W659 of CaD; the N-terminal domain of CaM appears associated with the region of CaD in the vicinity of W692, although sites I and II are relatively far away from this Trp residue. These findings are consistent with a model in which CaM binds to CaD in an antiparallel manner. Such a binding mode, however, may be flexible enough to accommodate alternative spatial arrangements when the preferred binding sites are either altered or rendered unavailable.

Amino Acid Sequence↗
PubMed1997

Involvement of protein kinase C in the activation of extracellular signal-regulated kinase 1/2 by UVC irradiation.

UVC irradiation activates mitogen-activated protein kinases (MAPKs), including ERK, JNK, and P38. This study examined the role of protein kinase C (PKC) in the regulation of UVC-stimulated MAPKs activation. Either the depletion of PKC by prolonged treatment of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) or the inhibition of PKC by a selective PKC inhibitor, UCN-01-ME, attenuated UVC-activation of ERK1/2, keeping the activation of JNK1/2 intact. However, K252a, a non-selective PKC inhibitor, inhibited the activation of both ERK1/2 and JNK1/2 by UVC. In three isoforms of PKC (alpha, delta, epsilon) examined, PKC epsilon shows the most evident translocation, a temporal association with cell membrane, upon the UVC irradiation of NIH 3T3 cells. These results suggest that PKC is acting in the UVC-dependent activation of ERK1/2, and PKC epsilon is one of the PKC isozymes playing such a role.

3T3 Cells↗
PubMed1997

Apoptin induces apoptosis in human transformed and malignant cells but not in normal cells.

The chicken anemia virus protein apoptin induces a p53-independent, Bcl-2-insensitive type of apoptosis in various human tumor cells. Here, we show that, in vitro, apoptin fails to induce programmed cell death in normal lymphoid, dermal, epidermal, endothelial, and smooth-muscle cells. However, when normal cells are transformed they become susceptible to apoptosis by apoptin. Long-term expression of apoptin in normal human fibroblasts revealed that apoptin has no toxic or transforming activity in these cells. In normal cells, apoptin was found predominantly in the cytoplasm, whereas in transformed and malignant cells it was located in the nucleus, suggesting that the localization of apoptin is related to its activity. These properties make apoptin a potential agent for the treatment of a large number of tumors, also those lacking p53 and/or overexpressing Bcl-2.

Apoptosis↗
PubMed1996

Heat treatment could affect the biochemical properties of caldesmon.

Smooth muscle caldesmon (CaD) exhibits apparent heat stability. A widely used purification procedure of CaD involves extensive heat treatment (Bretscher, A. (1984) J. Biol. Chem. 259, 12873-12880). CaD thus purified co-sediments with actin, inhibits actomyosin ATPase activity, and interacts with Ca2+/calmodulin, similarly to the unheated protein. On the other hand, heat-treated CaD binds to actin filaments in a tether-like fashion, whereas lengthwise binding dominates in vivo (Mabuchi, K., Lin, J. J.-C., and Wang, C.-L. A. (1993) J. Muscle Res. Cell Motil. 14, 54-64), suggesting that differences do exist between heat-purified CaD and the native protein. We have isolated, without heat treatment, full-length recombinant chicken gizzard CaD overexpressed in insect cells (High-FiveTM) using a baculovirus expression system. We found that such unheated CaD interacts with calmodulin 10 times stronger than does the heated CaD; its inhibitory action on actomyosin ATPase is reversed by a much lesser amount of calmodulin. Moreover, electron microscopic examination indicated that actin binding at the N-terminal region is more frequent in the unheated CaD, resulting in more lengthwise binding. These findings point to the fact that CaD is not entirely heat-stable; the C-terminal CaM-binding regions and the N-terminal actin-binding region are possibly affected by heat treatment.

Actins↗
PubMed1995

Identification of the functionally relevant calmodulin binding site in smooth muscle caldesmon.

The C-terminal region of smooth muscle caldesmon (CaD) interacts with calmodulin (CaM) and reverses CaD's inhibitory effect on the actomyosin ATPase activity. We have previously shown that the major CaM-binding site (site A) in this region is within the segment from Met-658 to Ser-666 (Zhan, Q., Wong, S. S., and Wang, C.-L. A. (1991) J. Biol. Chem. 266, 21810-21814). Recently, another segment (site B), Asn-675 to Lys-695, was reported to bind CaM (Mezgueldi, M., Derancourt, J., Calas, B., Kassab, R., and Fattoum, A. (1994) J. Biol. Chem. 269, 12824-12832). To assess the functional relevance of these two putative CaM-binding sites, we have examined three synthetic peptides regarding their effects on CaM's ability to reverse CaD-induced inhibition of actomyosin ATPase activity: GS17C (Gly-651 to Ser-667), VG29C (Val-685 to Gly-713), each containing one CaM-binding site, and MG56C (Met-658 to Gly-713), which contains both sites. We found that although VG29C did bind CaM, its affinity was weakened by GS17C, and it failed to compete with CaD for CaM under the conditions where GS17C effectively displaced CaD from CaM. MG56C had an effect similar to that of GS17C. These experiments demonstrated that site A for CaM binding is involved in regulating the inhibitory property of CaD.

Amino Acid Sequence↗
PubMed1995

Interaction between caltropin and the C-terminal region of smooth muscle caldesmon.

Caltropin (CaT) binds caldesmon (CaD) in a Ca(2+)-dependent manner with an affinity higher than that of calmodulin (CaM). Photo-crosslinking between CaT and a benzophenone-labeled C-terminal CaD fragment (27K) results in a 35-kDa protein that corresponds to the 1:1 adduct between CaT and 27K. In the absence of Ca2+, no crosslinking is obtained. This result is similar to that obtained with CaM and 27K. The apparent affinity of CaM for GS17C, a CaM-binding peptide of CaD, is weakened by CaT, suggesting CaT competes with CaM for the peptide. In contrast to CaM, CaT does not induce changes in the tryptophan fluorescence of GS17C. Thus although the two Ca(2+)-binding proteins behave similarly, there are differences in their interactions with CaD.

Animals↗