Biomedical subjects
Publications and source records attributed to L Zhu.
Self-assembled monolayers of a tripod-shaped conjugated-thiol grafted onot Au(111) substrates are found to show electrochemically reversible oxidation and reduction and to improve electroluminescence performances of organic light-emitting diodes.
Denaturation of the Saccharomyces cerevisiae prion protein Ure2 was investigated using hydrostatic pressure. Pressures of up to 600 MPa caused only limited perturbation of the structure of the 40-kDa dimeric protein. However, nondenaturing concentrations of GdmCl in combination with high pressure resulted in complete unfolding of Ure2 as judged by intrinsic fluorescence. The free energy of unfolding measured by pressure denaturation or by GdmCl denaturation is the same, indicating that pressure does not induce dimer dissociation or population of intermediates in 2 M GdmCl. Pressure-induced changes in 5 M GdmCl suggest residual structure in the denatured state. Cold denaturation under pressure at 200 MPa showed that unfolding begins below -5 degrees C and Ure2 is more susceptible to cold denaturation at low ionic strength. Results obtained using two related protein constructs, which lack all or part of the N-terminal prion domain, were very similar.
The antitumor efficiency of secondary lymphoid organ chemokine (SLC), a CC chemokine that chemoattracts both dendritic cells (DCs) and T lymphocytes,was evaluated in SV40 large T-antigen transgenic mice that develop bilateral multifocal pulmonary adenocarcinomas. Injection of recombinant SLC in the axillary lymph node region led to a marked reduction in tumor burden with extensive lymphocytic and DC infiltration of the tumors and enhanced survival. SLC injection led to significant increases in CD4 and CD8 lymphocytes as well as DC at the tumor sites, lymph nodes, and spleen. The cellular infiltrates were accompanied by the enhanced elaboration of Type 1 cytokines and the antiangiogenic chemokines IFN-gamma inducible protein 10, and monokine induced by IFN-gamma (MIG). In contrast, lymph node and tumor site production of the immunosuppressive cytokine transforming growth factor beta was decreased in response to SLC treatment. In vitro, after stimulation with irradiated autologous tumor, splenocytes from SLC-treated mice secreted significantly more IFN-gamma and granulocyte macrophage colony-stimulating factor, but reduced levels of interleukin 10. Significant reduction in tumor burden in a model in which tumors develop in an organ-specific manner provides a strong rationale for additional evaluation of SLC in regulation of tumor immunity and its use in lung cancer immunotherapy.
Mrf-2 is a member of a new class of DNA-binding proteins known as the AT-rich interaction domain family or ARID. Chemical shift indices and characteristic NOE values indicate that the three-dimensional structure of the Mrf-2 ARID in complex with DNA is nearly identical to that of the free protein. The backbone dynamics of the Mrf-2 domain free and in complex with DNA have been characterized by (15)N NMR relaxation measurements and model-free analysis. Chemical shift perturbations and dynamic studies suggest that two flexible interhelical loops, the flexible C-terminal tail, and one alpha-helix are involved in DNA recognition, indicating the importance of protein dynamics in DNA binding. Some well-structured regions, in particular the putative DNA-contacting helix, in Mrf-2 show a decrease in the order parameters (S(2)) upon complex formation. The less well-structured loops and the unstructured C-terminus show reduced flexibility upon DNA binding. In addition, the model-free analysis indicates motions on the picosecond to nanosecond and micro- to millisecond time scales at the DNA-binding surface of the bound Mrf-2 ARID, suggesting a model where interactions between the protein and DNA are highly dynamic.
The purpose of this study was to investigate the use of 6-carboxycellulose (OC), a biocompatible and bioresorbable polymer, as a prodrug carrier for amine drugs. Phenylpropanolamine hydrochloride (PPA.HCl) was used as a model drug. OC and PPA were reacted in dimethylformamide (DMF) in the presence of 1,3-dicyclohexylcarbodiimide (DCC) for 2.5 days at room temperature. Filtration, followed by washing with methanol, and subsequent drying under vacuum, produced the conjugate in 65-78% yield. The amount of PPA in the product, determined from the difference in the carboxylic content before and after the reaction, was 24.2% (w/w), corresponding to a degree of substitution (DS) value of 0.7. The Fourier transform-infra red (FT-IR) spectrum of the conjugate, compared with that of OC and PPA.HCl, showed a new band at about 1533 cm(-1) attributable to a C = O (amide II) stretching and N single bond H (amide I and amide II) bending vibrations, a decrease in intensity of the characteristic free carboxylic acid carbonyl stretching band at about 1748 cm(-1), and a strong band at 1663 cm(-1) due to C = O (amide I) stretching vibration, suggesting that the OC is linked to PPA via an amide bond. The solid-state carbon-13 cross polarization/magic angle spinning nuclear magnetic resonance ((13)CCP/MAS NMR) spectrum of the conjugate was also consistent with this structure. The release studies performed in pH 4.5, 5.5, and 7.4 buffer solutions and in rat liver homogenate (pH 7.4), showed the conjugate to be more susceptible to hydrolysis at a lower pH and in the presence of rat liver homogenate. In conclusion, the results presented show that OC can be covalently linked to amine drugs via an amide bond in DMF using DCC as a coupling agent, and provide a macromolecular prodrug delivery system.
Creatine kinase (CK) is a dimeric enzyme important in ATP regeneration in cells where energy demands are high. The folding of CK under equilibrium and transient conditions has been studied in detail and is found to be complex. At equilibrium in 0.8 M GuHCl, 90% of CK molecules are in the form of a partially structured, monomeric intermediate. We exploit this property to measure kinetics of refolding and unfolding to and from this equilibrium intermediate (EI), using far-UV circular dichroism and intrinsic fluorescence as structural probes. We are thus able to compare the properties of EI and the kinetic intermediate formed during the burst phase in refolding. Native CK and EI unfold with rate constants in seconds and milliseconds, respectively. As is observed for refolding of fully-denatured CK, refolding from EI to the native state shows a burst phase followed by two exponential phases. The burst phase refolding intermediate is inferred to have more structure and greater stability than the equilibrium intermediate. When refolding from the fully-denatured state in 0.8 M GuHCl, the equilibrium intermediate is formed within the dead-time of mixing in the stopped-flow apparatus. The equilibrium intermediate may thus represent a kinetic intermediate formed early during folding.
A simple liquid-liquid-liquid microextraction device utilizing a 2 cm x 0.6 mm I.D. hollow fiber membrane was used to preconcentrate nitrophenols from water sample prior to capillary liquid chromatography (cLC) analysis. The extraction procedure was induced by the pH difference inside and outside the hollow fiber. The donor phase outside the hollow fiber was adjusted to pH approximately 1 with HCl; the acceptor phase was NaOH solution used at various concentrations. Organic solvent was immobilized into the pores of the hollow fiber. With stirring, the neutral nitrophenols outside the fiber were extracted into the organic solvent, then back extracted into 2 microl of basic acceptor solution inside the fiber. The acceptor phase was then withdrawn into a microsyringe and injected into the cLC system directly. This technique used a low-cost disposable extraction "device" and is very convenient to operate. Up to 380-fold enrichment of analytes could be achieved. This procedure could also serve as a sample clean-up step because large molecules and basic compounds were not extracted into the acceptor phase. The RSD (n=6) was less than 6.2%, while the linear calibration range was from 1 to 200 microg/ml with r>0.998. The procedure was applied to the analysis of seawater.
The proteins Ku70 (69.8 kDa) and Ku80 (82.7 kDa) form a heterodimeric complex that is an essential component of the nonhomologous end joining DNA double-strand break repair pathway in mammalian cells. Interaction of Ku with DNA is central for the functions of Ku. Ku70, which is mainly responsible for the DNA binding activity of the Ku heterodimer, contains two DNA-binding domains. We have solved the solution structure of the Ku80-independent DNA-binding domain of Ku70 encompassing residues 536-609 using nuclear magnetic resonance spectroscopy. Residues 536-560 are highly flexible and have a random structure but form specific interactions with DNA. Residues 561-609 of Ku70 form a well defined structure with 3 alpha-helices and also interact with DNA. The three-dimensional structure indicates that all conserved hydrophobic residues are in the hydrophobic core and therefore may be important for structural integrity. Most of the conserved positively charged residues are likely to be critical for DNA recognition. The C-terminal DNA-binding domain of Ku70 contains a helix-extended strand-helix motif, which occurs in other nucleic acid-binding proteins and may represent a common nucleic acid binding motif.
A procedure that combines two common stacking techniques, field-amplified sample injection and water removal, with an electroosmotic flow pump, is used to separate phenoxy acid herbicides by capillary zone electrophoresis. Before sample loading, a long plug of water was hydrodynamically injected into the capillary both to serve as the medium to permit a high electric field strength and to contain sample anions. Because of this long length of water, the number of ions injected into the capillary was greatly increased. Electrokinetic injection at reversed voltage was then used for introducing negatively charged ions from the diluted sample into the column. The water was removed from the capillary using the electroosmotic flow (EOF) pump when the EOF of the background electrolyte was suppressed. This method afforded a sensitivity enhancement of greater than 3,000 times. Combined with solid-phase extraction, detection limits for the phenoxy acid herbicides as low as 0.01 ng/mL could be achieved.
Small angle x-ray analyses show that the shear-induced hexagonal perforated layer phase in a poly(ethylene oxide)- b-polystyrene diblock copolymer consists of trigonal (R3;m) twins and a hexagonal (P6(3)/mmc) structure, with trigonal twins being majority components. Transmission electron microscopy reveals that the hexagonal structure is generated through sequential intrinsic stacking faults on the second layer from a previous edge dislocation line, while the trigonal twins are formed by successive intrinsic stacking faults on neighboring layers due to the plastic deformation under mechanical shear.
Gingival fibromatosis (GINGF) is an oral disorder characterized by enlargement of the gingiva. It occurs either as the sole phenotype or combined with other symptoms. Thus far, one GINGF locus has been mapped on chromosome 2, at 2p21, and a second possible locus has been mapped to 2p13. However, the genes responsible for this disorder have not been elucidated. We identified a four-generation Chinese GINGF family in which the disease manifests within 1 year after birth. After exclusion of the two known GINGF loci in this family, we performed a genome-wide search to map the chromosome location of the responsible gene. We identified a new locus, GINGF2, on chromosome 5q13-q22 with a maximum two-point lod score of 4.31 at D5S1721 (theta = 0.00). Haplotype analysis placed the critical region in the interval defined by D5S1491 and D5S1453. Within this region, calcium/calmodulin-dependent protein kinase IV (CAMK4) is a strong candidate.
Highly ordered hexagonal mesoporous aluminosilicates (MAS-5) with uniform pore sizes have been successfully synthesized from assembly of preformed aluminosilcate precursors with cetyltrimethylammonium bromide (CTAB) surfactant. The aluminosilicate precursors were obtained by heating, at 100--140 degrees C for 2--10 h, aluminasilica gels at the Al(2)O(3)/SiO(2)/TEAOH/H(2)O molar ratios of 1.0/7.0--350/10.0--33.0/500--2000. Mesoporous MAS-5 shows extraordinary stability both in boiling water (over 300 h) and in steam (800 degrees C for 2 h). Temperature-programmed desorption of ammonia shows that the acidic strength of MAS-5 is much higher than that of MCM-41 and is comparable to that of microporous Beta zeolite. In catalytic cracking of 1,3,5-triisopropylbenzene and alkylation of isobutane with butene, MAS-5 exhibits greater catalytic activity and selectivity, as compared with MCM-41 and HZSM-5. The MAS-5 samples were characterized with infrared, UV--Raman, and NMR spectroscopy and numerous other techniques. The results suggest that MAS-5 consists of both mesopores and micropores and that the pore walls of MAS-5 contain primary and secondary structural building units, similar to those of microporous zeolites. Such unique structural features might be responsible for the observed strong acidity and high thermal stability of the mesoporous aluminosilicates with well-ordered hexagonal symmetry.
Cretinism is a disease characterized by neurological defects associated with severe iodine deficiency. In a rat model of severe iodine deficiency, we investigated the distribution pattern of trace elements (iodine [I], selenium [Se], and bromine [Br] in brain tissue samples; potassium [K], calcium [Ca], manganese [Mn], iron [Fe], copper [Cu], zinc [Zn], rubidium [Rb], and lead [Pb] in erythrocytes) after supplementing the rats with I and/or Se. Neutron activation analysis, proton induced x-ray emission and x-ray fluorescence were used. The serum levels of total and free thyroxine (T4, FT4), and of total, free, and reverse triiodothyronine (T3, FT3, rT3, respectively) were assessed by radioimmunoassay. The results were statistically evaluated by one-way analysis of variance and bivariate correlation. The study indicated that the levels of T4, FT4, and rT3 increased in the serum of iodine-deficient rats supplemented with I or I + Se. In the same animals, we documented alterations of the content of Br in the brain, and of Zn, Mn, Cu, and Rb in erythrocytes, whereas the brain content of I and Se was unchanged. Thus, I and I + Se supplementation improves thyroid hormone metabolism but affects the content of selected trace elements in erythrocytes and of Br in the brain. The data stimulate further clarification of the role of trace elements in the central nervous system.
The development of an effective vaccine against Mycobacterium tuberculosis is a research area of intense interest. Mounting evidence suggests that protective immunity to M. tuberculosis relies on both MHC class II-restricted CD4(+) T cells and MHC class I-restricted CD8(+) T cells. By purifying polypeptides present in the culture filtrate of M. tuberculosis and evaluating these molecules for their ability to stimulate PBMC from purified protein derivative-positive healthy individuals, we previously identified a low-m.w. immunoreactive T cell Ag, Mtb 8.4, which elicited strong Th1 T cell responses in healthy purified protein derivative-positive human PBMC and in mice immunized with recombinant Mtb 8.4. Herein we report that Mtb 8.4-specific T cells can be detected in mice immunized with the current live attenuated vaccine, Mycobacterium bovis-bacillus Calmette-Guérin as well as in mice infected i.v. with M. tuberculosis. More importantly, immunization of mice with either plasmid DNA encoding Mtb 8.4 or Mtb 8.4 recombinant protein formulated with IFA elicited strong CD4(+) T cell and CD8(+) CTL responses and induced protection on challenge with virulent M. tuberculosis. Thus, these results suggest that Mtb 8.4 is a potential candidate for inclusion in a subunit vaccine against TB.
A multi-residue LC-MS method was developed to determine avermectin residues in swine liver. Abamectin and ivermectin (22,23-dihydroabamectin) were extracted, and cleaned up by immunoaffinty columns with immobilized anti-avermectin polyclonal antibodies. The cleaned samples were separated by high-performance liquid chromatography (HPLC) with a C8 column and determined by negative-ion atmospheric pressure chemical ionization (APCI) mass spectrometry (MS) using selective ion monitoring (SIM) of [M-H]-. Recoveries of abamectin and ivermectin from fortified samples at 5-100 microg kg(-1) levels ranged from 74 to 94% and from 65 to 87%, respectively. The limits of detection were 5 microg of abamectin or ivermectin in 1 kg sample.
We present the first experimental observation of superpersistent chaotic transients. In particular, we investigate the effect of noise on phase synchronization in coupled chaotic electronic circuits and obtain the scaling relation that is characteristic of those extremely long chaotic transients.
Elevated tumor cyclooxygenase (COX-2) expression is associated with increased angiogenesis, tumor invasion, and suppression of host immunity. We have previously shown that genetic inhibition of tumor COX-2 expression reverses the immunosuppression induced by non-small cell lung cancer (NSCLC). To assess the impact of COX-2 expression in lung cancer invasiveness, NSCLC cell lines were transduced with a retroviral vector expressing the human COX-2 cDNA in the sense (COX-2-S) and antisense (COX-2-AS) orientations. COX-2-S clones expressed significantly more COX-2 protein, produced 10-fold more prostaglandin E(2), and demonstrated an enhanced invasive capacity compared with control vector-transduced or parental cells. CD44, the cell surface receptor for hyaluronate, was overexpressed in COX-2-S cells, and specific blockade of CD44 significantly decreased tumor cell invasion. In contrast, COX-2-AS clones had a very limited capacity for invasion and showed diminished expression of CD44. These findings suggest that a COX-2-mediated, CD44-dependent pathway is operative in NSCLC invasion. Because tumor COX-2 expression appears to have a multifaceted role in conferring the malignant phenotype, COX-2 may be an important target for gene or pharmacologic therapy in NSCLC.
The insulin signaling pathway is activated by tyrosine phosphorylation of the insulin receptor and key post-receptor substrate proteins and balanced by the action of specific protein-tyrosine phosphatases (PTPases). PTPase activity, in turn, is highly regulated in vivo by oxidation/reduction reactions involving the cysteine thiol moiety required for catalysis. Here we show that insulin stimulation generates a burst of intracellular H(2)O(2) in insulin-sensitive hepatoma and adipose cells that is associated with reversible oxidative inhibition of up to 62% of overall cellular PTPase activity, as measured by a novel method using strictly anaerobic conditions. The specific activity of immunoprecipitated PTP1B, a PTPase homolog implicated in the regulation of insulin signaling, was also strongly inhibited by up to 88% following insulin stimulation. Catalase pretreatment abolished the insulin-stimulated production of H(2)O(2) as well as the inhibition of cellular PTPases, including PTP1B, and was associated with reduced insulin-stimulated tyrosine phosphorylation of its receptor and high M(r) insulin receptor substrate (IRS) proteins. These data provide compelling new evidence for a redox signal that enhances the early insulin-stimulated cascade of tyrosine phosphorylation by oxidative inactivation of PTP1B and possibly other tyrosine phosphatases.