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Biomedical subjects

Z Rao

Publications and source records attributed to Z Rao.

At least 19 recordsLinked to original sources
PubMed2006

Human 8-oxoguanine DNA glycosylase increases resistance to hyperoxic cytotoxicity in lung epithelial cells and involvement with altered MAPK activity.

It is unknown whether base excision DNA repair (BER) proteins interact with mitogen-activated protein kinases (MAPK) under oxidation. Here, we explored roles of BER proteins in signaling transduction involving MAPK during hyperoxia. We demonstrated that ERK1/2 phosphorylation in A549 cells was increased in 95% O(2). p38 activity in A549 cells was also increased by exposure to 95% O(2). To evaluate regulatory roles of MAPK, we have transduced A549 cells and primary alveolar epithelial type II cells (AECII) to overexpress 8-oxoguanine DNA glycosylase (hOgg1). Overexpression of hOgg1 reduced hyperoxic toxicity in A549 and AECII cells. Furthermore, protection by BER against hyperoxia appeared to involve an upregulation of ERK1/2 and downregulation of p38. These observations demonstrate, for the first time, that reduction of hyperoxic toxicity by BER proteins may be involved with MAPK activity, thereby impacting cell survival. Furthermore, our studies suggest that modulation of MAPK may be used in combination with BER proteins to counteract hyperoxic toxicity.

Blotting, Western↗
PubMed2003

Purification, crystallization and preliminary X-ray analysis of human pirin.

Pirin is a novel highly conserved nuclear protein, but very little is known about its cellular function. Human pirin has been cloned, expressed, purified and crystallized using PEG as precipitant. The crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 42.3, b = 67.0, c = 107.3 A, alpha = beta = gamma = 90 A. It contains one molecule per asymmetric unit and diffracts to 2.0 A under cryoconditions (100 K) using an in-house Cu rotating-anode X-ray generator.

Amino Acid Sequence↗
PubMed2003

Biochemical and biophysical analysis of heptad repeat regions from the fusion protein of Menangle virus, a newly emergent paramyxovirus.

Menangle virus is a novel paramyxovirus isolated in Australia in 1997, but its classification position has not yet been finally settled. Here by using a computational program, LearnCoil-VMF, we determined the heptad repeat (HR) regions (HR1 and HR2) of Menangle virus F protein. Subsequently the HR1 and HR2 peptides were expressed as a single chain (named 2-Helix) connected by a six amino-acid linker as a GST fusion protein with an E. coli in vitro expression system. The GST-removed purified 2-Helix protein could form a stable trimer in vitro judging by gel-filtration and chemical cross-linking. CD spectra showed that the 2-Helix protein had a high percentage of alpha-helix and was very thermo-stable. Crystals of the 2-Helix protein preparations have been obtained in many conditions with hanging-drop diffusion method. These results indicated that Menangle virus has the common features of the fusion protein for other paramyxoviruses and should adopt a similar fusion mechanism to other members. As the HR regions of Menangle virus F protein could form stable six-helix bundle coiled coil structure, they should be used as drug target for the design of fusion inhibitors, as successfully used for other parmyxoviruses. This is especially relevant to such a newly emergent virus with zoonotic potentials.

Amino Acid Sequence↗
PubMed2003

Engineering substrate recognition in catalysis by cytochrome P450cam.

We have a continuing interest in applying the current knowledge of cytochrome P450cam substrate recognition to engineer the enzyme for the biotransformation of unnatural substrates with the long-term aim of applications in the synthesis of fine chemicals and bioremediation of environmental contaminants. Comparisons of the structure of target substrates with that of camphor, the natural substrate, led to the design of active-site mutants with greatly enhanced activity for the oxidation of chlorinated benzenes and selectivity of (+)-alpha-pinene oxidation. The crystal structures of the F87W/Y96F/V247L mutant with 1,3,5-trichlorobenzene or (+)-alpha-pinene bound have revealed the enzyme-substrate contacts and provided insights into the activity and selectivity patterns. The structures have also provided a novel basis for further engineering of P450cam for increased activity in the oxidation of the highly inert pentachlorobenzene and hexachlorobenzene, and increased selectivity of (+)-alpha-pinene oxidation.

Amino Acid Substitution↗
PubMed2003

Crystallization of the N-terminal domain of DFF45: the mutual chaperone mechanism is challenged.

DNA fragmentation factor 45 (DFF45) regulates DNase DFF40 as its inhibitor and chaperone. It was reported that the N-terminal domain (NTD) of DFF45 alone is disordered and DFF40 is necessary as a mutual chaperone for the folding of NTD. However, here we reported the crystallization of DFF45 NTD. These crystals diffract to 9A using a synchrotron radiation source. In spite of the low resolution, the demonstration of crystal formation indicates that DFF45 NTD itself is not unstructured, which strongly questions the mutual chaperone speculation about DFF45 and DFF40.

Apoptosis Regulatory Proteins↗
PubMed2002

Purification, crystallization and X-ray analysis of swine vesicular disease virus.

Swine vesicular disease virus (SVDV) is the etiological agent of swine vesicular disease, a highly contagious disease in pigs, and is related to coxsackie B virus. Crystalline arrays of SVDV can be observed in the cytoplasm of cells 4.5 h after inoculation to porcine kidney cells (IBRS-2 cells). Crystals of the JX/78 strain of SVDV were obtained from virus in two wells of crystallization conditions and present preliminary X-ray data to 3.6 A resolution.

Crystallization↗
PubMed2002

Crystallization and preliminary x-ray crystallographic studies of trichosanthin delta C7.

Trichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) which possesses rRNA N-glycosidase activity. TCS has various pharmacological properties. It is possible to reduce the antigenicity of TCS by deleting up to seven C-terminal residues of TCS (TCS-C7) with minimal effect on its activity. TCS-C7 has been crystallized and the crystal diffracted to 1.8 A. It belongs to space group P2(1), with unit-cell parameters a=71.6A, b=74.4A, c=87.6A, beta=97.0 degrees. It is given that there are four molecules per asymmetric unit.

Antineoplastic Agents, Phytogenic↗
PubMed2001

Crystallization and preliminary X-ray crystallographic studies of a macromolecular antitumour antibiotic, C1027.

C1027 is a macromolecular antitumour antibiotic produced by Streptomyces globispourus C1027 and consists of an apoprotein and a non-protein labile chromophore. Little is known about how the thermally unstable chromophore is stabilized by the apoprotein. The purified C1027 was monodisperse according to dynamic light-scattering measurements and crystallized in two different crystal forms from two different starting conditions using the vapour-diffusion method. Condition I yielded hexagonal prism crystals having space group P3(1)/P3(2) and unit-cell parameters a = b = 66.8, c = 55.4 A. Diffraction data were collected to 2.1 A resolution using an in-house Rigaku rotating Cu anode X-ray generator. Another condition produced rod-like crystals with space group P3(1)21/P3(2)21 and unit-cell parameters a = b = 55.15, c = 55.87 A. A data set to 1.8 A resolution was collected from a rod-like crystal using a MAR CCD detector at the SRS synchrotron source.

Aminoglycosides↗
PubMed2001

Crystallization and preliminary X-ray analysis of a Trx domain of human thioredoxin-like protein.

The Trx domain of human thioredoxin-like protein has been purified and crystallized using ammonium sulfate as precipitant. The crystal belongs to space group C2, with unit-cell parameters a = 87.5, b = 48.5, c = 29.8 A, beta = 99.59 degrees. It has one molecule per asymmetric unit and diffracts beyond 2.2 A under cryoconditions (100 K) using an in-house Cu rotating-anode X-ray generator.

Crystallization↗
PubMed2001

Crystallization and preliminary X-ray crystallographic analysis of native and selenomethionyl recombinant tabtoxin-resistance protein complexed with acetyl-coenzyme A.

Tabtoxin-resistance protein (TTR), an acetyltransferase from Pseudomonas syringae pv. tabaci, was overexpressed in Eschericha coli M15 and the TTR fusion protein complexed with acetyl-coenzyme A (AcCoA) was purified and crystallized. Diffraction data were collected to 3.0 A resolution in-house and the crystal was found to belong to space group P2(1), with unit-cell parameters a = 47.6, b = 66.6, c = 53.5 A, beta = 104.3 degrees. Furthermore, a selenomethionine (SeMet) TTR fusion protein derivative was overexpressed in the same expression system and its complex with AcCoA was purified in a reductive environment. The SeMet TTR derivative crystallized in two forms: the first was identical to that observed for native crystals and the second belonged to space group C2, with unit-cell parameters a = 101.7, b = 45.6, c = 84.2 A, beta = 105.8 degrees. Data from the P2(1) crystal form were collected in-house to 2.3 A resolution. Subsequently, three different wavelength data sets of the C2 crystal form to 1.55 A resolution were collected at the Advanced Photon Source at Argonne National Laboratory.

Acetyl Coenzyme A↗
PubMed2001

Evidence for involvement of the neural pathway containing the peripheral vagus nerve, medullary visceral zone and central amygdaloid nucleus in neuroimmunomodulation.

It is now evident that a bidirectional communication network exists between the central nervous system (CNS) and immune system (IS). However, the way in which the IS passes inform to the brain is not quite clear.In the present study, one of the neural pathways involved in the cytokine-to-brain communication was investigated in the rat. This pathway starts at the vagal nerve projecting to the medullary visceral zone (MVZ), an arc-shape band from the dorsomedial to ventrolateral area in the middle-caudal segment of the medulla oblongata, and terminates at the central amygdaloid nucleus (Ce) which receives projections from large catecholaminergic neurons in the MVZ. Animals were randomly divided into two experimental groups. Triple-labeling was used in Group I animals to combine wheat germ aggulutinin-conjugated horseradish peroxidase (WGA-HRP) retrograde tracing with anti-Fos and anti-tyrosine hydroxylase (TH) immunostaining. WGA-RP was stereotaxically injected into the unilateral Ce of the animals and, after a survival period of 48 h, intraperitoneal (IP) injection of lipopolysaccharide (LPS) was performed. Seven kinds of labeled neurons were observed in the MVZ, namely, HRP-, Fos- or TH-singly-labeled neurons; Fos/HRP-, Fos/TH- or HRP/TH-doubly-labeled neurons; and Fos/HRP/TH-triply-labeled neurons. As for Group II animals, bilateral subdiaphragmatic vagotomy (SDV) or sham operation was performed, followed 4 weeks later by IP injection of LPS. The number of Fos-positive neurons within the Ce and MVZ was significantly lower (P<0.01) in rats having SDV when compared with those receiving sham operation. Our results suggest that part of the peripheral immune information can be conveyed through the vagus to the catecholaminergic neurons in the MVZ, where it is transported to the Ce. The MVZ is a neural relay station in the immune-to-brain communication and might play a significant role in neuroimmuno-modulation via the vagus-MVZ-Ce pathway.

Amygdala↗
PubMed2001

Preliminary X-ray crystallographic analysis of a Ca2+-binding protein human S100A1.

S100A1, a Ca(2+)-binding protein from the S100 protein family, has been crystallized by the vapour-diffusion method using polyethylene glycol 4000 as the precipitant at pH 8.5. The crystal belongs to space group P6(3). The unit-cell parameters are a = b = 57.3, c = 104.7 A. There appear to be two S100A1 molecules in the asymmetric unit. The crystals were stable during exposure to X-rays and diffract to 2.6 A resolution in-house.

Calcium-Binding Proteins↗
PubMed2001

A new implementation of the molecular replacement method using a six-dimensional Patterson vector search.

The current molecular replacement programs are primarily implemented in reciprocal space. In this paper a new implementation in direct (real) space is proposed by matching the model atomic vectors with the vectors in the Patterson vector space using a six-dimensional exhaustive search method. It is shown that this implementation can find the correct rotations and translations of alpha helices in a myoglobin crystal structure using experimental diffraction data at 2 A resolution. A comparison with previous Patterson vector search methods is discussed.

Journal Article↗
PubMed2001

The experimental study on the effect calcitonin gene-related peptide on bone resorption mediated by interleukin-1.

To investigate the effect of calcitonin gene-related peptide (CGRP) on bone resorption mediated by interleukin-1 beta(IL-1 beta) in vitro, the osteoclasts isolated from the long bones of newborn SD rats were co-cultured with osteoblasts on ivory slices placed in 24-well plates. 24 h later, conditioned media containing CGRP and/or IL-1 beta were added to the wells respectively, and continued culturing for 48 h. After the cells were stripped off by ultrasonication, the ivory slices were stained in toludine blue. The number and the total area of resorption lacunae on each slice were measured by computer imaging analysis system. Our results showed that IL-1 beta significantly stimulated bone resorption, but CGRP inhibited the effect mediated by IL-1 beta in a dose-dependent manner. It is suggested that CGRP may inhibit osteoclastic bone resorption through two ways: One is that CGRP functions directly on osteoclasts to block their activation; the other is that CGRP regulates the release of cytokines by osteoblasts and indirectly affects the function of osteoclasts.

Animals↗
PubMed2001

Deriving robust operating rules for the interim control of the Kirazdere reservoir.

A methodology is developed for deriving robust operating rules for the interim control of a multipurpose reservoir. The methodology includes the generation of policies using optimisation models, the derivation of robust operating rules using artificial neural networks and the evaluation of the identified policies through simulation. The approach developed is demonstrated through an application to the Kirazdere Reservoir located on the Kiraz River in Turkey.

Conservation of Natural Resources↗
PubMed2000

Design and synthesis of new steroidal inhibitors of estrogen synthase (aromatase).

The estrogen synthase (aromatase) enzyme system is responsible for the biosynthesis of estrogen hormones in human females. Estrogens are vital for normal growth and development, but will promote the growth of certain breast cancers. Approximately 30-50% of breast cancers are considered to be hormone-dependent. Consequently regulation of estrogen biosynthesis has advanced as a potential therapeutic strategy. This has led to the development of active-site inhibitors, which may have potential for the control of breast cancer. We have recently prepared a number of new steroidal inhibitors that have been evaluated as aromatase inhibitors. These include steroidal A/B-ring isoxazoles and a series of A/B-ring pyrazoles with alkyl- and aryl-substituted nitrogen. In addition, we have developed new chemical procedures for the synthesis of 6beta-hydroxy steroids, which could be key intermediates in the preparation of C-19 inhibitors of aromatase activity.

Antineoplastic Agents↗
PubMed1998

An improved procedure for the preparation of X-ray diffraction-quality crystals of cytochrome p450cam.

A procedure for the crystallization of recombinant cytochrome P450cam has been developed which avoids the difficulties inherent in the glass-capillary free-interface diffusion method reported previously. The surface mutation, Cys334-->Ala (C334A), originally designed to prevent dimer formation and thus improve routine handling of the enzyme, facilitates crystallization by the hanging-drop vapour-diffusion technique. Crystals of (C334A)P450cam grow within 48 h and diffract to beyond 1.2 A at 100 K in-house on a Siemens multiwire area detector. Data have been collected from the camphor-bound form to 1.35 A.

Alanine↗