Biomedical subjects

Z Shao

Publications and source records attributed to Z Shao.

At least 55 records · Page 3Linked to original sources

PubMed2000

Thermal noise reduction of mechanical oscillators by actively controlled external dissipative forces

We show that the thermal fluctuations of very soft mechanical oscillators, such as the cantilever in an atomic force microscope (AFM), can be reduced without changing the stiffness of the spring or having to lower the environment temperature. We derive a theoretical relationship between the thermal fluctuations of an oscillator and an actively controlled external dissipative force. This relationship is verified by experiments with an AFM cantilever where the external active force is coupled through a magnetic field. With simple instrumentation, we have reduced the thermal noise amplitude of the cantilever by a factor of 3.4, achieving an apparent temperature of 25 K with the environment at 295 K. This active noise reduction approach can significantly improve the accuracy of static position or static force measurements in a number of practical applications.

Journal Article↗

PubMed2000

Probing the structure of monomers and dimers of the bacterial virus phi29 hexamer RNA complex by chemical modification.

All dsDNA viruses multiply their genome and assemble a procapsid, a protein shell devoid of DNA. The genome is subsequently inserted into the procapsid. The bacterial virus phi29 DNA translocating motor contains a hexameric RNA complex composed of six pRNAs. Recently, we found that pRNA dimers are building blocks of pRNA hexamers. Here, we report the structural probing of pRNA monomers and dimers by chemical modification under native conditions and in the presence or absence of Mg2+. The chemical-modification pattern of the monomer is compared to that of the dimer. The data strongly support the previous secondary-structure prediction of the pRNA concerning the single-stranded areas, including three loops and seven bulges. However, discrepancies between the modification patterns of two predicted helical regions suggest the presence of more complicated, higher-order structure in these areas. It was found that dimers were formed via hand-in-hand and head-to-head contact, as the interacting sequence of the right and left loops and all bases in the head loop were protected from chemical modification. Cryoatomic force microscopy revealed that the monomer displayed a check-mark shape and the dimer exhibited an elongated shape. The dimer was twice as long as the monomer. Direct observation of the shape and measurement of size and thickness of the images strongly support the conclusion from chemical modification concerning the head-to-head contact in dimer formation. Our results also suggest that the role for Mg2+ in pRNA folding is to generate a proper configuration for the right and head loops, which play key roles in this symmetrical head-to-head organization. This explains why Mg2+ plays a critical role in pRNA dimer formation, procapsid binding, and phi29 DNA packaging.

Bacillus Phages↗

PubMed2000

Optimizing the window size for multiple least squares fitting of electron energy loss spectra.

The purpose of this study was to determine the effect of the fitting window size used with the linear least squares fit method when quantitating trace elements with electron energy loss spectroscopy. Theory and computer simulation with a simple model of two 'signals' show that, when the background underlying the signal is slowly varying and the signal is localized, there exists a minimum window optimal for fitting raw spectra. The width of the minimum fitting window can be determined directly from the reference standard spectrum of the signal alone and the estimates of signal and background are related. Use of narrower than the minimum window will increase the fitting uncertainty of the signal and yield less reliable results. More complicated experimental spectra must be fitted to more than two standards and a simple analytical expression of the minimum fitting window cannot be derived, but can be determined empirically. Our study shows that the empirical value obtained from experimental spectra is only slightly larger than the theoretical value derived from the simple model, indicating that this conclusion is still valid. When fitting difference spectra with a slowly varying background, the estimates of signal and background are independent and windows wider than the size of the signal will yield the same fitting uncertainty. In the presence of a non-slowly varying background, common in difference spectra, the minimum window size depends on the fine structure of the signal and the background.

Calcium↗

PubMed2000

Atomic force microscopy in structural biology: from the subcellular to the submolecular.

Atomic force microscopy (AFM) is capable of generating images within ranges of resolution that are of particular interest in biology. Although atomic resolution may not be possible with biological samples, a great deal of information can still be obtained from images that provide structures at a slightly lower level of resolution. The submolecular resolution images of bacteriorhodopsin and the chaperonin GroES, which revealed, respectively, individual loops and beta-turns, confirmed and complemented other structural investigations, while the molecular-level features in images of membrane-bound VacA, a cytotoxin from Helicobacter pylori, immediately suggested the possibility, subsequently proven, of channel-forming ability. A series of images with macromolecular resolution directly provided details on the mechanisms by which RNA polymerase nonspecifically translocates along DNA, and images with subcellular resolving power of erythrocytic cellular membranes showed, with unambiguous clarity, linear arrays of molecular complexes. In this review, we will describe some of the most biologically relevant findings that have been obtained with AFM within ranges of resolution from the submolecular to the molecular, and from the macromolecular to the subcellular. Furthermore, we will describe some of the sample conditions and imaging environments that are likely important to achieve a particular level of resolution.

Bacterial Proteins↗

PubMed2000

PNA-dependent gene chemistry: stable coupling of peptides and oligonucleotides to plasmid DNA.

Two approaches are described for stably conjugating peptides, proteins and oligonucleotides onto plasmid DNA. Both methods use a peptide nucleic acid (PNA) clamp, which binds irreversibly and specifically to a binding site cloned into the plasmid. The first approach uses a biotin-conjugated PNA clamp that can be used to introduce functional biotin groups onto the plasmid to which streptavidin can bind. Atomic force microscopy images of linearized plasmid show streptavidin localized at the predicted PNA binding site on the DNA strand. Peptides and oligonucleotides containing free thiol groups were conjugated to maleimide streptavidin, and these streptavidin conjugates were bound to the biotin-PNA-labeled plasmid. In this way, peptides and oligonucleotides could be brought into stable association with the plasmid. A second approach used a maleimide-conjugated PNA clamp. Methods are described for conjugating thiolated peptides and oligonucleotides directly to the maleimide-PNA-DNA hybrid. This straightforward technology offers an easy approach to introduce functional groups onto plasmid DNA without disturbing its transcriptional activity.

Animals↗

PubMed2000

[Inner-hemangioma ligation and pingyangmycin injection in hemangioma (with 30 cases report)].

OBJECTIVE: To provide a simple and safe surgical approach in treating cavernous and racemose hemangioma. METHODS: First cross-ligate the hemangioma, as to divide the tumor into independent compartments, them inject pingyangmycin into the compartments respectively. RESULTS: There are 30 cases in this group (35 hemangiomas). Twenty-four cases (28 hemangiomas) were totally cured, accounting for 80%; Six cases were improved (7 hemangiomas) for 20%; There was no ineffective case. CONCLUSION: "Inner-hemangioma ligation and injection of pingyangmycin" has broad surgical indications, with desirable outcome in treating cavernous and racemose hemangioma, especially those unsuitable for surgery, or ineffective simply by inner-hemangioma injection.

Adolescent↗

PubMed2000

[Genistein exerts multiple suppressive effects on human breast carcinoma cells].

OBJECTIVE: To study the suppressive effects of genistein on progression of breast carcinoma. METHODS: In vitro cell culture, animal studies, zymography, Northern and Western blot were used in this study. RESULTS: Genistein inhibited in vitro invasion of MCF-7 and MDA-MB-231 cells. This inhibition was associated with down-regulation of MMP-9 and up-regulation of TIMP-1. These effects were also demonstrated in vivo. Genistein inhibited growth of the breast cancer cells transplanted in nude mice. The growth inhibitory effect was associated with increased tumor cell apoptosis, and up-regulated p21WAF1/CIP1 expression. Furthermore, genistein inhibited angiogenesis as shown by decreased vessel density and decreased production and release of VEGF and TGF-beta 1. CONCLUSION: Genistein exerts multiple suppressive effects on breast carcinoma. It may help halt its progression.

Animals↗

PubMed2000

[Clinical implication of apoptosis of breast cancer induced by preoperative chemotherapy].

OBJECTIVE: To investigate whether apoptosis could be induced by preoperative chemotherapy in breast cancer patients, and determine if it is correlated with clinical response and patients' survival. METHODS: Apoptosis was assayed by in situ DNA end labeling and receptors for estrogen and progesterone were examined immunohistochemically. Comparison of the differences in apoptotic index (AI) and clinical response was made between patients received preoperative chemotherapy and those did not, using the one tailed Student's t-test. The Kaplan-Meier method was utilized to calculate relapse-free and overall survival time. Cox proportional hazards model was used to conduct multivariate analysis of prognostic factors and differences in overall survival and relapse-free survival after adjustment for other variables. RESULTS: Apoptosis was induced by preoperative chemotherapy, especially in lymph node positive patients. Post-chemotherapy AI correlated with better clinical response and increased patient survival, including relapse (disease) free survival and overall survival. CONCLUSION: The extent of apoptosis induced by preoperative chemotherapy of primary breast cancer may possibly predict an individual patient's overall response to treatment.

Adult↗

PubMed2000

[Establishment and identification of B cell lines with PNH phenotype].

OBJECTIVE: To establish B cell lines from patients with paroxysmal nocturnal haemoglobinuria (PNH) and normal controls. METHODS: B lymphoid cells prepared from PNH patients and normal controls were infected with EB virus. Phenotype of the immortal cells was analyzed by flow cytometry. The immunomagnetic beads were used to select CD(55)(-) and CD(59)(-) B lymphoid cells, which were cloned by limiting dilution. The clonality of the cloned cells was confirmed by testing the pattern of Ig rearrangements using polymerase chain reaction (PCR). RESULTS: From 2 PNH patients and 2 normal controls, CD(55)(-) and CD(59)(-) B lymphoblastoid cell lines were established which had PNH phenotype and normal phenotype, respectively. CONCLUSION: B lymphoid cell lines from PNH patients can be obtained by EB virus infection.

B-Lymphocytes↗

PubMed2000

[Study of the expression and function of c-kit receptor on the bone marrow mononuclear cells of the patients with myelodysplastic syndromes].

OBJECTIVE: To determine the expression and function of c-kit receptor on bone marrow mononuclear cells (BMMNC) of the patients with myelodysplastic syndromes. METHODS: c-kit protein (CD(117)) was detected by direct immunofluorescence assay, c-kit mRNA by reverse transcriptive polymerase chain reaction (RT-PCR) and the function of c-kit receptor by cell culture. RESULTS: c-kit protein expression in MDS was significantly higher than that in control [(8.58 +/- 5.28)% vs (3.04 +/- 1.49)%, P < 0.05]. c-kit protein expression in RA patients was significantly lower than that in RAEB/RAEB-t patients [(5.12 +/- 2.13)% vs (10.01 +/- 5.07)%, P < 0.05]. The rate of c-kit protein expression was (32.43 +/- 18.16)% in AML cases transformed from MDS (t-AML). c-kit mRNA expression in MDS was correlated with c-kit protein expression. Addition of interleukin-3 (IL-3), erythropoietin (Epo) and stem cell factor (SCF) to the semi-solid culture of BMMNC showed significant stimulating effects on CFU-GM and BFU-E formation in normal controls, but did not in MDS patients. CONCLUSION: The protein and mRNA expressions of c-kit receptor on BMMNC in MDS are higher than those in normal control, and the function of this receptor in MDS is abnormal.

Adolescent↗

PubMed2000

[Study on clinical and laboratory features of preleukemia patients].

OBJECTIVE: To explore prospective diagnostic criteria for preleukemia. METHODS: A case control study of the discrepancies of clinical and laboratory features between patients with preleukemia and those with chronic aplastic anemia (CAA) or atypical paroxysmal nocturnal hemoglubinuria (a-PNH). RESULTS: There were eight variables of significance: (1) lymphocytoid micromegakaryocytes in marrow; (2) immature granulocytes in peripheral blood; (3) >or= 2% myeloblasts in marrow; (4) positive periodic acid schiff (PAS) staining of nucleated erythrocytes; (5) myeloid differentiation index >or= 1.8; (6) clonal karyotypic abnormalities; (7) negative sister chromatid differentiation; (8) > 4.0 cluster/colony ratio of granulocyte-macrophage colony-forming units (CFU-GM). The following criteria was assigned: A: To meet (1) and at least two of the other seven variables; B: To meet at least four of the eight variables. All of the patients with preleukemia met A or B and none of the patients with CAA or a-PNH did. CONCLUSION: Preleukemia is different from CAA or a-PNH. It has its own clinical and laboratory features, which may be useful for prospective diagnosis.

Adolescent↗

PubMed2000

[Expression and insecticidal characteristics of cry3A in Lepidoptera-specific Bacillus thuringiensis].

The cry3A gene coding Coleoptera-specific protein was transformed into a wild strain YBT803-1 by electroporation, A transformant BMBY-001 was obtained. SDS-PAGE analysis demonstrated that cry3A could express 64 kD protein in BMBY-001. However, the expression of cry1Ab and cry1Ac were effected. Bioassay showed that BMBY-001 was not only highly toxic to Phyllodecta vulgatissima larva(LC50 0.413 microL/mL) but also toxic to Plutella xylostella(LC50 3.319 microL/mL).

Animals↗

PubMed1999

A dominant negative mutant of Helicobacter pylori vacuolating toxin (VacA) inhibits VacA-induced cell vacuolation.

Most Helicobacter pylori strains secrete a toxin (VacA) that causes structural and functional alterations in epithelial cells and is thought to play an important role in the pathogenesis of H. pylori-associated gastroduodenal diseases. The amino acid sequence, ultrastructural morphology, and cellular effects of VacA are unrelated to those of any other known bacterial protein toxin, and the VacA mechanism of action remains poorly understood. To analyze the functional role of a unique strongly hydrophobic region near the VacA amino terminus, we constructed an H. pylori strain that produced a mutant VacA protein (VacA-(Delta6-27)) in which this hydrophobic segment was deleted. VacA-(Delta6-27) was secreted by H. pylori, oligomerized properly, and formed two-dimensional lipid-bound crystals with structural features that were indistinguishable from those of wild-type VacA. However, VacA-(Delta6-27) formed ion-conductive channels in planar lipid bilayers significantly more slowly than did wild-type VacA, and the mutant channels were less anion-selective. Mixtures of wild-type VacA and VacA-(Delta6-27) formed membrane channels with properties intermediate between those formed by either isolated species. VacA-(Delta6-27) did not exhibit any detectable defects in binding or uptake by HeLa cells, but this mutant toxin failed to induce cell vacuolation. Moreover, when an equimolar mixture of purified VacA-(Delta6-27) and purified wild-type VacA were added simultaneously to HeLa cells, the mutant toxin exhibited a dominant negative effect, completely inhibiting the vacuolating activity of wild-type VacA. A dominant negative effect also was observed when HeLa cells were co-transfected with plasmids encoding wild-type and mutant toxins. We propose a model in which the dominant negative effects of VacA-(Delta6-27) result from protein-protein interactions between the mutant and wild-type VacA proteins, thereby resulting in the formation of mixed oligomers with defective functional activity.

Amino Acid Sequence↗

PubMed1999

Recombination and chimeragenesis by in vitro heteroduplex formation and in vivo repair.

We describe a simple method for creating libraries of chimeric DNA sequences derived from homologous parental sequences. A heteroduplex formed in vitro is used to transform bacterial cells where repair of regions of non-identity in the heteroduplex creates a library of new, recombined sequences composed of elements from each parent. Heteroduplex recombination provides a convenient addition to existing DNA recombination methods ('DNA shuffling') and should be particularly useful for recombining large genes or entire operons. This method can be used to create libraries of chimeric polynucleotides and proteins for directed evolution to improve their properties or to study structure-function relationships. We also describe a simple test system for evaluating the performance of DNA recombination methods in which recombination of genes encoding truncated green fluorescent protein (GFP) reconstructs the full-length gene and restores its characteristic fluorescence. Comprising seven truncated GFP constructs, this system can be used to evaluate the efficiency of recombination between mismatches separated by as few as 24 bp and as many as 463 bp. The optimized heteroduplex recombination protocol is quite efficient, generating nearly 30% fluorescent colonies for recombination between two genes containing stop codons 463 bp apart (compared to a theoretical limit of 50%).

Cloning, Molecular↗

PubMed1999

Images of oligomeric Kv beta 2, a modulatory subunit of potassium channels.

The Shaker type voltage-gated potassium (K+) channel consists of four pore-forming Kv alpha subunits. The channel expression and kinetic properties can be modulated by auxiliary hydrophilic Kv beta subunits via formation of heteromultimeric Kv alpha-Kv beta complexes. Because each (Kv alpha)4 could recruit more than one Kv beta subunit and different Kv beta subunits could potentially interact, the stoichiometry of alpha-beta and beta-beta complexes is therefore critical for understanding the functional regulation of Shaker type potassium channels. We expressed and purified Kv beta 2 subunit in Sf9 insect cells. The purified Kv beta 2, examined by atomic force and electron microscopy techniques, is found predominately as a square-shaped tetrameric complex with side dimensions of 100 x 100 A2 and height of 51 A. Thus, Kv beta 2 is capable of forming a tetramer in the absence of pore-forming alpha subunits. The center of the Kv beta 2 complex was observed to be the most heavily stained region, suggesting that this region could be part of an extended tubular structure connecting the inner mouth of the ion permeation pathway to the cytoplasmic environment.

Amino Acid Sequence↗

PubMed1999

Stabilization of chromatin structure by PRC1, a Polycomb complex.

The Polycomb group (PcG) genes are required for maintenance of homeotic gene repression during development. Mutations in these genes can be suppressed by mutations in genes of the SWI/SNF family. We have purified a complex, termed PRC1 (Polycomb repressive complex 1), that contains the products of the PcG genes Polycomb, Posterior sex combs, polyhomeotic, Sex combs on midleg, and several other proteins. Preincubation of PRC1 with nucleosomal arrays blocked the ability of these arrays to be remodeled by SWI/SNF. Addition of PRC1 to arrays at the same time as SWI/SNF did not block remodeling. Thus, PRC1 and SWI/SNF might compete with each other for the nucleosomal template. Several different types of repressive complexes, including deacetylases, interact with histone tails. In contrast, PRC1 was active on nucleosomal arrays formed with tailless histones.

Animals↗

PubMed1999

VacA from Helicobacter pylori: a hexameric chloride channel.

VacA is a unique protein toxin secreted by the human pathogen Helicobacter pylori. At a neutral pH, the cytotoxin self-associates into predominantly dodecameric complexes. In this report, we show that at an acidic pH, VacA forms anion selective channels in planar phospholipid bilayers. Similar to several other chloride channels, the VacA channel exhibits a moderate selectivity for anions over cations (P(Cl):P(Na) = 4.2:1), inhibition by the blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and a permeability sequence, SCN- >> I- > Br- > Cl- > F, consistent with a 'weak field strength' binding site for the permeant anion. Single channel recordings reveal rapid transitions (486 s(-1)) between the closed state and a single open state of 24 pS (+60 mV, 1.5 M NaCl). Evaluation of the rate of increase in macroscopic current as well as atomic force microscopy suggest that this VacA channel is a hexamer, formed by the assembly of membrane-bound monomers. Not only are these VacA channels likely to play an important role in the pathological activity of this toxin, but they may also serve as a model system to further investigate the mechanism of anion selectivity in general.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1999

The vacuolating toxin from Helicobacter pylori forms hexameric pores in lipid bilayers at low pH.

Pathogenic strains of Helicobacter pylori secrete a cytotoxin, VacA, that in the presence of weak bases, causes osmotic swelling of acidic intracellular compartments enriched in markers for late endosomes and lysosomes. The molecular mechanisms by which VacA causes this vacuolation remain largely unknown. At neutral pH, VacA is predominantly a water-soluble dodecamer formed by two apposing hexamers. In this report, we show by using atomic force microscopy that below pH approximately 5, VacA associates with anionic lipid bilayers to form hexameric membrane-associated complexes. We propose that water-soluble dodecameric VacA proteins disassemble at low pH and reassemble into membrane-spanning hexamers. The surface contour of the membrane-bound hexamer is strikingly similar to the outer surface of the soluble dodecamer, suggesting that the VacA surface in contact with the membrane is buried within the dodecamer before protonation. In addition, electrophysiological measurements indicate that, under the conditions determined by atomic force microscopy for membrane association, VacA forms pores across planar lipid bilayers. This low pH-triggered pore formation is likely a critical step in VacA activity.

Bacterial Proteins↗