Enhanced analysis of human breast cancer proteomes using micro-scale solution isoelectrofocusing combined with high resolution 1-D and 2-D gels

Current methods for quantitatively comparing proteomes (protein profiling) have inadequate resolution and dynamic range for complex proteomes such as those from mammalian cells or tissues. More extensive profiling of complex proteomes would be obtained if the proteomes could be reproducibly divided into a moderate number of well-separated pools. But the utility of any prefractionation is dependent upon the resolution obtained because extensive cross contamination of many proteins among different pools would make quantitative comparisons impractical. The current study used a recently developed microscale solution isoelectrofocusing (musol-IEF) method to separate human breast cancer cell extracts into seven well-resolved pools. High resolution fractionation could be achieved in a series of small volume tandem chambers separated by thin acrylamide partitions containing covalently bound immobilines that establish discrete pH zones to separate proteins based upon their pIs. In contrast to analytical 2-D gels, this prefractionation method was capable of separating very large proteins (up to about 500 kDa) that could be subsequently profiled and quantitated using large-pore 1-D SDS gels. The pH 4.5-6.5 region was divided into four 0.5 pH unit ranges because this region had the greatest number of proteins. By using very narrow pH range fractions, sample amounts applied to narrow pH range 2-D gels could be increased to detect lower abundance proteins. Although 1.0 pH range 2-D gels were used in these experiments, further protein resolution should be feasible by using 2-D gels with pH ranges that are only slightly wider than the pH ranges of the musol-IEF fractions. By combining musol-IEF prefractionation with subsequent large pore 1-D SDS-PAGE (>100 kDa) and narrow range 2-D gels (<100 kDa), large proteins can be reliably quantitated, many more proteins can be resolved, and lower abundance proteins can be detected.     

Mechanism of action of RNase T. I. Identification of residues required for catalysis,substrate binding,and dimerization

Escherichia coli RNase T, an RNA-processing enzyme and a member of the DEDD exonuclease superfamily, was examined using sequence analysis and site-directed mutagenesis. Like other DEDD exonucleases, RNase T was found to contain three conserved Exo motifs that included four invariant acidic residues. Mutagenesis of these motifs revealed that they are essential for RNase T activity, indicating that they probably form the RNase T catalytic center in a manner similar to that found in other DEDD exonucleases. We also identified by sequence analysis three short, but highly conserved, sequence segments rich in positively charged residues. Site-directed mutagenesis of these regions indicated that they are involved in substrate binding. Additional analysis revealed that residues within the C-terminal region of RNase T are essential for RNase T dimerization and, consequently, for RNase T activity. These data define the domains necessary for RNase T action, and together with information in the accompanying article, have led to the formulation of a detailed model for the structure and mechanism of action of RNase T.     

In vitro reconstitution of the Schizosaccharomyces pombe alternative excision repair pathway

Schizosaccharomyces pombe alternative excision repair has been shown genetically and biochemically to be involved in the repair of a wide variety of DNA lesions. AER is initiated by a damage-specific endonuclease (Uve1p) that recognizes UV-induced photoproducts, base mispairs, abasic sites, and platinum G-G diadducts and cleaves the DNA phosphodiester backbone 5' to a lesion. Several models exist that employ various mechanisms for damage removal based on the activities of Rad2p, a nuclease thought to be responsible for damage excision in AER. This study represents the first report of the biochemical reconstitution of the AER pathway. A base mispair-containing substrate is repaired in a reaction requiring S. pombe Uve1p, Rad2p, DNA polymerase delta, replication factor C, proliferating cell nuclear antigen, and T4 DNA ligase. Surprisingly, damage is removed exclusively by the 5' to 3' exonuclease activity of Rad2p and not its "flap endonuclease" activity and is absolutely dependent upon the presence of the 5'-phosphoryl moiety at the Uve1p cleavage site.     

A method for global analysis of complex proteomes using sample prefractionation by solution isoelectrofocusing prior to two-dimensional electrophoresis

Two-dimensional electrophoresis is a critical technique for proteome research, but currently available methods are not capable of resolving the >10,000 protein components in most eukaryotic proteomes. We have developed and demonstrated the utility of a novel solution isoelectrofocusing device and method that can reproducibly prefractionate cell extracts into well-defined pools prior to 2D PAGE on a scale directly compatible with the high sensitivity of proteome studies. A prototype device was used to separate metabolically radiolabeled Escherichia coli extracts in method optimization and proof-of-principle experiments. Samples were loaded into separation chambers divided by thin polyacrylamide gels containing immobilines at specific pH values and isoelectrically focused for several hours, which resulted in well-resolved fractions. Total recoveries in the fractionated samples were greater than 80% and most protein spots in the original sample were recovered after this prefractionation step. Nonideal behavior (precipitation/aggregation), typically encountered when unfractionated samples at high protein loads were applied directly to either narrow- or broad-range IPG gels, was dramatically reduced. Hence this approach allows increases in overall protein loads, resolution, and dynamic detection range compared with either alternative prefractionation methods or direct use of parallel narrow pH range gels without sample prefractionation. The pH ranges and number of fractions can be readily adapted to the requirements of specific types of samples and projects. This method should allow quantitative comparisons of at least 10,000 protein components on a series of narrow pH range gels, and protein detection limits are estimated to be 1000 molecules per cell when mammalian proteomes are fractionated into five or more pools.     

疱疹病毒定量PCR的建立

A single pair of oligonucleatide primer selected within a highly conserved region of the DNA polymerase gene in herpesviruses was synthesized. The competitive template DNA purified from cytomegalovirus (CMV) DNA was used to carry out competiitve PCR amplification with herpes simplex virus type 1 (HSV1) DNA (target sequences). And anti-HSV1 effects of acyclovir (ACV) was investigated by the method.The results showed that the efficacy of PCR amplification was equal to each other(the ratio of the quantity of c…     

Evaluation of the Microcirculation in a Rabbit Hemorrhagic Shock Model Using Laser Doppler Imaging

The aim of this study is to evaluate the feasibility of Laser Doppler imaging (LDI) for noninvasive and dynamic assessment of hemorrhagic shock in a rabbit model. A rabbit model of hemorrhagic shock was generated and LDI of the microcirculation in the rabbit ears was performed before and at 0, 30, 60, and 90 min after hemorrhage. The CCD (Charge Coupled Device) image of the ears, the mean arterial pressure (MAP) and the heart rate (HR) were monitored. The mean LDI flux was calculated. The HR of rabbits was significantly (p < 0.05) elevated and the MAP was decreased after hemorrhage, compared to the pre-hemorrhage level. Within the initial 30 min after hemorrhage, the perfusion flux lineally dropped down. In contrast, the MAP values did not differ significantly between the time points of 0 and 30 after hemorrhage (p > 0.05). Both the flux numbers and the red-to-blue color changes on LDI imaging showed the reduction of the microcirculation. LDI imaging is a noninvasive and non-contact approach to evaluate the microcirculation and may offer benefits in the diagnosis and treatment of hemorrhage shock. Further studies are needed to confirm its effectiveness in clinical practice.     

Liquid-Solid Phase-Inversion PLGA Implant for the Treatment of Residual Tumor Tissue after HIFU Ablation

BackgroundHIFU has been shown to be a more suitable alternative for the treatment of primary solid tumors and metastatic diseases than other focal heat ablation techniques due to its noninvasive and extracorporeal nature. However, similar to other focal heat ablation techniques, HIFU is still in need of refinements due to tumor recurrence.MethodsIn this work, we investigated the effectiveness of an adjunct treatment regimen using doxorubicin (DOX)-loaded, injectable, in situ-forming, and phase-inverting PLGA as the second line of defense after HIFU ablation to destroy detrimental residual tumors and to prevent tumor recurrence. All of the statistical analyses were performed using the Statistical Package for the Social Sciences 18.0(SPSS, Inc., Chicago, IL, USA), and p< 0.05 was considered statistically significant. All of the results are presented as the means ± STDEV (standard deviation). For multiple comparisons, ANOVA (differences in tumor volumes, growth rates, apoptosis, proliferation indexes, and Bcl-2 and Bax protein levels) was used when the data were normally distributed with homogenous variance, and rank sum tests were used otherwise. Once significant differences were detected, Student-t tests were used for comparisons between two groups.ResultsOur results revealed that DOX diffused beyond the ablated tissue regions and entered tumor cells that were not affected by the HIFU ablation. Our results also show that HIFU in concert with DOX-loaded PLGA led to a significantly higher rate of tumor cell apoptosis and a lower rate of tumor cell proliferation in the areas beyond the HIFU-ablated tissues and consequently caused significant tumor volume shrinkage (tumor volumes:0.26±0.1,1.09±0.76, and 1.42±0.9cm³ for treatment, sham, and no treatment control, respectively).ConclusionsFrom these results, we concluded that the intralesional injection of DOX-loaded PLGA after HIFU ablation is significantly more effective than HIFU alone for the treatment of solid tumors.