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Biomedical subjects

Anthony T Yeung

Publications and source records attributed to Anthony T Yeung.

11 recordsLinked to original sources

Characterization of a periplasmic S1-like nuclease coded by the Mesorhizobium loti symbiosis island.

DNA sequences encoding hypothetical proteins homologous to S1 nuclease from Aspergillus oryzae are found in many organisms including fungi, plants, pathogenic bacteria, and eukaryotic parasites. One of these is the M1 nuclease of Mesorhizobium loti which we demonstrate herein to be an enzymatically active, soluble, and stable S1 homolog that lacks the extensive mannosyl-glycosylation found in eukaryotic S1 nuclease homologs. We have expressed the cloned M1 protein in M. loti and purified recombinant native M1 to near homogeneity and have also isolated a homogeneous M1 carboxy-terminal hexahistidine tag fusion protein. Mass spectrometry and N-terminal Edman degradation sequencing confirmed the protein identity. The enzymatic properties of the purified M1 nuclease are similar to those of S1. At acidic pH M1 is 25 times more active on single-stranded DNA than on double-stranded DNA and 3 times more active on single-stranded DNA than on single-stranded RNA. At neutral pH the RNase activity of M1 exceeds the DNase activity. M1 nicks supercoiled RF-I plasmid DNA and rapidly cuts the phosphodiester bond across from the nick in the resultant relaxed RF-II plasmid DNA. Therefore, M1 represents an active bacterial S1 homolog in spite of great sequence divergence. The biochemical characterization of M1 nuclease supports our sequence alignment that reveals the minimal 21 amino acid residues that are necessarily conserved for the structure and functions of this enzyme family. The ability of M1 to degrade RNA at neutral pH implies previously unappreciated roles of these nucleases in biological systems.

Alphaproteobacteria↗

Enzymatic mutation detection technologies.

Mutation is as necessary for life as fidelity is in DNA replication. The study of mutations reveals the normal functions of genes, messages, proteins, the causes of many diseases, and the variability of responses among individuals. Indeed, recent mutations that have not yet become polymorphisms are often deleterious and pertinent to the disease history of afflicted individuals. This review discusses the principles behind a variety of methods for the detection of mutations and factors that should be considered in future methods design. One enzymatic approach in particular using orthologs of the CEL I nuclease that show high specificity for all mismatches, appears to be easy and robust. Further developments of this and other methods will allow mutation detection to become an integral component of individualized medicine.

Base Pair Mismatch↗

Altered gene expression in phenotypically normal renal cells from carriers of tumor suppressor gene mutations.

BACKGROUND: The inherently complex signaling networks of tumors result from genetic and epigenetic alterations that occur during cancer initiation and progression. METHODS: In an attempt to identify early molecular changes associated with dominantly inherited predisposition to "two-hit" renal tumors, the expression profiles of primary cultures of phenotypically normal renal epithelial cells from individuals bearing a germline mutation in either the von Hippel-Lindau (VHL) or the tuberous sclerosis complex (TSC) gene were compared to that of renal epithelial cells from control nonmutation carriers by microarray analysis. RESULTS: Reliability of the microarray data from pooled samples was confirmed by real-time RT-PCR. Principal Component Analysis revealed substantial differences in the gene expression profiles of the renal epithelial cells from VHL and TSC mutation carriers. In several instances, the microarray data confirm our present knowledge of the cellular pathways affected by biallelic VHL and TSC mutations. CONCLUSIONS: These findings demonstrate that heterozygosity for a mutant tumor suppressor gene may alter the expression profiles of phenotypically normal epithelial cells in a gene-specific manner. Detectable effects of "one-hit" represent early molecular changes in tumorigenesis that may serve as targets for chemopreventive intervention.

Biomarkers, Tumor↗

Evaluation of dual-labeled fluorescent DNA probe purity versus performance in real-time PCR.

Real-time PCR technology using dual-labeled fluorescent oligonucleotide probes allows for sensitive, specific, and quantitative determination of mRNA or DNA targets. Historically, dual-labeled probes have been the most expensive reagent in real-time PCR because of the postsynthesis high-performance liquid chromatography (HPLC) and/or gel purification steps required due to limitations in traditional synthesis chemistry. The recent availability of quencher reagents that allow the 3' quencher incorporation as part of the on-machine synthesis has presented the possibility that probes, when carefully synthesized, may be used without extensive postsynthesis purification. This would substantially reduce cost, making the synthesis of dual-labeled fluorescent probes affordable to any DNA synthesis laboratory. The Nucleic Acids Research Group (NARG) of the Association of Biomolecular Resource Facilities (ABRF) (Santa Fe, NM, USA) tested the hypothesis that now any DNA synthesis laboratory is capable of making quality dual-labeled fluorescent probes suitable for real-time PCRs without the need for postsynthesis purification. Members of the DNA synthesis community synthesized dual-labeled human beta-actin probes and submitted them for quality and functional analysis. We found that probes that were at least 20% pure had the same efficiency as those near 100% purity, but the sensitivity of the assay was reduced as the level of purity decreased.

Chromatography, High Pressure Liquid↗

Enhanced cisplatin cytotoxicity by disturbing the nucleotide excision repair pathway in ovarian cancer cell lines.

Ovarian cancer is the leading cause of death among women from gynecological malignancies inthe United States. Resistance to the chemotherapeutic agent cisplatin isa major limitation for the successful treatment of ovarian cancer. In an effort to overcome the cisplatin resistance problem in ovarian cancer treatment, we have sought to enhance cisplatin cytotoxicity by perturbing the nucleotide excision repair (NER) pathway. The NER pathway is responsible for repairing cisplatin bound to DNA. Expression of one of the NER components, ERCC1, is correlated with cisplatin drug resistance. Hence, we targeted ERCC1 by antisense RNA methodologies, and we show that we could sensitize a relatively sensitive A2780 cell line and also the highly resistant OVCAR10 cell line to cisplatin by expressing antisense ERCC1 RNA in them as measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. The A2780 cell lines expressing antisense ERCC1 had 1.9-8.1-fold enhancements in cisplatin sensitivity. The OVCAR10 antisense ERCC1 cell lines had IC(50) values ranging from 2.28 microM to 2.7 microM cisplatin as compared with 9.52 micro M for control OVCAR10 cells. The OVCAR10 antisense ERCC1 cells also show reduced DNA-damage repair capacity as assessed by host cell reactivation. Furthermore, immunocompromised mice transplanted with the antisense cell lines survived longer than the mice bearing control cells after response to cisplatin treatment. These data suggest that it is possible to substantially enhance the cisplatin cytotoxicity by disturbing the NER pathway in cisplatin-resistant cell lines and to enhance the survival capacity of mice in an ovarian cancer xenograft model.

Antineoplastic Agents↗

Advances in endoscopic disc and spine surgery: foraminal approach.

Endoscopic spine surgery is evolving rapidly due to improvements in surgical technique, endoscope design, and instrumentation. The current technique expands on the basic features and principles of Kambin's access to the spine through the triangular zone. A standardized method for foraminal surgery, the Yeung Endoscopic Spine System (YESS) (Richard Wolf Surgical Instrument Company, Vernon Hills, Illinois, USA) technique is proposed: (1) A protocol for optimal instrument placement by identifying the skin window, annular window, anatomic disc center, and disc inclination plane through topographical coordinates calculated by lines drawn on the skin from the C-Arm image. Adjustments in the trajectory are made to accommodate individual anatomic considerations and the pathologic disorders to be accessed. (2) Evocative Chromo-Discography (Richard Wolf Surgical Instrument Company, Vernon Hills, Illinois, USA). (3) Selective Endoscopic Discectomy (Richard Wolf Surgical Instrument Company, Vernon Hills, Illinois, USA). (4) Thermal discoplasty and annuloplasty. (5) Endoscopic foraminoplasty. (6) Accessing the epidural space in the axilla between the traversing and exiting nerve root. (7) Partially resecting the posterior annulus to get beneath the herniated fragment, if needed. This technique allows access to the epidural space from the lumbar disc as far cephalad as the middle of the vertebral body or approximately 2-3 mm caudally. The foraminal approach is routinely accessible from T-10 to L4-5. L5-S1 can be accessed with special techniques that include foraminoplasty of the lateral facet. Surgical results continue to improve, consistent with refinement of indications and techniques for specific conditions treatable by this endoscopic method.

Cadaver↗

Use of fluorescent DNA-intercalating dyes in the analysis of DNA via ion-pair reversed-phase denaturing high-performance liquid chromatography.

SYBR Green 1 is an asymmetrical cyanine DNA-binding dye that provides an opportunity for increasing the sensitivity of nucleic acid detection when used in conjunction with gel electrophoresis. In this paper, we summarize the general properties and specific uses of SYBR green 1 in ion-pair reversed-phase denaturing high-performance liquid chromatography (IP DHPLC). We describe several applications for the WAVE DHPLC platform that illustrate the generic potential of such intercalating dyes in mutation detection and gene expression profiling. We show that SYBR Green 1 obviates the need to use end-labeled oligodeoxynucleotides for the sensitive detection of nucleic acids during chromatography. Moreover the incorporation of SYBR Green 1 into samples and elution buffers does not impair resolution and has no significant effect on the retention times of DNA fragments compared with dye-free DHPLC.

Base Pair Mismatch↗

Posterolateral transforaminal selective endoscopic discectomy and thermal annuloplasty for chronic lumbar discogenic pain: a minimal access visualized intradiscal surgical procedure.

BACKGROUND CONTEXT: Chronic lumbar discogenic pain (CLDP) impairs the patient's physical abilities to function within the normal physiologic loading ranges of activities of daily living. The pathogenesis of CLDP is multifactorial and not well understood. Conservative therapeutic regimens often fail to achieve sufficient pain relief. Surgical options vary greatly in surgical invasiveness as well as outcome. Definitive surgical treatment is often 360-degree fusion. The morbidity associated with this approach is significant, considering that only 65% to 80% of patients obtain satisfactory clinical results. This has spawned interest in minimally invasive surgical options, such as intradiscal electrothermal therapy (IDET; ORATEC Interventions, Inc., Menlo Park, CA), but results are conflicting. PURPOSE: The authors describe their surgical technique of minimal access posterolateral transforaminal selective endoscopic discectomy (SED) and bipolar radiofrequency thermal annuloplasty to treat CLDP. The procedure's rationale is based on the hypothesis that annular defects are the focal points of chronic exposure between neural sensory receptors in the defect and the inflammatogenic nucleus pulposus. In contrast to other percutaneous procedures, this technique allows direct visualization and targeting of the disc nucleus and annular fissures. Our 2-year clinical result is reported. STUDY DESIGN/SETTING: This is a retrospective review of consecutive surgical cases performed by one surgeon (ATY). The procedures were carried out from January 1997 to December 1999. Each patient has a minimum postoperative follow-up of 2 years. PATIENT SAMPLE: A total of 113 patients met the generally accepted clinical criteria for chronic lumbar discogenic pain and were selected for the procedure. OUTCOME MEASURES: Two outcome measures were used for clinical assessment: a surgeon-based modified MacNab method and a patient-based questionnaire. A mandatory poor result was given to any patient who had repeat spine surgery at the same level or has indicated dissatisfaction with the surgical result on the questionnaire response. METHOD: After meeting CLDP selection criteria, provocation contrast/indigo carmine dye discography was performed. This test was used to confirm the suspected discs as pain generators. The subject surgery then followed. Only cases with one and two levels of confirmed painful discs were entered into the study. The nonoperating author (PMT) analyzed the data. RESULTS: Using the surgeon assessment method, 17 patients (15%) had excellent results, 32 patients (28.3%) had good results, 34 patients (30.1%) had fair results and 30 patients (26.5%) had poor results. Of the 30 patients in the poor result group, 12 reported either no improvement or worsening, and refused further surgical treatment. Of the remaining 18 patients in the poor group, 8 had spinal fusion, 3 had laminectomy and 7 had repeat spinal endoscopic surgery. The patient-based questionnaire yielded similar percentages in each category. However, only 73.5% of the 113 patients returned the survey questionnaire. There were no aborted procedures, unexpected hemorrhage, device-related complications, neurologic deficits, perioperative deaths or late instability. CONCLUSIONS: Posterolateral transforaminal SED and radiofrequency thermal annuloplasty were used to interrupt the purported annular defect pain sensitization process, thought to be necessary in the genesis of chronic lumbar discogenic pain. Lack of clinical benefit from the subject procedure did not degrade any subsequent surgical or nonsurgical treatment options. The experience gained from this study warrants further investigation into the cellular and molecular processes that provided back pain relief in these patients.

Adult↗

Analyzing alkaline proteins in human colon crypt proteome.

Normal human colon crypt protein extract was resolved by two-dimensional gel electrophoresis using pH 6-11 immobilized pH gradient strips in the first dimension. The optimized isoelectric focusing protocol includes cup-loading sample application at the anode and 1.2% hydroxyethyl disulfide (DeStreak), 15% 2-propanol and 5% glycerol in the rehydration buffer. Spots were well resolved across the entire pH range up to 11. A total of 311 protein spots were identified by mass spectrometry and peptide mass mapping. After combining isoforms, 231 nonredundant proteins were grouped into 16 categories according to their subcellular locations, and 17 categories according to their physiological functions. Histone proteins, ribosomal proteins and mitochondrial proteins were among the well-resolved highest p/ proteins. Application of this protocol to the analysis of normal and neoplastic colon crypts will contribute to the proteomic study of colorectal tumorigenesis since a significant portion of the human proteins is in basic pH range.

Cell Extracts↗