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Biomedical subjects

K Hung

Publications and source records attributed to K Hung.

16 recordsLinked to original sources
PubMed2002

Detection of tumor mutations in the presence of excess amounts of normal DNA.

Mutations are important markers in the early detection of cancer. Clinical specimens such as bodily fluid samples often contain a small percentage of mutated cells in a large background of normal cells. Thus, assays to detect mutations leading to cancer need to be highly sensitive and specific. In addition, they should be possible to carry out in an automated and high-throughput manner to allow large-scale screening. Here we describe a screening method, termed PPEM (PNA-directed PCR, primer extension, MALDI-TOF), that addresses these needs more effectively than do existing methods. DNA samples are first amplified using peptide nucleic acid (PNA)-directed PCR clamping reactions in which mutated DNA is preferentially enriched. The PCR-amplified DNA fragments are then sequenced through primer extension to generate diagnostic products. Finally, mutations are identified using matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. This method can detect as few as 3 copies of mutant alleles in the presence of a 10,000-fold excess of normal alleles in a robust and specific manner. In addition, the method can be adapted for simultaneous detection of multiple mutations and is amenable to high-throughput automation.

Alleles↗
PubMed2002

A matrix-assisted laser desorption/ionization time-of-flight based method for screening the 1691G --> A mutation in the factor V gene.

A point mutation, 1691 G --> A in the coagulation factor V gene results in an Arg506 --> Gln amino acid substitution in the factor V molecule. This mutation, defined as factor VLEIDEN, results in activated protein C (APC) resistance and is the most common genetic risk factor for familial thrombophilia. A new mini-sequencing method using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was developed for the screening of the 1691G --> A substitution in factor V. In this method, a fragment of genomic DNA containing the 1691st base is first amplified, followed by mini-sequencing in the presence of dGTP and ddATP, ddCTP, and ddTTP. In this manner, the primer is extended by one base from one allele and two bases from the other allele. The extended products are analyzed using MALDI-TOF mass spectrometry. The base at position 1691 is identified based on the number of nucleotides added. We have used this method to genotype 16 APC-resistant patients previously identified by conventional methods and 11 normal control samples. The genotypes of all samples were correctly identified. This method is accurate, fast, and potentially allows for simultaneous multiplex genotyping of a number of mutation sites associated with thrombophilia and clot formation.

Activated Protein C Resistance↗
PubMed2001

Antisera against endogenous opioids increase the nocifensive response to formalin: demonstration of inhibitory beta-endorphinergic control.

The roles of endogenous opioid peptides in the brain in the modulation of nocifensive responses to formalin in ICR mice were studied. Mice were pretreated intracerebroventricularly (i.c.v.) with rabbit antiserum against beta-endorphin, [Leu5]enkephalin, [Met5]enkephalin or dynorphin A-(1-17) 1 h prior to intraplantar injection of formalin (0.5%, 25 microl) and the nocifensive licking responses were then observed. Pretreatment of mice with antiserum against beta-endorphin enhanced the second phase, but not the first phase of the nocifensive responses to formalin. Pretreatment with antiserum against [Leu5]enkephalin also caused a small but statistically significant enhancement of the second phase, but not the first phase of nocifensive responses to formalin. On the other hand, pretreatment with antiserum against [Met5]enkephalin or dynorphin A-(1-17) did not affect the nocifensive response to formalin. Our results indicate that beta-endorphinergic, and to a lesser extent, [Leu5]enkephalinergic systems are activated at the supraspinal sites to attenuate the nocifensive responses to formalin stimulation.

Animals↗
PubMed2000

A new MALDI-TOF based mini-sequencing assay for genotyping of SNPS.

A new MALDI-TOF based mini-sequencing assay termed VSET was developed for genotyping of SNPs. In this assay, specific fragments of genomic DNA containing the SNP site(s) are first amplified, followed by mini-sequencing in the presence of three ddNTPs and the fourth nucleotide in the deoxy form. In this way, the primer is extended by only one base from one allele, while it is typically extended by two bases from another allele. The products are then analyzed using MALDI-TOF mass spectrometry. The genotype of the SNP site is identified based on the number of nucleotides added. This assay has been examined using both synthetic and genomic DNA samples. In addition, multiplexed assays were successfully performed to genotype four SNP sites in a single tube. The main aspect of this assay is that it can overcome the key problems associated with the currently used mini-sequencing methods. First, it significantly reduces the stringent high-resolution and extensive desalting requirements that are essential to the pinpoint assay. Second, it avoids the long extension problem associated with the PROBE assay.

DNA Mutational Analysis↗
PubMed2000

Antigen-specific cancer immunotherapy using a GM-CSF secreting allogeneic tumor cell-based vaccine.

Granulocyte-macrophage colony-stimulating factor (GM-CSF)-transduced autologous tumor cell-based vaccines are currently one of the major forms of cancer vaccines. However, the preparation of GM-CSF-transduced autologous tumor vaccines is time-consuming and technically challenging. In addition, the host antigen presenting cells, rather than the tumor vaccine cells themselves, present tumor-specific antigens and prime the host T cells. Therefore, we tested the efficacy of antigen-specific allogeneic tumor vaccines. We used human papillomavirus 16 (HPV-16) E7 protein as a model tumor antigen, which is associated with the development of most cervical carcinoma. B16, a C57BL/6 (H-2(b)) derived melanoma cell line, was genetically engineered to produce GM-CSF alone (B16GM), HPV-16 E7 alone (B16E7), or both (B16GME7). These vaccine cells were injected into BALB/c (H-2(d)) mice (10(6) cells/mouse). Two weeks later, mice were challenged with 10(5) live HPV-16 E7(+) BL-1 (H-2(d)) tumor cells and monitored for tumor progression twice weekly. To determine the effective cell population in the antitumor immunity elicited by B16GME7, we carried out in vivo antibody depletion experiments using CD4 and CD8 specific antibodies. In addition, as a measure of the immune responses produced by B16GME7, we performed an in vitro cytotoxic T lymphocyte assay using a standard chromium release method. We found that all of the mice vaccinated with B16GME7 remained tumor free 49 days post-BL-1 challenge. In contrast, mice vaccinated with B16GM and B16E7 did not show any tumor protection against a similar dose of BL-1 cells. Furthermore, the antitumor immunity produced by B16GME7 was dependent on both CD4 and CD8 T cells. In addition, E7-specific cytotoxic T lymphocyte activity could be readily demonstrated in mice immunized with B16GME7. These results suggest that allogeneic tumor cells transduced with GM-CSF and the tumor antigen, HPV-16 E7, cannot only generate an E7-specific cytotoxic T lymphocytes response in vitro, but can also elicit a potent antitumor immune response against an E7 expressing tumor in vivo.

Animals↗
PubMed1999

Clinical findings and outcomes of intra-hemodialysis cardiopulmonary resuscitation.

Renal failure with severe uremia is still an important cause of mortality, despite effective renal replacement therapy. Cardiopulmonary arrest (CPA) is the most severe complication during hemodialysis (HD). To acquire more information about cardiopulmonary resuscitation (CPR) during HD, we retrospectively enrolled 24 patients (11 males and 13 females) who had CPR during HD in a medical center during a 3-year period. Their mean age was 66.8 +/- 16.8 years. The CPR rate of the patients from our outpatient department (0.02%) was significantly lower than that from general wards (0.11%), the intensive care unit (ICU, 0.16%), or the emergency room (ER, 0.38%). Eighteen patients (75%) were initially resuscitated successfully. Only 11 patients (45.8%) survived more than 24 h after CPR, and 2 patients (8.3%) survived more than 1 month, but none survived until discharge. The rates of surviving 24 h and surviving to discharge during HD were lower than those in the general wards, the ICU or the ER. Sepsis (33.3%) and cardiogenic shock (25%) were the two leading causes of death. For analyzing factors affecting the outcome of CPR, we divided the patients into 2 groups by survival time (<==24 vs. >24 h). Patients with heart disease or with prolonged CPR durations (>30 min) had shorter survival. No significant survival difference between the 2 groups was found due to factors of age, sex, diabetic nephropathy, pre-arrest morbidity scores, pre-arrest laboratory data, renal failure pattern, HD duration, the preceding HD time and ultrafiltrated volume.

Aged↗
PubMed1998

The central role of CD4(+) T cells in the antitumor immune response.

The induction of optimal systemic antitumor immunity involves the priming of both CD4(+) and CD8(+) T cells specific for tumor-associated antigens. The role of CD4(+) T helper cells (Th) in this response has been largely attributed to providing regulatory signals required for the priming of major histocompatibility complex class I restricted CD8(+) cytolytic T lymphocytes, which are thought to serve as the dominant effector cell mediating tumor killing. However, analysis of the effector phase of tumor rejection induced by vaccination with irradiated tumor cells transduced to secrete granulocyte/macrophage colony-stimulating factor indicates a far broader role for CD4(+) T cells in orchestrating the host response to tumor. This form of immunization leads to the simultaneous induction of Th1 and Th2 responses, both of which are required for maximal systemic antitumor immunity. Cytokines produced by these CD4(+) T cells activate eosinophils as well as macrophages that produce both superoxide and nitric oxide. Both of these cell types then collaborate within the site of tumor challenge to cause its destruction.

Animals↗
PubMed1995

Neuroprotective effects of gangliosides may involve inhibition of nitric oxide synthase.

Gangliosides are polar, sugar-containing lipids that are major constituents of neuronal membranes. Gangliosides are neuroprotective in animal models of neurotoxicity and may also be useful in patients with clinical conditions such as spinal cord injury. We show that a series of gangliosides inhibit nitric oxide synthase activity by binding calmodulin. The prevention of glutamate neurotoxicity in cortical cultures by gangliosides closely parallels their potencies in binding calmodulin and inhibiting nitric oxide synthase. Neuroprotective effects of gangliosides may arise from blockade of nitric oxide formation.

Amino Acid Oxidoreductases↗
PubMed1991

Effects of thymidine and uridine on the phosphorylation of 3'-azido-3'-deoxythymidine (zidovudine) in human mononuclear cells.

The effects of thymidine and uridine on the phosphorylation of 3'-azido-3'-deoxythymidine (AZT) were studied in various human mononuclear cell preparations. Thymidine suppressed [3H]AZT phosphorylation in the same concentration range (20 to 100 microM) in which it antagonizes the anti-human immunodeficiency virus activity of AZT. Uridine, in turn, had no influence on AZT phosphorylation, just as it has no effect on the anti-human immunodeficiency virus activity of AZT. These findings are consistent with a close relationship between the inhibition of AZT phosphorylation and the influence of physiological nucleosides on the antiviral activity of AZT.

Bone Marrow↗
PubMed1990

Effect of dipyridamole on transport and phosphorylation of thymidine and 3'-azido-3'-deoxythymidine in human monocyte/macrophages.

Dipyridamole (DPM), a commonly used coronary vasodilator and antithrombotic drug, was shown recently to potentiate the antiviral effect of 3'-azido-3'-deoxythymidine (AZT) in HIV-1 infected human monocyte-derived macrophages (M/M) in vitro. We report in the present study that in uninfected M/M, DPM markedly inhibited cellular uptake of [3H]thymidine (dThd) and its incorporation into the nucleotide pools, particularly the dThd-triphosphate pool. In contrast, DPM did not affect cellular uptake and phosphorylation of [3H]AZT. Since dThd counteracts the phosphorylation and antiviral action of AZT, these findings support the hypothesis that the potentiation of the anti-HIV effect of AZT is due, at least in part, to differential inhibition of nucleoside salvage.

Biological Transport↗