Treatment of acute paraquat intoxication using recommended megadose of vitamin C: a reappraisal.

Megadose of vitamin C (MVC) has been proposed for an emergent treatment of acute paraquat (PQ) poisoning. However, the safety issue of this treatment protocol has not been evaluated. Here, we present the first evidence that vitamin C can promote aggravated production of hydroxyl radical (OH(•)) via interacting with preexisting PQ(+•)/H2O2 system in a nonmetal-catalyzed manner. This enhanced oxidative stress would therefore expect to cause more deleterious effect during acute PQ intoxication. To lend support to this possibility, we set out to attest the effects of MVC on a simulated, PQ-intoxicated, Madin-Darby canine kidney (MDCK) cell model. First, PQ alone could trigger oxidative-nitrosative stress (ONS) through robust generation of reactive oxygen species and nitric oxide (NO) that could induce apoptotic killing via promoting effective release of mitochondrial cytochrome c, an apoptogenic factor. The percentage of apoptosis for MDCK cells treated with 1.0 mM PQ for 24 h was 16.3 ± 13.0%. However, when MDCK cells were treated with a combination of PQ (1.0 mM) and MVC (20 mM) for 24 h, the severity of apoptotic killing was further exacerbated as reflected by a nearly 7-fold increase in the release of mitochondrial cytochrome c and the percentage of apoptotic cell population rose sharply to 90.7 ± 5.1%. These data indicate that MVC apparently exacerbates further killing rather than cytoprotection on this simulated, PQ-intoxicated MDCK cell model and suggest that the treatment of PQ poisoning using MVC protocol should be cautious.

MicroRNA-17-5p post-transcriptionally regulates p21 expression in irradiated betel quid chewing-related oral squamous cell carcinoma cells.

BACKGROUND AND PURPOSE: Betel nut chewing is associated with oral cavity cancer in Taiwan. OC3 is an oral carcinoma cell line that was established from cells collected from a long-term betel nut chewer who does not smoke. After we found that microRNA-17-5p (miR-17-5p) is induced in OC3 cells, we used this cell line to examine the biological role(s) of this microRNA in response to exposure to ionizing radiation. MATERIALS AND METHODS: A combined SYBR green-based real-time PCR and oligonucleotide ligation assay was used to examine the expression of the miR-17 polycistron in irradiated OC3 cells. The roles of miR-17-5p and p21 were evaluated with specific antisense oligonucleotides (ODN) that were designed and used to inhibit their expression. Expression of the p21 protein was evaluated by Western blotting. The clonogenic assay and annexin V staining were used to evaluate cell survival and apoptosis, respectively. Cells in which miR-17-5p was stably knocked down were used to create ectopic xenografts to evaluate in vivo the role of miR-17-5p. RESULTS: A radiation dose of 5 Gy significantly increased miR-17-5p expression in irradiated OC3 cells. Inhibition of miR-17-5p expression enhanced the radiosensitivity of the OC3 cells. We found that miR-17-5p downregulates radiation-induced p21 expression in OC3 cells and, by using a tumor xenograft model, it was found that p21 plays a critical role in increasing the radiosensitivity of OC3 cells in vitro and in vivo. CONCLUSION: miR-17-5p is induced in irradiated OC3 cells and it downregulates p21 protein expression, contributing to the radioresistance of OC3 cells.

Comparing the relationship of Taxol-based chemotherapy response with P-glycoprotein and lung resistance-related protein expression in non-small cell lung cancer.

Our aim was to compare Taxol-based chemotherapy response of non-small cell lung cancer (NSCLC) with P-glycoprotein (Pgp) or lung resistance protein expression (LRP). Immunohistochemical analyses were performed on multiple nonconsecutive sections of the biopsy specimens to detect Pgp and LPR expressions in 40 patients with advanced NSCLC before Taxol-based chemotherapy. The chemotherapy response was evaluated by clinical and radiological methods in the third month after completion of treatment. No significant differences of prognostic factors (age, sex, body weight loss, performance status, tumor size, tumor stage, and tumor cell type) were found between the 20 patients with good and the 20 patients with poor responses. The incidence difference of positive Pgp expressions between good and poor responses was significant, however, the difference of LRP expression was not. We concluded that Taxol-based chemotherapy response of patients with NSCLC was related to Pgp but not LPR expression.

Rapid and specific detection of hydroxyl radical using an ultraweak chemiluminescence analyzer and a low-level chemiluminescence emitter: application to hydroxyl radical-scavenging ability of aqueous extracts of Food constituents.

With the availability of an ultraweak chemiluminescence analyzer, it is possible to monitor the production of a specific oxygen-derived reactive species, such as hydroxyl radical ((*)OH), whenever a suitable chemiluminescent probe is obtainable. Reported herein is the development of a rapid and specific method for detecting (*)OH production using a specific probe, indoxyl-beta-glucuronide (IBG), a low-level chemiluminescence emitter. Using the Fenton reagent as a source of (*)OH, it was shown that IBG could elicit a very strong intensity of chemiluminescence (CL) (16200 +/- 200 photon counts/s). Conversely, IBG was shown to be insensitive to either superoxide radical or hydrogen peroxide with their CL intensities nearly close to the background values (25 +/- 5 and 180 +/- 20 photon counts/s, respectively). Furthermore, it was also shown that this IBG-based CL production could be effectively quenched by the addition of (*)OH scavengers such as sodium salicylate, dimethyl sulfoxide, and penicillamine to the assay system. Taken together, these data indicate that IBG is a specific CL probe suitable for monitoring the production of (*)OH. This system demonstrated inhibitory activities of various aqueous extracts of food constituents on the CL of hydroxyl radicals generated by Fenton's reagents with the order of scavenging efficiencies being Prunus mume > Cordyceps sinensin > Lilium lancifolium > Astragalus membranceus.

Application of representational difference analysis to cloning a Mycoplasma arthritidis specific DNA fragment.

Representation difference analysis (RDA) was applied to isolate a Mycoplasma arthritidis specific DNA fragment. The DNA fragment obtained was verified to be M. arthritidis specific by polymerase chain reaction (PCR) and dot blot hybridization tests. The size of this fragment was 194 bp and the nucleotide sequence was also determined.

EBV DNA polymerase inhibition of tannins from Eugenia uniflora.

Nasopharyngeal carcinoma (NPC) is one of the high population malignant tumors among Chinese in southern China and southeast Asia. Epstein-Barr virus (EBV) is a human B lymphotropic herpes virus which is known to be closely associated with NPC. EBV DNA polymerase is a key enzyme during EBV replication and is measured by its radioactivity. The addition of phorbol 12-myristate 13-acetate to Raji cell cultures led to a large increase in EBV DNA polymerase, which was purified by sequential DEAE-cellulose, phosphocellulose and DNA-cellulose column chromatography. Four tannins were isolated from the active fractions of Eugenia uniflora L., which were tested for the inhibition of EBV DNA polymerase. The results showed the 50% inhibitory concentration (IC(50)) values of gallocatechin, oenothein B, eugeniflorins D(1) and D(2) were 26.5 62.3, 3.0 and 3.5 microM, respectively. Furthermore, when compared with the positive control (phosphonoacetic acid), an inhibitor of EBV replication, the IC(50) value was 16.4 microM. In view of the results, eugeniflorins D(1) and D(2) are the potency principles in the inhibition of EBV DNA polymerase from E. uniflora.

Elevated lipid peroxidation and disturbed antioxidant enzyme activities in plasma and erythrocytes of patients with uterine cervicitis and myoma.

OBJECTIVES: We investigated whether oxidative stress is associated with human uterine cervicitis and uterine myoma. DESIGN AND METHODS: We measured lipid peroxidation and antioxidant enzymes in plasma and erythrocytes of cervicitis patients and myoma patients in comparison with matched controls. Thiobarbituric acid-reactive substances (TBARS), a measure of lipid peroxidation, were determined in plasma; glutathione peroxidase (GSHPx) and catalase in erythrocytes; and superoxide dismutase (SOD) in both plasma and erythrocytes. RESULTS: We showed that plasma TBARS were significantly higher (p < 0.05) in both cervicitis patients and myoma patients than in controls. Plasma TBARS were significantly (and negatively) correlated with plasma and erythrocyte T-SOD activities in cervicitis patients only. Plasma T-SOD activity was significantly lower in both groups of patients than in controls whereas erythrocyte T-SOD activity was only significantly lower in myoma patients. The lowered plasma T-SOD activity in the cervicitis patients was attributed to decreased Mn-SOD activity whereas the lowered plasma T-SOD activity in myoma patients was attributed to decreased activities of both Cu,Zn-SOD and Mn-SOD. Erythrocyte GSHPx activity was 14% higher (p < 0.05) in cervicitis patients and 11% lower (p > 0.05) in myoma patients than in controls; catalase activity was 10% higher (p > 0.05) in cervicitis patients and 13% lower (p > 0.05) in myoma patients than in controls. Neither erythrocyte GSHPx nor catalase activity was significantly correlated with plasma TBARS. CONCLUSIONS: The elevated lipid peroxidation and disturbed antioxidant enzyme activities demonstrate the potential of oxidative injury in patients with uterine cervicitis and myoma.

Antiviral tannins from two Phyllanthus species.

Seven ellagitannins isolated from Phyllanthus myrtifolius and P. urinaria (Euphorbiaceae) have been shown, for the first time, to be active against Epstein-Barr virus DNA polymerase (EBV-DP) at the microM level. All these compounds have the same moiety of a corilagin, and differ from each other by different substitutions at C-2 and C-4 of the glucose core. SAR analysis and molecular modeling reveal that the essential pharmacophore of these tannins resides in the corilagin moiety. The outer complex carboxylic acid moieties appear to act only as auxopharmacore.

[Continuing hospice care of cancer--a three-year experience].

The hospice at Mackay Memorial Hospital was established in February 1990. A group of team workers including physicians, nurses, social workers and the clergy were involved in this holistic care program for terminal cancer patients. Four hundred and seventy-nine patients were eligible for the program up to February 1993. Regarding duration of stay, 62.5% of patients resided for 14 days. Those surviving under 90 days constituted 75.5% of patients. Fifty-one point eight percent of patients died in the hospice and 18.2% died at home soon after being discharged from the hospice. Pain is the most common symptom among the patients. Treatment strategies vary according to the three-step-ladder protocol designed by WHO. Total pain relief was achieved in 80% of patients. Opportune private talking and family conferences formed the basis of the "peer model". Through this model, treatment decisions including physical, psychosocial and spiritual issues were made. Before the peer model, only 36 (10.3%) patients agreed with the idea of hospice care, while 257 (73.6%) patients agreed after the model was established. Awareness of dying was evident in 412 (86%) patients. Two hundred and eighty (68%) patients became aware of the prospect of death through guessing, while the other 132 (32%) patients were informed by medical staff. Problems encountered by the team workers included 1) needs in education and training, 2) psychological pressure, 3) management of loss and grief, 4) needs in supportive system and 5) troubles caused by families' lying to patients. The team workers were satisfied with the quality of care in 38.4% of patients and fairly satisfied with 30.7% of patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Radiotherapy following simple hysterectomy in patients with invasive carcinoma of the uterine cervix.

From January 1980 to December 1988, a total of 73 cases of invasive cervical cancer after simple hysterectomy were treated with radiotherapy. Seven patients were excluded due to incomplete treatment or loss of follow-up. Among the 66 patients, 52 had squamous cell carcinoma and 14 had adenocarcinoma or adenosquamous cell carcinoma. The patients were grouped as follows: group A, patients without gross residual tumor (n = 32); group B, patients with gross residual tumor (n = 23); and group C, patients with gross recurrent tumor (n = 11). All patients in groups A and B received radiotherapy immediately (within 4 months) following their simple hysterectomy. Patients in group C were treated six months to five years later. Different methods of radiotherapy were delivered during the two consecutive time periods. Before 1985, patients (n = 30) were irradiated with a dose of 45-50 Gy in the midpelvic plane, followed by a transvaginal boost of 30 Gy. After 1985, patients (n = 36) were treated with the same midpelvic dose, and boosted with 30 Gy by high-dose-rate brachytherapy. The overall five-year survival rate was 67%. The five-year survival rates were 81% in group A, 56% in group B, and 45% in group C. A low complication rate (10%) was obtained in our series.(ABSTRACT TRUNCATED AT 250 WORDS)

High-dose-rate afterloading brachytherapy in carcinoma of the uterine cervix.

High-dose-rate (HDR) afterloading brachytherapy has been used at Mackay Memorial Hospital since 1984 for the treatment of gynecological malignancies. From October 1984 to October 1990, a total of 321 previously untreated patients with biopsy proven uterine cervical cancer were treated with radiation therapy with curative intent. According to FIGO staging, the patients were grouped into stage I (19 patients), stage IIA (13 patients), stage IIB (96 patients), stage III (158 patients) and stage IVA (35 patients). All patients received a combination of external beam irradiation plus intracavitary brachytherapy using the Buchler Remote Afterloading (RAL) system. For most cases, external beam irradiation using a Co-60 or Clinac 1800 photon beam (6 MV or 15 MV) of 3,060 cGy to 3,960 cGy was given to the whole pelvis (180 cGy/day, five days/week), followed by a midline shield after RAL. The total dose to the pelvic sidewall was 5,040 cGy to 5,400 cGy. The overall actuarial five-year survival rate was 55%. The total complication rate in a follow-up of two to eight years was 1% to 4%, and a good correlation existed between rectal complications and the calculated rectal dose. We conclude that fractionated HDR intracavitary therapy concurrent with teletherapy can achieve a high regional control rate with few complications, and can reduce the cost of hospitalization and the risk of anesthesia.

High-sequence conservation among the United States bluetongue viruses cognate M2 genes which encode the nonstructural NS1 tubule protein.

Full-length cDNA copies of the M2 gene of BTV-2, -11, and -13 serotypes obtained by a modified polymerase chain reaction (Clamp-R) were cloned into pUC19 plasmid. The entire nucleotide sequences of each M2 gene were determined and compared with BTV-10 and BTV-17, thus completing the sequencing of these cognate M2 gene segments from all five U.S. BTV serotypes. Each M2 segment contained 1769 nucleotides, a single open reading frame (ORF) with an initiation codon at nucleotides 35-37, and a termination codon at nucleotides 1691-1693. This ORF can encode the 552-amino-acid NS1 protein (64 KDa) which has an isoelectric point of 7. Analyses of the nucleotide and deduced amino acid sequences of the five U.S. BTV serotypes indicated that the most recently isolated BTV-2 serotype was more distantly related than BTV-10, -11, -13, or -17. Analyses of the evolutionary relatedness of the cognate M2 genes by codon positions indicate that the rate of mismatch accumulations in the first and second base codon positions are less than 4%. However, the mismatch accumulations in the third base codon position are quite evident (23%) when BTV-2 serotype was compared with the other U.S. BTV serotypes. This suggests that BTV-2 has separated from the other four U.S. serotypes long before they themselves diverged. These data also indicate that the five U.S. BTV serotypes were apparently derived from two distinct gene pools that reflected geographic distribution in North America.

Evolutionary analyses of five US bluetongue viruses using the cognate S2 genes.

Full-length cDNA copies of S2 genes (segment 8), coding for non-structural protein 2 (NS2) of bluetongue virus serotypes 2, 11, 13 and 17 were selectively synthesized by a modified polymerase chain reaction (Clamp-R) and cloned into the PstI site of the pUC19 plasmid. Each of these S2 cognate genes was 1125 nucleotides in length with an initiation and a termination codon at nucleotides 20-22 and 1082-1084, respectively, resulting in a long open reading frame capable of coding for a protein of 354 amino acids. The deduced amino acid sequence of NS2 protein had a high concentration of lysine and contained a relatively low number of tryptophan and histidine residues. There was a highly conserved hydrophilic region at the carboxyl termini of predicted NS2 proteins in all five BTV serotypes, even though the amino acid sequence in this region in BTV-2 was more variable than in the other four serotypes. There was significant sequence homology of the cognate S2 genes at both the nucleotide and the amino acid levels. Phylogenetic analyses using the S2 gene sequences indicated that BTV-10, -11, -13 and -17 were more closely related and BTV-2 was the most distantly related serotype among the five US bluetongue viruses.

Comparative sequence analyses of the cognate structural protein VP6 genes of five US bluetongue viruses.

The S3 segment (the small segment 3), encoding the structural protein, VP6, from the five United States (US) prototype bluetongue virus (BTV) serotypes were amplified by the Clamp-R method and cloned as full-length entities. The complete nucleotide sequence of each cognate gene segment was determined. Each cognate S3 segment of BTV-10, 11, 13 and 17 was 1049 nucleotides long and contained an open reading frame (ORF) capable of encoding a 325-amino acid protein. However, the S3 segment of BTV-2, which also contained 1049 nucleotides, had a longer 5'-non-coding region of 99-nucleotide and contained an ORF capable only of encoding a 301-amino acid protein. Comparative analyses of the predicted amino acid sequences of S3 segments of BTV-2, 10, 11, 13 and 17 revealed that VP6 was unusually high in glycine and contained few aromatic amino acids, but a high concentration of charged amino acids, which is a characteristic of a hydrophilic protein. Phylogenetic analyses indicated that BTV-11, 13 and 17 were more closely related than the other two US BTV serotypes. BTV-2 was the most distantly related.

Sequence conservation among the cognate nonstructural NS3/3A protein genes of six bluetongue viruses.

Full-length cDNA copies of segment 10 genes of bluetongue virus serotypes 2, 11, 13 and 17 were synthesized by the Clamp-R method and inserted into the plasmid pUC19. The complete nucleotide sequences of these four cognate genes were sequenced and determined to be 822 nucleotides in length, smallest of the 10 genes in the bluetongue virion. These four cognate gene segments contained two in-phase and overlapping open reading frames capable of coding for two non-structural proteins of 229 and 216 amino acids with net charges of +4.5 and +5.5, respectively, at neutral pH. Comparative analyses of the predicted amino acid sequences of bluetongue virus serotypes 1, 2, 10, 11, 13 and 17 revealed (i) a high degree of sequence homology and conservation, (ii) a single conserved tryptophan located at residue 159, (iii) the presence of two conserved cysteines at residues 137 and 181 and two potential N-linked glycosylation sites at residues 63-65 and 150-152, (iv) a cluster of six prolines within a 15-amino acid region near the amino terminus, and (v) the longest 3' noncoding sequence of 113 bases among the 10 bluetongue viral genes. Phylogenetic analyses indicated that BTV-10 and -11 are very closely related and BTV-2 is the distantly related serotype of the five US bluetongue virus serotypes.

Chromatographic purification and characterization of EBV DNase from chemically induced lymphoid cells.

Epstein-Barr virus-associated deoxyribonuclease (EBV-DNase) was purified to homogeneity, as determined by silver staining, sequential column chromatography, and FPLC from Raji and P3HR-1 cells treated with 12-O-tetradecanoyl-phorbol-13-acetate and sodium butyrate. This viral protein was immunogenic and elicited high neutralization titer sera in rabbits. By silver staining of SDS-PAGE, Western immunoblot, and radioimmunoprecipitation using NPC patient sera and both polyclonal and monoclonal antibodies, the EBV DNase was identified as a 58K protein. The potential presence of two EBV DNases was also discussed.

Purification of Epstein-Barr virus associated DNase with affinity chromatography of nasopharyngeal carcinoma patient serum.

Epstein-Barr virus associated DNase in the homogeneous form can be purified by chromatographys using CH-sepharose 4B column conjugated with nasopharyngeal carcinoma patient serum, DNA cellulose and phosphocellulose in that sequence. The molecular weight of this enzyme is shown to be 51 kilo-daltons in silver-staining and immunostains. Among various methods for keeping DNase activity, the addition of BSA and dialysis in glycerol or ethylene glycol immediately after the enzyme purification is suggested.

Rheumatic fever recurrences: controlled study of 3-week versus 4-week benzathine penicillin prevention programs.

To compare the merits of 3-week versus 4-week injections of benzathine penicillin G in preventing recurrence of rheumatic fever, 179 patients aged 4 to 19 years were assigned to one of the two programs. Age, weight, cardiac status, and streptococcal infections among the patients and their family members studied in each program were comparable. Eight-two patients and their family members were monitored for streptococcal infections. Compliance in the two programs was comparable. Of the 63 patients who stayed in the 4-week program, RF recurred in six, as a result of prophylaxis failure in five and associated with partial compliance in one. Of the 90 patients in the 3-week program, RF recurred in one, associated with partial compliance; no failures occurred (P = 0.01). We recommended that for RF chemoprophylaxis in individuals at great risk, regardless of age, benzathine penicillin injections should be administered every 3 rather than every 4 weeks.

Human cytomegalovirus-induced DNA polymerase and its interaction with the triphosphates of 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-methyluracil, -5-iodocytosine, and -5-methylcytosine.

Human cytomegalovirus-induced DNA polymerase and cellular DNA polymerase alpha were purified by successive chromatography on DEAE-cellulose, phosphocellulose, heparin agarose, and single-stranded DNA agarose columns. The purified virus-induced DNA polymerase was resolved to consist of two polypeptides corresponding to molecular weights of 140,000 and 58,000, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Virus-induced DNA polymerase and cellular alpha polymerase were examined for their sensitivities to the triphosphates of 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-methyluracil (FMAUTP), -5-iodocytosine (FIACTP), and -5-methylcytosine (FMACTP). The inhibitive effects of these triphosphates on the DNA polymerases were competitive with regard to the natural substrates; thus FMAUTP competes with dTTP, and FIACTP and FMACTP compete with dCTP. The inhibition constants (Ki) for FMAUTP, FIACTP, and FMACTP of virus-induced DNA polymerase are 0.06, 0.30, and 0.47 microM, respectively. Cellular DNA polymerase alpha is much less sensitive to these inhibitors, and its Ki values for FMAUTP, FIACTP, and FMACTP are 0.45, 3.10, and 2.90 microM, respectively. In addition, human cytomegalovirus-induced DNA polymerase, but not cellular DNA polymerase alpha, can utilize these analog triphosphates as alternate substrates for their corresponding natural deoxyribonucleoside triphosphates in in vitro DNA synthesis.