Epstein-Barr virus transcription activator R upregulates BARF1 expression by direct binding to its promoter, independent of methylation.

Epstein-Barr virus (EBV) BamHI-A rightward frame 1 (BARF1) is considered a major viral oncogene in epithelial cells and has immune-modulating properties. However, in B cells and lymphomas, BARF1 expression is restricted to the viral lytic replication cycle. In this report, the transcriptional regulation of BARF1 during lytic replication is unraveled. Bisulfite sequencing of various cell lines indicated a high level of methylation of the BARF1 gene control region. A BARF1 promoter luciferase reporter construct was created using a CpG-free vector, enabling true assessment of promoter methylation. Induction of the EBV lytic cycle is mediated by the immediate-early proteins BZLF1 (Z) and BRLF1 (R). R was found to activate expression of the BARF1 promoter up to 250-fold independently of Z and unaffected by BARF1 promoter methylation. Chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay (EMSA), and specific mutagenesis of the R-responsive elements (RREs) demonstrated direct binding of R to RREs between nucleotides -554 and -327 relative to the BARF1 transcriptional ATG start site. The kinetics of BARF1 expression upon transactivation by R showed that BARF1 mRNA was expressed within 6 h in the context of the viral genome. In conclusion, expression of the BARF1 protein during lytic replication is regulated by direct binding of R to multiple RREs in the gene control region and is independent of the promoter methylation status. The early kinetics of BARF1 upon transactivation by R confirm its status as an early gene and emphasize the necessity of early immune modulation during lytic reactivation.

Use of an automated circuit for isolated limb infusion for malignancy.

BACKGROUND: Isolated limb infusion (ILI) for recurrent or in-transit melanoma is an accepted technique that allows high-dose chemotherapy to be delivered to an extremity with minimal systemic toxicity. Current infusion systems have relied on manual delivery of drugs and circulation of blood during the treatment. Herein, we document our initial results with an automated circuit for ILI as an alternative to the manual technique. METHODS: Patients undergoing ILI with an automated circuit for recurrent or advanced malignancy were identified. ILI was performed utilizing a Sarns 8000 roller pump attached to a Cobe 4:1 cardioplegia set with heat exchanger with a total priming volume of 80 ml. Melphalan (7.5 mg/L) and Dactinomycin (75 µg/L) doses which were corrected for ideal body weight were delivered via the infusion circuit after limb temperature reached 38 °C. RESULTS: Fourteen lower extremity infusion procedures were performed in 10 patients. Successful infusion procedures were completed in all patients using the automated circuit. Constant flow rates of 50-70 cc/minute were achievable with the automated circuit. Acute toxicity and clinical results were similar to that reported with manual delivery systems. CONCLUSION: ILI for advanced malignancy utilizing an automated circuit is feasible and safe. This automated system offers a safe and reliable alternative to the manual infusion technique.

Lack of clinical utility of folate levels in the evaluation of macrocytosis or anemia.

PURPOSE: Folate levels are routinely ordered in the evaluation of macrocytosis with or without frank anemia, yet the value of these tests is questionable. We evaluated the clinical utility of folate testing in routine clinical practice. SUBJECTS AND METHODS: We conducted a retrospective review of all serum and erythrocyte folate assays performed over a one-year period at three hospitals. We determined the frequency of low values, then reviewed the medical records of all patients with low values to determine whether low folate levels changed clinician behavior. We also performed a cost analysis to determine the cost of testing per case in which behavior changed. RESULTS: Only 2.3% of the 2,998 folate levels obtained during the study period were low. The low levels were noted in the record in 53% of cases, and folic acid was prescribed or continued at discharge in only 24%. The cost analysis showed that nearly $10,000 was spent in folate testing per patient in which behavior changed. CONCLUSIONS: Folate values were rarely low in the population tested, and low values infrequently led to a change in clinician behavior. Given the limited clinical value of folate tests, we propose that, in cases of macrocytosis with or without anemia, to minimize cost and prevent missed cases of true folate deficiency, empirical supplementation with folic acid should be used in place of testing for deficiency.

Levels of five mycotoxins in grains harvested in Atlantic Canada as measured by high performance liquid chromatography.

An analytical protocol using high performance liquid chromatography (HPLC) was used to analyze samples of spring wheat, winter wheat, 2-row barley, and 6-row barley over a period of three years for the presence of five mycotoxins. These included deoxynivalenol, zearalenone, T-2 toxin, HT-2 toxin, and diacetoxyscirpenol. The protocol employed a single extraction step using acetonitrile-water and two cleanup procedures. One utilized a solid-phase extraction column and the other a charcoal-alumina column. Detection limits ranged from 0.02 to 0.15 micrograms of mycotoxin g-1 grain. Little T-2 toxin, HT-2 toxin, or diacetoxyscirpenol was found in the samples. Deoxynivalenol was detected in 53 to 62% of the samples tested and zearalenone in 25-29% of the samples. Several enzyme-linked immunosorbent assays were used for comparison purposes with deoxynivalenol, zearalenone, and T-2 toxin. These kits provided reliable qualitative, but not quantitative, data.

Screening for psychiatric symptoms and illness behaviour in a pain clinic.

Evolution of a multidisciplinary pain clinic in a rehabilitation hospital is described. The assessment process was facilitated, particularly with respect to psychiatric and psychological evaluation, by the preliminary administration of two well established but simple self-administered questionnaire instruments, the Illness Behaviour Questionnaire (IBQ) and the Crown-Crisp Experiential Index (CCEI). The characteristics of our pain clinic population are described. They demonstrate questionnaire profiles which are typical of pain patients. Attention is drawn to the CCEI profile now shown in three studies of pain patients, predominance of the somatic anxiety (S) scale occurring in all. Correlations between the IBQ and CCEI showed, inter alia, that the S scale is a measure of somatisation in pain patients. This paper aims to describe the changes in the clinic since previously published descriptions, to note the characteristics of our patient on the questionnaire instruments and to comment on the value of these questionnaires in the assessment of psychiatric symptoms and somatisation in pain clinic patients.

Heavy metal, pH, and total solid content of maple sap and syrup produced in eastern Canada.

Maple sap and syrups in eastern Canada were analyzed for pH, total solids, and the heavy metals Cu, Fe, Pb, and Zn. The levels of heavy metals found were within the range normally contained in food and water samples except for Pb. The concentration factor found in reducing sap to syrup did not reflect the same concentration change for the measured parameters. This indicates removal or conversion of heavy metals and organic acids with the sugar sands. There was no statistical difference among provinces with respect to the heavy metal, pH, and total solids content of sap. The only significant difference in syrup occurred with Cu and this appeared to be the result of the processing procedure. As the season progressed, the Cu, Pb, pH, and total solids content of the sap decreased while Zn increased and Fe showed little change. Syrups reflected a similar change. Statistical differences occurred in sap composition among sites within each province.

Turbulent jets and eddies in the california current and inferred cross-shore transports.

The instantaneous California Current is seen to consist of intense meandering current filaments (jets) intermingled with synoptic-mesoscale eddies. These quasi-geostrophic jets entrain cold, upwelled coastal waters and rapidly advect them far offshore; this behavior accounts for the elongated, cool surface features that are seen extending across the California Current region in satellite infrared imagery. The associated advective mechanism should provide significant cross-shore transports of heat, nutrients, biota, and pollutants. The dynamics of the current system should be crucially influenced by its highly variable structure.

Radioactive conversion products of intramuscularly injected [4-14C]formononetin including sulfates in the urine of hens.

The phytoestrogen formononetin was injected intramuscularly as [4-14C]formononetin into two adult hens. Radioactive materials in the urine for the succeeding 14 days (hen 1) or 16 days (hen 2) were fractionated on DEAE-Sephadex-25 columns by elution with a gradient of NaCl; the four major fractions thus separated were examined by solvent partition, thin-layer chromatography, and enzymic cleavage. The following seven radioactive components were identified in the urine, the average proportions of each being given in terms of percentage of total 14C recovered from the urine: [14C]formononetin (4.3%); [14C]diaidzein (11.4%); [14C]equol (6.8%); [14C]daidzein monosulfate (30.4%); [14C]equol monosulfate (5.8%); [14C]diadzein disulfate (19.8%); and [14C]equol disulfate (6.5%). Small proportions of sulfates of unidentified radioactive phenols were present. Tests for presence of glucosiduronates of 14C-labelled material gave negative results. Radioactive formononetin sulfate was not detected in the urine of either hen.

Identification of radioactive 17beta-estradiol-17-beta-D-glucuronide and 17alpha-estradiol-17-beta-D-glucuronide in the urine of the domestic fowl after intramuscular injection of [4-14C]estrone.

Two previously uncharacterized radioactive estrogen conjugates, 17beta-estradiol-17-beta-D-glucuronide (3-hydroxyestra-1,3,5(10)-trien-17beta-D-glucopyranosiduronate) and 17alpha-estradiol-17beta-D-glucuronide (3-hydroxyestra-1,3,5(10)-trien-17alpha-yl-beta-D-glucopyranosiduronate), have been identified in small but significant amounts in avian urine and in a ratio of approximately 2:1 after intramuscular injection of [4-14C]estrone.

Identifications of radioactive steroid estrogen conjugates in blood plasma of laying hens after intramuscular injection of (4--14C)-estrone.

[4--14C] Estrone was injected intramuscularly into six laying hens. Fifty minutes later the hens were exsanguinated. The plasmas were examined for conjugates of radioactive phenolic steroids by recovery on columns of Amberlite XAD-2 or by extraction with tetrahydrofuran followed by chromatography on a column of DEAE-Sephadex A-25 in a gradient of NaCl. The biggest Sephadex chromatographic fraction (50,4% of total) contained about 42% of its radioactivity as estradiol-17alpha-3-sulfate and 18% as estradiol-17beta-3-sulfate and the remaining 40% was identified tentatively as estradiol-17alpha-17-sulfate plus a small proportion of estradiol-17beta-17-sulfate. The second biggest Sephadex chromatographic fraction (12.7% of total) was a mixture of conjugates not further identified. Minor fractions identified comprised estrone-beta-glucuronide (2.8%), estradiol-17alpha-3-beta-glucuronide (2.8%), estradiol-17beta-3-beta-glucuronide (2.3%) and estrone sulfate (6.0%). Evidence was obtained for the presence of small proportions of estradiol-17alpha disulfate and estradiol-17beta disulfate.

Excretion of radioactive diadzein and equol as monosulfates and disulfates in the urine of the laying hen.

The phytoestrogen, diadzein, was injected intramuscularly as [4-14-C]daidzein into two laying hens. The radioactive materials in the urine for the succeeding 23 (hen 1) or 14 (hen 2) days were fractionated on a DEAE-Sephadex column by a gradient of NaCl and the fractions thus separated were further analyzed by solvent partition, susceptability to enzymic cleavage and thin-layer chromatography. The sic following components were identified and quantitated: [14-C]diadzein, [14-C]equol, [14-C]diadzein monosulfate, [14-C]equol monosulfate, [14-C]diazein disulfate, and [14-C]equol disulfate. The urine from hen 2 yielded also the sulfate of an unidentified conversion product of [14-C]daidzein. Repeared tests for glucuronides of [14-C]daidzein or its conversion products gave negative results, excluding the possibility that any appreciable proportion of the radioactivity in the urine was in the form of beta-glucuronide. It is concluded that the diadzein and the equol excretion in the urine of the laying hen are present for the most part as monosulfates and disulfates.

Bottom Velocity Observations Directly under the Gulf Stream.

Speeds as high as 44 centimeters per second were observed 200 meters above the ocean bottom under the Gulf Stream at 70 degrees W longitude and were associated with time-dependent motion that had a speed range of 40 centimeters per second and a time scale of about 30 days. These deep current fluctuations appear to be coupled with fluctuations in the surface position of the Stream and with surface and bottom current fluctuations 200 kilometers to the north.