Support programs for minority students at Ohio University College of Osteopathic Medicine.

The Ohio University College of Osteopathic Medicine ranks high among the nation's 19 osteopathic medical schools with respect to the percentage of underrepresented minorities (URMs) in the entering class. The college has strong recruitment and retention programs for URM and disadvantaged students. URM enrollment rose steadily from 11% in 1982-83 to 22% in 1997-98, despite the school's location in a rural, residential public university with few minorities as students or town residents. The college has six programs to support minority students through both undergraduate and medical school: the Summer Scholars Program (1983 to present), an intensive six-week summer program to prepare rising under-graduate seniors and recent graduates to apply to medical school; Academic Enrichment (1987 to present), to support first- and second-year medical students; the Prematriculation Program (1988 to present), an intensive six-week summer program for students who will matriculate in the college; Program ExCEL (1993 to present), a four-year program for undergraduates at Ohio University; the Summer Enrichment Program (1993 to present), an optional six-week program for students who will enter the premedical course at Ohio University; and the Post-baccalaureate Program (1993 to present), a year-long, individually tailored program for URM students who have applied to the medical college but have been rejected. The medical college first focused on supporting students already in the medical school curriculum, then expanded logically back through the undergraduate premedical programs, always targeting learning strategies and survival strategies, peer and faculty support, and mastery of the basic science content. The college plans to create an on-site MCAT preparation program and perhaps expand into secondary education.

Persistence of gentian violet and leucogentian violet in channel catfish (ictalurus punctatus) muscle after water-borne exposure.

Gentian violet is a triphenylmethane dye that is an antifungal/antiparastic agent. GV is similar to malachite green that has been used in the aquaculture industry for treatment or prevention of external fungal and parasitic infections in fish and fish eggs although it (MG) is not approved for this use. For these reasons, GV's potential for misuse by the aquaculture industry is high. The uptake and depletion of gentian violet (GV) were determined in channel catfish (Ictalurus punctatus) after water-borne exposure (100 ng ml(-1), 1 h) under simulated aquaculture farming conditions. Leucogentian violet (LGV) was rapidly formed, concentrated in the muscle tissue, and very slowly eliminated from muscle tissue. An isocratic (60% acetonitrile-40% water; 0.05 M ammonium acetate buffer, pH 4.5) HPLC system consisting of a 5 microm LC-CN 250x4.6 mm I.D. column, a 20x2.0 mm I.D. PbO2 oxidative post-column, and a UV-VIS detector set at 588 nm were used to determine uptake and depletion of tissue residues of GV and LGV with time. GV was rapidly depleted and converted to its major metabolite, LGV, which was detected out to 79 days. Therefore, LGV is the appropriate target analyte for monitoring exposure of channel catfish to GV.

Determination of calcium by inductively coupled plasma-atomic emission spectrometry, and lead by graphite furnace atomic absorption spectrometry, in calcium supplements after microwave dissolution or dry-ash digestion: method trial.

A 3-laboratory method trial was conducted to evaluate 2 sample digestion procedures and instrumental determination parameters for analysis of calcium and lead in Ca supplements. Calcium supplements were treated by dry-ash digestion or microwave dissolution prior to spectrometric analysis. In each case, Pb was determined by graphite furnace atomic absorption spectrometry and Ca by inductively coupled plasma-atomic emission spectrometry. Blind duplicates of 6 Ca supplement samples were analyzed after each sample treatment procedure. Matrix pairs contained dissimilar Pb levels to cover the analyte range encountered during method development. Calcium content of the Ca supplement samples also reflected the range seen during method development. Stock solutions of Ca and Pb were supplied to collaborators for preparation of quantitation standards to remove a variable external to the method. National Institute of Standards and Technology Standard Reference Material (NIST SRM) 1486, bone meal, was included to assess method accuracy and recovery at NIST certificate Ca and Pb levels for this material (26.58 +/- 0.24% Ca and 1.335 +/- 0.014 micrograms Pb/g). Analyses of the NIST SRM yielded 25.9 +/- 1.1 and 27.2 +/- 2.3% Ca and 1.53 +/- 0.19 and 1.26 +/- 0.19 micrograms Pb/g for dry-ash and microwave procedures, respectively. Statistical analyses of data indicated acceptable repeatability and reproducibility for determination of Pb and Ca in various Ca supplements. With either sample preparation technique, the method is appropriate for determining Pb or Ca in Ca supplements.

A bridging study between liquid chromatography and microbial inhibition assay methods for determining amoxicillin residues in catfish muscle.

A bridging study was conducted to establish the correlation between a liquid chromatographic (LC) method and a microbial inhibition (MI) method for analysis of amoxicillin residues in catfish muscle. The LC procedure involved precolumn derivatization with formaldehyde followed by LC separation with fluorescence detection. The MI procedure used Bacillus stearothermophilus as the test organism and was validated in this study before the bridging investigation. The 2 methods were compared for determination of both fortified and incurred samples. No significant differences were found between the methods when all data were included in statistical computations. The linear correlation of LC means versus MI means had a slope of 0.972 and a negligible intercept (1.0 ng/g), with a correlation coefficient of 0.9962. LC was more specific and showed better sensitivity than MI for amoxicillin residues at < or = 10 ng/g. For practical purposes, values obtained by the 2 methods can be considered equivalent.

Determination of sulfonamides in edible salmon tissue by liquid chromatography with postcolumn derivatization and fluorescence detection.

Fourteen sulfonamides-sulfanilamide, sulfadiazine, sulfathiazole, sulfapyridine, sulfamerazine, sulfamethazine, sulfamethizole, sulfamethoxypyridazine, sulfachloropyridazine, sulfamonomethoxine, sulfadoxine, sulfamethoxazole, sulfadimethoxine, and sulfaquinoxoline-residues of which could be found in aquacultured species, were separated in < 25 min by reversed-phase (C18) liquid chromatography (LC) with gradient elution. Analytes were extracted from edible salmon tissue (muscle and adhering skin) with acetonitrile-2% aqueous acetic acid, isolated with 2 liquid-liquid partitionings, and derivatized with fluorescamine after eluting from the column. The derivatives were detected by fluorescence. Recoveries (n = 4) from coho salmon fortified with sulfonamides at 5, 10, and 20 ng/g tissue averaged 79.7 +/- 7.3, 84.6 +/- 7.7, and 88.2 +/- 7.1%, respectively. Limits of quantitation were 5 ng/g tissue, for sulfanilamide, sulfamethoxypyridazine, and sulfaquinoxoline and 1 ng/g tissue for the remaining sulfonamides.

Rapid method for the determination of ampicillin residues in animal muscle tissues by high-performance liquid chromatography with fluorescence detection.

A rapid and sensitive HPLC method was developed for the determination of ampicillin residues in muscle tissues of beef, pork, chicken and catfish. Muscle tissues were blended with a food processor into paste. A 5-g aliquot of the blended tissues was homogenized with 14 ml of 0.01 M phosphate buffer (pH 4.5) using a tissue homogenizer. Proteins were precipitated with the addition of 1 ml trichloroacetic acid (75%, w/v) followed by centrifugation. After filtration, 1 ml of the supernatant was reacted with formaldehyde under acidic and heating conditions. The ampicillin fluorescent derivative was then analyzed by reverse phase HPLC with fluorescence detection. Recoveries of spiked ampicillin at 5, 10 and 20 ng/g were >85%, with coefficients of variation <5%. The limit of detection and limit of quantitation for ampicillin in the tissues were 0.6 ng/g and 1.5 ng/g, respectively. The method is also applicable to the analysis of ampicillin residue in dry milk powder.

Simultaneous determination of malachite green, gentian violet and their leuco metabolites in catfish or trout tissue by high-performance liquid chromatography with visible detection.

A sensitive analytical procedure for the determination of residues of leucomalachite green (LMG)-malachite green (MG) and leucogentian violet (LGV)-gentian violet (GV) in catfish or trout tissue is presented. Frozen (-20 degrees C) fish fillets were cut into small pieces and blended in a Waring blender. A 20-g amount of homogenized fish tissue was extracted with acetonitrile-buffer, partitioned against methylene chloride, and cleaned up on tandem neutral alumina and propylsulfonic acid cation-exchange solid-phase extraction cartridges. Samples of 100 microliters (0.8 g equiv.) were chromatographed isocratically in 10 min using an acetonitrile-buffer mobile phase on a short-chain deactivated (SCD) reversed-phase column (250 x 4.6 mm I.D.) in-line with a post-column PbO2 oxidation reactor. The PbO2 post-column reactor efficiently oxidized LMG to the chromatic MG, and LGV to the chromatic GV permitting visible detection at 588 nm for all four compounds. Linearity was demonstrated with standards over the range of 0.5-50 ng per injection. Recoveries of LMG, MG, LGV and GV from catfish tissues fortified at 10 ng/g were 75.4 +/- 3.0, 61.3 +/- 4.1, 72.6 +/- 3.7 and 87.9 +/- 2.5, respectively, while trout tissues fortified at 10 ng/g yielded recoveries of 82.6 +/- 2.3, 48.6 +/- 1.8, 72.1 +/- 2.1 and 83.8 +/- 4.6 (mean +/- S.D., n = 4), respectively.

Determination of lincomycin residues in salmon tissues by gas chromatography with nitrogen-phosphorus detection.

A sensitive method for the determination of lincomycin residues in fish tissues is described. Lincomycin was extracted from fish tissues with phosphate buffer (pH 4.5). The extract was concentrated with a C18 solid-phase extraction cartridge and further cleaned up by solvent extraction. Lincomycin was derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide to form a trimethylsilyl derivative before being analyzed by gas chromatography with nitrogen-phosphorus detection. Coumaphos was used as the internal standard. Assays showed good linearity in the range 25-250 ppb (ng/g) (r = 0.9994). Recoveries of fortified lincomycin at 50, 100 and 200 ppb were > 80% with relative standard deviations < 6%. The limit of detection of the method was 1.7 ppb and the limit of quantitation was 3.8 ppb.

Determination of lincomycin residue in salmon tissues by ion-pair reversed-phase liquid chromatography with electrochemical detection.

A method is described for detecting and quantitating lincomycin residue in salmon muscle and skin tissues by ion-pair reversed-phase liquid chromatography (LC) with electrochemical detection at +0.9 V. Lincomycin was extracted from tissues by homogenizing with 0.01 M KH2PO4 buffer (pH 4.5) and centrifuging the mixture. Water-soluble proteins were precipitated by adding sodium tungstate and sulfuric acid and removed by centrifugation. The buffer extract was then passed through a C18 solid-phase extraction cartridge. Lincomycin was eluted with 50% acetonitrile in water, and the eluate containing lincomycin was extracted with ethyl acetate. After the solvent had evaporated, the residue was redissolved in mobile phase and analyzed by LC. The method had a limit of detection of 7 ng/g lincomycin for salmon muscle and 12 ng/g for salmon skin. The limit of quantitation was 17 ng/g for salmon muscle and 24 ng/g for salmon skin. Average recoveries of lincomycin spiked at 50, 100, and 200 ng/g were > or = 85% for salmon muscle and > or = 80% for salmon skin.

Analysis of fumonisin B1 in rodent feed by CE with fluorescence detection of the FMOC derivative.

A fast, simple, and environmentally friendly method has been developed for the analysis of fumonisin B1 (FB1) in rodent feed using CE. FB1 is the major fumonisin metabolite produced by the fungus Fusarium moniliforme and has been implicated in human and animal diseases. FB1 was extracted from rodent feed with acetonitrile/water (50/50) (vol/vol) and cleaned up with a C18 Sep-Pak Vac cartridge (Waters Corp., Milford, MA, U.S.A.). FB1 was quantitated after elution from the column using capillary electrophoresis with a 25-mM sodium borate buffer (pH 9.0) containing 10% acetonitrile and fluorescence detection of the (9-fluorenylmethyl) chloroformate (FMOC) derivative. The minimum detectable amount in rodent feed was 0.5 pmm. Recovery values in spiked rodent feed averaged more than 87% over the 2-20 ppm range.

Determination of amoxicillin in catfish and salmon tissues by liquid chromatography with precolumn formaldehyde derivatization.

A liquid chromatographic (LC) method with fluorescence detection was developed for analysis of amoxicillin in catfish and salmon tissues. The tissue was extracted with phosphate buffer (pH 4.5), followed by trichloroacetic acid (TCA) precipitation of proteins and solid-phase (C18) extraction. Trace amounts of nonpolar interfering substances present after solid-phase extraction were removed by ether liquid-liquid extraction. The extract was reacted with formaldehyde and TCA at 100 degrees C for 30 min. A fluorescent derivative was extracted with ether, concentrated, and analyzed by reversed-phase LC with fluorescence detection. Average recoveries of amoxicillin spiked at 2.5-20 ppb were > 80% for catfish and > 75% for salmon muscle tissue, with coefficients of variation of < 6%. Limits of detection (LOD) and quantitation (LOQ) for catfish tissue were 0.5 and 1.2 ppb, respectively. LOD and LOQ for salmon muscle tissue were 0.8 and 2.0 ppb, respectively.

Determination of underivatized fumonisin B1 and related compounds by HPLC.

A method is presented for determining the purity of the mycotoxin fumonisin B1 (FB1) by high performance liquid chromatography (HPLC) with evaporative light scattering detection (ELSD). The ELSD is a universal HPLC detector that exhibits a non-linear relationship between analyte amount and the resulting response. A log-log plot of ELSD response with the mass of FB1 injected was used as a calibration curve for determining the quantities of both FB1 and also individual impurities present in samples. Assumptions related to the uniformity of ELSD response for different but related compounds and other issues implied in this use of ELSD data were examined. One potential error produced by use of this method for purity analysis comes from the ELSD's decreased sensitivity for low-concentration analytes. Because analytes become more dilute the longer they remain on a chromatographic column, this sensitivity discrimination can be related to the retention times at which they appear. The ELSD response for FB1 at retention time 15.5 minutes was used to construct a general purpose calibration curve. Whenever a peak appeared at any time other than 15.5 minutes, the discrimination effect was corrected using a an empirically determined weighting factor and a proportion calculated from the retention time difference compared to 15.5 minutes. Purities for two fumonisin samples were calculated using both the ELSD method described above and an electrospray/mass spectrometric method. The quantitative assumptions underlying each method were discussed in order to understand and reconcile differences between the two sets of purity values obtained.

Determination of leucogentian violet and gentian violet in catfish tissue by high-performance liquid chromatography with visible detection.

A sensitive analytical procedure for the determination of residues of leucogentian violet (LGV) and gentian violet (GV) in catfish tissue is presented. Frozen (-20 degrees C) catfish fillets were cut into chunks and then blended in a Waring blender. A 10-g amount of catfish muscle tissue was homogenized and extracted with acetonitrile-buffer, partitioned against methylene chloride, and cleaned up on tandem neutral alumina and propylsulfonic acid cation-exchange solid-phase extraction cartridges. Samples of 100 microliters (0.5 g equiv.) were chromatographed isocratically in 15 min using an acetonitrile-buffer mobile phase on a cyano phase column in-line with a post-column PbO2 oxidation reactor. The PbO2 post-column reactor efficiently oxidized the LGV to the chromatic GV permitting visible detection at 588 nm for both LGV and GV. Linearity was demonstrated with standards over the range 0.5-50 ng per injection. Recoveries of LGV and GV from catfish tissues fortified at 20, 10, and 1 ng/g were 83.1 +/- 1.2, 78.4 +/- 4.0, 84 +/- 8 and 92.7 +/- 1.8, 95.0 +/- 2.2, 93 +/- 2 (mean +/- S.D., n = 4), respectively.

Interactions of calf thymus DNA polymerase alpha with primer/templates.

The interactions of calf thymus DNA polymerase alpha (pol alpha) with primer/templates were examined. Simply changing the primer from DNA to RNA had little effect on primer/template binding or dNTP polymerization (Km, Vmax and processivity). Surprisingly, however, adding a 5'-triphosphate to the primer greatly changed its interactions with pol alpha (binding, Vmax and Km and processivity). While changing the primer from DNA to RNA greatly altered the abilit of pol alpha to discriminate against nucleotide analogs, it did not compromise the ability of pol alpha to discriminate against non-cognate dNTPs. Thus the nature of the primer appears to affect 'sugar fidelity', without altering 'base fidelity'. DNase protection assays showed that pol alpha strongly protected 9 nt of the primer strand, 13 nt of the duplex template strand and 14 nt of the single-stranded template from hydrolysis by DNase I and weakly protected several bases outside this core region. This large DNA binding domain may account for the ability of a 5'-triphosphate on RNA primers to alter the catalytic properties of pol alpha.

Liquid chromatographic determination of sulfadiazine in salmon by postcolumn derivatization and fluorescence detection.

A reversed-phase (ODS-2) liquid chromatographic method was developed to determine low nanogram-per-gram levels of sulfadiazine (SDZ) in salmon muscle tissue. SDZ was extracted with acetonitrile-aqueous 2% acetic acid (pH 3.0), partitioned into methylene chloride, and cleaned up by using a strong-cation-exchange, solid-phase extraction cartridge. SDZ was derivatized postcolumn with fluorescamine and detected by fluorescence. The limit of detection was 0.2 ng SDZ/g tissue. Recoveries from coho salmon tissue fortified with 1, 5, 10, and 20 ng SDZ/g tissue averaged 84.5, 85.0, 83.6, and 83.9%, respectively; recoveries from Atlantic salmon tissue fortified with 10 ng SDZ/g tissue averaged 82.6%.

Arabinofuranosyl nucleotides are not chain-terminators during initiation of new strands of DNA by DNA polymerase alpha-primase.

Polymerization of NTPs and arabinofuranosyladenosine triphosphate (araATP) during DNA polymerase alpha catalyzed elongation of primase-synthesized primers was examined. After primase synthesizes a primer, pol alpha normally polymerizes multiple dNTPs onto this primer. In the absence of a required dNTP, however, primers were still elongated by up to 35 nucleotides via polymerization of the corresponding NTP in place of the missing dNTP. During the elongation of exogenously added primer/templates, however, NTPs were not readily polymerized. AraATP was readily incorporated into products during elongation of primase-synthesized primers. Importantly, polymerization of araATP did not result in chain termination; rather, the next correct nucleotide was added such that araATP was simply an alternate substrate. In contrast, polymerization of araATP during elongation of exogenously added primer/templates resulted in strong chain termination. Thus, elongation of primase-synthesized primers by pol alpha-primase is fundamentally different than elongation of exogenously added primer/templates with respect to interactions with dNTP analogs. Furthermore, these data provide a rationale for how araNMPs are efficiently incorporated into internucleotide linkages of DNA in whole cells and suggest that the initiation of new strands of DNA by pol alpha-primase may be a unique target for inhibiting replication.

High-performance liquid chromatographic analysis of the antituberculosis drugs aconiazide and isoniazid.

Reversed-phase HPLC methods are described for determining the stability and concentration certification of the antituberculosis prodrug aconiazide (ACON) in aqueous dosing solution and for assessing the concentrations of ACON and isoniazid (INH) in plasma from ACON-treated male and female Fischer-344 rats. ACON was analyzed in plasma by direct injection; it was separated on a 250 x 4.6 mm I.D. 5 microns C18 column using a 40% aqueous methanol mobile phase containing 5 g/l ammonium formate, and detected at 313 nm. INH was determined in the plasma of treated rats after a two-step precipitation of plasma proteins; it was separated on a 250 mm x 4.6 mm I.D. 5 microns CN column, eluted with 5% aqueous isopropanol containing 5 g/l ammonium formate, and detected with an electrochemical detector at +0.8 V. These methods allow a simple, rapid, and reliable determination of ACON and INH in plasma down to 0.1 micrograms/ml.

Liquid chromatographic determination of thiamine in rodent feed by postcolumn derivatization and fluorescence detection.

A liquid chromatographic method was developed for determination of the essential nutrient thiamine (vitamin B1) in rodent feed. Thiamine was extracted with hydrochloric acid, separated by reversed-phase liquid chromatography, derivatized postcolumn to thiochrome with potassium hydroxide and potassium ferricyanide, and detected by fluorescence. Excitation and emission wavelengths were 370 and 430 nm, respectively. Detector response was linear in the range of 2.58 to 15.5 ng of thiamine injected. Instrument detection limit was 5 pg of thiamine injected.

Analysis of calcium and lead in calcium supplements by inductively coupled plasma-atomic emission spectrometry and graphite furnace atomic absorption spectrophotometry.

A method was developed to analyze various calcium supplements for Ca and Pb content. The analysis involves a dry ash of the supplements followed by wet digestion. The Pb is determined by graphite furnace atomic absorption spectrophotometry (GFAAS). Analysis of Ca is by inductively coupled plasma-atomic emission spectrometry (ICP-AES). Ca supplements fortified with Pb at levels ranging from 0.25 to 10.0 micrograms/g yielded recoveries ranging from 82.7 +/- 4.2 to 105.0 +/- 1.7%. To test accuracy, the method was applied to National Institute of Standards and Technology standard reference materials (NIST SRMs) 1572 citrus leaves and 1486 bone meal. GFAAS analysis of SRM 1572 averaged 13.1 +/- 0.6 micrograms Pb per g (certificate value, 13.3 +/- 2.4 micrograms Pb per g), and analysis of SRM 1486 averaged 1.34 +/- 0.11 micrograms Pb per g (certificate value, 1.335 +/- 0.014 micrograms Pb per g). ICP-AES analysis of SRM 1572 averaged 3.12 +/- 0.01% Ca (certificate value, 3.15 +/- 0.10% Ca by weight), and analysis of SRM 1486 averaged 27.63 +/- 0.27% Ca (certificate value, 26.58 +/- 0.24% Ca). The method's limit of quantitation (LOQ), on supplement Ca basis and a 1 g sample, averaged 0.75 micrograms Pb per 1 g Ca for supplements containing 9 to 35% Ca by weight. At a Pb level of 0.663 micrograms/g Ca, the reproducibility relative standard deviation (RSDr) averaged 7.3% and the repeatability relative standard deviation (RSDR) averaged 8.0%. It is recommended that the method be studied collaboratively.

Incidence of BK virus and JC virus viruria in human immunodeficiency virus-infected and -uninfected subjects.

BK virus (BKV) and JC virus (JCV) are present within the renal system of most adults. Reactivation may be linked to immunodeficiency, since many of the extant virus strains have been isolated from urine or kidney tissue of patients who were receiving immunosuppressive therapy or who had disorders of the immune system. To more critically evaluate the relationship between immunodeficiency and viruria, urine samples from individuals infected with human immunodeficiency virus (HIV) with various degrees of immunodeficiency were screened for the presence of viral DNA. JCV viruria occurred in 24%-27% of immunocompetent control subjects and was not increased with immunodeficiency. By contrast, there were both qualitative and quantitative changes in BKV viruria in immunodeficient subjects. The incidence of BKV viruria was increased, and some immunodeficient subjects shed BKV at levels up to 3000 times greater than levels shed by any of the nonimmunodeficient controls. DNA sequence rearrangements in the viral regulatory region did not appear to be required for shedding of virus, although they were present in approximately 20% of samples.