Extraction and analysis of lead in sweeteners by flow-injection Donnan dialysis with flame atomic absorption spectroscopy.

Flow-injection Donnan dialysis is demonstrated for the extraction of lead in sweeteners using flame atomic absorption spectroscopy (FAAS). For spiked concentrations in the low microgram per gram range, recoveries were greater than 90%, and the relative standard deviation was typically less than 10% for a 15-min dialysis procedure. The method detection limit is 350 ng/g. Donnan dialysis is shown to be successful for the extraction of lead in sucrose, corn syrup, and honey but limited in performance for molasses and artificial syrup. This paper also includes a comparison to other procedures for the determination of lead in sweeteners and presents options for realizing improved method performance with Donnan dialysis.

Plant-derived and semi-synthetic calanolide compounds with in vitro activity against both human immunodeficiency virus type 1 and human cytomegalovirus.

Plant-derived and semi-synthetic calanolide compounds with anti-human immunodeficiency virus type 1 (HIV-1) activity were tested for anti-human cytomegalovirus (HCMV) activity in both cytopathic effect inhibition and plaque reduction assays. The results indicated that the anti-HCMV activity of calanolide compounds does not correlate with their activity against HIV-1. The semi-synthetic 12-keto derivatives tended to be more active against HCMV than the corresponding 12-OH congeners, which were more active against HIV-1. It appeared that the 7,8-unsaturated double bond in the chromene ring played a certain role in maintaining activities against both HCMV and HIV-1. Saturation of the double bond increased the EC50 values against both viruses, with concomitant increase in toxicity. The calanolide compounds reported here are the first non-nucleoside analogues capable of inhibiting both HIV-1 and HCMV and, therefore, may be useful chemoprophylactic agents for HCMV in HIV-infected people or vice versa.

Adolescent health care experience of gay, lesbian, and bisexual young adults.

METHODS: Subjects were 102 self-identified gay, lesbian, and bisexual youth aged 18-23 years. A confidential self-administered survey elicited demographic information, sexual orientation information, health care experiences, subjects' understanding of medical confidentiality during ages 14-18 years, and their suggestions for improving care to gay and lesbian adolescents. RESULTS: Two-thirds of subjects never discussed sexual orientation with their provider but reported a desire to do so. Fewer than one-half of subjects remembered being informed about their right to medical confidentiality; those who reported being so informed were three times more likely to have discussed their sexual orientation with their provider. Over 70% of subjects who reported not being informed about their right to medical confidentiality stated that they would have been more likely to discuss sexual orientation with their provider had they been so informed. Only 13 of subjects had disclosed their sexual orientation to their health care providers. Of these, only half of the males received information on human immunodeficiency virus prevention. CONCLUSIONS: Health care providers may be failing to fully address issues of confidentiality and sexual orientation with adolescents, despite a decade of increased information on adolescent homosexuality.

Evaluation of anti-AIDS drugs in conventional mice implanted with a permeable membrane device containing human T cells infected with HIV.

We now report the confirmation of the work of Hollingshead et al. (1995) on development of a cell based hollow fiber (HF) system for evaluating potential anti-AIDS drugs in vivo using conventional mice rather than SCID mice. CD4 +, CEM-SS cells infected with HIV/1, strain RF, at a multiplicity of infection of 0.1 were placed into HFs. The fibers were implanted into the peritoneal cavity of outbred Swiss mice. Using this model, the antiviral activity of azidothymidine (AZT) at doses of approximately 150, 75 and 37.5 mg/kg/day was evaluated by administering AZT to the mice in drinking water. Upon fiber removal on day 6, AZT treatment was shown to significantly increase CEM cell viability over the untreated, virus control group and significantly reduced the levels of HIV p24 and HIV RT activity.

In vitro and in vivo enhancement of ddI activity against Rauscher murine leukemia virus by ribavirin.

Ribavirin has been reported to enhance the activity of ddI against HIV. We explored this enhancement of antiviral activity in Rauscher murine leukemia virus (RMuLV) models in vitro and in vivo. The significant finding in these studies was that combinations of the drugs exhibited virus titer reductions that were greater than would be expected if the drug interactions were simply additive. These effects were designated synergistic by the method of Prichard and Shipman (Prichard, M.N. and Shipman, C., Jr. (1990). A three-dimensional model to analyze drug-drug interaction, Antiviral Res. 14, 181-206). In addition to the antiviral synergy, we also observed some synergistic toxicity in the animal model.

Differential antiviral activity of two TIBO derivatives against the human immunodeficiency and murine leukemia viruses alone and in combination with other anti-HIV agents.

R82913 and R86183, two derivatives of tetrahydroimidazo[4,5,1-jk][1,4]-benzodiazepin-2(1H)-thione (TIBO), were found to potently and selectively inhibit the replication and cell killing effects of a panel of biologically diverse laboratory and clinical strains of HIV-1. The two compounds exhibited significant activity in all human cell lines tested, as well as in fresh human peripheral blood lymphocytes and macrophages. One of these two compounds (R82913) was found to significantly inhibit the replication of a murine retrovirus (Rauscher murine leukemia virus) in both UV-XC plaque formation and virus yield reduction assays. R86183, despite differing from R82913 only in the positioning of a single chlorine molecule, was not active against the murine retrovirus but was 10-fold more potent in inhibiting HIV-1 replication. Combination antiviral assays with other reverse transcriptase inhibitors, including AZT, ddC, and carbovir, yielded synergistic anti-HIV activity with both TIBO derivatives. Additive to slightly synergistic results were obtained in combinations with ddI and phosphonoformic acid whereas additive to antagonistic activity was detected in combination with dextran sulfate.

Thiazolobenzimidazole: biological and biochemical anti-retroviral activity of a new nonnucleoside reverse transcriptase inhibitor.

Thiazolobenzimidazole (NSC 625487) was a highly potent inhibitor of human immunodeficiency virus-induced cell killing and viral replication in a variety of human cell lines, as well as fresh human peripheral blood lymphocytes and macrophages. The compound was active against a panel of biologically diverse laboratory and clinical strains of HIV-1, including the AZT-resistant strain G910-6. However, the agent was inactive against HIV-2 and a pyridinone-resistant strain (A17) of HIV-1, a strain which is cross-resistant to several structurally diverse members of a common pharmacologic class of nonnucleoside reverse transcriptase inhibitors. The compound selectively inhibited HIV-1 reverse transcriptase but not HIV-2 reverse transcriptase. Combinations of thiazolobenzimidazole with either AZT or ddI synergistically inhibited HIV-1 induced cell killing in vitro. Thiazolobenzimidazole also inhibited the replication of the Rauscher murine leukemia retrovirus. Thus, thiazolobenzimidazole is a new active anti-HIV-1 chemotype and may represent a subclass of nonnucleoside reverse transcriptase inhibitors with an enhanced range of anti-retroviral activity.

An ELISA system for evaluating antiretroviral activity against Rauscher murine leukemia virus.

A system for evaluating the activity of antiviral agents against Rauscher murine leukemia virus (R-MuLV) has been developed using an enzyme linked immunosorbent assay technique. The activity of various antiviral compounds demonstrated in this assay system has been compared to their activity in the UV-XC plaque reduction assay, which has been used historically for evaluating anti-R-MuLV compounds. The assay is based upon detection of R-MuLV encoded p30 protein production in virus infected murine cells. The assay reagents are readily available and the assay system is amenable to automated data collection systems. Cytotoxicity evaluations are conducted in parallel to the Rauscher MuLV ELISA assay in order to assess drug-induced reductions in cell viability. Cytotoxicity evaluations are important to interpretation of the ELISA results since reductions in cell viability reduce viral protein production which would indicate an antiviral drug effect. This system is less sensitive than the classical UV-XC plaque reduction assay; however, it does offer an alternative to the time-consuming and labor-intensive plaque assay.

Novel method for evaluating antiviral drugs against human cytomegalovirus in mice.

A virus-host cell system in which human cytomegalovirus-infected human cells are entrapped in agarose plugs has been developed. This model provides an inexpensive method for the in vivo evaluation (with outbred, immunocompetent mice) of antiviral drugs against human viruses such as cytomegalovirus that replicate primarily or only in human cells.

Effect of the estrous cycle on susceptibility of female mice to intravaginal inoculation of herpes simplex virus type 2 (HSV-2).

In order to explore the effect of sexual maturity on the susceptibility of mice to genital herpesvirus infections, mice were separated into the four stages of the estrous cycle and inoculated intravaginally with varying doses of HSV-2, strain 186. Deaths were observed as indicators of susceptibility and were recorded as follows: proestrous, 33%; estrous, 16%; metestrous, 9% and diestrous, 75%. To determine the course of infection in animals inoculated at different stages of estrous, cotton swabs were used to collect vaginal specimens at various times post-virus inoculation for virus titration. All mice inoculated during diestrous were positive for virus as early as 6 hours post-virus inoculation and had titers that increased over a 3 day period. Mice inoculated in other stages of estrus were positive only briefly (at 6 h) or had no detectable virus. In order to verify the susceptibility associated with diestrous, mice were ovariectomized to produce a continuous diestrous (pseudodiestrous) and when inoculated greater than or equal to 66.7% died. In contrast, none of the mice which had been ovariectomized and treated with estrogen to simulate the estrus stage died. We postulate that in stages other than diestrous virus may adsorb to epithelial cells in the lumen of the vagina and/or be expelled from the body by nonspecific resistance functions, thus reducing the likelihood of vaginal infection.

Sulphoevernan, a polyanionic polysaccharide, and the narcissus lectin potently inhibit human immunodeficiency virus infection by binding to viral envelope protein.

Sulphoevernan is a sulphated alpha-1----3, 1----4 polyglucan (Mr 20,000) with a helical structure. This compound effectively inhibits both human immunodeficiency virus type 1 (HIV-1) and type 2 infection of cells in vitro at concentrations around 0.5 micrograms/ml. Moreover, the compound completely inhibits HIV-1-induced syncytium formation at a concentration of 1 microgram/ml. Competition experiments with 35S-labelled sulphoevernan revealed that the mannose-specific lectin from Narcissus pseudonarcissus prevented binding of sulphoevernan to HIV-1, whereas the antibody OKT4A did not reduce the amount of sulphoevernan bound to MT-2 cells. These data indicate that the non-cytotoxic polymer sulphoevernan binds to the virus rather than to the host cell. In vivo studies, using Rauscher leukaemia virus in NMRI mice, revealed that, at a daily dose of 20 mg/kg, the animals were protected against virus-induced increases in spleen weight. From these in vitro and in vivo data we conclude that sulphoevernan has potential in the treatment of acquired immunodeficiency syndrome.

Synthesis, antitumor activity, and antiviral activity of 3-substituted 3-deazacytidines and 3-substituted 3-deazauridines.

Novel 3-substituted analogues of 4-amino-1-beta-D-ribofuranosyl-2(1H)-pyridinone (3-deazacytidine, 3) and 4-hydroxy-1-beta-D-ribofuranosyl-2(1H)-pyridinone (3-deazauridine, 4) have been synthesized and tested for antitumor and antiviral activity. Thus the 3-chloro (9a), 3-bromo (9b), and 3-nitro (9c) analogues of 3 and the 3-chloro (9d), 3-bromo (9e), and 3-nitro (9f) analogues of 4 were prepared by standard glycosylating procedures. Novel requisite heterocycles 4-amino-3-chloro-2(1H)-pyridinone (7a) and 4-amino-3-bromo-2(1H)-pyridinone (7b) were prepared by halogenating 4-amino-2(1H)-pyridinone (5). Requisite heterocycles 4-amino-3-nitro-2(1H)-pyridinone (7c), 3-chloro-4-hydroxy-2(1H)-pyridinone (7d), 3-bromo-4-hydroxy-2(1H)-pyridinone (7e), and 4-hydroxy-3-nitro-2(1H)-pyridinone (7f) were synthesized by known procedures from 4-hydroxy-2(1H)-pyridinone (6). Structure proof of target nucleosides was provided by independent synthesis, 1H NMR, and UV. Compounds 9a-f were devoid of activity against intraperitoneally implanted L1210 leukemia in mice. Compound 9f displayed significant activity against rhinovirus type 34 grown in WISH cells. 4-Amino-3-fluoro-1-beta-D-ribofuranosyl-2(1H)-pyridinone (1) displayed good activity against intraperitoneally implanted P388 leukemia in mice, but it was devoid of activity against M5076 sarcoma, amelanotic (LOX) melanoma xenograft, and subrenal capsule human mammary carcinoma MX-1 xenograft in mice. Compound 1 also displayed significant activity against rhinovirus type 34.

Antiviral and cytotoxicity evaluation of 3-nitro-3-deazauridine.

3-Nitro-3-deazauridine (3N-3DU) is a new synthetic nucleoside having activity against members of 5 RNA virus families including: paramyxoviruses (parainfluenza, PIV), picornaviruses (rhino-, RV), rhabdoviruses (vesicular stomatitis, VSV), togaviruses (Semliki Forest, SFV) and bunyaviruses (Punta Toro, PTV). In this report, we evaluate and compare its activity with the parent nucleoside, 3-deazauridine (3DU) and ribavirin as drug standards. Comparison of drug activities utilizes observations of antiviral indices, which are determined by the following formula: maximum tolerated dose (MTD)/minimum inhibitory concentration (MIC). The antiviral index (AI) of 3N-3DU (AI 15.3) was comparable to ribavirin and much higher than 3DU when evaluated against PIV. The 3N-3DU was the most active of the three when tested against RV (AI 24.1), SFV (AI 76.9) or VSV (AI 50). In contrast to the RV activity, 3N-3DU (AI 0.5) and 3DU (AI less than 0.1) were less active than ribavirin (AI 1.3) when evaluated against poliovirus, type 1 (PoV). Ribavirin (AI 10.0) was more active than 3N-3DU (AI 2.4) and 3DU (AI less than 0.1) against PTV. 3N-3DU exhibited comparable toxicity to ribavirin in KB cells, was 4-fold less toxic in WISH cells and 4-fold more toxic in LLC-MK2 cells. Overall, 3N-3DU is markedly less toxic than its parent nucleoside, 3DU. It appears from this study that the structural modification of 3DU resulting from the addition of the nitro group in the 3 position of the base reduces toxicity and enhances the antiviral activity.

Evaluation of continuous cell lines in antiviral studies with murine cytomegalovirus.

Cell culture systems were developed for rapid antiviral drug screening, using murine cytomegalovirus (MCMV) as an alternative to the slower growing human CMV. Since previous assay methods with MCMV employed mouse embryo fibroblasts (MEF cells), which are labor intensive to prepare and die off after 3-4 passages from primary culture, identification of virus-susceptible continuous cell lines was desirable. Three cell lines were found useful for assaying MCMV: C127I, SC-1, and 3T3. The antiviral agents acyclovir, ganciclovir, 5-fluoroarabinofuranosylcytosine, and 2'-fluoro-2'-deoxy-5-iodoarabinofuranosylcytosine were evaluated in the 3 continuous cell lines and in MEF cells. The 50% virus- or cell-inhibitory concentration values determined for each compound did not vary much from cell to cell. MEF cells were 10-fold more sensitive than the other cell lines to quantify virus from mouse organs, however. Virus propagated in 3T3 and SC-1 cells were as virulent to mice as salivary gland virus, whereas virus from MEF and C127I cells was more attenuated. Overall, C127I cells were judged to be the best for large scale antiviral screening in vitro, but MEF was the cell type of choice for titration of viruses from mouse organs and tissues.

A simple method of drying virus on inanimate objects for virucidal testing.

A simple method for drying virus on inanimate objects (cover slips) under vacuum in the cold is described. Following this procedure virus maintains high titers (10(6-7)) for periods of 1-3 wk at -70 degrees C depending on the virus. For virucidal assay of disinfectants, cover slips are exposed to medium simulating the disinfectant (virus control) or disinfectant in an upright position in an Ultra-Vu cuvette. Cover slips are readily removed and placed in tissue culture medium for dilution of virus and determination of virus titer. Cytotoxicity of disinfectant is determined by exposing cover slip without virus to disinfectant, then placing it in medium, diluting the medium and incubating with the indicator cells. The use of this technique results in high titers of virus on cover slips, which are inanimate objects requiring minimal manipulation. The titration of virus or cytotoxicity in microplates is cell, medium, serum, and labware economical.

Cytostatic and antiherpesvirus type 1 and 2 activities of 1-beta-D-arabinofuranosylthymine (ara T) prodrugs.

A series of derivatives of the antibiotic 1-beta-D-arabinofuranosylthymine (ara T) was synthesized by esterification of the hydroxy group in the 5'-position of the arabinose moiety of the nucleoside with straight-chain and branched-chain carboxylic acids: acetyl-ara T, butyryl-ara T, valeroyl-ara T, pivaloyl-ara T and palmitoyl-ara T. These ara T prodrugs were evaluated for their effect on growth of L5178y mouse lymphoma cells and noninfected BHK-21 cells as well as for their antiviral activity in Herpes simplex virus type 1 and 2 infected BHK-21 cells. All compounds exhibited a marked antiherpes virus activity, whereas the cytostatic activity of two of them, the pivaleric ester and the palmitic ester, was extremely weak. The relative antiviral indices of the 5'-pivaloyl-ara T and 5'-palmitoyl-ara T were found to be much better than the index of ara T itself.

Acyclic pyrimidine nucleoside analogues: influence on growth of L5178y mouse lymphoma cells and antiherpes activity in KB cells.

Several uracil and cytosine nucleoside analogues with 2-hydroxyethoxymethyl, 2-aminoethoxymethyl or 1,3-dihydroxypropoxymethyl side chains were synthesized and evaluated for cytostatic (L5178y mouse lymphoma cells) and antiviral (herpes simplex virus type 1 infected KB cells) activity. Two compounds exhibited antiherpesvirus activity. These were 1-(2'-hydroxyethoxymethyl)-5-aza-cytosine and 1-(1',3'-dihydroxypropoxymethyl)-5-iodouracil. The MIC values were 25.8 and 325.7 micrograms/ml, respectively.

Evaluation of the anti-herpesvirus drug combinations: virazole plus arabinofuranosylhypoxanthine and virazole plus arabinofuranosyladenine.

Combinations of Virazole plus arabinofuranosylhypoxanthine (ara-Hx) and Virazole plus arabinofuranosyladenine (ara-A) were investigated in KB or BHK cells infected with types 1 or 2 herpes viruses. Combinations of Virazole and ara-Hx exhibited significant synergy as evaluated graphically (isobolograms) or by fractional inhibitory concentration (FIC) indices. Optimal ratios for the combination were 1:1 to 1:10 for Virazole to ara-Hx. At these ratios, FIC indices in the range of 0.5-0.2 were commonly observed. Combinations of Virazole and ara-A were antagonistic when observed in the presence of pentostatin, an adenosine deaminase inhibitor. In the absence of pentostatin, the minimum inhibitory concentration (MIC) of ara-A and degree of synergy with Virazole were variable.

Phosphorus in swine. V. Interrelationships of various feedlot performance, serum minerals, structural soundness and bone parameters in barrows, boars and gilts.

Daily gain, daily feed intake, feed per unit of gain, serum Ca, serum P, serum Mg, structural soundness scores, foot pad scores, metacarpal breaking force and metacarpal ash values from five Ca P trials with barrows, gilts or boars were subjected by sex in each trial to multivariate analysis of variance. Correlation coefficients were obtained from the residual sums of squares and sum of products; thus, coefficients were corrected for treatment effects. Individual values were used for all comparisons except those involving daily feed and feed/gain, for which pen means were used. High positive correlations were observed between daily gain and daily feed; there was no relationship between daily gain and feed/gain, but daily feed was positively correlated with feed/gain. Serum Ca levels were not highly correlated with daily gain, daily feed or feed/gain. Although all coefficients were less than .5, serum P was positively (P less than .01 or less than .05) related to daily gain. Serum Ca and serum Mg concentrations were unrelated to serum P concentrations, but serum Ca was positively correlated with serum Mg. There was no relationship between daily gain and soundness or pad scores. Although there were some inconsistencies, a positive relationship was observed between daily gain and metacarpal dried weight, and between daily gain and breaking strength. There appeared to be little, if any, relationship between daily feed and feed/gain and bone parameters. Ca, P and Mg were not consistently related to metacarpal dried weight, breaking strength or ash. Dried weight was positively correlated to breaking strength in four trials and to ash in two trials. Breaking strength was correlated to ash in only one trial. These results offer no support for the belief that stronger, denser bones produce more structurally sound animals, because soundness and pad scores were not related to bone parameters.

Synthesis and antitumor and antiviral activities of 1-beta-D-arabinofuranosyl-2-amino-1,4(2H)-iminopyrimidine and its derivatives.

1-beta-D-Arabinofuranosyl-2-amino-1,4(2H)-imino-5-fluoropyrimidine (10), 1-beta-D-arabinofuranosyl-2-amino-1,4(2H)-imino-5-fluoropyrimidine 3'-phosphate (9), and 1-beta-D-arabinofuranosyl-2-amino-1,4(2H)-imino-5-chloropyrimidine (11) have been synthesized from 2,2'-anhydro-1-beta-D-arabinofuranosyl-5-fluorocytosine (5), 2,2'-anhydro-1-beta-D-arabinofuranosyl-5-fluorocytosine 3'-phosphate (4), and 2,2'-anhydro-1-beta-D-arabinofuranosyl-5-chlorocytosine (6), respectively. 2,2'-Anhydro-1-beta-D-arabinofuranosylcytosine 3'-phosphate (7), 1-beta-D-arabinofuranosyl-2-amino-1,4-(2H)-iminopyrimidine (13), 1-beta-D-arabinofuranosyl-2-amino-1,3(2H)-iminopyrimidine 3'-phosphate (12), and compounds 4, 5, and 9 showed significant in vitro activity against a number of DNA viruses. Compounds 7 and 12 were also effective in vivo against type 1 herpes simplex virus. Compounds 7, 12, and 13 were extremely effective in the treatment of mice bearing leukemia L1210.