Influence of Acyclic Nucleoside Phosphonate Antivirals on Gene Expression of Chemokine Receptors CCR5 and CXCR4.

Acyclic nucleoside phosphonates (ANPs) are potent antiviral agents effective against replication of DNA viruses and retroviruses including human immunodeficiency virus (HIV). Prototype compound 9-(R)-[2-(phosphonomethoxy)propyl]adenine (tenofovir) is a principal component of drugs widely used in the treatment of HIV infection (Viread, Truvada). Besides their antimetabolic mode of action, ANPs possess immunomodulatory properties. A number of them have been previously found to stimulate secretion of cytokines and anti-HIV effective chemokines. In the present pilot experiments we analysed the in vitro effects of ANPs on the expression of chemokine receptors CCR5 and CXCR4 that are co-receptors of HIV-1 entry in cells. The impact of ANPs was investigated at the level of gene transcription of mRNA in mouse lymphocytes and macrophages using the RT-PCR method. The following compounds were included in the study: 9-(R)-[2-(phosphonomethoxy) propyl]adenine (tenofovir), N6-cyclopropyl-(R)- 9-[2-(phosphonomethoxy)-propyl]2,6-diaminopurine, N6-cyclopentyl-(R)-9-[2-(phosphonomethoxy) propyl]2,6-diaminopurine, N6-dimethylaminoethyl-(R)-9-[2-(phosphonomethoxy)propyl]2,6-diaminopurine, N6-cyclopentyl-9-[2-(phosphonomethoxy) ethyl]2,6-diaminopurine, N6-isobutyl-9-[2-(phosphonomethoxy) ethyl]2,6-diaminopurine. Gene transcription of chemokine receptors CCR5 and CXCR4 was not affected after application of these acyclic nucleoside phosphonate antivirals.

Fracture strength of root filled premolar teeth restored with silorane and methacrylate-based resin composite.

OBJECTIVES: To compare fracture characteristics of root-filled teeth with variable cavity design restored with a low shrinkage silorane and methacrylate-based resin composite. METHODS: 77 extracted maxillary premolars were divided randomly into seven groups: (Group 1) intact teeth; (Groups 2-4) MOD plus endodontic access with the buccopalatal width of the occlusal isthmus equals one third of the intercuspal width; (Groups 5-7) MOD plus endodontic access with the buccopalatal width of the occlusal isthmus equals one half of the intercuspal width. Groups 2 and 5 were left unrestored, Groups 3 and 6 were restored with a silorane-based resin composite (Filtek P90) and Groups 4 and 7 with a methacrylate-based resin composite (Z250). Teeth were loaded in a universal testing machine; load and fracture patterns were recorded and compared statistically using 2-way ANOVA and t-test for pairwise comparisons and 1-way ANOVA with Dunnett test for multiple comparisons. RESULTS: Unrestored teeth became progressively weaker with more extensive preparations, Group 5 (unfilled ½) showed the lowest fracture load among the groups (71±22N, P<0.001). Restorations increased the fracture strength of unrestored teeth regardless of cavity size (P<0.001), but was still significantly weaker than sound teeth, with no significant difference between silorane and methacrylate groups. Failure of restored teeth was mostly adhesive at the tooth restoration interface. CONCLUSIONS: Silorane-based resin composite have no superior strengthening effect over the conventional methacrylate-based resin composite in restoration of root filled teeth. Both materials showed similar fracture patterns. CLINICAL SIGNIFICANCE: Root filled teeth are considerably weakened via restorative and endodontic procedures. A direct adhesive restoration will aid in preserving tooth structure as far as it provides enough strength.

New in vitro method for evaluating antiviral activity of acyclic nucleoside phosphonates against plant viruses.

A new method was developed for testing antiviral compounds against plant viruses based on rapidly growing brassicas in vitro on liquid medium. This method enables exchange of media containing tested chemicals in various concentrations and simultaneous evaluation of their phytotoxicity and antiviral activity. While using ribavirin as a standard for comparison, phytotoxicity and ability of the acyclic nucleotide analogues (R)-PMPA, PMEA, PMEDAP, and (S)-HPMPC to eliminate ssRNA Turnip yellow mosaic virus (TYMV) were evaluated by this method. Double antibody sandwich ELISA and real-time PCR were used for relative quantification of viral protein and nucleic acid in plants. Ribavirin had the most powerful antiviral effect against TYMV. On the other hand, (R)-PMPA and PMEA had no antiviral effect and almost no phytotoxicity compared to the control. (S)-HPMPC and PMEDAP showed moderate antiviral effect, accompanied by higher phytotoxicity. The tested compounds can be screened within 6-9 weeks in contrast to the 6 months for traditionally used explants on solid medium. The method enables large-scale screening of potential antivirals for in vitro elimination of viruses from vegetatively propagated crops and ornamentals.

[Acyclic nucleoside phosphonates as potential antineoplastic agents]. / Acyklické nukleosidfosfonáty jako potenciální antineoplastika.

Recently, Gilead Sciences (Foster City, CA, USA) presented a potential cytostatic drug GS-9219. It is a novel lipophilic prodrug of cyprPMEDAP, in vivo releasing the active compound PMEG in a two-step process. GS-9219 has shown a substantial therapeutic potential in treatment of spontaneous non-Hodgkin's lymphoma in dogs and its utilization in the human medicine is prospective. Hence, cyprPMEDAP represents a key intermediate in the intracellular activation of GS-9219. Both acyclic nucleoside phosphonates PMEG and cyprPMEDAP, serving as the basis for development of GS-9219, were discovered and their mechanism of action was investigated in detail at the Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic. The biological studies using the rat lymphoma were carried out at the First Faculty of Medicine, Charles University.

In vivo study of the effect of antiviral acyclic nucleotide phosphonate (R)-9-[2-(phosphonomethoxy)propyl]adenine (PMPA, tenofovir) and its prodrug tenofovir disoproxil fumarate on rat microsomal cytochrome P450.

The total content of rat liver microsomal cytochrome P450 (CYP) significantly decreased after repeated i.p. administration of the antiviral agent tenofovir ((R)-9-[2-(phosphonomethoxy)propyl] adenine) and tenofovir disoproxil at a daily dose 25 mg/kg, although the content of liver microsomal protein did not change. The decrease of the CYP content was accompanied by concomitant increase of the amount of inactive CYP form, cytochrome P420. This effect was confirmed by a parallel study of the activities of selected CYP forms, CYP2E1 (p-nitrophenol hydroxylation) and CYP1A2 (7-ethoxyresorufin deethylation). The activity (expressed relatively to the protein content) of both CYP forms decreased significantly following the decrease of the total CYP. On the other hand, the CYP2E1 activity expressed relatively to the decreasing total CYP content remained unchanged. However, CYP1A2 activity also decreased when calculated relatively to the total native CYP content indicating lower stability of this form. Semiquantitative RT-PCR showed no significant changes in expression of major rat liver microsomal CYP forms after tenofovir treatment. In conclusion, repeated administration of tenofovir in higher doses led to significant decrease of the relative proportion of active liver microsomal CYPs accompanied by a conversion of these enzymes to the inactive form (CYP420) maintaining the sum of CYP proteins unchanged.

Cleaning and handling implements as potential reservoirs for bacterial contamination of some ready-to-eat foods in retail delicatessen environments.

This study assessed the association of bacteria with cleaning tools, such as floor mops (n = 25) and cleaning cloths (n = 39), and handling devices, such as disposable plastic gloves (n = 20), used during filled baguette and assorted salad preparation in four selected retail delicatessens in Johannesburg, South Africa. Samples of each cleaning or handling tool were prepared for aerobic (APC), coliform (CC), Escherichia coli (EC), Bacillus cereus (BCC), and Staphylococcus aureus (SAC) counts, as well as tested for the incidence of Listeria monocytogenes (LM) and Salmonella (SALM) by standard plating methods. Bacterial populations attached to the cleaning and handling tools were observed by scanning electron microscopy (SEM). Ten selected gram-positive isolates were further analyzed by 16S rRNA sequence analysis and compared with isolates from filled baguettes and assorted salads. The floor mops consistently yielded the highest APCs, CCs, and ECs (5.7, 4.1, and 3.0 log CFU/g, respectively), while gloves had the lowest corresponding counts (3.6, 2.0, and 1.0 log CFU/g, respectively). Low BCCs and SACs were recorded in this study (ca. 1.2 log CFU/g), while SALM and LM were each detected in five cleaning tool samples. SEM showed rods and cocci attached to handling and cleaning tools. Furthermore, results of 16S rRNA sequence analysis indicated that several gram-positive isolates were identified as S. aureus, Staphylococcus pasteuri, Staphylococcus sciuri, and Enterococcus faecalis. Genetically similar strains (100% similarity) were isolated from cleaning and handling tools and associated ready-to-eat (RTE) foods. Cleaning and handling tools may act as reservoirs of contamination for RTE foods during preparation in retail delicatessens in South Africa. The transfer of potential pathogens, such as S. aureus, to foods from cleaning and handling tools may hold food safety implications.

Experimental therapy with 9-[2-(phosphonomethoxy)ethyl]-2,6-diaminopurine (PMEDAP): origin of resistance.

The role of MRP4 and MRP5 transporters in the acyclic nucleoside phosphonate PMEDAP efflux was studied in vitro (CCRF-CEM cells) and in vivo (spontaneous transplantable T-cell lymphoma of SD/Cub inbred rats). The increased resistance against the cytostatic agent PMEDAP during longterm treatment was found to be associated with overexpression of MRP4 and MRP5 genes. The course of both gene activation differs significantly. While the MRP5 function is important in the onset of PMEDAP resistance, the intensity of the relative MRP4 gene expression increases rather continuously. Our data indicate cooperative acting of both MRP4 and MRP5 genes during the PMEDAP resistance development.

In vitro activity of 2,4-diamino-6-[2-(phosphonomethoxy)ethoxy]-pyrimidine against multidrug-resistant hepatitis B virus mutants.

The susceptibilities of drug-resistant hepatitis B virus (HBV) mutants to lamivudine, adefovir, tenofovir, entecavir, and 2,4-diamino-6-[2-(phosphonomethoxy)ethoxy]-pyrimidine (PMEO-DAPym), a novel acyclic pyrimidine analogue, were assessed in vitro. Most drug-resistant mutants, including multidrug-resistant strains, remained sensitive to tenofovir and PMEO-DAPym. Therefore, the latter molecule deserves further evaluation for the treatment of HBV infection.

Towards the mechanism of trimeric purine nucleoside phosphorylases: stopped-flow studies of binding of multisubstrate analogue inhibitor - 2-amino-9-[2-(phosphonomethoxy)ethyl]-6-sulfanylpurine.

The binding of multisubstrate analogue inhibitor - 2-amino-9-[2-(phosphonomethoxy)ethyl]-6-sulfanylpurine (PME-6-thio-Gua) to purine nucleoside phosphorylase from Cellulomonas sp. at 20 degrees C, in 20 mM Hepes buffer with ionic strength adjusted to 50 mM using KCl, at several pH values between 6.5 and 8.2, was investigated using a stopped-flow spectrofluorimeter. The kinetic transients registered after mixing a protein solution with ligand solutions of different concentrations were simultaneously fitted by several association reaction models using nonlinear least-squares procedure based on numerical integration of the chemical kinetic equations appropriate for given model. It is concluded that binding of a PME-6-thio-Gua molecule by each of the binding sites is sufficiently well described by one-step process, with a model assuming interacting binding sites being more probable than a model assuming independent sites. The association rate constants derived from experimental data, assuming one step binding and independent sites, are decreasing with an increase in pH, changing from 30 to 6 microM(-1)s(-1) per binding site. The dissociation rate constants are in the range of 1-3 s(-1), and they are rather insensitive of changes in pH. Interestingly, for each pH value, the one-step binding model with interacting sites results in the association rate constant per site 1.5-4 times smaller for the binding of the first ligand molecule than that for the binding of the second one. Decrease of association constants with pH indicate that the enzyme does not prefer binding of the naturally occurring anionic form of the 6-thioguanine ring (pK(a) 8.7) resulting from a dissociation of N(1)-H. This finding supports the mechanism in which hydrogen bond interaction of N(1)-H with Glu204 (Glu 201 in mammalian PNPs) is crucial in the catalytic process. Results obtained also indicate that, in contrast to transition-state analogues, for which binding is followed by a conformational change, binding of multisubstrate analogue inhibitors to trimeric PNPs is a one-step process.

Inhibition of human liver microsomal cytochrome P450 activities by adefovir and tenofovir.

Adefovir (PMEA) and tenofovir (PMPA) and their prodrugs, adefovir dipivoxil (bisPOM-PMEA) and tenofovir disoproxil (bisPOC-PMPA), were subjected to a detailed study of their potential to inhibit the activities of human liver microsomal cytochromes P450 (CYP). The inhibition of marker enzyme activities of CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2D6, CYP2E1 and CYP3A4 was examined with high-performance liquid chromatography (HPLC) or spectroscopic (fluorescence, luminescence) detection. Adefovir and adefovir dipivoxil did not significantly influence activities of most CYP enzymes. The activity of CYP3A4 was inhibited by adefovir dipivoxil at concentrations over 100 microM. Adefovir and its prodrug inhibited CYP2C9 at concentrations below 100 microM; inhibition by adefovir was of the uncompetitive (at the lower inhibitor concentrations) or of the competitive nature with a Ki = 420 microM. Tenofovir and tenofovir disoproxil influenced the activity of CYP2C9, and competitive inhibition was found with Ki = 580 and 395 microM, respectively. Tenofovir disoproxil was shown to inhibit microsomal CYP2E1 activities by a mixed-type inhibition with Ki values at about 140 microM. The results indicate the possibility of an influence of the compounds tested on the respective CYP activities when used at high doses.

Bacterial biofilms within the clinical setting: what healthcare professionals should know.

Bacterial biofilm formation is the prevailing microbial lifestyle in natural and manmade environments and occurs on all surface types. Biofilm formation develops in several phases and is influenced by various parameters, both environmental and inherent to the attaching cell. Biofilms also serve as protective niches for particular pathogens when outside a host. Although it is accepted that biofilms are ubiquitous in nature, the significance of biofilms in clinical settings, especially with regard to their role in medical-related infections, is often underestimated. It has been found that several aspects of human pathogenesis within a clinical context are directly related to biofilm development. Various types of surfaces in clinical settings are prone to biofilm development and an increased risk of disease may be a direct consequence of their formation. This review describes the process of biofilm formation, highlights the importance of bacterial associations with surfaces in clinical settings and describes various methods for biofilm visualization and control.

Carbon : nitrogen : phosphorus ratios influence biofilm formation by Enterobacter cloacae and Citrobacter freundii.

AIMS: To test the effects of C : N : P ratio modification of a well-known nutrient medium formulation, the Endo formulation on biofilm formation by Enterobacter cloacae Ecl and Citrobacter freundii Cf1 in both single-species and binary species biofilms. METHODS AND RESULTS: The C : N : P atom : atom ratio of a well-known nutrient medium formulation, the Endo formulation, that has been applied in fermentative biohydrogen studies, was modified to include two different C concentrations, one containing 17.65 g l(-1) and the other 8.84 g l(-1) sucrose, each containing four different C : N : P ratios, two at higher C : N : P ratios (334 : 84 : 16.8 and 334 : 84 : 3) and two at lower C : N : P ratios (334 : 28 : 5.6 and 334 : 28 : 1). Attached cells were enumerated after dislodging the biofilms that had formed on granular activated carbon (GAC). The modified medium containing 17.65 g l(-1) sucrose and having a C : N : P ratio of 334 : 28 : 5.6 resulted in significantly (P < 0.05) higher counts of attached cells for both single-species biofilms at 7.73 log(10) CFU g(-1) GAC and 9.3 log(10)CFU g(-1) GAC for Ent. cloacae Ecl and Cit. freundii Cf1, respectively, and binary species biofilms at 8.2 log(10) CFU g(-1) GAC and 6.34 log(10) CFU g(-1) GAC for Ent. cloacae Ecl and Cit. freundii Cf1, respectively. Scanning electron micrographs showed qualitative evidence that the 334 : 28 : 5.6 ratio encouraged more complex and extensive biofilm growth for both single-species and binary species biofilms. CONCLUSIONS: The differences in the attachment numbers between the different ratios were found not to be a result of the individual actions of the bacterial isolates involved but rather because of the effects of the various C : N : P ratios. The 334 : 28 : 5.6 ratio showed significantly (P < 0.05) higher counts of attached cells for both single-species and binary species biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates that C : N : P ratios should be a key consideration with regard to maximizing biofilm formation in shake flask and fluidized bed bioreactor studies as well as understanding fundamental factors affecting biofilm growth in natural environments.

Improving street food vending in South Africa: achievements and lessons learned.

Until the late 1990s there were limited scientific data on the microbiological quality and safety of street-vended foods in South Africa, while information was already available in other developing countries, including those within the African region. At that time street-vended foods were perceived as unsafe and street food vending in South Africa was regarded as a practice, which should be outlawed. The first comprehensively documented scientific research into the safety of street-vended foods in South Africa was carried out through university-based research. This research found that street food vendors in South Africa were capable of producing relatively safe foods, with low bacterial counts, although there was still a need for proper hygiene conditions and access to basic sanitary facilities. The Department of Health of South Africa, when coordinating an FAO Technical Cooperation Programme (TCP) project on Improving Street Foods in South Africa, drew similar conclusions. This article provides information of the efforts by universities and health authorities in South Africa towards improving the safety and promoting the sale of street-vended foods. It is shown that a successful transition from street food vending being perceived as a nuisance by health authorities can be made to these authorities promoting and improving street food vending instead.

Biofilm-spore response in Bacillus cereus and Bacillus subtilis during nutrient limitation.

This study aimed to trace the dynamics of biofilm formation by vegetative cells and endospores of Bacillus cereus DL5 and Bacillus subtilis 168. Counts of B. cereus DL5 and B. subtilis 168 vegetative cells and spores either attached to glass wool or, correspondingly, planktonic cells were determined by standard plate-counting methods. Results from this study highlighted the biofilm-forming potential of both spores and vegetative cells of two different Bacillus species. It was shown that once Bacillus spores had attached to a surface, the spores germinated under favorable (B. cereus DL5) and even unfavorable (B. subtilis 168) nutrient conditions, resulting in biofilms containing both spores and vegetative populations. Furthermore, it was suggested that vegetative B. cereus DL5 cells exhibited a low propensity for spore formation in attached and planktonic growth forms in nutrient-limited growth medium. By contrast, vegetative B. subtilis 168 cells readily formed spores in planktonic and attached microcosms when exposed to nutrient-limited growth conditions. Sporulation in attached Bacillus populations is an important practical consideration for many food industries, such as dairy processing, where bacilli are routinely isolated from populations attached to processing-equipment surfaces.

Activities of acyclic nucleoside phosphonates against Orf virus in human and ovine cell monolayers and organotypic ovine raft cultures.

Orf virus, a member of the Parapoxvirus genus, causes a contagious pustular dermatitis in sheep, goats, and humans. Previous studies have demonstrated the activity of (S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine (HPMPC; cidofovir; Vistide) against orf virus in cell culture and humans. We have evaluated a broad range of acyclic nucleoside phosphonates (ANPs) against several orf virus strains in primary lamb keratinocytes (PLKs) and human embryonic lung (HEL) monolayers. HPMPC, (S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]-2,6- diaminopurine (HPMPDAP), and (R)-9-[3-hydroxy-2-(phosphonomethoxy)propoxy]-2,4-diaminopyrimidine (HPMPO-DAPy) were three of the most active compounds that were subsequently tested in a virus yield assay with PLK and HEL cells by virus titration and DNA quantification. HPMPC, HPMPDAP, and HPMPO-DAPy were evaluated for their activities against orf virus replication in organotypic epithelial raft cultures from differentiated PLK cells. At the highest concentrations (50 and 20 microg/ml), full protection was provided by the three drugs, while at 5 microg/ml, only HPMPDAP and HPMPC offered partial protection. The activities of the three compounds in the raft culture system were confirmed by quantification of infectious virus and viral DNA. These findings provide a rationale for the use of HPMPC and other ANPs in the treatment of orf (contagious ecthyma) in humans and animals.

Antiviral potential of a new generation of acyclic nucleoside phosphonates, the 6-[2-(phosphonomethoxy)alkoxy]-2,4-diaminopyrimidines.

Three acyclic nucleoside phosphonates (ANPs) have been formally approved for clinical use in the treatment of 1) cytomegalovirus retinitis in AIDS patients (cidofovir, by the intravenous route), 2) chronic hepatitis B virus (HBV) infections (adefovir dipivoxil, by the oral route), and 3) human immunodeficiency virus (HIV) infections (tenofovir disoproxil fumarate, by the oral route). The activity spectrum of cidofovir {(S)- 1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine [(S)-HPMPC)]}, like that of (S)-HPMPA [(S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine) and (S)-HPMPDAP [(S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]-2, 6-diaminopurine), encompasses a broad spectrum of DNA viruses, including polyoma-, papilloma-, adeno-, herpes-, and poxviruses. Adefovir {9-[2-(phosphonomethoxy)ethyl]adenine (PMEA)} and tenofovir [(R)-9-[2-(phosphonomethoxy) propyl]adenine [(R)-PMPA)]} are particularly active against retroviruses (ie., HIV) and hepadnaviruses (ie., HBV); additionally, PMEA also shows activity against herpes- and poxviruses. We have recently identified a new class of ANPs, namely 6-[2-(phosphonomethoxy)alkoxy]-2,4-diaminopyrimidines, named, in analogy with their alkylpurine counterparts, HPMPO-DAPy, PMEO-DAPy, and (R)-PMPO-DAPy. These compounds exhibit an antiviral activity spectrum and potency that is similar to that of (S)-HPMPDAP, PMEA, and (R)-PMPA, respectively. Thus, PMEO-DAPy and (R)-PMPO-DAPy, akin to PMEA and (R)-PMPA, proved particularly active against HIV- 1, HIV-2, and the murine retrovirus Moloney sarcoma virus (MSV). PMEO-DAPy and (R)-PMPO-DAPy also showed potent activity against both wild-type and lamivudine-resistant strains of HBV. HPMPO-DAPy was found to inhibit different poxviruses (ie., vaccinia, cowpox, and orf) at a similar potency as cidofovir. HPMPO-DAPy also proved active against adenoviruses. In vivo, HPMPO-DAPy proved equipotent to cidofovir in suppressing vaccinia virus infection (tail lesion formation) in immunocompetent mice and promoting healing of disseminated vaccinia lesions in athymic-nude mice. The 6-[2-(phosphonomethoxy)alkoxy]-2,4-diaminopyrimidines offer substantial potential for the treatment of a broad range of retro-, hepadna-, herpes-, adeno-, and poxvirus infections.

Spore formation in Bacillus subtilis biofilms.

Spore formation by a Bacillus strain (Bacillus subtilis SpoIVFB-GFP) engineered with a green fluorescent protein (GFP) fused to a polytopic membrane protein (SpoIVF) that fluoresces during sporulation was observed. Biofilms of B. subtilis SpoIVFB-GFP containing ca. 8 log CFU/ml vegetative cells and spores below the lower detection limit (i.e., <1 log CFU/ ml) were allowed to develop on glass wool (37 degrees C). These biofilms were subsequently exposed to nutrient limitation to stimulate spore formation, which was monitored for fluorescence by confocal scanning laser microscopy. Sporulation in corresponding planktonic cells was also monitored for comparative purposes. Planktonic B. subtilis SpoIVFB-GFP cells began fluorescing after 5 h, while B. subtilis SpoIVFB-GFP biofilm cells began fluorescing after 30 h. Results suggested that an existing biofilm of vegetative B. subtilis cells may be stimulated to form spores when exposed to conditions of nutrient limitation. From a practical point of view, it may be suggested that a window of time does exist before sporulation occurs in attached Bacillus biofilms highlighting the need for shorter operating runs between cleaning and sanitation of food-processing equipment surfaces.

Novel acyclic nucleoside phosphonate analogues with potent anti-hepatitis B virus activities.

Novel acyclic nucleoside phosphonates with a pyrimidine base preferentially containing an amino group at C-2 and C-4 and a 2-(phosphonomethoxy)ethoxy or (R)-2-(phosphonomethoxy)propoxy group at C-6 selectively inhibit the replication of wild-type and lamivudine-resistant hepatitis B viruses. The activity of the most potent compounds was comparable to that of adefovir.

6-[2-phosphonomethoxy)alkoxy]-2,4-diaminopyrimidines: a new class of acyclic pyrimidine nucleoside phosphonates with antiviral activity.

Acyclic nucleoside phosphonate derivatives containing a pyrimidine base preferably bearing amino groups at C-2 and C-4 (DAPym), and linked at the C-6 position to (S)-[3-hydroxy-2-(phosphonomethoxy)propoxy] (HPMPO), 2-(phosphonomethoxy) ethoxy (PMEO) or (R)-[2-(phosphonomethoxy)propoxy] (PMPO), display an antiviral sensitivity spectrum that closely mimic that of the parental (S)-HPMP-, PME- and (R)-PMP-purine derivatives. Several PMEO-DAPym derivatives proved as potent as PMEA (adefovir) and (R)-PMPA (tenofovir) in inhibiting Moloney murine sarcoma virus (MSV)-induced tumor formation in newborn NMRI mice. The HPMPO-, PMEO- and PMPO-DAPym derivatives represent a novel well-defined subclass among the acyclic nucleoside phosphonates endowed with potent and selective antiviral activity.

The presence of Enterococcus, coliforms and E. coli in a commercial yeast manufacturing process.

This study evaluated a typical commercial yeast manufacturing process for bacterial contamination. Product line samples of a commercial yeast manufacturing process and the corresponding seed yeast manufacturing process were obtained upstream from the final compressed and dry yeast products. All samples were analysed before (non-PI) and after preliminary incubation (PI) at 37 degrees C for 24 h. The PI procedure was incorporated for amplification of bacterial counts below the lower detection limit. Enterococcus, coliform and Escherichia coli counts were quantified by standard pour-plate techniques using selective media. Presence at all stages and progressive increases in counts of Enterococcus, coliforms and E. coli during processing in the commercial manufacturing operation suggested that the primary source of contamination of both compressed and dry yeast with these bacteria was the seed yeast manufacturing process and that contamination was amplified throughout the commercial yeast manufacturing process. This was confirmed by surveys of the seed yeast manufacturing process which indicated that contamination of the seed yeast with Enterococcus, coliforms and E. coli occurred during scale up of seed yeast biomass destined as inoculum for the commercial fermentation.