Design and Production of a Novel Recombinant Chimeric IL2-Omp31 Antigen against Brucella Infection.

Brucellosis is a zoonotic disease in human and animals. Brucella melitensis is one of the most pathogenic species of Brucella in goat and sheep. Omp31 is an outer membrane protein of Brucella that acts as an immunogenic protein. Cytokines are glycoproteins with low molecular weight that play the role of an immune adjuvant and regulate immune responses. Interleukin-2 is one of the most important cytokines, which are secreted by the white blood cells and involved in T cell immune responses. In the present study, a chimeric Omp31-Interleukin2 recombinant protein was generated by means of genetic engineering techniques. This chimeric coding sequence was amplified by using specific primers and using Splicing Overlap Extension (SOE) PCR technique. The fusion of the two mentioned proteins was accomplished using a rigid linker. The generated chimeric IL2-Omp31 fragment was TA cloned, and then subcloned into pEt22b vector as an expression vector. The chimeric protein was successfully expressed in E. coli BL21 (DE3) and confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and also Western-blotting analysis. Finally, in order to assess the antigenic features of the recombinant chimeric IL2-Opm31 protein, its secondary structure and antigenicity were predicted in silico.

Production and characterization of egg yolk antibody (IgY) against recombinant VP8-S2 antigen.

Bovine Rotavirus and Bovine Coronavirus are the most important causes of diarrhea in newborn calves and in some other species such as pigs and sheep. VP8 subunit of rotavirus is the major determinant of the viral infectivity and neutralization. Spike glycoprotein of coronavirus is responsible for induction of neutralizing antibody response. Studies showed that immunoglobulin of egg yolk (IgY) from immunized hens has been identified to be a convenient source for specific antibodies for using in immunotherapy and immunodiagnostic to limit the infections. In this study, chimeric VP8-S2 gene was designed using by computational techniques. The chimeric VP8-S2 gene was cloned and sub-cloned into pGH and pET32a (+) vectors. Then, recombinant pET32a-VP8-S2 vector was transferred into E. coli BL21 CodonPlus (DE3). The expressed protein was purified by Ni-NTA chromatography column. Hens were immunized with the purified VP8-S2 protein three times. IgY was purified from egg yolks using polyethylene glycol precipitation method. Activity and specificity of anti-VP8-S2 IgY were detected by dot-blotting, Western-blotting and indirect ELISA. We obtained anti-VP8-S2 IgY by immunizing hens with the recombinant VP8-S2 protein. The anti-VP8-S2 IgY was showed to bind specifically to the chimeric VP8-S2 protein by dot-blotting, Western-blotting analyses and indirect ELISA. The result of this study indicated that such construction can be useful to investigate as candidates for development of detection methods for simultaneous diagnosis of both infections. Specific IgY against the recombinant VP8-S2 could be recommended as a candidate for passive immunization against bovine rotavirus and bovine coronavirus.

Lentiviral mediated overexpression of Insulin like Growth Factor-1 in mouse myoblast.

Insulin-like growth factor gene (IGF-1) is one of the most important growth factors that plays a key role in the proliferation and differentiation of muscle cells. IGF-1 is also a radial stimulant in muscle hypertrophy in mammals. In our study, we constructed a lentiviral vector inducing an overexpression of IGF-1 in order to study the regulating mechanisms of this gene. The IGF-1 gene was cloned into the lentiviral shuttle plasmid pCDH-cGFP and the recombinant lentiviral vector was transducted into myoblast C2C12 cell line. The overexpression of IGF-1 was confirmed by RT-PCR and western blotting for IGF-1 receptor gene. Additionally, chemiluminescence results also showed that the concentration of IGF-1 in the transduced cells significantly increased compared to the control group. The results of our study suggests that constructed recombinant lentiviral vector can potentially be used for regulating the expression of IGF-1 in myoblast C2C12 cells.

The suitability of some blood gas and biochemical parameters as diagnostic tools or early indicators of ascites syndrome in broiler sire lines.

In recent few years, there have been some attempts to find a reliable indicator trait as a selection criterion against susceptibility to ascites syndrome (AS). Blood parameters were of great interest as they could be measured in live animals without implementing an ascites-inducing challenge (AIC). In this work, the suitability of some blood parameters was evaluated for diagnosing AS-susceptible chicks in later steps of the disease in trial 1 as well as their early predictive ability in trial 2. In the first trial, one hundred 1-day-old chicks from two pure broiler lines namely S1 and S2 and, in the second trial, 226 1-day-old chicks from line S2 were subjected to AIC. Saline drinking water (1200 mg/l) and lower-than-standard ambient temperatures were the implemented AICs in trials 1 and 2 respectively. The blood parameters including pH, partial pressure of O2 (pO2 ), partial pressure of CO2 (pCO2 ), bicarbonate ion concentration (BIC), percentage of haematocrit (HCT) and saturated haemoglobin (SaO2 ) were measured twice per each bird at days 28 and 35 in trial 1 and once in trial 2 at day 21. The results of the first trial revealed that in line S2 some of the blood parameters differed significantly between the ascitic and non-ascitic groups following exposure to AIC. In this line, the incidence of AS was accompanied by a lower pO2 , SaO2 and BIC, while with higher pCO2 and HCT values. In the second trial, however, although almost all of the parameters showed meaningful differences between the ascitic and non-ascitic broilers, only mean difference of BIC parameter was statistically significant. The general conclusion of this study is that the blood parameters can somewhat have diagnostic ability in the condition in which the AIC is already present, whereas the results did not approve their usefulness as early predictors of AS.

BosFinder: a novel pre-microRNA gene prediction algorithm in Bos taurus.

MicroRNAs (miRNAs) are small non-coding RNAs that modulate gene expression transcriptionally (transcriptional activation or inactivation) and/or post-transcriptionally (translation inhibition or degradation of their target mRNAs). This phenomenon has significant roles in growth and developmental processes in plants and animals. Bos taurus is one of the most important livestock animals, having great importance in food and economical sciences and industries. However, limited information is available on Bos taurus constituent miRNAs because its whole genome assembly has been only recently published. Therefore, computational methods have been essential tools in miRNA gene prediction and discovery. Among these, machine-learning-based approaches are used to characterize genome scale pre-miRNAs from expressed sequence tags (ESTs). In this study, a support vector machine model was used to classify 33 structural and thermodynamic features of pre-miRNA genes. Public bovine EST data were obtained from different tissues in various developmental stages. A new algorithm, called BosFinder, was developed to identify and annotate the whole genome's derived pre-miRNAs. We found 18 776 highly potential pre-miRNA sequences. This is the first genome survey report of Bos taurus based on a machine-learning method for pre-miRNA gene finding. The bosfinder program is freely available at http://lbb.ut.ac.ir/Download/LBBsoft/BosFinder/.

Impact of milk components on recovery of viral RNA from MS2 bacteriophage.

Noroviruses are responsible for approximately 44 % of outbreaks involving dairy products for which causative agents are reported. Recovery of viruses from milk and dairy products is a difficult task. The role of different components of milk in the recovery of viral RNA was evaluated in this study. Four model milk formulations (A-D) were prepared by mixing different combinations of lactose, whey protein, casein, and fat in water. Each model formulation was spiked with five concentrations of bacteriophage MS2. The phenol-guanidine thiocyanate-chloroform protocol was used for extracting viral RNA from the model milk formulations and then extracted RNA was measured by a nanodrop spectrophotometer in ng/µl. The results showed that casein and whey protein had the highest negative impact on RNA yield, especially when the number of MS2 was less than 1.3 pfu/ml. The highest RNA recovery was obtained from the model milk formulation containing all four components; lactose, whey protein, casein, and fat. The amount of extracted RNA was closely correlated with the dry matter content of each formulation and the spiked concentration of coliphage using response surface modeling (R²:0.93). It was determined that milk fat is the most effective component in facilitating RNA extraction and the highest RNA yield can be achieved via elimination of whey protein and casein from milk by centrifugation at 40,000×g for 60 min. To achieve the highest viral RNA recovery efficiency by the proposed method, milk fat must be recombined with the supernatant of the centrifuged sample and then homogenized before performing the extraction protocol.

Single nucleotide polymorphisms and mRNA expression of VEGF-A in papillary thyroid carcinoma: potential markers for aggressive phenotypes.

BACKGROUND AND OBJECTIVES: Polymorphisms of the VEGF gene are known to affect the biological behaviour of cancers but have seldom been studied in thyroid cancer. The aim of the current study is to evaluate the prevalence and relevance of VEGF-A polymorphisms and mRNA expression in papillary thyroid carcinoma (PTC). MATERIALS AND METHODS: Genomic DNA and total RNA were isolated from paraffin-embedded tissue from 91 PTC (51 conventional PTC and 40 follicular variant) and 78 control thyroid tissues. Three DNA polymorphisms (+936C > T, +405C > G and -141A > C) in the 3' and 5' untranslated region (3'-UTR, 5'-UTR) of VEGF-A were studied using PCR and RFLP. Also, the mRNA expression of VEGF-A in these tissues was studied by real-time PCR. RESULTS: Distribution of polymorphisms in the 5'-UTR (VEGF-VEGF -141A > C and +405C > G) and 3'-UTR (VEGF +936C > T) were all significantly different in PTC and benign thyroid tissue (p = 0.0001, 0.001 and 0.028 respectively). The VEGF -141 C allele was more common in PTC with lymph node metastases (p = 0.026). VEGF + 405 Galleles andVEGF +936 CC genotype were more common in PTC of advanced pathological staging (p = 0.018 and 0.017 respectively). Also, increased expression of VEGF-A mRNA was noted in PTC compared to control (p = 0.009). Within the group of patients with conventional PTC, those with lymph nodal metastases had a higher level of VEGF-A mRNA expression than other patients (p = 0.0003). CONCLUSION: These findings suggest that VEGF polymorphisms and mRNA expression may predict the aggressiveness behaviour of thyroid cancer.

Impact of milk components in recovery of the MS2 bacteriophage as an indicator of enteric viruses.

The objective of this study was to characterize the role of milk components in the recovery of viral particles from raw milk. For such characterization, four model milk formulations (A-D) were constituted by mixing different combinations of lactose, whey protein, casein, and fat into water. Each model formulation was spiked with six concentrations of bacteriophage MS2. The soluble and insoluble components of each model milk formulation were separated by centrifugation at 40,000 x g and viruses were enumerated in each supernatant fluid and pellet by the double agar layer (DAL) method. When samples were spiked with MS2 at concentrations lower than 4.8 x 10(5) pfu/ml, milk components did not significantly impact the overall recovery. However, the impact of milk components was measurable at higher concentrations. In general, higher numbers of MS2 were recovered from supernatant fluids of model milk formulations containing no fat. The highest number of viral particles were recovered from the pellet of model C (lactose+whey protein+casein). The recovery efficiency of MS2 was correlated with the dry matter contents of each model milk formulation and the initial spiking concentration of coliphage using response surface modeling.

[Mitochondrial DNA polymorphism in populations of the Caspian region and southeastern Europe]. / Polimorfizm mitokhondrial'noi DNK v populiatsiiakh Kaspiiskogo regiona i iuzhnoi chasti Vostochnoi Evropy.

Mitochondrial DNA (mtDNA) restriction polymorphism was examined in Turkmens, Eastern Iranians, and Ukrainians. The gene pools of all populations studied were characterized by the presence of European mtDNA lineages. Mongoloid component observed in Turkmen and Iranian populations with the frequencies of about 20% was represented by groups C, D, and E/G in Turkmens, and by M*, D, A, and B in Iranians. The relative positions of the populations studied, of populations from the Caucasus, Western Iran, and Russian populations from the Krasnodar krai and Belgorod oblast in the space of principal components revealed a geographically specific pattern of the population clustering. The data on mtDNA polymorphism indicated pronounced differentiation of Eastern and Western Iranians. The latter were characterized by a mtDNA group composition similar to that in Eastern Slavs. The historical role of the Caspian populations in the formation of the population of Southeastern Europe is discussed.

Human bone marrow fibroblasts infected by cytomegalovirus: ultrastructural observations.

Human cytomegalovirus (HCMV) is capable of infecting human bone marrow fibroblastic stromal cells (HBMF-sc). This infection is important to assess in regard to the pathogenesis of HCMV, particularly in immunocompromised patients. Stromal fibroblastic cells were infected by Towne strain of cytomegalovirus (CMV) in vitro. Several ultrastructural features of uninfected HBMF-sc were also described. The CMV-infected cells exhibited significant mitochondrial enlargement, production of dense bodies by the Golgi apparatus and cytoplasmic accumulation. Ultrastructural aspects of HCMV entry in host cells, capture by endosomes, penetration of genetic material in the nucleoplasm, assembly and formation of nucleocapsids were detected and described. Viral fusion and transit through the nuclear envelope were shown along with envelope proliferations. Trafficking of virions, maturation and completion of their cytoplasmic coating were also illustrated. Fully developed virions, defective virions, other apparently-emptied vesicles, multivesicular bodies as well as cytoplasmic dense bodies were illustrated along arrays of microtubules organized by defective centrosomes and constituted a peculiar structure that we termed 'viral field'. While some viral and dense bodies were carried to adjacent sites of the plasmalemma, in order to be extruded from the infected cells, others were concentrated into black holes--dense heterogenous bodies accumulated at the periphery of viral fields. This study further described the functional aspects of HBMF-sc and summarized the unknown aspects of ultrastructural characteristics of HCMV-infected fibroblastic stromal cells which may serve as harmful reservoir for the replication of virus. In addition, the findings of this study may stimulate further investigations about the basic cell biology and functions of the bone marrow stromal cells and may also generate some interests to bone marrow transplantation medicine as to how HBMF-sc can serve as a reservoir in the pathogenesis of CMV infections.

Antiviral activity of magnesium and magnesium/poly r(A-U) combinations against two RNA viruses.

Magnesium (Mg2+) potentiated the anti-vesicular stomatitis virus (VSV) activity of poly r(A-U) or poly r(G-C) and the anti-HIV-1 activity of poly r(A-U). Mg2+ did not affect the anti-VSV activity of poly (rI).poly (rC), poly (dA-dT).poly (dA-dT) or poly (dG-dC).poly (dG-dC). Modulation of one or more nuclear (nucleolar) processes of the host cell may be responsible for the synergistic antiviral activity.

Nonnucleoside pyrrolopyrimidines with a unique mechanism of action against human cytomegalovirus.

Based upon a prior study which evaluated a series of nonnucleoside pyrrolo[2,3-d]pyrimidines as inhibitors of human cytomegalovirus (HCMV), we have selected three active analogs for detailed study. In an HCMV plaque-reduction assay, compounds 828, 951, and 1028 had 50% inhibitory concentrations (IC(50)s) of 0.4 to 1.0 microM. Similar results were obtained when 828 and 951 were examined by HCMV enzyme-linked immunosorbent assay (IC(50)s = 1.9 and 0.4 microM, respectively) and when 828 was tested in a viral DNA-DNA hybridization assay (IC(50) = 1.3 microM). In yield-reduction assays with a low multiplicity of infection (MOI), all three compounds caused multiple log(10) reductions in virus titer, and the activities of these compounds were comparable to the activity of ganciclovir (GCV; IC(90) = 0.2 microM). In contrast to the reduction of viral titers by GCV, the reduction of viral titers by 828, 951, and 1028 decreased with increasing MOI. Cytotoxicity in human foreskin fibroblasts and KB cells ranged from 32 to >100 microM. In addition, 828 (the only compound tested) was less toxic against human bone marrow progenitor cells than GCV. Time-of-addition and time-of-removal studies established that the three pyrrolopyrimidines inhibited HCMV replication before GCV had an effect on viral DNA synthesis but after viral adsorption. Compound 828 was equally effective against GCV-sensitive and GCV-resistant HCMV clinical isolates. Combination studies with 828 and GCV showed that the effects of the two compounds on HCMV were additive but not synergistic. Taken together, the data indicate that these pyrrolopyrimidines target a viral protein that is required in an MOI-dependent manner and that is expressed early in the HCMV replication cycle.

Ultrastructural aspects of cytomegalovirus-infected fibroblastic stromal cells of human bone marrow.

Human cytomegalovirus (HCMV) and bone marrow interactions are important in the pathogenesis of HCMV infections. Human bone marrow fibroblastic stromal cells (HBFM-sc) were infected by Towne strain of cytomegalovirus (CMV) in cell culture. Several cytostructural features of control bone marrow stromal cells are described and compared with those of CMV-infected cells. Under these experimental conditions, HBFM-sc are cell types that can be successfully infected by CMV in vitro. The CMV-infected cells displayed typical features characteristic of DNA virus infections, such as cellular swelling, intranuclear inclusions, nucleolar condensation and disappearance (at the end stage), nuclear envelope proliferation as redundant folds. Other characteristics of CVM-infected cells include mitochondrial enlargement and vacuolization, cytoplasmic dense bodies associated or not with viral particles, accumulation and extrusion of viral particles and dense bodies. These preliminary observations shed some light on human bone-marrow stromal-cell morphology and function, one of the latter being that of a potentially harmful reservoir for CMV virus.

Infection and replication of human cytomegalovirus in bone marrow stromal cells: effects on the production of IL-6, MIP-1alpha, and TGF-beta1.

We have investigated the mechanisms by which hematopoiesis is suppressed in patients suffering from human cytomegalovirus (HCMV) infections. Mixed populations of human bone marrow stromal and hematopoietic progenitor cells were inoculated with the Towne strain of HCMV to determine whether these populations could be infected and support HCMV replication. We found that the Towne strain of HCMV was capable of infecting and replicating in a mixed population of bone marrow stromal cells. We observed no significant alterations in bone marrow stromal cell proliferation or the production of IL-6, GM-CSF, soluble c-kit ligand and TNF-alpha following HCMV replication in either stimulated lipopolysaccharide (LPS) or unstimulated conditions. In samples of culture supernatants from LPS-stimulated HCMV-infected stromal cells, significant elevations in MIP-1alpha were observed. TGF-beta1 levels on the other hand exhibited two patterns following HCMV exposure; either TGF-beta1 levels decreased regardless of LPS stimulation or there was no effect. In addition, we observed that exposure to the Towne strain of HCMV resulted in significant inhibition of both granulocytic and erythrocytic colony formation in methylcellulose progenitor assays. Thus, both the direct effect of HCMV on hematopoietic progenitors as well as altered cytokine production by bone marrow stromal cells (including MIP-1alpha and TGF-beta1, but not IL-6) could contribute to hematopoietic failure during HCMV infection.

Comparison of benzimidazole nucleosides and ganciclovir on the in vitro proliferation and colony formation of human bone marrow progenitor cells.

Recently we have shown that certain benzimidazole ribonucleosides are potent and selective inhibitors of human cytomegalovirus (HCMV) replication. Because antiviral drugs used to treat HCMV and human immunodeficiency virus (HIV) infections can suppress marrow progenitors, we have evaluated the most promising of the new benzimidazoles for their effects on human bone marrow cells in vitro. In an initial study of the bone marrow toxicity of one of the most active compounds, 100 microM 2-bromo-5,6-dichloro-1-(beta-D-ribofuranosyl)-benzimidazole (BDCRB) inhibited cell proliferation by 20% over a 10 d period compared to 52% inhibition by 100 microM ganciclovir, the drug currently most used to treat HCMV infections. The effects of these drugs and selected other benzimidazole nucleosides were evaluated more extensively in haemopoietic progenitor cell colony formation assays. Colony formation was determined at 2 weeks and scored as either burst forming units-erythroid (BFU-E), or colony forming units-granulocyte/macrophage (CFU-GM). At the highest concentration tested, 100 microM BDCRB only moderately affected BFU-E or CFU-GM formation (31% and 47% inhibition, respectively). This concentration is 10-fold higher than that required to produce a 10000-fold reduction in virus titre. Evaluation of the 2-chloro analog of BDCRB (TCRB) which is less potent against HCMV, its 5'-deoxy analog (5'-dTCRB) which is more potent, and the 2-unsubstituted compound (DRB) gave the following order of haemopoietic toxicity: DRB > TCRB > or = 5'-dTCRB > BDCRB. In contrast to the benzimidazoles, ganciclovir decreased colony formation by 84% for BFU-E and 86% for CFU-GM at 100 microM. These results establish that certain benzimidazole nucleosides are less toxic to haemopoietic progenitors than the preferred drug now being used clinically for HCMV infections. The results also establish that different structure-activity relationships exist for antiviral activity and progenitor cell toxicity, thereby suggesting that different mechanisms are involved in the two types of drug action.

Design, synthesis, and antiviral activity of certain 2,5,6-trihalo-1-(beta-D-ribofuranosyl)benzimidazoles.

A new series of 2-substituted 5,6-dichlorobenzimidazole ribonucleosides has been synthesized and tested for activity against two human herpes viruses and for cytotoxicity. 2,5,6-Trichloro-1-(beta-D-ribofuranosyl)benzimidazole (TCRB) was prepared by ribosylation of the heterocycle 2,5,6-trichlorobenzimidazole followed by a removal of the protecting groups. The 2-bromo derivative (BDCRB) was made in a similar fashion from 2-bromo-5,6-dichlorobenzimidazole. In contrast, the 2-iodo derivative presented a more difficult problem since the appropriate heterocycle was unavailable. This prompted us to prepare the 2-amino derivative followed by nonaqueous diazotization and removal of the blocking groups. Biological evaluation revealed marked differences in the activities of these compounds and the closely related known compound 5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole (DRB). DRB was weakly active against both human cytomegalovirus (HCMV) and herpes simplex virus type 1 (HSV-1), (IC50's = 42 and 30 microM, respectively) but was cytotoxic to uninfected human foreskin fibroblasts and KB cells in the same dose range. Similar results were obtained with the heterocycle 2,5,6-trichlorobenzimidazole. In marked contrast, the ribonucleoside of 2,5,6-trichlorobenzimidazole (TCRB) was active against HCMV (IC50 = 2.9 microM, plaque assay; IC90 = 1.4 microM, yield assay) but only weakly active against HSV-1 (IC50 = 102 microM, plaque assay). Little to no cytotoxicity was observed in HFF and KB cells at concentrations up to 100 microM. By changing the substituent at the 2-position from chlorine to bromine (BDCRB), a 4-fold increase in activity against HCMV was observed without any significant increase in cytotoxicity. In contrast, the 2-I and 2-NH2 derivatives were only weakly active against HCMV and HSV-1 with activity not well-separated from cytotoxicity. These data establish that for maximum activity against HCMV with separation from cytotoxicity, ribose is preferred at the 1-position and that Cl or Br is apparently preferred at the 2-position. The activity and selectivity of both TCRB and BDCRB were better than that observed with either ganciclovir or foscarnet.

Synthesis and antiproliferative and antiviral activity of 2'-deoxy-2'-fluoroarabinofuranosyl analogs of the nucleoside antibiotics toyocamycin and sangivamycin.

The glycosylation of 3,4-dicyano-2-[(ethoxymethylene)amino]pyrrole (7) with 2-deoxy-2-fluoro-alpha-D-erythro-pentofuranosyl bromide (2) furnished an anomeric mixture of nucleosides (8a,b). This mixture was separated, and the individual anomers were treated with methanolic ammonia to effect a concomitant deblocking and ring closure. This furnished both anomers of 2'-deoxy-2'-fluoro-ara-toyocamycin (9a,b). The cyano moiety of 9b was converted to the carboxamide moiety to furnish 2'-deoxy-2'-fluoro-ara-sangivamycin (10) and to the thiocarboxamide moiety to furnish 2'-deoxy-2'-fluoro-ara-thiosangivamycin (11). The target compounds 10 and 11 showed similar antiproliferative activity against L1210 cells in vitro, with IC50's of 3 and 5 microM. Antiviral evaluation revealed a somewhat different pattern of activity. All analogs, both alpha and beta anomers, were active against human cytomegalovirus (HCMV), albeit the beta anomers were most active. The beta anomers also were active against herpes simplex virus type 1 (HSV-1) and human immunodeficiency virus (HIV). Compound 10 was most active in the series, ca. 10-fold more potent than 11; IC50's for 10 ranged from 4 to 25 nM for HCMV, HIV, and varicella zoster virus (VZV) and from 30 to 500 nM for HSV-1. Even though compound 10 was cytotoxic, which will probably preclude its use as an antiviral drug (IC50's = 0.2-5.5 microM), the difference between cytotoxicity and activity against HCMV, HIV, and VZV was sufficient to indicate specific activity against a viral target.

Synthesis and evaluation of certain thiosangivamycin analogs as potential inhibitors of cell proliferation and human cytomegalovirus.

A series of 7-substituted 4-aminopyrrolo[2,3-d]pyrimidines related to the nucleosides toyocamycin and thiosangivamycin were prepared and tested for their activity against human cytomegalovirus (HCMV). The nucleosides 2'-deoxytoyocamycin (1), xylo-toyocamycin (2), 3'-deoxytoyocamycin (3), 2',3'-dideoxy-2',3'-didehydrotoyocamycin (4), 2',3'-dideoxytoyocamycin (5), ara-toyocamycin (6), 2'-deoxy-2'-amino-ara-toyocamycin (7), and 5'-deoxytoyocamycin (8) were treated with sodium hydrogen sulfide generated in situ to afford the corresponding thiosangivamycin analogs (9-16). The cyano derivatives 1-8 were synthesized by modifications of literature procedures. All of the thioamide derivatives (9-16) were active against HCMV with IC50's ranging from 0.5 to 6 microM. Most also were active against herpes simplex virus type 1 (HSV-1) but at higher concentrations. The antiviral activity was not completely separated from cytotoxicity in two human cell lines. The antiproliferative activity was strongly influenced by the position of the modification on the carbohydrate moiety. The xylosyl and 3'-deoxy derivatives were significantly more potent than those with modifications at the 2', 5', or 2',3' position(s). Interestingly, 5'-deoxythiosangivamycin (16) possessed both antiviral and antiproliferative activity suggesting that phosphorylation of the 5'-hydroxyl may not be required for these compounds to have biological activity.

Application of flow cytometry to determine the cytotoxicity of urethane dimethacrylate in human cells.

The effects of an oligomer, urethane dimethacrylate (UDMA), on two human cell lines were studied using flow cytometry (FCM). Untreated and treated cultures of propidium iodine-stained KB (epidermal oral carcinoma cells) and human foreskin fibroblast (HFF) cells were analyzed for cellular DNA content. Concentrations of 10 and 25 microM of UDMA slightly perturbed the KB cell cycle progression at 24 and 48 h of incubation. However, the effect of 50 microM was more pronounced at the latter incubation time period. In cell growth experiments, the sublethal concentrations (10 and 25 microM) produced inhibition of KB cell growth rate at a moderate level, which resulted in the prolongation of cell population doubling time. Significant inhibition of cell growth occurred when 50 microM (lethal concentration) was used. Data obtained from the cell cycle perturbation analysis, evidenced by FCM, correlated with the extent of inhibition in KB cell growth rates. The effects of sublethal concentrations were reversible during a 24 h period of oligomer withdrawal from culture medium. In contrast, the effects of 50 microM were not reversible. In HFF cells the depletion of S phase in the cell cycle was the major effect of 50 microM of UDMA. It was concluded that FCM technology is an ideal and practical approach for studying the cytotoxicity of components of dental composites.