Human Embryonic Lung Fibroblast Cells Research Articles

Screening of Anticancer Materials from Myxobacteria and Evaluation of Their Bioactivity in Vivo

Myxobacteria are a class of prokaryotes with complex multi-cellular behavior and could produce various bioactive materials. Forty-eight strains stored in Key Lab of Microbial Diversity Research and Application of Hebei Province were screened by MTT methods with L1210, Hela and normal diploid human embryonic lung fibroblast cell lines as screening models. The results demonstrated that most of the strains could inhibit cancer cells. The inhibition rates to L1210 and Hela are about 78.5% and 59%, respectively. Strain 910018 and 920036 showed higher inhibition activity to cancer cells L1210 and Hela in compare with lower inhibition activity to normal diploid human embryonic lung fibroblast cell line. Therefore, the extracts from the two strains have the potentials as anticancer drugs and were undertaken in vivo anticancer experiments. The results showed that strain 910018 and 920036 could inhibit mice transplanted liver cancer cells H22. The inhibition rate of low doses group of strain 910018 is 69.37% and that of strain 920036 is 54.98%. Therefore, the two strains could be good candidates for producing of anticancer drugs.

In vitro and in vivo anticancer activity evaluation of ursolic acid derivatives

Twenty-three ursolic acid (1) derivatives 2– 24 (ten novel compounds 8– 10, 14– 17 and 22– 24) modified at the C-3 and the C-28 positions were synthesized, and their structures were confirmed by IR, 1H NMR, MS, and elemental analysis. The single crystals of compounds 15 and 17 were obtained. The cytotoxic activity of the derivatives was evaluated against HepG2, BGC-823, SH-SY5Y, HeLa and HELF cells by the MTT assay. The induction of apoptosis and affects on the cell cycle distribution with compound 14 were assessed by fluorescence microscopy, flow cytometry and the activity of caspase-3 in HepG2 cells. Compounds 14– 17 had more significant antiproliferative ability against the four cancer cell lines and low cytotoxicity to human embryonic lung fibroblast cells (HELF). Compounds 11, 14– 16, 21 and 23 were particularly active against HepG2 cell growth. Compound 14 was selected to investigate cell apoptosis and cell cycle distribution. Flow cytometric analysis and morphologic changes of the cell exhibited that treatment of HepG2 cells with compound 14 led to cell apoptosis accompanied by cell cycle arrest at the S phase in a dose-dependent manner. Furthermore, the activity of the caspase-3 enzyme was increased in the treated cells. In vivo studies using H22 xenografts in Kunming mice were conducted with compound 14 at doses of 50, 100 and 150 mg/kg body weight. The results revealed that the medium dosage group (100 mg/kg) showed significant anticancer activity (45.6 ± 4.3%) compared to the control group.

A novel single‐chain‐Fv antibody against connective tissue growth factor attenuates bleomycin‐induced pulmonary fibrosis in mice

Connective tissue growth factor (CTGF) has been identified as playing critical roles in fibrosis and is a promising therapeutic target. In a previous study, we used a phage display library to develop a humanized single-chain variable fragment antibody (scFv) against CTGF. In the present study, the protective effect of anti-CTGF scFv against bleomycin (BL)-induced pulmonary fibrosis was investigated in mice. The expression of α-smooth muscle actin in human embryonic lung fibroblast (HELF) cells was analysed by western blotting. A mouse model of pulmonary fibrosis was established by tracheal injection of BL (5 mg/kg). Mice received anti-CTGF scFv (4 mg/kg, three times a week) by i.v. injection. The effects of anti-CTGF scFv were evaluated by leukocyte counts in BAL fluid, hydroxyproline measurements in lung tissue and pathological examination. α-Smooth muscle actin expression was decreased in HELF cells treated with anti-CTGF scFv. Anti-CTGF scFv significantly reduced the numbers of inflammatory leukocytes (total and differential count) in BAL fluid, as well as the hydroxyproline content of lung tissue. The severity of alveolitis and fibrosis in the mouse model was markedly attenuated by treatment with anti-CTGF scFv. Anti-CTGF scFv may potentially be developed as a useful inhibitor of pulmonary fibrosis.

Inhibitory Effects of Statins on Cytomegalovirus Production in Human Cells: Comprehensive Analysis of Gene Expression Profiles

Background/Purpose Inhibitory effects of statins on human immunodeficiency virus-1 or poliovirus replication were reported. Our aim was to clarify whether statins could inhibit the replication of cytomegalovirus (CMV) in human cells and to determine the changes in gene expression profiles in host cells treated with statins using a DNA microarray. Methods Human embryonic lung (HEL) fibroblast cells were infected with CMV (Towne strain) at a multiplicity of infection of 1 and were simultaneously treated with mevastatin, simvastatin, lovastatin, or pravastatin (0.001–10μM). HEL cells were incubated for 6 days, and progeny viral titers were quantified by plaque assay. Time-dependent effects of mevastatin or simvastatin (1μM) on CMV replication were also examined. We determined the effects of mevastatin or simvastatin at concentrations ranging from 0.1μM to 10μM on the expressions of CMV immediate-early-1 (IE-1) and late proteins using Western blotting. Comprehensive analysis of gene expression profiles in HEL cells treated with mevastatin (1μM) was performed with a DNA microarray 1 day after infection. Results The 50% effective concentration values for the inhibition of CMV titers by mevastatin, simvastatin, lovastatin, and pravastatin were 0.0006μM, 0.0055μM, 0.04μM, and 2.55μM, respectively. Inhibition of viral replication by mevastatin was observed when added 24 hours after infection, whereas that by simvastatin was observed when added 48 hours after infection. Mevastatin decreased the expression of the IE-1 protein, and simvastatin inhibited the expression of the late protein. We observed significant changes of cellular growth/differentiation-associated gene expressions (e.g., downregulated cdk2 mRNA) in HEL cells treated with mevastatin. Conclusion Our data suggest that treatment with mevastatin could inhibit CMV replication at IE phase through altered expressions of cellular growth/differentiation-associated genes.

2-Aminophenoxazine-3-one and 2-amino-4,4.ALPHA.-dihydro-4.ALPHA.,7-dimethyl-3H-phenoxazine-3-one cause cellular apoptosis by reducing higher intracellular pH in cancer cells

We examined intracellular pH (pHi) of ten cancer cell lines derived from different organs and two normal cell lines including human embryonic lung fibroblast cells (HEL) and human umbilical vein endothelial cells (HUVEC) in vitro, and found that pHi of most of these cancer cells was evidently higher (pH 7.5 to 7.7) than that of normal cells (7.32 and 7.44 for HEL and HUVEC, respectively) and that of primary leukemic cells and erythrocytes hitherto reported (≤7.2). Higher pHi in these cancer cells could be related to the Warburg effect in cancer cells with enhanced glycolytic metabolism. Since reversal of the Warburg effect may perturb intracellular homeostasis in cancer cells, we looked for compounds that cause extensive reduction of pHi, a major regulator of the glycolytic pathway and its associated metabolic pathway. We found that phenoxazine compounds, 2-aminophenoxazine-3-one (Phx-3) and 2-amino-4,4α-dihydro-4α,7-dimethyl-3H-phenoxazine-3-one (Phx-1) caused a rapid and drastic dose-dependent decrease of pHi in ten different cancer cells within 30 min, though the extent of the decrease of pHi was significantly larger for Phx-3 (ΔpHi = 0.6 pH units or more for 100 µM Phx-3) than for Phx-1 (ΔpHi = 0.1 pH units or more for 100 µM Phx-1). This rapid and drastic decrease of pHi in a variety of cancer cells caused by Phx-3 and Phx-1 possibly perturbed their intracellular homeostasis, and extensively affected the subsequent cell death, because these phenoxazines exerted dose-dependent proapoptotic and cytotoxic effects on these cells during 72 h incubation, confirming a causal relationship between ΔpHi and cytotoxic effects due to Phx-3 and Phx-1. Phx-3 and Phx-1 also reduced pHi of normal cells including HEL and HUVEC, although they exerted less proapoptotic and cytotoxic effects on these cells than on cancer cells. Drugs such as Phx-3 and Phx-1 that reduce pHi and thereby induce cellular apoptosis might serve as benevolent anticancer drugs.

Identification ofLegionella feeleiiCellulitis

Identification of<i>Legionella feeleii</i>Cellulitis

New Potential Anticancer Agent of Carborane Derivatives: Selective Cellular Interaction and Activity of Ferrocene-Substituted Dithio-o-carborane Conjugates

The large diversity of structures and unique bonding modes of organometallic complexes make them possible to act as promising candidate therapeutic agents. In this study, the new type of ferrocene-substituted dithio-o-carborane conjugates (FcSB1, FcSB2, and FcSBCO) has been synthesized, and their in vitro antineoplastic activities have been explored by means of the electrochemical study, the real time cell electronic sensing (RT-CES) system, and biological assays. The conjugate-cell interactions were first monitored by electrochemistry, and the results show different cell uptake efficiency for FcSB1, FcSB2, and FcSBCO toward target cells. Both the highly hydrophobic ferrocenyl and carboranyl groups render the conjugates able to rapidly enter cells and exert acute cytotoxicity after 4 h incubation in serum-free media. However, FcSB1, FcSB2, and FcSBCO display different inhibition efficiencies toward SMMC-7721 and HepG2 cancer cells via the G(0)/G(1) arrest mechanism in a physiological environment. The anticancer activity is in the order FcSB2 > FcSB1 > FcSBCO, which is parallel to the order of the redox potentials of the ferrocenyl groups in the three complexes. In particular, FcSB1 and FcSB2 display a potent selective inhibition effect on the proliferation of the cancer cell lines SMMC-7721 and HepG2, but almost no effect on the normal cell line, the human embryonic lung fibroblast (HELF) cells. Thus, these results may provide some clues for use of the ferrocene-carborane conjugates in developing anticancer drugs.

B-MYB delays cell aging by repressing p16 (INK4α) transcription.

p16 ( INK4α ), an inhibitor of cyclin-dependent kinase 4 and 6, has been proposed to play an important role in cellular aging and in premature senescence. The expression of the p16 ( INK4α ) is primarily under transcriptional control. Our previous data showed that a negative regulation element lies in its promoter. In that element, a MYB-binding site (MBS) was uncovered by transcription analysis. Here, we report that MBS is a negative regulation element and B-MYB binds to this site in vivo. In human embryonic lung fibroblast cells, B-MYB downregulated p16 ( INK4α ) expression, whereas knocking down of B-MYB upregulated it. Evidence also showed that overexpression of B-MYB in cells could increase the number of utmost passage and decrease G1 block, whereas knocking down of B-MYB could impair their replicative ability. This study provides evidence of the capacity of B-MYB not only to regulate p16 ( INK4α ) expression but also the phenotypic consequence on cellular senescence.

Transcription pattern of UL131A-128 mRNA in clinical strains of human cytomegalovirus

Human cytomegalovirus (HCMV) mRNA was obtained from human embryonic lung fibroblast cells infected by HCMV clinical strains from urine samples of infants at different kinetic periods. The cDNA of UL131A-128 mRNAs was amplified using reverse transcription-polymerase chain reaction (RT-PCR) and analysed by sequencing. Meanwhile, clones containing UL131A-128 transcripts in an HCMV cDNA library of a clinical strain were selected and sequenced. It was demonstrated that UL131A-128 mRNA was expressed with immediately early, early and late kinetics. Sequences obtained by RT-PCR showed that the UL131A gene consisted of two exons and the coding region of the UL130 gene was not interrupted by any intron in the region as reported earlier. However, the transcript of the UL128 gene showed two patterns: one pattern consisted of three exons as reported earlier; the other contained the three exons and also the first intron. Moreover, the above characteristics of UL131A-128 spliced transcripts were confirmed by the sequences of clones selected from the HCMV cDNA library. Our results demonstrated that the UL131A, UL130 and UL128 genes were transcribed with the 3'-coterminal, although the initiation points of their mRNA may be different. The variation in the transcripts found in our study indicated the complex nature of transcription of UL131A-128 genes in clinical strains of HCMV.

Characterization of the interaction between fibroblasts and tumor cells on a microfluidic co‐culture device

Fibroblasts and tumor cells have been involved in the process of cancer development, progression and therapy. Here, we present a simple microfluidic device which enables to study the interaction between fibroblasts and tumor cells by indirect contact co-culture. The device is composed of multiple cell culture chambers which are connected by a parallel of cell migration regions, and it enables to realize different types of cells to communicate each other on the single device. In this work, human embryonic lung fibroblasts cells were observed to exhibit obvious migration towards tumor cells instead of normal epithelial cells on the co-culture device. Moreover, transdifferentiation of human embryonic lung fibroblast cells was recognized by the specific expression of alpha-smooth muscle actin, indicating the effect of tumor cells on the behavior of fibroblasts. Furthermore, multiple types of cell co-culture can be demonstrated on the single device which enables to mimic the complicated microenviroment in vivo. The device is simple and easy to operate, which enables to realize real-time observation of cell migration after external stimulus. This microfluidic device allows for the characterization of various cellular events on a single device sequentially, facilitating the better understanding of interaction between heterotypic cells in a more complex microenvironment.

Human cytomegalovirus tropism for endothelial/epithelial cells: scientific background and clinical implications

Human cytomegalovirus (HCMV) has been routinely isolated from and propagated in vitro in human embryonic lung fibroblast (HELF) cell cultures, while in vivo it is known to infect predominantly endothelial and epithelial cells. In recent years, genetic determinants of the HCMV tropism for endothelial/epithelial cells were identified in the UL131A/UL130/UL128 locus of HCMV genome of wild-type strains. UL131A-UL128 gene products form a complex with glycoprotein H (gH) and L (gL) resulting in a gH/gL/UL131A-UL128 complex that is required for HCMV entry into endothelial/epithelial cells. In contrast, virus entry into fibroblasts has its genetic determinants in the complex gH/gL/gO (or gH/gL). During primary HCMV infection, the neutralising antibody response measured in endothelial cells (EC) is potent, occurs very early and is directed mostly against combinations of two or three gene products of the UL131A-128 locus. On the contrary, neutralising antibodies measured in fibroblasts appear late, are relatively weak in potency and are directed against gH and gB. The T-cell immune response to UL131A-UL128 gene products remains to be investigated. Recently, a role has been proposed for neutralising antibody in conferring prevention/protection against HCMV infection/disease in pregnant women with primary HCMV infection. However, the level of cooperation between humoral immunity and the well-established T-cell protection remains to be defined.

A One-Step, Real-Time PCR Assay for Rapid Detection of Rhinovirus

One-step, real-time PCR assays for rhinovirus have been developed for a limited number of PCR amplification platforms and chemistries, and some exhibit cross-reactivity with genetically similar enteroviruses. We developed a one-step, real-time PCR assay for rhinovirus by using a sequence detection system (Applied Biosystems; Foster City, CA). The primers were designed to amplify a 120-base target in the noncoding region of picornavirus RNA, and a TaqMan (Applied Biosystems) degenerate probe was designed for the specific detection of rhinovirus amplicons. The PCR assay had no cross-reactivity with a panel of 76 nontarget nucleic acids, which included RNAs from 43 enterovirus strains. Excellent lower limits of detection relative to viral culture were observed for the PCR assay by using 38 of 40 rhinovirus reference strains representing different serotypes, which could reproducibly detect rhinovirus serotype 2 in viral transport medium containing 10 to 10,000 TCID(50) (50% tissue culture infectious dose endpoint) units/ml of the virus. However, for rhinovirus serotypes 59 and 69, the PCR assay was less sensitive than culture. Testing of 48 clinical specimens from children with cold-like illnesses for rhinovirus by the PCR and culture assays yielded detection rates of 16.7% and 6.3%, respectively. For a batch of 10 specimens, the entire assay was completed in 4.5 hours. This real-time PCR assay enables detection of many rhinovirus serotypes with the Applied Biosystems reagent-instrument platform.

Determination, characterization and cytotoxicity on HELF cells of ZnO nanoparticles

Three kinds of ZnO nanoparticles (NPs) with different size were characterized with transmission electron microscopy (TEM), X-ray diffraction (XRD). The potential cytotoxicity of ZnO NPs with various concentrations has been investigated using human embryonic lung fibroblasts (HELF) cells. The cytotoxicity of ZnO NPs on the normal HELF cell was evaluated by 3-(4,5-dimethylthiazol)-2,5-diphenltetrazoliumhromide (MTT) assay and characterized with photo microscopy and scanning electron microscopy (SEM). The results indicated that ZnO NPs aqueous suspensions with concentrations varying from 2.5 to 150 mg L −1 were harmful to the HELF cells and the cytotoxicity to HELF showed obviously concentration dependence. With concentrations higher than 20 mg L −1, ZnO NPs led to the livability of the tested cells lower than 10%. These results highlighted the need for caution during the use of ZnO NPs above 1.2–1.6 mg L −1 for safety. Finally, an UV–vis spectroscopic approach was developed for the determination of ZnO NPs concentration in the linear range 2–50 mg L −1 (correlation coefficients more than 0.9990), and the detection limit was 1.0 mg L −1.

Effects of respiratory syncytial virus upon expressions of mRNA and protein of intercellular adhesion molecule-1 in human embryonic lung fibroblast cells

To explore the effects of respiratory syncytial virus (RSV) upon the expressions of mRNA and protein of intercellular adhesion molecule-1 (ICAM-1) in human embryonic lung fibroblast cells. The expressions of mRNA and protein of ICAM-1 were determined in human embryonic lung fibroblast cells after being infected by the LONG strain type A RSV and in normal fibroblast cells with real-time PCR and flow cytometry. The mRNA of ICAM-1 expression in human embryonic lung fibroblast cells was 2.51 times at 24 h post-infection as that in normal fibroblast cells (P < 0.05). The protein of ICAM-1 expression of RSV control group (1.25 +/- 0.09, 1.87 +/- 0.18, 4.78 +/- 0.52, 13.34 +/- 0.64, 1.58 +/- 0.37) were significantly higher than those of normal cell group (0.21 +/- 0.06, 0.30 +/- 0.06, 0.29 +/- 0.07, 0.35 +/- 0.17, 0.35 +/- 0.14) at 12, 24, 48, 72 and 96 h post-infection (all P < 0.01). The protein of ICAM-1 expression of RSV control group achieved a peak value between 48 h and 72 h, and then it decreased significantly at 96 h. Lung fibroblast cell and ICAM-1 may play some roles in pathogenic mechanism of RSV viral pneumonia.

Anti-parasitic activity and cytotoxicity of selected medicinal plants from Kenya

Indigenous rural communities in the tropics manage parasitic diseases, like malaria and leishmaniasis, using herbal drugs. The efficacy, dosage, safety and active principles of most of the herbal preparations are not known. Extracts from 6 selected plant species, used as medicinal plants by indigenous local communities in Kenya, were screened for in vitro anti-plasmodial and anti-leishmanial activity, against 2 laboratory-adapted Plasmodium falciparum isolates (D6, CQ-sensitive and W2, CQ-resistant) and Leishmania major (IDU/KE/83 = NLB-144 strain), respectively. The methanol extract of Suregada zanzibariensis leaves exhibited good anti-plasmodial activity (IC 50 4.66 ± 0.22 and 1.82 ± 0.07 μg/ml for D6 and W2, respectively). Similarly, the methanol extracts of Albizia coriaria (IC 50 37.83 ± 2.11 μg/ml for D6) and Aspergillus racemosus (32.63 ± 2.68 and 33.95 ± 2.05 μg/ml for D6 and W2, respectively) had moderate anti-plasmodial activity. Acacia tortilis (IC 50 85.73 ± 3.36 μg/ml for W2) and Albizia coriaria (IC 50 71.17 ± 3.58 μg/ml for W2) methanol extracts and Aloe nyeriensis var kedongensis (IC 50 87.70 ± 2.98 and 67.84 ± 2.12 μg/ml for D6 and W2, respectively) water extract exhibited mild anti-plasmodial activity. The rest of the extracts did not exhibit any anti-plasmodial activity. Although the leishmanicidal activity of extracts were lower than for pentosam (80%), reasonable activity was observed for Aloe nyeriensis methanol (68.4 ± 6.3%), Albizia coriara water (66.7 ± 5.0%), Maytenus putterlickoides methanol (60.0 ± 6.23%), Asparagus racemosus methanol and water (58.3 ± 8.22 and 56.8 ± 6.58%, respectively), Aloe nyeriensis water (53.3 ± 5.1%) and Acacia tortilis water (52.9 ± 6.55%) extracts at 1000 μg/ml. Leishmania major infected macrophages treated with methanol extracts of Suregada zanzibariensis and Aloe nyeriensis var kedongensis and Pentostam ® had infection rates of 28 ± 2.11, 30 ± 1.22 and 40 ± 3.69%, respectively at 1000 μg/ml, indicating better anti-leishmanial activity for the extracts. The methanol extract of Albizia coriara (44.0 ± 3.69%) and aqueous extracts of Asparagus racemosus (42 ± 3.84%) and Acacia tortilis (44 ± 5.59%) had similar activity to pentosam ®. Multiplication indices for Leishmania major amastigotes treated with methanol extracts of Albizia coriaria, Suregada zanzibariensis and Aloe nyeriensis var kedongensis, aqueous extract of Acacia tortilis and pentosam ® were 28.5 ± 1.43, 29.4 ± 2.15, 31.1 ± 2.22, 35.9 ± 3.49 and 44.0 ± 3.27%, respectively, at 1000 μg/ml, confirming better anti-leishmanial activity for the extracts. Aqueous extracts of Aloe nyeriensis (46.7 ± 3.28%) and Albizia coriaria (47.5 ± 3.21%) had similar activity level to pentosam ®. The plant extracts have better inhibitory activity while pentosam ® has better leishmanicidal activity. All extracts exhibited very low cytotoxicity (CC 50 > 500 μg/ml) against human embryonic lung fibroblast (HELF) cells. The investigations demonstrated the efficacy and safety of some extracts of plants that are used by rural indigenous communities for the treatment of parasitic diseases.

Inhibition of Herpes Simplex Virus Type 1 Infection by Some Selected Antitumor Titanium(III)-Based Coordination Compounds

ABSTRACTUsing a standard plaque assay and clinical strains of Herpes Simplex virus type 1 (HSV 1), we have tested the ability of two titanium (Ti) - content complexes—Titanocene Dichloride (Cp2TiCl2) and Titanocene Y. Virus was treated by incubation at 37°C with both complexes in cell culture media and was then diluted and plated onto human embryonic lung fibroblast cells MRC-5 for detection and quantitation of remaining infectious virus. Of one randomly chosen clinical HSV 1 (TM strain), sensitive to acyclovir (ACV), was inactivated > 98% by treatment in vitro with 0.01 μM Titanocene Y for 2h. The effect was concentration dependent. With an HSV 1 strain, 0.01 μM Titanocene Y or Cp2TiCl2 caused 99% inactivation, 0.001μM caused 98 to 99% inactivation, and 0.0001μM caused 63 to 86% inactivation. Short (5 min) treatments of selected isolate with Titanocene Y or Cp2TiCl2 yielded inactivation rates of 0 to 55%. We assumed also that Titanocene Y or Cp2TiCl2 could be able to affect negatively not only virus DNA synthesis but also another virus target. The data obtained show that: (i) the 300bp relatively stable region during a short period of viral replication corresponding to ReIV is amplified in non-treated viral control, but not in mock cells and in negative control and (ii) the region was not amplified in Titanocene Y treated samples. The data show that the effect of the suppressive therapy of virus replication by Titanocene Y could be due to the suppression of immediate early Us1 and Us12 genes encoding essential for virus replication products α22—the protein affecting virus ability to replicate, and α47—the protein inhibiting MHC I antigen presentation.

Clinical aplications of Trioxolane derivatives

The aqueous extracts of three medicinal plants, Carissa edulis (Forssk.) Vahl (Apocynaceae), Prunus africana (Hook.f.) Kalkm (Rosaceae) and Melia azedarach L. (Meliaceae) have shown significant reduction in the replication of human cytomegalovirus (HCMV) in human embryonic lung (HEL) fibroblasts cells in vitro. Using the plaque inhibition assay for the determination of anti-viral activity, the HEL fibroblast cells cultured in 24 well plates were infected with 1 x 102 PFU 91S HCMV and treated with various concentrations of the extracts. The plaques formed were counted after 7 days incubation at 37°C in 5% CO2 and the percent plaques inhibited were calculated against infected untreated control. The effective concentrations inhibiting plaque formation by 50% (EC50) was found between 40 to 80 &#956;g/ml for all the extracts. The cell cytotoxic concentrations (CC50) for each of the three extracts, by the trypan blue exclusion test, gave a safe therapeutic index. These results have demonstrated the potential anti-viral activities of the extracts of the three medicinal plants at non-cytotoxic concentrationsAfrican Journal of Health Sciences Vol. 15 (1&2) 2008: pp. 1-5

In vitro anti-viral activity of aqueous extracts of Kenyan Carissa edulis Prunus africana and Melia azedarach against human cytomegalovirus.

The aqueous extracts of three medicinal plants, Carissa edulis (Forssk.) Vahl (Apocynaceae), Prunus africana (Hook.f.) Kalkm (Rosaceae) and Melia azedarach L. (Meliaceae) have shown significant reduction in the replication of human cytomegalovirus (HCMV) in human embryonic lung (HEL) fibroblasts cells in vitro. Using the plaque inhibition assay for the determination of anti-viral activity, the HEL fibroblast cells cultured in 24 well plates were infected with 1 x 102 PFU 91S HCMV and treated with various concentrations of the extracts. The plaques formed were counted after 7 days incubation at 370C in 5% CO2 and the percent plaques inhibited were calculated against infected untreated control. The effective concentrations inhibiting plaque formation by 50% (EC50) was found between 40 to 80 &#956;g/ml for all the extracts. The cell cytotoxic concentrations (CC50) for each of the three extracts, by the trypan blue exclusion test, gave a safe therapeutic index. These results have demonstrated the potential anti-viral activities of the extracts of the three medicinal plants at non-cytotoxic concentrations. African Journal of Health Sciences Vol. 14 (3-4) 2007: pp. 143-148

Benzo (a) pyrene induces quiescent diploid human embryonic lung fibroblast cells undergoing a reversible G1 arrest

To study the influence of Benzo (a) pyrene on cell cycle distribution of quiescent diploid human embryonic lung fibroblast (HELF) cells. HELF cells were synchronized at G0 phase of cell cycle by 0.5% serum starvation for 48 hours and identified by flow cytometry (FCM). Cells were treated with 20 micromol/L benzo (a) pyrene for 4 h and detected for the changes of cell cycle distribution 0 h, 24 h, 48 h after treatment respectively. HELF cells were treated with 0, 5, 10, 20 micromol/L Benzo (a) pyrene for 24 h and detected for cell cycle regulators p53, p21 and p16 expression changes using Western Blotting method. On the other hand, the dynamic changes of these regulators were also been detected within 24 h after 20 micromol/L Benzo (a) pyrene treated for 4h. 0.5% Serum starvation for 48 hours could effectively synchronize HELF at G0 stage and G0 reached 78%. Well-modulated control cells entered into cell cycle to synthesize DNA and cells at S phase reached 43.9% 24 h after serum re-stimulate, while 20 micromol/L B (a) P treated cells were arrested in G1 stage. Control cells entered into the G1 stage of next cell cycle another 24 h later, B (a) P treated cells recovered from G1 arrest, 26.5% of which reached S phase, having a delay of almost 24 h compared with controls. After a series of B (a) P concentrations acting for 24 h, we found that P53 and P21 expression increased dramatically. On the other hand, P53 and P21 increased 4 h after treatment, P53 recovered to normal level after 12 h while P21 kept increasing in 24 h. P16 initially decreased and became normal at 24 h. B (a) P induced quiescent HELF cells undergoing a reversible G1 arrest related with p53-p21 pathway.

Human cytomegalovirus UL145 gene is highly conserved among clinical strains

Human cytomegalovirus (HCMV), a ubiquitous human pathogen, is the leading cause of birth defects in newborns. A region (referred to as UL/b') present in the Toledo strain of HCMV and low-passage clinical isolates) contains 22 additional genes,which are absent in the highly passaged laboratory strain AD169. One of these genes,UL145 open reading frame (ORF), is located between the highly variable genes UL144 and UL146. To assess the structure of the UL145 gene,the UL145 ORF was amplified by PCR and sequenced from 16 low-passage clinical isolates and 15 non-passage strains from suspected congenitally infected infants. Nine UL145 sequences previously published in the GenBank were used for sequence comparison. The identities of the gene and the similarities of its putative protein among all strains were 95.9 -100% and 96.6-100%, respectively. The post-translational modification motifs of the UL145 putative protein in clinical strains were conserved,comprising the protein kinase C phosphorylation motif (PKC)and casein kinase II phosphorylation site (CK-II). We conclude that the structure of the UL145 gene and its putative protein are relatively conserved among clinical strains, irrespective of whether the strains come from patients with different manifestations, from different areas of the world, or were passaged or not in human embryonic lung fibroblast (HELF) cells.