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PubMed2006

Discovery of imidazolidine-2,4-dione-linked HIV protease inhibitors with activity against lopinavir-resistant mutant HIV.

A new series of HIV protease inhibitors has been designed and synthesized based on the combination of the (R)-(hydroxyethylamino)sulfonamide isostere and the cyclic urea component of lopinavir. The series was optimized by replacing the 6-membered cyclic urea linker with an imidazolidine-2,4-dione which readily underwent N-alkylation to incorporate various methylene-linked heterocycle groups that bind favorably in site 3 of HIV protease. Significant improvements compared to lopinavir were seen in cell culture activity versus wild-type virus (pNL4-3) and the lopinavir-resistant mutant virus A17 (generated by in vitro serial passage of HIV-1 (pNL4-3) in MT-4 cells). Select imidazolidine-2,4-dione containing PIs were also more effective at inhibiting highly resistant patient isolates Pt1 and Pt2 than lopinavir. Pharmacokinetic data collected for compounds in this series varied considerably when coadministered orally in the rat with an equal amount of ritonavir (5 mg/kg each). The AUC values ranged from 0.144 to 12.33 microg h/mL.

Crystallography, X-Ray↗

PubMed2003

Aging is associated with decreased maximal life span and accelerated senescence of bone marrow stromal cells.

Age-related decrease in bone formation is well described. However, the cellular causes are not known. Thus, we have established cultures of bone marrow stromal cells (MSC) from young (aged 18-29 years, n = 6) and old (aged 68-81 years, n = 5) donors. MSC were serially passaged until reaching maximal life span. Cell growth, markers of cellular senescence, and osteogenic and adipogenic potential were determined in early-passage and late-passage cells established from young and old donors. MSC from old donors exhibited a decreased maximal life span compared with cells from young donors (24 +/- 11 population doublings [PD] vs 41 +/- 10 PD, P < 0.05) and mean PD rate was lower in old donor cells (0.05 +/- 0.02 PD/day) compared with young donor cells (0.09 +/- 0.02 PD/day) (P < 0.05). No differences were detected in number of senescence-associated beta-galactosidase positive (SA beta-gal+) cells and mean telomere length in early-passage cells obtained from young and old donors. However, MSC from old donors exhibited accelerated senescence evidenced by increased number of SA beta-gal+ cells per PD as compared with young (4% per PD vs 0.4% per PD, respectively). MSC from young and old donors were able to form similar amounts of mineralized matrix in vitro and of normal lamellar bone in vivo. In adipogenic medium similar numbers of adipocytes formed in cultures of young and old donors. In conclusion, aging is associated with decreased proliferative capacity of osteoprogenitor cells, suggesting that decreased osteoblastic cell number, and not function, leads to age-related decrease in bone formation.

Adipocytes↗

PubMed2004

Mammalian cells cycle without the D-type cyclin-dependent kinases Cdk4 and Cdk6.

Cdk4 and Cdk6 are thought to be essential for initiation of the cell cycle in response to mitogenic stimuli. Previous studies have shown that Cdk4 is dispensable for proliferation in most cell types, an observation attributed to a putative compensatory role by Cdk6. Cdk6-null mice are viable and develop normally although hematopoiesis is slightly impaired. Embryos defective for Cdk4 and Cdk6 die during the late stages of embryonic development due to severe anemia. However, these embryos display normal organogenesis and most cell types proliferate normally. In vitro, embryonic fibroblasts lacking Cdk4 and Cdk6 proliferate and become immortal upon serial passage. Moreover, quiescent Cdk4/Cdk6-null cells respond to serum stimulation and enter S phase with normal kinetics although with lower efficiency. These results indicate that D-type cyclin-dependent kinases are not essential for cell cycle entry and suggest the existence of alternative mechanisms to initiate cell proliferation upon mitogenic stimulation.

Animals↗

PubMed2006

Challenging successive mosquito generations with a densonucleosis virus yields progressive survival improvement but persistent, innocuous infections.

Research on cultivated shrimp suggests that they have the capability to tolerate viral pathogens in a highly specific manner by mechanisms currently unknown. The phenomenon is difficult to study in detail because they have a generation time of 1-2yr and lack continuous cell lines. Thus, we developed a mosquito-densovirus model to examine whether similar phenomena occur in insects. Serial challenge of five generations with a stock densovirus (AThDNV) resulted in progressive survival increases from 15% to 58%. Prevalence of AThDNV infection in surviving mosquito larvae (confirmed by PCR, histology, in situ hybridization and transmission electron microscopy) was relatively high (e.g. 36% in F2) but they grew normally to establish each succeeding generation. At the end of five generations, comparison of deduced amino acid sequences from genome fragments revealed a significantly higher (p=0.02) estimated prevalence of defective targets in the survivor virus population (29.7%+/-10.0 SD) than in the stored viral population (3.3%+/-5.8 SD). The results paralleled those reported for serially passaged C6/36 mosquito cell cultures infected with a densovirus. There, reduced infection rates are ascribed to the production of defective interfering particles (DIP). Thus, it is possible that the presence of prior AThDNV infections with a high level of DIP contributed to improved survival in our challenged F4 mosquito population. If so, it suggests that persistent viral infections in arthropods may serve in a specific, adaptive manner to reduce the incidence and severity of disease.

Aedes↗

PubMed2005

Clinical isolates of Staphylococcus aureus from 1987 and 1989 demonstrating heterogeneous resistance to vancomycin and teicoplanin.

Fifty isolates of Staphylococcus aureus, obtained during a multicenter clinical trial evaluating the efficacy of teicoplanin that was performed between 1987 and 1992, underwent glycopeptide susceptibility testing, and 2 isolates were found to be capable of growth on agar containing 4 or 8 mg/L of vancomycin. Both of these isolates were from patients that had received prolonged teicoplanin therapy and were deemed clinical failures. Extended susceptibility testing combined with mecA gene probing revealed that one isolate was susceptible to oxacillin, the other was resistant, and both were susceptible to a variety of nonglycopeptide agents. Population analyses revealed heterogeneous vancomycin- and teicoplanin-susceptibility profiles. Both strains were differentiated from recent glycopeptide intermediately resistant Staphylococcus aureus isolates by pulse-field analysis and by the fact that the resistance phenotype was stable to multiple serial passages. To our knowledge, this is the earliest report of S. aureus clinical isolates having reduced vancomycin and teicoplanin susceptibility.

Analysis of Variance↗

PubMed2004

Clonogenic assay with established human tumour xenografts: correlation of in vitro to in vivo activity as a basis for anticancer drug discovery.

Pluripotent cells can be grown in clonogenic assays. The tumour stem-cell fraction, which accounts for <0.4% of the total cells, and which is considered the most relevant cell type in the development of metastases and recurrences, is able to divide and to form colonies in a semisolid matrix (agar or methylcellulose). Major applications of the tumour clonogenic assay (TCA) are chemosensitivity testing of tumours and xenografts, and for assessments within drug discovery programmes. Of critical relevance for the usefulness of the TCA is whether it can predict sensitivity or resistance towards clinically used agents. When we compared the response of human tumours established as xenografts in nude mice in the TCA in vitro to that of the clinical response, 62% of the comparisons for drug sensitivity, and 92% of the comparisons for drug resistance were correct. The same percentage of true/false observations was found when tumours were tested after serial passage in nude mice in the TCA in vitro and their response compared to in vivo activity in corresponding xenografts (60% and 90%, respectively). The highest correct predictive values were, however, found when the clinical response of tumours was compared to their explants established in the nude mouse and treated in vivo. Of 80 comparisons performed, we observed a correct prediction for tumour resistance in 97% and for tumour sensitivity in 90%. In our opinion, the TCA with established human tumour xenografts has an important role in current drug discovery strategies. We therefore included the TCA as secondary assay in our approach to anticancer drug discovery and found that a number of novel agents were active; these are now in advanced preclinical development or clinical trials. Thus, the tumour clonogenic assay has proven predictive value in the chemosensitivity testing of standard and experimental anticancer drugs.

Animals↗

PubMed2005

Ca++-switch induction of RPE differentiation.

Cultured retinal pigment epithelial (RPE) cells are commonly used as a model of the tissue to study their involvement in visual diseases. Unfortunately, cultured RPE often lose their differentiated phenotype reducing their usefulness as a model of the RPE in vivo. In this study, we used a Ca++-switch protocol to initiate the patterned expression of several phenotypic and functional markers of RPE differentiation. Cultured RPE cells from adult donors were maintained through at least six serial passages prior to assay to minimize their differentiated properties. The cells were then subjected to the Ca++-switch protocol and maintained at confluence for up to 4 months. Paired control and Ca++-switch cells were examined for phenotype, pigmentation, and the expression of tyrosinase, CRABP, myocilin, and bestrophin by western blot analysis. The Ca++-switch protocol led to a rapid restriction of N-cadherin to lateral cell borders, and to expression of tyrosinase by day 4. After 8 weeks, the experimental RPE monolayers began to accumulate visible pigment, and after 12 weeks CRABP expression was observed. Myocilin was observed at 4 months after the Ca++-switch but bestrophin was not detected at any time point. Our results suggest this protocol may drive epithelial morphogenesis in RPE cells. We note two specific differences in cells plated in low Ca++, reduced spreading on the substrate and coordinated development of cadherin adhesion when the Ca++-concentration is returned to normal. Thus, we suggest that this method produces phenotypic changes through multiple cell signalling pathways.

Adolescent↗

PubMed2005

Induced resistance to the antimicrobial peptide lactoferricin B in Staphylococcus aureus.

This study was designed to investigate inducible intrinsic resistance against lactoferricin B in Staphylococcus aureus. Serial passage of seven S. aureus strains in medium with increasing concentrations of peptide resulted in an induced resistance at various levels in all strains. The induced resistance was unstable and decreased relatively rapidly during passages in peptide free medium but the minimum inhibitory concentration remained elevated after thirty passages. Cross-resistance to penicillin G and low-level cross-resistance to the antimicrobial peptides indolicidin and Ala(8,13,18)-magainin-II amide [corrected] was observed. No cross-resistance was observed to the human cathelicidin LL-37. In conclusion, this study shows that S. aureus has intrinsic resistance mechanisms against antimicrobial peptides that can be induced upon exposure, and that this may confer low-level cross-resistance to other antimicrobial peptides.

Anti-Bacterial Agents↗

PubMed2005

Induction of macrolide resistance in Mycoplasma gallisepticum in vitro and its resistance-related mutations within domain V of 23S rRNA.

Antibiotic-resistant mutants of Mycoplasma gallisepticum were selected in vitro from the susceptible strains S6 and BG44T by serial passages in stepwise concentrations of erythromycin, tylosin, or tilmicosin. High resistance to erythromycin or tilmicosin developed readily, whereas resistance to tylosin developed only after greater numbers of passages. Three mutants selected by each selector antibiotic were cloned and detected, and all cloned mutants exhibited cross-resistance to the three selector antibiotics as well as to lincomycin. Portions of the genes encoding domain V of 23S rRNA of the cloned mutants were amplified by PCR, and their nucleotide sequences were compared to those of the susceptible parent strains. Five of the six mutants selected by erythromycin harbored an A2058G (Escherichia coli numbering) mutation in one of the two 23S rRNA. One of the six mutants selected by erythromycin harbored a G2057A mutation and an A2059G mutation in the other 23S rRNA. In tilmicosin-selected mutants, two mutations, A2058G and A2503U, occurred in one of the two 23S rRNA. No mutation was detected in the two 23S rRNA of tylosin-selected mutants with low-level resistance. Mutations at homologous locations in the 23S rRNA of other macrolide-resistant bacteria indicate that the phenotype of macrolide resistance occurring in M. gallisepticum is strongly associated with point mutations in domain V of 23S rRNA.

Anti-Bacterial Agents↗

PubMed2005

Taf1: a class II transposon of Aspergillus fumigatus.

Aspergillus fumigatus is an important medical pathogen that lacks a known sexual cycle. Transposons may provide an important mechanism for the generation of genetic diversity in this organism. Here, we describe Taf1, the first class II transposon to be identified in A. fumigatus. Taf1, a member of the mariner superfamily and pogo family of transposons, is distinguished by the presence of extremely long (89 bp) inverted repeats that flank the transposase coding sequence. Taf1 is present in different locations and copy number among clinical strains of A. fumigatus and is transcribed. Analysis of multiple insertion sequences within a single strain suggests that Taf1 elements undergo inactivation by a repeat induced polymorphism-like mechanism. Taf1 insertion patterns were extremely stable despite multiple stressors including heat shock, serial passage, and infection in mice. Thus Taf1 may be useful for strain identification and molecular typing.

Amino Acid Sequence↗

PubMed2004

Glucose-6-phosphate dehydrogenase-deficient cells show an increased propensity for oxidant-induced senescence.

Glucose-6-phosphate dehydrogenase (G6PD) is involved in the generation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and the maintenance of cellular redox balance. We previously showed that G6PD-deficient fibroblasts undergo growth retardation and premature cellular senescence. In the present study, we demonstrate abatement of both the intracellular G6PD activity and the ratio NADPH/NADP(+) during the serial passage of G6PD-deficient cells. This was accompanied by a significant increase in the level of 8-hydroxy-2-deoxyguanosine (8-OHdG). This suggests that the lowered resistance to oxidative stress and accumulative oxidative damage may account for the premature senescence of these cells. Consistent with this, the G6PD-deficient cells had an increased propensity for hydrogen peroxide (H(2)O(2))-induced senescence; these cells exhibited such senescent phenotypes as large, flattened morphology and increased senescence-associated beta-galactosidase (SA-beta-Gal) staining. Decreases in both the intracellular G6PD activity and the NADPH/NADP(+) ratio were concomitant with an increase in 8-OHdG level in H(2)O(2)-induced senescent cells. Exogenous expression of G6PD protected the deficient cells from stress-induced senescence. No significant telomere shortening occurred upon repetitive treatment with H(2)O(2). Simultaneous induction of p16(INK4a) and p53 was detected in G6PD-deficient but not in normal fibroblasts during H(2)O(2)-induced senescence. Our findings support the notion that G6PD status, and thus proper redox balance, is a determinant of cellular senescence.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed2006

Characterisation of laboratory-generated vancomycin intermediate resistant Staphylococcus aureus strains.

Vancomycin has been the drug of choice for 30 years for the treatment of methicillin-resistant Staphylococcus aureus (MRSA). Emergence of decreased vancomycin susceptibility in MRSA strains presents a significant clinical problem with few therapeutic options. This study was performed to generate and characterise S. aureus strains with reduced susceptibility to vancomycin. Eighteen S. aureus strains were subjected to serial passaging on vancomycin to generate vancomycin intermediate resistant S. aureus (VISA) strains. Minimum inhibitory concentration (MIC) determination was performed for the parent and the passaged cultures with 13 different antibiotics. The strains were tested by the following five methods: simplified population analysis; CDC method; modified vancomycin agar screen; population analysis profile (PAP); and modified population analysis (PAP-area under the curve (AUC) ratio). Phenotypic changes such as doubling time, synergy with beta-lactam antibiotics and effect on norA efflux pumps were also studied for these strains. The result indicated that 8 VISA mutants (vancomycin MICs, 8-16 microg/mL) were generated in vitro from the 18 S. aureus strains. The CDC and modified agar methods proved to be the most sensitive and specific methods for detection of VISA strains. The PAP for all the VISA strains ranged from 12 microg/mL to > 16 microg/mL, with a PAP-AUC ratio of > 1.3. All mutants showed increased doubling time compared with their parent isolate. Synergism of the vancomycin and beta-lactam combinations was observed for all methicillin-resistant mutants. Upon acquisition of vancomycin resistance, a few mutants showed decreased oxacillin resistance. Two VISA strains were chosen for molecular characterisation of the mecA gene and one mutant showed genotypic changes with deletion of mecA. Loss of norA efflux pumps leading to fluoroquinolone sensitivity was also observed in four mutants.

Laboratories↗

PubMed2006

Mechanisms of daptomycin resistance in Staphylococcus aureus.

Daptomycin resistance in Staphylococcus aureus emerged during therapy of tricuspid endocarditis. Susceptibility to daptomycin of the parent strain (SA-675), other daptomycin-susceptible strains and the non-susceptible mutant (SA-684) was heterogeneous; however, subpopulations growing at concentrations above the minimum inhibitory concentration (MIC) were not stably resistant. Stable resistance was produced only by serial passage on daptomycin-containing media. Daptomycin dissipated the membrane potential of SA-675 but not SA-684, which also lost an 81 kDa membrane protein. Whole cells and membranes of SA-684 bound a reduced amount of daptomycin. Reduced drug binding in SA-684 correlates with daptomycin resistance, possibly as a result of the loss of a membrane protein 'chaperone' with which daptomycin interacts. Heterogeneity of daptomycin MICs in susceptible strains may be an important factor in the development of stable, clinically relevant resistance.

Daptomycin↗

PubMed2006

Characterisation of in vitro-selected mutants of Ureaplasma parvum resistant to macrolides and related antibiotics.

Resistant mutants of Ureaplasma parvum were selected by serial passages of a susceptible strain in subinhibitory concentrations of different macrolides and related antibiotics (erythromycin, azithromycin, josamycin, quinupristin, quinupristin/dalfopristin, pristinamycin and telithromycin). Mechanisms of resistance were characterised by sequencing portions of genes encoding 23S rRNA and ribosomal proteins L4 and L22. Mutants with significantly increased minimum inhibitory concentrations could be selected with all the selector antibiotics, except quinupristin and pristinamycin. Mutants harboured mutations in domain V of the 23S rRNA gene at nucleotides G2056, G2057 or A2058 (Escherichia coli numbering) and in conserved portions of ribosomal proteins L4 and L22. Most of the mutations were associated with complete loss of macrolide and ketolide activity, whereas streptogramin combinations were less affected.

Anti-Bacterial Agents↗

PubMed2005

Characterization of human nasal septal chondrocytes cultured in alginate.

BACKGROUND: After serial passages in monolayer, chondrocytes dedifferentiate into a fibroblast-like phenotype. Our objective was to determine if culture in alginate affects the phenotype of dedifferentiated human nasal septal chondrocytes. STUDY DESIGN: Human nasal septal chondrocytes were seeded at low density and passaged in monolayer culture. At passages (P) 1, 2, and 3 a portion of cells were cultured in alginate. Collagen, glycosaminoglycan (GAG), and DNA production were assessed. RESULTS: Chondrocytes in alginate proliferated less yet produced higher levels of GAG and collagen than those in monolayer culture. Alginate encapsulated P1 chondrocytes stained strongly for GAG and collagen type II, and minimally for collagen type I. Monolayer cells at P0 and P1 stained positively for collagen type II. All monolayer passages stained positive for collagen type I with minimal GAG staining. CONCLUSIONS: Compared with monolayer culture, alginate stimulates deposition of GAG and collagen type II, and supports the chondrocyte phenotype through P1, but does not promote redifferentiation.

Alginates↗

PubMed2004

Tumour lines from a spontaneous rat endometrial stromal sarcoma, showing dendritic cell-like and myofibroblastic cell-like phenotypes.

A transplantable tumour (RY) and cell lines (RY-PB and clone RY-B-E3 isolated from RY-PB) were established from a naturally occurring endometrial stromal sarcoma (ESS) found in a 24-month-old female F344 rat. The primary tumour and RY tumours, which had been serially passaged in syngeneic female rats up to the 10th generation, consisted of spindle or round cells arranged in ill-defined bundles or sheets. Neoplastic cells of the primary and RY tumours, as well as cultured cells of RY-PB and RY-B-E3, showed positive reactions to vimentin, ED1/ED2 (both for rat macrophages/histiocytes), OX6 (for dendritic cells expressing rat MHC class II antigens), and lysosomal enzymes such as acid phosphatase and non-specific esterase, in varying degrees. Ultrastructurally, neoplastic cells characteristically had tubulovesicular system-like structures and variously developed lysosomes in the cytoplasm. Neoplastic cells also exhibited immunoexpression to an alpha-smooth muscle actin (alpha-SMA). The addition of transforming growth factor (TGF)-beta1 to RY-PB and RY-B-E3 cultures increased the number of alpha-SMA-positive cells, whilst the positive cell number was decreased by anti-TGF-beta antibody. The RT-PCR method revealed the expression of TGF-beta1 mRNA in the cultured cells. The present study showed that rat ESS-derived cells exhibited dendritic cell-like and myofibroblastic cell-like phenotypes. The histogenesis of ESSs in human beings and rats remains poorly understood, and these tumour lines may therefore become useful tools for further research.

Animals↗

PubMed2005

Chronic infection with hepatitis B viruses and antiviral drug evaluation in uPA mice after liver repopulation with tupaia hepatocytes.

BACKGROUND/AIMS: Transplantation of primary human hepatocytes and establishment of hepatitis B virus (HBV) infection in immunodeficient urokinase plasminogen activator (uPA) transgenic mice was shown. However, the availability of usable primary human hepatocytes is very limited. Therefore, alternative and more accessible sources of hepatocytes permissive for HBV infection are highly desirable. Here we investigated the potential of primary hepatocytes from the tree shrew Tupaia belangeri that were shown to be susceptible to HBV infection. METHODS: Freshly isolated or cryopreserved primary tupaia hepatocytes were transplantated via intrasplenic injection into immunodeficient uPA/RAG-2 mice. Engrafted mice were then infected with HBV and woolly monkey (WM)-HBV positive sera. RESULTS: Extensive proliferation of xenografted cells was demonstrated by the stable production of tupaia alpha1-antitrypsin in serum and liver of transplanted mice. Quantitative PCR assays demonstrated the presence of circulating viral particles as well as intracellular viral DNA, including covalently closed circular (ccc) DNA, in transplanted mice. Viral infection could be serially passaged in mice. Furthermore, viral replication was strongly inhibited by treating mice with adefovir dipivoxil. CONCLUSIONS: uPA mice repopulated with tupaia hepatocytes represent a useful and more accessible model for HBV infection studies, including the evaluation of antiviral therapy and cccDNA.

Adenine↗

PubMed2004

Effect of serial transfer of three strains of Paecilomyces fumosoroseus on growth in vitro, virulence, and host specificity.

Serial passage of entomopathogenic Hyphomycetes has been shown to alter virulence and host specificity. We evaluated virulence, host specificity, biomass production, conidiation, conidial germination, and a genetic fingerprint of 3 strains of Paecilomyces fumosoroseus after passage in vitro or in vivo in Diuraphis noxia or Plutella xylostella. Strain 4461 did not change in virulence toward D. noxia or P. xylostella after 30 passages in vitro nor after 15 passages in D. noxia. However, it lost virulence toward D. noxia after 15 passages in P. xylostella and did not regain virulence after 5 passages in D. noxia. Passage in D. noxia did result in a loss in conidiation for strain 4461, and passage in vitro resulted in a reduction in the speed of germination. Strain 4481 was the least variable and did not change in any of our tests. Strain 4491 did not change in virulence after passage in vitro nor after passage in D. noxia. It lost virulence toward D. noxia after passage in P. xylostella but regained virulence after re-passage in D. noxia. Mycelial dry weight and conidiation were both reduced after passage in vitro, but were increased after passage in D. noxia. These two traits did not change after passage in P. xylostella. Germination speed was reduced after in vitro passage of strain 4491. No change in banding pattern was observed for any strain using 14 primers for RAPD-PCR. These results demonstrate the intraspecific variability and phenotypic plasticity of strains of P. fumosoroseus. Stability of virulence after in vitro passage is clearly a desirable trait for a mass-produced biocontrol agent. However, a change in host specificity or productivity in vitro, as we observed for some strains, must be monitored and minimized.

Animals↗