The role of xenobiotic metabolizing enzymes in arylamine toxicity and carcinogenesis: Functional and localization studies

In both animal models and humans, the first and obligatory step in the activation of arylamines is N-hydroxylation. This pathway is primarily mediated by the phase-I enzymes CYP1A1, CYP1A2 and CYP4B1. In the presence of flavonoids such as alpha-naphthoflavone and flavone, both CYP3A4 and CYP3A5 have also been shown to play a minor role in the activation of food-derived heterocyclic amines. The further activation of N-hydroxyarylamines by phase-II metabolism can involve both N,O-acetylation and N,O-sulfonation catalyzed by N-acetyltransferases (NAT1 and NAT2) and sulfotransferases, respectively. Using an array of techniques, we have been unable to detect constitutive CYP1A expression in any segments of the human gastrointestinal tract. This is in contrast to the rabbit where CYP1A1 protein was readily detectable on immunoblots in microsomes prepared from the small intestine. In humans, CYP3A3/3A4 expression was detectable in the esophagus and all segments of the small intestine. Northern blot analysis of eleven human colons showed considerable heterogeneity in CYP3A mRNA between individuals, with the presence of two mRNA species in same subjects. Employing the technique of hybridization histochemistry (also known as in situ hybridization), CYP4B1 expression was observed in some human colons but not in the liver or the small intestine. Hybridization histochemistry studies have also demonstrated variable NAT1 and NAT2 expression in the human gastrointestinal tract. NAT1 and NAT2 mRNA expression was detected in the human liver, small intestine, colon, esophagus, bladder, ureter, stomach and lung. Using a general aryl sulfotransferase riboprobe (HAST1), we have demonstrated marked sulfotransferase expression in the human colon, small intestine, lung, stomach and liver. These studies demonstrate that considerable variability exists in the expression of enzymes involved in the activation of aromatic amines in human tissues. The significance of these results in relation to a role for heterocyclic amines in colon cancer is discussed.

Cumulative sedimentation analysis of Escherichia coli debris size

A new method to measure Escherichia coil cell debris size after homogenization is presented. It is based on cumulative sedimentation analysis under centrifugal force, coupled with Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis of sedimented proteins. The effects that fermentation and homogenization conditions have on the resulting debris distributions were investigated using this method. Median debris size decreased significantly from approximately 0.5 mu m to 0.3 mu m as the number of homogenization passes increased from 2 to 10. Under identical homogenization conditions, uninduced host cells in stationary phase had a larger debris size than exponential cells after 5 homogenizer passes. This difference was not evident after 2 or in passes, possibly because of confounding intact cells and the existence of a minimum debris size for the conditions investigated. Recombinant cells containing protein inclusion bodies had the smallest debris size following homogenization. The method was also used to measure the size distribution of inclusion bodies. This result compared extremely well with an independent determination using centrifugal disc photosedimentation (CDS), thus validating the method. This is the first method that provides accurate size distributions of E. coli debris without the need for sample pretreatment, theoretical approximations (e.g. extinction coefficients), or the separation of debris and inclusion bodies prior to analysis. (C) 1997 John Wiley & Sons, Inc.

PCR-mediated detection of acidophilic, bioleaching-associated bacteria

The detection of acidophilic microorganisms from mining environments by culture methods is time consuming and unreliable. Several PCR approaches were developed to amplify small-subunit rRNA sequences from the DNA of six bacterial phylotypes associated with acidic mining environments, permitting the detection of the target DNA at concentrations as low as 10 fg.

Endostatin- and interleukin-2-expressing retroviral bicistronic vector for gene therapy of metastatic renal cell carcinoma

Background Metastatic renal cell carcinoma (mRCC) is one of the most treatment-resistant malignancies. Despite all new therapeutic advances, almost all patients develop resistance to treatment and cure is rarely seen. In the present study, we evaluated the antitumor effect of a bicistronic retrovirus vector encoding both endostatin (ES) and interleukin (IL)-2 using an orthotopic metastatic RCC mouse model. Methods Balb/C-bearing Renca cells were treated with NIH/3T3-LendIRES-IL-2-SN cells. In the survival studies, mice were monitored daily until they died. At the end of the in vivo experiment, serum levels of IL-2 and ES were measured, the lung was weighed, and the number of metastatic nodules, nodule area, tumor vessels and proliferation of tumor-infiltrating Renca cells were determined. Results Inoculation of NIH/3T3-LendIRES-IL-2-SN cells resulted in an increase in ES and IL-2 levels in the treated group (p < 0.05). There was a significant decrease in lung wet weight, lung nodule area and tumor vessels in the treated group compared to the control group (p < 0.001). The proliferation of Renca cells in the bicistronic-treated group was significantly reduced compared to the control group (p < 0.05). Kaplan-Meier survival curves showed that the probability of survival was significantly higher for mice submitted to bicistronic therapy (log-rank test, p = 0.0016). Bicistronic therapy caused an increase in the infiltration of CD4, CD4 interferon (IFN)gamma-producing, CD8, CD8 IFN gamma-producing and natural killer (CD49b) cells. Conclusions Retroviral bicistronic gene transfer led to the secretion of functional ES and IL-2 that was sufficiently active to: (i) inhibit tumor angiogenesis and tumor cell proliferation and (ii) increase the infiltration of immune cells (C) Copyright. 2011 John Wiley & Sons, Ltd.

Characterization of Adherent Umbilical Cord Blood Stromal Cells Regarding Passage, Cell Number, and Nano-biomarking Utilization

Adherent umbilical cord blood stromal cells (AUCBSCs) are multipotent cells with differentiation capacities. Therefore, these cells have been investigated for their potential in cell-based therapies. Quantum Dots (QDs) are an alternative to organic dyes and fluorescent proteins because of their long-term photostability. In this study we determined the effects of the cell passage on AUCBSCs morphology, phenotype, and differentiation potential. QDs labeled AUCBSCs in the fourth cell passage were differentiated in the three mesodermal lineages and were evaluated using cytochemical methods and transmission electron microscopy (TEM). Gene and protein expression of the AUCBSCs immunophenotypic markers were also evaluated in the labeled cells by real-time quantitative PCR and flow cytometry. In this study we were able to define the best cellular passage to work with AUCBSCs and we also demonstrated that the use of fluorescent QDs can be an efficient nano-biotechnological tool in differentiation studies because labeled cells do not have their characteristics compromised.

Expression of heterochromatin protein 1 in the primary tumor of breast cancer patients in the presence or absence of occult metastatic cells in the bone marrow

Gene silencing may occur in breast cancer samples from patients presenting with occult metastatic cells in the bone marrow and one mechanism regulating gene suppression is heterochromatin formation. We have studied whether members of the heterochromatin protein 1 family Hp1(Hs alpha), Hp1(Hs beta) and Hp1(Hs gamma) which take part in chromatin packaging and gene expression regulation, were differentially expressed in tumors from patients with and without occult metastatic cells in their bone marrow. Tumor samples and bone marrow aspirates were obtained from 37 breast cancer patients. Median age was 63 years and 68% of the patients presented with clinical stage I/II disease. Presence of occult metastatic cells in bone marrow was detected through keratin-19 expression by nested RT-PCR in samples from 20 patients (54.1%). The presence of occult metastatic cells in bone marrow was not associated with node involvement, histological grade, estrogen receptor and ERBB2 immunoexpression. Relative gene expression of HP1(Hs alpha), HP1(Hs beta) and HP1(Hs gamma) was determined by real-time RT-PCR and did not vary according to the presence of occult metastatic cells in bone marrow. In addition, the combined expression of these three transcripts could not be used to classify samples according to the presence of bone marrow micrometastasis. Our work indicates that regulation of heterochromatin formation through HP1 family members may not be the sole mechanism implicated in the metastatic process to the bone marrow. (Int J Biol Markers 2008; 23: 219-24)

Expression of recombinant human follicle stimulating hormone mRNA in Chinese hamster ovary cells under different promoters

Levels of recombinant human follicle stimulating hormone (r-hFSH) mRNA expressed under butyrate and zinc treatment were compared in two CHO-K1 derived cell lines. In King cells under the metallothionein promoter, butyrate induced the increase in both r-hFSH productivity (q(FSH)) and mRNA levels proportionally. In the presence of 1 mM butyrate and 40 mu M zinc, a 4-fold increase in q(FSH) and mRNA levels was achieved as compared to zinc (40) alone; this wasa approximately 6 times higher than in serum free medium. In Darren cells under the beta-actin promotor butyrate induced an increase in q(SFH) but not in mRNA levels.

Acute intermittent porphyria: the in vitro expression of mutant hydroxymethylbilane synthase

Acute intermittent porphyria (AIP) is an inborn error of haem biosynthesis caused by a variety of mutations in the gene coding for hydroxymethylbilane synthase (HMB-S). The entire coding sequence of this gene, from each of three South African AIP patients, was therefore screened for mutations using chemical cleavage mismatch (CCM) analysis and any changes detected characterized by DNA sequencing. Three single base changes were identified; a G(77) to A in exon 3, a C-346 to T in exon 8 and a G(518) to A in exon 10. These missense mutations, previously reported to be present in other populations, are known to be responsible for the structurally deleterious amino acid replacements R26H, R116W and R173Q, respectively. The in vitro expression of the enzymes containing these mutations and the subsequent measurement of their specific activities revealed a reduction to approximately 4% of normal activity. (C) 1997 Academic Press Limited.

A high-resolution genetic map of the familial Mediterranean fever candidate region allows identification of haplotype-sharing among ethnic groups

Familial Mediterranean fever (FMF) is a recessive disorder of inflammation caused by mutations in a gene (designated MEFV) on chromosome 16p13.3, We have recently constructed a 1-Mb cosmid contig that includes the FMF critical region. Here we show genotype data for 12 markers from our physical map, including 5 newly identified microsatellites, in FMF families. Intrafamilial recombinations placed MEFV in the similar to 285 kb between D16S468/D16S3070 and D16S3376. We observed significant linkage disequilibrium in the North African Jewish population, and historical recombinants in the founder haplotype placed MEFV between D16S3082 and D16S3373 (similar to 200 kb). In smaller panels of Iraqi Jewish, Arab, and Armenian families, there were significant allelic associations only for D16S3370 and D16S2617 among the Armenians. A sizable minority of Iraqi Jewish and Armenian carrier chromosomes appeared to be derived from the North African Jewish ancestral haplotype. We observed a unique FMF haplotype common to Iraqi Jews, Arabs, and Armenians and two other haplotypes restricted to either the Iraqi Jewish or the Armenian population. These data support the view that a few major mutations account for a large percentage of the cases of FMF and suggest that same of these mutations arose before the affected Middle Eastern populations diverged from one another. (C) 1997 Academic Press.

Cell cycle model to describe animal cell size variation and lag between cell number and biomass dynamics

The use of cell numbers rather than mass to quantify the size of the biotic phase in animal cell cultures causes several problems. First, the cell size varies with growth conditions, thus yields expressed in terms of cell numbers cannot be used in the normal mass balance sense. Second, experience from microbial systems shows that cell number dynamics lag behind biomass dynamics. This work demonstrates that this lag phenomenon also occurs in animal cell culture. Both the lag phenomenon and the variation in cell size are explained using a simple model of the cell cycle. The basis for the model is that onset of DNA synthesis requires accumulation of G1 cyclins to a prescribed level. This requirement is translated into a requirement for a cell to reach a critical size before commencement of DNA synthesis. A slower gl-owing cell will spend more time in G1 before reaching the critical mass. In contrast, the period between onset of DNA synthesis and mitosis, tau(B), is fixed. The two parameters in the model, the critical size and tau(B), were determined from eight steady-state measurements of mean cell size in a continuous hybridoma culture. Using these parameters, it was possible to predict with reasonable accuracy the transient behavior in a separate shift-up culture, i.e., a culture where cells were transferred from a lean environment to a rich environment. The implications for analyzing experimental data for animal cell culture are discussed. (C) 1997 John Wiley & Sons, Inc.

Poly(3-hydroxybutyrate) production from whey using recombinant Escherichia coli

Recombinant Escherichia coli strains harboring the genes from Alcaligenes eutrophus for polyhydroxyalkanoate biosynthesis were constructed and compared for their ability to synthesize poly(3-hydroxybutyrate) in a defined medium with whey as the sole carbon source. The highest PHB concentration and PHB content obtained were 5.2 g/L and 81% of dry cell weight, respectively.

A polydnavirus-encoded protein of an endoparasitoid wasp is an immune suppressor

The molecular mechanism by which polydnaviruses of endoparasitoid wasps disrupt cell-mediated encapsulation reactions of host insects is largely unknown. Here we show that a polydnavirus-encoded protein, produced from baculovirus and plasmid expression vectors, prevents cell surface exposure of lectin-binding sites and microparticle formation during immune stimulation of haemocytes. The inactivation of immune-related cellular processes by this protein was analysed using a specific lectin and annexin V and shown to be virtually identical to polydnavirus-mediated effects on haemocytes. Cytochalasin D application has similar effects on haemocytes, suggesting that the immune suppression by the polydnavirus protein is caused by the destabilization of actin filaments. Since the exposure of cell surface glycoproteins and the formation of microparticles are part of an immune response to foreign objects or microorganisms and a prerequisite for cell-mediated encapsulation of microorganisms and parasites, the virus-encoded protein may become an important tool for the inactivation of cellular immune reactions in insects and an essential component in understanding immune suppression in parasitized host insects.

Molecular evidence of horizontal transmission of hepatitis C virus within couples

Hepatitis C virus (HCV) transmission has decreased with the adoption of universal blood donor screening and social policies to reduce the risk of infection in intravenous drug users, but remains a worldwide health problem. The objective of this study was to evaluate the phylogenetic relationships among sequences from different HCV genomic regions from sexual partners of infected patients. Nine couples with a stable relationship and without other risk factors for HCV infection and 42 control patients were selected, and the NS3 and NS5B regions were analysed. Phylogenetic analysis showed that viruses from five of the couples had a common origin, clustering in the same monophyletic group, with bootstrap values greater than 70. For the other couples, monophyletic groups were observed, but without bootstrap support. Thus, using two different viral genome regions, a common source of infection was observed in both members of five couples. These data strongly support HCV transmission within couples.

Novel fed-batch strategy for the production of insulin-like growth factor 1 (IGF-1)

The production of Long-R-3-IGF-1 (an IGF-1 fusion analog) by constant-rate, fed-batch fermentation of Escherichia coli yielded 2.6 g fusion protein/L, corresponding to an actual IGF-1 concentration of 2.2 g/L. A novel strategy employing three distinct feeding stages was developed which raised product concentration to 4.3 g/L (3.6 g/L of IGF-1) while minimising glucose and acetate accumulation. This improved productivity was not accompanied by an increase in inclusion body size.

Molecular epidemiology and genetic diversity of hepatitis B virus genotype E in an isolated Afro-Colombian community

Hepatitis B virus (HBV) infection is a significant public health concern with 350 million chronic carriers worldwide. Eight HBV genotypes (A-H) have been described so far. Genotype E (HBV/E) is widely distributed in West Africa and has rarely been found in other continents, except for a few cases in individuals with an African background. In this study, we characterized HBV genotypes in Quibdo, Colombia, by partial S/P gene sequencing, and found, for the first time, HBV/E circulating in nine Afro-Colombian patients who had no recent contact with Africa. The presence of HBV/E in this community as a monophyletic group suggests that it was a result of a recent introduction by some Afro-descendent contact or, alternatively, that the virus came with slaves brought to Colombia. By using sequences with sampling dates, we estimated the substitution rate to be about 3.2x10(-4) substitutions per site per year, which resulted in a time to the most recent common ancestor (TMRCA) of 29 years. In parallel, we also estimated the TMRCA for HBV/E by using two previously estimated substitution rates (7.7x10(-4) and 1.5x10(-5) substitutions per site per year). The TMRCA was around 35 years under the higher rate and 1500 years under the slower rate. In sum, this work reports for the first time the presence of an exclusively African HBV genotype circulating in South America. We also discuss the time of the entry of this virus into America based on different substitution rates estimated for HBV.