Release and uptake of gene transfer agent by Rhodopseudomonas capsulata.

Many strains of Rhodopseudomonas capsulata are capable of exchanging genetic information via a recently discovered gene transfer process involving the release and subsequent uptake from the medium of particles containing genetic information (gene transfer agents, GTAs). No viral activities are observed to be associated with this system. An assay has been developed to quantitate gene transfer in R. capsulata. Conditions are described for which the number of cells acquiring a new genetic trait is direcly proportional to the number of GTAs and independent of the number of receipient cells. These conditions were used for the assay of the uptake and release of GTAs by cells. The maximum fraction of recipients that acquire a given genetic marker is approximately 4 X 10(-4). Free GTA appears in a growing culture in one or two abrupt waves near the time of transition from exponential to stationary phase. During these waves, the titer of GTA for a given marker may reach 2 X 103/ml. A comparison of the frequency of single- and double-marker transfers suggests that most of the cells in early-stationary-phase cultures are active recipients. The ultraviolet inactivation spectrum of GTA resembles that of the small ribonucleic acid phages. The inactivation cross section section beta, for GTA was calculated to be 1.7 X 10(-16) cm2/photon at 265 nm.     

Elevated BCRP/ABCG2 expression confers acquired resistance to gefitinib in wild-type EGFR-expressing cells

Background

The sensitivity of non-small cell lung cancer (NSCLC) patients to EGFR tyrosine kinase inhibitors (TKIs) is strongly associated with activating EGFR mutations. Although not as sensitive as patients harboring these mutations, some patients with wild-type EGFR (wtEGFR) remain responsive to EGFR TKIs, suggesting that the existence of unexplored mechanisms renders most of wtEGFR-expressing cancer cells insensitive.

Methodology/Principal Findings

Here, we show that acquired resistance of wtEGFR-expressing cancer cells to an EGFR TKI, gefitinib, is associated with elevated expression of breast cancer resistance protein (BCRP/ABCG2), which in turn leads to gefitinib efflux from cells. In addition, BCRP/ABCG2 expression correlates with poor response to gefitinib in both cancer cell lines and lung cancer patients with wtEGFR. Co-treatment with BCRP/ABCG2 inhibitors enhanced the anti-tumor activity of gefitinib.

Conclusions/Significance

Thus, BCRP/ABCG2 expression may be a predictor for poor efficacy of gefitinib treatment, and targeting BCRP/ABCG2 may broaden the use of gefitinib in patients with wtEGFR.     

Impact of dried skim milk in production diets on Lactobacillus and pathogenic bacterial shedding in growing-finishing swine

AIMS: To determine the possible effects of inclusion of dried skim milk (DSM) in swine diets on indigenous Lactobacillus spp. and Escherichia coli, and its potential for controlling pathogen shedding and affect animal growth in growing-finishing swine. METHODS AND RESULTS: Animals were fed over three dietary phases to match production needs from age 10-14 weeks, 14-18 weeks and 18-22 weeks. For each feeding phase, diets were formulated to contain 0 or 10% DSM (balanced for metabolizable energy and true ileal digestible amino acids). Animals were weighed every 2 weeks and faecal samples were collected from 40 animals (20 with DSM and 20 without DSM) at week 10 (d 0 on diets), 14, 18 and 22 of age, and were analysed for Lactobacillus spp., Enterobacteriaceae, coliforms, E. coli, Salmonella, Campylobacter and E. coli O157:H7. At the start of the study (week 10), faecal bacterial counts (log10 CFU g(-1) faeces) were 9.55, 7.26, 7.01 and 6.93 for Lactobacillus, Enterobacteriaceae, coliforms and E. coli populations respectively. The Enterobacteriaceae, coliform and E. coli populations decreased through week 14 and 18, but were higher in animals fed with the DSM diet compared with the basal diet without DSM. The Lactobacillus populations at weeks 14 and 18 were lower in the animals fed the diet without DSM, whereas feeding DSM maintained the Lactobacillus counts from week 10. At week 22, populations of Enterobacteriaceae, coliforms and E. coli were >week 18 for the animals fed the diet without DSM, less change was observed with the feeding of DSM, and no differences between the diets were observed at week 22. However, in week 22 the animal gain was positively correlated with Lactobacillus numbers and negatively correlated with E. coli numbers. Subtraction of the E. coli population (log10) from the Lactobacillus population (log10) yielded a positive value termed 'effective'Lactobacillus that correlated well with animal gain and may better define a beneficial function in the intestine. Salmonella were detected in over 60% of the animals at week 10 and 14, and <20% at week 18 and 22. Campylobacter were detected rarely at weeks 10, 14 and 18, but were found in 25% of the animals at week 22. The DSM did not affect Salmonella or Campylobacter shedding, but examination of individual animals over the entire experiment indicated that fewer recurring incidences of Salmonella shedding occurred in animals that maintained higher Lactobacillus. In addition, at week 22, Salmonella and Campylobacter shedding was associated with lower levels of effective Lactobacillus and lower animal weight gains. CONCLUSIONS: The DSM did not directly affect the animal performance or pathogen shedding via the Lactobacillus spp. population at any phase of production. However, analysis of data from all animals revealed that faecal Lactobacillus affected Salmonella shedding and in the finishing phase, animal growth and pathogen shedding also were affected, as reflected by the 'effective'Lactobacillus-associated observations. SIGNIFICANCE AND IMPACT OF THE STUDY: In the swine intestine, any benefits from gastrointestinal Lactobacillus may be compromized by the E. coli population, and this antagonism may explain responses observed with prebiotics or probiotics in some swine.     

Export of L-isoleucine from Corynebacterium glutamicum: a two-gene-encoded member of a new translocator family

Bacteria possess amino acid export systems, and Corynebacterium glutamicum excretes L-isoleucine in a process dependent on the proton motive force. In order to identify the system responsible for L-isoleucine export, we have used transposon mutagenesis to isolate mutants of C. glutamicum sensitive to the peptide isoleucyl-isoleucine. In one such mutant, strong peptide sensitivity resulted from insertion into a gene designated brnF encoding a hydrophobic protein predicted to possess seven transmembrane spanning helices. brnE is located downstream of brnF and encodes a second hydrophobic protein with four putative membrane-spanning helices. A mutant deleted of both genes no longer exports L-isoleucine, whereas an overexpressing strain exports this amino acid at an increased rate. BrnF and BrnE together are also required for the export of L-leucine and L-valine. BrnFE is thus a two-component export permease specific for aliphatic hydrophobic amino acids. Upstream of brnFE and transcribed divergently is an Lrp-like regulatory gene required for active export. Searches for homologues of BrnFE show that this type of exporter is widespread in prokaryotes but lacking in eukaryotes and that both gene products which together comprise the members of a novel family, the LIV-E family, generally map together within a single operon. Comparisons of the BrnF and BrnE phylogenetic trees show that gene duplication events in the early bacterial lineage gave rise to multiple paralogues that have been retained in alpha-proteobacteria but not in other prokaryotes analyzed.     

A new synnematous species of Penicillium from soil in Taiwan

A synnematous species of Penicillium, P. calidicanium, is described and illustrated. The fungus was isolated from soil in Taiwan. Penicillium calidicanium can be placed in subgenus Biverticillium because of its symmetrical, biverticillate penicilli, ampulliform to acerose phialides, and ability to produce abundant synnemata in Czapek yeast extract agar, malt extract agar, and Czapek's solution agar. It is close to P. duclauxii and P. vulpinum, but differs in colony morphology, growth rate, morphology of the synnemata, and ornamentation of the conidial wall.     

Population dynamics of Meloidogyne incognita on black pepper plants in two agro-ecological regions in Vietnam

The objective of our study was to examine the effect of local environmental conditions, especially air temperature and rainfall, in two agro-ecological regions in Vietnam on the population dynamics of Meloidogyne incognita on black pepper plants and on percentage root galling. The two study sites were situated in Cam Lo, Quang Tri province (North Central Coast) and Buon Ma Thuot, Dac Lac province (Central Highlands). At the study sites, 13 plant-parasitic nematode taxa belonging to 12 genera were identified. Ten of these taxa were present in both study sites. Helicotylenchus certus, Hemicriconemoides cocophilus and Pratylenchus coffeae were only found in Buon Ma Thuot. Based on nematode population densities, M. incognita was the most abundant taxon present. Rainfall and air temperature differed significantly between the two study sites. The most important climatic difference was rainfall: in Buon Ma Thuot, the rainy season lasted 2 months longer and the monthly rainfall during the rainy season was much higher compared to Cam Lo. Although this difference resulted in some differences in the population dynamics of M. incognita in the soil and roots of the black pepper variety Vinh Linh, the highest root population densities were observed in both study sites during the first half of the dry season. In Cam Lo, the highest percentage root galling was observed during the first half of the dry season. In Buon Ma Thuot, the highest percentage root galling was observed towards the end of the rainy season. Estimating the population densities if M. incognita to decide on the application of a management strategy should be done during the first half of the dry season.     

A simple and sensitive ribonucleotide reductase assay

Ribonucleotide reductase (RR) is a key regulatory enzyme in the DNA synthesis pathway and is the target of the cancer chemotherapeutic agent hydroxyurea. The study of RR is significantly hindered by the tedious and labor-intensive nature of enzymatic assay. In this report, we present a novel RR assay in which detection of the deoxyribonucleotides produced by RR occurs via coupling to the DNA polymerase reaction, and is enhanced by using RNase to degrade endogenous RNA. Cell extracts from various cell lines were treated with RNase and then reacted with ATP and radioactive ribonucleotide diphosphate as the substrate. Incorporation of the radioactive substrate [¹⁴C]CDP into DNA was linear over 30 min and was linear with the amount of extract, which provided RR activity. The reaction was inhibited by hydroxyurea and required Mg²⁺ and ATP, suggesting that the assay is specific to RR activity. While RR activities determined by our method and by a conventional method were comparable, this novel method proved to be simpler, faster, more sensitive and less expensive. In addition, assay of the RR activity for multiple samples can easily be performed simultaneously. It is superior to other RR assays in all aspects.     

Heme oxygenase is involved in nitric oxide- and auxin-induced lateral root formation in rice

Lateral root (LR) development performs the essential tasks of providing water, nutrients, and physical support to plants. Therefore, understanding the regulation of LR development is of agronomic importance. In this study, we examined the effect of nitric oxide (NO), auxin, and hemin (Hm) on LR formation in rice. Treatment with Hm [a highly effective heme oxygenase (HO) inducer], sodium nitroprusside (SNP, an NO donor), or indole-3-butyric acid (IBA, a naturally occurring auxin) induced LR formation and HO activity. LR formation and HO activity induced by SNP and IBA but not Hm was reduced by the specific NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. As well, Hm, SNP, and IBA could induce OsHO1 mRNA expression. Zn protoporphyrin IX (the specific inhibitor of HO) and hemoglobin (the carbon monoxide/NO scavenger) reduced LR number and HO activity induced by Hm, SNP, and IBA. Our data suggest that HO is required for Hm-, auxin-, and NO-induced LR formation in rice.     

Quantitative profiling of drug-associated proteomic alterations by combined 2-nitrobenzenesulfenyl chloride (NBS) isotope labeling and 2DE/MS identification

The identification of drug-responsive biomarkers in complex protein mixtures is an important goal of quantitative proteomics. Here, we describe a novel approach for identifying such drug-induced protein alterations, which combines 2-nitrobenzenesulfenyl chloride (NBS) tryptophan labeling with two-dimensional gel electrophoresis (2DE)/mass spectrometry (MS). Lysates from drug-treated and control samples are labeled with light or heavy NBS moiety and separated on a common 2DE gel, and protein alterations are identified by MS through the differential intensity of paired NBS peptide peaks. Using NBS/2DE/MS, we profiled the proteomic alterations induced by tamoxifen (TAM) in the estrogen receptor (ER) positive MCF-7 breast cancer cell line. Of 88 protein spots that significantly changed upon TAM treatment, 44 spots representing 23 distinct protein species were successfully identified with NBS-paired peptides. Of these 23 TAM-altered proteins, 16 (70%) have not been previously associated with TAM or ER activity. We found the NBS labeling procedure to be both technically and biologically reproducible, and the NBS/2DE/MS alterations exhibited good concordance with conventional 2DE differential protein quantitation, with discrepancies largely due to the comigration of distinct proteins in the regular 2DE gels. To validate the NBS/2DE/MS results, we used immunoblotting to confirm GRP78, CK19, and PA2G4 as bona fide TAM-regulated proteins. Furthermore, we demonstrate that PA2G4 expression can serve as a novel prognostic factor for disease-free survival in two independent breast cancer patient cohorts. To our knowledge, this is the first report describing the proteomic changes in breast cancer cells induced by TAM, the most commonly used selective estrogen receptor modulator (SERM). Our results indicate that NBS/2DE/MS may represent a more reliable approach for cellular protein quantitation than conventional 2DE approaches.     

Investigating mechanisms of collagen thermal denaturation by high resolution second-harmonic generation imaging

We apply the technique of second-harmonic generation (SHG) microscopy to obtain large area submicron resolution image of Type I collagen from rat tail tendon as it is heated from 40 degrees C to 70 degrees C for 0-180 min. The change in the collagen structure as reflected in its SHG image is observed at length scales from submicron to hundreds of microns. We observed that heating the tendon below the temperature of 54 degrees C does not produce any change in the averaged SHG intensity. At the heating temperature of 54 degrees C and above, we find that increasing the heating temperature and time leads to decreasing SHG intensity. As the tendon is heated above 54 degrees C, the regions where the SHG signal vanish and form a tiger-tail like pattern. In addition, a decrease in the SHG signal occurs uniformly throughout the tendon. By comparing the relative SHG intensities in small and large areas, we found that the denaturation process responsible for forming the tiger-tail like pattern occurs at a higher rate than the global denaturation process occurring throughout the tendon. We also measured the fibril spacing and found that it remains constant at 1.61 +/- 0.04 micron for all heating temperature and times. The constant fibril density shows that the global denaturation process occurs at a length scale smaller than the size of the fibril. Our results show that second-harmonic generation microscopy is effective in monitoring the thermal damage to collagen and has potential applications in biomedicine.