Lrp Gene Research Articles

Global Lrp regulator protein from Haloferax mediterranei: Transcriptional analysis and structural characterization

Haloferax mediterranei, an extreme halophilic archaeon thriving in hypersaline environments, has acquired significant attention in biotechnological and biochemical research due to its remarkable ability to flourish in extreme salinity conditions. Transcription factors, essential in regulating diverse cellular processes, have become focal points in understanding its adaptability. This study delves into the role of the Lrp transcription factor, exploring its modulation of glnA, nasABC, and lrp gene promoters in vivo through β-galactosidase assays. Remarkably, our findings propose Lrp as the pioneering transcriptional regulator of nitrogen metabolism identified in a haloarchaeon. This study suggests its potential role in activating or repressing assimilatory pathway enzymes (GlnA and NasA). The interaction between Lrp and these promoters is analyzed using Electrophoretic Mobility Shift Assay and Differential Scanning Fluorimetry, highlighting l-glutamine's indispensable role in stabilizing the Lrp-DNA complex. Our research uncovers that halophilic Lrp forms octameric structures in the presence of l-glutamine. The study reveals the three-dimensional structure of the Lrp as a homodimer using X-ray crystallography, confirming this state in solution by Small-Angle X-ray Scattering. These findings illuminate the complex molecular mechanisms driving Hfx. mediterranei's nitrogen metabolism, offering valuable insights about its gene expression regulation and enriching our comprehension of extremophile biology.

Lrp as a potential transcriptional regulator involved in stress response in Haloferax mediterranei

The Archaea domain consists of a heterogeneous group of microorganisms with unique physiological properties that occupy a wide variety of niches in nature. Haloferax mediterranei is an extremely halophilic archaeon classified in the Phylum Euryarchaeota, which requires a high concentration of inorganic salts for optimal growth. In haloarchaea, transcription factors play a fundamental role in an adequate adaptation to environmental and nutritional changes, preserving the survival and integrity of the organism. To deepen knowledge of the Lrp/AsnC transcriptional regulator family, a lrp gene (HFX_RS01210) from this family has been studied. Site-directed mutagenesis has allowed us to identify the TATA-box and two potential sites of the transcriptional factor (TF) to its own promoter and autoregulate itself. Several approaches were carried out to elucidate whether this transcriptional regulator is involved in stresses due to heavy metals and limited nitrogen conditions. Characterization of the lrp deletion mutant and the Lrp overexpressed strain, suggests that the level of lrp expression depends on the nitrogen source and the presence of cobalt. The most striking results were obtained in the presence of nitrate as a nitrogen source due to the inability of the deletion mutant to grow. All these results confirm that Lrp is a powerful candidate for a regulatory role in the stress response, particularly under N-limiting conditions and the presence of cobalt.

Analysis of Haloferax mediterranei Lrp Transcriptional Regulator.

Haloferax mediterranei is an extremely halophilic archaeon, able to live in hypersaline environments with versatile nutritional requirements, whose study represents an excellent basis in the field of biotechnology. The transcriptional machinery in Archaea combines the eukaryotic basal apparatus and the bacterial regulation mechanisms. However, little is known about molecular mechanisms of gene expression regulation compared with Bacteria, particularly in Haloarchaea. The genome of Hfx. mediterranei contains a gene, lrp (HFX_RS01210), which encodes a transcriptional factor belonging to Lrp/AsnC family. It is located downstream of the glutamine synthetase gene (HFX_RS01205), an enzyme involved in ammonium assimilation and amino acid metabolism. To study this transcriptional factor more deeply, the lrp gene has been homologously overexpressed and purified under native conditions by two chromatographic steps, namely nickel affinity and gel filtration chromatography, showing that Lrp behaves asa tetrameric protein of approximately 67 kDa. Its promoter region has been characterized under different growth conditions using bgaH as a reporter gene. The amount of Lrp protein was also analyzed by Western blotting in different nitrogen sources and under various stress conditions. To sum up, regarding its involvement in the nitrogen cycle, it has been shown that its expression profile does not change in response to the nitrogen sources tested. Differences in its expression pattern have been observed under different stress conditions, such as in the presence of hydrogen peroxide or heavy metals. According to these results, the Lrp seems to be involved in a general response against stress factors, acting as a first-line transcriptional regulator.

USAGE OF TRANSCRIPTIONAL REGULATOR LRP BASED BIOSENSOR FOR RESEARCH OF VALINE BIOSYNTHESIS PATHWAYS IN CORYNEBACTERIUM GLUTAMICUM CELLS

The biosensor construction based on Lrp gene was obtained, what contains a fluorescence protein gene under control of BrnFE gene promotor. It was displayed that the fluorescence of Corynebacterium glutamicum cells carrying the biosensor correlates with the production of valine, and with its help it is possible to study the biosynthesis of valine.

Validation of qPCR assays for the detection of citrus canker

Citrus canker, a serious bacterial disease affecting the citrus industry worldwide, is caused by Xanthomonas citri subsp. citri (Xcc) pathotypes A, A* and Aw, and to a lesser extent by X. fuscans subsp. aurantifolii (Xfa). The recent citrus canker outbreak in Australia has emphasised the need to re-evaluate the efficiency of molecular assays used for detecting citrus canker bacteria. Two published probe-based qPCR assays targeting the pth and lrp genes were tested for Xcc, whereas a SYBR Greenbased qPCR assay was tested for Xfa. The Xcc pth gene and Xfa qPCR assays were shown to be specific towards all pathotypes of Xcc and Xfa, respectively. The detection limit for both assays were 1 pg of genomic DNA or 103 CFU in bacteria-spiked leaf sample. The Xcc lrp gene qPCR assay was able to discriminate Xcc pathotypes with a detection limit of 1 ng of genomic DNA or 106 CFU in bacteriaspiked leaf sample, but this assay showed cross-reaction with Xfa. To allow rapid high-throughput detection of all Xcc pathotypes, a duplex probe-based qPCR assay was developed by incorporating COX primers as an internal control for plant DNA into the pth gene qPCR assay.

Competitive Repression of the artPIQM Operon for Arginine and Ornithine Transport by Arginine Repressor and Leucine-Responsive Regulatory Protein in Escherichia coli.

Two out of the three major uptake systems for arginine in Escherichia coli are encoded by the artJ-artPIQM gene cluster. ArtJ is the high-affinity periplasmic arginine-specific binding protein (ArgBP-I), whereas artI encodes the arginine and ornithine periplasmic binding protein (AO). Both ArtJ and ArtI are supposed to combine with the inner membrane-associated ArtQMP2 transport complex of the ATP-binding cassette-type (ABC). Transcription of artJ is repressed by arginine repressor (ArgR) and the artPIQM operon is regulated by the transcriptional regulators ArgR and Leucine-responsive regulatory protein (Lrp). Whereas repression by ArgR requires arginine as corepressor, repression of PartP by Lrp is partially counteracted by leucine, its major effector molecule. We demonstrate that binding of dimeric Lrp to the artP control region generates four complexes with a distinct migration velocity, and that leucine has an effect on both global binding affinity and cooperativity in the binding. We identify the binding sites for Lrp in the artP control region, reveal interferences in the binding of ArgR and Lrp in vitro and demonstrate that the two transcription factors act as competitive repressors in vivo, each one being a more potent regulator in the absence of the other. This competitive behavior may be explained by the partial steric overlap of their respective binding sites. Furthermore, we demonstrate ArgR binding to an unusual position in the control region of the lrp gene, downstream of the transcription initiation site. From this unusual position for an ArgR-specific operator, ArgR has little direct effect on lrp expression, but interferes with the negative leucine-sensitive autoregulation exerted by Lrp. Direct arginine and ArgR-dependent repression of lrp could be observed with a 25-bp deletion mutant, in which the ArgR binding site was artificially moved to a position immediately downstream of the lrp transcription initiation site. This finding is reminiscent of a previous observation made for the carAB operon encoding carbamoylphosphate synthase, where ArgR bound in overlap with the downstream promoter P2 does not block transcription initiated 67 bp upstream at the P1 promoter, and further supports the hypothesis that ArgR does not act as an efficient roadblock.

Engineering of leucine-responsive regulatory protein improves spiramycin and bitespiramycin biosynthesis

BackgroundBitespiramycin (BT) is produced by recombinant spiramycin (SP) producing strain Streptomyces spiramyceticus harboring a heterologous 4″-O-isovaleryltransferase gene (ist). Exogenous l-Leucine (l-Leu) could improve the production of BT. The orf2 gene found from the genomic sequence of S. spiramyceticus encodes a leucine-responsive regulatory protein (Lrp) family regulator named as SSP_Lrp. The functions of SSP_Lrp and l-Leu involved in the biosynthesis of spiramycin (SP) and BT were investigated in S. spiramyceticus.ResultsSSP_Lrp was a global regulator directly affecting the expression of three positive regulatory genes, bsm23, bsm42 and acyB2, in SP or BT biosynthesis. Inactivation of SSP_Lrp gene in S. spiramyceticus 1941 caused minor increase of SP production. However, SP production of the ΔSSP_Lrp-SP strain containing an SSP_Lrp deficient of putative l-Leu binding domain was higher than that of S. spiramyceticus 1941 (476.2 ± 3.1 μg/L versus 313.3 ± 25.2 μg/L, respectively), especially SP III increased remarkably. The yield of BT in ΔSSP_Lrp-BT strain was more than twice than that in 1941-BT. The fact that intracellular concentrations of branched-chain amino acids (BCAAs) decreased markedly in the ΔSSP_Lrp-SP demonstrated increasing catabolism of BCAAs provided more precursors for SP biosynthesis. Comparative analysis of transcriptome profiles of the ΔSSP_Lrp-SP and S. spiramyceticus 1941 found 12 genes with obvious differences in expression, including 6 up-regulated genes and 6 down-regulated genes. The up-regulated genes are related to PKS gene for SP biosynthesis, isoprenoid biosynthesis, a Sigma24 family factor, the metabolism of aspartic acid, pyruvate and acyl-CoA; and the down-regulated genes are associated with ribosomal proteins, an AcrR family regulator, and biosynthesis of terpenoid, glutamate and glutamine.ConclusionSSP_Lrp in S. spiramyceticus was a negative regulator involved in the SP and BT biosynthesis. The deletion of SSP_Lrp putative l-Leu binding domain was advantageous for production of BT and SP, especially their III components.

Lrp, a global regulator, regulates the virulence of Vibrio vulnificus

BackgroundAn attenuated mutant (designated NY303) of Vibrio vulnificus, which causes serious wound infection and septicemia in humans, was isolated fortuitously from a clinical strain YJ016. This mutant was defective in cytotoxicity, migration on soft agar and virulence in the mouse. The purpose of this study was to map the mutation in this attenuated mutant and further explore how the gene thus identified is involved in virulence.MethodsThe whole genome sequence of mutant NY303 determined by next-generation sequencing was compared with that of strain YJ016 to map the mutations. By isolating and characterizing the specific gene-knockout mutants, the gene associated with the phenotype of mutant NY303 was identified. This gene encodes a global regulator, Lrp. A mutant, YH01, deficient in Lrp was isolated and examined in vitro, in vivo and ex vivo to find the affected virulence mechanisms. The target genes of Lrp were further identified by comparing the transcriptomes, which were determined by RNA-seq, of strain YJ016 and mutant YH01. The promoters bound by Lrp were identified by genome footprinting-sequencing, and those related with virulence were further examined by electrophoretic mobility shift assay.ResultsA mutation in lrp was shown to be associated with the reduced cytotoxicity, chemotaxis and virulence of mutant NY303. Mutant YH01 exhibited a phenotype resembling that of mutant NY303, and was defective in colonization in the mouse and growth in mouse serum, but not the antiphagocytosis ability. 596 and 95 genes were down- and up-regulated, respectively, in mutant YH01. Many of the genes involved in secretion of the MARTX cytotoxin, chemotaxis and iron-acquisition were down-regulated in mutant YH01. The lrp gene, which was shown to be negatively autoregulated, and 7 down-regulated virulence-associated genes were bound by Lrp in their promoters. A 14-bp consensus sequence, mkCrTTkwAyTsTG, putatively recognized by Lrp was identified in the promoters of these genes.ConclusionsLrp is a global regulator involved in regulation of cytotoxicity, chemotaxis and iron-acquisition in V. vulnificus. Down-regulation of many of the genes associated with these properties may be responsible, at least partly, for loss of virulence in mutant NY303.

Frameshift Mutation Confers Function as Virulence Factor to Leucine-Rich Repeat Protein from Acidovorax avenae.

Many plant pathogens inject type III (T3SS) effectors into host cells to suppress host immunity and promote successful infection. The bacterial pathogen Acidovorax avenae causes brown stripe symptom in many species of monocotyledonous plants; however, individual strains of each pathogen infect only one host species. T3SS-deleted mutants of A. avenae K1 (virulent to rice) or N1141 (virulent to finger millet) caused no symptom in each host plant, suggesting that T3SS effectors are involved in the symptom formation. To identify T3SS effectors as virulence factors, we performed whole-genome and predictive analyses. Although the nucleotide sequence of the novel leucine-rich repeat protein (Lrp) gene of N1141 had high sequence identity with K1 Lrp, the amino acid sequences of the encoded proteins were quite different due to a 1-bp insertion within the K1 Lrp gene. An Lrp-deleted K1 strain (KΔLrp) did not cause brown stripe symptom in rice (host plant for K1); by contrast, the analogous mutation in N1141 (NΔLrp) did not interfere with infection of finger millet. In addition, NΔLrp retained the ability to induce effector-triggered immunity (ETI), including hypersensitive response cell death and expression of ETI-related genes. These data indicated that K1 Lrp functions as a virulence factor in rice, whereas N1141 Lrp does not play a similar role in finger millet. Yeast two-hybrid screening revealed that K1 Lrp interacts with oryzain α, a pathogenesis-related protein of the cysteine protease family, whereas N1141 Lrp, which contains LRR domains, does not. This specific interaction between K1 Lrp and oryzain α was confirmed by Bimolecular fluorescence complementation assay in rice cells. Thus, K1 Lrp protein may have acquired its function as virulence factor in rice due to a frameshift mutation.

Effect of Expression of Multidrug Resistance Genes in Newly Diagnosed Multiple Myeloma on the Clinical Course of the Disease

Multidrug resistance (MDR) is the biological phenomenon, significantly reduces the survival of patients with multiple myeloma (MM) to the conventional chemotherapy. Overexpression of MDR gene (MDR1, MRP1, LRP, BCRP) by plasma cells may be clinically manifest of disease progression during therapy. Proteasome inhibitor Bortezomib for the treatment of MM in clinical practice has greatly improved the survival of patients with this malignant disease, but at the same time, there is a group of patients resistant to the treatment with Bortezomib. The common (total, summary) effect of the expression of 4 MDR genes on the development of drug resistance to the treatment of Bortezomib have not been studied earlier.Aim. Determination of the expression of mRNA genes MDR1, MRP1, LRP, BCRP, responsible for the development of MDR in bone marrow aspirates in patients with newly diagnosed MM before the Bortezomib-containing therapy on the clinical course of the disease and response to the treatment.Materials and methods. Was investigated a group of 15 patients with newly diagnosed stage III MM classification Durie-Salmon before the start of cytostatic therapy. The expression of MDR genes was studied in bone marrow mononuclear cells fraction containing plasma cells, by RT-PCR (polymerase chain reaction reverse transcription). The degree of expression was assessed by semi-quantitative visual assessment from 0 (no electrophoretic strips) to 4 points (bright glow of the transcript).Results. The expression of MDR gene was found in all patients with newly diagnosed MM before the start of cytostatic therapy: MDR 1 gene is expressed in 14 patients (93%), genes MRP 1 and LRP were detected in 11 patients (73%), the expression of BCRP gene was found in 15 patients (100%). The mean value of expression of MRP 1 and BCRP genes according to visual assessment was 1. The mean value of gene expression MDR 1 (M ± SE) is 1.5 ± 0.27 points and LRP gene is 1.47 ± 0.14 points. The common (summary) mean expression of 4 genes was 5.0 ± 0.76 points.According to the expression level - above or below the mean value, we have identified two subgroups of the patients: subgroup A - Patients with higher overall expression of 4 genes and subgroup B - Patients with lower overall MDR genes expression. In these subgroups was analyzed the clinical parameters of disease at the moment of diagnosis, such as the level of hemoglobin, red blood cells, paraprotein, creatinine, calcium, LDH and the effectiveness of the 6 courses of induction therapy with Bortezomib- containing treatment. No significant differences in clinical scores in the subgroups A and B were not found. After induction treatment, in the subgroup A with higher overall MDR genes expression is not detected significant reduction in the absolute value of paraprotein (32,0 ± 3,7 g / l before the treatment, and 19.34 ± 6.47 g /l after the treatment). Whereas, in the subgroup B with lower overall expression, in the same conditions of the treatment, registered a significant reduction of paraprotein value (41,9 ± 5,0 g /l before the treatment, and 14.85 ± 6.53 g / l after the treatment p <0.05).Conclusion. We the first time identified the group of patients with newly diagnosed MM associated with high common expression of MDR genes before to the cytostatic treatment. The common overexpression of 4 MDR genes not associated with such clinical parameters of the disease as the level of hemoglobin, red blood cells, paraprotein, creatinine, calcium, LDH, but it decreases the immediate response to the Bortezomib-containing induction treatment. DisclosuresNo relevant conflicts of interest to declare.

MULTIPLE DRUG RESISTANCE PROTEINS OF PULMONARY SOMATIC CELLS AND THEIR SPECIFIC EXPRESSION IN FIBROUS CAVERNOUS TUBERCULOSIS

Cells of the host containing special proteins-transporters, so-called proteins of multiple drug resistance (MDR proteins) can contribute to the drug resistance formation. Goal of the study: to define specific expression of gene and distribution of main MDR proteins (MDR1/Pgp, MRP1, lRP, BCRP) in the pulmonary cells in case of active tuberculosis. Materials and methods. Expression of MDR protein genes was evaluated by RT-PCR for mRNA isolated from the surgical specimen of fibrous cavernous tuberculosis patients. Localization of MDR proteins was performed by immunohistochemical staining and confocal laser microscopy. Main results. Intensity of MDR protein genes expression varies in different zones of tuberculous lesions: MDR1 and BCRP are characterized by the highest level and MRP1 gene is characterized by the minimum level. The level of lRP gene expression depends on the inflammation zone and it is maximum in perifocal zone where protein is detected in the cells of alveolar epithelium and macrophages. High expression of MDR protein genes in various parts of tuberculous lesions witnesses about the potential involvement of these proteins in the development of drug resistance to anti-tuberculosis drugs.

Increasingl-isoleucine production inCorynebacterium glutamicumby overexpressing global regulator Lrp and two-component export system BrnFE

To increase the l-isoleucine production in Corynebacterium glutamicum by overexpressing the global regulator Lrp and the two-component export system BrnFE. The brnFE operon and the lrp gene were cloned into the shuttle vector pDXW-8 individually or in combination. The constructed plasmids were transformed into an l-isoleucine-producing strain C. glutamicum JHI3-156, and the l-isoleucine production in these different strains was analysed and compared. More l-isoleucine was produced when only Lrp was expressed than when only BrnFE was expressed. Significant increase in l-isoleucine production was observed when Lrp and BrnFE were expressed in combination. Compared to the control strain, l-isoleucine production in JHI3-156/pDXW-8-lrp-brnFE increased 63% in flask cultivation, and the specific yield of l-isoleucine increased 72% in fed-batch fermentation. Both Lrp and BrnFE are important to enhance the l-isoleucine production in C. glutamicum. The results provide useful information to enhance l-isoleucine or other branched-chain amino acid production in C. glutamicum.

Expression of genes related to multiple drug resistance and apoptosis in acute leukemia: response to induction chemotherapy

Resistance to chemotherapy is a major impediment to the successful treatment of acute leukemia (AL). Expression of genes involved in drug resistance and apoptosis may be responsible for this. This study aimed to investigate the expression of drug resistance (MDR1, MRP1, LRP, BCRP, GSTP1, DHFR) and apoptotic genes (p53, BCL-2, Survivin) in adult acute leukemias and compare them with clinical and hematological findings and response to induction chemotherapy. Eighty-five patients with AL [45 with acute myeloid leukemia (AML) and 40 with acute lymphoblastic leukemia (ALL)] were used as a study group. Real-time PCR results showed that expression level of MDR1 was significantly higher in AML whereas expression of DHFR, BCRP and Survivin was significantly higher in ALL patients. In AML, significant correlation was observed between LRP and MRP1 (rs=0.44, p=0.016), LRP and DHFR (rs=0.41, p=0.02), MDR1 and BCL-2 (rs=0.38, p=0.03). Expression of GSTP1 and LRP correlated with high white blood count (p=0.03 and p=0.03) and BCL-2 with high peripheral blast count (p=0.009). MDR1 expression was significantly associated with the expression of immature stem cell marker CD34 (p=0.002). In ALL, significant association was found between LRP gene and female sex (p<0.0001), LRP and B-ALL patients (p=0.04) and LRP and BCR/ABL positive patients (p=0.004). High expression of MDR1 and BCL-2 in AML and MRP1 gene in ALL was associated with response to induction chemotherapy (p=0.001, p=0.02 and p=0.007 respectively). These results showed the potential clinical relevance of MDR1, MRP1 and BCL-2 in adult patients with acute leukemia in the context of induction chemotherapy.

Characterization of Wnt/β‐catenin signalling in osteoclasts in multiple myeloma

We recently showed that increasing Wnt/beta-catenin signalling in the bone marrow microenvironment or in multiple myeloma (MM) cells clearly suppresses osteoclastogenesis in SCID-hu mice; however, this regulation of osteoclastogenesis could result directly from activation of Wnt/beta-catenin signalling in osteoclasts or indirectly from effects on osteoblasts. The present studies characterized Wnt/beta-catenin signalling and its potential role in osteoclasts. Systematic analysis of expression of WNT, FZD, LRP and TCF gene families demonstrated that numerous Wnt-signalling components were expressed in human osteoclasts from patients with MM. Functional Wnt/beta-catenin signalling was identified by accumulation of total and active beta-catenin and increases in Dvl-3 protein in response to Wnt3a or LiCl. Furthermore, Wnt-induced increases in beta-catenin and Dvl-3 were attenuated by Wnt antagonists Dkk1 and sFRP1. Finally, Wnt3a-induced TCF/LEF transcriptional activity suggests that canonical Wnt signalling is active in osteoclasts. Supernatants from dominant-negative-beta-catenin-expressing osteoblast clones significantly stimulated tartrate-resistant acid phosphatase-positive osteoclast formation from primary MM-derived osteoclasts, compared with supernatants from control cells. These results suggested that Wnt/beta-catenin signalling is active in osteoclasts in MM and is involved in osteoclastogenesis in bone marrow, where it acts as a negative regulator of osteoclast formation in an osteoblast-dependent manner in MM.

Autoregulated expression of the gene coding for the leucine-responsive protein, Lrp, a global regulator in Salmonella enterica serovar Typhimurium

In this study, the lrp gene encoding the leucine-responsive regulatory protein (Lrp) in Salmonella enterica serovar Typhimurium was found to be negatively autoregulated. Its transcription start site was determined by primer extension analysis, showing that the lrp promoter is located at a different site to that inferred previously from the S. Typhimurium genome sequence. Chromosomal DNA fragments that include the promoter region were bound by purified Lrp protein in vitro, producing up to four distinct protein-DNA complexes. DNase I footprinting identified regions that were protected by the protein in vitro as well as bases that became hypersensitive to DNase I treatment following Lrp binding. A clear pattern of periodic hypersensitivity was detected between positions -130 and +15 that was consistent with wrapping of the DNA around Lrp in a nucleoprotein complex that includes the putative promoter region. Lrp-DNA interaction in this region was fully consistent with the observed repression of lrp transcription by this protein. Leucine was found to modulate Lrp-mediated autorepression by remodelling the Lrp-DNA nucleoprotein complex.

CD34+ leukemic subpopulation predominantly displays lower spontaneous apoptosis and has higher expression levels of Bcl-2 and MDR1 genes than CD34− cells in childhood AML

In view of obscure clinical and biological significance of leukemic cells heterogeneity, we studied the efficacy of apoptosis, proliferation, and expression levels of the Bcl-2, MDR1, LRP, and BCRP genes in sorted CD34+ and CD34- subpopulations of childhood AML leukemic samples. In five out of nine cases, CD34+ cells were less sensitive to spontaneous apoptosis and had from 1.2- to 5.0-fold higher expression levels of Bcl-2 (eight of ten) and from 1.5- to 28.7-fold higher expression levels of MDR1 (eight of ten). The expression levels of the LRP gene were from 1.1- to 1.8-fold higher in CD34+ subpopulations (five of ten cases), and the expression levels of the BCRP gene were from 1.1- to 22.4-fold higher in CD34+ leukemic cells (six of ten). In all M4 cases, the expression levels of LRP were higher in the CD34- subpopulation. Significant differences in the patterns of genes expression between patients do not allow us to conclude that the CD34+ fractions have more resistant phenotype than the CD34- subpopulations. Nevertheless, distinctions between CD34+ and CD34- cells may lead to different chemosensitivities between leukemic subpopulations in vivo and may determine the alteration of the leukemic immunophenotype during treatment and in relapse.

Transcription Regulation by Feast/Famine Regulatory Proteins, FFRPs, in Archaea and Eubacteria

Feast/famine regulatory proteins (FFRPs) comprise a single group of transcription factors systematically distributed throughout archaea and eubacteria. In the eubacterial domain in Escherichia coli, autotrophic pathways are activated and heterotrophic pathways are repressed by an FFRP, the leucine-responsive regulatory protein (Lrp), in some cases in interaction with other transcription factors. By sensing the concentration of leucine, Lrp changes its association state between hexadecamers and octamers to adapt the autotrophic or heterotrophic mode. The lrp gene is regulated so that the concentration of Lrp decreases in the presence of rich nutrition. In the archaeal domain a large part of the metabolism of Pyrococcus OT3 is regulated by another FFRP, FL11. In the presence of rich nutrition, the metabolism is released from repression by FL11; transcription of fl11 is terminated by FL11 forming octamers in interaction with lysine. When the nutrient is depleted, the metabolism is arrested by a high concentration of FL11; FL11 disassembles to dimers in the absence of lysine, and repression of transcription of fl11 is relaxed. Common characteristics of the master regulations by FL11 and Lrp hint at the prototype regulation once achieved in the common ancestor of all extant organisms. Mechanisms of discrimination by FFRPs between DNA sequences and also between co-regulatory molecules, mostly amino acids, and variations of transcription regulations observed with archaea and eubacteria are reviewed.

Detection of drug-resistance genes using single bronchoscopy biopsy specimens

Expression of three major resistance genes MDR1, MRP1 and LRP was investigated in small cell lung cancer, non-small cell lung cancer and metastasis. Single biopsies of bronchoscopy from 73 patients were performed to investigate expression of these three resistance genes by reverse transcriptase-polymerase chain reaction. Relations between gene expression and patient age, smoking status, histology, and chemotherapy were evaluated. A more frequent expression of MDR1 (77 versus 66%), MRP1 (91 versus 72%) and LRP (77 versus 63%) genes was detected in the malignant biopsies than in the non-malignant, respectively. In the metastasis biopsies, expression of these genes was markedly increased. No significant difference was observed between specimens before and after chemotherapy. Biopsies from progressing cancer showed higher MDR1, MRP1 and LRP gene expression. In conclusion, these data reveal a major role of MRP1 in intrinsic resistance and the high gene expression of MDR1 and MRP1 in relapsed diseases.

Immunolocalization and Cell Expression of Lung Resistance-related Protein (LRP) in Normal and Tumoral Human Respiratory Cells

Lung resistance-related protein (LRP) is an integral part of the multidrug resistance (MDR) phenotype involved in cell resistance toward xenobiotics or chemotherapy. The aim of this study was to compare the intracellular localization and cell expression of LRP in normal bronchial cells and their tumoral counterparts from non-small cell lung cancer (NSCLC). LRP expression was also investigated concurrently with DNA ploidy and chromosome 16 (lrp gene locus) aberrations. Confocal microscopy showed that LRP localization was exclusively intracytoplasmic regardless of the cell type and was never observed in the nuclear pore complex. Flow cytometry demonstrated a similar level of LRP expression in normal bronchial cells and in cancer cells from NSCLC samples. FISH analysis, performed to evaluate the number of chromosome 16 and lrp loci, demonstrated a significant gain of chromosome 16 in DNA aneuploid tumors. Furthermore, we did not find any link between LRP expression and DNA ploidy status or chromosome 16 number. These results suggest that LRP expression observed in NSCLC, maintained through the carcinogenesis process of respiratory cells, is not altered by the increased number of copies of chromosome 16 and probably controlled by mechanisms different from those of MRP1 expression, whereas both proteins are associated with the MDR phenotype.

Avian apolipoprotein A-V binds to LDL receptor gene family members

Apolipoprotein A-V (apoA-V) affects plasma triglyceride (TG) levels; however, the properties of apoA-V that mediate its action(s) are still incompletely understood. It is unclear how apoA-V, whose plasma concentration is extremely low, can affect the pronounced TG differences observed in individuals with various apoA-V dysfunctions. To gain novel insights into apoA-V biology, we expanded our previous studies in the chicken to this apolipoprotein. First, we characterized the first avian apoA-V, revealing its expression not only in liver and small intestine but also in brain, kidney, and ovarian follicles and showing its presence in the circulation. Second, we demonstrate directly that galline apoA-V binds to the major LDL receptor family member (LR) of the laying hen and that this interaction does not depend on the association of the apolipoprotein with lipid or lipoproteins. We propose that a direct interaction with LRs may represent a novel, additional mechanism for the modulation of TG levels by apoA-V.