Real-time PCR investigation on the expression of sboA and ituD genes in Bacillus spp

Aims: To investigate the expression of sboA and ituD genes among strains of Bacillus spp. at different pH and temperature. Methods and Results: Different Bacillus strains from the Amazon basin and Bacillus subtilis ATCC 19659 were investigated for the production of subtilosin A and iturin A by qRT-PCR, analysing sboA and ituD gene expression under different culture conditions. Amazonian strains presented a general gene expression level lower than B. subtilis ATCC 19659 for sboA. In contrast, when analysing the expression of ituD gene, the strains from the Amazon, particularly P40 and P45B, exhibited higher levels of expression. Changes in pH (6 and 8) and temperature (37 and 42 degrees C) caused a decrease in sboA expression, but increased ituD expression among strains from Amazonian environment. Conclusions: Temperature and pH have an important influence on the expression of genes sboA (subtilosin A) and ituD (iturin A) among Bacillus spp. The strains P40 and P45B can be useful for the production of antimicrobial peptide iturin A. Significance and Impact of the Study: Monitoring the expression of essential biosynthetic genes by qRT-PCR is a valuable tool for optimization of the production of antimicrobial peptides.

EIN3-like gene expression during fruit ripening of Cavendish banana (Musa acuminata cv. Grande naine)

Ethylene signal transduction initiates with ethylene binding at receptor proteins and terminates in a transcription cascade involving the EIN3/EIL transcription factors. Here, we have isolated four cDNAs homologs of the Arabidopsis EIN3/EIN3-like gene, MA-EILs (Musa acuminata ethylene insensitive 3-like) from banana fruit. Sequence comparison with other banana EIL gene already registered in the database led us to conclude that, at this day, at least five different genes namely MA-EIL1, MA-EIL2/AB266318, MA-EIL3/AB266319, MA-EIL4/AB266320 and AB266321 exist in banana. Phylogenetic analyses included all banana EIL genes within a same cluster consisting of rice OsEILs, a monocotyledonous plant as banana. However, MA-EIL1, MA-EIL2/AB266318, MA-EIL4/AB266320 and AB266321 on one side, and MA-EIL3/AB266319 on the other side, belong to two distant subclusters. MA-EIL mRNAs were detected in all examined banana tissues but at lower level in peel than in pulp. According to tissues, MA-EIL genes were differentially regulated by ripening and ethylene in mature green fruit and wounding in old and young leaves. MA-EIL2/AB266318 was the unique ripening- and ethylene-induced gene; MA-EIL1, MA-EIL4/Ab266320 and AB266321 genes were downregulated, while MA-EIL3/AB266319 presented an unusual pattern of expression. Interestingly, a marked change was observed mainly in MA-EIL1 and MA-EIL3/Ab266319 mRNA accumulation concomitantly with changes in ethylene responsiveness of fruit. Upon wounding, the main effect was observed in MA-EIL4/AB266320 and AB266321 mRNA levels, which presented a markedly increase in both young and old leaves, respectively. Data presented in this study suggest the importance of a transcriptionally step control in the regulation of EIL genes during banana fruit ripening.

Identification of residues involved in binding of IL5 to beta(com) using beta(IL3) and beta(com) chimeras

In mice there are two forms of the beta chain used in the IL3 receptor system, beta(com) and beta(IL3). beta(com) is used by the IL3, ILS and GM-CSF receptors whereas Pns is only used in the IL3 receptor. In this work an assay was developed to identify residues of beta(IL3) that restrict IL5 activity. It was found that such residues reside within the 2nd CRM of the molecule. Furthermore, when residues in the beta(IL3) B'-C' loop were replaced with beta(com) sequence a form of beta(IL3) was produced that was able to respond to IL5. This region is also responsible for IL3 binding to beta(IL3) in the absence of alpha chain. It is therefore an important structural motif of beta(com) and beta(IL3) responsible for both ligand interaction and specificity. (C) 1999 Federation of European Biochemical Societies.

Myeloproliferative disorder and leukaemia in mice induced by different classes of constitutive mutants of the human IL-3/IL-5/GM-CSF receptor common beta subunit

Several constitutively active mutant forms of the common β subunit of the human IL-3, IL-5 and GM-CSF receptors (hβc), which enable it to signal in the absence of ligand, have recently been described. Two of these, V449E and I374N, are amino acid substitutions in the transmembrane and extracellular regions of hβc, respectively. A third, FIΔ, contains a 37 amino acid duplication in the extracellular domain. We have shown previously that when expressed in primary murine haemopoietic cells, the extracellular mutants confer factor-independence on cells of the neutrophil and monocyte lineages only, whereas V449E does so on all cell types of the myeloid and erythroid compartments. To study the in vivo effects and leukaemic potential of these mutants, we have expressed all three in mice by bone marrow reconstitution using retrovirally infected donor cells. Expression of the extracellular mutants leads to an early onset, chronic myeloproliferative disorder marked by elevations in the neutrophil, monocyte, erythrocyte and platelet lineages. In contrast, expression of V449E leads to an acute leukaemia-like syndrome of anaemia, thrombocytopaenia and blast cell expansion. These data support the possibility that activating mutations in hβc are involved in haemopoietic disorders in man.

Expression of the estrogen receptor-associated immunophilins, cyclophilin 40 and FKBP52, in breast cancer

The estrogen receptor alpha (ER alpha) is implicated in the development of breast cancer. The immunophilins, cyclophilin 40 (CyP40) and FKBP52, are associated with ER alpha and other steroid receptors in mutually exclusive heterocomplexes and may differentially modulate receptor activity. Since previous studies have not assessed the levels of these immunophilins in breast cancer, we examined 10 breast cancer cell lines for mRNA and protein expression of CyP40 and FKBP52 and for amplification of the CyP40 gene. In addition, 26 breast carcinomas, including seven with matched normal breast tissue, were examined for mRNA expression of both immunophilins. CyP40 and FKBP52 were ubiquitously expressed in breast cancer cell lines, but there were significant differences in their pattern of expression. FKBP52 protein levels were generally an order of magnitude greater than those for CyP40. FKBP52 mRNA expression correlated strongly with protein expression and was significantly higher in ER alpha-positive compared with ER alpha-negative cell lines. However, CyP40 mRNA expression did not correlate with protein expression, nor did expression of this immunophilin correlate with ER alpha status. Relatively high expression of CyP40 in one cell line (BT-20) could be attributed to amplification of the CyP40 gene. Both immunophilins were also ubiquitously expressed in breast carcinomas, and we demonstrate for the first time that both CyP40 and FKBP52 mRNA are overexpressed in breast tumors compared to matched normal breast controls. The overexpression of CyP40 and FKBP52, coupled with relative differences in their expression in tumors, may have important functional implications for ER alpha and other steroid receptors in breast cancer.

Mutation screening of the c-MYB negative regulatory domain in acute and chronic myeloid leukaemia

Over-expression of the c-myb gene and expression of activated forms of myb are known to transform haemopoietic cells, particularly cells of the myeloid lineage. Truncations or mutations that disrupt the negative regulatory domain (NRD) of the Myb protein confer an increased ability to transform cells. Although it has proved difficult to link mutations in c-MYB to human leukaemia, no studies investigating the presence of mutations within the c-MYB NRD have been reported. Therefore, we have performed mutational analysis of this region, using polymerase chain reaction-single-stranded conformation polymorphism and sequence analysis, in 26 patients with acute or chronic myeloid leukaemia, No mutations were detected, indicating that mutation of this region of the Myb protein is not common in the pathogenesis or progression of these diseases.

Directed evolution of mammalian cytochrome P450 enzymes involved in xenobiotic metabolism

Directed evolution of cytochrome P450 enzymes represents an attractive means of generating novel catalysts for specialized applications. Xenobiotic-metabolizing P450s are particularly well suited to this approach due to their inherent wide substrate specificity. In the present study, a novel method for DNA shuffling was developed using an initial restriction enzyme digestion step, followed by elimination of long parental sequences by size-selective filtration. P450 2C forms were subjected to a single round of shuffling then coexpressed with reductase in E. coli. A sample (54 clones) of the resultant library was assessed for sequence diversity, hemo- and apoprotein expression, and activity towards the substrate indole. All mutants showed a different RFLP pattern compared to all parents, suggesting that the library was free from contamination by parental forms. Haemoprotein expression was detectable in 45/54 (83%) of the mutants sampled. Indigo production was less than or comparable to the activities of one or more of the parental P450s, but three mutants showed indirubin production in excess of that seen with any parental form, representing a gain of function. In conclusion, a method is presented for the effective shuffling of P450 sequences to generate diverse libraries of mutant P450s containing a high proportion of correctly folded hemoprotein, and minimal contamination with parental forms.

Quantification of prolactin-releasing peptide (PrRP) mRNA expression in specific brain regions of the rat during the oestrous cycle and in lactation

Real-time Taqman(TM) RT-PCR was used to make quantitative comparisons of the levels of PrRP mRNA expression in micropunch brain samples from rats at different stages of the oestrous cycle and in lactation. The nucleus of the solitary tract and ventrolateral reticular nuclei of the medulla oblongata contained significantly (P < 0.05) greater levels of PrRP mRNA than any hypothalamic region. Within the hypothalamus, the highest level of PrRP expression was localised to the dorsomedial aspect of the ventromedial hypothalamus. All other hypothalamic regions exhibited significantly (P < 0.05) lower levels of expression, including the rostral and caudal dorsomedial hypothalamus. Very low levels of PrRP expression were observed in the arcuate nucleus, paraventricular nucleus, medial preoptic nucleus and ventrolateral aspect of the ventromedial hypothalamus. No significant changes in PrRP expression were noted in any sampled region between proestrus, oestrus or dioestrus. Similarly, PrRP expression in hypothalamic regions did not differ between lactating and non-lactating (dioestrous) animals. During validation of RT-PCR techniques we cloned and sequenced a novel splice variant of PrRP from the hypothalamus. This variant arises from alternative splicing of the donor site within exon 2, resulting in an insert of 64 base pairs and shift in the-codon:reading frame with the introduction of an early stop codon. In the hypothalamus and brainstem, mRNA expression of the variant was restricted to regions that expressed PrRP. These results suggest that PrRP expression in the hypothalamus may be more Widespread than previously reported. However, the relatively low level of PrRP in the hypothalamus and the lack of significant changes in expression during the oestrous cycle and lactation provides further evidence that PrRP is unlikely to be involved in the regulation of prolactin, secretion. (C) 2003 Elsevier Science B.V. All rights reserved.

Regulation of the gene encoding glutathione S-transferase M1 (GSTM1) by the Myb oncoprotein

The identification of Myb 'target' genes will not only aid in the understanding of how overexpression of Myb, or expression of activated forms of Myb, leads to cellular transformation but will also shed light on its role in normal cells. Using a combination of an estrogen-regulated Myb-transformed cell line (ERMYB) and PCR-based subtractive hybridization, we have identified the gene (GSTM1) encoding the detoxification enzyme glutathione S-transferase M1 as being transcriptionally upregulated by Myb. Functional analysis of the GSTM1 promoter using reporter assays indicated that both the DNA binding and transactivation domains of Myb were required for transcriptional activation. Mutational analysis of consensus Myb-binding sites (MBS) in the promoter and electrophoretic mobility gel shift analysis indicated that one of the three potential MBS can bind Myb protein, and is the primary site involved in the regulation of this promoter by Myb.

Effects of reversible inactivation of the medial septum on rat exploratory behavior in the elevated plus-maze using a test-retest paradigm

The effect of intraseptal injections of lidocaine before a first or a second session in the elevated plus-maze, in a test-retest paradigm, was investigated. In addition to gross session analyses, a minute-by-minute analysis of the sessions was used to evaluate both anxiety and memory. Lidocaine injections before the test session produced increases in the frequency of entries, time spent and distance run in the open arms without affecting activity occurring in the closed arms. During the retest session, saline- and lidocaine-treated rats exhibited increased indices of anxiety and lidocaine-treated rats exhibited decreased closed-arm entries. The minute-by-minute analysis showed a faster decrease in anxiety-related behaviors during the test session by saline- than by lidocaine-treated rats and a significant decrease in closed-arm exploration by saline-treated rats, but not by lidocaine-treated ones. Lidocaine injection before the retest session produced increases in the frequency of entries, time spent and distance run in the open arms in the second session when compared with saline-treated rats. Minute-by-minute analysis showed an increase in the time spent in the open arms by lidocaine animals at the beginning of the retest session in comparison to saline animals and a significant decrease in closed-arm exploration by both groups. These results suggest that inactivation of the medial septum by lidocaine affects the expression of unconditioned and conditioned forms of anxiety in the elevated plus-maze and, in a lesser way, the acquisition and retention of spatial information. (C) 2010 Elsevier B.V. All rights reserved.

Midazolam reduces the selective activation of the rhinal cortex by contextual fear stimuli

Independent brain circuits appear to underlie different forms of conditioned fear, depending on the type of conditioning used, such as a context or explicit cue paired with footshocks. Several clinical reports have associated damage to the medial temporal lobe (MTL) with retrograde amnesia. Although a number of studies have elucidated the neural circuits underlying conditioned fear, the involvement of MTL components in the aversive conditioning paradigm is still unclear. To address this issue, we assessed freezing responses and Fos protein expression in subregions of the rhinal cortex and ventral hippocampus of rats following exposure to a context, light or tone previously paired with footshock (Experiment 1). A comparable degree of freezing was observed in the three types of conditioned fear, but with distinct patterns of Fos distribution. The groups exposed to cued fear conditioning did not show changes in Fos expression, whereas the group subjected to contextual fear conditioning showed selective activation of the ectorhinal (Ect), perirhinal (Per), and entorhinal (Ent) cortices, with no changes in the ventral hippocampus. We then examined the effects of the benzodiazepine midazolam injected bilaterally into these three rhinal subregions in the expression of contextual fear conditioning (Experiment 2). Midazolam administration into the Ect, Per, and Ent reduced freezing responses. These findings suggest that contextual and explicit stimuli endowed with aversive properties through conditioning recruit distinct brain areas, and the rhinal cortex appears to be critical for storing context-, but not explicit cue-footshock, associations. (C) 2010 Elsevier B.V. All rights reserved.

Elevated expression of MeCP2 in cardiac and skeletal tissues is detrimental for normal development

MeCP2 plays a critical role in interpreting epigenetic signatures that command chromatin conformation and regulation of gene transcription. In spite of MeCP2`s ubiquitous expression, its functions have always been considered in the context of brain physiology. In this study, we demonstrate that alterations of the normal pattern of expression of MeCP2 in cardiac and skeletal tissues are detrimental for normal development. Overexpression of MeCP2 in the mouse heart leads to embryonic lethality with cardiac septum hypertrophy and dysregulated expression of MeCP2 in skeletal tissue produces severe malformations. We further show that MeCP2`s expression in the heart is developmentally regulated; further suggesting that it plays a key role in regulating transcriptional programs in non-neural tissues.

DNA binding-independent transcriptional activation of the vascular endothelial growth factor gene (VEGF) by the Myb oncoprotein

Myb is a key transcription factor that can regulate proliferation, differentiation, and apoptosis, predominantly in the haemopoietic system. Abnormal expression of Myb is associated with a number of cancers, both haemopoietic and non-haemopoietic. In order to better understand the role of Myb in normal and tumorigenic processes, we undertook a cDNA array screen to identify genes that are regulated by this factor. In this way, we identified the gene encoding vascular endothelial growth factor (VEGF) as being potentially regulated by the Myb oncoprotein in myeloid cells. To determine whether this was a direct effect on VEGF gene transcription, we examined the activity of the murine VEGF promoter in the presence of either wild-type (WT) or mutant forms of Myb. It was found that WT Myb was able to activate the VEGF promoter and that a minimal promoter region of 120 bp was sufficient to confer Myb responsiveness. Surprisingly, activation of the VEGF promoter was independent of DNA binding by Myb. This was shown by the use of DNA binding-defective Myb mutants and by mutagenesis of a potential Myb-binding site in the minimal promoter. Mutation of Sp1 sites within this region abolished Myb-mediated regulation of a reporter construct, suggesting that Myb DNA binding-independent activation of VEGF expression occurs via these Sp1 binding elements. Regulation of VEGF production by Myb has implications for the potential role of Myb in myeloid leukaemias and in solid tumours where VEGF may be functioning as an autocrine growth factor. (c) 2006 Elsevier Inc. All rights reserved.

In situ hybridization detection of homeobox genes reveals distinct expression patterns in oral squamous cell carcinomas

Aims: To analyse the expression of three homeobox genes (HOXA7, PITX1 and PRRX1) in oral squanous cell carcinomas (OSCC) and the relationship of such expression to certain distinct histopathological features of OSCC and in comparison to adjacent non-neoplastic epithelium (NT). Methods and results: Digoxigenin-labelled riboprobes that are specific for each homeobox gene were generated and in situ hybridization was carried out on frozen sections. In NT samples, HOXA7 and PITX1 transcripts were found more frequently in all epithelial layers, while PRRX1 was expressed in the basal layer. With OSCC samples, expression of the three genes was associated with all histological features. However, the HOXA7 and PITX1 signals were more intense in sheets and nests and PRRX1 in small nests and isolated cells. Conclusion: HOXA7, PIXT1 and PRRX1 homeobox genes have different patterns of expression in OSCC depending on its histological features.

Immunohistochemical detection of polyductin and co-localization with liver progenitor cell markers during normal and abnormal development of the intrahepatic biliary system and in adult hepatobiliary carcinomas

The longest open reading frame of PKHD1 (polycystic kidney and hepatic disease 1), the autosomal recessive polycystic kidney disease (ARPKD) gene, encodes a single-pass, integral membrane protein named polyductin or fibrocystin. A fusion protein comprising its intracellular C-terminus, FP2, was previously used to raise a polyclonal antiserum shown to detect polyductin in several human tissues, including liver. In the current study, we aimed to investigate by immunohistochemistry the detailed polyductin localization pattern in normal (ductal plate [DP], remodelling ductal plate [RDP], remodelled bile ducts) and abnormal development of the primitive intrahepatic biliary system, known as ductal plate malformation (DPM). This work also included the characterization of polyductin expression profile in various histological forms of neonatal and infantile cholestasis, and in cholangiocellular carcinoma (CCC) and hepatocellular carcinoma (HCC). We detected polyductin expression in the intrahepatic biliary system during the DP and the RDP stages as well as in DPM. No specific staining was found at the stage of remodelled bile ducts. Polyductin was also detected in liver biopsies with neonatal cholestasis, including mainly biliary atresia and neonatal hepatitis with ductular reaction as well as congenital hepatic fibrosis. In addition, polyductin was present in CCC, whereas it was absent in HCC. Polyductin was also co-localized in some DP cells together with oval stem cell markers. These results represent the first systematic study of polyductin expression in human pathologies associated with abnormal development of intrahepatic biliary tree, and support the following conclusions: (i) polyductin expression mirrors developmental properties of the primitive intrahepatic biliary system; (ii) polyductin is re-expressed in pathological conditions associated with DPM and (iii) polyductin might be a potential marker to distinguish CCC from HCC.