Alkaline phosphatase as a reporter of sigma(S) levels and rpoS polymorphisms in different E-coli strains

sigma(S) is responsible for the transcriptional regulation of genes related to protection against stresses and bacterial survival and it accumulates in the cell under conditions of stress, such as nutrient limitation. An increase in the levels of sigma(S) causes a reduction in the expression of genes that are transcribed by RNA polymerase associated with the principal sigma factor, sigma(70). phoA, that encodes alkaline phosphatase (AP) is expressed under phosphate shortage conditions, and is also repressed by sigma(S). Here we show that in a Pi-limited chemostat, accumulation of rpoS mutations is proportional to the intrinsic level of sigma(S) in the cells. Acquisition of mutations in rpoS relieves repression of the PHO genes. We also devised a non-destructive method based on the rpoS effect on AP that differentiates between rpo(S+) and rpoS mutants, as well as between high and low-sigma(S) producers. Using this method, we provide evidence that sigma(S) contributes to the repression of AP under conditions of Pi excess and that AP variation among different strains is at least partly due to intrinsic variation in sigma(S) levels. Consequently, a simple and non-destructive AP assay can be employed to differentiate between strains expressing different levels of sigma(S) on agar plates.

Leptin Targets in the Mouse Brain

The central actions of leptin are essential for homeostatic control of adipose tissue mass, glucose metabolism, and many autonomic and neuroendocrine systems. In the brain, leptin acts on numerous different cell types via the long-form leptin receptor (LepRb) to elicit its effects. The precise identification of leptin`s cellular targets is fundamental to understanding the mechanism of its pleiotropic central actions. We have systematically characterized LepRb distribution in the mouse brain using in situ hybridization in wildtype mice as well as by EYFP immunoreactivity in a novel LepRb-IRES-Cre EYFP reporter mouse line showing high levels of LepRb mRNA/EYFP coexpression. We found substantial LepRb mRNA and EYFP expression in hypothalamic and extrahypothalamic sites described before, including the dorsomedial nucleus of the hypothalamus, ventral premammillary nucleus, ventral tegmental area, parabrachial nucleus, and the dorsal vagal complex. Expression in insular cortex, lateral septal nucleus, medial preoptic area, rostral linear nucleus, and in the Edinger-Westphal nucleus was also observed and had been previously unreported. The LepRb-IRES-Cre reporter line was used to chemically characterize a population of leptin receptor-expressing neurons in the midbrain. Tyrosine hydroxylase and Cre reporter were found to be coexpressed in the ventral tegmental area and in other midbrain dopaminergic neurons. Lastly, the LepRbI-RES-Cre reporter line was used to map the extent of peripheral leptin sensing by central nervous system (CNS) LepRb neurons. Thus, we provide data supporting the use of the LepRb-IRES-Cre line for the assessment of the anatomic and functional characteristics of neurons expressing leptin receptor. J. Comp. Neurol. 514:518-532, 2009. (C) 2009 Wiley-Liss, Inc.

Expression of alpha-synuclein is increased in the hippocampus of rats with high levels of innate anxiety

A genomic region neighboring the alpha-synuclein gene, on rat chromosome 4, has been associated with anxiety- and alcohol-related behaviors in different rat strains. In this study, we have investigated potential molecular and physiological links between alpha-synuclein and the behavioral differences observed between Lewis (LEW) and Spontaneously Hypertensive (SHR) inbred rats, a genetic model of anxiety. As expected, LEW rats appeared more fearful than SHR rats in three anxiety models: open field, elevated plus maze and light/dark box. Moreover, LEW rats displayed a higher preference for alcohol and consumed higher quantities of alcohol than SHR rats. alpha-Synuclein mRNA and protein concentrations were higher in the hippocampus, but not the hypothalamus of LEW rats. This result inversely correlated with differences in dopamine turnover in the hippocampus of LEW and SHR rats, supporting the hypothesis that alpha-synuclein is important in the downregulation of dopamine neurotransmission. A novel single nucleotide polymorphism was identified in the 30-untranslated region (3`-UTR) of the alpha-synuclein cDNA between these two rat strains. Plasmid constructs based on the LEW 3`-UTR sequence displayed increased expression of a reporter gene in transiently transfected PC12 cells, in accordance with in-vivo findings, suggesting that this nucleotide exchange might participate in the differential expression of alpha-synuclein between LEW and SHR rats. These results are consistent with a novel role for alpha-synuclein in modulating rat anxiety- like behaviors, possibly through dopaminergic mechanisms. Since the behavioral and genetic differences between these two strains are the product of independent evolutionary histories, the possibility that polymorphisms in the alpha-synuclein gene may be associated with vulnerability to anxiety- related disorders in humans requires further investigation. Molecular Psychiatry (2009) 14, 894-905; doi: 10.1038/mp.2008.43; published online 22 April 2008

Adipocyte differentiation is inhibited by melatonin through the regulation of C/EBP beta transcriptional activity

Considering that melatonin has been implicated in body weight control, this work investigated whether this effect involves the regulation of adipogenesis. 3T3-L1 preadipocytes were induced to differentiate in the absence or presence of melatonin (10(-3) m). Swiss-3T3 cells ectopically and conditionally (Tet-off system) over-expressing the 34 kDa C/EBP beta isoform (Swiss-LAP cells) were employed as a tool to assess the mechanisms of action at the molecular level. Protein markers of the adipogenic phenotype were analyzed by Western blot. At 36 hr of differentiation of 3T3-L1 preadipocytes, a reduction of PPAR gamma expression was detected followed by a further reduction, at day 4, of perilipin, aP2 and adiponectin protein expression in melatonin-treated cells. Real-time PCR analysis also showed a decrease of PPAR gamma (60%), C/EBP alpha (75%), adiponectin (30%) and aP2 (40%) mRNA expression. Finally, we transfected Swiss LAP cells with a C/EBP alpha gene promoter/reporter construct in which luciferase expression is enhanced in response to C/EBP beta activity. Culture of such transfected cells in the absence of tetracycline led to a 2.5-fold activation of the C/EBP alpha promoter. However, when treated with melatonin, the level of C/EBP alpha promoter activation by C/EBP beta was reduced by 50% (P = 0.05, n = 6). In addition, this inhibitory effect of melatonin was also reflected in the phenotype of the cells, since their capacity to accumulate lipids droplets was reduced as confirmed by the poor staining with Oil Red O. In conclusion, melatonin at a concentration of 10(-3) m works as a negative regulator of adipogenesis acting in part by inhibiting the activity of a critical adipogenic transcription factor, C/EBP beta.

A Metal-Repressed Promoter From Gram-Positive Bacillus subtilis Is Highly Active and Metal-Induced in Gram-Negative Cupriavidus metallidurans

A synthetic version of the metal-regulated gene A (mrgA) promoter from Bacillus subtilis, which in this Gram-positive bacterium is negatively regulated by manganese, iron, cobalt, or copper turned out to promote high level of basal gene expression that is further enhanced by Co(II), Cd(II), Mn(II), Zn(II), Cu(II), or Ni(II), when cloned in the Gram-negative bacterium Cupriavidus metallidurans. Promoter activity was monitored by expression of the reporter gene coding for the enhanced green fluorescent protein (EGFP), and cellular intensity fluorescence was quantified by flow cytometry. Expression levels in C. metallidurans driven by the heterologous promoter, here called pan, ranged from 20- to 53-fold the expression level driven by the Escherichia coli lac promoter (which is constitutively expressed in C. metallidurans), whether in the absence or presence of metal ions, respectively. The pan promoter did also function in E. coli in a constitutive pattern, regardless of the presence of Mn(II) or Fe(II). In conclusion, the pan promoter proved to be a powerful tool to express heterologous proteins in Gram-negative bacteria, especially in C. metallidurans grown upon high levels of toxic metals, with potential applications in bioremediation. Biotechnol. Bioeng. 2010; 107: 469-477. (C) 2010 Wiley Periodicals, Inc.

CztR, a LysR-Type Transcriptional Regulator Involved in Zinc Homeostasis and Oxidative Stress Defense in Caulobacter crescentus

Caulobacter crescentus is a free-living alphaproteobacterium that has 11 predicted LysR-type transcriptional regulators (LTTRs). Previously, a C. crescentus mutant strain with a mini-Tn5lacZ transposon inserted into a gene encoding an LTTR was isolated; this mutant was sensitive to cadmium. In this work, a mutant strain with a deletion was obtained, and the role of this LTTR (called CztR here) was evaluated. The transcriptional start site of this gene was determined by primer extension analysis, and its promoter was cloned in front of a lacZ reporter gene. beta-Galactosidase activity assays, performed with the wild-type and mutant strains, indicated that this gene is 2-fold induced when cells enter stationary phase and that it is negatively autoregulated. Moreover, this regulator is essential for the expression of the divergent cztA gene at stationary phase, in minimal medium, and in response to zinc depletion. This gene encodes a hypothetical protein containing 10 predicted transmembrane segments, and its expression pattern suggests that it encodes a putative zinc transporter. The cztR strain was also shown to be sensitive to superoxide (generated by paraquat) and to hydrogen peroxide but not to tert-butyl hydroperoxide. The expression of katG and ahpC, but not that of the superoxide dismutase genes, was increased in the cztR mutant. A model is proposed to explain how CztR binding to the divergent regulatory regions could activate cztA expression and repress its own transcription.

Cellular and molecular characterization of an embryonic cell line (BME26) from the tick Rhipicephalus (Boophilus) microplus

The cellular and molecular characteristics of a cell line (BME26) derived from embryos of the cattle tick Rhipicephalus (Boophilus) microplus were studied. The cells contained glycogen inclusions, numerous mitochondria, and vesicles with heterogeneous electron densities dispersed throughout the cytoplasm. Vesicles contained lipids and sequestered palladium meso-porphyrin (Pd-mP) and rhodamine-hemoglobin, suggesting their involvement in the autophagic and endocytic pathways. The cells phagocytosed yeast and expressed genes encoding the antimicrobial peptides (microplusin and defensin). A cDNA library was made and 898 unique mRNA sequences were obtained. Among them, 556 sequences were not significantly similar to any sequence found in public databases. Annotation using Gene Ontology revealed transcripts related to several different functional classes. We identified transcripts involved in immune response such as ferritin, serine proteases, protease inhibitors,. antimicrobial peptides, heat shock protein, glutathione S-transferase, peroxidase, and NADPH oxidase. BME26 cells transfected with a plasmid carrying a red fluorescent protein reporter gene (DsRed2) transiently expressed DsRed2 for up to 5 weeks. We conclude that BME26 can be used to experimentally analyze diverse biological processes that occur in R. (B.) microplus such as the innate immune response to tick-borne pathogens. (C) 2008 Elsevier Ltd. All rights reserved.

Sugarcane Phosphoribosyl Pyrophosphate Synthetase: Molecular Characterization of a Phosphate-independent PRS

Phosphoribosyl pyrophosphate synthetase (PRS-EC:2.7.6.1) is an important enzyme present in several metabolic pathways, thus forming a complex family of isoenzymes. However, plant PRS enzymes have not been extensively investigated. In this study, a sugarcane prs gene has been characterized from the Sugar Cane Expressed Sequence Tag Genome Project. This gene contains a 984-bp open reading frame encoding a 328-amino acid protein. The predicted amino acid sequence has 77% and 78% amino acid sequence identity to Arabidopsis thaliana and Spinacia oleracea PRS4, respectively. The assignment of sugarcane PRS as a phosphate-independent PRS isoenzyme (Class II PRS) is verified following enzyme assay and phylogenetic reconstruction of PRS homologues. To gain further insight into the structural framework of the phosphate independence of sugarcane PRS, a molecular model is described. This model reveals the formation of two conserved domains elucidating the structural features involved in sugarcane PRS phosphate independence. The recombinant PRS retains secondary structure elements and a quaternary arrangement consistent with known PRS homologues, based on circular dichroism measurements.

Bioluminescent sensor for naphthalene in air: Cell immobilization and evaluation with a dynamic standard atmosphere generator

A dynamic atmosphere generator with a naphthalene emission source has been constructed and used for the development and evaluation of a bioluminescence sensor based on the bacteria Pseudomonas fluorescens HK44 immobilized in 2% agar gel (101 cell mL(-1)) placed in sampling tubes. A steady naphthalene emission rate (around 7.3 nmol min(-1) at 27 degrees C and 7.4 mLmin(-1) of purified air) was obtained by covering the diffusion unit containing solid naphthalene with a PTFE filter membrane. The time elapsed from gelation of the agar matrix to analyte exposure (""maturation time"") was found relevant for the bioluminescence assays, being most favorable between 1.5 and 3 h. The maximum light emission, observed after 80 min, is dependent on the analyte concentration and the exposure time (evaluated between 5 and 20 min), but not on the flow rate of naphthalene in the sampling tube, over the range of 1.8-7.4 nmol min(-1). A good linear response was obtained between 50 and 260 nmol L-1 with a limit of detection estimated in 20 nmol L-1 far below the recommended threshold limit value for naphthalene in air. (c) 2008 Elsevier B.V. All rights reserved.