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PubMed2002

The genome of bacteriophage phiKZ of Pseudomonas aeruginosa.

Bacteriophage phiKZ is a giant virus that efficiently infects Pseudomonas aeruginosa strains pathogenic to human and, therefore, it is attractive for phage therapy. We present here the complete phiKZ genome sequence and a preliminary analysis of its genome structure. The 280,334 bp genome is a linear, circularly permutated and terminally redundant, A+T-rich double-stranded DNA molecule. The phiKZ DNA has no detectable sequence homology to other viruses and microorganisms, and it does not contain NotI, PstI, SacI, SmaI, XhoI, and XmaIII endonuclease restriction sites. The genome has 306 open reading frames (ORFs) varying in size from 50 to 2237 amino acid residues. According to the orientation of transcription, ORFs are apparently organized into clusters and most have a clockwise direction. The phiKZ genome also encodes six tRNAs specific for Met (AUG), Asn (AAC), Asp (GAC), Leu (TTA), Thr (ACA), and Pro (CCA). A putative promoter sequence containing a TATATTAC block was identified. Most potential stem-loop transcription terminators contain the tetranucleotide UUCG loops. Some genes may be assigned as phage-encoded RNA polymerase subunits. Only 59 phiKZ gene products exhibit similarity to proteins of known function from a diversity of organisms. Most of these conserved gene products, such as dihydrofolate reductase, ribonucleoside diphosphate reductase, thymidylate synthase, thymidylate kinase, and deoxycytidine triphosphate deaminase are involved in nucleotide metabolism. However, no virus-encoded DNA polymerase, DNA replication-associated proteins, or single-stranded DNA-binding protein were found based on amino acid homology, and they may therefore be strongly divergent from known homologous proteins. Fifteen phiKZ gene products show homology to proteins of pathogenic organisms, including Mycobacterium tuberculosis, Haemophilus influenzae, Listeria sp., Rickettsia prowazakeri, and Vibrio cholerae that must be considered before using this phage as a therapeutic agent. The phiKZ coat contains at least 40 polypeptides, and several proteins are cleaved during virus assembly in a way similar to phage T4. Eleven phiKZ-encoded polypeptides are related to proteins of other bacteriphages that infect a variety of hosts. Among these are four gene products that contain a putative intron-encoded endonuclease harboring the H-N-H motif common to many double-stranded DNA phages. These observations provide evidence that phages infecting diverse hosts have had access to a common genetic pool. However, limited homology on the DNA and protein levels indicates that bacteriophage phiKZ represents an evolutionary distinctive branch of the Myoviridae family.

Amino Acid Sequence↗

PubMed2004

p68 DEAD box RNA helicase expression in keratinocytes. Regulation, nucleolar localization, and functional connection to proliferation and vascular endothelial growth factor gene expression.

Nitric oxide (NO) represents a short lived mediator that pivotally drives keratinocyte movements during cutaneous wound healing. In this study, we have identified p68 DEAD box RNA helicase (p68) from an NO-induced differential keratinocyte cDNA library. Subsequently, we have analyzed regulation of p68 by wound-associated mediators in human and murine keratinocytes. NO, serum, growth factors, and pro-inflammatory cytokines were potent inducers of p68 expression in the cells. p68 was constitutively expressed in the epithelial compartment of murine skin. Upon injury, we found a transient down-regulation of overall p68 protein in wound tissue. However, p68 did not completely disappear during early wound repair, as we found an expression of p68 protein in isolated wound margin tissue 24 h after wounding. Moreover, immunohistochemistry and cell fractionation analysis revealed a restricted localization of p68 in keratinocyte nuclei of the developing epithelium. Accordingly, cultured keratinocytes also showed a nuclear localization of the helicase. Moreover, confocal microscopy revealed a strong localization of p68 protein within the nucleoli of the cells. Functional analyses demonstrated that p68 strongly participated in keratinocyte proliferation and gene expression. Keratinocytes that constitutively overexpressed p68 protein were characterized by a marked increase in serum-induced proliferation and vascular endothelial growth factor expression, whereas down-regulation of endogenous p68 using small interfering RNA markedly attenuated serum-induced proliferation and vascular endothelial growth factor expression. Altogether, our results suggest a tightly controlled expression and nucleolar localization of p68 in keratinocytes in vitro and during skin repair in vivo that functionally contributes to keratinocyte proliferation and gene expression.

Animals↗

PubMed2007

Altered expression of SOCS3 in the hypothalamic arcuate nucleus during seasonal body mass changes in the field vole, Microtus agrestis.

We have previously shown that cold-acclimated (8 degrees C) male field voles (Microtus agrestis) transferred from short day (SD, 8 h light) to long day (LD, 16 h light) photoperiod exhibit an increase in body mass lasting 4 weeks, after which they stabilise at a new plateau approximately 7.5 g (24.8%) higher than animals maintained in SD. By infusing voles with exogenous leptin, we have also demonstrated that SD voles respond to the hormone by reducing body mass and food intake, whereas LD animals increasing body mass are resistant to leptin treatment. In the present study, we investigated whether seasonal changes in body mass could be linked to modulation of the leptin signal by suppressor of cytokine signalling-3 (SOCS3). We used in situ hybridisation to examine hypothalamic arcuate nucleus (ARC) expression of SOCS3, neuropeptide Y (NPY), agouti-related peptide (AgRP), pro-opiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) genes in 90 voles exposed to either SD or LD for up to 11 weeks. LD voles increasing body mass had significantly higher levels of SOCS3 mRNA than SD or LD voles with a stable body mass. There were no associated changes in expression of NPY, AgRP, POMC and CART genes. These results suggest that voles that regulate body mass at either the lower (SD) or upper (LD) plateau remain sensitive to leptin action, whereas SOCS3-mediated leptin resistance is a short-term mechanism that enables animals to move between the stable body mass plateaus. Our data provide evidence that expression of SOCS3 in the ARC is involved in the modulation of the strength of the leptin signal to facilitate seasonal cycles in body mass and adiposity.

Acclimatization↗

PubMed2001

Duplication and concerted evolution of the mitochondrial control region in the parrot genus Amazona.

We report a duplication and rearrangement of the mitochondrial genome involving the control region of parrots in the genus Amazona. This rearrangement results in a gene order of cytochrome b/tRNA(Thr)/pND6/pGlu/CR1/tRNA(Pro)/NADH dehydrogenase 6/tRNA(Glu)/CR2/tRNA(Phe)/12s rRNA, where CR1 and CR2 refer to duplicate control regions, and pND6 and pGlu indicate presumed pseudogenes. In contrast to previous reports of duplications involving the control regions of birds, neither copy of the parrot control region shows any indications of degeneration. Rather, both copies contain many of the conserved sequence features typically found in avian control regions, including the goose hairpin, TASs, the F, C, and D boxes, conserved sequence box 1 (CSB1), and an apparent homolog to the mammalian CSB3. We conducted a phylogenetic analysis of homologous portions of the duplicate control regions from 21 individuals representing four species of Amazona (A. ochrocephala, A. autumnalis, A. farinosa, and A. amazonica) and Pionus chalcopterus. This analysis revealed that an individual's two control region copies (i.e., the paralogous copies) were typically more closely related to one another than to corresponding segments of other individuals (i.e., the orthologous copies). The average sequence divergence of the paralogous control region copies within an individual was 1.4%, versus a mean value of 4.1% between control region orthologs representing nearest phylogenetic neighbors. No differences were found between the paralogous copies in either the rate or the pattern in which the two copies accumulated base pair changes. This pattern suggests concerted evolution of the two control regions, perhaps through occasional gene conversion events. We estimated that gene conversion events occurred on average every 34,670 +/- 18,400 years based on pairwise distances between the paralogous control region sequences of each individual. Our results add to the growing body of work indicating that under some circumstances duplicated mitochondrial control regions are retained through evolutionary time rather than degenerating and being lost, presumably due to selection for a small mitochondrial genome.

Animals↗

PubMed1995

1-Methylguanosine deficiency of tRNA influences cognate codon interaction and metabolism in Salmonella typhimurium.

1-Methylguanosine (m1G) is present next to the 3' end of the anticodon (position 37) in tRNA(1,2,3,Leu), tRNA(1,2,3,Pro), and tRNA(3Arg). A mutant of Salmonella typhimurium lacks m1G in these seven tRNAs when grown at or above 37 degrees C, as a result of a mutation (trmD3) in the structural gene (trmD) for the tRNA(m1G37)methyltransferase. The m1G deficiency induced 24 and 26% reductions in the growth rate and polypeptide chain elongation rate, respectively, in morpholinepropanesulfonic acid (MOPS)-glucose minimal medium at 37 degrees C. The expression of the leuABCD operon is controlled by the rate with which tRNA(2Leu) and tRNA(3Leu) read four leucine codons in the leu-leader mRNA. Lack of m1G in these tRNAs did not influence the expression of this operon, suggesting that m1G did not influence the efficiency of tRNA(2,3Leu). Since the average step time of the m1G-deficient tRNAs was increased 3.3-fold, the results suggest that the impact of m1G in decoding cognate codons may be tRNA dependent. The trmD3 mutation rendered the cell more resistant or sensitive to several amino acid analogs. 3-Nitro-L-tyrosine (NT), to which the trmD3 mutant is sensitive, was shown to be transported by the tryptophan-specific permease, and mutations in this gene (mtr) render the cell resistant to NT. Since the trmD3 mutation did not affect the activity of the permease, some internal metabolic step(s), but not the uptake of the analog per se, is affected. We suggest that the trmD3-mediated NT sensitivity is by an abnormal translation of some mRNA(s) whose product(s) is involved in the metabolic reactions affected by the analog. Our results also suggest that tRNA modification may be a regulatory device for gene expression.

3-Isopropylmalate Dehydrogenase↗

PubMed2006

Human in vivo dose-response to controlled, low-dose low linear energy transfer ionizing radiation exposure.

PURPOSE: The effect of low doses of low-linear energy transfer (photon) ionizing radiation (LDIR, <10 cGy) on human tissue when exposure is under normal physiologic conditions is of significant interest to the medical and scientific community in therapeutic and other contexts. Although, to date, there has been no direct assessment of the response of human tissue to LDIR when exposure is under normal physiologic conditions of intact three-dimensional architecture, vasculature, and cell-cell contacts (between epithelial cells and between epithelial and stromal cells). EXPERIMENTAL DESIGN: In this article, we present the first data on the response of human tissue exposed in vivo to LDIR with precisely controlled and calibrated doses. We evaluated transcriptomic responses to a single exposure of LDIR in the normal skin of men undergoing therapeutic radiation for prostate cancer (research protocol, Health Insurance Portability and Accountability Act-compliant, Institutional Review Board-approved). Using newly developed biostatistical tools that account for individual splice variants and the expected variability of temporal response between humans even when the outcome is measured at a single time, we show a dose-response pattern in gene expression in a number of pathways and gene groups that are biologically plausible responses to LDIR. RESULTS: Examining genes and pathways identified as radiation-responsive in cell culture models, we found seven gene groups and five pathways that were altered in men in this experiment. These included the Akt/phosphoinositide-3-kinase pathway, the growth factor pathway, the stress/apoptosis pathway, and the pathway initiated by transforming growth factor-beta signaling, whereas gene groups with altered expression included the keratins, the zinc finger proteins and signaling molecules in the mitogen-activated protein kinase gene group. We show that there is considerable individual variability in radiation response that makes the detection of effects difficult, but still feasible when analyzed according to gene group and pathway. CONCLUSIONS: These results show for the first time that low doses of radiation have an identifiable biosignature in human tissue, irradiated in vivo with normal intact three-dimensional architecture, vascular supply, and innervation. The genes and pathways show that the tissue (a) does detect the injury, (b) initiates a stress/inflammatory response, (c) undergoes DNA remodeling, as suggested by the significant increase in zinc finger protein gene expression, and (d) initiates a "pro-survival" response. The ability to detect a distinct radiation response pattern following LDIR exposure has important implications for risk assessment in both therapeutic and national defense contexts.

Biopsy↗

PubMed1999

Seasonal regulation of food intake and body weight in the male Siberian hamster: studies of hypothalamic orexin (hypocretin), neuropeptide Y (NPY) and pro-opiomelanocortin (POMC).

The aim of these experiments was to investigate the relationship between hypothalamic expression of orexin (also called hypocretin), neuropeptide Y (NPY) and pro-opiomelanocortin (POMC) mRNA and seasonal cycles of body weight and food intake in the Siberian hamster. Adult males were transferred from long days of 16 h light and 8 h dark to short days of 8 h light and 16 h dark, a procedure known to induce major reductions in food intake and body weight in this species. After 8 weeks of exposure to short days, while body weight was declining, hypothalamic NPY mRNA levels as assessed by in situ hybridization were slightly lower (P < 0.05) than in age-matched controls exposed to long days. After 12 weeks with short days, when body weight would be expected to have reached its seasonal nadir, POMC mRNA levels were lower (P < 0.05) than in hamsters under long days. At no stage did orexin mRNA levels in hamsters under short days differ significantly from levels in those under long days. To investigate further the role of these peptide systems in seasonal changes in body weight and food intake, two provocative tests were carried out. Firstly, a 48-h fast induced a significant increase (P < 0.025) in hypothalamic NPY mRNA levels in both long- and short-day conditions, but did not change hypothalamic POMC or orexin mRNA levels. Secondly, systemic (intraperitoneal) treatment with recombinant murine leptin (5 mg/kg body weight) significantly decreased (P < 0.01) food intake over a 6-h post-treatment period in both long- and short-day conditions. However, this acute leptin treatment did not induce significant changes in hypothalamic orexin, NPY or POMC mRNA abundance. The increase in NPY expression in both long- and short-day conditions following food restriction and the suppression of food intake by leptin in both conditions suggests that acute homeostatic mechanisms operate in both long-day (obese) and short-day (nonobese) conditions. The lack of major changes in orexin, NPY and POMC in such different metabolic states suggest that other central systems must play a greater role in generating these states. Such findings are consistent with the 'sliding set-point' hypothesis, that is, seasonal cycles in food intake and fat metabolism are brought about by as yet unknown central mechanisms that chronically alter the level ('set point') around which homeostasis occurs, rather than resulting from changes in the potency of the acute feedback mechanisms themselves.

Animals↗

PubMed2002

Sterol carrier protein-2: structure reveals function.

The multiple actions of sterol carrier protein-2 (SCP-2) in intracellular lipid circulation and metabolism originate from its gene and protein structure. The SCP-x/pro-SCP-2 gene is a fusion gene with separate initiation sites coding for 15-kDa pro-SCP-2 (no enzyme activity) and 58-kDa SCP-x (a 3-ketoacyl CoA thiolase). Both proteins share identical cDNA and amino acid sequences for 13-kDa SCP-2 at their C-termini. Cellular 13-kDa SCP-2 derives from complete, posttranslational cleavage of the 15-kDa pro-SCP-2 and from partial posttranslational cleavage of 58-kDa SCP-x. Putative physiological functions of SCP-2 have been proposed on the basis of enhancement of intermembrane lipid transfer (e.g., cholesterol, phospholipid) and activation of enzymes involved in fatty acyl CoA transacylation (cholesterol esters, phosphatidic acid) in vitro, in transfected cells, and in genetically manipulated animals. At least four important SCP-2 structural domains have been identified and related to specific functions. First, the 46-kDa N-terminal presequence present in 58-kDa SCP-x is a 3-ketoacyl-CoA thiolase specific for branched-chain acyl CoAs. Second, the N-terminal 20 amino acid presequence in 15-kDa pro-SCP-2 dramatically modulates the secondary and tertiary structure of SCP-2 as well as potentiating its intracellular targeting coded by the C-terminal peroxisomal targeting sequence. Third, the N-terminal 32 amino acids form an amphipathic a-helical region, one face of which represents a membrane-binding domain. Positively charged amino acid residues in one face of the amphipathic helices allow SCP-2 to bind to membrane surfaces containing anionic phospholipids. Fourth, the hydrophobic faces of the N-terminal amphipathic a helices along with beta strands 4, 5, and helix D form a ligand-binding cavity able to accommodate multiple types of lipids (e. g., fatty acids, fatty acyl CoAs, cholesterol, phospholipids, isoprenoids). Two-dimensional 1H-15N heteronuclear single quantum coherence spectra of both apo-SCP-2 and of the 1:1 oleate-SCP-2 complex, obtained at pH 6.7, demonstrated the homogenous formation of holo-SCP-2. While comparison of the apo- and holoprotein amide fingerprints revealed about 60% of the resonances remaining essentially unchanged, 12 assigned amide residues underwent significant chemical-shift changes upon oleic acid binding. These residues were localized in three regions: the juncture of helices A and B, the mid-section of the beta sheet, and the interface formed by the region of beta strands 4, 5, and helix D. Circular dichroism also showed that these chemical-shift changes, upon oleic acid binding, did not alter the secondary structure of SCP-2. The nuclear magnetic resonance chemical shift difference data, along with mapping of the nearby hydrophobic residues, showed the oleic acid-binding site to be comprised of a pocket created by the face of the beta sheet, helices A and B on one end, and residues associated with beta strands 4, 5, and helix D at the other end of the binding cavity. Furthermore, the hydrophobic nature of the previously ill-defined C-terminus suggested that these 20 amino acids may form a 'hydrophobic cap' which closes around the oleic acid upon binding. Thus, understanding the structural domains of the SCP-x/pro-SCP-2 gene and its respective posttranslationally processed proteins has provided new insights into their functions in intracellular targeting and metabolism of lipids.

Acetyl-CoA C-Acetyltransferase↗