Significance: A highly interactive serine protease/plasmin/matrix metalloproteinase axis regulates stromal remodeling in the wound microenvironment. Current findings highlight the importance of stringent controls on protease expression and their topographic activities in cell proliferation, migration, and tissue homeostasis. Targeting elements in this cascading network may lead to novel therapeutic approaches for fibrotic diseases and chronic wounds.
Recent Advances: Matrix-active proteases and their inhibitors orchestrate wound site tissue remodeling, cell migration, and proliferation. Indeed, the serine proteases urokinase plasminogen activator and tissue-type plasminogen activator (uPA/tPA) and their major phsyiological inhibitor, plasminogen activator inhibitor-1 (PAI-1; serine protease inhibitor clade E member 1 [SERPINE1]), are upregulated in several cell types during injury repair. Coordinate expression of proteolytic enzymes and their inhibitors in the wound bed provides a mechanism for fine control of focal proteolysis to facilitate matrix restructuring and cell motility in complex environments.
Critical Issues: Cosmetic and tissue functional consequences of wound repair anomalies affect the quality of life of millions of patients in the United States alone. The development of novel therapeutics to manage individuals most affected by healing anomalies will likely derive from the identification of critical, translationally accessible, control elements in the wound site microenvironment.
Future Directions: Activation of the PAI-1 gene early after wounding, its prominence in the repair transcriptome and varied functions suggest a key role in the global cutaneous injury response program. Targeting PAI-1 gene expression and/or PAI-1 function with molecular genetic constructs, neutralizing antibodies or small molecule inhibitors may provide a novel, therapeutically relevant approach, to manage the pathophysiology of wound healing disorders associated with deficient or excessive PAI-1 levels.
During development of TGF-β1-initiated fibroproliferative disorders, NADPH oxidases (NOX family members) generate reactive oxygen species (ROS) resulting in downstream transcription of a subset genes encoding matrix structural elements and profibrotic factors. Prominent among the repertoire of disease-implicated genes is the TGF-β1 target gene encoding the potent profibrotic matricellular protein plasminogen activator inhibitor-1 (PAI-1 or SERPINE1). PAI-1 is the major physiologic inhibitor of the plasmin-based pericellular cascade and a causative factor in the development of vascular thrombotic and fibroproliferative disorders. ROS generation in response to TGF-β1 stimulation is rapid and precedes PAI-1 induction; engagement of non-SMAD (e.g., EGFR, Src kinase, MAP kinases, p53) and SMAD2/3 pathways are both required for PAI-1 expression and are ROS-dependent. Recent findings suggest a novel role for p53 in TGF-β1-induced PAI-1 transcription that involves ROS generation and p53/SMAD interactions. Targeting ROS and ROS-activated cellular events is likely to have therapeutic implications in the management of fibrotic disorders, particularly in the context of prolonged TGF-β signaling.
TGF-β; NOX; NADPH oxidases; PAI-1; ROS; p53; EGFR; SMADs
Transcriptional regulation is often the convergence point of oncogenic signaling. It is not surprising, therefore, that aberrant gene expression is a hallmark of cancer. Transformed cells often develop a dependency on such a reprogramming highlighting the therapeutic potential of rectifying cancer-associated transcriptional abnormalities in malignant cells. Although transcription is traditionally considered as undruggable, agents have been developed that target various levels of transcriptional regulation including DNA binding by transcription factors, protein-protein interactions, and epigenetic alterations. Some of these agents have been approved for clinical use or entered clinical trials. While artificial transcription factors have been developed that can theoretically modulate expression of any given gene, the emergence of reliable reporter assays greatly facilitate the search for transcription-targeted agents. This review provides a comprehensive overview of these developments, and discusses various strategies applicable for developing transcription-targeted therapeutic agents.
Cancer; targeted therapy; transcription; transcriptional regulation; transcription therapy; drug development
Focal Proteolysis: Regulation of Cell Migration and Signaling by the Serine Protease-Matrix Metalloproteinase Cascade
We investigated the mechanism of carbapenem resistance in 10 Acinetobacter baumannii strains isolated from the United States and Mexico between 2005 and 2009. The detection of known metallo-β-lactamase or carbapenem-hydrolyzing oxacillinase (OXA) genes by PCR was negative. The presence of plasmid-encoded carbapenem resistance genes was investigated by transformation of A. baumannii ATCC 17978. Shotgun cloning experiments and sequencing were performed, followed by the expression of a novel β-lactamase in A. baumannii. Three novel OXA enzymes were identified, OXA-235 in 8 isolates and the amino acid variants OXA-236 (Glu173-Val) and OXA-237 (Asp208-Gly) in 1 isolate each. The deduced amino acid sequences shared 85% identity with OXA-134, 54% to 57% identities with the acquired OXA-23, OXA-24, OXA-58, and OXA-143, and 56% identity with the intrinsic OXA-51 and, thus, represent a novel subclass of OXA. The expression of OXA-235 in A. baumannii led to reduced carbapenem susceptibility, while cephalosporin MICs were unaffected. Genetic analysis revealed that blaOXA-235, blaOXA-236, and blaOXA-237 were bracketed between two ISAba1 insertion sequences. In addition, the presence of these acquired β-lactamase genes might result from a transposition-mediated mechanism. This highlights the propensity of A. baumannii to acquire multiple carbapenem resistance determinants.
Previously, we showed that transient inhibition of TGF- β1 resulted in correction of key aspects of diabetes-induced CD34+ cell dysfunction. In this report, we examine the effect of transient inhibition of plasminogen activator inhibitor-1 (PAI-1), a major gene target of TGF-β1 activation. Using gene array studies, we examined CD34+ cells isolated from a cohort of longstanding diabetic individuals, free of microvascular complications despite suboptimal glycemic control, and found that the cells exhibited reduced transcripts of both TGF-β1 and PAI-1 compared to age, sex, and degree of glycemic control-matched diabetic individuals with microvascular complications. CD34+ cells from diabetic subjects with microvascular complications consistently exhibited higher PAI-1 mRNA than age-matched non-diabetic controls. TGF- β1 phosphorodiamidate morpholino oligo (PMO) reduced PAI-1 mRNA in diabetic (p<0.01) and non-diabetic (p=0.05) CD34+ cells. To reduce PAI-1 in human CD34+ cells, we utilized PAI-1 siRNA, lentivirus expressing PAI-1 shRNA or PAI-1 PMO. We found that inhibition of PAI-1 promoted CD34+ cell proliferation and migration in vitro, likely through increased PI3(K) activity and increased cGMP production. Using a retinal ischemia reperfusion injury model in mice, we observed that recruitment of diabetic CD34+ cells to injured acellular retinal capillaries was greater after PAI-1-PMO treatment compared with control PMO-treated cells. Targeting PAI-1 offers a promising therapeutic strategy for restoring vascular reparative function in defective diabetic progenitors.
The difficulties in quantifying the 3D form and spatial relationships of the skeletal components of the ribcage present a barrier to studies of the growth of the thoracic skeleton. Thus, most studies to date have relied on traditional measurements such as distances and indices from single or few ribs. It is currently known that adult-like thoracic shape is achieved early, by the end of the second postnatal year, with the circular cross-section of the newborn thorax transforming into the ovoid shape of adults; and that the ribs become inclined such that their anterior borders come to lie inferior to their posterior. Here we present a study that revisits growth changes using geometric morphometrics applied to extensive landmark data taken from the ribcage. We digitized 402 (semi) landmarks on 3D reconstructions to assess growth changes in 27 computed tomography-scanned modern humans representing newborns to adults of both sexes. Our analyses show a curved ontogenetic trajectory, resulting from different ontogenetic growth allometries of upper and lower thoracic units. Adult thoracic morphology is achieved later than predicted, by diverse modifications in different anatomical regions during different ontogenetic stages. Besides a marked increase in antero-posterior dimensions, there is an increase in medio-lateral dimensions of the upper thorax, relative to the lower thorax. This transforms the pyramidal infant thorax into the barrel-shaped one of adults. Rib descent is produced by complex changes in 3D curvature. Developmental differences between upper and lower thoracic regions relate to differential timings and rates of maturation of the respiratory and digestive systems, the spine and the locomotor system. Our findings are relevant to understanding how changes in the relative rates of growth of these systems and structures impacted on the development and evolution of modern human body shape.
Cooperative interactions between growth factor signaling pathways are important elements in carcinoma progression. A model system combining transforming growth factor-β1 (TGF-β1) and EGF was developed to investigate mechanisms underlying induced epithelial-to-mesenchymal transition (EMT) in ras-transformed human (HaCaT II-4) keratinocytes. Dual stimulation with TGF-β1+EGF resulted in keratinocyte “plasticity” and pronounced colony dispersal. The most highly expressed transcript, identified by mRNA profiling, encoded plasminogen activator inhibitor-1 (PAI-1; SERPINE1). PAI-1 negatively regulates plasmin-dependent matrix degradation, preserving a stromal scaffold permissive for keratinocyte motility. Mitogen-activated extracellular kinase (MEK)/extracellular signal-regulated kinase (ERK) and p38 signaling were required for maximal PAI-1 upregulation and TGF-β1+EGF-stimulated cell locomotion, as pharmacologic disruption of MEK/p38 activity ablated both responses. Moreover, PAI-1 knockdown alone effectively inhibited TGF-β1+EGF-dependent cell scattering, indicating a functional role for this SERPIN in the dual-growth factor model of induced motility. Moreover, EGFR signaling blockade or EGFR knockdown attenuated TGF-β1-induced PAI-1 expression, implicating EGFR transactivation in TGF-β1-stimulated PAI-1 expression, and reduced colony dispersal in TGF-β1+EGF-treated cultures. Identification of such cooperative signaling networks and their effect on specific invasion-promoting target genes, such as PAI-1, may lead to the development of pathway-specific therapeutics that affect late-stage events in human tumor progression.
How animals cope with increases in body size is a key issue in biology. Here, we consider scaling of xenarthrans, particularly how femoral form and function varies to accommodate the size range between the 3 kg armadillo and its giant relative the 300 kg glyptodont. It has already been noted that femoral morphology differs between these animals and suggested that this reflects a novel adaptation to size increase in glyptodont. We test this idea by applying a finite element analysis of coronal plane forces to femoral models of these animals, simulating the stance phase in the hind limb; where the femur is subject to bending owing to longitudinal compressive as well as abduction loads on the greater trochanter. We use these models to examine the hypothesis that muscles attaching on the third trochanter (T3) can reduce this bending in the loaded femur and that the T3 forces are more effective at reducing bending in glyptodont where the T3 is situated at the level of the knee. The analysis uses traditional finite element methods to produce strain maps and examine strains at 200 points on the femur. The coordinates of these points before and after loading are also used to carry out geometric morphometric (GM) analyses of the gross deformation of the model in different loading scenarios. The results show that longitudinal compressive and abductor muscle loading increases bending in the coronal plane, and that loads applied to the T3 reduce that bending. In the glyptodont model, the T3 loads are more effective and can more readily compensate for the bending owing to longitudinal and abductor loads. This study also demonstrates the usefulness of GM methods in interpreting the results of finite element analyses.
third trochanter; armadillo; glyptodont femur; xenarthran; finite element analysis; geometric morphometrics
Plasminogen activator inhibitor-1 (PAI-1; SERPINE1) is the major physiologic regulator of the plasmin-based pericellular proteolytic cascade, a modulator of vascular smooth muscle cell (VSMC) migration and a causative factor in cardiovascular disease and restenosis, particularly in the context of increased vessel transforming growth factor- β1 (TGF-β1) levels. PAI-1 limits conversion of plasminogen to plasmin (and, thereby, fibrin degradation) by inhibiting its protease targets urokinase and tissue-type plasminogen activators (uPA, tPA). PAI-1 also has signaling functions and binds to the low density lipoprotein receptor-related protein 1 (LRP1) to regulate LRP1-dependent cell motility that, in turn, contributes to neointima formation. PAI-1/uPA/uPA receptor/LRPI/integrin complexes are endocytosed with subsequent uPAR/LRP1/integrin redistribution to the leading edge, initiating an “adhesion-detachment-readhesion” cycle to promote cell migration. PAI-1 also interacts with LRP1 in a uPA/uPAR-independent manner triggering Jak/Stat1 pathway activation to stimulate cell motility. PAI-1 itself is a substrate for extracellular proteases and exists in a “cleaved” form which, while unable to interact with uPA and tPA, retains LRP1-binding and migratory activity. These findings suggest that there are multiple mechanisms through which inhibition of PAI-1 may promote cardiovascular health. Several studies have focused on the design, synthesis and preclinical assessment of PAI-1 antagonists including monoclonal antibodies, peptides and low molecular weight (LMW) antagonists. This review discusses the translational impact of LMW PAI-1 antagonists on cardiovascular disease addressing PAI-1-initiated signaling, PAI-1 structure, the design and characteristics of PAI-1-targeting drugs, results of in vitro and in vivo studies, and their clinical implications.
Accumulation of neurotoxic amyloid peptides (Aβ) in the brain, generated by β-site proteolytic processing of the amyloid precursor protein (APP), is the hallmark pathophysiologic feature of Alzheimer's disease. The plasmin-activating cascade, in which urokinase (uPA) and tissue-type (tPA) plasminogen activators convert plasminogen to the broad-spectrum protease plasmin, appears to serve a protective, Aβ-clearing, role in the central nervous system. Plasmin degrades Aβ and catalyzes α- site APP proteolysis generating nontoxic peptides. Plasmin activation in the brain is negatively regulated by the fast-acting clade E serine protease inhibitor (SERPIN) plasminogen activator inhibitor type-1 (PAI-1; SERPINE1) resulting in Aβ accumulation. PAI-1 and its major physiological inducer TGF-β1, moreover, are both increased in Alzheimer's disease models and implicated in the etiology and progression of human neurodegenerative disorders. Current findings support the hypothesis that targeting of PAI-1 function (by small molecule drugs) and/or gene expression (by histone deacetylase inhibitors) may constitute a clinically-relevant molecular approach to the therapy of neurodegenerative diseases associated with increased PAI-1 levels.
Computer-based simulation techniques such as multi-body dynamics analysis are becoming increasingly popular in the field of skull mechanics. Multi-body models can be used for studying the relationships between skull architecture, muscle morphology and feeding performance. However, to be confident in the modelling results, models need to be validated against experimental data, and the effects of uncertainties or inaccuracies in the chosen model attributes need to be assessed with sensitivity analyses. Here, we compare the bite forces predicted by a multi-body model of a lizard (Tupinambis merianae) with in vivo measurements, using anatomical data collected from the same specimen. This subject-specific model predicts bite forces that are very close to the in vivo measurements and also shows a consistent increase in bite force as the bite position is moved posteriorly on the jaw. However, the model is very sensitive to changes in muscle attributes such as fibre length, intrinsic muscle strength and force orientation, with bite force predictions varying considerably when these three variables are altered. We conclude that accurate muscle measurements are crucial to building realistic multi-body models and that subject-specific data should be used whenever possible.
bite force; multi-body dynamics analysis; skull; feeding; validation; Tupinambis
Depth of invasion, a quantifier of vertical growth, is a major cutaneous melanoma staging factor. Stromal penetrance requires pericellular proteolysis regulated by the serine protease and matrix metalloproteinase cascades. The serine protease inhibitor SERPINE1, a poor prognosis biomarker in various cancers, promotes tumor progression likely by titrating the extent and local of plasmin-initiated matrix remodeling. SERPINE1 in human melanoma was assessed using tissue arrays that included primary/ metastatic tumors and normal skin. SERPINE1 was basal layer-restricted in the normal epidermis. SERPINE1 immunoreactivity was evident in 27/28 primary (96%) and 24/26 metastatic tumors (92%); cutaneous metastases (80%) had significantly elevated SERPINE1 levels compared to low signals characteristic of lymph node lesions. Moderate SERPINE1 expression was a general finding in primary melanoma whereas reduced or increased SERPINE1 immunolocalization typified metastatic deposits. The amplitude of SERPINE1 expression may impact melanoma site-specific dissemination, with cutaneous metastases representing a high-SERPINE1 tumor subtype.
Dermatopathology; Melanoma; PAI-1; SERPINE1; Metastasis
The modern human face differs from that of our early ancestors in that the facial profile is relatively retracted (orthognathic). This change in facial profile is associated with a characteristic spatial distribution of bone deposition and resorption: growth remodeling. For humans, surface resorption commonly dominates on anteriorly-facing areas of the subnasal region of the maxilla and mandible during development. We mapped the distribution of facial growth remodeling activities on the 900–800 ky maxilla ATD6-69 assigned to H. antecessor, and on the 1.5 My cranium KNM-WT 15000, part of an associated skeleton assigned to African H. erectus. We show that, as in H. sapiens, H. antecessor shows bone resorption over most of the subnasal region. This pattern contrasts with that seen in KNM-WT 15000 where evidence of bone deposition, not resorption, was identified. KNM-WT 15000 is similar to Australopithecus and the extant African apes in this localized area of bone deposition. These new data point to diversity of patterns of facial growth in fossil Homo. The similarities in facial growth in H. antecessor and H. sapiens suggest that one key developmental change responsible for the characteristic facial morphology of modern humans can be traced back at least to H. antecessor.
Using a repetitive-sequence-based (rep)-PCR (DiversiLab), we have molecularly typed Acinetobacter nosocomial bloodstream isolates (Acinetobacter
baumannii [n = 187], Acinetobacter
pittii [n = 23], and Acinetobacter
nosocomialis [n = 61]) obtained from patients hospitalized in U.S. hospitals over a 10-year period (1995-2004) during a nationwide surveillance study (Surveillance and Control of Pathogens of Epidemiological Importance [SCOPE]). Patterns of A. baumannii rep-PCR were compared to those of previously identified international clonal lineages (ICs) and were further investigated by multilocus sequence typing (MLST) to compare the two typing methods. Forty-seven of the A. baumannii isolates clustered with the previously defined IC 2. ICs 1, 3, 6, and 7 were also detected. The remaining 81 isolates were unrelated to the described ICs. In contrast, A. pittii and A. nosocomialis isolates were more heterogeneous, as determined by rep-PCR. Our MLST results were in good correlation with the rep-PCR clusters. Our study confirms previous data indicating the predominance of a few major clonal A. baumannii lineages in the United States, particularly IC 2. The presence in the United States of A. baumannii ICs 1, 2, and 3 from as early as 1995 suggests that global dissemination of these lineages was an early event.
Chronic kidney disease constitutes an increasing medical burden affecting 26 million people in the United States alone. Diabetes, hypertension, ischemia, acute injury, and urological obstruction contribute to renal fibrosis, a common pathological hallmark of chronic kidney disease. Regardless of etiology, elevated TGF-β1 levels are causatively linked to the activation of profibrotic signaling pathways initiated by angiotensin, glucose, and oxidative stress. Unilateral ureteral obstruction (UUO) is a useful and accessible model to identify mechanisms underlying the progression of renal fibrosis. Plasminogen activator inhibitor-1 (PAI-1), a major effector and downstream target of TGF-β1 in the progression of several clinically important fibrotic disorders, is highly up-regulated in UUO and causatively linked to disease severity. SMAD and non-SMAD pathways (pp60c-src, epidermal growth factor receptor [EGFR], mitogen-activated protein kinase, p53) are required for PAI-1 induction by TGF-β1. SMAD2/3, pp60c-src, EGFR, and p53 activation are each increased in the obstructed kidney. This review summarizes the molecular basis and translational significance of TGF-β1-stimulated PAI-1 expression in the progression of kidney disease induced by ureteral obstruction. Mechanisms discussed here appear to be operative in other renal fibrotic disorders and are relevant to the global issue of tissue fibrosis, regardless of organ site.
Fibrosis; PAI-1; TGF-β1; p53; Transcription
We have investigated the reproducibility of DiversiLab rep-PCR fingerprints between two laboratories with the aim of determining if the fingerprints and clustering are laboratory-specific or portable. One-hundred non-duplicate Acinetobacter baumannii isolates were used in this study. DNA isolation and rep-PCR were each performed separately in two laboratories and rep-PCR patterns generated in laboratory A were compared with those from laboratory B. Twelve A. baumannii isolates processed in laboratory A showed ≥98 % pattern similarity with the corresponding 12 isolates tested in laboratory B and were considered identical. Sixty-four isolates showed 95–97.9 % similarity with their corresponding isolates. Twenty-three isolates showed 90–94 % similarity with the corresponding isolates, while one isolate showed only 87.4 % similarity. However, intra-laboratory clustering was conserved: isolates that clustered in laboratory A also clustered in laboratory B. While clustering was conserved and reproducible at two different laboratories, demonstrating the robustness of rep-PCR, interlaboratory comparison of individual isolate fingerprints showed more variability. This comparison allows conclusions regarding clonality to be reached independent of the laboratory where the analysis is performed.
The polyphenol quercetin (Quer) represses expression of the cardiovascular disease risk factor plasminogen activator inhibitor-1 (PAI-1) in cultured endothelial cells (ECs). Transfection of PAI-1 promoter-luciferase reporter deletion constructs identified a 251-bp fragment (nucleotides −800 to −549) responsive to Quer. Two E-box motifs (CACGTG), at map positions −691 (E-box1) and −575 (E-box2), are platforms for occupancy by several members of the c-MYC family of basic helix-loop-helix leucine zipper (bHLH-LZ) proteins. Promoter truncation and electrophoretic mobility shift/supershift analyses identified upstream stimulatory factor (USF)-1 and USF-2 as E-box1/E-box2 binding factors. ECs co-transfected with a 251 bp PAI-1 promoter fragment containing the two E-box motifs (p251/luc) and a USF-2 expression vector (pUSF-2/pcDNA) exhibited reduced luciferase activity versus p251/luc alone. Overexpression of USF-2 decreased, while transfection of a dominant-negative USF construct increased, EC growth consistent with the known anti-proliferative properties of USF proteins. Quer-induced decreases in PAI-1 expression and reduced cell proliferation may contribute, at least in part, to the cardioprotective benefit associated with daily intake of polyphenols.
PAI-1; ENDOTHELIAL CELLS; TRANSFECTION; PROLIFERATION; TRANSCRIPTION FACTOR; USF
This study investigated the correlation between blaOXA-51 variants and Acinetobacter baumannii worldwide clonal lineages 1 to 8 (WW1 to -8). The blaOXA-51-like genes of 102 A. baumannii isolates were sequenced. Using DiversiLab repetitive-sequence-based PCR (rep-PCR) typing, 92 of these isolates had previously been assigned to WW1 to -8 and 10 were unclustered. Clustering of DNA sequences was performed using the neighbor-joining method and the Jukes-Cantor phylogenetic correction. blaOXA-51 variants were in good correlation with DiversiLab-defined clonal lineages. Sequence-based typing of blaOXA-51 variants has the potential to be applied for epidemiologic characterization of A. baumannii and to identify worldwide clonal lineages 1 to 8.
Sea turtles (Chelonoidea) are a charismatic group of marine reptiles that occupy a range of important ecological roles. However, the diversity and evolution of their feeding anatomy remain incompletely known.
Using computed tomography and classical comparative anatomy we describe the cranial anatomy in two sea turtles, the loggerhead (Caretta caretta) and Kemp’s ridley (Lepidochelys kempii), for a better understanding of sea turtle functional anatomy and morphological variation. In both taxa the temporal region of the skull is enclosed by bone and the jaw joint structure and muscle arrangement indicate that palinal jaw movement is possible. The tongue is relatively small, and the hyoid apparatus is not as conspicuous as in some freshwater aquatic turtles. We find several similarities between the muscles of C. caretta and L. kempii, but comparison with other turtles suggests only one of these characters may be derived: connection of the m. adductor mandibulae internus into the Pars intramandibularis via the Zwischensehne. The large fleshy origin of the m. adductor mandibulae externus Pars superficialis from the jugal seems to be a characteristic feature of sea turtles.
In C. caretta and L. kempii the ability to suction feed does not seem to be as well developed as that found in some freshwater aquatic turtles. Instead both have skulls suited to forceful biting. This is consistent with the observation that both taxa tend to feed on relatively slow moving but sometimes armoured prey. The broad fleshy origin of the m. adductor mandibulae externus Pars superficialis may be linked to thecheek region being almost fully enclosed in bone but the relationship is complex.
The metabolic syndrome is common in populations exposed to a typical Western diet. There is a lack of an animal model that mimics this condition.
We fed 15 cynomolgus monkeys ad libitum a high sugar high fat (HSHF) diet for 33 weeks. Body weight, body composition, serum lipids and insulin were measured at baseline and at 33 weeks.
The animals tolerated the HSHF diet very well. In the intervention group, total serum cholesterol and LDL-C were 3- and 5-fold higher, respectively, at 33 weeks as compared to their baseline levels. Serum HDL-C and triglycerides were not significantly affected. Dual-energy X-ray absorptiometry (DXA) analysis of the intervention group indicated that the trunk fat mass increased by 187% during this period.
Cynomolgus monkeys should be a useful model for investigating the interactions of diet and other factors such as genetics in the development of the metabolic syndrome.
Dual X-ray absorptiometry; LDL-cholesterol; triglyceride; insulin
Transcriptional regulators, such as SoxS, RamA, MarA, and Rob, which upregulate the AcrAB efflux pump, have been shown to be associated with multidrug resistance in clinically relevant Gram-negative bacteria. In addition to the multidrug resistance phenotype, these regulators have also been shown to play a role in the cellular metabolism and possibly the virulence potential of microbial cells. As such, the increased expression of these proteins is likely to cause pleiotropic phenotypes. Klebsiella pneumoniae is a major nosocomial pathogen which can express the SoxS, MarA, Rob, and RamA proteins, and the accompanying paper shows that the increased transcription of ramA is associated with tigecycline resistance (M. Veleba and T. Schneiders, Antimicrob. Agents Chemother. 56:4466–4467, 2012). Bioinformatic analyses of the available Klebsiella genome sequences show that an additional AraC-type regulator is encoded chromosomally. In this work, we characterize this novel AraC-type regulator, hereby called RarA (Regulator of antibiotic resistance A), which is encoded in K. pneumoniae, Enterobacter sp. 638, Serratia proteamaculans 568, and Enterobacter cloacae. We show that the overexpression of rarA results in a multidrug resistance phenotype which requires a functional AcrAB efflux pump but is independent of the other AraC regulators. Quantitative real-time PCR experiments show that rarA (MGH 78578 KPN_02968) and its neighboring efflux pump operon oqxAB (KPN_02969_02970) are consistently upregulated in clinical isolates collected from various geographical locations (Chile, Turkey, and Germany). Our results suggest that rarA overexpression upregulates the oqxAB efflux pump. Additionally, it appears that oqxR, encoding a GntR-type regulator adjacent to the oqxAB operon, is able to downregulate the expression of the oqxAB efflux pump, where OqxR complementation resulted in reductions to olaquindox MICs.
The delta-5 and delta-6 desaturases (D5D and D6D), encoded by fatty acid desaturase 1 (FADS1) and 2 (FADS2) genes, respectively, are rate-limiting enzymes in the metabolism of ω-3 and ω-6 fatty acids. The objective of this study was to identify genes influencing variation in estimated D5D and D6D activities in plasma and erythrocytes in Alaskan Eskimos (n = 761) participating in the genetics of coronary artery disease in Alaska Natives (GOCADAN) study. Desaturase activity was estimated by product: precursor ratio of polyunsaturated fatty acids. We found evidence of linkage for estimated erythrocyte D5D (eD5D) on chromosome 11q12-q13 (logarithm of odds score = 3.5). The confidence interval contains candidate genes FADS1, FADS2, 7-dehydrocholesterol reductase (DHCR7), and carnitine palmitoyl transferase 1A, liver (CPT1A). Measured genotype analysis found association between CPT1A, FADS1, and FADS2 single-nucleotide polymorphisms (SNPs) and estimated eD5D activity (p-values between 10−28 and 10−5). A Bayesian quantitative trait nucleotide analysis showed that rs3019594 in CPT1A, rs174541 in FADS1, and rs174568 in FADS2 had posterior probabilities > 0.8, thereby demonstrating significant statistical support for a functional effect on eD5D activity. Highly significant associations of FADS1, FADS2, and CPT1A transcripts with their respective SNPs (p-values between 10−75 and 10−7) in Mexican Americans of the San Antonio Family Heart Study corroborated our results. These findings strongly suggest a functional role for FADS1, FADS2, and CPT1A SNPs in the variation in eD5D activity.
essential fatty acids; single-nucleotide polymorphisms; bayesian quantitative trait nucleotide analysis