Periplasmic flagella are essential for the distinctive morphology, motility, and infectious life cycle of the Lyme disease spirochete Borrelia burgdorferi . In this study, we genetically trapped ...intermediates in flagellar assembly and determined the 3D structures of the intermediates to 4-nm resolution by cryoelectron tomography. We provide structural evidence that secretion of rod substrates triggers remodeling of the central channel in the flagellar secretion apparatus from a closed to an open conformation. This open channel then serves as both a gateway and a template for flagellar rod assembly. The individual proteins assemble sequentially to form a modular rod. The hook cap initiates hook assembly on completion of the rod, and the filament cap facilitates filament assembly after formation of the mature hook. Cryoelectron tomography and mutational analysis thus combine synergistically to provide a unique structural blueprint of the assembly process of this intricate molecular machine in intact cells.
Obesity and vascular dysfunction Stapleton, Phoebe A.; James, Milinda E.; Goodwill, Adam G. ...
Pathophysiology (Amsterdam),
08/2008, Letnik:
15, Številka:
2
Journal Article
Recenzirano
Odprti dostop
One of the most profound challenges facing public health and public health policy in Western society is the increased incidence and prevalence of both overweight and obesity. While this condition can ...have significant consequences for patient mortality and quality of life, it can be further exacerbated as overweight/obesity can be a powerful stimulus for the development of additional risk factors for a negative cardiovascular outcome, including increased insulin resistance, dyslipidemia and hypertension. This manuscript will present the effects of systemic obesity on broad issues of vascular function in both afflicted human populations and in the most relevant animal models. Among the topics that will be covered are alterations to vascular reactivity (both dilator and constrictor responses), adaptations in microvascular network and vessel wall structure, and alterations to the patterns of tissue/organ perfusion as a result of the progression of the obese condition. Additionally, special attention will be paid to the contribution of chronic inflammation as a contributor to alterations in vascular function, as well as the role of perivascular adipose tissue in terms of impacting vessel behavior. When taken together, it is clearly apparent that the development of the obese condition can have profound, and frequently difficult to predict, impacts on integrated vascular function. Much of this complexity appears to have its basis in the extent to which other co-morbidities associated with obesity (e.g., insulin resistance) are present and exert contributing effects.
Center for Interdisciplinary Research in Cardiovascular Sciences, Department of Physiology and Pharmacology, West Virginia University School of Medicine, Morgantown, West Virginia
Submitted 7 June ...2008
; accepted in final form 1 August 2008
This study determined if altered vascular prostacyclin (PGI 2 ) and/or thromboxane A 2 (TxA 2 ) production with reduced P O 2 contributes to impaired hypoxic dilation of skeletal muscle resistance arterioles of obese Zucker rats (OZRs) versus lean Zucker rats (LZRs). Mechanical responses were assessed in isolated gracilis muscle arterioles following reductions in P O 2 under control conditions and following pharmacological interventions inhibiting arachidonic acid metabolism and nitric oxide synthase and alleviating elevated vascular oxidant stress. The production of arachidonic acid metabolites was assessed using pooled arteries from OZRs and LZRs in response to reduced P O 2 . Hypoxic dilation, endothelium-dependent in both strains, was attenuated in OZRs versus LZRs. Nitric oxide synthase inhibition had no significant impact on hypoxic dilation in either strain. Cyclooxygenase inhibition dramatically reduced hypoxic dilation in LZRs and abolished responses in OZRs. Treatment of arterioles from OZRs with polyethylene glycol-superoxide dismutase improved hypoxic dilation, and this improvement was entirely cyclooxygenase dependent. Vascular PGI 2 production with reduced P O 2 was similar between strains, although TxA 2 production was increased in OZRs, a difference that was attenuated by treatment of vessels from OZRs with polyethylene glycol-superoxide dismutase. Both blockade of PGH 2 /TxA 2 receptors and inhibition of thromboxane synthase increased hypoxic dilation in OZR arterioles. These results suggest that a contributing mechanism underlying impaired hypoxic dilation of skeletal muscle arterioles of OZRs may be an increased vascular production of TxA 2 , which competes against the vasodilator influences of PGI 2 . These results also suggest that the elevated vascular oxidant stress inherent in metabolic syndrome may contribute to the increased vascular TxA 2 production and may blunt vascular sensitivity to PGI 2 .
skeletal muscle microcirculation; endothelium-dependent dilation; vascular reactivity; rodent models of obesity
Address for reprint requests and other correspondence: J. C. Frisbee, Center for Interdisciplinary Research in Cardiovascular Science, Dept. of Physiology and Pharmacology, Robert C. Byrd Health Sciences Center, PO Box 9105, West Virginia Univ. School of Medicine, Morgantown, WV 26505 (e-mail: jfrisbee{at}hsc.wvu.edu )
Spirochaetes are bacteria responsible for several serious diseases, including Lyme disease (Borrelia burgdorferi), syphilis (Treponema pallidum) and leptospirosis (Leptospira interrogans), and ...contribute to periodontal diseases (Treponema denticola)(1). These spirochaetes employ an unusual form of flagella-based motility necessary for pathogenicity; indeed, spirochaete flagella (periplasmic flagella) reside and rotate within the periplasmic space(2-11). The universal joint or hook that links the rotary motor to the filament is composed of ∼120-130 FlgE proteins, which in spirochaetes form an unusually stable, high-molecular-weight complex(9,12-17). In other bacteria, the hook can be readily dissociated by treatments such as heat(18). In contrast, spirochaete hooks are resistant to these treatments, and several lines of evidence indicate that the high-molecular-weight complex is the consequence of covalent crosslinking(12,13,17). Here, we show that T. denticola FlgE self-catalyses an interpeptide crosslinking reaction between conserved lysine and cysteine, resulting in the formation of an unusual lysinoalanine adduct that polymerizes the hook subunits. Lysinoalanine crosslinks are not needed for flagellar assembly, but they are required for cell motility and hence infection. The self-catalytic nature of FlgE crosslinking has important implications for protein engineering, and its sensitivity to chemical inhibitors provides a new avenue for the development of antimicrobials targeting spirochaetes.
Microcirculation (2010) 17, 47–58. doi: 10.1111/j.1549‐8719.2009.00003.x
Genetic familial hypercholesterolemia (FH) and combined hyperlipidemia (FCH) are characterized by elevated plasma low‐density ...lipoprotein (LDL) (FH) and LDL/triglycerides (FCH), with mouse models represented by LDL receptor (LDLR) and apolipoprotein E (ApoE) gene deletion mice, respectively. Given the impact of FH and FCH on health outcomes, we determined the impact of FH/FCH on vascular structure in LDLR and ApoE mice. LDLR, ApoE and control mice were utilized at 12–13 and 22–23 weeks when gracilis arteries were studied for wall mechanics and gastrocnemius muscles were harvested for microvessel density measurements. Conduit arteries and plasma samples were harvested for biochemical analyses. Arteries from ApoE and LDLR exhibited blunted expansion versus control, reduced distensibility and left‐shifted stress versus strain relation (LDLR > ApoE). Microvessel density was reduced in ApoE and LDLR (ApoE > LDLR). Secondary analyses suggested that wall remodeling in LDLR was associated with cholesterol and MCP‐1, while rarefaction in ApoE was associated with tumor necrosis factors‐α, triglycerides and vascular production of TxA2. Remodeling in ApoE and LDLR appears distinct; as that in LDLR is preferential for vascular walls, while that for ApoE is stronger for rarefaction. Remodeling in LDLR may be associated with cellular adhesion, while that in ApoE may be associated with pro‐apoptotsis and constrictor prostanoid generation.
ABSTRACT
Objective: The obese Zucker rat (OZR) model of the metabolic syndrome is partly characterized by moderate hypercholesterolemia, in addition to other contributing comorbidities. Previous ...results suggest that vascular dysfunction in OZR is associated with chronic reduction in vascular nitric-oxide (NO) bioavailability and chronic inflammation, both frequently associated with hypercholesterolemia. As such, we evaluated the impact of chronic cholesterol-reducing therapy on the development of impaired skeletal muscle arteriolar reactivity and microvessel density in OZR and its impact on chronic inflammation and NO bioavailability.
Materials and Methods: Beginning at seven weeks of age, male OZR were treated with gemfibrozil, probucol, atorvastatin, or simvastatin (in chow) for 10 weeks. Subsequently, plasma and vascular samples were collected for biochemical/molecular analyses, while arteriolar reactivity and microvessel network structure were assessed by using established methodologies after 3, 6, and 10 weeks of drug therapy.
Results: All interventions were equally effective at reducing total cholesterol, although only the statins also blunted the progressive reductions to vascular NO bioavailability, evidenced by greater maintenance of acetylcholine-induced dilator responses, an attenuation of adrenergic constrictor reactivity, and an improvement in agonist-induced NO production. Comparably, while minimal improvements to arteriolar wall mechanics were identified with any of the interventions, chronic statin treatment reduced the rate of microvessel rarefaction in OZR. Associated with these improvements was a striking statin-induced reduction in inflammation in OZR, such that numerous markers of inflammation were correlated with improved microvascular reactivity and density. However, using multivariate discriminant analyses, plasma RANTES (regulated on activation, normal T-cell expressed and secreted), interleukin-10, monocyte chemoattractant protein-1, and tumor necrosis factor alpha were determined to be the strongest contributors to differences between groups, although their relative importance varied with time.
Conclusions: While the positive impact of chronic statin treatment on vascular outcomes in the metabolic syndrome are independent of changes to total cholesterol, and are more strongly associated with improvements to vascular NO bioavailability and attenuated inflammation, these results provide both a spatial and temporal framework for targeted investigation into mechanistic determinants of vasculopathy in the metabolic syndrome.
1 Division of Exercise Physiology, 2 Department of Physiology and Pharmacology, and 3 Center for Interdisciplinary Research in Cardiovascular Sciences, West Virginia University School of Medicine, ...Morgantown, West Virginia
Submitted 11 June 2007
; accepted in final form 9 July 2007
With most cardiovascular disease risk factors, endothelium-dependent dilation of skeletal muscle resistance arterioles is compromised, although with hypercholesterolemia, impairments to reactivity are not consistently observed. Using apolipoprotein E (ApoE) and low-density lipoprotein receptor (LDLR) gene deletion male mouse models of hypercholesterolemia at 20 wk of age, we tested the hypothesis that arteriolar dilation would be maintained due to an increased stimulus-induced production of dilator metabolites via cyclooxygenase and cytochrome P -450 epoxygenase pathways. Arterioles from both strains demonstrated mild reductions in dilation to hypoxia and acetylcholine versus responses in C57/Bl/6J (C57) controls. However, although inhibition of nitric oxide synthase (NOS) attenuated dilation in arterioles from C57 controls, this effect was absent in ApoE or LDLR strains. In contrast, cyclooxygenase-dependent portions of dilator reactivity were maintained across the three strains. Notably, although combined NOS and cyclooxygenase inhibition abolished arteriolar responses to hypoxia and acetylcholine in C57 controls, significant reactivity remained in ApoE and LDLR strains. Whereas inhibition of cytochrome P -450 -hydroxylase and epoxygenases had no effect on this residual reactivity in ApoE and LDLR strains, inhibition of 12/15-lipoxygenase with nordihydroguaiaretic acid abolished the residual reactivity. With both hypoxic and methacholine challenges, arteries from ApoE and LDLR strains demonstrated an increased production of both 12( S )- and 15( S )-hydroxyeicosatetraenoic acid, end products of arachidonic acid metabolism via 12/15-lipoxygenase, a response that was not present in C57 controls. These results suggest that with development of hypercholesterolemia, mechanisms contributing to dilator reactivity in skeletal muscle arterioles are modified such that net reactivity to endothelium-dependent stimuli is largely intact.
skeletal muscle microcirculation; mouse models of cardiovascular disease
Address for reprint requests and other correspondence: J. C. Frisbee, Center for Interdisciplinary Research in Cardiovascular Science, Dept. of Physiology and Pharmacology, Robert C. Byrd Health Sciences Center, PO Box 9105, West Virginia Univ. School of Medicine, Morgantown, WV 26505 (e-mail: jfrisbee{at}hsc.wvu.edu )
Objective: The aim of this study was to determine if arachidonic acid (AA)-induced skeletal muscle arteriolar dilation is altered with hypercholesterolemia in ApoE and low-density lipoprotein ...receptor (LDLR) gene deletion mice fed a normal diet. This study also determined contributors to altered AA-induced dilation between dyslipidemic mice and controls, C57/Bl/6J (C57).
Methods: Gracilis muscle arterioles were isolated, with mechanical responses assessed following a challenge with AA under control conditions and after elements of AA metabolism pathways were inhibited. Conduit arteries from each strain were used to assess AA-induced production of PGI2 and TxA2.
Results: Arterioles from ApoE and LDLR exhibited a blunted dilation to AA versus C57. While responses were cyclo-oxygenase-dependent in all strains, inhibition of thromboxane synthase or blockade of PGH2/TxA2 receptors improved dilation in ApoE and LDLR only. AA-induced generation of PGI2 was comparable across strains, although TxA2 generation was increased in ApoE and LDLR. Arteriolar reactivity to PGI2 and TxA2 was comparable across strains. Treatment with TEMPOL improved dilation and reduced TxA2 production with AA in ApoE and LDLR.
Conclusions: These results suggest that AA-induced arteriolar dilation is constrained in ApoE and LDLR via an increased production of TxA2. While partially due to elevated oxidant stress, additional mechanisms contribute that are independent of acute alterations in oxidant tone.
Abstract only
Our studies have demonstrated that rarefaction of the skeletal muscle microcirculation in OZR with evolution of the metabolic syndrome is well predicted by a chronic reduction to ...vascular nitric oxide (NO) bioavailability. However, prior to any loss in NO, skeletal muscle vessels from OZR exhibit a shift in arachidonic acid metabolism, with an increased production of TxA
2
over PGI
2
. Temporal analyses indicate that this early increase in vascular TxA
2
production/action is associated with an initial early rarefaction of 5–7%. If NO bioavailability is maintained (TEMPOL) after the initial increase in TxA
2
and microvessel loss, the total rarefaction magnitude is blunted. Alternately, if actions of TxA
2
are chronically inhibited, the initial rarefaction never develops and the subsequent secondary pulse (from NO reductions) is strongly muted. Chronic treatment with pentoxyfillene (to prevent TNF‐α production) or HMG CoA reductase inhibitors (atorvastatin/simvastatin) prevented much of the secondary pulse of rarefaction (associated with maintenance of NO bioavailability), yet did not alter either the shift in TxA
2
production or the initial rarefaction. These results suggest that microvessel rarefaction in OZR represents the integration of an early mild vessel loss that is dependent on TxA
2
, which creates an environment for the extensive rarefaction that is dependent on low NO bioavailability. (NIH DK64668)
Our studies have demonstrated that rarefaction of the skeletal muscle microcirculation in OZR with evolution of the metabolic syndrome is well predicted by a chronic reduction to vascular nitric ...oxide (NO) bioavailability. However, prior to any loss in NO, skeletal muscle vessels from OZR exhibit a shift in arachidonic acid metabolism, with an increased production of TxA2 over PGI2. Temporal analyses indicate that this early increase in vascular TxA2 production/action is associated with an initial early rarefaction of 5–7%. If NO bioavailability is maintained (TEMPOL) after the initial increase in TxA2 and microvessel loss, the total rarefaction magnitude is blunted. Alternately, if actions of TxA2 are chronically inhibited, the initial rarefaction never develops and the subsequent secondary pulse (from NO reductions) is strongly muted. Chronic treatment with pentoxyfillene (to prevent TNF‐α production) or HMG CoA reductase inhibitors (atorvastatin/simvastatin) prevented much of the secondary pulse of rarefaction (associated with maintenance of NO bioavailability), yet did not alter either the shift in TxA2 production or the initial rarefaction. These results suggest that microvessel rarefaction in OZR represents the integration of an early mild vessel loss that is dependent on TxA2, which creates an environment for the extensive rarefaction that is dependent on low NO bioavailability. (NIH DK64668)
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