The folate pathway is a target for resistance to the drug para-aminosalicylic acid (PAS) in mycobacteria

The increasing rate of multidrug-resistant tuberculosis has led to more use of second-line antibiotics such as para-aminosalicylic acid (PAS). The mode of action of PAS remains unclear, and mechanisms of resistance to this drug are undefined. We have isolated PAS-resistant transposon mutants of Mycobacterium bovis BCG with insertions in the thymidylate synthase (thyA) gene, a critical determinant of intracellular folate levels. BCG thyA mutants have reduced thymidylate synthase activity and are resistant to known inhibitors of the folate pathway. We also find that mutations in thyA are associated with clinical PAS resistance. We have identified PAS-resistant Mycobacterium tuberculosis isolates from infected patients, which harbour mutations in thyA and show reduced activity of the encoded enzyme. Thus, PAS acts in the folate pathway, and thyA mutations probably represent a mechanism of developing resistance not only to PAS but also to other drugs that target folate metabolism.     

Skin tight: cell adhesion in the epidermis of Caenorhabditis elegans

The powerful genetics, genomics and microscopy tools available for C. elegans make it well suited to studying how epithelial cells adhere to one another and the extracellular matrix, and how the integrated, simultaneous activities of multiple cell adhesion complexes function to shape an organism. Recent studies using forward and reverse genetics have shed light on how phylogenetically conserved cell adhesion complexes, such as the cadherin/catenin complex, claudins, the Discs large complex and hemidesmosome-like attachment structures, regulate epithelial cell adhesion, providing new insights into conserved cell adhesion mechanisms in higher eukaryotes.     

Intensive statin therapy in acute coronary syndromes: clinical benefits and vascular biology

PURPOSE OF REVIEW: The results of a landmark clinical study comparing intensive statin therapy with conventional statin therapy, in patients with acute coronary syndromes (ACS), are reviewed. The mechanisms behind these results are analysed drawing data from vascular and cell biology. RECENT FINDINGS: The Pravastatin or Atorvastatin Evaluation and Infection Therapy-Thrombolysis in Myocardial Infarction (PROVE IT-TIMI 22) study showed that intensive statin therapy with 80 mg of atorvastatin to achieve a low-density lipoprotein cholesterol of 62 mg/dl resulted in a 3.9% absolute and a 16% relative risk reduction in death or major cardiovascular events up to 2 years, compared to 40 mg of pravastatin, in patients with ACS. The results were especially significant as intensive statin therapy resulted in a very early benefit (<30 days) and occurred against a background of percutaneous coronary intervention (69%) for the index admission and high use of medications for secondary prevention. The PROVE IT and the Myocardial Ischaemia Reduction with Aggressive Cholesterol Lowering (MIRACL) C-reactive protein sub-study also showed that atorvastatin (80 mg) resulted in a significant reduction in markers of inflammation, whilst the Reversal of Atherosclerosis with Aggressive Lipid Lowering (REVERSAL) study showed that intensive statin therapy was associated with reduced progression of atherosclerosis compared with conventional doses of statins. SUMMARY: Intensive statin therapy results in a significant early reduction in adverse cardiac events in ACS patients which are sustained over 2 years. The early benefits seen are likely to result from modulation of inflammation, endothelial function and coagulation, i.e. the pleiotropic effects, whereas the greater reduction in low-density lipoprotein cholesterol results in reduced long-term events.     

Comparison of effects of exercise and diuretic on left ventricular geometry, mass, and insulin resistance in older hypertensive adults

To compare the effects of exercise training and hydrochlorothiazide on left ventricular (LV) geometry and mass, blood pressure (BP), and hyperinsulinemia in older hypertensive adults, we studied 28 patients randomized either to a group (age 66.4 +/- 1.3 yr; n = 16) that exercised or to a group (age 65.3 +/- 1.2 yr; n = 12) that received hydrochlorothiazide for 6 mo. Endurance exercise training induced a 15% increase in peak aerobic power. The reduction in systolic BP was twofold greater with thiazide than with exercise (26.6 +/- 12.2 vs. 11.5 +/- 10.9 mmHg). Exercise and thiazide reduced LV wall thickness, LV mass index (14% in each group), and the LV wall thickness-to-radius ratio (h/r) similarly (exercise: before 0.48 +/- 0.2, after 0.42 +/- 0.01; thiazide: before 0.47 +/- 0.04, after 0.40 +/- 0.04; P = 0.017). The reductions in systolic BP and h/r were correlated in the exercise group (r = 0.70, P = 0.005) but not in the thiazide group. Exercise training reduced glucose-stimulated hyperinsulinemia (before: 13.65 +/- 2.6 vs. 9.84 +/- 1.5 mU.ml(-1).min; P = 0.04) and insulin resistance. Thiazide did not affect plasma insulin levels. The results suggest that although exercise is less effective in reducing systolic BP than thiazide, it can induce regression of LV hypertrophy similar in magnitude to thiazide. Unlike hydrochlorothiazide, exercise training can improve insulin resistance and aerobic capacity in older hypertensive people.     

Coverslip hypoxia: a novel method for studying cardiac myocyte hypoxia and ischemia in vitro

In vitro experimental models designed to study the effects of hypoxia and ischemia typically employ oxygen-depleted media and/or hypoxic chambers. These approaches, however, allow for metabolites to diffuse away into a large volume and may not replicate the high local concentrations that occur in ischemic myocardium in vivo. We describe herein a novel and simple method for creating regional hypoxic and ischemic conditions in neonatal rat cardiac myocyte monolayers. This method consists of creating a localized diffusion barrier by placing a glass coverslip over a portion of the monolayer. The coverslip restricts covered myocytes to a thin film of media while leaving uncovered myocytes free to access the surrounding bulk media volume. Myocytes under the coverslip undergo marked morphology changes over time as assessed by video microscopy. Fluorescence microscopy shows that these changes are accompanied by alterations in mitochondrial membrane potential and plasma membrane dynamics and eventually result in myocyte death. We also show that the metabolic activity of myocytes drives cell necrosis under the coverslip. In addition, the intracellular pH of synchronously contracting myocytes under the coverslip drops rapidly, which further implicates metabolic activity in regulating cell death under the coverslip. In contrast with existing models of hypoxia/ischemia, this technique provides a simple and effective way to create hypoxic/ischemic conditions in vitro. Moreover, we conclude that myocyte death is hastened by the combination of hypoxia, metabolites, and acidosis and is facilitated by a reduction in media volume, which may better represent ischemic conditions in vivo.     

Mechanical anisotropy of adherent cells probed by a three-dimensional magnetic twisting device

We describe a three-dimensional magnetic twisting device that is useful in characterizing the mechanical properties of cells. With the use of three pairs of orthogonally aligned coils, oscillatory mechanical torque was applied to magnetic beads about any chosen axis. Frequencies up to 1 kHz could be attained. Cell deformation was measured in response to torque applied via an RGD-coated, surface-bound magnetic bead. In both unpatterned and micropatterned elongated cells on extracellular matrix, the mechanical stiffness transverse to the long axis of the cell was less than half that parallel to the long axis. Elongated cells on poly-L-lysine lost stress fibers and exhibited little mechanical anisotropy; disrupting the actin cytoskeleton or decreasing cytoskeletal tension substantially decreased the anisotropy. These results suggest that mechanical anisotropy originates from intrinsic cytoskeletal tension within the stress fibers. Deformation patterns of the cytoskeleton and the nucleolus were sensitive to loading direction, suggesting anisotropic mechanical signaling. This technology may be useful for elucidating the structural basis of mechanotransduction. cytoskeleton; prestress; stress fibers; mechanotransduction; mechanical deformation     

Role of immunoreceptor tyrosine-based inhibitory motifs of PECAM-1 in PECAM-1-dependent cell migration

Platelet endothelial cell adhesion molecule (PECAM-1), a transmembrane glycoprotein, has been implicated in angiogenesis, with recent evidence indicating the involvement of PECAM-1 in endothelial cell motility. The cytoplasmic domain of PECAM-1 contains two tyrosine residues, Y663 and Y686, that each fall within an immunoreceptor tyrosine-based inhibitory motif (ITIM). When phosphorylated, these residues together mediate the binding of the protein tyrosine phosphatase SHP-2. Because SHP-2 has been shown to be involved in the turnover of focal adhesions, a phenomenon required for efficient cell motility, the association of this phosphatase with PECAM-1 via its ITIMs may represent a mechanism by which PECAM-1 might facilitate cell migration. Studies were therefore done with cell transfectants expressing wild-type PECAM or mutant PECAM-1 in which residues Y663 and Y686 were mutated. These mutations eliminated PECAM-1 tyrosine phosphorylation and the association of PECAM-1 with SHP-2 but did not impair the ability of the molecule to localize at intercellular junctions or to bind homophilically. However, in vitro cell motility and tube formation stimulated by the expression of wild-type PECAM-1 were abrogated by the mutation of these tyrosine residues. Importantly, during wound-induced migration, the number of focal adhesions as well as the level of tyrosine phosphorylated paxillin detected in cells expressing wild-type PECAM-1 were markedly reduced compared with control cells or transfectants with mutant PECAM-1. These data suggest that, in vivo, the binding of SHP-2 to PECAM-1, via PECAM-1’s ITIM domains, promotes the turnover of focal adhesions and, hence, endothelial cell motility. platelet endothelial cell adhesion molecule-1; endothelial cells; angiogenesis     

5-lipoxygenase and cyclooxygenase regulate wound closure in NIH/3T3 fibroblast monolayers

Wound healing involves multiple cell signaling pathways, including those regulating cell-extracellular matrix adhesion. Previous work demonstrated that arachidonate oxidation to leukotriene B₄ (LTB₄) by 5-lipoxygenase (5-LOX) signals fibroblast spreading on fibronectin, whereas cyclooxygenase-2 (COX-2)-catalyzed prostaglandin E₂ (PGE₂) formation facilitates subsequent cell migration. We investigated arachidonate metabolite signaling in wound closure of perturbed NIH/3T3 fibroblast monolayers. We found that during initial stages of wound closure (0–120 min), all wound margin cells spread into the wound gap perpendicularly to the wound long axis. At regular intervals, between 120 and 300 min, some cells elongated to project across the wound and meet cells from the opposite margin, forming distinct cell bridges spanning the wound that act as foci for later wound-directed cell migration and resulting closure. 5-LOX inhibition by AA861 demonstrated a required LTB₄ signal for initial marginal cell spreading and bridge formation, both of which must precede wound-directed cell migration. 5-LOX inhibition effects were reversible by exogenous LTB₄. Conversely, COX inhibition by indomethacin reduced directed migration into the wound but enhanced early cell spreading and bridge formation. Exogenous PGE₂ reversed this effect and increased cell migration into the wound. The differential effects of arachidonic acid metabolites produced by LOX and COX were further confirmed with NIH/3T3 fibroblast cell lines constitutively over- and underexpressing the 5-LOX and COX-2 enzymes. These data suggest that two competing oxidative enzymes in arachidonate metabolism, LOX and COX, differentially regulate sequential aspects of fibroblast wound closure in vitro. leukotriene B₄; prostaglandin E₂; spreading; migration; bridges     

Chronic beta-adrenoreceptor activation increases cardiac cavity size through chamber remodeling and not via modifications in myocardial material properties

Chronic beta-adrenoreceptor (beta-AR) activation increases left ventricular (LV) cavity size by promoting a rightward shift in LV diastolic pressure-volume (P-V) relations in association with increases in low-tensile strength myocardial (non-cross-linked) collagen concentrations. Because diastolic P-V relations are determined by chamber remodeling as well as by myocardial material properties (indexed by myocardial stiffness), both of which are associated with modifications in myocardial collagen cross-linking, we evaluated whether chamber remodeling or alterations in myocardial material properties govern beta-AR-mediated modifications in diastolic P-V relations. The effects of chronic administration of isoproterenol (Iso; 0.04 mg.kg(-1).day(-1) from 12 to 19 mo of age) to spontaneously hypertensive rats (SHRs) on LV cavity dimensions, LV diastolic P-V relations, myocardial collagen characteristics, myocardial stiffness constants [e.g., the slope of the LV diastolic stress-strain relation (k)], and LV chamber and myocardial systolic function were assessed. SHRs at 19 mo of age had normal LV diastolic P-V relations, marked myocardial fibrosis (using a pathological score), increased myocardial cross-linked (insoluble to cyanogen bromide digestion) type I and type III collagen concentrations, and enhanced myocardial k values. Iso administration to SHRs resulted in enlarged LV cavity dimensions mediated by a rightward shift in LV diastolic P-V relations, increased volume intercept of the LV diastolic P-V relation, decreased LV relative wall thickness despite a tendency to augment LV hypertrophy, and increased non-cross-linked type I and type III myocardial collagen concentrations. Iso administration resulted in reduced pump function without modification of intrinsic myocardial systolic function. However, despite increasing myocardial non-cross-linked concentrations, Iso failed to alter myocardial k in SHRs. These results suggest that beta-AR-mediated rightward shifts in LV diastolic P-V relations, which induce decreased pump function, are mediated by chamber remodeling but not by modifications in myocardial material properties.