We have used adenoviral-mediated gene transfer of a constitutively active (V12rac1) and dominant negative (N17rac1) isoform of rac1 to assess the role of this small GTPase in cardiac myocyte hypertrophy. Expression of V12rac1 in neonatal cardiac myocytes results in sarcomeric reorganization and an increase in cell size that is indistinguishable from ligand-stimulated hypertrophy. In addition, V12rac1 expression leads to an increase in atrial natriuretic peptide secretion. In contrast, expression of N17rac1, but not a truncated form of Raf-1, attenuated the morphological hypertrophy associated with phenylephrine stimulation. Consistent with the observed effects on morphology, expression of V12rac1 resulted in an increase in new protein synthesis, while N17rac1 expression inhibited phenylephrine-induced leucine incorporation. These results suggest rac1 is an essential element of the signaling pathway leading to cardiac myocyte hypertrophy. ( J. Clin. Invest. 1998. 102:929-937.)

We demonstrate that adenoviral-mediated gene transfer of a dominant negative rac1 gene product (N17rac1) inhibits the intracellular burst of reactive oxygen species (ROS) that occurs after reoxygenation of vascular smooth muscle cells. In contrast, expression of a dominant negative ras gene (N17ras) had no effect. Challenge of control cells and cells expressing N17rac1 with a direct oxidant stress produced an equivalent increase in intracellular ROS levels and subsequent cell death. This suggests that N17rac1 expression appears to block production of harmful oxygen radicals and does not act directly or indirectly to scavenge ROS generated during reoxygenation. Expression of N17rac1 results in protection from hypoxia/reoxygenation-induced cell death in a variety of cell types including vascular smooth muscle cells, fibroblasts, endothelial cells, and ventricular myocytes. These results suggest that reoxygenation injury requires the activation of rac proteins, and that inhibition of rac-dependent pathways may be a useful strategy for the prevention of reperfusion injury in ischemic tissues.

We have transiently expressed a dominant negative form of rac1 (N17rac1) using adenoviral-mediated gene transfer. The level of N17rac1 expression is demonstrated to be proportional to the multiplicity of infection. Expression of N17rac1 in Rat 2 fibroblasts results in cytostatic growth arrest. Cell-cycle analysis demonstrates that cells expressing N17rac1 accumulate in G2/M. These results suggest that rac1 is required for cell proliferation and provide the first demonstration in mammalian cells of a role for small GTP-binding proteins in the G2/M transition.

We have constructed a recombinant adenovirus (Ad.Id1) that allows for efficient expression of the helixloop-helix protein Id1. After infection with Ad.Id1, neonatal cardiac myocytes display a significant reduction in viability, which was proportional to the level of Id1 expression. A similar effect was observed in adult myocytes. Morphological and biochemical assays demonstrated that Id1 expression resulted in myocyte apoptosis. In contrast, expression of Id1 in endothelial cells, vascular smooth muscle cells, or fibroblasts did not affect the viability of these cells. Along with the induction of apoptosis, the expression of Id1 in neonatal cardiac myocytes resulted in an increase in the level of intracellular reactive oxygen species. The source of these reactive oxygen species appears to be the mitochondria. Reducing the ambient oxygen concentration or treatment with a cell-permeant H 2 O 2 scavenger prevented Id1-stimulated apoptosis in cardiac myocytes. These results suggest that the expression of Id1 leads to the induction of apoptosis in cardiac myocytes through a redox-dependent mechanism.The Id family of proteins belongs to a class of nuclear proteins known as helix-loop-helix (HLH) 1 proteins, which regulate differentiation and tissue-specific gene expression. To date, four different Id family members have been identified (1-6). Although these four proteins share a high degree of homology, especially within the HLH domain, evidence suggests that they exert nonoverlapping functions (7-9).The HLH domain acts as a protein dimerization motif. Members of the HLH protein family which act to regulate transcription positively also contain an additional basic domain that mediates binding to DNA. In contrast, members of the Id family lack this domain and therefore are unable to bind DNA. These structural differences have led to a model in which Id proteins act in a dominant negative fashion to inhibit differentiation (1, 2). Under these circumstances, expression of high levels of Id1 would result in the binding and subsequent inactivation of basic domain HLH proteins.Consistent with this model of Id function is the observation that Id expression decreases when skeletal muscle cells differentiate (10). In addition, forced expression of Id has been shown to inhibit tissue-specific gene expression or differentiation of a variety of cell types (1, 10 -12). Previous reports have demonstrated that Id levels are high in embryonic hearts and decrease after birth (13-15). Such a shift in Id expression is consistent with its role as an inhibitor of differentiation. Although Id expression is low or absent in adult cardiac myocytes in culture, its expression can be reactivated by certain stimuli, such as ␣-adrenergic agonists (14, 15). A similar induction of Id expression has been demonstrated in skeletal muscle after treatment with the cardiotoxic chemotherapeutic agent doxorubicin (16,17).Although previous studies have demonstrated that forced expression of Id proteins can block differentiation, relatively little is known a...

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A case of pulmonary alveolar microlithiasis (PAM) is reported wherein total lung lavage was performed for relief of dyspnea. Characterization of the lavage material and examination of the microliths isolated from the lavage fluid confirmed previous reports of their spherical-ovoid shape and a 2:1 calcium to phosphate composition. The microliths contained considerable amounts of ionizable iron and generated oxidants in an in vitro system. A detailed biochemical analysis of the lavage fluid reflected elevations in total protein, phosphatidylserine, phosphatidylglycerol and the ratio of phosphatidylglycerol to phosphatidylinositol. Surfactant apoprotein-A levels approximated that of normal patients. The potential roles of oxidant generation and alterations in surfactant metabolism are discussed in the context of the pathogenesis of PAM.

Objective: To compare economic and clinical outcomes of barbed sutures versus conventional sutures alone in wound closure for patients undergoing spinal surgery. Method: A retrospective study using the Premier Healthcare Database. The database was searched for patients who underwent elective inpatient spinal surgery (fusion or laminectomy) for a spinal disorder between 1 January 2014 and 30 June 2018 (first=index admission). Using billing records for medical supplies used during the index admission, patients were classified into mutually-exclusive groups: patients with any use of STRATAFIX (Ethicon, US) knotless tissue control devices (barbed sutures group); or patients with use of conventional sutures alone (conventional sutures group). Outcomes included the index admission's length of stay, total and subcategories of hospital costs, non-home discharge, operating room time (ORT, minutes), wound complications and readmissions within ≤90 days. Propensity score matching and generalised estimating equations were used to compare outcomes between the study groups. Results: After matching, 3705 patients were allocated to each group (mean age=61.5 years [standard deviation, SD±12.9]; 54% were females). Compared with the conventional suture group, the barbed suture group had significantly lower mean ORT (239±117 minutes, versus 263±79 minutes conventional sutures, p=0.015). Operating room costs were also siginificantly lower in the barbed suture group ($6673±$3976 versus $7100±$2700 conventional sutures, p=0.020). Differences were statistically insignificant for other outcomes (all p>0.05). Subanalysis of patients undergoing fusions of ≥2 vertebral joints yielded consistent results. Conclusion: In this study, wound closure incorporating barbed sutures was associated with lower ORT and operating room costs, with no significant difference in wound complications or readmissions, when compared with conventional sutures alone.

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