Intronic enhancement of angiotensin II type 2 receptor transgene expression in vitro and in vivo

The angiotensin II type 2 receptor (AT2R) can influence a variety of intracellular signaling molecules and cellular functions. However, its physiological functions and the roles of introns in the regulation of its expression have not been well determined. We first demonstrated that both intron 1 and intron 2 of AT2R could significantly enhance AT2R overexpression. Thus, we have provided some new prerequisites for further studies on the mechanisms that control AT2R gene expression. Next, we established a highly efficient method of delivering this receptor in vitro and in vivo using an AT2R recombinant adenoviral vector containing two introns of the AT2R. The vector may be useful in helping to uncover AT2R physiological functions and also for gene therapy related to AT2R. Moreover, we determined the important role of introns in gene expression cassettes and the inconsistency of expression between the targeted gene and the reporter gene.     

ACE2: A novel therapeutic target for cardiovascular diseases

Hypertension afflicts over 65 million Americans and poses an increased risk for cardiovascular morbidity such as stroke, myocardial infarction and end-stage renal disease resulting in significant mortality. Overactivity of the renin-angiotensin system (RAS) has been identified as an important determinant that is implicated in the etiology of these diseases and therefore represents a major target for therapy. In spite of the successes of drugs inhibiting various elements of the RAS, the incidence of hypertension and cardiovascular diseases remain steadily on the rise. This has lead many investigators to seek novel and innovative approaches, taking advantage of new pathways and technologies, for the control and possibly the cure of hypertension and related pathologies. The main objective of this review is to forward the concept that gene therapy and the genetic targeting of the RAS is the future avenue for the successful control and treatment of hypertension and cardiovascular diseases. We will present argument that genetic targeting of angiotensin-converting enzyme 2 (ACE2), a newly discovered member of the RAS, is ideally poised for this purpose. This will be accomplished by discussion of the following: (i) summary of our current understanding of the RAS with a focus on the systemic versus tissue counterparts as they relate to hypertension and other cardiovascular pathologies; (ii) the newly discovered ACE2 enzyme with its physiological and pathophysiological implications; (iii) summary of the current antihypertensive pharmacotherapy and its limitations; (iv) the discovery and design of ACE inhibitors; (v) the emerging concepts for ACE2 drug design; (vi) the current status of genetic targeting of the RAS; (vii) the potential of ACE2 as a therapeutic target for hypertension and cardiovascular disease treatment; and (viii) future perspectives for the treatment of cardiovascular diseases.     

Microbial and enzymatic improvement of anaerobic digestion of waste biomass

Metabolic activities of different microorganisms (Bacillus subtilis, B. licheniformis and Aspergillus niger) and hydrolytic enzymes (concentrations: 1 to 200 mg enzyme solids g^–1 feed) were studied individually and in combinations with respect to H₂ and methane production from damaged wheat grains. Bacillus subtilis, B. licheniformis and pre-existing hydrogen producers (control) produced 45 to 64 l H₂ kg^–1 total solids and subsequently, with the help of added methanogens, 155 to 220 l methane kg^–1 total solids could be produced. H₂ production from damaged wheat grains could be decreased to 28% or enhanced up to 152% with respect to control, by employing various microbial and enzymatic treatments. Similarly, it has been made possible to vary methane production capacities from as low as 17% to as high as 110% with respect to control.     

Critical role of mitochondrial dysfunction and impaired mitophagy in diabetic nephropathy

Mitochondrial dynamics play a critical role in deciding the fate of a cell under normal and diseased condition. Recent surge of studies indicate their regulatory role in meeting energy demands in renal cells making them critical entities in the progression of diabetic nephropathy. Diabetes is remarkably associated with abnormal fuel metabolism, a basis for free radical generation, which if left unchecked may devastate the mitochondria structurally and functionally. Impaired mitochondrial function and their aberrant accumulation have been known to be involved in the manifestation of diabetic nephropathy, indicating perturbed balance of mitochondrial dynamics, and mitochondrial turnover. Mitochondrial dynamics emphasize the critical role of mitochondrial fission proteins such as mitochondrial fission 1, dynamin-related protein 1 and mitochondrial fission factor and fusion proteins including mitofusin-1, mitofusin-2 and optic atrophy 1. Clearance of dysfunctional mitochondria is aided by translocation of autophagy machinery to the impaired mitochondria and subsequent activation of mitophagy regulating proteins PTEN-induced putative kinase 1 and Parkin, for which mitochondrial fission is a prior event. In this review, we discuss recent progression in our understanding of the molecular mechanisms targeting reactive oxygen species mediated alterations in mitochondrial energetics, mitophagy related disorders, impaired glucose transport, tubular atrophy, and renal cell death. The molecular cross talks linking autophagy and renoprotection through an intervention of 5′-AMP-activated protein kinase, mammalian target of rapamycin, and SIRT1 factors are also highlighted here, as in-depth exploration of these pathways may help in deriving therapeutic strategies for managing diabetes provoked end-stage renal disease.     

Insulin-like growth factor I receptors in neuronal and glial cells. Characterization and biological effects in primary culture

Primary cultures of neuronal and glial cells from 1-day-old neonatal rats contain high affinity receptors for insulin-like growth factor I (IGF-I). The IC50 for displacement of 125I-IGF-I binding by unlabeled IGF-I was 3 nM for neuronal cells and 4 nM for glial cells. Unlabeled insulin was 20-50 times less potent. Apparent molecular mass of the alpha subunits of the IGF-I receptor was 125 kDa in neuronal and 135 kDa in glial cells. IGF-I induced autophosphorylation of the IGF-I receptor beta subunit in lectin-purified membrane preparations in a dose-dependent manner. The major phosphoamino acid of the beta subunit in both cell types was tyrosine in the IGF-I-stimulated state and serine in the basal state. Apparent molecular mass of the beta subunits of the IGF-I receptors was 91 kDa for neuronal and 95 kDa for glial cells. Tyrosine kinase activity of the IGF-I receptors was demonstrated by IGF-I-induced phosphorylation of the exogenous substrate poly(Glu, Tyr) 4:1 in both cell types. IGF-I had no effect on 2-deoxyglucose uptake in neuronal cells. In contrast, in glial cells, IGF-I stimulated 2-deoxyglucose uptake at very high doses, presumably acting via the insulin receptor. The effect of IGF-I as a neurotrophic growth factor in both neuronal and glial cells was demonstrated by its stimulation of [3H]thymidine incorporation. These findings suggest the IGF-I is an important growth factor in nervous tissue-derived cells.