Absorption of intact albumin across rat alveolar epithelial cell monolayers

Transport characteristics of intact albumin were investigated using primary cultured rat alveolar epithelial cell monolayers. The apical-to-basolateral (ab) flux of intact fluorescein isothiocyanate (FITC)-labeled albumin (F-Alb) is greater than basolateral-to-apical (ba) flux at the same upstream [F-Alb]. Net absorption of intact F-Alb occurs with half-maximal concentration of approximately 1.6 microM and maximal transport rate of approximately 0.15 fmol.cm(-2).s(-1). At 15 and 4 degrees C, both ab and ba F-Alb fluxes are not different from zero, collapsing net absorption. The presence of excess unlabeled albumin (but not other macromolecule species) in either the apical or basolateral fluid significantly reduces both ab and ba unidirectional F-Alb fluxes. Photoaffinity labeling of apical cell membranes revealed an approximately 60-kDa protein that exhibits specificity for albumin. These data indicate that net absorption of intact albumin takes place via saturable receptor-mediated transcellular endocytotic processes recognizing albumin, but not other macromolecules, that may play an important role in alveolar homeostasis in the mammalian lung.     

Central leptin gene therapy suppresses body weight gain, adiposity and serum insulin without affecting food consumption in normal rats: a long-term study

The weight-reducing effects of leptin are predominantly mediated through the hypothalamus in the brain. Gene therapy strategies designed for weight control have so far tested the short-term effect of peripherally delivered viral vectors encoding the leptin gene. In order to circumvent the multiple peripheral effects of hyperleptinemia and to overcome the age-related development of leptin resistance due to multiple factors, including defective leptin transport across the blood brain barrier, we determined whether delivery of viral vectors directly into the brain is a viable therapeutic strategy for long-term weight control in normal wild-type rats. A recombinant adeno-associated virus (rAAV) vector encoding rat leptin (Ob) cDNA was generated (rAAV-betaOb). When administered once intracerebroventricularly (i.c.v.), rAAV-betaOb suppressed the normal time-related weight gain for extended periods of time in adult Sprague-Dawley rats. The vector expression was confirmed by immunocytochemical localization of GFP and RT-PCR analysis of leptin in the hypothalamus. This sustained restraint on weight gain was not due to shifts in caloric consumption because food-intake was similar in rAAV-betaOb-treated and rAAV-GFP-treated control rats throughout the experiment. Weight gain suppression, first apparent after 2 weeks, was a result of reduced white fat depots and was accompanied by drastically reduced serum leptin and insulin concentrations in conjunction with normoglycemia. Additionally, there was a marked increase in uncoupling protein-1 (UCP1) mRNA expression in brown adipose tissue, thereby indicating increased energy expenditure through thermogenesis. Seemingly, a selective enhancement in energy expenditure following central delivery of the leptin gene is a viable therapeutic strategy to control the age-related weight gain and provide protection from the accompanying multiple peripheral effects of hyperleptinemia and hyperinsulinemia.     

Effect of endothelial cell polarity on beta-amyloid-induced migration of monocytes across normal and AD endothelium

During normal aging and amyloid beta-peptide (Abeta) disorders such as Alzheimer's disease (AD), one finds increased deposition of Abeta and activated monocytes/microglial cells in the brain. Our previous studies show that Abeta interaction with a monolayer of normal human brain microvascular endothelial cells results in increased adherence and transmigration of monocytes. Relatively little is known of the role of Abeta accumulated in the AD brain in mediating trafficking of peripheral blood monocytes (PBM) across the blood-brain barrier (BBB) and concomitant accumulation of monocytes/microglia in the AD brain. In this study, we showed that interaction of Abeta(1--40) with apical surface of monolayer of brain endothelial cells (BEC), derived either from normal or AD individuals, resulted in increased transendothelial migration of monocytic cells (HL-60 and THP-1) and PBM. However, transmigration of monocytes across the BEC monolayer cultivated in a Transwell chamber was increased 2.5-fold when Abeta was added to the basolateral side of AD compared with normal individual BEC. The Abeta-induced transmigration of monocytes was inhibited in both normal and AD-BEC by antibodies to the putative Abeta receptor, receptor for advanced glycation end products (RAGE), and to the endothelial cell junction molecule, platelet-endothelial cell adhesion molecule-1 (PECAM-1). We conclude that interaction of Abeta with the basolateral surface of AD-BEC induces cellular signaling, promoting transmigration of monocytes from the apical to basolateral direction. We suggest that Abeta in the AD brain parenchyma or cerebrovasculature initiates cellular signaling that induces PBM to transmigrate across the BBB and accumulate in the brain.     

Fermentation of enzymatically saccharified sunflower stalks for ethanol production and its scale up

Pretreated sunflower stalks saccharified with a Trichoderma reesei Rut-C 30 cellulase showed 57.8% saccharification. Enzyme hydrolysate concentrated to 40 g/l reducing sugars was fermented under optimum conditions of fermentation time (24 h), pH (5.0), temperature (30 degrees C) and inoculum size (3% v/v) and, showed a maximum ethanol yield of 0.444 g/g ethanol. Ethanol production scaled up in a 1 l and a 15 l fermenter under optimum conditions revealed maximum ethanol yields of 0.439 and 0.437 g/g respectively.     

Central LIF gene therapy suppresses food intake,body weight,serum leptin and insulin for extended periods

Leukemia inhibitory factor (LIF) overexpression, induced by the intracerebroventricular (i.c.v.) injection of an recombinant adeno-associated viral vector encoding LIF (rAAV-LIF), resulted in a dose-dependent reduction in body weight (BW) gain, food intake (FI) and adiposity, evidenced by suppression of serum leptin and free fatty acids for an extended period in outbred adult female rats. A dose-dependent reduction in serum insulin levels and unchanged serum glucose, energy expenditure through thermogenesis as indicated by uncoupling protein-1 (UCP-1) mRNA expression in brown adipose tissue (BAT), and metabolism as indicated by serum T3 and T4, accompanied the blockade of weight gain. Thus, central rAAV-LIF therapy is a viable strategy to voluntarily reduce appetite and circumvent leptin resistance, a primary factor underlying age-dependent weight gain and obesity in rodents and humans.     

Polyester and polycarbonate synthesis by in vitro enzyme catalysis

Enzyme technology has significantly expanded in scope and impact over the past 10 years to include organic transformations in non-traditional environments. This is in part due to an increased understanding and capability of using enzyme catalysis in a wide variety of organic solvents, at interfaces, and at high temperatures and pressures. This review focuses on a relatively new but rapidly expanding research activity where in vitro enzyme catalysis is used for the synthesis of non-natural polyesters and polycarbonates. The inclination to use of enzymes for polymer synthesis has been fueled by a desire to carry out these reactions in the absence of heavy metals, at lower temperatures, and with increased selectivity. Aspects of this work that include enzyme-catalyzed step-growth condensation reactions, chain-growth ring-opening polymerizations, and corresponding transesterification of macromolecular substrates are discussed.     

A GABA-neuropeptide Y (NPY) interplay in LH release

Neuropeptide Y (NPY) stimulates and gamma-amino butyric acid (GABA) inhibits LH release in the rat. Since a sub-population of NPY-producing neurons in the arcuate nucleus (ARC) of the hypothalamus co-express GABA, the possibility of an interplay between NPY and GABA in the release of LH was investigated in two ways. First by employing light and electron microscopic double staining for NPY and GABA, using pre and post-immunolabeling on rat brain sections, we detected GABA in NPY immunoreactive axon terminals in the MPOA, one of the primary sites of action of these neurotransmitters/neuromodulators in the regulation of LH release. These morphological findings raised the possibility that inhibitory GABA co-released with NPY may act to restrain the excitatory effects of NPY on LH release. Muscimol (MUS, 0.44 or 1.76 nmol/rat), a GABA(A) receptor agonist, administered intracerebroventricularly (icv), alone failed to affect LH release, but NPY (0.47 nmol/rat icv) alone stimulated LH release in ovarian steroid-primed ovariectomized rats. On the other hand, administration of MUS blocked the NPY-induced stimulation of LH release in a dose-dependent manner. Similarly, administration of MUS abolished the excitatory effects on LH release of 1229U91, a selective NPY Y4 receptor agonist. These results support the possibility that in the event of co-release of these neurotransmitters/neuromodulators, GABA may act to restrain stimulation of LH release by NPY during the basal episodic and cyclic release of LH in vivo.     

beta-amyloid-induced migration of monocytes across human brain endothelial cells involves RAGE and PECAM-1

In patients withamyloid -related cerebrovascular disorders, e.g., Alzheimer'sdisease, one finds increased deposition of amyloid peptide (A) andincreased presence of monocyte/microglia cells in the brain. However,relatively little is known of the role of A in the trafficking ofmonocytes across the blood-brain barrier (BBB). Our studies show thatinteraction of A_1-40 with monolayer of human brainendothelial cells results in augmented adhesion and transendothelialmigration of monocytic cells (THP-1 and HL-60) and peripheral bloodmonocytes. The A-mediated migration of monocytes was inhibited byantibody to A receptor (RAGE) and platelet endothelial cell adhesionmolecule (PECAM-1). Additionally, A-induced transendothelialmigration of monocytes were inhibited by protein kinase C inhibitor andaugmented by phosphatase inhibitor. We conclude that interaction ofA with RAGE expressed on brain endothelial cells initiates cellularsignaling leading to the transendothelial migration of monocytes. Wesuggest that increased diapedesis of monocytes across the BBB inresponse to A present either in the peripheral circulation or in thebrain parenchyma may play a role in the pathophysiology of A-relatedvascular disorder.