Stabilization of poly-L-lysine/DNA polyplexes for in vivo gene delivery to the liver.
We are developing a self-assembling non-viral in vivo gene delivery vehicle based on poly-l-lysine and plasmid DNA. We have characterized poly-l-lysines of different chain lengths for DNA condensation and strength of DNA binding. Poly-l-lysine chains >20 residues bound DNA efficiently in physiological saline, while shorter chains did not. Attachment of asialoorosomucoid to PLL increased the PLL chain length required for efficient DNA binding in saline and for efficient DNA condensation. By electron microscopy, poly-l-lysine/DNA polyplexes appeared as toroids 25-50 nm in diameter or rods 40-80 nm long; conjugation of asialoorosomucoid to the polylysine component increased the size of resulting polyplexes to 50-90 nm. In water, poly-l-lysine and asialoorosomucoid-PLL polyplexes have effective diameters of 46 and 87.6 nm, respectively. Polyplexes containing only poly-l-lysine and DNA aggregated in physiological saline at all charge ratios and aggregated at neutral charge ratios in water. Attachment of asialoorosomucoid lessened, but did not eliminate, the aggregation of PLL polyplexes, and did not result in efficient delivery of polyplexes to hepatocytes. Conjugation of polyethylene glycol to poly-l-lysine sterically stabilized resulting polyplexes at neutral charge ratios by shielding the surfaces. For efficient in vivo gene delivery, polyplexes will need to be sterically stabilized to prevent aggregation and interaction with serum components. (+info)
The effect of age and teat order on alpha1-acid glycoprotein, neutrophil-to-lymphocyte ratio, cortisol, and average daily gain in commercial growing pigs.
The objectives of the study were to evaluate age and teat order on a performance trait, average daily gain, and on physiological stress indicators, alpha1-acid glycoprotein (AGP), neutrophil-to-lymphocyte ratio (N:L), and cortisol in commercial growing pigs from weaning to market age. Pigs (n = 129) from five commercial California farms were weighed and blood-sampled at 28-d intervals from 28 to 168 d of age. Laboratory assays were performed from blood samples to quantify cortisol, AGP, and N:L. Age and facility effects (P<.001), but not teat order effects (P>.05), were found for all three physiological traits and ADG. Pigs that routinely suckled from teats 1, 4, or 6 (numbered from anterior to posterior on the upper teat bank) had similar (P>.05) ADG and BW throughout the production cycle. No correlation (P> .05) was found between cortisol, AGP, and N:L. The use of these physiological and production traits as stress and health indices of growing pigs in commercial facilities has limitations in comparing data between facilities or different ages of pigs. (+info)
Altered pharmacokinetics of a novel anticancer drug, UCN-01, caused by specific high affinity binding to alpha1-acid glycoprotein in humans.
The large species difference in the pharmacokinetics/pharmacodynamics of 7-hydroxystaurosporine (UCN-01) can be partially explained by the high affinity binding of UCN-01 to human alpha1-acid glycoprotein (AGP) (Fuse et al, Cancer Res., 58: 3248-3253, 1998). To confirm whether its binding to human AGP actually changes the in vivo pharmacokinetics, we have studied the alteration in its pharmacokinetics after simultaneous administration of human AGP to rats: (a) the protein binding of UCN-01 was evaluated by chasing its dissociation from proteins using dextran-coated charcoal. The UCN-01 remaining 0.1 h after adding dextran-coated charcoal to human plasma or AGP was approximately 80%, although the values for other specimens, except monkey plasma (approximately 20%), were <1%, indicating that the dissociation from human AGP was specifically slower than from other proteins; and (b) the pharmacokinetics of UCN-01 simultaneously administered with human AGP has been determined. The plasma concentrations after i.v. administration of UCN-O1 with equimolar human AGP were much higher than those after administration of UCN-01 alone. The steady-state distribution volume and the systemic clearance were reduced to about 1/100 and 1/200, respectively. Human AGP thus reduced the distribution and elimination of UCN-01 substantially. On the other hand, dog AGP, which has a low binding affinity for UCN-01, did not change the pharmacokinetics of UCN-01 so much. Furthermore, human AGP markedly reduced the hepatic extraction ratio of UCN-01 from 0.510 to 0.0326. Also, human AGP (10 microM) completely inhibited the initial uptake of UCN-01 (1 microM) into isolated rat hepatocytes, whereas the uptake of UCN-01 was unchanged in the presence of human serum albumin (10 microM). In conclusion, the high degree of binding of UCN-01 to human AGP causes a reduction in the distribution and clearance, resulting in high plasma concentrations in humans. (+info)
Sequential deglycosylation and utilization of the N-linked, complex-type glycans of human alpha1-acid glycoprotein mediates growth of Streptococcus oralis.
Streptococcus oralis is the agent of a large number of infections in immunocompromised patients, but little is known regarding the mechanisms by which this fermentative organism proliferates in vivo. Glycoproteins are widespread within the circulation and host tissues, and could provide a source of fermentable carbohydrate for the growth of those pathogenic organisms with the capacity to release monosaccharides from glycans via the production of specific glycosidases. The ability of acute phase serum alpha1-acid glycoprotein to support growth of S.oralis in vitro has been examined as a model for growth of this organism on N-linked glycoproteins. Growth was accompanied by the production of a range of glycosidases (sialidase, N-acetyl-beta-D-glucosaminidase, and beta-D-galactosidase) as measured using the 4-methylumbelliferone-linked substrates. The residual glycoprotein glycans remaining during growth of this organism were released by treatment with hydrazine and their analysis by HPAEC-PAD and MALDI demonstrated extensive degradation of all glycan chains with only terminal N-acetylglucosamine residues attached to asparagines of the protein backbone remaining when growth was complete. Monosaccharides were released sequentially from the glycans by S.oralis glycosidases in the order sialic acid, galactose, fucose, nonterminal N-acetylglucosamine, and mannose due to the actions of exo-glycosidic activities, including mannosidases which have not previously been reported for S.oralis. All released monosaccharides were metabolized during growth with the exception of fucose which remained free in culture supernatants. Direct release of oligosaccharides was not observed, indicating the absence of endo-glycosidases in S.oralis. We propose that this mechanism of deglycosylation of host glycoproteins and the subsequent utilization of released monosaccharides is important in the survival and persistence of this and other pathogenic bacteria in vivo. (+info)
Serum alpha 1-acid glycoprotein, sialic acid, and protein binding of disopyramide in normal subjects and cardiac patients.
AIM: To study influence of congestive heart failure (CHF) and acute myocardial infarction (AMI) on alpha 1-acid glycoprotein (AGP) and sialic acid (SA) concentration, and binding of AGP to disopyramide (Dis). METHODS: Sera from 85 healthy subjects, 6 patients with CHF, and 6 patients with AMI were determined by immunochemistry for AGP, by HPLC method for sialic acid (SA), and by ultrafiltration and HPLC for the free fraction of Dis. RESULTS: Serum AGP concentrations (g.L-1) were 0.74 +/- 0.16 (healthy), 1.18 +/- 0.40 (d 1, CHF) and 0.90 +/- 0.24 (d 14, CHF), 1.53 +/- 0.26 (d 5, AMI) and 1.08 (d 14, AMI). The free Dis were 1.76 +/- 0.62 (d 1) and 2.14 +/- 0.48 (d 14), in CHF patients, 1.66 +/- 0.52 (d 5) and 1.77 (d 14) in AMI patients. The changes of serum SA and AGP concentrations showed the same tendency. CONCLUSION: The free Dis in serum was affected by the change of AGP binding in CHF and AMI patients. (+info)
Effects of individual housing design and size on behavior and stress indicators of special-fed Holstein veal calves.
The objectives of this study were to determine effects of housing design (calves tethered in open stalls vs untethered in individual pens) and widths of 56, 66, and 76 cm (2 x 3 factorial arrangement of treatments) on indicators of stress and behavior in special-fed veal calves. Three production cycles (groups) were used, each with 36 Holstein bull calves. Calves (n = 108) were randomly allotted to treatments upon arrival at the facility. Blood samples were collected four times (wk 4, 9, 13, and 18) during the 18-wk production cycle. Blood serum values for cortisol and alpha1-acid glycoprotein (AGP) exhibited few treatment differences. Blood leukocyte differential counts at 4 and 18 wk (segmented neutrophils [N], banded neutrophils, lymphocytes [L], basophils, and the N:L ratio) were not different (P > .05) among housing designs or widths. However, there were differences (P < .05) in monocytes and eosinophils during the 28-d period after arrival; calves in stalls 76 cm wide had the greatest percentage of both leukocytes, and calves in the 66-cm stalls had the lowest monocyte percentage. Calves were recorded on videotape during wk 4, 13.5, and 18 to determine frequencies and durations of postures and behaviors (e.g., lying, standing, chewing, tongue playing, grooming, and investigative activities). There were no consistent differences (P > .05) in postures or behaviors among calves in different housing designs or widths. Calves spent approximately 71 and 31% in lying and standing positions, with no preference for the right or left side while recumbent. There was a tendency for calves in wider stalls or pens at wk 9 and 18 to exhibit more self-grooming activities. Tongue playing and investigative and chewing activities were exhibited in all treatments, but no differences (P > .05) were observed. However, calves housed in the 56-cm pens displayed difficulty in changing from lying to a standing position and were unable to extend one or more legs while recumbent. Even though there were few differences in behavioral, physiological, growth, or anatomical traits in this study, further increases in age and(or) weight of finished calves will require a reassessment of the appropriateness of individual veal calf housing design and dimensions. (+info)
Inducible expression and regulation of the alpha 1-acid glycoprotein gene by alveolar macrophages: prostaglandin E2 and cyclic AMP act as new positive stimuli.
We have reported that alpha 1-acid glycoprotein (AGP) gene expression was induced in lung tissue and in alveolar type II cells during pulmonary inflammatory processes, suggesting that local production of this immunomodulatory protein might contribute to the modulation of inflammation within the alveolar space. Because AGP may also be secreted by other cell types in the alveolus, we have investigated the expression and the regulation of the AGP gene in human and rat alveolar macrophages. Spontaneous AGP secretion by alveolar macrophages was increased 4-fold in patients with interstitial lung involvement compared with that in controls. In the rat, immunoprecipitation of [35S]methionine-labeled cell lysates showed that alveolar macrophages synthesize and secrete AGP. IL-1 beta had no effect by itself, but potentiated the dexamethasone-induced increase in AGP production. RNase protection assay demonstrated that AGP mRNA, undetectable in unstimulated cells, was induced by dexamethasone. Conditioned medium from LPS-stimulated macrophages as well as IL-1 beta had no effect by themselves, but potentiated the dexamethasone-induced increase in AGP mRNA levels. In addition to cytokines, PGE2 as well as dibutyryl cAMP increased AGP mRNA levels in the presence of dexamethasone. When AGP expression in other cells of the monocyte/macrophage lineage was examined, weak and no AGP production by human blood monocytes and by rat peritoneal macrophages, respectively, were observed. Our data showed that 1) AGP expression is inducible specifically in alveolar macrophages in vivo and in vitro; and 2) PGE2 and cAMP act as new positive stimuli for AGP gene expression. (+info)
Potentiation of the cytotoxicity of thymidylate synthase (TS) inhibitors by dipyridamole analogues with reduced alpha1-acid glycoprotein binding.
Dipyridamole has been shown to enhance the in vitro activity of antimetabolite anticancer drugs through the inhibition of nucleoside transport. However, the clinical potential of dipyridamole has not been realized because of the avid binding of the drug to the plasma protein alpha1-acid glycoprotein (AGP). Dipyridamole analogues that retain potent nucleoside transport inhibitory activity in the presence of AGP are described and their ability to enhance the growth inhibitory and cytotoxic effects of thymidylate synthase (TS) inhibitors has been evaluated. Three dipyridamole analogues (NU3026, NU3059 and NU3060) were shown to enhance the growth inhibitory activity of the TS inhibitor CB3717 and block thymidine rescue in L1210 cells. The extent of potentiation at a fixed analogue concentration (10 microM) was related to the potency of inhibition of thymidine uptake. A further analogue, NU3076, was identified, which was more potent than dipyridamole with a Ki value for inhibition of thymidine uptake of 0.1 microM compared to 0.28 microM for dipyridamole. In marked contrast to dipyridamole, inhibition of thymidine uptake by NU3076 was not significantly affected by the presence of AGP (5 mg ml(-1)). NU3076 and dipyridamole produced equivalent potentiation of the cytotoxicity of the non-classical antifolate TS inhibitor, nolatrexed, in L1210 cells with both compounds significantly reducing the LC90, by > threefold in the absence of salvageable thymidine. Thymidine rescue of L1210 cells from nolatrexed cytotoxicity was partially blocked by both 1 microM NU3076 and 1 microM dipyridamole. NU3076 also caused a significant potentiation of FU cytotoxicity in L1210 cells. These studies demonstrate that nucleoside transport inhibition can be maintained in the absence of AGP binding with the dipyridamole pharmacophore and that such analogues can enhance the cytotoxicity of TS inhibitors. (+info)