ABSTRACT
UNLABELLED: Dietary intervention studies in COPD patients often are short-term inpatient studies where a certain amount of extra energy is guaranteed. The aim of this study was to evaluate the effect of an 1 year individual multifaceted dietary intervention during multidisciplinary rehabilitation. Eighty-seven patients with severe COPD, not demanding oxygen therapy were included, 24 of them served as controls. A dietary history interview was performed at baseline and at study end. Dietary advice given were based on results from the dietary history and socio-economic status. The intervention group was divided into three parts; NW: normal weight (dietary advice given aiming to weight maintenance), OW: overweight (weight-reducing advice) and UW: underweight (dietary advise based on an energy- and protein-rich diet). RESULTS: UW-group: Eighty-one per cent of the patients gained weight or kept a stable weight. OW-group: Fifty-seven per cent lost more than 2 kg NW-group: Seventy-six per cent kept a stable weight or gained weight. Increased dietary intake from baseline was seen for energy protein, carbohydrates and certain micronutrients (P < 0.05) in the UW group. Six minutes walking distance increased by approximately 20 m in both NW (P < 0.05) and UW patients. To conclude, slight, but uniform, indications of positive effects of dietary intervention during multidisciplinary rehabilitation was seen. Dietary intervention in underweight COPD patients might be a prerequisite for physical training.
Subject(s)
Pulmonary Disease, Chronic Obstructive/diet therapy , Aged , Body Composition , Body Weight , Combined Modality Therapy , Energy Intake , Humans , Micronutrients/administration & dosage , Middle Aged , Nutrition Assessment , Prospective Studies , Pulmonary Disease, Chronic Obstructive/physiopathology , Pulmonary Disease, Chronic Obstructive/rehabilitationABSTRACT
Pokeweed antiviral protein (PAP)-I from the spring leaves of Phytolacca americana is a naturally occurring RNA-depurinating enzyme with broad-spectrum antiviral activity. Interest in PAP is growing due to its use as a potential anti-HIV agent. However, the clinical use of native PAP is limited due to inherent difficulties in obtaining sufficient quantities of homogeneously pure active PAP without batch-to-batch variation from its natural resource. Here, we report the expression of mature PAP (residues 23 to 284) with a C-terminal hexahistidine tag in the methylotrophic yeast Pichia pastoris, as a secreted soluble protein. The final yield of the secreted PAP is greater than 10 mg/L culture in shaker flasks. The secreted recombinant protein is not toxic to the yeast cells and has an apparent molecular mass of 33-kDa on SDS-PAGE gels. The in vitro enzymatic activity and cellular anti-HIV activity of recombinant PAP were of the same magnitude as those of the native PAP purified from P. americana. To our knowledge, this is the first large-scale expression and purification of soluble and biologically active recombinant mature PAP from yeast.
Subject(s)
Anti-HIV Agents/metabolism , N-Glycosyl Hydrolases , Plant Proteins/metabolism , Anti-HIV Agents/pharmacology , Cloning, Molecular , Genetic Vectors , HIV-1/drug effects , Humans , Immunoblotting , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/virology , Pichia/metabolism , Plant Proteins/genetics , Plant Proteins/pharmacology , Protein Biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Ribosome Inactivating Proteins, Type 1 , Transformation, Genetic , Virus ReplicationABSTRACT
Pokeweed antiviral protein (PAP) from the leaves of the pokeweed plant, Phytolacca americana, is a naturally occurring single-chain ribosome-inactivating protein, which catalytically inactivates both prokaryotic and eukaryotic ribosomes. The therapeutic potential of PAP has gained considerable interest in recent years due to the clinical use of native PAP as the active moiety of immunoconjugates against cancer and AIDS. The clinical use of native PAP is limited due to inherent difficulties in obtaining sufficient quantities of a homogenously pure and active PAP preparation with minimal batch to batch variability from its natural source. Previous methods for expression of recombinant PAP in yeast, transgenic plants and Escherichia coli have resulted in either unacceptably low yields or were too toxic to the host system. Here, we report a successful strategy which allows high level expression of PAP as inclusion bodies in E. coli. Purification of refolded recombinant protein from solubilized inclusion bodies by size-exclusion chromatography yielded biologically active recombinant PAP (final yield: 10 to 12 mg/L). The ribosome depurinating in vitro N-glycosidase activity and cellular anti-HIV activity of recombinant PAP were comparable to those of the native PAP. This expression and purification system makes it possible to obtain sufficient quantities of biologically active and homogenous recombinant PAP sufficient to carry out advanced clinical trials. To our knowledge, this is the first large-scale expression and purification of biologically active recombinant PAP from E. coli.
