Evidence for L-dopa incorporation into cell proteins in patients treated with levodopa

Levodopa (L-dopa) is the most widely used agent for the symptomatic relief of Parkinson's disease. There is concern that chronic L-dopa treatment may be detrimental, with some studies suggesting that L-dopa may be neurotoxic. A potentially important mechanism whereby L-dopa may exert neurotoxic effects has been overlooked: that of the incorporation of L-dopa into proteins by protein synthesis. L-Dopa competes with tyrosine as a substrate in protein synthesis in vitro. We provide evidence that L-dopa can also be incorporated into proteins in vivo. Blood from L-dopa-treated and -non-treated patients was separated into protein, erythrocyte and lymphocyte fractions and levels of protein-incorporated dopa quantified by HPLC. Levels of protein-incorporated dopa were significantly increased in lymphocyte cell proteins from L-dopa-treated patients. This has not arisen from oxidative pathways as there was no evidence of oxidative damage to proteins. In addition, there was no increase in protein-incorporated dopa in erythrocytes, which are not actively synthesizing proteins. We suggest that protein-incorporated dopa could also be generated in the CNS. The accumulation of protein-incorporated dopa in cells is associated with oxidative stress and impaired function and could contribute to some of the problems associated with long-term L-dopa treatment.     

Bro1 stimulates Vps4 to promote intralumenal vesicle formation during multivesicular body biogenesis

Endosomal sorting complexes required for transport (ESCRT-0, -I, -II, -III) execute cargo sorting and intralumenal vesicle (ILV) formation during conversion of endosomes to multivesicular bodies (MVBs). The AAA-ATPase Vps4 regulates the ESCRT-III polymer to facilitate membrane remodeling and ILV scission during MVB biogenesis. Here, we show that the conserved V domain of ESCRT-associated protein Bro1 (the yeast homologue of mammalian proteins ALIX and HD-PTP) directly stimulates Vps4. This activity is required for MVB cargo sorting. Furthermore, the Bro1 V domain alone supports Vps4/ESCRT–driven ILV formation in vivo without efficient MVB cargo sorting. These results reveal a novel activity of the V domains of Bro1 homologues in licensing ESCRT-III–dependent ILV formation and suggest a role in coordinating cargo sorting with membrane remodeling during MVB sorting. Moreover, ubiquitin binding enhances V domain stimulation of Vps4 to promote ILV formation via the Bro1–Vps4–ESCRT-III axis, uncovering a novel role for ubiquitin during MVB biogenesis in addition to facilitating cargo recognition.     

Indirect nontarget effects of host-specific biological control agents: Implications for biological control

Classical biological control of weeds currently operates under the assumption that biological control agents are safe (i.e., low risk) if they do not directly attack nontarget species. However, recent studies indicate that even highly host-specific biological control agents can impact nontarget species through indirect effects. This finding has profound implications for biological control. To better understand the causes of these interactions and their implications, we evaluate recent case studies of indirect nontarget effects of biological control agents in the context of theoretical work in community ecology. We find that although particular indirect nontarget effects are extremely difficult to predict, all indirect nontarget effects of host specific biological control agents derive from the nature and strength of the interaction between the biological control agent and the pest. Additionally, recent theoretical work suggests that the degree of impact of a biological control agent on nontarget species is proportional to the agent’s abundance, which will be highest for moderately successful control agents. Therefore, the key to safeguarding against indirect nontarget effects of host-specific biological control agents is to ensure the biological control agents are not only host specific, but also efficacious. Biological control agents that greatly reduce their target species while remaining host-specific will reduce their own populations through density-dependent feedbacks that minimize risks to nontarget species.     

Differential expression, function and response to inflammatory stimuli of 11β-hydroxysteroid dehydrogenase type 1 in human fibroblasts: a mechanism for tissue-specific regulation of inflammation

Stromal cells such as fibroblasts play an important role in defining tissue-specific responses during the resolution of inflammation. We hypothesized that this involves tissue-specific regulation of glucocorticoids, mediated via differential regulation of the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). Expression, activity and function of 11β-HSD1 was assessed in matched fibroblasts derived from various tissues (synovium, bone marrow and skin) obtained from patients with rheumatoid arthritis or osteoarthritis. 11β-HSD1 was expressed in fibroblasts from all tissues but mRNA levels and enzyme activity were higher in synovial fibroblasts (2-fold and 13-fold higher mRNA levels in dermal and synovial fibroblasts, respectively, relative to bone marrow). Expression and activity of the enzyme increased in all fibroblasts following treatment with tumour necrosis factor-α or IL-1β (bone marrow: 8-fold and 37-fold, respectively, compared to vehicle; dermal fibroblasts: 4-fold and 14-fold; synovial fibroblasts: 7-fold and 31-fold; all P < 0.01 compared with vehicle). Treatment with IL-4 or interferon-γ was without effect, and there was no difference in 11β-HSD1 expression between fibroblasts (from any site) obtained from patients with rheumatoid arthritis or osteoarthritis. In the presence of 100 nmol/l cortisone, IL-6 production – a characteristic feature of synovial derived fibroblasts – was significantly reduced in synovial but not dermal or bone marrow fibroblasts. This was prevented by co-treatment with an 11β-HSD inhibitor, emphasizing the potential for autocrine activation of glucocorticoids in synovial fibroblasts. These data indicate that differences in fibroblast-derived glucocorticoid production (via the enzyme 11β-HSD1) between cells from distinct anatomical locations may play a key role in the predeliction of certain tissues to develop persistent inflammation.     

Irrelevance of the Power Stroke for the Directionality,Stopping Force,and Optimal Efficiency of Chemically Driven Molecular Machines

A simple model for a chemically driven molecular walker shows that the elastic energy stored by the molecule and released during the conformational change known as the power-stroke (i.e., the free-energy difference between the pre- and post-power-stroke states) is irrelevant for determining the directionality, stopping force, and efficiency of the motor. Further, the apportionment of the dependence on the externally applied force between the forward and reverse rate constants of the power-stroke (or indeed among all rate constants) is irrelevant for determining the directionality, stopping force, and efficiency of the motor. Arguments based on the principle of microscopic reversibility demonstrate that this result is general for all chemically driven molecular machines, and even more broadly that the relative energies of the states of the motor have no role in determining the directionality, stopping force, or optimal efficiency of the machine. Instead, the directionality, stopping force, and optimal efficiency are determined solely by the relative heights of the energy barriers between the states. Molecular recognition—the ability of a molecular machine to discriminate between substrate and product depending on the state of the machine—is far more important for determining the intrinsic directionality and thermodynamics of chemo-mechanical coupling than are the details of the internal mechanical conformational motions of the machine. In contrast to the conclusions for chemical driving, a power-stroke is very important for the directionality and efficiency of light-driven molecular machines and for molecular machines driven by external modulation of thermodynamic parameters.     

Strategies for genetic conservation of trees in the Peruvian Amazon

Forestry practices and high rates of land clearance for agriculture are causing genetic erosion of valuable tree species in the Peruvian Amazon, thereby endangering the economic sustainability of rural communities and limiting Peru's opportunities for the development of new timber and non-timber forest products. The potential utility and limitations of six low-input interventions to help forestall further genetic erosion in the region are discussed, with a focus on local community involvement. Improved agroforestry systems may help reduce deforestation by increasing farm productivity, although methods to increase the currently low adoption rate of these technologies need to be developed. Use of strategic tree domestication techniques can also improve farm productivity and prevent inadvertent genetic drift and inbreeding associated with traditional domestication practices, although to have a major impact, current programs need to be extended across the region. Woodlot forestry could supplant selective extraction of timber and offers an attractive opportunity for poverty alleviation if appropriate credit and land tenure policies can be developed. However, it may also result in increased deforestation if activities on public land cannot be controlled. The implementation of improved seed collection systems and simple seed transfer guidelines would help to reduce the collection of seed of poor quality and low genetic diversity, and avoid maladapted plantings, although such programs are difficult to monitor and seed costs may increase. Strategic identification and design of in situ conservation areas would help to ensure the viability of conserved populations, but requires the forfeiture of significant revenue from timber concessions.     

Carboxylate composition of root exudates does not relate consistently to a crop species' ability to use phosphorus from aluminium, iron or calcium phosphate sources

* The relationship between carboxylate release from roots and the ability of the species to utilize phosphorus from sparingly soluble forms was studied by comparing Triticum aestivum, Brassica napus, Cicer arietinum, Pisum sativum, Lupinus albus, Lupinus angustifolius and Lupinus cosentinii. * Plants were grown in sand and supplied with 40 mg P kg(-1) in the sparingly soluble forms AlPO(4), FePO(4) or Ca(5)OH(PO(4))(3), or as soluble KH(2)PO(4); control plants received no P. * The ability to utilize sparingly soluble forms of P differed between forms of P supplied and species. Pisum sativum and C. arietinum did not access AlPO(4) or FePO(4) despite releasing carboxylates into the rhizosphere. * Species accessed different forms of sparingly soluble P, but no species was superior in accessing all forms. We conclude that a single trait cannot explain access to different forms of sparingly soluble P, and hypothesize that in addition to carboxylates, rhizosphere pH and root morphology are key factors.     

The molecular basis for the lack of immunostimulatory activity of vertebrate DNA

Macrophages and B cells are activated by unmethylated CpG-containing sequences in bacterial DNA. The lack of activity of self DNA has generally been attributed to CpG suppression and methylation, although the role of methylation is in doubt. The frequency of CpG in the mouse genome is 12.5% of Escherichia coli, with unmethylated CpG occurring at approximately 3% the frequency of E. coli. This suppression of CpG alone is insufficient to explain the inactivity of self DNA; vertebrate DNA was inactive at 100 micro g/ml, 3000 times the concentration at which E. coli DNA activity was observed. We sought to resolve why self DNA does not activate macrophages. Known active CpG motifs occurred in the mouse genome at 18% of random occurrence, similar to general CpG suppression. To examine the contribution of methylation, genomic DNAs were PCR amplified. Removal of methylation from the mouse genome revealed activity that was 23-fold lower than E. coli DNA, although there is only a 7-fold lower frequency of known active CpG motifs in the mouse genome. This discrepancy may be explained by G-rich sequences such as GGAGGGG, which potently inhibited activation and are found in greater frequency in the mouse than the E. coli genome. In summary, general CpG suppression, CpG methylation, inhibitory motifs, and saturable DNA uptake combined to explain the inactivity of self DNA. The immunostimulatory activity of DNA is determined by the frequency of unmethylated stimulatory sequences within an individual DNA strand and the ratio of stimulatory to inhibitory sequences.     

A sensitive, specific assay for tissue collagenase using telopeptide-free [3H]acetylated collagen

Collagenase is assayed by incubation with soluble, telopeptide-free collagen extracted from rat skin and labeled with [2-3H]acetic anhydride. Collagen is cleaved by collagenase and the resulting fragments are digested with trypsin and chymotrypsin. Undigested collagen is recovered by precipitation with trichloroacetic acid, collected on glass-fiber filters, and quantitated by liquid scintillation spectrometry. This procedure combines features of the Cawston and Barrett (T.E. Cawston and A.J. Barrett, 1979, Anal. Biochem. 99, 340-345) and the Ryh?nen et al. (L. Ryh?nen et al., 1982, Collagen Rel. Res. 2, 117-130) methods. The first method provides a simple way to prepare large quantities of uniform substrate, while the second increases the specificity of the assay by removal of the labeled telopeptides. The assay is reproducible and linear with time and enzyme concentration. It is approximately 10X more sensitive than the Cawston and Barrett method and can readily detect 1-8 mU collagenase (1 unit equals 1 microgram collagen cleaved/min at 30 degrees C). The substrate is resistant to elastase, trypsin, and chymotrypsin and is completely degraded by bacterial collagenase. Collagenase is the only tissue metalloprotease found, to date, that cleaves the substrate.