Mammalian GRIP domain proteins differ in their membrane binding properties and are recruited to distinct domains of the TGN

The four mammalian golgins, p230/golgin-245, golgin-97, GCC88 and GCC185 are targeted to trans-Golgi network ITGN) membranes by their C-terminal GRIP domain in a G-protein-dependent process. The Arf-like GTPase, Arl1, has been shown to mediate TGN recruitment of p230/golgin245 and golgin-97 by interaction with their GRIP domains; however, it is not known whether all the TGN golgins bind to Arl1 and whether they are all recruited to the same or different TGN domains. Here we demonstrate differences in membrane binding properties and TGN domain recruitment of the mammalian GRIP domain proteins. Overexpression of full-length GCC185 resulted in the appearance of small punctate structures dispersed in the cytoplasm of transfected cells that were identified as membrane tubular structures by immunoelectron microscopy. The cytoplasmic GCC185-labelled structures were enriched for membrane binding determinants of GCC185 GRIP, whereas the three other mammalian GRIP family members did not colocalize with the GCC185-labelled structures. These GCC185-labelled structures included the TGN resident protein alpha2,6 sialyltransferase and excluded the recycling TGN protein, TGN46. The Golgi stack was unaffected by overexpression of GCC185. Overexpression of both full-length GCC185 and GCC88 showed distinct and nonoverlapping structures. We also show that the GRIP domains of GCC185 and GCC88 differ in membrane binding properties from each other and, in contrast to p230/golgin245 and golgin-97, do not interact with Arl1 in vivo. Collectively these results show that GCC88, GCC185 and p230/golgin245 are recruited to functionally distinct domains of the TGN and are likely to be important for the maintenance of TGN subdomain structure, a critical feature for mediating protein sorting and membrane transport.

Identification of a Golgi-localised GRIP domain protein from Arabidopsis thaliana

A family of Golgi-localised molecules was recently described in animals and fungi possessing extensive coiled regions and a short (similar to40 residues) conserved C-terminal domain, called the GRIP domain, which is responsible for their location to this organelle. Using the model plant Arabidopsis thaliana, we identified a gene (AtGRIP) encoding a putative GRIP protein. We demonstrated that the C-terminal domain from AtGRIP functions as a Golgi-targeting sequence in plant cells. Localisation studies in living cells expressing the AtGRIP fused to a DsRed2 fluorescent probe, showed extensive co-location with the Golgi marker alpha-mannosidase I in transformed tobacco protoplasts. GRIP-like sequences were also found in genomic databases of rice, maize, wheat and alfalfa, suggesting that this domain may be a useful Golgi marker for immunolocalisation studies. Despite low sequence identity amongst GRIP domains, the plant GRIP sequence was able to target to the Golgi of mammalian cells. Taken together, these data indicate that GRIP domain proteins might be implicated in a targeting mechanism that is conserved amongst eukaryotes.

Validation of the Action Research Arm Test using item response theory in patients after stroke

Objective: To validate the unidimensionality of the Action Research Arm Test (ARAT) using Mokken analysis and to examine whether scores of the ARAT can be transformed into interval scores using Rasch analysis. Subjects and methods: A total of 351 patients with stroke were recruited from 5 rehabilitation departments located in 4 regions of Taiwan. The 19-item ARAT was administered to all the subjects by a physical therapist. The data were analysed using item response theory by non-parametric Mokken analysis followed by Rasch analysis. Results: The results supported a unidimensional scale of the 19-item ARAT by Mokken analysis, with the scalability coefficient H = 0.95. Except for the item pinch ball bearing 3rd finger and thumb'', the remaining 18 items have a consistently hierarchical order along the upper extremity function's continuum. In contrast, the Rasch analysis, with a stepwise deletion of misfit items, showed that only 4 items (grasp ball'', grasp block 5 cm(3)'', grasp block 2.5 cm(3)'', and grip tube 1 cm(3)'') fit the Rasch rating scale model's expectations. Conclusion: Our findings indicated that the 19-item ARAT constituted a unidimensional construct measuring upper extremity function in stroke patients. However, the results did not support the premise that the raw sum scores of the ARAT can be transformed into interval Rasch scores. Thus, the raw sum scores of the ARAT can provide information only about order of patients on their upper extremity functional abilities, but not represent each patient's exact functioning.

Molecular characterization of the microbial species that colonize human ileal and colonic mucosa by using 16S rDNA sequence analysis

Aim: The aim of this study was to characterize the bacterial community adhering to the mucosa of the terminal ileum, and proximal and distal colon of the human digestive tract. Methods and Results: Pinch samples of the terminal ileum, proximal and distal colon were taken from a healthy 35-year-old, and a 68-year-old subject with mild diverticulosis. The 16S rDNA genes were amplified using a low number of PCR cycles, cloned, and sequenced. In total, 361 sequences were obtained comprising 70 operational taxonomic units (OTU), with a calculated coverage of 82.6%. Twenty-three per cent of OTU were common to the terminal ileum, proximal colon and distal colon, but 14% OTU were only found in the terminal ileum, and 43% were only associated with the proximal or distal colon. The most frequently represented clones were from the Clostridium group XIVa (24.7%), and the Bacteroidetes (Cytophaga-Flavobacteria-Bacteroides ) cluster (27.7%). Conclusion: Comparison of 16S rDNA clone libraries of the hindgut across mammalian species confirms that the distribution of phylogenetic groups is similar irrespective of the host species. Lesser site-related differences within groups or clusters of organisms, are probable. Significance and Impact: This study provides further evidence of the distribution of the bacteria on the mucosal surfaces of the human hindgut. Data contribute to the benchmarking of the microbial composition of the human digestive tract.

Domains of the TGN: Coats, tethers and G proteins

The trans-Golgi network is the major sorting compartment of the secretory pathway for protein, lipid and membrane traffic. There is a constant flow of membrane and cargo to and from this compartment. Evidence is emerging that the trans-Golgi network has multiple biochemically and functionally distinct subdomains, each of which contributes to the combined sorting and transport requirements of this dynamic compartment. The recruitment of distinct arrays of protein complexes to trans-Golgi network membranes is likely to produce the diversity of structure and biochemistry observed amongst subdomains that serve to generate different carriers or maintain resident trans-Golgi network components. This review discusses how these subdomains may be formed and examines the molecular players involved, including G proteins, clathrin adaptors and golgin tethers. Diversity within these protein families is highlighted and shown to be critical for the functionality of the trans-Golgi network, as a mediator of protein sorting and membrane transport, and for the maintenance of Golgi structure.

Performance of the Norwegian version of AUSCAN - a disease-specific measure of hand osteoarthritis

Objective: To examine the performance of the Norwegian version of the AUSCAN Index as a disease-specific health status measure in patients with hand osteoarthritis (OA). Methods: One hundred and ninety-nine patients with clinical hand OA (mean (SD) age 61.7 (5.7) years, 18 (9%) males) underwent a comprehensive examination including joint status, examination of grip strength and completion of several self-reported health status questionnaires. The Australian/Canadian OA hand index (AUSCAN) captures three different dimensions of hand OA: pain (5 items), stiffness (1 item), and difficulties with daily activities (9 items). Our pre-study hypothesis was to identify AUSCAN as a specific hand measure with strong correlations to hand measures and lower correlations to other general measures of health. Results: Patient completion of the AUSCAN Index was similar or better than other measures. The internal consistency of the AUSCAN was excellent. The pain and physical dimension of AUSCAN correlated substantially to, each other and moderately to the stiffness scale. The AUSCAN physical scale correlated moderately to substantially to other measures, the highest correlation being seen with the Arthritis Impact Measurement Scale (AIMS) 2 hand and finger function scale (r= 0.73). The standardised differences between patients with and without radiographic abnormalities were numerically larger for the AUSCAN pain and physical scales than for other measures. Conclusion: The Norwegian version of the AUSCAN has an acceptable clinimetric performance and is a suitable tool for assessment of hand OA. (C) 2005 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Three-dimensional simulation of the rolling of a saturated fibro-porous media

This paper describes recent advances made in computational modelling of the sugar cane liquid extraction process. The saturated fibro-porous material is rolled between circumferentially grooved rolls, which enhance frictional grip and provide a low-resistance path for liquid flow during the extraction process. Previously reported two-dimensional (2D) computational models, account for the large deformation of the porous material by solving the fully coupled governing fibre stress and fluid-flow equations using finite element techniques. While the 2D simulations provide much insight into the overarching cause-effect relationships, predictions of mechanical quantities such as roll separating force and particularly torque as a function of roll speed and degree of compression are not satisfactory for industrial use. It is considered that the unsatisfactory response in roll torque prediction may be due to the stress levels that exist between the groove tips and roots which have been largely neglected in the geometrically simplified 2D model. This paper gives results for both two- and three-dimensional finite element models and highlights their strengths and weaknesses in predicting key milling parameters. (c) 2005 Elsevier B.V. All rights reserved.

Estrogen replacement, muscle composition, and physical function: The health ABC study

Purpose: Although the beneficial effects of estrogen use on cardiovascular and cognitive function in postmenopausal women have been recently discredited, controversy remains regarding its usefulness for maintaining skeletal muscle mass or strength. Therefore, the purpose of this study was to determine whether estrogen use is associated with enhanced muscle composition and, if so, whether this translates into improved strength and physical function. Methods: Cross-sectional analysis of 840 well-functioning community-dwelling white women (current estrogen replacement therapy (ERT) users = 259, nonusers = 581) aged 70-79 yr participating in the Health, Aging and Body Composition Study. Muscle composition of the midthigh by computed tomography included cross-sectional area (CSA) of the quadriceps, hamstrings, intermuscular fat and subcutaneous fat, and muscle attenuation in Hounsfield units (HU) as a measure of muscle density. Isometric hand grip and isokinetic knee extensor strength were assessed by dynamometry. Physical function was assessed using a summary scale that included usual 6-m walk and narrow walk speed, repeated chair stands, and standing balance. Results: In analyses of covariance adjusted for relevant confounders. quadriceps muscle CSA and HU were greater in Current ERT than non-ERT women (P < 0.05). Grip strength was also greater (P < 0.05) in women taking ERT while knee extensor strength approached significance (P < 0.10). However, differences in muscle composition and strength were modest at <= 3.3%. There was no difference by ERT status for the hamstring, muscles. fat CSA. or for physical function. Conclusion: The associations between ERT and muscle composition and strength were minor and did not translate into improved physical function. Initiation of ERT for preservation of muscle composition and function may not be indicated.

E-cadherin transport from the trans-Golgi network in tubulovesicular carriers is selectively regulated by Golgin-97

E-cadherin is a cell-cell adhesion protein that is trafficked and delivered to the basolateral cell surface. Membrane-bound carriers for the post-Golgi exocytosis of E-cadherin have not been characterized. Green fluorescent protein (GFP)-tagged E-cadherin (Ecad-GFP) is transported from the trans-Golgi network (TGN) to the recycling endosome on its way to the cell surface in tubulovesicular carriers that resemble TGN tubules labeled by members of the golgin family of tethering proteins. Here, we examine the association of golgins with tubular carriers containing E-cadherin as cargo. Fluorescent GRIP domains from golgin proteins replicate the membrane binding of the full-length proteins and were coexpressed with Ecad-GFP. The GRIP domains of p230/golgin-245 and golgin-97 had overlapping but nonidentical distributions on the TGN; both domains were on TGN-derived tubules but only the golgin-97 GRIP domain coincided with Ecad-GFP tubules in live cells. When the Arl1-binding endogenous golgins, p230/golgin-245 and golgin-97 were displaced from Golgi membranes by overexpression of the p230 GRIP domain, trafficking of Ecad-GFP was inhibited. siRNA knockdown of golgin-97 also inhibited trafficking of Ecad-GFP. Thus, the GRIP domains of p230/golgin-245 and golgin-97 bind discriminately to distinct membrane subdomains of the TGN. Golgin-97 is identified as a selective and essential component of the tubulovesicular carriers transporting E-cadherin out of the TGN.

TRAP220 is modulated by the antineoplastic agent 6-Mercaptopurine, and mediates the activation of the NR4A subgroup of nuclear receptors

The NR4A1-3 (Nur77, NURR1 and NOR-1) subfamily of nuclear hormone receptors (NRs) has been implicated in Parkinson's disease, schizophrenia, manic depression, atherogenesis, Alzheimer's disease, rheumatoid arthritis, cancer and apoptosis. This has driven investigations into the mechanism of action, and the identification of small molecule regulators, that may provide the platform for pharmaceutical and therapeutic exploitation. Recently, we found that the purine antimetabolite 6-Mercaptopurine (6-MP), which is widely used as an anti-neoplastic and anti-inflammatory drug, modulated the NR4A1-3 subfamily. Interestingly, the agonist-mediated activation did not involve modulation of primary coactivators' (e.g. p300 and SRC-2/GRIP-1) activity and/or recruitment. However, the role of the subsequently recruited coactivators, for example CARM-1 and TRAP220, in 6-MP-mediated activation of the NR4A1-3 subfamily remains obscure. In this study we demonstrate that 6-MP modulates the activity of the coactivator TRAP220 in a dose-dependent manner. Moreover, we demonstrate that TRAP220 potentiates NOR-1-mediated transactivation, and interacts with the NR4A1-3 subgroup in an AF-1-dependent manner in a cellular context. The region of TRAP220 that mediated 6-MP activation and NR4A interaction was delimited to amino acids 1-800, and operates independently of the critical PKC and PKA phosphorylation sites. Interestingly, TRAP220 expression does not increase the relative induction by 6-MP, however the absolute level of NOR-1-mediated trans-activation is increased. This study demonstrates that 6-MP modulates the activity of the NR4A subgroup, and the coactivator TRAP220.

Developmental changes in the response to obstacles during prehension

Adults are proficient at reaching to grasp objects of interest in a cluttered workspace. The issue of concern, obstacle avoidance, was studied in 3 groups of young children aged 11-12, 9-10, and 7-8 years (n = 6 in each) and in 6 adults aged 18-24 years. Adults slowed their movements and decreased their maximum grip aperture when an obstacle was positioned close to a target object (the effect declined as the distance between target and obstacle increased). The children showed the same pattern, but the magnitude of the effect was quite different. In contrast to the adults, the obstacle continued to have a large effect when it was some distance from the target (and provided no physical obstruction to movement).

Pendant drop formation of shear-thinning and yield stress fluids

A volume-of-fluid numerical method is used to predict the dynamics of shear-thinning liquid drop formation in air from a circular orifice. The validity of the numerical calculation is confirmed for a Newtonian liquid by comparison with experimental measurements. For particular values of Weber number and Froude number, predictions show a more rapid pinch-off, and a reduced number of secondary droplets, with increasing shear-thinning. Also a minimum in the limiting drop length occurs for the smallest Weber number as the zero-shear viscosity is varied. At the highest viscosity, the drop length is reduced due to shear-thinning, whereas at lower viscosities there is little effect of shear-thinning. The evolution of predicted drop shape, drop thickness and length, and the configuration at pinch-off are discussed for shear-thinning drops. The evolution of a drop of Bingham yield stress liquid is also considered as a limiting case. In contrast to the shear-thinning cases, it exhibits a plug flow prior to necking, an almost step-change approach to pinch-off of a torpedo shaped drop following the onset of necking, and a much smaller neck length; no secondary drops are formed. The results demonstrate the potential of the numerical model as a design tool in tailoring the fluid rheology for controlling drop formation behaviour. (c) 2006 Elsevier Inc. All rights reserved.