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Abstract: The objective of this work is to identify proteins of the human and porcine parasite, Taenia solium, which may be exploited for control of the parasite. Through screening a cDNA library of T. solium metacestodes, we have identified a novel Sec-14-like Taenia lipid-binding protein that may play an important role in membrane trafficking. The Sec14-like sequence is a single copy gene, encoding a putative polypeptide of 320 amino acids and 36.1 kDa (sec14Tsol protein). Secondary amino acid structural analysis suggested that the sec14Tsol protein might contain two distinct structural domains, an amino-terminal α-helix rich domain and a mixed α-helix/β-stand carboxy-terminal zone, showing homology with the conserved SEC14 domain found in a great number of proteins that bind lipids, as the regulators of membrane trafficking between Golgi membrane bilayers. Significantly, therefore, in a phosphoinositide-binding assay, sec14Tsol purified recombinant protein specifically interacted with important lipid regulators of membrane trafficking, with a preference for PI(3)P2, PI(3,4)P2, PI(4,5)P2 and phosphatidic acid. Moreover, the sec14Tsol protein was localized in the Golgi apparatus of transfected cells and in the spiral canal region of T. solium metacestode tegument. As sec14Tsol protein may play an important role in membrane trafficking, its demonstrated localisation in the intact parasite tegument suggests its involvement in the function of the tegument and thus perhaps interaction with the host. [Copyright &y& Elsevier]

Abstract: The skeletal muscle-specific ClC-1 is a voltage-gated chloride channel protein. Specific antibodies against ClC-1 revealed in muscle sections a sarcolemmal staining that was absent in the myotonic arrested development of righting response (ADR) mouse muscle. The intensity of the sarcolemmal staining varied from one type of muscle to another and in lateral sections showed a typical mosaic pattern that colocalized with β-dystroglycan and left the transverse tubule openings clear. Surprisingly, in isolated myofibers, the ClC-1 protein was absent from the sarcolemma. Instead, it localized to intracellular I band areas as soon as the myofibers were isolated. When the isolated myofibers were incubated with the kinase inhibitor staurosporine, the ClC-1 protein shifted back to the sarcolemma. Electric stimulation of the cultivated fibers had a similar effect. Also, myofibers infected with a recombinant Semliki Forest virus (SFV) expressing myc-tagged ClC-1 showed intracellular localization of the protein. The virally expressed mycClC-1 reached the Golgi apparatus but sarcolemmal staining remained nondetectable, and addition of staurosporine into the growth medium recruited the mycClC-1 to the sarcolemma. These data indicate that sarcolemmal targeting of the ClC-1 requires specific signals that are provided by the physiological environment. [Copyright &y& Elsevier]

Abstract: Embryonic stem (ES) cells are genetically manipulable pluripotential cells that can be differentiated in vitro into neurons, oligodendrocytes, and astrocytes. Given their potential utility as a source of replacement cells for the injured nervous system and the likelihood that transplantation interventions might include co-application of growth factors, we examined the effects of neurotrophin and GDNF family ligands on the survival and excitotoxic vulnerability of ES cell-derived neurons (ES neurons) grown in vitro. ES cells were differentiated down a neural lineage in vitro using the 4−/4+ protocol (Bain et al., Dev Biol 168:342–57, 1995). RT-PCR demonstrated expression of receptors for neurotrophins and GDNF family ligands in ES neural lineage cells. Neuronal expression of GFRα1, GFRα2, and ret was confirmed by immunocytochemistry. Exposure to 30–100 ng/ml GDNF or neurturin (NRTN) resulted in activation of ret. Addition of NT-3 and GDNF did not increase cell division but did increase the number of neurons in the cultures 7 days after plating. Pretreatment with NT-3 enhanced the vulnerability of ES neurons to NMDA-induced death (100 μM NMDA for 10 min) and enhanced the NMDA-induced increase in neuronal [Ca2+]i, but did not alter expression of NMDA receptor subunits NR2A or NR2B. In contrast, pretreatment with GDNF reduced the vulnerability of ES neurons to NMDA-induced death while modestly enhancing the NMDA-induced increase in neuronal [Ca2+]i. These findings demonstrate that the response of ES-derived neurons to neurotrophins and GDNF family ligands is largely similar to that of other cultured central neurons. [Copyright &y& Elsevier]

Cdc42 is a member of the Rho family of GTPases and plays an important role in the regulation of actin cytoskeletal organization. Activation of Cdc42 and associated signal transduction cascades are dependent upon proper localization of this GTPase. The studies described herein address the hypothesis that Rho GDP-dissociation inhibitor, RhoGDI, plays an essential role in the translocation of Cdc42 to signaling complexes at the plasma membrane and is essential for Cdc42-mediated actin cytoskeletal rearrangements. An activating mutant of Cdc42 that is RhoGDI-binding defective (Cdc42(G12V/R66E)) is characterized and used as a tool to study the functional importance of the Cdc42–RhoGDI interaction. Overexpression of mycCdc42(G12V/R66E) in COS-7 cells results in actin cytoskeletal rearrangements that are indistinguishable from those stimulated by overexpression of mycCdc42(G12V). In addition, the G12V activating mutant of Cdc42 was overexpressed in mesangial cells that are null for RhoGDI expression. MycCdc42(G12V) stimulation of filopodia formation in these cells was indistinguishable from that observed in wild-type mesangial cells. Taken together, the results presented herein indicate that although RhoGDI is a critical regulator of guanine nucleotide binding, cycling of Cdc42 between membranes and the cytosol and cellular transformation, it is not essential for Cdc42-mediated organization of the actin cytoskeleton. [Copyright &y& Elsevier]

In order to facilitate locomotion and limb movement many animals store energy elastically in their tendons. In the turkey, much of the force generated by the gastrocnemius muscle is stored as elastic energy during tendon deformation and not within the muscle. As limbs move, the tendons are strained causing the collagen fibers in the extracellular matrices to be strained. During growth, avian tendons mineralize in the portions distal to the muscle and show increased tensile strength, modulus, and energy stored per unit strain as a result. In this study the energy stored in unmineralized and mineralized collagen fibers was measured and compared to the amount of energy stored in molecular models. Elastic energy storage values calculated using the molecular model were slightly higher than those obtained from collagen fibers, but display the same increases in slope as the fiber data. We hypothesize that these increases in slope are due to a change from the stretching of flexible regions of the collagen molecule to the stretching of less flexible regions. The elastic modulus obtained from the unmineralized molecular model correlates well with elastic moduli of unmineralized collagen from other studies. This study demonstrates the potential importance of molecular modeling in the design of new biomaterials. [Copyright &y& Elsevier]

We have cloned, expressed and characterized catfish αB-crystallin (FαB). Genomic sequence comparison has revealed conservation of intron splicing sites and coding regions, however, the two intron sequences, 5′- and 3′-untranslated regions of FαB gene are shorter than those reported for other vertebrates. In contrast to mammalian homologues with a subunit association ratio (αA-crystallin/αB-crystallin) of 3:1, α-crystallin from catfish lens showed a ratio of 19:1. The biophysical properties and chaperone-like activity of recombinant FαB and porcine αB-crystallin (PαB) were studied and compared by heat denaturation, circular dichroism, intrinsic and dye-binding fluorescence, gel-filtration, and analytical ultracentrifugation. FαB shows 50% precipitation occurring at 72°C that is higher than PαB at 66°C. Even though FαB also possesses more surface hydrophilic regions than PαB, FαB still possesses higher chaperone activity to prevent aggregation of alcohol dehydrogenase at 60°C. The molecular mass of FαB showed a smaller size (450 kDa) than PαB (550 kDa), which is also confirmed by analytical ultracentrifugation. In addition, FαB possesses better refolding potential after preheating treatment than PαB. FαB also exhibits higher chaperone-like activity than PαB to prevent insulin aggregation induced by dithiothreitol. In contrast to the prevalent notion that fish crystallins generally denature easily, FαB with chaperone-like activity appears to be more stable than mammalian homologues towards thermal and chemical denaturation. [Copyright &y& Elsevier]

Interest in the biochemical detection of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein followed soon after cloning of the gene and prediction of the protein structure. Ever since, antibodies (Abs) have been produced and used to detect CFTR in both heterologously and endogenously expressing cells and tissues. Although designed to be sensitive and specific, these Abs produce, in most cases, unsatisfactory results when used for the biochemical detection of CFTR either by Western blot or by immunoprecipitation. The lack of Abs that can reliably detect the CFTR protein is a major constraint to studies of CF. We compared 14 different Abs for their ability to detect CFTR in both stably transfected and endogenously expressing cell lines. [Copyright &y& Elsevier]

Subcellular translocation of phototransduction proteins in response to light has previously been detected by immunocytochemistry. This movement is consistent with the hypothesis that migration is part of a basic cellular mechanism regulating photoreceptor sensitivity. In order to monitor the putative migration of arrestin in response to light, we expressed a functional fusion between the signal transduction protein arrestin and green fluorescent protein (GFP) in rod photoreceptors of transgenic Xenopus laevis. In addition to confirming reports that arrestin is translocated, this alternative approach generated unique observations, raising new questions regarding the nature and time scale of migration. Confocal fluorescence microscopy was performed on fixed frozen retinal sections from tadpoles exposed to three different lighting conditions. A consistent pattern of localization emerged in each case. During early light exposure, arrestin-GFP levels diminished in the inner segments (ISs) and simultaneously increased in the outer segments (OSs), initially at the base and eventually at the distal tips as time progressed. Arrestin-GFP reached the distal tips of the photoreceptors by 45–75 min at which time the ratio of arrestin-GFP fluorescence in the OSs compared to the ISs was maximal. When dark-adaptation was initiated after 45 min of light exposure, arrestin-GFP rapidly re-localized to the ISs and axoneme within 30 min. Curiously, prolonged periods of light exposure also resulted in re-localization of arrestin-GFP. Between 150 and 240 min of light adaptation the arrestin-GFP in the ROS gradually declined until the pattern of arrestin-GFP localization was indistinguishable from that of dark-adapted photoreceptors. This distribution pattern was observed over a wide range of lighting intensity (25–2700 lux). Immunocytochemical analysis of arrestin in wild-type Xenopus retinas gave similar results. [Copyright &y& Elsevier]

Atlantic halibut were injected intraperitoneally with human gamma globulin suspended in either phosphate buffered saline, Freunds complete adjuvant or Montanide ISA711 to test the long-term effects of adjuvants. Every month for 12 months up to five animals from each group were sampled. The peritoneal cavity was examined and the adhesion level scored on an arbitrary scale. Serum was also collected and analysed by ELISA for antibodies to human gamma globulin. Results show that whilst FCA produced the highest and fastest antibody response, it also produced the fastest intraperitoneal adhesions, persisting through 11 months. However, the adhesions were not very severe and did not appear to affect the halibut. Crown [Copyright &y& Elsevier]

gp41 is the protein responsible for the process of membrane fusion that allows primate lentiviruses (HIV and SIV) to enter into their host cells. gp41 ectodomain contains an N-terminal and a C-terminal heptad repeat region (NHR and CHR) connected by an immunodominant loop. In the absence of membranes, the NHR and CHR segments fold into a protease-resistant core with a trimeric helical hairpin structure. However, when the immunodominant loop is not present (either in a complex formed by HIV-1 gp41-derived NHR and CHR peptides or by mild treatment with protease of recombinant constructs of HIV-1 gp41 ectodomain, which also lack the N-terminal fusion peptide and the C-terminal Trp-rich region) membrane binding induces a conformational change in the gp41 core structure. Here, we further investigated whether covalently linking the NHR and CHR segments by the immunodominant loop affects this conformational change. Specifically, we analyzed a construct corresponding to a fragment of SIVmac239 gp41ectodomain (residues 27–149, named e-gp41) by means of surface plasmon resonance, Trp and rhodamine fluorescence, ATR-FTIR spectroscopy, and differential scanning calorimetry. Our results suggest that the presence of the loop stabilizes the trimeric helical hairpin both when e-gp41 is in aqueous solution and when it is bound to the membrane surface. Bearing in mind possible differences between HIV-1 and SIV gp41, and considering that the gp41 ectodomain constructs analyzed to date lack the N-terminal fusion peptide and the C-terminal Trp-rich region, we discuss our observations in relation to the mechanism of virus-induced membrane fusion. [Copyright &y& Elsevier]

The expression of splice variants of FGF receptors, which differ in the third Ig domain, was investigated in retinal pigment epithelium (RPE) cells in vitro and in vivo. This region of the protein determines ligand-binding specificity. Additionally, the expression of potential ligands for these receptors was investigated. Expression of FGF receptor transcript alternative splicing was analyzed by RT-PCR/Southern analysis in RPE cells in vitro and in vivo. The expression of FGFs by RT-PCR, in situ hybridization, and immunohistochemistry in sections of the human posterior pole was also investigated. The ARPE-19 cell line expresses only the FGFR2IIIc splice variant and does not express any FGFR3 splice variants in vitro. Two in vivo samples exhibited expression of the FGFR2IIIc and FGFR3IIIc splice variants and no evidence of the corresponding IIIb splice variant. The results from previous studies for these receptors imply that FGF9 or FGF4 could act as ligands. We demonstrated that FGF9 is expressed in a subpopulation of the RPE, as well as photoreceptors and other neurons of the retina. FGF4 was not detected by RT-PCR analysis in RPE cells in vitro. These data suggest that FGF9 may be an autocrine/paracrine factor in the outer retina. [Copyright &y& Elsevier]

Human topoisomerase I interacts with and phosphorylates the SR-family of RNA splicing factors, including ASF/SF2, and has been suggested to play an important role in the regulation of RNA splicing. Here we present evidence to support the theory that the regulation can go the other way around with the SR-proteins controlling topoisomerase I DNA activity. We demonstrate that the splicing factor ASF/SF2 inhibits relaxation by interfering with the DNA cleavage and/or DNA binding steps of human topoisomerase I catalysis. The inhibition of relaxation correlated with the ability of various deletion mutants of the two proteins to interact directly, suggesting that an interaction between the RS-domain of ASF/SF2 and a region between amino acid residues 208–735 on topoisomerase I accounts for the observed effect. Consistently, phosphorylation of the RS-domain with either topoisomerase I or a human cell extract reduced the inhibition of relaxation activity. Taken together with the previously published studies of the topoisomerase I kinase activity, these observations suggest that topoisomerase I activity is shifted from relaxation to kinasing by specific interaction with SR-splicing factors. [Copyright &y& Elsevier]

The primary component of amyloid plaque in the brains of Alzheimer''s patients is the 42 residue amyloid-β-peptide (Aβ42). Although the amino acid residue sequence of Aβ42 is known, the molecular determinants of Aβ amyloidogenesis have not been elucidated. To facilitate an unbiased search for the sequence determinants of Aβ aggregation, we developed a genetic screen that couples a readily observable phenotype in E. coli to the ability of a mutation in Aβ42 to reduce aggregation. The screen is based on our finding that fusions of the wild-type Aβ42 sequence to green fluorescent protein (GFP) form insoluble aggregates in which GFP is inactive. Cells expressing such fusions do not fluoresce. To isolate variants of Aβ42 with reduced tendencies to aggregate, we constructed and screened libraries of Aβ42–GFP fusions in which the sequence of Aβ42 was mutated randomly. Cells expressing GFP fusions to soluble (non-aggregating) variants of Aβ42 exhibit green fluorescence. Implementation of this screen enabled the isolation of 36 variants of Aβ42 with reduced tendencies to aggregate. The sequences of most of these variants are consistent with previous models implicating hydrophobic regions as determinants of Aβ42 aggregation. Some of the variants, however, contain amino acid substitutions not implicated in pre-existing models of Aβ amyloidogenesis. [Copyright &y& Elsevier]

The innate arm of the immune system responds to inflammatory stimuli by the activation of phagocytes, and by altered levels of several plasma proteins. These changes in plasma proteins comprise a major component of the acute phase response, which is thought to be an adaptive response that contributes to regaining homeostasis after tissue injury or infection. In this study, rainbow trout (Oncorhynchus mykiss) were injected with a variety of potential inflammatory agents, and changes in the concentrations of plasma proteins were sought in polyacrylamide gels in which plasma proteins had been electrophoresed. Bacteria, viruses and yeast all induced changes in plasma protein profiles. Increases were first evident 2 days after injections, and most were evident within 1 week. The greatest number of changes occurred after injection with aVibrio bacterin emulsified in Freund''s incomplete adjuvant. While some proteins increased and others decreased following several treatments, other proteins changed only in response to injections of viruses or viral proteins, and others changed in response to bacterial components. Some proteins that increased after yeast injection decreased after injection of viral components. The partial amino acid sequence of one increased protein identified it as haptoglobin. [Copyright &y& Elsevier]

The cytokines IL-6, initially recognized as a regulator of immune and inflammatory response and IL-8, a potential regulator of angiogenesis, also regulate the growth of many tumor cells. Human cancer cells selected for multidrug resistance to common chemotherapeutic agents demonstrate increased expression of IL-6 and IL-8. To determine whether IL-6 or IL-8 overexpression contributes directly to the drug resistant phenotype, IL-6 or IL-8 cDNA were introduced into the paclitaxel sensitive human osteosarcoma cell line U-2OS using the pIRESneo bicistronic expression vector. Interleukin-6 and IL-8 transfectants were selected for either high IL-6 or IL-8 secretion and evaluated in drug resistance assays. Two IL-6 and two IL-8 secreting clones express IL-6 or IL-8 levels of 10 ng/ml and 1 ng/ml in culture, while parental U-2OS and pIRESneo vector transfected control cells express IL-6 and IL-8 levels of 0.005 ng/ml and 0.1 ng/ml, respectively. MTT cytotoxicity with IL-6 transfected cells demonstrates a five-fold increase in resistance to paclitaxel and a four-fold increase in resistance to doxorubicin as compared to U-2OS. There are no changes in mitoxantrone or topotecan resistance in the IL-6 transfectants as compared to parental U-2OS. Northern analysis of IL-6 transfectants demonstrates that the resistant phenotype is not related to increased levels of MDR-1, MRP-1, or LRP. Western analysis also confirms that P-glycoprotein levels are not altered in IL-6 transfectants. Further supporting an MDR-1 independent mechanism of drug resistance, verapamil cannot reverse paclitaxel resistance in transfected cells, findings further supported by rhodamine 123 exclusion data. Treatment of IL-6 transfected cells with paclitaxel, compared with drug-sensitive parental U-2OS, shows U-2OSIL-6 are significantly more resistant to apoptosis induced by paclitaxel and exhibit decreased proteolytic activation of caspase-3. In contrast U-2OSIL-8 transfectants demonstrate no appreciable increase in paclitaxel resistance when compared with parental cells. In summary, while both IL-6 and IL-8 are overexpressed in paclitaxel resistant cell lines, only IL-6 has the potential to contribute directly to paclitaxel and doxorubicin resistance in U-2OS. This resistance is through a non-MDR-1 pathway. [Copyright &y& Elsevier]