Deletion of small ankyrin 1 (sAnk1) isoforms results in structural and functional alterations in aging skeletal muscle fibers.

Muscle-specific ankyrins 1 (sAnk1) are a group of small ankyrin 1 isoforms, of which sAnk1.5 is the most abundant. sAnk1 are localized in the sarcoplasmic reticulum (SR) membrane from where they interact with obscurin, a myofibrillar protein. This interaction appears to contribute to stabilize the SR close to the myofibrils. Here we report the structural and functional characterization of skeletal muscles from sAnk1 knockout mice (KO). Deletion of sAnk1 did not change the expression and localization of SR proteins in 4- to 6-mo-old sAnk1 KO mice. Structurally, the main modification observed in skeletal muscles of adult sAnk1 KO mice (4-6 mo of age) was the reduction of SR volume at the sarcomere A band level. With increasing age (at 12-15 mo of age) extensor digitorum longus (EDL) skeletal muscles of sAnk1 KO mice develop prematurely large tubular aggregates, whereas diaphragm undergoes significant structural damage. Parallel functional studies revealed specific changes in the contractile performance of muscles from sAnk1 KO mice and a reduced exercise tolerance in an endurance test on treadmill compared with control mice. Moreover, reduced Qγ charge and L-type Ca(2+) current, which are indexes of affected excitation-contraction coupling, were observed in diaphragm fibers from 12- to 15-mo-old mice, but not in other skeletal muscles from sAnk1 KO mice. Altogether, these findings show that the ablation of sAnk1, by altering the organization of the SR, renders skeletal muscles susceptible to undergo structural and functional alterations more evident with age, and point to an important contribution of sAnk1 to the maintenance of the longitudinal SR architecture.

Chitosan-coated alginate micro-particles delivery of active principles through conventional pelleted food - A study in Tilapia (Oreochromis niloticus).

The search for alternatives to antibiotics in aquaculture has focused on the use of vaccines for immune-prophylaxis. The purpose of this study was to examine the feasibility and characteristics of chitosan-alginate microparticles for the oral delivery of immune-prophylactics to finfish. The microparticles, which incorporate fluorescent-labelled lysozyme, were produced by spray-drying method; their structural properties and uptake from the gastrointestinal tract of Tilapia (Oreochromis niloticus) were assessed by microscopy. The main findings show that the microparticles are able to retain their content in an acidic environment and to release it later in slightly alkaline conditions such as those found in the intestines. Moreover, both the microencapsulation procedure and the biopolymers used have no deleterious impact on the lysozyme lytic activity, which is maintained after the protein has been released from the microparticles. Administered in vivo in Tilapia by medicated food, the microparticles transit unaffected through the stomach, and reach the anterior intestines, in particular the villum sectum and the basal lamina of epithelial cells, 2 and 4 h after feeding. Overall, the evidence obtained here supports the potential of these chitosan-alginate microparticles as agents for oral immune-prophylaxis in the management of fish diseases.

DSP1, an HMG-like protein, is involved in the regulation of homeotic genes.

The Drosophila dsp1 gene, which encodes an HMG-like protein, was originally identified in a screen for corepressors of Dorsal. Here we report that loss of dsp1 function causes homeotic transformations resembling those associated with loss of function in the homeotic genes Sex combs reduced (Scr), Ultrabithorax (Ubx), and Abdominal-B. The expression pattern of Scr is altered in dsp1 mutant imaginal discs, indicating that dsp1 is required for normal expression of this gene. Genetic interaction studies reveal that a null allele of dsp1 enhances trithorax-group gene (trx-G) mutations and partially suppresses Polycomb-group gene (Pc-G) mutations. On the contrary, overexpression of dsp1 induces an enhancement of the transformation of wings into halteres and of the extra sex comb phenotype of Pc. In addition, dsp1 male mutants exhibit a mild transformation of A4 into A5. Comparison of the chromatin structure at the Mcp locus in wild-type and dsp1 mutant embryos reveals that the 300-bp DNase I hypersensitive region is absent in a dsp1 mutant context. We propose that DSP1 protein is a chromatin remodeling factor, acting as a trx-G or a Pc-G protein depending on the considered function.

A hormone-dependent post-translationally regulated mutant for investigating type I cadherin function.

Type I cadherins are Ca2+-dependent cell adhesion molecules. Their function in early Xenopus laevis development has been extensively studied in recent years, by injecting synthetic mRNAs encoding dominant negative mutants with deletions of the extracellular domain into embryos. However, studies at post-gastrula stages have been hampered by the inabilityto progress through post-gastrula development in embryos expressing these mutant proteins. This problem has been partly overcome by injecting into a few targeted blastomeres in stage 6 N.F. embryos, but only restricted studies are possible with this technique. Several studies have made use of the hormone-binding domain (HBD), which is activated by hormones. In this study, we used this method to analyze the activity of dominant negative cadherins. We generated a mutant E-cadherin (AE-Cad, consisting of the cytoplasmic domain and transmembrane domain) fused to the hormone-binding domain of estradiol receptor (HBDER) and we validated this technique with functional analyses. The function of the mutant deltaE-HBDER was strictly dependent on hormone induction. This conditional mutant had the same effects and exerted the same dominant negative function as the corresponding constitutive mutant.

Centrifugal assay for fluorescence-based cell adhesion adapted to the analysis of ex vivo cells and capable of determining relative binding strengths.

Cell adhesion assays are widely used to identify novel cellular ligands, novel cell surface receptors for these ligands and to elucidate the mechanisms responsible for the underlying cellular and molecular interactions. We report here the development of a novel centrifugal assay for fluorescence-based cell adhesion (CAFCA) that offers a number of advantages over the currently available assays. CAFCA is based on two centrifugation steps: one to allow for the synchronization of the initial cell-substratum contact and one to enable both a defined removal force to be exerted onto the cells for displacement of unbound cells and determination of the relative binding strengths of adhering cells. The fluorescently tagged cells are monitored in specifically devised, disposable microplate assemblies by a two-sided fluorescence detection through the computer-interfaced SPECTRAFLUOR microplate fluorometer. The assay is rapid, accurate, reproducible and adaptable to small numbers of delicate primary cells that can ideally be labeled with the fluorochrome calcein AM (or analogous vital fluorescent dyes). Most uniquely, CAFCA provides (i) means of assessing the precise number of cells bound to a given substratum out of the total amount of cells contained within the population to be analyzed and (ii) a means of establishing the attachment strengths (i.e., dynes/cell) in a high number of samples/conditions simultaneously. CAFCA is therefore expected to make a substantial methodological and conceptual contribution to the range of available assays aimed at examining cellular interactions in vitro and promises the potential of being able to transpose automated versions of these tests for routine use in laboratories.

Stimulation of GALT and activation of mesenteric lymph node lymphocytes by a modified lysozyme in CBA mice with MCa mammary carcinoma.

Lysozyme (hen egg-white lysozyme) and its derivative mPEG-lyso (lysozyme coupled with polyoxyethyleneglycol) were tested in CBA mice bearing MCa mammary carcinoma for their effects on intestinal mucosal immunity (GALT) and mesenteric lymph node lymphocytes (MLNL), after oral administration. Following a cycle of administration of 100 mg/kg/day lysozyme or 350 mg/kg/day mPEG-lyso for 9 consecutive days, GALT was analyzed by using optical histology, and mesenteric lymph node lymphocytes were studied by cytofluorimetric analysis of CD3, CD4 and CD8 antigens, and of DNA and RNA content following in vitro culture with concanavalin A. Both lysozymes significantly increase the number of lymphatic nodules on gut epithelium as determined by histological analysis of sections of small bowel. mPEG-lyso, unlike native lysozyme, gives protection from the decline of the blastogenic activity of MLNL observed at early stages of tumor growth, as shown by the increased nucleic acid content of these cells. On the same cells, both lysozyme and mPEG-lyso also seem to prevent the decline of CD4+ cells observed during tumor growth in control animals. These data confirm the effects of lysozyme on GALT and show that the new lysozyme derivative mPEG-lyso has effects on host immunity greater than those of the native molecule.

Cytofluorimetric analysis of gut-intraepithelial and mesenteric lymph node lymphocytes of tumour bearing mice fed with egg-white lysozyme.

The effects of the oral administration of 100 mg/kg/day egg-white lysozyme (EWL) on the expression of CD3, CD4, CD8 and CD25 antigens of lymphocytes harvested from IEL and mesenteric lymph nodes (MLNL) were tested in mice bearing MCa mammary carcinoma. Lysozyme, after oral administration, retains its enzymatic activity along the entire small bowel and almost 10% of the administered dose is recovered 1 hr after treatment in the middle of the jejunum. Correspondingly, the number of cells expressing the test antigens in MLNL is greater than in controls after a few days of treatment and is maintained high up to the end of treatment but returns to control values after treatment withdrawal; CD4:CD8 ratio is decreased by EWL in favour of CD8 positive cells. Treatment with EWL does not modify the ratio between CD4+ and CD8+ cells vs controls in IEL nor does it change the % of CD3 positive cells or the expression of IL-2 receptor at this level. These data support the existence of the induction of an immunity communication by EWL along the axis GALT-mesenteric lymph nodes which is in agreement with the reported effects of the oral administration of EWL on tumour growth in experimental systems and on host immunity in humans.

Antimetastatic action and lymphocyte activation by the modified lysozyme mPEG-Lyso in mice with MCa mammary carcinoma.

The effects of Lysozyme (hen egg-white lysozyme) and of its modified derivative mPEG-Lyso, (Lysozyme coupled with monomethoxypolyethylenglycol) were tested on CBA mice bearing MCa mammary carcinoma. mPEG-Lyso, given by the oral route at a dose comparable to 100 mg/kg/day of native Lysozyme, is at least as active as Lysozyme for the activation of lymphocytes obtained from different districts along the axis GALT-spleen. These effects were evidenced by measuring the in vitro response of lymphocytes of animals treated in vivo with ConA and LPS using the SRB test, and measuring the content of nucleic acids by cytofluorimetric analysis. Lymphocytes obtained from the mesenteric lymph nodes of animals treated with mPEG-Lyso, show a response to ConA and to LPS at early stages of treatment, when tumor growth reduces the response to controls. mPEG-Lyso, was also effective on lung metastasis formation. Considering that mPEG-Lyso,, compared to the native Lysozyme, completely lost its enzymatic action on Micrococcus lysodehycticus cell walls, this data suggest that the effects of lysozyme on immunity and on tumour growth are unrelated to the production of immunoactive peptidoglycans in the gut.

The KSR2-calcineurin complex regulates STIM1-ORAI1 dynamics and store-operated calcium entry (SOCE).

Store-operated calcium entry (SOCE) is the predominant Ca(2+) entry mechanism in nonexcitable cells and controls a variety of physiological and pathological processes. Although significant progress has been made in identifying the components required for SOCE, the molecular mechanisms underlying it are elusive. The present study provides evidence for a direct involvement of kinase suppressor of Ras 2 (KSR2) in SOCE. Using lymphocytes and fibroblasts from ksr2(-/-) mice and shKSR2-depleted cells, we find that KSR2 is critical for the elevation of cytosolic Ca(2+) concentration. Specifically, our results show that although it is dispensable for Ca(2+)-store depletion, KSR2 is required for optimal calcium entry. We observe that KSR2 deficiency affects stromal interaction molecule 1 (STIM1)/ORAI1 puncta formation, which is correlated with cytoskeleton disorganization. Of interest, we find that KSR2-associated calcineurin is crucial for SOCE. Blocking calcineurin activity impairs STIM1/ORAI1 puncta-like formation and cytoskeleton organization. In addition, we observe that calcineurin activity and its role in SOCE are both KSR2 dependent.

Expression and localization of ATP binding cassette transporter A1 (ABCA1) in first trimester and term human placenta.

ATP binding cassette transporter A1 (ABCA1) is a membrane transporter which performs cellular efflux of cholesterol and phospholipid. ABCA1's cholesterol transporting role in human placenta appears to be crucial for normal fetal development. Despite the critical importance of cholesterol in fetal development, expression of ABCA1 in the human placenta throughout gestation and its specific cellular localization have not been known yet. We therefore investigated ABCA1 expression in human placenta at first trimester and term by western blot and quantitative real-time PCR (qRT-PCR) analysis. Furthermore, its localization was investigated by immunohistochemistry and confocal microscopy. Expression of ABCA1 did not differ significantly between first trimester and term placenta at both protein and mRNA levels. Immunohistochemical data demonstrated that ABCA1 was widely localized in the villous and extravillous cytotrophoblast as well as in some stromal and endothelial cells. Confocal microscopy imaging data showed that ABCA1 was localized largely at the basolateral and to some extent at the apical side of first trimester villous cytotrophoblast cell membranes. Placental expression of ABCA1 throughout the gestation and its specific cellular localization indicate that this transporter may play an important role in materno-fetal cholesterol transfer.

Cloning and characterization of three Xenopus slug promoters reveal direct regulation by Lef/beta-catenin signaling.

In amphibians and birds, one of the first steps of neural crest cell (NCC) determination is expression of the transcription factor Slug. This marker has been used to demonstrate that BMP and Wnt molecules play a major role in NCC induction. However, it is unknown whether Slug expression is directly or indirectly regulated by these signals. We report here the cloning and characterization of three Xenopus Slug promoters: that of the Xenopus tropicalis slug gene and those of two Xenopus laevis Slug pseudoalleles. Although the three genes encode proteins with almost identical amino acid sequences and are expressed with similar spatiotemporal patterns, their 5'-flanking regions are quite different. A striking difference is a deletion in the X. tropicalis gene located precisely at the transcription initiation site that results in the X. tropicalis promoter being inefficient in X. laevis. Additionally, we identified two regions common to the three promoters that are necessary and sufficient to drive specific expression in NCCs. Interestingly, one of the common regulatory regions presents a functional Lef/beta-catenin-binding site necessary for specific expression. As the Lef.beta-catenin complex is a downstream effector of Wnt signaling, these results suggest that Xenopus Slug is a direct target of NCC determination signals.

Self-assembly and supramolecular organization of EMILIN.

The primary structure of human Elastin microfibril interface-located protein (EMILIN), an elastic fiber-associated glycoprotein, consists of a globular C1q domain (gC1q) at the C terminus, a short collagenous stalk, a long region with a high potential for forming coiled-coil alpha helices, and a cysteine-rich N-terminal sequence. It is not known whether the EMILIN gC1q domain is involved in the assembly process and in the supramolecular organization as shown for the similar domain of collagen X. By employing the yeast two-hybrid system the EMILIN gC1q domains interacted with themselves, proving for the first time that this interaction occurs in vivo. The gC1q domain formed oligomers running as trimers in native gels that were less stable than the comparable trimers of the collagen X gC1q domain since they did not withstand heating. The collagenous domain was trypsin-resistant and migrated at a size corresponding to a triple helix under native conditions. In reducing agarose gels, EMILIN also migrated as a trimer, whereas under non-reducing conditions it formed polymers of many millions of daltons. A truncated fragment lacking gC1q and collagenous domains assembled to a much lesser extent, thus deducing that the C-terminal domain(s) are essential for the formation of trimers that finally assemble into large EMILIN multimers.

EMILIN, a component of the elastic fiber and a new member of the C1q/tumor necrosis factor superfamily of proteins.

EMILIN (elastin microfibril interface located protein) is an extracellular matrix glycoprotein abundantly expressed in elastin-rich tissues such as blood vessels, skin, heart, and lung. It occurs associated with elastic fibers at the interface between amorphous elastin and microfibrils. Avian EMILIN was extracted from 19-day-old embryonic chick aortas and associated blood vessels and purified by ion-exchange chromatography and gel filtration. Tryptic peptides were generated from EMILIN and sequenced, and degenerate inosine-containing oligonucleotide primers were designed from some peptides. A set of primers allowed the amplification of a 360-base pair reverse transcription polymerase chain reaction product from chick aorta mRNA. A probe based on a human homologue selected by comparison of the chick sequence with EST data base was used to select overlapping clones from both human aorta and kidney cDNA libraries. Here we present the cDNA sequence of the entire coding region of human EMILIN encompassing an open reading frame of 1016 amino acid residues. There was a high degree of homology (76% identity and 88% similarity) between the chick C terminus and the human sequence as well as between the N terminus of the mature chick protein where 10 of 12 residues, as determined by N-terminal sequencing, were identical or similar to the deduced N terminus of human EMILIN. The domain organization of human EMILIN includes a C1q-like globular domain at the C terminus, a collagenous stalk, and a longer segment in which at least four heptad repeats and a leucine zipper can be identified with a high potential for forming coiled-coil alpha helices. At the N terminus there is a cysteine-rich sequence stretch similar to a region of multimerin, a platelet and endothelial cell component, containing a partial epidermal growth factor-like motif. The native state of the recombinantly expressed EMILIN C1q-like domain to be used in cell adhesion was determined by CD spectra analysis, which indicated a high value of beta-sheet conformation. The EMILIN C1q-like domain promoted a high cell adhesion of the leiomyosarcoma cell line SK-UT-1, whereas the fibrosarcoma cell line HT1080 was negative.

Cooperative activity of alpha4beta1 and alpha4beta7 integrins in mediating human B-cell lymphoma adhesion and chemotaxis on fibronectin through recognition of multiple synergizing binding sites within the central cell-binding domain.

We have quantitated the relative contributions of the constitutively active alpha4beta1 and alpha4beta7 integrins and the domains embodying their cognate binding sites in mediating human B-cell lymphoma adhesion and chemotaxis on fibronectin. By cooperating, the central cell-binding and IIICS carboxy-terminal domains were entirely responsible for the adhesion activity displayed by fibronectin, and their relative contribution to this process was estimated to be 30% versus 70%. Assessment of the leukocyte-substrate binding strength (ie, dynes/cell) indicated a 10-fold higher avidity of the cell-IIICS domain interaction. The two integrins interchangeably recognized both domains, but differed quantitatively in their participation in the adhesive event, as well as in domain preference. The use of 3Fn (according to the nomenclature proposed by Bork and Koonin [Curr Opin Struct Biol 6:366, 1996] for the type III fibronectin modules) module-specific antibodies and recombinant polypeptides showed that alpha4 integrins recognized both the RGD sequence (3Fn10) and an apparently novel synergistic site located within the 3Fn8 module; even in this case, the integrins displayed a distinct binding site preference. Interleukin-1beta (IL-1beta)/IL-2-induced chemotaxis also involved cooperative function of the central cell-binding and IIICS domains, but the mechanisms regulating this phenomenon differed markedly from those controlling cell adhesion. First, the relative contribution of the individual domains was comparable, but neither of the individual domains promoted migration to the extent observed on intact fibronectin. Secondly, alpha4beta1 and alpha4beta7 integrins were both involved in the domain-binding necessary for initiation of migration, but the relative contribution of each receptor in the chemotactic process was less disparate than for initial cell adhesion. Thirdly, the mode by which chemotactic B-lymphoma movement was supported by the central cell-binding domain differed from that sustaining cell adhesion in that it involved independent recognition of either the 3Fn8 or the 3Fn9 module, which acted in synergy with the 3Fn10 module. Our data provide novel evidence concerning the relative importance of the constitutively active alpha4beta1 and alpha4beta7 integrins for the interaction of B-cell lymphoma cells with fibronectin, and they emphasize a multiple and diverse recognition of sites responsible for either anchorage or locomotion of tumor leukocytes on this matrix molecule.