Biomedical subjects

M A Alonso

Publications and source records attributed to M A Alonso.

At least 19 recordsLinked to original sources

PubMed2001

MAL mediates apical transport of secretory proteins in polarized epithelial Madin-Darby canine kidney cells.

The MAL proteolipid is an integral membrane protein identified as a component of the raft machinery for apical sorting of membrane proteins in Madin-Darby canine kidney (MDCK) cells. Previous studies have implicated lipid rafts in the transport of exogenous thyroglobulin (Tg), the predominant secretory protein of thyroid epithelial cells, to the apical surface in MDCK cells. We have examined the secretion of recombinant Tg and gp80/clusterin, a major endogenous secretory protein not detected in Triton X-100 insoluble rafts, for the investigation of the involvement of MAL in the constitutive apical secretory pathway of MDCK cells. We show that MAL depletion impairs apical secretion of Tg and causes its accumulation in the Golgi. Cholesterol sequestration, which blocks apical secretion of Tg, did not alter the levels of MAL in rafts but created a block proximal to Tg entrance into rafts. Apical secretion of gp80/clusterin was also inhibited by elimination of endogenous MAL. Our results suggest a role for MAL in the transport of both endogenously and exogenously expressed apical secretory proteins in MDCK cells.

Animals↗

PubMed2001

BENE, a novel raft-associated protein of the MAL proteolipid family, interacts with caveolin-1 in human endothelial-like ECV304 cells.

The MAL proteolipid, an integral protein present in glycolipid- and cholesterol-enriched membrane (GEM) rafts, is an element of the machinery necessary for apical sorting in polarized epithelial Madin-Darby canine kidney cells. MAL was the first member identified of an extended family of proteins that have significant overall sequence identity. In this study we have used a newly generated monoclonal antibody to investigate an unedited member of this family, named BENE, which was found to be expressed in endothelial-like ECV304 cells and normal human endothelium. Human BENE was characterized as a proteolipid protein predominantly present in GEM rafts in ECV304 cells. Coimmunoprecipitation experiments revealed that BENE interacted with caveolin-1. Confocal immunofluorescence and electron microscopic analyses indicated that BENE mainly accumulated into intracellular vesicular/tubular structures that partially colocalize with internal caveolin-1. In response to cell surface cholesterol oxidation, BENE redistributed to the dilated vesicular structures that concentrate most of the caveolin-1 originally on the cell surface. After cessation of cholesterol oxidation, a detectable fraction of the BENE molecules migrated to the plasmalemma accompanying caveolin-1 and then returned progressively to its steady state distribution. Together, these features highlight the BENE proteolipid as being an element of the machinery for raft-mediated trafficking in endothelial cells.

Amino Acid Sequence↗

PubMed2001

Segregation of co-stimulatory components into specific T cell surface lipid rafts.

The glycosylphosphatidylinositol (GPI)-anchored protein CD59 and the ganglioside GM1 are present on lipid rafts that can be isolated in a detergent-insoluble membrane (DIM) fraction. TCR engagement promotes integration of components of the TCR/CD3 signaling machinery into DIM. As DIM are isolated as a heterogeneous mixture of coalescent membranes, it is uncertain whether the cofractionation of GPI-anchored proteins and GM1 reflects the existence of an association between these molecules within the same lipid rafts in the cell. We have studied the surface distribution of the co-stimulatory CD59 and GM1 molecules and their role in the recruitment of components of the TCR signaling machinery in DIM. Although both CD59 and GM1 are present in rafts, these molecules occur in a steady state, mainly clustered in different membrane subdomains. Multimerization of either molecule did not induce cocapping or co-internalization of the other. Aggregation of GM1, CD59 or TCR/CD3 increased tyrosine phosphorylation but only in the latter case was a significant increase observed in both tyrosine phosphorylation and recruitment of elements of the signaling machinery in DIM. Our results show the existence of specific co-stimulatory membrane microdomains that require a direct TCR/CD3 engagement for efficient recruitment of signaling machinery into rafts.

Animals↗

PubMed2001

An intact dilysine-like motif in the carboxyl terminus of MAL is required for normal apical transport of the influenza virus hemagglutinin cargo protein in epithelial Madin-Darby canine kidney cells.

The MAL proteolipid, a component of the integral protein sorting machinery, has been demonstrated as being necessary for normal apical transport of the influenza virus hemagglutinin (HA) and the overall apical membrane proteins in Madin-Darby canine kidney (MDCK) cells. The MAL carboxy terminus ends with the sequence Arg-Trp-Lys-Ser-Ser (RWKSS), which resembles dilysine-based motifs involved in protein sorting. To investigate whether the RWKSS pentapeptide plays a role in modulating the distribution of MAL and/or its function in apical transport, we have expressed MAL proteins with distinct carboxy terminus in MDCK cells whose apical transport was impaired by depletion of endogenous MAL. Apical transport of HA was restored to normal levels by expression of MAL with an intact but not with modified carboxyl terminal sequences bearing mutations that impair the functioning of dilysine-based sorting signals, although all the MAL proteins analyzed incorporated efficiently into lipid rafts. Ultrastructural analysis indicated that compared with MAL bearing an intact RWKSS sequence, a mutant with lysine -3 substituted by serine showed a twofold increased presence in clathrin-coated cytoplasmic structures and a reduced expression on the plasma membrane. These results indicate that the carboxyl-terminal RWKSS sequence modulates the distribution of MAL in clathrin-coated elements and is necessary for HA transport to the apical surface.

Adaptor Protein Complex gamma Subunits↗

PubMed2001

The role of lipid rafts in signalling and membrane trafficking in T lymphocytes.

Combinatorial association of different lipid species generates microheterogeneity in biological membranes. The association of glycosphingolipids with cholesterol forms membrane microdomains--lipid rafts--that are involved in specialised pathways of protein/lipid transport and signalling. Lipid rafts are normally dispersed in cellular membranes and appear to require specialised machinery to reorganise them to operate. Caveolin-1 and MAL are members of two different protein families involved in reorganisation of lipid rafts for signalling and/or intracellular transport in epithelial cells. T cell activation induces a rapid compartmentalisation of signalling machinery into reorganised rafts that are used as platforms for the assembly of the signalling complex. Costimulatory molecules participate in this process by providing signals that mobilise raft lipids and proteins, and remodel the cytoskeleton to the contact site. As in epithelial cells, rafts are used also as vesicular carriers for membrane trafficking in T lymphocytes. Furthermore, there are potential similarities between the specialised protein machinery underlying raft-mediated processes in T lymphocytes and polarised epithelial cells.

Animals↗

PubMed2001

Using rays better. I. Theory for smoothly varying media.

We present a method for computing ray-based approximations to optical fields that not only offers unprecedented accuracy but is also accompanied by accessible error estimates. The basic elements of propagation through smooth media, refraction and reflection at interfaces, and diffraction by obstacles give the foundations for the new framework, and the first of these is treated here. The key in each case is that the wave field and any relevant derivatives are expressed consistently as a superposition of delocalized ray contributions. In this way, the mysteries surrounding the sometimes perplexing tenaciousness of ray-based estimates are clearly resolved. Further, an essential degree of freedom in this approach offers an attractive resolution of part of the apparent conflict of particle/wave duality.

Journal Article↗

PubMed2001

Using rays better. II. Ray families to match prescribed wave fields.

A key step in any ray-based method for propagating waves is the choice of a family of rays to be associated with the initial wave field. We develop some basic prescriptions for constructing initial ray families to match two particular types of waves. Various Gaussian and Bessel beams are separately given special treatment because of their general interest. These ideas are directly useful for a newly developed method for ray-based wave modeling. The new method expresses the wave as a superposition of ray contributions that is independent of the width of the field element associated with each ray. This insensitivity is investigated here even when the elemental width varies from ray to ray. The results increase the applicability of the new wave-modeling scheme.

Journal Article↗

PubMed2001

Using rays better. III. Error estimates and illustrative applications in smooth media.

A new method for computing ray-based approximations to optical wave fields is demonstrated through simple examples involving wave propagation in free space and in a gradient-index waveguide. The analytic solutions that exist for these cases make it easy to compare the new estimates with exact results. A particularly simple RMS error estimate is developed here, and corrections to the basic field estimate are also discussed and tested. A key step for any ray-based method is the choice of a family of rays to be associated with the initial wave field. We show that, for maximal accuracy, not only must the initial field be considered in choosing the rays, but so too must the medium that is to carry the wave.

Journal Article↗

PubMed2001

Radiometry and wide-angle wave fields III: partial coherence.

The analogs of the generalized radiances introduced in two previous manuscripts [J. Opt. Soc. Am. A 18, 902 (2001) and J. Opt. Soc. Am. A 18, 910 (2001)] for fully coherent fields in two- and three-dimensional free space are given here for the case of partial coherence. These functions are exactly conserved along rays and are suitable for the description of fields with components propagating in any direction. Also defined here is a global measure of coherence, which can be expressed in terms of the new functions. The cases of radiation in a blackbody cavity and partially coherent focused waves are considered as examples.

Journal Article↗

PubMed2000

Thyroglobulin is selected as luminal protein cargo for apical transport via detergent-resistant membranes in epithelial cells.

Thyroid hormone synthesis by thyrocytes depends upon apical secretion of thyroglobulin (Tg), the glycoprotein prohormone. In stably transfected MDCK cells, recombinant Tg is also secreted apically. All secreted Tg has undergone Golgi carbohydrate modification, whereas most intracellular Tg (which is slow to exit the endoplasmic reticulum) is sensitive to digestion with endoglycosidase H. However, in MDCK cells and PC Cl3 thyrocytes, a subpopulation of newly synthesized recombinant and endogenous Tg, respectively, is recovered in a Triton X-100 insoluble, glycosphingolipid/cholesterol-enriched (GEM/raft) fraction, and this small subpopulation is overwhelmingly endoglycosidase H resistant. Upon apical secretion, Tg solubility is restored. Apical secretion of Tg is inhibited by cellular cholesterol depletion. In FRT cells, recombinant Tg becomes Triton X-100 insoluble within 60 min after synthesis and a portion is actually endoglycosidase H-sensitive, suggesting early Tg entry into GEMs/rafts. Interestingly in FRT cells, Tg remains associated with the apical plasma membrane upon exocytosis, and all surface Tg is GEM/raft-associated. Thus, Tg is the first secretory protein demonstrated to enter Triton X-100 insoluble membranes en route to the apical surface of epithelial cells. The data imply that Tg utilizes a cargo-selective mechanism for apical sorting.

Animals↗

PubMed2000

Rhodium complexes with the chelating and binucleating ligands P(CH2CH2Py)nPh3-n (Py = 2-pyridyl; n = 1,2): structures and fluxional behavior.

Several rhodium(I) complexes of the type [RhX(CO)(PePy2)], [Rh(diene)(PePy)]+, and [Rh(diene)(PePy2)]+ (PePyn = P(CH2CH2Py)nPh3-n; Py = 2-pyridyl; n = 1, 2) have been prepared. The two former are square planar; the latter are pentacoordinated for diene = tetrafluorobenzobarrelene or norbornadiene (confirmed by X-ray diffraction), but an equilibrium of 4- and 5-coordinate isomers exists in solution for diene = 1,5-cyclooctadiene. The fluxional behavior of all these complexes is studied by NMR spectroscopy. The complex [Rh(NBD)(PePy2)]PF6.Cl2CH2 crystallizes in the monoclinic space group P21/n with a = 8.455(1) A, b = 18.068(3) A, c = 19.729(3) A, beta = 99.658(3)degrees, and Z = 4. The complexes [Rh(diene)(PePy2)]+ react with CO to give the dimeric complex [Rh2(CO)2[P(CH2CH2Py)2Ph]2](BF4)2 with the pyridylphosphine acting as P,N-chelating and P,N-bridging.

Journal Article↗

PubMed2000

The amino-terminal nine amino acid sequence of poliovirus capsid VP4 protein is sufficient to confer N-myristoylation and targeting to detergent-insoluble membranes.

The confinement of membrane proteins by lipid-lipid interactions into specialized detergent-insoluble membrane (DIM) microdomains has been proposed as a general mechanism to recruit selectively lipid-modified proteins and specific transmembrane proteins. Poliovirus capsid VP4 protein and its precursors are myristoylated at the NH(2)-terminal Gly residue. To determine whether poliovirus uses DIMs during its replicative cycle, we isolated DIMs from poliovirus-infected HeLa cells and identified the presence of capsid proteins and their precursors, proteinases 2A and 3C, and other viral proteins involved in poliovirus RNA replication such as protein 2C and the polymerase 3D. The morphology of these DIMs was similar to that of the previously described rosette-like vesicles associated with replication complexes isolated from poliovirus-infected cells. To examine the possible role of the myristoyl moiety in the targeting of poliovirus structural proteins to DIMs, we generated a chimeric protein consisting of the nine amino-terminal amino acids from VP4 fused to the amino terminus of the green fluorescent protein (GFP). The selected VP4 sequence was sufficient to confer N-myristoylation and targeting to DIMs to the GFP chimera. Mutations within this sequence known to affect both myristoylation and poliovirus assembly abrogated the targeting of the GFP chimera. These results indicate that the myristoylated amino-terminal nonapeptide from poliovirus VP4 protein constitutes a signal for incorporation into DIMs.

Animals↗

PubMed2000

The MAL proteolipid is necessary for the overall apical delivery of membrane proteins in the polarized epithelial Madin-Darby canine kidney and fischer rat thyroid cell lines.

The MAL proteolipid has been recently demonstrated as being necessary for correct apical sorting of the transmembrane influenza virus hemagglutinin (HA) in Madin-Darby canine kidney (MDCK) cells. The fact that, in contrast to MDCK cells, Fischer rat thyroid (FRT) cells target the majority of glycosylphosphatidylinositol (GPI)-anchored proteins to the basolateral membrane provides us with the opportunity to determine the role of MAL in apical transport of membrane proteins under conditions in which the majority of GPI-anchored proteins are (MDCK cells) or are not (FRT cells) targeted to the apical surface. Using an antisense oligonucleotide-based strategy to deplete endogenous MAL, we have observed that correct transport of apical transmembrane proteins associated (HA) or not (exogenous neurotrophin receptor and endogenous dipeptidyl peptidase IV) with lipid rafts, as well as that of the bulk of endogenous apical membrane, takes place in FRT cells by a pathway that requires normal MAL levels. Even transport of placental alkaline phosphatase, a GPI-anchored protein that is targeted apically in FRT cells, was dependent on normal MAL levels. Similarly, in addition to the reported effect of MAL on HA transport, depletion of MAL in MDCK cells caused a dramatic reduction in the apical delivery of the GPI-anchored gD1-DAF protein, neurotrophin receptor, and the bulk of membrane proteins. These results suggest that MAL is necessary for the overall apical transport of membrane proteins in polarized MDCK and FRT cells.

Animals↗

PubMed2000

Phase-space distributions for high-frequency fields.

The Wigner distribution function and various windowed Fourier transforms are examples of phase-space distributions that are used, among other things, to formalize the link between ray and wave optics. It is well known that, in the limit of high frequencies, these distributions become localized for simple wave fields and therefore that the localization can be used to define the associated ray families. This localized form is characterized here for both the Wigner distribution function and a Gaussian windowed Fourier transform. Aside from the greater understanding of the distributions themselves, these results promise a clearer intuition of phase-space-based methods for optical modeling. In particular, regardless of the context, the geometric construction that is presented for estimating the Wigner distribution function gives a valuable appreciation of its highly structured and sometimes surprising form.

Journal Article↗

PubMed2000

Uncertainty products for nonparaxial wave fields.

Although maximal localization is a basic notion in the consideration of phase-space representations of fields, it has not yet been pursued for general wave fields. We develop measures of spatial and directional spreads for nonparaxial waves in free space. These measures are invariant under translation and rotation and are shown to reduce to the conventional ones when applied to paraxial fields. The associated uncertainty relation sets limits to joint localization in coordinate and frequency space. This relation provides a basis for the definition of a joint localization measure that is analogous to the beam propagation factor (i.e., M2) of paraxial optics. The results are first developed for two-dimensional fields and then generalized to three dimensions.

Journal Article↗

PubMed1999

The N-terminal 209-aa domain of high molecular-weight 4.1R isoforms abrogates 4.1R targeting to the nucleus.

An extensive repertoire of protein 4.1R isoforms is predominantly generated by alternative pre-mRNA splicing and differential usage of two translation initiation sites. The usage of the most upstream ATG (ATG-1) generates isoforms containing N-terminal extensions of up to 209 aa compared with those translated from the downstream ATG (ATG-2). To characterize nonerythroid 4.1R proteins translated from ATG-1 and analyze their intracellular localization, we cloned 4.1R cDNAs containing this translation initiation site. Six different clones were isolated from the nucleated human MOLT-4 T-cell line by reverse transcriptase-PCR techniques. Transient expression of the six ATG-1-translated 4.1R isoforms tagged with a c-Myc epitope revealed that all of them predominantly distributed to the plasma membrane and the endoplasmic reticulum. Staining of MOLT-4 cell plasma membranes but not nuclei was also observed by immunofluorescence microscopy by using an antibody specific to the N-terminal extension. Consistent with this, the antibody reacted with a major endogenous protein of approximately 145 kDa present in nonnuclear but absent from nuclear fractions prepared from MOLT-4 cells. Because these data suggested that ATG-1-translated 4.1R isoforms were predominantly excluded from the nucleus, we fused the 209-aa domain to nuclear 4.1R isoforms encoded from ATG-2 and observed that this domain inhibited their nuclear targeting. All these results indicate that the N-terminal domain of ATG-1-translated 4.1R isoforms plays a pivotal role in differential targeting of proteins 4.1R.

Biological Transport↗

PubMed1999

Incorporation of MAL, an integral protein element of the machinery for the glycolipid and cholesterol-mediated apical pathway of transport, into artificial membranes requires neither of these lipid species.

The MAL proteolipid, an integral membrane protein with selective residence in glycolipid- and cholesterol-enriched membrane (GEM) microdomains, has recently been identified as being an element of the integral protein machinery necessary for apical transport in MDCK cells. With the use of a recombinant baculovirus, we have expressed and purified polyhistidine-tagged MAL to determine whether MAL has special lipid requirements for becoming incorporated into membranes. In contrast with caveolin-1, a component of GEMs that requires cholesterol for its integration into artificial membranes, MAL incorporation took place with dimyristoylphosphatidylcholine as the only lipid component. The presence of cholesterol, sphingomyelin, or galactocerebrosides did not affect the efficiency of this process. These results indicated that MAL is compatible with membranes containing either only phospholipids or also glycolipids and cholesterol and are consistent with the reported requirement of a sorting event for the specific targeting of MAL to GEM microdomains.

Animals↗

PubMed1999

The MAL gene is expressed in primary mediastinal large B-cell lymphoma.

Primary mediastinal large B-cell lymphoma (PMBL) appears to be a distinct clinicopathologic entity among diffuse large B-cell lymphomas (DLBLs). To find molecular alterations associated with this disease, we compared the mRNAs expressed in 3 PMBLs and 3 peripheral DLBLs by differential display-reverse transcription (DDRT) and identified a mRNA specifically expressed in PMBLs. Sequence analysis showed that this mRNA is encoded by the MAL gene, the expression of which was shown to be restricted to the T-cell lineage during hematopoiesis. MAL gene expression was demonstrated by Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) in 8 of 12 PMBLs. However, there was little or no MAL gene expression in 8 peripheral DLBLs. Immunohistochemical analysis evidenced expression of MAL protein in tumoral B cells restricted to the PMBL subtype. Finally, Southern blot studies did not demonstrate rearrangement of the MAL gene. Altogether, our results indicate that MAL expression is recurrent in PMBLs, providing further evidence that PMBL represents a distinct entity among DLBLs. Because MAL protein is located in detergent-insoluble glycolipid-enriched membrane (GEM) domains involved in lymphocyte signal transduction, abnormal expression of MAL protein in the B-lymphoid lineage may have significant implications in PMBL lymphomagenesis.

Adult↗