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PubMed2000

Isolation and characterization of myrmexins, six isoforms of venom proteins with anti-inflammatory activity from the tropical ant, Pseudomyrmex triplarinus.

Venom from the tropical ant, Pseudomyrmex triplarinus, has anti-inflammatory properties demonstrated by the carrageenin-induced edema animal mode. A multi-protein complex that inhibits edema was isolated from the venom and was further characterized by high performance liquid chromatography (HPLC), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and amino acid sequencing. Although the complex exhibited a single band in SDS-PAGE electrophoresis, six proteins (isoforms) were resolved and purified to homogeneity and were designated myrmexin I-VI. They have very similar molecular masses between 6998 and 7142 Da. Each myrmexin is a heterodimer consisting of a small subunit (SS1 or SS2 or SS3) disulfide-linked to a larger, quite structurally unrelated subunit (LS1 or LS2). Thus, the myrmexin complex consists of six isoforms of venom proteins: myrmexin I (SS1/LS2), myrmexin II (SS1/LS1), myrmexin III (SS2/LS2), myrmexin IV (SS3/LS2), myrmexin V (SS2/LS1), myrmexin VI (SS3/LS1). Subunit SS1 is highly homologous to SS2 (96% of identity) and SS3 (87% of identity) and LS1 is highly homologous to LS2 (79% of identity). Our study suggests that myrmexins may represent a new class of anti-inflammatory proteins.

Amino Acid Sequence↗
PubMed2005

The anatomical arrangement of the myocardial cells making up the ventricular mass.

The architectural arrangement of the myocytes within the ventricular mass remains a highly contentious topic. It has recently been suggested by several distinguished surgeons that the overall myocardial structure is disposed in the form of a 'ventricular myocardial band'. There are, however, major anatomic deficiencies in this hypothesis, because the heart is formed on the basis of a modified blood vessel, rather than a collection of discrete muscular entities resembling the skeletal musculature. There is ample alternative evidence, nonetheless, already existing to provide a suitable explanation for the 'forceful reciprocal twisting' of the ventricular mass that is seen by cardiac surgeons during operative procedures. We provide here, therefore, a review of the anatomical studies we have performed separately and conjointly over a period of nearly 30 years. As before, we show that there is no anatomic evidence to support the concept of the 'ventricular myocardial band'. The overall arrangement is for the myocytes to be supported as the muscular components of a continuous and complex mass, the supporting collagenous fibrous matrix possessing epimysial, perimysial, and endomysial components. It had already been discussed at length during the previous century why there was no anatomic evidence to support the existence of separate 'muscles' within the ventricular continuum. There are no fibrous sheaths within the ventricular walls that permit the myofibres to be dissected on the basis of muscle bundles having a discrete origin and insertion, as is the case with the arrangement of the skeletal muscles. We have never sought ourselves, however, to deny the central helical nature of the overall architecture of the ventricular walls. The anatomic evidence supporting an overall helical nature for the ventricular myocardium has existed for over 150 years. All the available evidence, nonetheless, shows that these helical patterns are to be found throughout the walls, and in no way constitute a unique myocardial band.

Collagen↗
PubMed2003

Altered expression of matrix metalloproteinases within mineralizing bone cells in vitro in the presence of fluoride.

Fluoride is known to alter mineralization within bone, although the mechanism for its action is unclear. An important stage in the formation of mineralized tissues is the remodeling of the osteoid, facilitating mineral deposition. Using a bone mineralizing culture system derived from rat femur washes, this study investigated the influence of fluoride on MMP expression at a developmental stage relating to the onset of mineralization. Bone cells cultured in the absence of fluoride synthesized an active form of a 45-kD MMP, which was immunoreactive with an antibody to human MMP-1 (although full characterization of this MMP was not achieved), trace levels of an MMP immunoreactive with anti-MMP-3, and a 66-kD proteolytic species. Incubation in 10(-7) and 10(-5) M fluoride resulted in a decrease in expression of the 45-kD MMP, sharp increases in the expression of MMP-3, and the appearance of a band at 110 kD, which showed immunoreactivity for MMP-9. The influence of fluoride on MMP expression is likely to influence the composition of the remodeling matrix and subsequent mineralization and offers a potential mechanism by which fluoride alters mineralization.

Animals↗
PubMed2006

New insights into the glycosylation of the surface layer protein SgsE from Geobacillus stearothermophilus NRS 2004/3a.

The surface of Geobacillus stearothermophilus NRS 2004/3a cells is covered by an oblique surface layer (S-layer) composed of glycoprotein subunits. To this S-layer glycoprotein, elongated glycan chains are attached that are composed of [-->2)-alpha-l-Rhap-(1-->3)-beta-l-Rhap-(1-->2)-alpha-L-Rhap-(1-->] repeating units, with a 2-O-methyl modification of the terminal trisaccharide at the nonreducing end of the glycan chain and a core saccharide as linker to the S-layer protein. On sodium dodecyl sulfate-polyacrylamide gels, four bands appear, of which three represent glycosylated S-layer proteins. In the present study, nanoelectrospray ionization time-of-flight mass spectrometry (MS) and infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry were adapted for analysis of this high-molecular-mass and water-insoluble S-layer glycoprotein to refine insights into its glycosylation pattern. This is a prerequisite for artificial fine-tuning of S-layer glycans for nanobiotechnological applications. Optimized MS techniques allowed (i) determination of the average masses of three glycoprotein species to be 101.66 kDa, 108.68 kDa, and 115.73 kDa, (ii) assignment of nanoheterogeneity to the S-layer glycans, with the most prevalent variation between 12 and 18 trisaccharide repeating units, and the possibility of extension of the already-known -->3)-alpha-l-Rhap-(1-->3)-alpha-l-Rhap-(1--> core by one additional rhamnose residue, and (iii) identification of a third glycosylation site on the S-layer protein, at position threonine-590, in addition to the known sites threonine-620 and serine-794. The current interpretation of the S-layer glycoprotein banding pattern is that in the 101.66-kDa glycoprotein species only one glycosylation site is occupied, in the 108.68-kDa glycoprotein species two glycosylation sites are occupied, and in the 115.73-kDa glycoprotein species three glycosylation sites are occupied, while the 94.46-kDa band represents nonglycosylated S-layer protein.

Bacillaceae↗
PubMed2005

Luminescence spectroscopy of matrix-isolated atomic manganese: excitation of the "forbidden" a6D(J)<-->a6S transitions.

Laser-induced excitation spectra recorded for the electric-quadrupole 3d(6)4s a6D(J)<--3d(5)4s2a6S(5/2) transitions of atomic Mn, allow assignment of the red emission features, previously observed in Mn/RG (RG=Ar, Kr and Xe) matrices with resonance 3d(5)4s4pz6P(5/2)<--3d(5)4s2 a6S(5/2) excitation, to the metastable a6D(9/2) state. Narrow excitation bands recorded for the red site in the Mn/Kr system allow identification of all five spin-orbit levels (J=1/2, 3/2, 5/2, 7/2 and 9/2) in the a6D state. The coincidence of the lowest energy excitation band and the observed 585.75 nm (17,072 cm(-1)) emission band of atomic Mn in Kr matrices, yielded a definitive assignment of this emission to a transition from the J=9/2 spin-orbit level. Temperature dependent emission scans lead to the identification of the zero phonon line for the a6D(9/2)-->a6S(5/2) transition at 585.75 nm. The identified matrix-shift of +20 cm(-1) allows an assessment of the extent of the ground state stabilization in the red (secondary) site of atomic Mn isolation in solid Kr. Emission produced with direct a6D state excitation yielded both the 585.75 and 626 nm features. The former band arises for Mn atoms occupying the red site--the latter from blue site occupancy in solid Kr. The excitation linewidths recorded for these two sites differ greatly, with the blue site yielding a broad featureless profile, in contrast to the narrow, structured features of the red site. The corresponding red site a6D(J)<-->a6S(5/2) transitions in Ar and Xe matrices are broader than in Kr--a difference considered to originate from the site sizes available in these hosts and the interatomic Mn x RG potentials. The millisecond decay times recorded for the red emission bands in the Mn/RG systems are all much shorter than the 3 s value predicted for the gas phase a6D(9/2)-->a6S(5/2) transition. This enhancement allows optical pumping of the forbidden a6D(J)<-->a6S transitions with low laser powers when atomic manganese is isolated in the solid state. However all the emission decays are complex, exhibiting triple exponential decays. This behavior may be related to the dependence of the excitation linewidths on the J value, indicating removal of the J degeneracy due to weak matrix-induced, crystal field splitting.

Journal Article↗
PubMed2006

Influence of temperature and pressure on shape and shift of impurity optical bands in polymer glasses.

The shape, broadening, and shift of optical absorption spectra of molecular impurity centers in polymer glasses are considered in terms of inhomogeneous energy distributions and coupling of electronic transitions to vibrations. Persistent spectral hole burning was applied for frequency-selective probing of zero-phonon lines. The shift and broadening of spectral holes were studied between 5 and 50 K and by applying a hydrostatic He gas pressure up to 200 bar. Broadband absorption spectra were recorded between 5 and 300 K in poly(methyl methacrylate) and polyethylene. In addition to "normal" thermal broadening, due to the first- and second-order electron phonon coupling, several narrowing components were predicted on the basis of frequency dependent hole behavior. Thermal expansion of the matrix and the relaxation of local strains, previously accumulated on cooling below the glass temperature can lead to shrinking of the inhomogeneous width. A Voigt treatment of absorption band shapes reveals that the Gaussian component can indeed suffer remarkable narrowing. Inhomogeneous band shapes and the frequency-dependent thermal and baric line shifts were rationalized with the aid of a pair of two-body Lennard-Jones potentials. The shift of potential well minima is a crucial factor influencing solvent shifts, inhomogeneous band shapes, pressure shift coefficients, and quadratic electron phonon coupling constants.

Journal Article↗
PubMed1987

Transferrin mRNA level in the mouse mammary gland is regulated by pregnancy and extracellular matrix.

We have isolated an almost full length cDNA to transferrin from a mouse mammary tumor virus-induced tumor cDNA library. On Northern blots of RNA isolated from liver and mammary glands, the cDNA hybridized to a single band of 2.4 kilobases. The authenticity of the probe is shown further by 83% sequence homology to human cDNA and identical amino acid sequence to a small cDNA probe isolated from a mouse liver library. The level of transferrin mRNA is very low in the glands from virgin mice, but is as abundant in the glands from pregnant and lactating mice as in the liver. We further show that the steady-state transferrin mRNA level in culture is relatively insensitive to lactogenic hormones compared to that of beta-casein mRNA. Culturing the cells on extracellular matrix, however, markedly affects its expression. These findings raise the possibility that the composition of the basement membrane in the mammary gland may play a role in regulation of transferrin levels in vivo.

Amino Acid Sequence↗
PubMed2005

Intercalation and photophysical characterization of 1-pyrenemethylamine in zirconium phosphate layered materials.

The ion exchange of the luminescent probe 1-pyrenemethylamine (PYMA) into zirconium phosphate (ZrP) layered materials has been accomplished. The matrices used were the hexahydrated 10.3 A phase of ZrP (10.3 A ZrP, where 10.3 A represents the interlayer distance) and butylammonium-exchanged ZrP (BAZrP) with an expanded 18.6 A interlayer distance. The XRPD patterns for the 10.3 A ZrP after PYMA exchange (PYMA-exchanged ZrP), at high PYMA concentrations, show an increase in the interlayer distance from 10.3 A in unexchanged 10.3 A ZrP to 23.5 A in PYMA-exchanged ZrP, indicating PYMA intercalation. The luminescence spectrum for the PYMA-exchanged ZrP exhibits an excimer band at 458 nm that is absent in the luminescence spectrum of PYMA in aqueous solution at low concentrations. The intensity of the excimer emission increased at low PYMA concentrations. These results are in contrast to experiments using the BAZrP matrix. The XRPD patterns for PYMA-exchanged BAZrP do not show changes in the interlayer distance, which suggests that PYMA is not being intercalated and is only surface bound. The luminescence spectrum for PYMA-exchanged BAZrP exhibits a lower emission intensity in its excimer band, at different PYMA concentrations, compared with the PYMA-exchanged ZrP excimer band. For PYMA-exchanged ZrP, we propose a process in which exchange at low PYMA concentrations occurs at external surface sites with clustering promoting excimer formation followed by exchange at high PYMA concentrations occurring at interior sites reducing excimer formation.

Journal Article↗
PubMedDate not supplied

Cytokines and growth factors positively and negatively regulate interstitial collagen gene expression in human vascular smooth muscle cells.

Human atheromas accumulate extracellular matrix proteins such as collagen types I and III. We tested whether cytokines or growth factors produced by cells found in human atherosclerotic plaques alter collagen gene expression in vascular smooth muscle cells (VSMCs), which produce the blood vessel matrix. Interleukin-1 (IL-1, 1-10 ng/ml) modestly increased the synthesis of collagens I and III (measured by tritiated proline incorporation into specific electrophoretic bands), whereas transforming growth factor-beta (TGF-beta) or platelet-derived growth factor (PDGF) markedly stimulated production of these interstitial collagens. Interferon gamma (IFN-gamma), a product of activated T cells found in atheromas, selectively alters several VSMC functions. For example, this cytokine reduces growth of VSMCs, decreases alpha-actin gene expression, and induces VSMC expression of class II histocompatibility antigens. We report here that IFN-gamma also inhibits basal as well as IL-1-, PDGF-, or TGF-beta-stimulated collagen I and III synthesis by human VSMCs. TGF-beta, the most potent stimulator of collagen synthesis studied here, raised the level of collagen III mRNA in VSMCs 4.8-fold (determined by densitometry of Northern blots), whereas exposure to both TGF-beta and IFN-gamma reduced this mRNA to 0.5 of basal level. Locally produced cytokines and growth factors may thus modify matrix accumulation during atherogenesis by stimulating or suppressing expression of interstitial collagen mRNA and protein by VSMCs.

Arteriosclerosis↗
PubMed1999

Organic matrix composition and ultrastructure of eggshell: a comparative study.

1. The avian eggshell is a biomineralised composite ceramic consisting of calcium carbonate embedded in an organic matrix. Matrix components are supposed to be involved in the control of mineralisation, crystallographic texture and biomechanical properties of eggshell. 2. The structure and eggshell matrix composition of various domesticated bird species were compared to gain insight into the universality of the eggshell mineralisation process. 3. The SDS-PAGE profiles of soluble eggshell matrix were specific within groups of birds (a: laying hen, breeder hen, quail, pheasant and possibly turkey; b: guinea fowl; c: duck and goose) but some of the protein bands were common to all groups. 4. Analogies between species were confirmed by Western blotting using hen protein antibodies. Ovocleidin-17 (OC-17) and ovalbumin were revealed in all species (except quail for OC-17). Lysozyme was present only in hen eggshell. Another egg white protein: ovotransferrin showed a positive signal in hens, turkey and quail. Osteopontin was observed in laying and breeder hens and quail. 5. Different proteoglycans were localised to discrete regions within the eggshell. Dermatan sulphate was observed within the matrix of the calcified shell of all species except quail which contained chondroitin-6-sulfate. Keratan sulphate was observed in mammillary bodies of breeder and laying hen, quail, pheasant and turkey while chondroitin sulphate was also present in guinea fowl and duck. 6. The general structural organisation of the different avian eggshells was similar but specific differences were observed in the ultrastructure of the mammillary layer. Species of the same taxonomic family could be grouped according to their structural analogies: breeder hen, turkey and pheasant resembled that of the domestic fowl. Guinea fowl was unique. Goose and duck were quite similar with large and confluent mammillary bodies. 7. Some matrix components are therefore common to eggshells of various species but more information is needed to relate differences in matrix composition between taxonomic groups with differences in ultrastructure.

Animals↗
PubMed1986

Isolation and characterization of a metalloprotease associated with chicken epiphyseal cartilage matrix vesicles.

A metalloprotease has been isolated from matrix vesicles of chicken epiphyseal cartilage and subsequently characterized. Matrix vesicles obtained by collagenase digestion and differential centrifugation were further purified by Sepharose CL2B gel filtration. The protease was solubilized from the vesicles by treatment with deoxycholate and freeze-thawing, and then isolated by Sephadex G150 gel filtration. Disc electrophoresis of the enzyme, which displayed protease activity toward azocasein substrate, gave a single protein band. Based on molecular weight (MW) determination, lack of immunocross reactivity, and differences in electrophoretic migration, there is little possibility of any contamination with external protease from the commercial collagenase used for vesicle preparation. The matrix vesicle protease had a MW of 33,000 and a pH optimum of 7.2 and was completely inhibited by 0.1 mM EDTA and 0.2 mM o-phenanthroline. alpha 2-Macroglobulin, ovalbumin, cysteine, penicillamine, ethane-1-hydroxy-1, 1-diphosphonate (EHDP) and pyrophosphate at higher concentrations were also inhibitory. The inhibition by omicron-phenanthroline was reversed by Co2+, Zn2+, Fe2+, and Cu2+. Protease activity was most abundant in the heavy fraction of matrix vesicles fractionated by discontinuous sucrose density gradient centrifugation. Release of this protease at the calcifying front could degrade noncollagenous protein moieties that inhibit precipitation of minerals in the extravesicular matrix and thus facilitate mineralization.

Animals↗
PubMed1999

Transforming growth factor-alpha and rhinitis.

OBJECTIVES: Transforming growth factor-alpha (TGF-alpha) has been implicated in diverse physiologic and pathophysiologic functions including immunological, inflammatory, and neoplastic processes. TGF-alpha has been localized in the hyperproliferative, inflammatory environment of chronic otitis media, cholesteatoma, and asthmatic airways. TGF-beta1, which must be present with TGF-alpha to transform fibroblasts, has been found in rhinitic mucosa and in asthma in prior studies. The authors sought to identify whether TGF-alpha also played a role in the inflammatory cascade and fibrosis of rhinitis. STUDY DESIGN: A nonrandomized, prospective study was carried out in which samples of inferior turbinate and nasal polyps from rhinitic and nonrhinitic patients were subjected to immunohistochemistry and Western blotting to determine the presence of TGF-alpha. METHODS: Twenty-seven subjects undergoing surgery for rhinitis, obstructive sleep apnea, nasal fracture, and rhinoplasty were recruited for this study, the latter three groups acting as controls. Immunohistochemical and Western blotting techniques were employed to identify the presence of TGF-alpha in inferior-turbinate and nasal-polyp samples of rhinitic subjects. RESULTS: Immunohistochemistry demonstrated the selective staining of TGF-alpha in the basement membrane and extracellular matrix, including lymphatic, vascular, and glandular structures, in most turbinate samples and the absence of staining in corresponding controls. Further, TGF-alpha was isolated to a discrete 30-kD band in both inferior turbinate and polyp tissues by Western blotting without staining in the corresponding controls. CONCLUSIONS: These results suggest that TGF-alpha may play a role in the inflammatory derangement of rhinitis.

Adolescent↗
PubMed1992

Identification of a factor in fetal bovine serum that stabilizes the cumulus extracellular matrix. A role for a member of the inter-alpha-trypsin inhibitor family.

Fetal bovine serum (FBS) has been widely used in the culture of cumulus-oocyte complexes, since it is believed that FBS stabilizes the expanding cumulus extracellular matrix. In this study, we have identified a factor in FBS that is responsible for this effect. FBS was first fractionated by stepwise precipitation with ammonium sulfate followed by high performance liquid chromatography (HPLC) on a gel filtration column. Active fractions, as determined by their ability to stabilize the cumulus extracellular matrix in a bioassay system, were further purified by HPLC on DEAE ion-exchange and gel filtration columns. The purified factor was exhibited as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent Mr 150,000 and a single peak on a C-8 reverse-phase HPLC. The sequence of the first 14 NH2-terminal amino acids was determined by Edman degradation, revealing significant sequence identity of the bovine serum factor with the light chain shared by members of the inter-alpha-trypsin inhibitor family. Western blot analysis using anti-human I alpha I IgG shows that the antibody reacts positively with the purified factor, and immunodepletion of FBS with anti-I alpha I antibody agarose eliminated its ability to stabilize the extracellular matrix. This factor is found in mouse follicular fluid collected 6 h following human chorionic gonadotropin injection to stimulate ovulation, but not in unstimulated mice. Anti-I alpha I-positive epitopes were also localized within the cumulus extracellular matrix of mouse preovulatory follicles in immunocytochemical preparations using anti-human I alpha I IgG, supporting the possibility that this molecule or molecules may diffuse into follicular fluid after an ovulatory stimulus to act as structural linkers that ensure normal cumulus expansion, through stabilization of the cumulus extracellular matrix thus supporting the process of ovulation.

Alpha-Globulins↗
PubMed1998

Rapid identification of comigrating gel-isolated proteins by ion trap-mass spectrometry.

In the search for novel nuclear binding proteins, two bands from a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel were analyzed and each was found to contain a number of proteins that subsequently were identified by tandem mass spectrometry (MS/MS) on a quadrupole ion trap instrument. The bands were digested with trypsin in situ on a polyvinylidene difluoride (PVDF) membrane following electroblot transfer. Analysis of a 2.5% aliquot of each peptide mixture by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) followed by an initial database search with the peptide masses failed to identify the proteins. The peptides were separated by reversed-phase capillary high performance liquid chromatography (HPLC) in anticipation of subsequent Edman degradation, but mass analysis of the chromatographic fractions by MALDI-MS revealed multiple, coeluting peptides that precluded this approach. Selected fractions were analyzed by capillary HPLC-electrospray ionization-ion trap mass spectrometry. Tandem mass spectrometry provided significant fragmentation from which full or partial sequence was deduced for a number of peptides. Two stages of fragmentation (MS3) were used in one case to determine additional sequence. Database searches, each using a single peptide mass plus partial sequence, identified four proteins from a single electrophoretic band at 45 kDa, and four proteins from a second band at 60 kDa. Many of these proteins were derived from human keratin. The protein identifications were corroborated by the presence of multiple matching peptide masses in the MALDI-MS spectra. In addition, a novel sequence, not found in protein or DNA databases, was determined by interpretation of the MS/MS data. These results demonstrate the power of the quadrupole ion trap for the identification of multiple proteins in a mixture, and for de novo determination of peptide sequence. Reanalysis of the fragmentation data with a modified database searching algorithm showed that the same sets of proteins were identified from a limited number of fragment ion masses, in the absence of mass spectral interpretation or amino acid sequence. The implications for protein identification solely from fragment ion masses are discussed, including advantages for low signal levels, for a reduction of the necessary interpretation expertise, and for increased speed.

Amino Acid Sequence↗
PubMed1997

A strategy for rapid, high-confidence protein identification.

A procedure is described for rapid, high-confidence identification of proteins using matrix-assisted laser desorption/ionization tandem ion trap mass spectrometry in conjunction with a genome database searching strategy. The procedure involves excision of copper-stained bands or spots from electrophoretic gels, in-gel trypsin digestion of the proteins, single-stage mass spectrometric analysis of the resultant mixture of tryptic peptides, followed by tandem ion trap mass spectrometric analysis of selected individual peptides, and database searching of the relevant genomic database using the program PepFrag. The scheme provides sensitive, real-time protein identification as well as facile identification of modifications. A single operator can unambiguously identify 5-10 proteins/day from an organism whose genome is known at a level of > 0.5 pmol of protein loaded on a gel. The utility of the technique was demonstrated by the identification and characterization of a band from a human HTLV-I preparation and 11 different proteins from a yeast RNA polymerase II C-terminal repeat domain-affinity preparation. The technology has great potential for postgenome biological science, where it promises to facilitate the dissection and anatomy of macromolecular assemblages, the definition of disease state markers, and the investigation of protein targets in biological processes such as the cell cycle and signal transduction.

Amino Acid Sequence↗
PubMed2006

Degradation of extracellular matrix proteins (fibronectin, vitronectin and laminin) by serine-proteinases isolated from Lonomia achelous caterpillar hemolymph.

Lonomia achelous is a caterpillar distributed in southern Venezuela and in northern Brazil that causes an acute hemorrhagic syndrome in people who have contact with its bristles. The effect of the crude hemolymph and its chromatographic fractions (FDII, Lonomin V and Lonomin V-2) on extracellular matrix proteins was studied. The chromatographic fractions show activities similar to plasmin and urokinase. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, both lonomins appear as a protein band of 25 kDa under reduced conditions. By exclusion chromatography, the molecular weights of Lonomin V and Lonomin V-2 were 26.5 and 24.5 kDa, respectively. Fibronectin, laminin and vitronectin were degraded by all venom components. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under reduced conditions, shows that lonomins degrade fibronectin in four main fragments of 116, 60, 50 and 30 kDa. Molecular exclusion chromatography in native conditions shows that the molecular masses of these fragments are > or = 300, 62 and 27 kDa. The proteolytic effect of lonomins was abolished by benzamidine/HCl, iodoacetic acid and aprotinin. The extracellular matrix protein degradation together with the fibrino(geno)lytic activity of hemolymph and its fractions could explain, in part, the hemorrhagic syndrome, and the wound dehiscence in persons who have had contact with the L. achelous caterpillar.

Animals↗
PubMed1998

Transitions in cell organization and in expression of contractile and extracellular matrix proteins during development of chicken aortic smooth muscle: evidence for a complex spatial and temporal differentiation program.

Whereas the understanding of the mechanisms underlying skeletal and cardiac muscle development has been increased dramatically in recent years, the understanding of smooth muscle development is still in its infancy. This paper summarizes studies on the ontogeny of chicken smooth muscle cells in the wall of the aorta and aortic arch-derived arteries. Employing immunocytochemistry with antibodies against smooth muscle contractile and extracellular matrix proteins we trace smooth muscle cell patterning from early development throughout adulthood. Comparing late stage embryos to young and adult chickens we demonstrate, for all the stages analyzed, that the cells in the media of aortic arch-derived arteries and of the thoracic aorta are organized in alternating lamellae. The lamellar cells, but not the interlamellar cells, express smooth muscle specific contractile proteins and are surrounded by basement membrane proteins. This smooth muscle cell organization of lamellar and interlamellar cells is fully acquired by embryonic day 11 (ED 11). We further show that, during earlier stages of embryogenesis (ED3 through ED7), cells expressing smooth muscle proteins appear only in the peri-endothelial region of the aortic and aortic arch wall and are organized as a narrow band of cells that does not demonstrate the lamellar-interlamellar pattern. On ED9, infrequent cells organized in lamellar-interlamellar organization can be detected and their frequency increases by ED10. In addition to changes in cell organization, we show that there is a characteristic sequence of contractile and extracellular matrix protein expression during development of the aortic wall. At ED3 the peri-endothelial band of differentiated smooth muscle cells is already positive for smooth muscle alpha actin (alphaSM-actin) and fibronectin. By the next embryonic day the peri-endothelial cell layer is also positive for smooth muscle myosin light chain kinase (SM-MLCK). Subsequently, by ED5 this peri-endothelial band of differentiated smooth muscle cells is positive for alphaSM-actin, SM-MLCK, SM-calponin, fibronectin, and collagen type IV. However, laminin and desmin (characteristic basement membrane and contractile proteins of smooth muscle) are first seen only at the onset of the lamellar-interlamellar cell organization (ED9 to ED10). We conclude that the development of chicken aortic smooth muscle involves transitions in cell organization and in expression of smooth muscle proteins until the adult-like phenotype is achieved by mid-embryogenesis. This detailed analysis of the ontogeny of chick aortic smooth muscle should provide a sound basis for future studies on the regulatory mechanisms underlying vascular smooth muscle development.

Age Factors↗
PubMed1999

Hensin remodels the apical cytoskeleton and induces columnarization of intercalated epithelial cells: processes that resemble terminal differentiation.

Intercalated epithelial cells exist in a spectrum of phenotypes; at one extreme, beta cells secrete HCO3 by an apical Cl/HCO3 exchanger and a basolateral H+ ATPase. When an immortalized beta cell line is seeded at high density it deposits in its extracellular matrix (ECM) a new protein, hensin, which can reverse the polarity of several proteins including the Cl/HCO3 exchanger (an alternately spliced form of band 3) and the proton translocating ATPase. When seeded at low density and allowed to form monolayers these polarized epithelial cells maintain the original distribution of these two proteins. Although these cells synthesize and secrete hensin, it is not retained in the ECM, but rather, hensin is present in a large number of intracellular vesicles. The apical cytoplasm of low density cells is devoid of actin, villin, and cytokeratin19. Scanning electron microscopy shows that these cells have sparse microvilli, whereas high density cells have exuberant apical surface infolding and microvilli. The apical cytoplasm of high density cells contains high levels of actin, cytokeratin19, and villin. The cell shape of these two phenotypes is different with high density cells being tall with a small cross-sectional area, whereas low density cells are low and flat. This columnarization and the remodeling of the apical cytoplasm is hensin-dependent; it can be induced by seeding low density cells on filters conditioned by high density cells and prevented by an antibody to hensin. The changes in cell shape and apical cytoskeleton are reminiscent of the processes that occur in terminal differentiation of the intestine and other epithelia. Hensin is highly expressed in the intestine and prostate (two organs where there is a continuous process of differentiation). The expression of hensin in the less differentiated crypt cells of the intestine and the basal cells of the prostate is similar to that of low density cells; i.e., abundant intracellular vesicles but no localization in the ECM. On the other hand, as in high density cells hensin is located exclusively in the ECM of the terminally differentiated absorptive villus cells and the prostatic luminal cell. These studies suggest that hensin is a critical new molecule in the terminal differentiation of intercalated cell and perhaps other epithelial cells.

Animals↗