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Modulation of the photophysical properties of C60 by electronic confinement effect.

This research work reports on the incorporation of fullerene C60 in diverse inorganic and organic matrixes and how these different environments induce changes on the photophysical properties of the molecule depending on the cavity dimensions of the host. Indeed the fluorescence emission band of C60 experiences a progressive bathochromic shift with respect to C60 in solution as the cavity dimensions of the host decrease in going from the mesoporous material MCM41 to UTD-1 and Na-Y zeolites. This experimental observation, which has been documentarily confirmed by theoretical predictions and recent experimental results, is a reflection of the confinement effect imposed by the host. However, the most striking result reported in this work is that the fluorescence range accessible to this occluded species can be extraordinarily extended by confinement inside the neutral cages of a "dendritic box". The ability of the dendritic shell to create a microenvironment, modifying the properties of its functional core, allows the emission bands of C60 incorporated into a dendrimer to be effectively red-shifted with respect to their emission in solution, and, contrarily to other confined spaces of considerable hardness such as zeolites or the high surface material MCM41, the magnitude of this shifting is maximum and can be modulated under appropriate experimental conditions. This phenomenon has an enormous relevance since it can be exploited in future technological applications.

Journal Article↗

Immunolocalization of urokinase and its receptor in prematurely fused cranial sutures of infants.

In cranial sutural samples derived from five children with premature cranial suture fusion we have performed immunostaining for the urokinase plasminogen activator (uPA) and urokinase receptor (uPAR). We have found a strong reactivity for cell- or matrix-bound uPA and uPAR in the sutural connective tissue and associated with the osteoblasts and osteocytes lining the calvarial bone. The sutural tissue itself showed a banding with different intensity of urokinase and uPAR staining concerning connective tissue. It is proposed that the components of the plasminogen activating system are involved in tissue turnover of sutural tissue and in sutural growth.

Connective Tissue↗

Isolation of the N-terminal globular protein domains from cartilage proteoglycans. Identification of G2 domain and its lack of interaction with hyaluronate and link protein.

The N-terminal fragment (G1-G2) of cartilage proteoglycan protein core contains two globular domains, binding region (G1) and a second globular domain (G2), G1-G2 was isolated after mild trypsin digestion of purified proteoglycan aggregates followed by chromatography first on Sepharose CL-2B under associative conditions and then on a TSK-4000 column in 4 M-guanidinium chloride. It migrated as a single band (apparent Mr 150,000) on SDS/polyacrylamide-gel electrophoresis. G2 was isolated by V8-proteinase digestion of G1-G2 followed by aggregation of the G1-containing fragments with hyaluronate and chromatography on TSK-4000. It migrated as a single band on SDS/polyacrylamide-gel electrophoresis of apparent Mr 66,000 after digestion with keratanase. G2 did not interact with proteoglycan monomer, hyaluronate, link protein or other extractable cartilage matrix proteins. A polyclonal antibody raised against G2 did not cross-react with G1 or link protein. These data show that, despite a high degree of sequence similarity, G1 and G2 do not share any functional properties nor have major antigenic sites in common.

Aggrecans↗

Additive effects of Helicobacter pylori lipopolysaccharide and proteins in monocyte inflammatory responses.

BACKGROUND: Chronic active gastritis associated with Helicobacter pylori infection is characterized by both a neutrophil and a mononuclear leukocyte infiltrate. While neutrophil functions in relation to H. pylori are well described, the interactions between Helicobacter bacterial factors and monocytes are poorly understood in relation to the mucosal inflammatory process. METHODS: Sonicates of a clinical strain as well as of a type strain of H. pylori were prepared in vitro. Monocytes from healthy donors were induced to release L-selectin (CD62L), to upregulate the adherence molecules (CD11a, CD11b. CD11c) and to produce toxic oxygen radicals. The inducing activities were assessed by flow cytometry and chemiluminescence. RESULTS: A dose-dependent shedding of CD62L and upregulation of CD11b and CD11c were observed with both bacterial strains as well as PMA and fMLP. CD11a remained unchanged. Activity could be attributed to bacterial factors of both lipopolysaccharide (LPS) and protein characteristics. The alterations observed for CD11b, CD11c and CD62L were induced by the same protein fractions in parallel. suggesting a common component and mechanism of action. A major protein component was urease, although other minor protein bands were found as well. Monoclonal antibodies to CD14-inhibited monocyte inflammatory responses induced by H. pylori sonicate at low concentration, whereas further LPS pierce-matrix reduction was necessary at high sonicate concentrations to reduce monocyte-inducing activity. CONCLUSIONS: Monocyte inflammatory activation is induced by H. pylori sonicate components. Factors of both LPS and protein characteristics are involved and an additive effect was demonstrated. Urease appears to be a major component in the protein preparations of highest inducing capacity. Further studies are warranted to assess whether the monocyte activation properties described here are related to the diversity of clinical gastroduodenal outcome for the chronic type B gastritis associated with H. pylori infection.

Bacterial Proteins↗

Electrophoretic properties of sodium dodecyl sulfate and related changes in its concentration in SDS-polyacrylamide gel electrophoresis.

Sodium dodecyl sulfate(SDS) in a protein sample solution migrates in SDS-polyacrylamide gel electrophoresis as a band with a mobility higher than those of protein bands. Behind this band, which is mostly composed of SDS micelles, SDS concentration is raised uniformly in a gel column as a result of the retardation effect of the gel matrix on SDS micelles. Electrophoretic patterns of SDS were obtained when SDS was omitted from various portions of the gel electrophoretic system.

Binding Sites↗

Excitation spectrum and staggering transformations in lattice quantum models.

We consider the energy-momentum excitation spectrum of diverse lattice Hamiltonian operators: the generator of the Markov semigroup of Ginzburg-Landau models with Langevin stochastic dynamics, the Hamiltonian of a scalar quantum field theory, and the Hamiltonian associated with the transfer matrix of a classical ferromagnetic spin system at high temperature. The low-lying spectrum consists of a one-particle state and a two-particle band. The two-particle spectrum is determined using a lattice version of the Bethe-Salpeter equation. In addition to the two-particle band, depending on the lattice dimension and on the attractive or repulsive character of the interaction between the particles of the system, there is, respectively, a bound state below or above the two-particle band. We show how the existence or nonexistence of these bound states can be understood in terms of a nonrelativistic single-particle lattice Schrödinger Hamiltonian with a delta potential. A staggering transformation relates the spectra of the attractive and the repulsive cases.

Journal Article↗

Identification of extractable proteins from the bovine ocular zonule: major zonular antigens of 32kD and 250kD.

Using Western immunoblotting, the extractable proteins of the bovine zonular fibers were examined for reactivity with two zonular antisera known to have strong affinity for zonular fibers in tissues, in order to identify the antigenic components. The extracts were also tested with antisera to several matrix proteins that have been reported to be associated with zonular fibers. Proteins reactive with antisera to bovine serum albumin, serum immunoglobulins and fibronectin were present. No bands reactive with antisera to a-elastin, prealbumin, amyloid P component, collagen VI, lysyl oxidase or monoclonal antibody to fibrillin were demonstrated. The major nonserum protein band identified by both antisera was a 32kD polypeptide. An equally strong 250kD polypeptide was shown by the antiserum to guanidine-dithiothreitol extracted zonular fibers. Both of these proteins were PAS-positive and were demonstrated also by the antisera in extracts of bovine elastic neck ligament. Whether the two glycoproteins are related to each other, with the higher molecular weight protein either a precursor or aggregate form, is not yet clear. They appear to bear a close relationship to the elusive core microfibrillar protein.

Animals↗

Growth of mineral crystals in turkey tendon collagen fibers.

Bone and several other vertebrate mineralized tissues are formed by the organized growth of crystals of carbonated apatite within a matrix of type 1 collagen fibers. The development of this process in isolated fibrils of young turkey leg tendons has been studied by transmission electron microscopy. Collagen banding, presumably due to ion concentration, precedes the appearance of any crystals. The smallest crystals observed are short needles in bands near the surface of the fibrils. Longer needles, up to the length of the collagen gap regions, were also seen, and, evidently at a later stage, single crystal belts extending partly or wholly through the fibrils. Finally, in mature tendon crystal platelets, seemingly derived from the cracking of belts, extend partly into the collagen overlap zone. In the least mineralized tendon, extrafibrillar mineral-containing vesicles have occasionally been observed adjacent to regions of radiating needle crystal growth in the fibrils, and, more commonly, smaller particles adjacent to bands of very small needles.

Aging↗

Ultrastructural description of a "cylinder organelle" in the outer plexiform layer of human retinas.

A cylinder-like organelle ("cylinder organelle") present in the outer plexiform layer of the retina and apparently confined to the horizontal cell processess is described. This structure was studied in detail in three normal human retinas from eyes enucleated for ciliary body melanomas. The cylinder organelle, when cut longitudinally, showed an electron-lucent area bound by two bands of electron-dense filaments. In transverse section, the cylinder organelle revealed a circular profile of electron-dense crisscrossing filaments about an electron-lucent matrix. Since this structure was only found in the rod spherule synaptic complex, it is hypothesized that it may be involved in transmitting impulses.

Aged↗

Fibro-osteochondroma of the uterus.

A very rare case of fibro-osteochondroma arising in uterine musculature was studied histologically. The tumor exhibited three types of cellular proliferation--fibroblastic, cartilaginous, and osseous--all within an abundant collagenous matrix. The three components were intermingled, and varied in the degree of differentiation of cartilaginous and osseous tissue. The more differentiated tissues were usually circumscribed by fibrocellular bands of spindle-shaped cells that seemed to have transformed into rounded cartilaginous cells. Presumably this tumor arose from undifferentiated mesenchymal cells in the myometrium, which proliferated to form fibroblasts, chondrocytes, and osteoblasts. The tumor may be designated fibrio-osteochondroma. It is histologically benign and exceedingly rare.

Adult↗

Borderline malignant serous tumors of the ovary maintained on extracellular matrix: evidence for clonal evolution and invasive potential.

Four cases of advanced stage (II or III) and one case of early stage (IC) borderline malignant serous cystadenocarcinomas of the ovary were maintained on culture dishes coated with an extracellular matrix (ECM) produced by bovine corneal endothelial cells. Cells harvested for chromosomal analysis after 2-3 days showed diploid or near-diploid modalities in all cases. Banded chromosome studies in two cases revealed nonrandom clonal abnormalities with trisomy 2, 7, and 12 in seven of 13 metaphases. No structural abnormalities were noted. These cytogenetic findings differ from those found in malignant serous tumors of the ovary. In addition, borderline tumor cells digested the ECM in all cases and formed a cribiform pattern within a few days of primary culture. This study suggests clonal progression from early to advanced stages of borderline malignant serous tumors; readily distinguishable from overtly malignant serous tumors of the ovary. Ability of tumor cells derived from both primary tumors and metastatic implants to digest the ECM implies the possibility that borderline serous tumors have invasive potential.

Adult↗

Tamoxifen down-regulates CD36 messenger RNA levels in normal and neoplastic human breast tissues.

Tamoxifen (TAM) exerts a long-term suppressive effect on human breast cancer cell proliferation. To determine whether the effects of TAM are mediated by specific gene activation or repression, normal and tumoral human breast tissues obtained before and during TAM treatment were analyzed by differential display technique. Total RNA for differential display analysis was obtained from breast tissues from two women with the diagnosis of estrogen receptor-positive stage II (T2N1M0) infiltrating ductal carcinoma, made by incisional biopsy, followed by modified radical mastectomy performed after a 30-day treatment with TAM (20 mg/day). One 202-bp cDNA band, AP5-1, was present in normal and tumoral biopsy samples, but was absent in breast tissue obtained during TAM treatment, and was confirmed by Northern hybridization, which showed a 2.7-kb band in both patients. The differentially expressed cDNA fragment showed 99% homology to Homo sapiens CD36 gene, a glycoprotein that acts as a receptor for the extracellular matrix proteins thrombospondin-1, collagen types I and IV, and oxidized low-density lipoprotein. These results indicate that the down-regulation of CD36 induced by TAM might represent alternative or additional mechanisms of action of this drug affecting the functions of thrombospondin-1, which is involved in hematogenous tumor spread, invasion and angiogenesis, and oxidized low-density lipoprotein, playing a role in inhibition of arteriosclerosis. The multiple functions affected by the down-regulation of CD36 by TAM warrant the need for additional studies.

Antineoplastic Agents, Hormonal↗

Cloning and molecular characterization of the basic peroxidase isoenzyme from Zinnia elegans, an enzyme involved in lignin biosynthesis.

The major basic peroxidase from Zinnia elegans (ZePrx) suspension cell cultures was purified and cloned, and its properties and organ expression were characterized. The ZePrx was composed of two isoforms with a M(r) (determined by matrix-assisted laser-desorption ionization time of flight) of 34,700 (ZePrx34.70) and a M(r) of 33,440 (ZePrx33.44). Both isoforms showed absorption maxima at 403 (Soret band), 500, and 640 nm, suggesting that both are high-spin ferric secretory class III peroxidases. M(r) differences between them were due to the glycan moieties, and were confirmed from the total similarity of the N-terminal sequences (LSTTFYDTT) and by the 99.9% similarity of the tryptic fragment fingerprints obtained by reverse-phase nano-liquid chromatography. Four full-length cDNAs coding for these peroxidases were cloned. They only differ in the 5'-untranslated region. These differences probably indicate different ways in mRNA transport, stability, and regulation. According to the k(cat) and apparent K(m)(RH) values shown by both peroxidases for the three monolignols, sinapyl alcohol was the best substrate, the endwise polymerization of sinapyl alcohol by both ZePrxs yielding highly polymerized lignins with polymerization degrees > or =87. Western blots using anti-ZePrx34.70 IgGs showed that ZePrx33.44 was expressed in tracheary elements, roots, and hypocotyls, while ZePrx34.70 was only expressed in roots and young hypocotyls. None of the ZePrx isoforms was significantly expressed in either leaves or cotyledons. A neighbor-joining tree constructed for the four full-length cDNAs suggests that the four putative paralogous genes encoding the four cDNAs result from duplication of a previously duplicated ancestral gene, as may be deduced from the conserved nature and conserved position of the introns.

Amino Acid Sequence↗

AM67, a secretory component of the guinea pig sperm acrosomal matrix, is related to mouse sperm protein sp56 and the complement component 4-binding proteins.

The guinea pig sperm acrosomal matrix is the dense core of the acrosome and is likely to be important in acrosome biogenesis and fertilization. Isolated acrosomal matrices are composed of a limited number of major bands when analyzed by SDS-polyacrylamide gel electrophoresis, among which is a Mr 67,000 protein that we have termed AM67. Indirect immunofluorescence demonstrated that AM67 is localized to the apical segment of the cauda epididymal sperm acrosome. Immunoelectron microscopy further refined the localization of AM67 to the M1 (dorsal bulge) domain within the acrosome. Using a polymerase chain reaction product based upon tryptic peptide sequences from AM67, a lambdagt11 guinea pig testis cDNA library was screened to yield two cDNA clones that encode the AM67 peptides. Northern analysis revealed that AM67 is transcribed as a 1. 9-kilobase testis-specific mRNA. The complete AM67 sequence encodes a prepropolypeptide of 533 amino acids with a calculated Mr of 59, 768. Following cleavage of a probable signal sequence, the polypeptide was predicted to have a Mr of 56,851 and seven consensus sites for asparagine-linked glycosylation. The deduced amino acid sequence of AM67 is most similar to those of the mouse sperm protein sp56 and the alpha-subunits of complement component 4-binding proteins from various mammalian species. Although mouse sp56 has been reported to be a cell-surface receptor for the murine zona pellucida glycoprotein ZP3, standard immunoelectron microscopy using the anti-sp56 monoclonal antibody 7C5 detected sp56 within the mouse sperm acrosome, but failed to detect sp56 on the surface of acrosome-intact mouse sperm. Furthermore, acrosomal labeling was detected in mouse sperm prepared for immunofluorescence using paraformaldehyde fixation, but was not observed with live unfixed sperm. Thus, the finding that sp56 is present within the acrosome provides further support that sp56 and AM67 are orthologues and suggests that sp56 may function in acrosomal matrix-zona pellucida interactions during and immediately following the acrosome reaction in the mouse.

Acrosome↗

Pulmonary lung surfactant synthetic peptide concentration-dependent modulation of DPPC and POPG acyl chain order in a DPPC:POPG:palmitic acid lipid mixture.

Lung surfactant-associated protein interaction with lipid matrices and the effects on lipid thermotropic phase behavior are areas of active research. Many studies limit the lipids to a single or two-component system. The current investigation utilizes a three-lipid component matrix (DPPC:POPG:palmitic acid) to investigate the impact of a synthetic surfactant protein B fragment (SP-B 53-78 DiACM) on the dynamic surface activity of the lipid admixture as measured by a Wilhelmy surface balance. Also, the modulation of the individual lipid acyl chain order by the peptide within the lipid matrix is studied through the use of thermal perturbation FTIR spectroscopy. The data clearly demonstrate a concentration-dependent effect of the peptide on the surface activity with an improvement in the dynamic surface tension diagram characteristics (decreased surface tension and increased collapse plateau) especially at low, 0.36 M%, peptide concentrations. These effects are diminished upon further addition of the peptide. FTIR spectral data demonstrate that the peptide addition results in a significant increase in the acyl chain order of the DPPC and POPG components as measured by the position of the methylene stretching vibrational bands. DPPC is most sensitive to the peptide presence, while the palmitic acid is least affected. The transition temperatures of the individual lipids are also increased with the addition of the peptide. The presence of POPG in the matrix achieves the surface activity similarly seen with natural lung surfactant relative to a DPPC/palmitic acid lipid matrix alone. Its presence increases the sensitivity of the DPPC acyl chains to the presence of the peptide. These effects on the chain order are most probably related to the increased acyl chain fluidity which POPG imparts to the lipid matrix because of the presence of the cis double bond. The phosphatidylglycerol headgroup also adds a negative charge to the lipid matrix which enhances the peptide-lipid interaction. Although the palmitic acid is minimally affected by the peptide, its presence, as suggested by surface balance measurements, results in the establishment of a stable lipid film with DPPC, capable of achieving low surface tension values.

1,2-Dipalmitoylphosphatidylcholine↗

Interphase nuclear matrix and metaphase scaffolding structures.

The protein compositions of purified metaphase chromosomes, nuclei and their residual scaffold and matrix structures, are reported. The protein pattern of nuclei on sodium dodecyl sulphate/polyacrylamide gels is considerably more complex and rich in non-histone proteins than that of chromosomes. Nuclei contain about three to four times more non-histone proteins relative to their histones than chromosomes. Besides the protein components of the peripheral lamina, several protein bands are specific or at least highly enriched in nuclei. Conversely, two proteins X0 (33 X 10(3) Mr) and X1 (37 X 10(3) Mr) are highly enriched in the pattern of metaphase chromosomes. We have compared morphologically the previously defined nuclear matrices type I and II. The type I nuclear matrix is composed of the known lamina proteins, which form the peripheral lamina structure, and a complex series of proteins that form the internal network of the matrix as observed by electron microscopy. This internal network is stabilized similarly to the metaphase scaffolding by metalloprotein interaction. Both the scaffolding and the internal network of the matrix dissociate if thiols or certain metal chelators are used in the extraction buffer. Under these conditions the resulting nuclear structure, called matrix type II, appears empty in the electron microscope, with the exception of some residual nucleolar material. This latter material can be extracted from the internal network by exhaustive treatment of the nuclei with RNase before extraction with high salt. Immunoblotting and activity studies show RNA polymerase II to be tightly bound to the type I, but not to the type II matrix, or to the scaffolding structure. No polymerase II enzyme was detected in isolated metaphase chromosomes. Another nuclear enzyme, poly(ADP-ribose) polymerase is not bound to either of the residual nuclear matrices or to the scaffolding structures. The association of RNA polymerase with the internal network of the nuclear matrix is consistent with the idea that transcription occurs in close association with this structure.

Cell Nucleus↗

Matrix metalloproteinase activities in avian tibial dyschondroplasia.

Tibial dyschondroplasia (TD) in poultry is a disorder of growth plate cartilage that fails to resorb and consequently prevents bone formation. Matrix metalloproteinases (MMP) contribute to the process of resorption through the degradation of extracellular matrices and facilitating vascularization, growth plate remodeling, and maturation. In order to understand the cause of the failure of cartilage degradation in TD, the gelatinase and collagenase activities, and the levels of collagen and glycosaminoglycans of conditioned media derived from cartilage-explant cultures of normal and TD growth plates were measured. Substrate zymography exhibited two prominent gelatinolytic and collagenolytic bands corresponding to MW 63, 59, and a broad but fuzzy band of activity between 100 and 200 kDa. On treatment with 4-aminophenylmercuric acetate, a compound that converts proenzyme forms of MMP, the 63 kDa MW gelatinolytic band migrated as a approximately 60 kDa band and contributed to the broadening of the 59 kDa band. The TD-growth plate-conditioned media had significantly lower collagenolytic-gelatinolytic activities. The sulfated glycosaminoglycans, but not the collagen contents of the conditioned media from TD-explant cultures, were also reduced significantly. It is likely that the decreased matrix metalloproteinase activities of growth plate chondrocyte may contribute to a reduced turnover of extracellular matrices (ECM), leading to the retention of cartilage and its lack of vascularity in TD-affected growth plates.

Animals↗

Glycoprotein 115, a glycoprotein isolated from chick blood vessels, is widely distributed in connective tissue.

An extracellular glycoprotein (gp 115) with an apparent Mr = 115,000 isolated from chick aortas (Bressan, G. M., I. Castellani, A. Colombatti, and D. Volpin, 1983, J. Biol. Chem., 258:13262-13267), was used to immunize mice. The antisera were shown to specifically recognize gp 115 by numerous criteria: a major band around Mr = 115,000 plus minor bands of lower Mr were visible by immunoblotting on aorta extracts, and a similar pattern was observed with a monoclonal antibody; no cross-reactivity was detected by radioimmunobinding with other extracellular proteins, namely, fibronectin, laminin, and collagen types I, III, IV, V, and VI. Antigen distribution on frozen tissue sections from newborn chicks was investigated by using affinity-purified antibody. Strong immunoreactivity was always found in blood vessels. In the digestive tract, the fluorescent staining was localized both at the level of muscular layers and in the stromal matrix of the villi. Within skeletal muscle and myocardium, staining was associated with large connective tissue bundles and the matrix around each muscle fiber. Intense fluorescence was observed in the kidney, in smooth muscle cells rich areas of parabronchi, and within the portal space and along liver sinusoids. The antigen was not detected at the epidermal-dermal junction; immunoreactivity in the dermis was present as a diffuse fibrillar pattern. That the antigen detected by immunofluorescence in the various organs was indeed gp 115 was demonstrated by immunoblotting analysis: as in aorta extracts, a major band around Mr = 115,000 was detected in several tissues. Antibody-reacting material was also incorporated into the extracellular matrix produced by embryo smooth muscle cells grown in vitro and was organized as a meshwork of fine fibrils.

Animals↗