Elimination of breast tumor-associated chondroitin sulfate promotes metastasis.

Breast cancer is one of the leading causes of cancer-related deaths amongst women in the USA. The tumor microenvironment has been suggested to be an attractive therapeutic target for treatment of cancers. The glycosaminoglycan chondroitin sulfate, as part of the cellular microenvironment, consists of long linear chains of repeating disaccharide units, which are covalently attached to core proteins to form chondroitin sulfate-proteoglycans. In vitro studies have implicated chondroitin sulfate in various aspects of carcinogenesis, whereas the in vivo roles of chondroitin sulfate are less clear. Drastically elevated levels of chondroitin sulfate have been observed within the stromal compartment of many solid tumors, including human breast carcinomas, the significance of which is unknown. We examined the role of tumor-associated chondroitin sulfate in breast cancer progression. Enzymatic elimination of endogenous chondroitin sulfate by intra-tumor injections of chondroitinase ABC leads to the development of secondary tumors and increased lung metastases, while primary orthotopic tumor growth was not affected. These results establish a metastasis-inhibiting effect of primary breast tumor-associated chondroitin sulfate, which may open novel carbohydrate-based therapeutic strategies to combat breast cancer.

Structural dynamics and interfacial properties of filler-reinforced elastomers.

The combined effect of filler networking and reduced chain mobility close to the filler interface is analyzed based on investigations of the relaxation dynamics of a solution of styrene butadiene rubber filled with different loadings and types of nanostructured carbon blacks. Dynamic-mechanical and dielectric spectra are studied in a wide frequency and temperature range. By referring to a tunneling process of charge carriers over nanoscopic gaps between adjacent carbon black particles the gap distance is evaluated from the dielectric spectra. This distance corresponds to the length of glassy-like polymer bridges forming flexible bonds between adjacent filler particles of the filler network. It is found that the gap distance decreases with increasing filler loading and specific surface area which correlates with an increase of the apparent activation energy of the filler network evaluated from dynamic-mechanical data. Due to the thermal activation of glassy-like polymer bridges the time-temperature superposition principle is not fulfilled for filled elastomers and the introduction of vertical shift factors is necessary to obtain viscoelastic master curves. The change in the low frequency viscoelastic properties by the incorporation of fillers is shown to be related to the superimposed dynamics of the filler network governed by the viscoelastic response of the glassy-like polymer bridges. This effect is distinguished from the reduced chain mobility close to the filler surface which results in a broadening of the glass transition on the high temperature or low frequency side. The microstructure-based interpretation of viscoelastic data is supported by an analysis of the relaxation time spectra.

Identification and characterization of TGFbeta-dependent and -independent cis-regulatory modules in the C4ST-1/CHST11 locus.

Chondroitin-4-sulfotransferase-1(C4ST-1)/carbohydrate sulfotransferase 11 (CHST11) is a Golgi-bound enzyme involved in the biosynthesis of the glycosaminoglycan chondroitin sulfate. The sulfation pattern of chondroitin is tightly regulated during development, injury and disease, with the temporal and spatial expression of chondroitin sulfotransferase genes believed to be a crucial determinant of the fine balance of chondroitin sulfation. We have previously identified mouse C4st-1 as a target gene of ligands of the TGFbeta superfamily of growth factors, which could positively regulate C4st-1 expression in a number of cell types. We have also shown that a gene trap loss-of-function mutation in C4st-1 leads to severe skeletal abnormalities during mouse embryogenesis. In addition, we described a highly specific temporal and spatial expression pattern of C4st-1 during mouse embryogenesis. However, the transcriptional regulatory mechanisms that control C4st-1 gene expression remain unexplored. In order to gain knowledge on the transcriptional regulation of C4ST-1, we used a bioinformatical approach to identify conserved putative long-range cis-regulatory modules in a region of 120 kb spanning the 5' end of the C4ST-1 gene. Luciferase reporter assays in human HEK293T and mouse NmuMG cells identified a functional C4ST-1 promoter, as well as a number of cis-regulatory modules able to positively and negatively regulate C4ST-1 expression. Moreover, we identified TGFbeta- responsive regulatory modules that can function in a cell type-specific fashion. Taken together, our results identify TGFbeta-dependent and -independent cis-regulatory modules of the C4ST-1 gene.

Evaluation of viscoelastic master curves of filled elastomers and applications to fracture mechanics.

The viscoelastic response of filler-reinforced elastomers has been investigated by dielectric- and dynamic-mechanical spectroscopy. Horizontal and vertical shifting factors are evaluated, which are used for the construction of viscoelastic master curves. They are discussed in the framework of filler network effects and the slowed-down dynamics of a polymer layer close to the filler surface. The observed shifting behaviour is shown to be related to the superposition of two relaxation processes, i.e. that of the polymer matrix and the filler network, leading to a failure of the time-temperature superposition principle. While the matrix transforms according to the Vogel-Fulcher equation, the filler network exhibits an Arrhenius dependence, which results from the thermal activation of filler-filler bonds, i.e. glassy-like polymer bridges between adjacent filler particles. Based on the viscoelastic master curves relaxation time spectra are evaluated. By referring to a recently developed theory of crack propagation in viscoelastic solids it is demonstrated that the behaviour of the scaling exponent of the relaxation time spectra correlates fairly well with that of the crack propagation rates measured under moderate severity conditions.

[Operation of the floppy eyelid. Symptomatic cases require surgical eyelid stabilization]. / Die Operation des Floppy Eyelids. Symptomatische Fälle erfordern eine chirurgische Lidstabilisierung.

BACKGROUND: Floppy Eyelid is a rare condition causing chronic papillary conjunctivitis and chronic corneal disorders (superficial punctate keratitis, epithelial and stromal ulcers). It is characterized by an extremely enlarged and "floppy" upper eyelid which can be easily everted by slight elevation. Usually obese men are affected who use to sleep face down either on the right or on the left side. Pushing the eyelid against the pillow, the lid is intermittently everted at sleep. This lagophthalmus with rubbing of the exposed eye and lid structures causes all pathologic disorders. Shielding the eye at night may help temporarily. PATIENTS: We performed surgery on 7 men with symptomatic floppy eyelid in the age of 42 to 61 years. The patients had been symptomatic between 1 month and 4 years prior to the definite diagnosis. Follow-up time has been 7 months to 4.5 years. RESULTS: In all cases surgery achieved improvement. CONCLUSIONS: Cases of unclear conjunctival or corneal damage and inflammation should led consider Floppy Eyelid as a possible cause. The typical clinical findings make diagnosis easy. As complete stopping of eyelid-rubbing by changing the patient's sleeping habits is mostly not easily possible, for acute therapy of threatening damages to cornea and conjunctiva a surgical shortening of the lid is necessary which stabilizes the lid in order to avoid nightly spontaneous eversion for a long time.

Lacrimal gland choristoma of the iris.

Intraocular lacrimal gland tissue is an extremely rare choristoma. A newborn girl was seen with a fleshy, vascular tumor arising from the peripheral iris and the anterior chamber angle. The tumor was treated with topical steroids on suspicion of a juvenile xanthogranuloma; later, it grew slightly and a secondary glaucoma developed. Histopathological examination of the resected tumor showed lacrimal gland tissue in the iris. Twelve cases of intraocular lacrimal gland tissue have been reported in the literature.

recessive spotting: a linked locus that interacts with W/Kit but is not allelic.

BACKGROUND: The murine coat-colour mutation recessive spotting (rs) maps very closely to the W/Kit locus, encoding the proto-oncoprotein Kit, the protein tyrosine kinase receptor for stem cell factor. Kit is important in the development of melanocytes, germ cells, interstitial cells of Cajal (ICC) and haemopoietic lineages, including mast cells. rs has never been genetically separated from Kit, and interacts with Kit mutations, suggesting that it is a recessive allele of Kit. Here we have tested this possibility. We have shown previously that diploid rs/rs melanocytes proliferated more slowly than did +/+ melanocytes, as did an immortal line of rs/rs melanocytes, melan-rs. RESULTS: The Kit mRNA level in rs/rs melanocytes was indistinguishable from that of other melanocyte lines. The Kit cDNA sequence from rs/rs melanocytes and the kinase activity of Kit in rs/rs mast cells appeared to be normal. No deficiency of mast cells or ICC was observed in rs/rs mice. Moreover, following the overexpression of a normal Kit cDNA, proliferation of rs/rs melanocytes was retarded further, but that of +/+ melanocytes was increased, indicating an intracellular interaction between rs and Kit. Of other closely linked tyrosine kinase genes, melanocytes and melanoblasts did not express mRNA for Pdgfra, Flk-1 or Txk, but both expressed Tec, encoding a nonreceptor kinase that interacts with Kit. CONCLUSIONS: rs is not a mutation in Kit, although we have confirmed that rs interacts with Kit. It seems unlikely that rs affects Pdgfra, Flk-1 or Txk, but Tec remains a candidate for rs.

Aquarius, a novel gene isolated by gene trapping with an RNA-dependent RNA polymerase motif.

In a retinoic acid (RA) gene trap screen of mouse embryonic stem (ES) cells, a novel gene, named Aquarius (Aqr), was identified and characterized. The promoterless lacZ marker was used to trap the genomic locus and to determine the expression pattern of the gene. Aqr transcripts are strongly induced in response to RA in vitro. During embryogenesis, Aqr is expressed in mesoderm, in the neural crest and its target tissues, and in neuroepithelium. Expression was first detected at 8.5 days postcoitum, when neural crest cells are visible at the lateral ridges of the neural plate. The gene-trapped Aqr locus was transmitted through the mouse germ line in three genetic backgrounds. In the F2 generation, the expected mendelian ratio of 1:2:1 was observed in all backgrounds, indicating that homozygous mice are viable. Homozygotes are normal in size and weight and breed normally. The gene trap insertion, however, does not seem to generate a null mutation, because Aqr transcripts are still present in the homozygous mutant animals. The Aqr open reading frame has weak homology to RNA-dependent RNA polymerases (RRPs) of the murine hepatitis viruses and contains an RRP motif. Aqr was mapped to mouse chromosome 2 between regions E5 through F2 by using fluorescence in situ hybridization analysis.

Mechanism of citrinin-induced dysfunction of mitochondria. VI. Effect on iron-induced lipid peroxidation of rat liver mitochondria and microsomes.

The inhibition by citrinin (CTN) of lipid peroxidation of mitochondria, sub-mitochondrial particles (SMP) and microsomes was studied. This effect was reversed by the presence of high concentrations of Fe3+ (0.4 and 0.5 mM), suggesting chelation of the mycotoxin with iron or interference in the reduction of Fe3+.

Developmental origin and Kit-dependent development of the interstitial cells of cajal in the mammalian small intestine.

Interstitial cells of Cajal (ICCs) form a network of cells between the external longitudinal and circular muscle layers at the level of the Auerbach's plexus in the mammalian small intestine. These cells express the Kit receptor tyrosine kinase and are essential for intestinal pacemaker activity. W mutant mice carrying structural mutations in the Kit gene lack both the network of ICCs and intestinal pacemaker activity. We were interested in the developmental origin of the cells that make up the network of ICCs. In addition, the specific stages of ICC development that require a functional Kit receptor have not been characterized. We show that ICCs originate from mesenchymal progenitor cells that coexpress both Kit and smooth muscle myosin heavy chain, a marker specific for smooth muscle, during embryogenesis. ICC and longitudinal smooth muscle lineages begin to diverge late in gestation. Embryos homozygous for the regulatory Wbanded (Wbd) mutation do not express Kit in these mesenchymal progenitor cells. Nevertheless, Wbd/Wbd mice display a normal network of ICCs and normal smooth muscle layers at postnatal day 5 (p5). Adult Wbd/Wbd mice lack a functional ICC network and intestinal pacemaker activity due to a failure of the ICCs to increase in numbers after p5. These data suggest a common developmental origin of the ICCs and the longitudinal smooth muscle layers in the mammalian small intestine and show that Kit expression is necessary for the postnatal development and proliferation of ICCs but not for the initial cell lineage decision toward an ICC fate during embryogenesis or for smooth muscle development.

Effect of amiodarone (AMD) on the antioxidant enzymes, lipid peroxidation and mitochondrial metabolism.

The effects of amiodarone (AMD) on lipid peroxidation of rat liver mitochondria, the formation of superoxide anions at the respiratory chain level, and the cytosolic and mitochondrial enzymatic protective mechanisms of oxidative stress were studied. An attempt of classify AMD according to its toxic ability to interfere with the integrated function of electron transport enzymes was also investigated. The results confirm the effects of AMD on complex I and permit the placing of this drug in class A of the classification of Knobeloch, together with rotenone, amytal and chaotropic agents. AMD has no effect on the activity of the enzymes superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase, nor on glucose 6-phosphate dehydrogenase. AMD did not promote an increase in the formation of anion superoxide at the respiratory chain level. Pre-incubation with AMD (16.6 microM) inhibited about 70 per cent of lipid peroxidation. The results suggest a protective effect of AMD against lipid peroxidation in mitochondrial membranes by iron-dependent systems.

Mechanism of citrinin-induced dysfunction of mitochondria. V. Effect on the homeostasis of the reactive oxygen species.

The effects of citrinin in the maintenance of the homeostasis of the reactive oxygen species in rat liver cells were evaluated. Citrinin (CTN) modifies the antioxidant enzymatic defences of cells through the inhibition of GSSG-reductase and transhydrogenase. No effect was observed on GSH-peroxidase, catalase, glucose 6-phosphate and 6 phosphogluconate dehydrogenases, and superoxide dismutase. The mycotoxin increased the generation of reactive oxygen species, stimulating the production of the superoxide anion in the respiratory chain. The results suggest that oxidative stress is an important mechanism, side by side with other effects previously shown, in the establishment of the cytotoxicity and cellular death provoked by CTN in several tissues.

[Characterization of mitochondrial myopathies through the evaluation of the enzymatic activities involved in energy metabolism]. / Caracterização de miopatias mitocondriais através da avaliação das atividades enzimáticas envolvidas no metabolismo energético.

The activities of the enzymes NADH dehydrogenase, NADH cytochrome e reductase, succinate dehydrogenase, succinate cytochrome e reductase, cytochrome c oxidase and citrate synthase in normal and sick human skeletal muscle mitochondria were determined. A control group was formed by 13 normal people and without using continuous medication. The patient group was formed by 10 people whose pathological diagnosis indicated suspicion of mitochondrial myopathy. A decrease in the activity of the enzymes in all patient was observed: 7 with abnormality in all the tested enzymes; 2 with deficiencies in all the enzymes except cytochrome e oxidase; and 1 with dysfunction only in the activities of succinate dehydrogenase and succinate cytochrome e reductase. The results indicate multiple or combined deficiencies in the respiratory chain, besides dysfunction of citrate synthase in 9 patients. In one exceptional case, the enzymatic deficiency was restricted to complex II. It is possible to conclude that the methodology used herein is adequate and easily applicable to clinical objectives, and that the results obtained allow characterization of the deficient mitochondrial enzymatic complexes, thus showing that the origin of the diseases is an energetic metabolic dysfunction.

[Therapy of advanced amoeba keratitis with keratoplasty à chaud and adjuvant cryotherapy]. / Therapie der fortgeschrittenen Amöbenkeratitis mit Keratoplastik à chaud und adjuvanter Kryoapplikation.

BACKGROUND: Since the mid-1980s acanthamoeba keratitis has been diagnosed with increasing frequency, especially in contact lens wearers. The assignment to specialized centers is often delayed many months and there is hardly any chance of controlling the disease by conservative treatment alone. In these cases, penetrating keratoplasty offers the only chance for rehabilitation. The therapeutic role of penetrating keratoplasty and supportive intraoperative cryotherapy is demonstrated by the courses of six patients with unilateral acanthamoeba keratitis. PATIENTS AND METHODS: The data of six patients aged 41.2 (22-63) years with medically uncontrollable acanthamoeba keratitis were evaluated retrospectively. The diagnosis was confirmed histologically in all cases. All patients were contact lens wearers. They underwent a total of ten keratoplasties between November 1986 and January 1995. The donors were 44.8 (23-58) years of age. The transplant diameters varied between 7.7 and 9.5 mm. The margins of the host cornea were intraoperatively frozen by a cryoprobe in three patients with a far advanced stage of corneolimbal parasitic infiltration. RESULTS: During a follow-up period of +/-10.2 (1-22) months, five of six eyes were rehabilitated with visual acuities between 0.4 and 1.0. One eye went blind after the fourth keratoplasty because of uncontrollable secondary glaucoma. After three keratoplasties with simultaneous application of cryocoagulation because of an especially high risk of persisting acanthamoeba infection, all corneae remained clear and free of recurrences. CONCLUSIONS: In advanced acanthamoeba keratitis which has not responded to conservative treatment, penetrating keratoplasty not only provides elimination of the pathogen, but also good functional results. In far advanced stages, the intraoperative application of cryocoagulation for parasite elimination in the host cornea seems to be a very effective measure against local recurrences of the infection.

Long-range genomic rearrangements upstream of Kit dysregulate the developmental pattern of Kit expression in W57 and Wbanded mice and interfere with distinct steps in melanocyte development.

Mutations in the murine dominant white spotting (W) locus cause pleiotropic developmental defects that affect hematopoietic cells, melanocytes, germ cells and the interstitial cells of Cajal in the gut. W mutations either alter the coding sequence of the Kit receptor tyrosine kinase, resulting in a receptor with impaired kinase activity, or affect Kit expression. Here we describe the molecular and cell-type-specific developmental defects of two of the latter class of regulatory W alleles, W57 and Wbanded(bd). In both mutants, the temporal and spatial patterns of Kit expression are dysregulated during embryogenesis and in adult animals. In Wbd mice, ectopic expression of Kit in the dermatome of the somites at days 10.8 and 11.8 of development seemed to interfere with melanoblast development. In contrast, the W57 allele leads to an intrinsic pigmentation defect by downregulating developmental Kit expression in trunk melanoblasts, but not melanoblasts around the otic vesicle. Both mutations affect transcriptional initiation of the Kit gene. The W57 allele is associated with a 80 kb deletion 5' of the Kit-coding region while Wbd is associated with a 2.8 Mb genomic inversion of chromosome 5 with the distal breakpoint between Kit and the platelet-derived growth factor receptor alpha (Pdgfra) gene, and the proximal breakpoint between the genes for the GABA receptor beta 1 (Gabrb1) and the Tec tyrosine kinase, juxtaposing the Kit and Tec tyrosine kinase genes. Neither W57 nor Wbd affect genomic sequences previously suggested in in vitro experiments to control cell-type-specific expression of Kit. These results link specific mechanisms of cellular and developmental defects to long-range genomic rearrangements that positively and negatively affect Kit transcription in different cell lineages as well as in different subpopulations of the same lineage.

Embryonic and adult expression patterns of the Tec tyrosine kinase gene suggest a role in megakaryocytopoiesis, blood vessel development, and melanogenesis.

The Tec cytoplasmic tyrosine kinase is a member of a family of src-like proteins that are thought to play important roles in hematopoiesis. Here we describe the temporal and spatial expression of the Tec gene during embryogenesis and in the adult. Our data demonstrate that embryonic Tec expression is restricted to distinct hematopoietic cells as well as structures and cell types that share a common feature of containing fluid in an enclosed cavity, e.g., endothelial cells. In addition, Tec is expressed in melanocytes late in gestation. The observed developmental expression pattern of Tec suggests a role for this gene in several aspects of hematopoiesis and/or blood vessel development as well as in late stages of melanogenesis.

[Surgical management of Marfan-associated and idiopathic lens dislocations]. / Operative Versorgung Marfan-assoziierter und idiopathischer Linsensubluxationen.

BACKGROUND: The ideal and safe surgical method for Marfan-associated or idiopathic lens subluxations is still a matter of debate. PATIENTS AND METHODS: Between 1990 and 1995, 23 eyes were operated for lens subluxations, mainly because of decreased visual acuity, but also because of conservatively uncontrolled secondary glaucoma. Marfan patients were 27.0 (5-62) years old at surgery; patients with idiopathic lens subluxations were 38.5 (11-63) years old. Surgical procedure depended on patient age and anatomical conditions. RESULTS: All patients achieved an increase in visual acuity. Amblyopia existed in six patients. All problems due to glaucoma were controlled postoperatively. Our greatest concern was rhegmatogenous retinal detachment. It occurred in only one eye of a non-Marfan patient. CONCLUSION: The prognosis for lens surgery in Martan-associated and idiopathic lens subluxations is good. The implantation of a posterior chamber lens provides a good and secure means of optical rehabilitation. Our preferred primary transscleral suture technique guarantees high security and stability of position. Previously feared surgical risks have been reduced by modern surgical procedures.

Effect of methotrexate (MTX) on NAD(P)+ dehydrogenases of HeLa cells: malic enzyme, 2-oxoglutarate and isocitrate dehydrogenases.

The effects of methotrexate (MTX) on oxygen uptake by permeabilized HeLa cells were evaluated. MTX did not inhibit state III respiration when the oxidizable substrate was succinate, but when the substrates were 2-oxoglutarate or isocitrate the respiration decreased about 50 per cent at 1.0 mM concentration of the drug. This effect was explained by inhibition of 2-oxoglutarate and isocitrate dehydrogenases by MTX. No effect was observed on succinate dehydrogenase. An evaluation of the effects of MTX on malic enzyme activity as measured by pyruvate plus lactate production in intact cells supplied with malate showed a decrease of about 40 per cent in metabolite production using 0.4 mM MTX. HeLa cell malic enzyme, as observed for other tumour cells, is compartmentalized in mitochondria and cytosol, and is another example of a dehydrogenase inhibited by MTX.