Heparan sulfate proteoglycans as trastuzumab targets in anoikis-resistant endothelial cells

Anoikis is a form of programmed cell death induced by loss of contact from neighboring cells or from their extracellular matrix (ECM). Many tumorigenic cells are anoikis resistant, facilitating cancer progression and metastasis. Trastuzumab is a monoclonal antibody used for the treatment of breast and gastric cell cancer, but its mechanism of action is not well elucidated and its target molecules not well defined. Heparan sulfate proteoglycans (HSPGs) and glycosaminoglycans (GAGs) play important roles in tumor development and in response of cancer cells to drugs. This study investigates the effect of trastuzumab on the expression of HSPGs and sulfated glycosaminoglycans (SGAGs) in anoikis-resistant endothelial cells. After trastuzumab treatment, endothelial cells resistant to anoikis show an increase in adhesion to fibronectin followed by a decrease in invasion, proliferation, and angiogenic capacity. In addition, a significant increase in the number of cells in the S phase of the cell cycle was also observed. In relation to HSPGs and SGAGs expression, we observed a decrease in syndecan-4 and perlecan expression, as well as in the heparan sulfate biosynthesis in anoikis-resistant endothelial cells after exposure to trastuzumab. Our results suggest that trastuzumab interacts with GAGs and proteoglycans of the cell surface and ECM and through this interaction controls cellular events in anoikis-resistant endothelial cells.     

Cell surface localization of heparanase on macrophages regulates degradation of extracellular matrix heparan sulfate

Extravasation of peripheral blood monocytes through vascular basement membranes requires degradation of extracellular matrix components including heparan sulfate proteoglycans (HSPGs). Heparanase, the heparan sulfate-specific endo-beta-glucuronidase, has previously been shown to be a key enzyme in melanoma invasion, yet its involvement in monocyte extravasation has not been elucidated. We examined a potential regulatory mechanism of heparanase in HSPG degradation and transmigration through basement membranes in leukocyte trafficking using human promonocytic leukemia U937 and THP-1 cells. PMA-treated cells were shown to degrade 35S-sulfated HSPG in endothelial extracellular matrix into fragments of an approximate molecular mass of 5 kDa. This was not found with untreated cells. The gene expression levels of heparanase or the enzyme activity of the amount of cell lysates were no different between untreated and treated cells. Immunocytochemical staining with anti-heparanase mAb revealed pericellular distribution of heparanase in PMA-treated cells but not in untreated cells. Cell surface heparanase capped into a restricted area on PMA-treated cells when they were allowed to adhere. Addition of a chemoattractant fMLP induced polarization of the PMA-treated cells and heparanase redistribution at the leading edge of migration. Therefore a major regulatory process of heparanase activity in the cells seems to be surface expression and capping of the enzyme. Addition of the anti-heparanase Ab significantly inhibited enzymatic activity and transmigration of the PMA-treated cells, suggesting that the cell surface redistribution of heparanase is involved in monocyte extravasation through basement membranes.     

Heparan sulfate proteoglycans and heparin regulate melanoma cell functions

Background

The solid melanoma tumor consists of transformed melanoma cells, and the associated stromal cells including fibroblasts, endothelial cells, immune cells, as well as, soluble macro- and micro-molecules of the extracellular matrix (ECM) forming the complex network of the tumor microenvironment. Heparan sulfate proteoglycans (HSPGs) are an important component of the melanoma tumor ECM. Importantly, there appears to be both a quantitative and a qualitative shift in the content of HSPGs, in parallel to the nevi–radial growth phase–vertical growth phase melanoma progression. Moreover, these changes in HSPG expression are correlated to modulations of key melanoma cell functions.

Scope of review

This review will critically discuss the roles of HSPGs/heparin in melanoma development and progression.

Major conclusions

We have correlated HSPGs' expression and distribution with melanoma cell signaling and functions as well as angiogenesis.

General significance

The current knowledge of HSPGs/heparin biology in melanoma provides a foundation we can utilize in the ongoing search for new approaches in designing anti-tumor therapy. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Self-assembly in vitro of the 68,000 molecular weight component of the mammalian neurofilament triplet proteins into intermediate-sized filaments

The mammalian neurofilament triplet proteins (210, 160 and 68 × 10³M_r proteins) are resolved by anion exchange chromatography in the presence of urea. Upon dialysis against physiological buffers at 37 °C only the 68 × 10³M_r protein shows self-assembly into morphologically normal intermediate-sized filaments. Addition of 210 × 10³M_r protein to 68 × 10₃M_r protein leads to shorter filaments, which upon embedding reveal a rough surface and whisker-like protrusions that are not present on the smooth surface of filaments assembled from 68 × 10₃M_r protein alone. Certain emerging principles of neurofilament structure are discussed, emphasizing a possible relation between neurofilaments and other intermediate-sized filaments.     

Murine macrophage heparanase: inhibition and comparison with metastatic tumor cells

Circulating macrophages and metastatic tumor cells can penetrate the vascular endothelium and migrate from the circulatory system to extravascular compartments. Both activated murine macrophages and different metastatic tumor cells (B16-BL6 melanoma; ESb T-lymphoma) attach, invade, and penetrate confluent vascular endothelial cell monlayer in vitro, by degrading heparan sulfate proteoglycans in the subendothelial extracellular matrix. The sensitivity of the enzymes from the various sources degrading the heparan sulfate proteoglycan was challenged and compared by a series of inhibitors. Activated macrophages demonstrate a heparanase with an endoglycosidase activity that cleaves from the [35S]O4 = -labeled heparan sulfate proteoglycans of the extracellular matrix 10 kDa glycosaminoglycan fragments. The macrophages do not store the heparanase intracellularly but it is instead found pericellularly and requires a continuous cell-matrix contact at the optimal pH for maintaining cell growth. The degradation of [35S]O4 = -labeled extracellular matrix proteoglycans by the macrophages' heparanase is significantly inhibited in the presence of heparan sulfate (10 micrograms/ml), arteparon (10 micrograms/ml), and heparin at a concentration of 3 micrograms/ml. In contrast, other glycosaminoglycans such as hyaluronic acid, dermatan sulfate, and chondroitin sulfate as well as the specific inhibitor of exo-beta-glucuronidase D-saccharic acid 1,4-lactone failed to inhibit the degradation of sulfated proteoglycans in the subendothelial extracellular matrix. Degradation of this heparan sulfate proteoglycan is a two-step sequential process involving protease activity followed by heparanase activity. However, the following antiproteases--alpha 2-macroglobulin, antithrombin III, leupeptin, and phenylmethylsulfony fluoride (PMSF)--failed to inhibit this degradation process, and only alpha 1-antitrypsin inhibited the heparanase activity. B16-BL6 metastatic melanoma cell heparanase, which is also a cell-associated enzyme, was inhibited by heparin to the same extent as the macrophage heparanase. On the other hand, heparanase of the highly metastatic variant (ESb) of a methylcholanthrene-induced T lymphoma, which is an extracellular enzyme released by the cells to the incubation medium, was more sensitive to heparin and arteparon than the macrophages' heparanase, inhibited at concentrations of 1 and 3 micrograms/ml, respectively. These results may indicate the potential use of heparin or other glycosaminoglycans as specific and differential inhibitors for the formation in certain cases of blood-borne tumor metastasis.     

Structural polypeptides and products of late genes of bacteriophage Mu: Characterization and functional aspects

This paper describes the identification and functional role of late gene products of bacteriophage Mu, including an analysis of the structural proteins of the Mu virion.In vitro reconstitution of infectious phage particles has shown that four genes (E, D, I, J) control the formation of phage heads and that a cluster of eight genes (K, L, M, N, P, Q, R, S) controls the formation of phage tails.Sodium dodecyl sulphate/polyacrylamide gel electrophoresis of Mu polypeptides synthesized in Escherichia coli minicells infected by Mu phages carrying amber mutations in various late genes has resulted in the identification of the products of gene C (15.5 × 10³M_r); H (64 × 10³M_r); F (54 × 10³M_r); G (16 × 10³M_r); L (55 × 10³M_r); N (60 × 10³M_r); P (43 × 10³M_r) and S (56 × 10³M_r). Minicells infected with λpMu hybrid phages and deletion mutants of Mu were used to identify polypeptides encoded by the V-β region of the Mu genome. These are the products of genes V, W or R (41.5 × 10³M_r, and 45 × 10³M_r); U (20.5 × 10³M_r) and of genes located in the β region (24 × 10³M_r (gpgin) and 37 × 10³M_r (possibly gpmom)).Analytical separation of the proteins of the Mu virion revealed that it consists of a major head polypeptide with a molecular weight of 33 × 10³, a second head polypeptide of 54 × 10³ (gpF) and two major tail polypeptides with molecular weights of 55 × 10³ and 12.5 × 10³ (gpL and gpY, respectively). In addition, there are five minor components of the tail (including gpN, gpS and gpU) and approximately seven minor components of the head structure of the virion (including gpH).     

Role of heparanase on hepatic uptake of intestinal derived lipoprotein and fatty streak formation in mice

Background

Heparanase modulates the level of heparan sulfate proteoglycans (HSPGs) which have an important role in multiple cellular processes. Recent studies indicate that HSPGs have an important function in hepatic lipoprotein handling and processes involving removal of lipoprotein particles.

Principal Findings

To determine the effects of decreased HSPGs chain length on lipoprotein metabolism and atherosclerosis, transgenic mice over-expressing the human heparanase gene were studied.Hepatic lipid uptake in hpa-Tg mice were evaluated by giving transgenic mice oral fat loads and labeled retinol. Sections of aorta from mice over-expressing heparanase (hpa-Tg) and controls (C57/BL6) fed an atherogenic diet were examined for evidence of atherosclerosis. Heparanase over-expression results in reduced hepatic clearance of postprandial lipoproteins and higher levels of fasting and postprandial serum triglycerides. Heparanase over-expression also induces formation of fatty streaks in the aorta. The mean lesion cross-sectional area in heparanase over-expressing mice was almost 6 times higher when compared to control mice (23,984 µm²±5,922 vs. 4,189 µm²±1,130, p<0.001).

Conclusions

Over-expression of heparanase demonstrates the importance of HSPGs for the uptake of intestinal derived lipoproteins and its role in the formation of fatty streaks.     

Identification of the cell surface and nuclear receptors for NGF in a breast carcinoma cell line

¹²⁵I-nerve growth factor (NGF) was found to be internalized and translocated to the nucleus of SKBr5 breast carcinoma cells. The cytoplasm and chromatin isolated from nonmitotic cells accumulated two-and five-fold, respectively, more of ¹²⁵I-NGF than the cells undergoing mitosis. MAb 20.4 developed against the NGF cell surface receptor immunoprecipitated the 80,000 M_r receptor from plasma membrane and two protein species from the chromatin; 90,000 M_r (major band) and 200,000 M_r (minor band). In SKBr5 cells, binding of NGF to the chromatin did not affect synthesis of rRNA. Proliferation of SKBr5 cells was slightly stimulated by NGF. In control melanoma A875 cells, which express the 230,000 M_r chromatin receptor, NGF inhibited both rRNA synthesis and cell proliferation. We suggest that the 90,000 M_r chromatin receptor expressed by SKBr5 cells represents a “nonactive”, ligand-binding subunit of the high molecular weight receptor for NGF. The critical role of the chromatin receptor for NGF in rRNA-dependent cell proliferation is discussed. © 1993 Wiley-Liss, Inc.     

Heparanase mechanisms of melanoma metastasis to the brain: Development and use of a brain slice model

Heparanase (HPSE-1) is an endo-beta-D-glucuronidase that cleaves heparan sulfate (HS) chains of proteoglycans (HSPG), and its expression has been associated with increased cell growth, invasion, and angiogenesis of tumors as well as with embryogenesis and tissue development. Since metastatic cancer cells express HPSE-1, we have developed an orthotopic brain slice model to study HPSE-1 involvement in brain-metastatic melanoma. This model allows for the characterization of tumor cell invasion at both quantitative and qualitative levels. Brain-metastatic melanoma cells (B16B15b) showed augmenting levels of HPSE-1 protein expression in a time-dependent manner. Secondly, B16B15b cells pre-treated with HPSE-1 showed a significant increase in the number of cells that invaded into the brain tissue. Finally, HPSE-1 exposure-augmented invasion depth in brain sections by brain-metastatic melanoma cells. We concluded that applying this brain slice model can be beneficial to investigate HPSE-1- related in vivo modalities in brain-metastatic melanoma and brain invasion in general. These results also further emphasize the potential relevance of using this model to design therapies for controlling this type of cancer by blocking HPSE-1 functionality.     

Heparan Sulfate Proteoglycan Modulation of Wnt5A Signal Transduction in Metastatic Melanoma Cells

Heparan sulfate proteoglycans (HSPGs) are important modulators for optimizing signal transduction of many pathways, including the Wnt pathways. We demonstrate that HSPG glycosaminoglycan levels increased with increasing metastatic potential of melanoma cells. Previous studies have demonstrated that Wnt5A increases the invasiveness of melanoma cells. We further demonstrate that HSPGs potentiate Wnt5A signaling, since enzymatic removal of the HSPG backbone resulted in a decrease in cellular Wnt5A levels, an increase in secreted Wnt5A in cell media, a decrease in downstream signaling, and ultimately, a decrease in invasiveness. Specifically, syndecan 1 and syndecan 4 expression correlated to Wnt5A expression and melanoma malignancy. Knockdown of syndecan 1 or 4 caused decreases in cell invasion, which could be restored by treating the cells with recombinant Wnt5A. These data indicate that syndecan 1 and 4 correlate to increased metastatic potential in melanoma patients and are an important component of the Wnt5A autocrine signaling loop, the activation of which leads to increased metastasis of melanoma.The American Cancer Society estimates that in 2009 there will be 68,720 new cases of melanoma in this country with ∼8,650 deaths. Recent studies have demonstrated that the non-canonical Wnt pathway, also known as the Wnt/Ca²⁺ pathway, plays an important role in increasing the metastatic potential of melanoma cells (1–5). Studies from our laboratory demonstrated that increasing Wnt5A, which mediates the non-canonical Wnt/Ca²⁺ signaling pathway, increased melanoma metastasis (1–3), and silencing Wnt5A levels via siRNA³ decreased invasion (2, 3). In addition, we have shown that Wnt5A acts via protein kinase C (PKC) to mediate the motility of melanoma cells via the inhibition of metastasis suppressors and an initiation of the epithelial to mesenchymal transition, characterized by the loss of E-cadherin and the up-regulation of Snail (2).Wnt signaling can be mediated by heparan sulfate proteoglycans (HSPGs) which are important signal transduction modulators. They mediate fibroblast growth factor, Hedgehog, epidermal growth factor, transforming growth factor-β, and WNT signaling pathways (6–11). HSPGs consist of two types, cell surface and basement membrane-associated HSPGs (12). Cell surface HSPGs are glycoproteins with covalently attached unbranched and modified sugar chains known as glycosaminoglycans (GAGs). There are two types of cell surface HSPGs, known as glypicans and syndecans (11, 13). Glypicans are attached to the cell surface via a glycosylphosphatidylinositol anchor, whereas syndecans are type 1 transmembrane proteins. HSPG GAG side chains are unbranched chains of modified repeating disaccharide units of N-acetylglucosamine and glucuronic acid. They are joined to the core protein via a tetrasaccharide linker attached to a serine residue. Following synthesis, these chains undergo modification with the addition of sulfates by N- and O-sulfation (14). The sulfation status determines to which specific portion of the GAG chains ligands, such as Wnt, will attach. The heparan sulfate endosulfatases Sulf1 and Sulf2 are cell surface enzymes that control growth factor signaling. The regulation of the 6-O-sulfation states by these endosulfatases changes the affinity of the GAG chains for ligand binding (15–17). Following sulfation modification, HSPGs can regulate signaling by dimerization (with other HSPGs or canonical signaling receptors), stabilization, or transport of the ligand to or away from the high affinity receptors (18–20). In addition, studies have suggested that the core proteins themselves may also play an important role in cell phenotype and function (21).HSPGs have been implicated in a number of pathological conditions, such as Simpson-Golabi-Behmel overgrowth syndrome (22), fibrodysplasia ossificans progressiva (23), and Alzheimer disease (24). In addition, HSPGs are overexpressed in many forms of cancer, including prostate cancer and melanoma (25, 26). Importantly, in cancer, proteoglycans can have both tumor-promoting and tumor-suppressing activities. This depends on the type of protein core, the GAGs attached, and the localization of the proteoglycan and the molecules they associate with. In addition, the tumor subtype, stages, and degree of tumor differentiation also affect the function of HSPGs (27). HSPGs are cleaved by heparanases or heparin lyases (heparinases), which have been shown to have differing effects on tumor cell activity. For example, treating cancer cells with heparanase-1, which cleaves heparin-like regions (specifically HLGAG sites with O-sulfated l-iduronic acid residues), results in an increase in both tumor growth and metastatic dissemination (28). However, treating tumor cells with heparinase III, which more specifically cleaves HSPGs (i.e. unsulfated d-glucorinic acid, heparan-sulfate-like regions) results in an inhibition of their metastatic capacity (29). Importantly, heparanase I cleaves only certain side chains, where heparinase III treatment cleaves the entire backbone of the HSPG. It is likely that cleavage of specific side chains facilitates cell motility by releasing cells from adhesion to neighboring cells, whereas cleavage of the entire molecule decreases the availability of secreted ligands to their receptors, especially those involved in autocrine signaling, such as Wnt5A.Historically, Wnt5A has been quite difficult to purify from cell culture media, despite the fact that it is a secreted protein. Further, in melanoma cells, Wnt5A appears to be signaling in an autocrine fashion (1, 2). These two observations, together with the fact that Wnt5A undergoes glycosylation (30), led us to hypothesize that HSPGs might be involved in increasing the availability of Wnt5A to its receptor, resulting in an increase in autocrine signaling and ultimately an increase in cellular invasion. In this study, we explore this hypothesis and investigate the role of HSPGs in the Wnt5A signaling cascade in metastatic melanoma cells.     

Heparanase is expressed in osteoblastic cells and stimulates bone formation and bone mass

Heparan sulfate proteoglycans (HSPGs) are ubiquitous macromolecules. In bone, they are associated with cell surfaces and the extracellular matrix (ECM). The heparan sulfate (HS) chains of HSPGs bind a multitude of bioactive molecules, thereby controlling normal and pathologic processes. The HS-degrading endoglycosidase, heparanase, has been implicated in processes such as inflammation, vascularization associated with wound healing and malignancies, and cancer metastasis. Here we show progressive mRNA expression of the hpa gene (encoding heparanase) in murine bone marrow stromal cells undergoing osteoblastic (bone forming) differentiation and in primary calvarial osteoblasts. Bone marrow stromal cells derived from transgenic mice expressing recombinant human heparanase (rh-heparanase) and MC3T3 E1 osteoblastic cells exposed to soluble rh-heparanase spontaneously undergo osteogenic differentiation. In addition, the transgenic bone marrow stromal cells degrade HS chains. In wild-type (WT) and hpa-transgenic (hpa-tg) mice, heparanase is weakly expressed throughout the bone marrow with a substantial increase in osteoblasts and osteocytes, especially in the hpa-tg mice. Heparanase expression was absent in osteoclasts. Micro-computed tomographic and histomorphometric skeletal analyses in male and female hpa-tg versus WT mice show markedly increased trabecular bone mass, cortical thickness, and bone formation rate, but no difference in osteoclast number. Collectively, our data suggest that proteoglycans tonically suppress osteoblast function and that this inhibition is alleviated by HS degradation with heparanase.     

Purification and Characterization of a Proteinase Inhibitor from Field Bean, Dolichos lablab perpureus L.

A proteinase inhibitor resembling Bowman-Birk family inhibitors has been purified from the seeds of cultivar HA-3 of Dolichos lablab perpureus L. The protein was apparently homogeneous as judged by SDS–PAGE, PAGE, IEF, and immunodiffusion. The inhibitor had 12 mole% 1/2-cystine and a few aromatic amino acids, and lacks tryptophan. Field bean proteinase inhibitor (FBPI) exhibited a pI of 4.3 and an M _r of 18,500 Da. CD spectral studies showed random coiled secondary structure. Conformational changes were detected in the FBPI–trypsin/chymotrypsin complexes by difference spectral studies. Apparent K _a values of complexes of inhibitor with trypsin and chymotrypsin were 2.1 × 10⁷ M^?1 and 3.1 × 10⁷ M^?1, respectively. The binary and ternary complexes of FBPI with trypsin and chymotrypsin have been isolated indicating 1:1 stoichiometry with independent sites for cognate enzymes. Amino acid modification studies showed lysine and tyrosine at the reactive sites of FBPI for trypsin and chymotrypsin, respectively.     

Synthesis of lactosaminoglycan-containing glycoproteins by vascular endothelial cells

Human vascular endothelial cells synthesize lactosaminoglycan-type glycoproteins which are found both associated with cells and secreted into the culture medium. Pronase-derived glycopeptides prepared from [³H]glucosamine-labeled glycoproteins were found to contain about 10% of the labeled products as a large size (M_r > 5000) ³H-labeled glycopeptide. Digestion of these ³H-labeled glycopeptides with endo-β-galactosidase resulted in the release of smaller size saccharides, which were characterized as having the structure sialic acid → Gal → GlcNAc → Gal. Treatment of [³H]glucosamine-labeled cells with melittin caused ³H-labeled glycoconjugates to be released from the cells. Separation of released glycoproteins from proteoglycans by DEAE-cellulose chromatography indicated that melittin had released 25% of the total ³H-labeled glycoproteins from the cell and 3% of the ³H-labeled proteoglycans. The ³H-labeled glycoproteins were digested with Pronase and the resulting ³H-labeled glycopeptides were fractionated on Sephadex G-50. The large size fraction (M_r > 5000) now comprised about 30% of these released ³H-labeled glycopeptides. These high molecular weight ³H-labeled glycopeptides were degraded with endo-β-galactosidase but not with testicular hyaluronidase. Analysis of the released ³H-labeled glycoproteins indicated a preferential release of glycoproteins of 70–90 kDa enriched in lactosaminoglycan-type oligosaccharides.     

The early synthesis and possible function of a 0.5 X 10(6) Mr RNA after transfer of dark-grown Spirodela plants to light.

A 0.5 × 10⁶$\text{M}$_r RNA found in plastids of the aquatic angiosperm $\text{Spirodela}$, is synthesized at a much higher rate than any other rapidly labeling RNA species about $\text{3–3}\text{1}\text{2}$ h after dark-grown plants are transferred to light. The pulse labeling kinetics of the 0.5 × 10⁶$\text{M}$_r RNA after transfer to light, argue against its involvement in the biogenesis of plant rRNAs. Although poly(A) RNA is found in $\text{Spirodela}$, poly(A) sequences are not detected in the 0.5 × 10⁶$\text{M}$_r RNA; yet a sucrose gradient fraction which includes RNA of this $\text{M}$_r stimulates amino acid incorporation by an $\text{E. coli}$ cell free extract more than other RNA fractions. The possible involvement of the 0.5 × 10⁶$\text{M}$_r RNA as a chloroplast messenger is discussed.     

Modulation of sulfated glycosaminoglycan and small proteoglycan synthesis by the extracellular matrix

Cell culture in collagen lattice is known to be a more physiological model than monolayer for studying the regulation of extracellular matrix protein deposition. The synthesis of sulfated glycosaminoglycans (GAG) and dermatan sulfate (DS) proteoglycans by 3 cell strains were studied in confluent monolayers grown on plastic surface, in comparison to fully retracted collagen lattices. Cells were labelled with³⁵S-sulfate, followed by GAG and proteoglycan analysis by cellulose acetate and SDS-polyacrylamide gel electrophoresis, respectively. The 3 cell strains contracted the lattice in a similar way. In monolayer cultures, the major part of GAG was secreted into culture medium whereas in lattice cultures of dermal fibroblasts and osteosarcoma MG-63 cells but not fibrosarcoma HT-1080 cells, a higher proportion of GAGs, including dermatan sulfate, was retained within the lattices. Small DS proteoglycans, decorin and biglycan, were detected in fibroblasts and MG-63 cultures. They were preferentially trapped within the collagen gel. In retracted lattices, decorin had a higher M_r than in monolayer. Biglycan was detected in monolayer and lattice cultures of MG-63 cells but in lattice cultures only in the case of fibroblasts. In this last case, an up regulation of biglycan mRNA steady state level and down regulation of decorin mRNA was observed, in comparison to monolayers, indicating that collagen can modulate the phenotypical expression of small proteoglycan genes.Supported by a fellowship from the Centre National de la Recherche Scientifique     

Occurrence in vivo of "hidden breaks" at specific sites of 26 S ribosomal RNA of Musca carnaria.

A fragmentation process occurs in 26 S ribosomal RNA of mature cytoplasmic ribosomes of Musca carnaria. It consists of the sequential appearance of three “hidden breaks” that fragment 26 S rRNA (M_r = 1.42 × 10⁶) into four pieces with approximate molecular weights of 0.68 × 10⁶, 0.35 × 10⁶, 0.29 × 10⁶ and 0.096 × 10⁶, respectively. This fragmentation was not observed in 17 S rRNA (M_r = 0.74 × 10⁶).Extremely mild treatment of newly assembled ribosomes with pancreatic RNAase reproduces the 26 S rRNA fragmentation phenomenon in vitro in the same way as it occurs in vivo.This evidence is discussed in relation to the secondary structure of 26 S rRNA and its binding with specific ribosomal proteins.     

Stabilities,solubility, and kinetics and mechanism for formation and hydrolysis of some palladium(II)and platinum(II) iodo complexes in aqueous solution

Complex formation between Pd(II), Pt(II) and iodide has been studied at 25 °C for an aqueous 1.00 M perchloric acid medium. Measurements of the solubility of PdI₂(s) in aqueous mercury(II) perchlorate and of AgI(s) and PdI₂(s) in aqueous solutions of Pd²⁺(aq) and Ag⁺(aq) gave the solubility product of PdI₂(s) as K_s^o=(7±3) × 10⁻³² M³, which is much smaller than previous literature values.The stability constants β₁=[MI(H₂O)₃⁺]/([M(H₂O)₄²⁺][I⁻]) for the two systems were obtained as the ratio between rate constants for the forward and reverse reactions of (i).

The following values of k₁ (s⁻¹ M⁻¹), k₋₁ (s⁻¹) and β₁ (M⁻¹) were obtained at 25 °C: (1.14±0.11) × 10⁶, (0.92±0.18), (12±4) × 10⁵ for MPd, and (7.7±0.4), (8.0±0.7) × 10⁻⁵, (9.6±1.3) × 10⁴ for MPt. Combination with previous literature data gives the following values of log(β₁ (M⁻¹)) to log(β₄ (M⁻⁴)): 6.08, ∼22, 25.8 and 28.3 for MPd, and 4.98, ∼25, ∼28, and ∼30 for MPt. The present results show that the large overall stability constants β₄ observed for the M²⁺I⁻ systems are most likely due to a very large stability of the second complex MI₂(H₂O)₂, which is probably a cis-isomer. A distinct plateau in the formation curve for mean ligand number 2 is obtained both for MPd and Pt. The other iodo complexes are not especially stable compared to those of chloride and bromide.ΔH^≠ (kJ mol⁻¹) and ΔS^≠ (JK⁻¹ mol⁻¹) for the forward reaction of (i), MPd, are (17.3±1.7) and (−71±5), and for the reverse reaction of (i) MPd, (45±3) and (−95±6), respectively. The kinetics are compatible with associative activation (I_a). The contribution from bond-breaking in the formation of the transition state seems to be less important for Pd than for Pt.     

Modulation of synthesis of specific proteins in endothelial cells by copper,cadmium, and disulfiram: An early response to an angiogenic inducer of cell migration

Copper, cadmium, and disulfiram (an ionophore for copper) modulate the synthesis of several polypeptides in two clonal lines of bovine aortal endothelial cells. After treatment of type 1 endothelial cells with 10^?3 M CuSO₄ or 10^?5 M CdCl₂ four cell-associated polypeptides (M_r = 28,000, 32,000, 73,000, and 83,000 daltons) were induced. In contrast, in Type 2 endothelial cells, which have cultural characteristics distinct from Type 1, only one new cell-associated protein (M_r = 32,000 and 40,000 daltons) was induced. Other differences are revealed by analyses of proteins secreted into the growth medium. In particular low levels of only CuSO₄ (10^?6 M) enhanced the synthesis in Type 2 cells of a protein (M_r = 220,000 daltons) identified as fibronectin. Since only copper ions induced fibronectin, we propose that the mechanism of induction of fibronectin synthesis, in contrast to the induction of cell?associated polypeptides, does not involve a sulphydryl?containing receptor molecule. It is suggested that the specific enhancement of fibronectin synthesis by copper ions may be a controlling event in the stimulation by copper ions of endothelial cell migration and angiogenesis.     

p75 neurotrophin receptor functions as a survival receptor in brain-metastatic melanoma cells

The p75 neurotrophin receptor (p75(NTR)), a common receptor for members of the neurotrophins (NT) family, was previously identified as a molecular determinant of brain metastasis. We have also reported that NT treatment of murine and human brain-metastatic melanoma cells affects their invasive capacities and increases the production of heparanase, an important and unique extracellular matrix (ECM) degradative enzyme. Neurotrophism can be a survival-support mechanism for brain-metastatic cells and a survival assay was devised to mimic the growth limiting conditions of rapidly expanding metastatic tumors prior to neoangiogenesis. We report that p75(NTR) promoted the survival of brain-metastatic melanoma cells but not melanocytes in stress cultures conditions. Secondly, melanoma cells fluorescently sorted for high p75(NTR) expression (p75(NTR-H) cells) had an up to a 15-fold greater survival than those sorted for low p75(NTR) expression (p75(NTR-L) cells). Thirdly, cells overexpressing p75(NTR) associated with the growth fraction and provided these cells with an inherent growth advantage. Finally, we observed an increased survival of sorted p75(NTR-L) cells, dependent upon treatment of NT members whose functional receptors are present on these cells. Together, these results delineate that p75(NTR)-mediated trophic support profoundly affects competitive melanoma-cell survival when the tumor cell microenvironment becomes growth limiting.     

Muscle Contraction : (Outline Studies in Biology) by C.R. Bagshaw Chapman & Hall; London,New York, 1982 79 pages. £2.75

On incubation of B. subtilis RM125(arg15 leuA8 r_M^? m_M^?) with DNA from alkalophilic Bacillus, the transformants (Arg⁺Leu^? or Leu^?Arg⁺) appeared at pH 10. The transformants were able to grow even at pH 7. Alkalophilic Bacillus was resistant to bacteriophages π105D1C2·1012 grown on B. subtilis 1012(r^-m_M⁺) and π105D1C2·ISMR4 grown on B. subtilis ISMR4r_M⁺r_R⁺m_M⁺m_R⁺), but the recipient B. subtilis and the transformant(Arg⁺Leu^?) were susceptible to both the of the bacteriophages. The results indicate that the transformant is a B. subtilis derivative and that alkalophilicity of alkalophilic Bacillus was transferred to B. subtilis.