Collagen triple helix repeat containing 1 promotes melanoma cell adhesion and survival.

BACKGROUND: The extracellular protein collagen triple helix repeat containing 1 (CTHRC1) is aberrantly upregulated in melanoma and most human solid cancers. However, its role in cancer remains unknown. OBJECTIVE: In this study, we investigated the functional impact of CTHRC1 on melanoma cells in vitro. METHODS: Stable clones of cultured melanoma cells expressing different amounts of CTHRC1 protein were generated and evaluated to characterize their growth, survival, and attachment ability as well as their sensitivity to chemotherapy. RESULTS: In cultured MMAN and MMRU melanoma cells, increased expression of CTHRC1 protein resulted in morphologic cell changes, enhanced cell adhesion to culture surfaces, increased cell proliferation, and decreased apoptosis. Furthermore, decreased CTHRC1 expression through antisense inhibition enhanced temozolomide sensitivity. CONCLUSION: CTHRC1 expression influences cellular processes, including cell adhesion and survival. Additionally, CTHRC1 inhibition may represent a potential method for decreasing melanoma resistance to conventional chemotherapy.

Antimicrobial activity of lipophilic avian eggshell surface extracts.

The avian eggshell cuticle is the waxy outermost layer of the mineralized eggshell in direct contact with the environment. In this study, lipophilic eggshell surface extracts from three domestic species were evaluated for their antimicrobial activity. Chicken and goose extracts demonstrated potent bactericidal activity against both Gram-positive and Gram-negative bacteria, while activity could not be detected for duck eggshell surface extracts. Using the chicken as a model species, evaluation of albumen, fecal material, and uropygial gland extracts eliminated these as a potential source of the observed activity. Results suggest that lipophilic components are incorporated into the egg during its formation and play a role in antimicrobial defense. This study represents the first successful extraction and evaluation of lipophilic antimicrobial components from the avian egg.

The US Orphan Drug Act: rare disease research stimulator or commercial opportunity?

OBJECTIVES: This study investigates issues associated with the United States Orphan Drug Act. METHODS: A comprehensive orphan drug database was compiled from FDA data and corporate annual reports of major pharmaceutical companies. Analysis allowed the generation of a descriptive orphan drug portrait as well as documentation of orphan drugs along their lifecycle. RESULTS: Currently, 2002 products have obtained orphan drug designation with 352 drugs obtaining FDA approval. Approximately 33% of orphan drugs are oncology products. On average, products obtain 1.7 orphan designations with approximately 70% obtaining a single designation. At least 9% of orphan drugs have reached blockbuster status with two-thirds having two or more designations. An additional 25 orphan drugs had sales exceeding US$ 100 million in 2008 alone. Since 1983, at least 14 previously discontinued products have been recycled as orphan drugs. CONCLUSIONS: The United States Orphan Drug Act has created issues which, in some cases, have led to commercial and ethical abuses. Orphan Drug Act reform is necessary but current incentives, including 7 year market exclusivity, should be maintained in order to favour patients as well as economic prosperity. Suggested reforms include price regulation, subsidy paybacks for profitable drugs and the establishment of an International Orphan Drug Office.

Antimicrobial effects of H4-(86-100), histogranin and related compounds--possible involvement of DNA gyrase.

Histone-derived antimicrobial peptides have been identified in various organisms from plants to humans. The rat histone H4 mRNA variants, H4-v.1 and rat histogranin (HNr) mRNAs, were recently reported to be involved in the synthesis of H4-(86-100) and its related peptide HNr, respectively. Herein, the two peptides were investigated for putative antimicrobial activity and found to inhibit growth of gram-negative (Escherichia coli, Pseudomonas aeruginosa) and gram-positive (Bacillus subtilis, Staphylococcus aureus) bacteria. Their inhibitory potencies in E. coli (LD(50): 3.48 and 4.34 microg x mL(-1)) are comparable to that of the antimicrobial peptide LL-37 (LD(50): 4.10 microg x mL(-1)). The antimicrobial activities of H4-(86-100) and HNr depend upon the integrity of the molecules, as precursors [H4-(84-102), pro-HNr] and fragments [bovine histogranin (HNb)-(1-13), HNb-(3-13), H4-(89-102) or OGP] are at least five times less potent than the parent peptides. Among various HN-like compounds, cyclo-(-Gly-pCl-Phe-Tyr-D-Arg) (compound 3) and N-5-guanidino pentanamide-(2R)-yl-2-N-(p-hydroxyphenylacetyl)-4-(p-chlorobenzoyl)-phenylene diamine (compound 8) display antimicrobial activities comparable to that of HNr. Interestingly, the antimicrobial activities of H4-(86-100), HNr and compound 3, like those of quinolone antibiotics acting as DNA gyrase poisons, are potentiated by ATP (1 mM) and coumermycin A1 (a DNA gyrase-linked ATPase inhibitor) and blocked by 2,4-dinitrophenol (DNP, an uncoupler of oxidative phosphorylation) and fluoroacetic acid (a metabolic poison). Finally, in vitro experiments indicate that H4-(86-100), HNr, compound 3 and compound 8, but not HNb-(1-13) or HNb-(3-13), inhibit DNA gyrase-mediated supercoiling of pBR322 DNA. These data indicate that the naturally occurring H4-(86-100) and HNr display antimicrobial effects that involve a modulation of ATP-dependent DNA gyrase.

Antimicrobial properties of avian eggshell-specific C-type lectin-like proteins.

C-type lectin-like proteins are major components of the calcified eggshell of multiple avian species. In this study, two representative avian C-type lectin-like proteins, ovocleidin-17 and ansocalcin, were purified from decalcified chicken and goose eggshell protein extracts and investigated for carbohydrate binding activity as well as antimicrobial activity. Purified ovocleidin-17 and ansocalcin were found to bind bacterial polysaccharides, and were bactericidal against Bacillus subtilis, Staphylococcus aureus and Pseudomona aeruginosa. Bactericidal activity was found to be enhanced in the presence of calcium but was not dependent on its presence. The results suggest that avian C-type lectin-like proteins may play an important antimicrobial role in defence of the avian embryo.

Antimicrobial activity of the Anseriform outer eggshell and cuticle.

The avian eggshell is a complex, multifunctional biomineral composed of a calcium carbonate mineral phase and an organic phase of lipids and proteins. The outermost layer of the eggshell, the eggshell cuticle, is an organic layer of variable thickness composed of polysaccharides, hydroxyapatite crystals, lipids and glycoprotein. In addition to regulating gas exchanges, the eggshell cuticle may contain antimicrobial elements. In this study, we investigated the antimicrobial activity of eggshell cuticle and outer eggshell protein extracts from four Anseriform species: wood duck (Aix sponsa), hooded merganser (Lophodytes cucullatus), Canada goose (Branta canadensis) and mute swan (Cygnus olor). Cuticle and outer eggshell protein was extracted by urea or HCl treatment of eggs. C-type lysozyme, ovotransferrin and an ovocalyxin-32-like protein were detected in all extracts. Cuticle and outer eggshell protein extracts inhibited the growth of Staphylococcus aureus, Escherichia coli D31, Pseudomonas aeruginosa and Bacillus subtilis. The presence of active antimicrobial proteins within the avian cuticle and outer eggshell suggests a role in antimicrobial defense. Protein extracts from the cavity nesting hooded merganser were especially potent. The unique environmental pressures exerted on cavity-nesting species may have led to the evolution of potent antimicrobial defenses.

Enhanced C-type lysozyme content of wood duck (Aix sponsa) egg white: an adaptation to cavity nesting?

Abstract Wild waterfowl species often nest in conditions where high humidity and microbial contamination may influence egg survival and quality. Albumen is traditionally regarded as the major impediment to microbial contamination of eggs, and its composition and activity may be selected by environmental pressures. Egg white protein from the eggs of wood duck (Aix sponsa), hooded merganser (Lophodytes cucullatus), Canada goose (Branta canadensis), and mute swan (Cygnus olor) was evaluated in order to compare the antimicrobial defenses of these species. Ovotransferrin and ovalbumin were identified in all species, but c-type lysozyme was present only in wood duck and hooded merganser egg white samples. Wood duck egg white showed the greatest bacterial activity as well as the highest lysozyme content. Egg white from wood duck and hooded merganser possessed greater lysozyme activity under acidic conditions, suggesting a c-type lysozyme with a pH optimum lower than that of Gallus gallus c-type lysozyme or the presence of g-type lysozyme. Ovotransferrin bacteriostatic activity appeared to be similar across the species investigated. The results suggest that lysozyme and ovotransferrin play a role in the antimicrobial defense of the avian egg. High levels of the broad-acting c-type lysozyme appear to have evolved in the albumen of the wood duck in order to ensure proper development of the embryo in the humid conditions of the cavity nest.

Recombinant eggshell ovocalyxin-32: expression, purification and biological activity of the glutathione S-transferase fusion protein.

The avian eggshell is a highly ordered biomineral composed mainly of calcium carbonate associated with an organic matrix composed of proteins, glycoproteins and proteoglycans. This structure provides the developing embryo with protection from physical damage and microbial invasion. Ovocalyxin-32 (OCX-32) is a 32 kDa eggshell-specific matrix protein which has been cloned and demonstrates 30% identity with the mammalian carboxypeptidase inhibitor, latexin. In order to further study its function, recombinant OCX-32 protein was expressed in E. coli. The protein was extracted from inclusion bodies and purified by sequential DEAE Sepharose and Ni2+ metal ion affinity chromatographies as a 58 kDa GST-fusion protein. The refolded GST-OCX-32 significantly inhibited bovine carboxypeptidase and also inhibited the growth of Bacillus subtilis. The results suggest that OCX-32 may show similar activity to the fusion protein and reinforce the antimicrobial properties of the eggshell by providing protection to the developing avian embryo. OCX-32 is the first example of an eggshell specific protein to be successfully cloned and expressed in a prokaryotic system. The association of an antimicrobial protease inhibitor with the outer eggshell and cuticle of the table egg may enhance the food safety of this product.