Regulated production of recombinant echistatin by yeast

Summary The -mating factor pre-pro-leader sequence under the regulation of theGAL10 promoter was used to direct the secretion of echistatin by recombinant yeasts. Optimization of the culture medium and host strain increased the productivity of shake flask cultures twenty-fold to 8 mg/L. In fermentors, the production of echistatin was greater than 40 mg/L.     

Interferon-γ inhibits interleukin-1β-induced matrix metalloproteinase production by synovial fibroblasts and protects articular cartilage in early arthritis

Introduction  

The first few months after symptom onset represents a pathologically distinct phase in rheumatoid arthritis (RA). We used relevant experimental models to define the pathological role of interferon-γ (IFN-γ) during early inflammatory arthritis.     

The many faces of ubiquitinated histone H2A: insights from the DUBs

Increasing knowledge on the cell cycle deregulations in cancers has promoted the introduction of phytochemicals, which can either modulate signaling pathways leading to cell cycle regulation or directly alter cell cycle regulatory molecules, in cancer therapy. Most human malignancies are driven by chromosomal translocations or other genetic alterations that directly affect the function of critical cell cycle proteins such as cyclins as well as tumor suppressors, e.g., p53. In this respect, cell cycle regulation and its modulation by curcumin are gaining widespread attention in recent years. Extensive research has addressed the chemotherapeutic potential of curcumin (diferuloylmethane), a relatively non-toxic plant derived polyphenol. The mechanisms implicated are diverse and appear to involve a combination of cell signaling pathways at multiple levels. In the present review we discuss how alterations in the cell cycle control contribute to the malignant transformation and provide an overview of how curcumin targets cell cycle regulatory molecules to assert anti-proliferative and/or apoptotic effects in cancer cells. The purpose of the current article is to present an appraisal of the current level of knowledge regarding the potential of curcumin as an agent for the chemoprevention of cancer via an understanding of its mechanism of action at the level of cell cycle regulation. Taken together, this review seeks to summarize the unique properties of curcumin that may be exploited for successful clinical cancer prevention.     

Purification and mutagenesis of LpxL, the lauroyltransferase of Escherichia coli lipid A biosynthesis

Escherichia coli lipid A is a hexaacylated disaccharide of glucosamine with secondary laurate and myristate chains on the distal unit. Hexaacylated lipid A is a potent agonist of human Toll-like receptor 4, whereas its tetra- and pentaacylated precursors are antagonists. The inner membrane enzyme LpxL transfers laurate from lauroyl-acyl carrier protein to the 2'- R-3-hydroxymyristate moiety of the tetraacylated lipid A precursor Kdo 2-lipid IV A. LpxL has now been overexpressed, solubilized with n-dodecyl beta- d-maltopyranoside (DDM), and purified to homogeneity. LpxL migration on a gel filtration column is consistent with a molecular mass of 80 kDa, suggestive of an LpxL monomer (36 kDa) embedded in a DDM micelle. Mass spectrometry showed that deformylated LpxL was the predominant species, noncovalently bound to as many as 12 DDM molecules. Purified LpxL catalyzed not only the formation in vitro of Kdo 2-(lauroyl)-lipid IV A but also a slow second acylation, generating Kdo 2-(dilauroyl)-lipid IV A. Consistent with the Kdo dependence of crude LpxL in membranes, Kdo 2-lipid IV A is preferred 6000-fold over lipid IV A by the pure enzyme. Sequence comparisons suggest that LpxL shares distant homology with the glycerol-3-phosphate acyltransferase (GPAT) family, including a putative catalytic dyad located in a conserved H(X) 4D/E motif. Mutation of H132 or E137 to alanine reduces specific activity by over 3 orders of magnitude. Like many GPATs, LpxL can also utilize acyl-CoA as an alternative acyl donor, albeit at a slower rate. Our results show that the acyltransferases that generate the secondary acyl chains of lipid A are members of the GPAT family and set the stage for structural studies.     

The morphological and spectral phenotype of apoptosis in HeLa cells varies following exposure to UV-C and the addition of inhibitors of ICE and CPP32

Numerous extra- and intracellular factors, including UV radiation, can initiate a programme of cell death by apoptosis. While apoptosis is commonly defined morphologically, the relationships between morphology and molecular events are not well established. To investigate these relationships in HeLa cells, eight morphometric criteria for cell proliferation and damage and 10 criteria for apoptotic phenotype were examined using light microscopy, and corroborated by ultrastructure and spectral imaging. They were identified (1) during a time course after irradiation with 0, 10 or 30 J/m² UV-C; (2) after separation of apoptotic from normal cells on a Percoll gradient; and (3) after irradiation with UV-C plus perturbation of the apoptotic pathway by treatment with inhibitors of two caspases, ICE and CPP32. The number of cells in apoptosis increased in a dose-dependent manner after UV-C treatment. Centrifugation of irradiated cells on a Percoll gradient increased the collection of apoptotic cells tenfold. The stereotypical apoptotic phenotype, in which cells have deep cytoplasmic blebbing and highly condensed DNA, comprised only a few percent of all apoptoses, and was rarely seen in groups receiving caspase inhibitors. The most common apoptotic phenotype was a rounded cell with large spherical nucleolus and associated DNA. After treatment with UV-C plus inhibitors the apoptotic index was decreased by about 30% compared to UV-C radiation alone. These apoptotic cells had dark spherical cytoplasm with small blebs, greatly increased numbers of cytoplasmic ribosomes, abundant nucleolar material with a large separate granular component, and chromatin condensed at the nuclear membrane. Using the technique of spectral imaging, it was found that the spectrum obtained from the granular component of the nucleolus, which was elevated in apoptotic cells treated with UV-C plus inhibitors, was similar to the dense accumulation of ribosomes in the apoptotic cytoplasm. The data indicate that spectral imaging may be a useful tool for identifying and characterizing variations in the apoptotic process, and that the caspase inhibitors used here do not completely abolish UV-C induced apoptosis, but rather alter its incidence and progression.     

Conformation of the metastasis-inhibiting laminin pentapeptide

The binding of cancer cells to the basement membrane glycoprotein laminin appears to be a critical step in the metastatic process. This binding can be inhibited competitively by a specific pentapeptide sequence (Tyr-Ile-Gly-Ser-Arg) of the laminin B1 chain, and this peptide can prevent metastasis formationin vivo. However, other similar pentapeptide sequences (e.g., Tyr-Ile-Gly-Ser-Glu) have been found to be much less active in metastasis inhibition, raising the possibility that such amino acid substitutions produce structural changes responsible for altering binding to the laminin receptor. In this study, conformational energy analysis has been used to determine the three-dimensional structures of these peptides. The results indicate that the substitution of Glu for the terminal Arg produces a significant conformational change in the peptide backbone at the middle Gly residue. These results have important implications for the design of drugs that may be useful in preventing metastasis formation and tumor spread.     

Synthesis and biological activity of piperazine-based dual MMP-13 and TNF-alpha converting enzyme inhibitors

A series of novel MMP-13 and TNF-alpha converting enzyme inhibitors based on piperazine 2-hydroxamic acid scaffolds are described. The TACE, MMP-1 and MMP-13 activity of these inhibitors as well as the effect of substitution of the piperazine nitrogen and the P-1' benzyloxy tailpiece is discussed. Moderate in vivo activity is observed with several members of this group.