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Considerable interest is currently focused on fish haemoglobins in order to identify the structural basis for their diversity of functional behaviour. The armored catfish Lip osarcus anisitsi presents accessorial air breathing through a modified stomach, which allows this species to survive in waters with low oxygen content. The analysis of its haemolysate has shown the presence of four main haemoglobins, with this work focusing on haemoglobin IV (LaHb-IV). LaHb-IV was crystallized and X-ray diffraction data were collected to 2.4 Å resolution using a synchrotron-radiation source. The crystal belongs to the orthorhombic space group P212121, with unit-cell parameters a = 52.6 (1), b = 104.8 (2), c =113.9 (2) Å; preliminary structural analysis revealed the presence of one tetramer in the asymmetric unit. The structure was determined using the standard molecular-replacement technique. [ABSTRACT FROM AUTHOR]

Haemoglobin, the `honorary enzyme' [Brunori (1999), Trends Biochem. Sci. 24, 158-161], constitutes a prime prototype for allosteric models. Here, the crystallization and preliminary X-ray analysis of haemoglo bin I from the South American fish Brycon cephalus are reported. X-ray diffraction data have been collected to 2.5 Å resolution using synchrotron radiation (LNLS). Crystals were determined to belong to the space group P6122 and preliminary structural analysis revealed the presence of one dimer (αβ) in the asymmetric unit. The structure was determined using standard molecular-replacement techniques. [ABSTRACT FROM AUTHOR]

Liposarcus anisitsi is an armoured catfish that presents accessorial air oxygenation through a modified stomach, which allows this species to survive in waters with very low oxygen content. Analysis of its haemolysate has shown the presence of four haemoglobins; this work focuses on the main component, haemoglobin I. It has been crystallized in two different forms and X-ray diffraction data have been collected to 2.77 and 2.86 Å resolution using synchrotron radiation. Crystals were determined to belong to the space groups C2 and P21 and preliminary structural analysis revealed the presence of one tetramer in the asymmetric unit in both crystal forms. The structure was determined using a standard molecular-replacement technique. [ABSTRACT FROM AUTHOR]

Sugarcane is an important crop that has recently become subject to attacks from the weevil Sphenophorus levis, which is not efficiently controlled with chemical insecticides. This demands the development of new control devices for which digestive physiology data are needed. In the present study, ion-exchange chromatography of S. levis whole midgut homogenates, together with enzyme assays with natural and synthetic substrates and specific inhibitors, demonstrated that a cysteine proteinase is a major proteinase, trypsin is a minor one and chymotrypsin is probably negligible. Amylase, maltase and the cysteine proteinase occur in the gut contents and decrease throughout the midgut; trypsin is constant in the entire midgut, whereas a membrane-bound aminopeptidase predominates in the posterior midgut. The cysteine proteinase was purified to homogeneity through ion-exchange chromatography. The purified enzyme had a mass of 37 kDa and was able to hydrolyze Z-Phe-Arg-MCA and Z-Leu-Arg-MCA with k(cat)/K(m) values of 20.0±1.1 μM(-1)s(-1) and 30.0±0.5 μM(-1)s(-1), respectively, but not Z-Arg-Arg-MCA. The combined results suggest that protein digestion starts in the anterior midgut under the action of a cathepsin L-like proteinase and ends on the surface of posterior midgut cells. All starch digestion takes place in anterior midgut. These data will be instrumental to developing S. levis-resistant sugarcane., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: Cystatins are inhibitors of cysteine proteases. The majority are only weak inhibitors of human cathepsin B, which has been associated with cancer, Alzheimer's disease and arthritis., Results: Starting from the sequences of oryzacystatin-1 and canecystatin-1, a shuffling library was designed and a hybrid clone obtained, which presented higher inhibitory activity towards cathepsin B. This clone presented two unanticipated point mutations as well as an N-terminal deletion. Reversing each point mutation independently or both simultaneously abolishes the inhibitory activity towards cathepsin B. Homology modeling together with experimental studies of the reverse mutants revealed the likely molecular determinants of the improved inhibitory activity to be related to decreased protein stability., Conclusion: A combination of experimental approaches including gene shuffling, enzyme assays and reverse mutation allied to molecular modeling has shed light upon the unexpected inhibitory properties of certain cystatin mutants against Cathepsin B. We conclude that mutations disrupting the hydrophobic core of phytocystatins increase the flexibility of the N-terminus, leading to an increase in inhibitory activity. Such mutations need not affect the inhibitory site directly but may be observed distant from it and manifest their effects via an uncoupling of its three components as a result of increased protein flexibility.

Using cDNA microarrays with 3800 cDNA fragments, we determined the expression profile of normal thyroid tissue, goiter, adenoma and papillary carcinoma (10 samples from each class). After background correction and statistical analysis, we identified a set of 160 genes as being differentially expressed in all pair-wise comparisons. Here we demonstrate that, at least on the basis of these differentially expressed genes, a positive correlation between goiter and papillary carcinomas could be observed. We identified a common set of genes whose expression is diminished in both goiter and papillary carcinomas as compared to normal thyroid tissue. Moreover, no genes with inverse correlation in samples from goiter and papillary carcinomas could be detected. Using Real-Time PCR and/or tissue microarrays, we confirmed the altered expression of some of the identified genes. Of notice, we demonstrate that the reduced mRNA levels of p27(kip1) observed in papillary carcinomas as compared to either goiter or normal thyroid tissues (P<0.001) is accompanied by an altered protein distribution within the cell. In papillary carcinomas, P27(KIP1) is preferentially cytoplasmic as opposed to goiter or normal thyroid tissue, where P27(KIP1) is preferentially located in the nucleus. The exploitation of the data presented here could contribute to the understanding of the molecular events related to thyroid diseases and gives support to the notion that common molecular events might be related to the frequent observation of areas of papillary carcinomas in the gland of patients with goiter.

Whereas genome sequencing defines the genetic potential of an organism, transcript sequencing defines the utilization of this potential and links the genome with most areas of biology. To exploit the information within the human genome in the fight against cancer, we have deposited some two million expressed sequence tags (ESTs) from human tumors and their corresponding normal tissues in the public databases. The data currently define approximately 23,500 genes, of which only approximately 1,250 are still represented only by ESTs. Examination of the EST coverage of known cancer-related (CR) genes reveals that <1% do not have corresponding ESTs, indicating that the representation of genes associated with commonly studied tumors is high. The careful recording of the origin of all ESTs we have produced has enabled detailed definition of where the genes they represent are expressed in the human body. More than 100,000 ESTs are available for seven tissues, indicating a surprising variability of gene usage that has led to the discovery of a significant number of genes with restricted expression, and that may thus be therapeutically useful. The ESTs also reveal novel nonsynonymous germline variants (although the one-pass nature of the data necessitates careful validation) and many alternatively spliced transcripts. Although widely exploited by the scientific community, vindicating our totally open source policy, the EST data generated still provide extensive information that remains to be systematically explored, and that may further facilitate progress toward both the understanding and treatment of human cancers.

Carboxyhaemoglobin-II isolated from the pacu (Piaractus mesopotamicus) has been crystallized and X-ray diffraction data were collected to 2.0 A resolution using synchrotron radiation. Crystals were characterized as belonging to the space group I23; preliminary structural analysis reveals the presence of one dimer in the asymmetric unit.