Correcting eggshell indices of raptor eggs for hole size and eccentricity

Eggshell thickness is often expressed by means of an index based on the length, breadth and weight of the shell. The effect of the blow-hole and egg eccentricity on Ratcliffe's shell thickness index was investigated in a sample of 585 eggs from six raptor species. Corrections for the size of the hole and egg eccentricity are proposed, as is a combined formula to correct both sources of error at the same time. It is shown that by using these formulae, considerable improvements in estimates of shell thinning are obtained. These may be especially useful when sample sizes are small, which is often the case when working with species that have been reduced in numbers.     

Anion- and pH-linked conformational transition in horseradish peroxidase

In a previous study we have shown that bringing horseradish peroxidase to pH 3.0 induces a spectroscopic transition (G. Smulevich et al., Biochemistry 36 (1997) 640). We have extended the investigation on this pH-linked conformational change to different experimental conditions, such as (i) in phosphate alone, (ii) in HCl alone and (iii) in phosphate + NaCl. The data obtained allow a number of conclusions to be drawn, namely: (a) the exposure to pH 3.0 under all conditions brings about an alteration of the distal portion of the heme pocket, leading to the rapid formation of a hexa-coordinated species; (b) only in the presence of phosphate is the hexa-coordination followed by a slow cleavage (or severe weakening) of the proximal Fe-His bond, and (c) the rate of this second process is speeded up in the presence of Cl- ions. Such observations underline the presence of a communication pathway between the two opposite sides of the heme pocket, such that any alteration of the structural arrangement on one side of the heme cavity is transmitted to the other, inducing a corresponding conformational change.     

The truncated hemoglobin from Mycobacterium leprae

Truncated hemoglobins (trHb's) form a family of low molecular weight O2 binding hemoproteins distributed in eubacteria, protozoa, and plants. TrHb's branch in a distinct clade within the hemoglobin (Hb) superfamily. A unique globin gene has recently been identified from the complete genome sequence of Mycobacterium leprae that is predicted to encode a trHb (M. leprae trHbO). Sequence comparison and modelling considerations indicate that monomeric M. leprae trHbO has structural features typical of trHb's, such as 20-40 fewer residues than conventional globin chains, Gly-based sequence consensus motifs, likely assembling into a 2-on-2 alpha-helical sandwich fold, and hydrophobic residues recognized to build up the protein matrix ligand diffusion tunnel. The ferrous heme iron atom of deoxygenated M. leprae trHbO appears to be hexacoordinated, like in Arabidopsis thaliana trHbO-3 (A. thaliana trHbO-3). Accordingly, the value of the second-order rate constant for M. leprae trHbO carbonylation (7.3 x 10(3) M(-1) s(-1)) is similar to that observed for A. thaliana trHbO-3 (1.4 x 10(4) M(-1) s(-1)) and turns out to be lower than that reported for carbon monoxide binding to pentacoordinated Mycobacterium tuberculosis trHbN (6.7 x 10(6) M(-1) s(-1)). The lower reactivity of M. leprae trHbO as compared to M. tuberculosis trHbN might be related to the higher susceptibility of the leprosy bacillus to toxic nitrogen and oxygen species produced by phagocytic cells.     

Inhibitors of protein synthesis increase the agglutinability mediated by concanavalin A of the unfertilized mouse oocyte

The agglutinability mediated by concanavalin A has been studied in the zona-free mouse oocyte before and after fertilization or treatment with inhibitors of protein synthesis. At lectin concentrations of 10 μg/ml ot higher the zygote is clearly more agglutinable than the unfertilized oocyte. Treatment with 10 μg/ml of cycloheximide or puromycin, which causes parthenogenetic activation, greatly increases the agglutinability of the unfertilized oocyte; the agglutinability of the zygote and of the early blastocyst is not increased by such treatment. The possibility is discussed that the repression of cell division requires the synthesis of unstable protein molecules of the cell surface which are also involved in lectinmediated agglutinability.     

Structural Bases for the Regulation of CO Binding in the Archaeal Protoglobin from Methanosarcina acetivorans

Studies of CO ligand binding revealed that two protein states with different ligand affinities exist in the protoglobin from Methanosarcina acetivorans (in MaPgb*, residue Cys(E20)101 was mutated to Ser). The switch between the two states occurs upon the ligation of MaPgb*. In this work, site-directed mutagenesis was used to explore the role of selected amino acids in ligand sensing and stabilization and in affecting the equilibrium between the “more reactive” and “less reactive” conformational states of MaPgb*. A combination of experimental data obtained from electronic and resonance Raman absorption spectra, CO ligand-binding kinetics, and X-ray crystallography was employed. Three amino acids were assigned a critical role: Trp(60)B9, Tyr(61)B10, and Phe(93)E11. Trp(60)B9 and Tyr(61)B10 are involved in ligand stabilization in the distal heme pocket; the strength of their interaction was reflected by the spectra of the CO-ligated MaPgb* and by the CO dissociation rate constants. In contrast, Phe(93)E11 is a key player in sensing the heme-bound ligand and promotes the rotation of the Trp(60)B9 side chain, thus favoring ligand stabilization. Although the structural bases of the fast CO binding rate constant of MaPgb* are still unclear, Trp(60)B9, Tyr(61)B10, and Phe(93)E11 play a role in regulating heme/ligand affinity.     

Agrin and Perlecan Mediate Tumorigenic Processes in Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma is the most common type of cancer in the oral cavity, representing more than 90% of all oral cancers. The characterization of altered molecules in oral cancer is essential to understand molecular mechanisms underlying tumor progression as well as to contribute to cancer biomarker and therapeutic target discovery. Proteoglycans are key molecular effectors of cell surface and pericellular microenvironments, performing multiple functions in cancer. Two of the major basement membrane proteoglycans, agrin and perlecan, were investigated in this study regarding their role in oral cancer. Using real time quantitative PCR (qRT-PCR), we showed that agrin and perlecan are highly expressed in oral squamous cell carcinoma. Interestingly, cell lines originated from distinct sites showed different expression of agrin and perlecan. Enzymatically targeting chondroitin sulfate modification by chondroitinase, oral squamous carcinoma cell line had a reduced ability to adhere to extracellular matrix proteins and increased sensibility to cisplatin. Additionally, knockdown of agrin and perlecan promoted a decrease on cell migration and adhesion, and on resistance of cells to cisplatin. Our study showed, for the first time, a negative regulation on oral cancer-associated events by either targeting chondroitin sulfate content or agrin and perlecan levels.     

Non-covalent and covalent modifications modulate the reactivity of monomeric mammalian globins

Multimeric globins (e.g., hemoglobin) are considered to be the prototypes of allosteric enzymes, whereas monomeric globins (e.g., myoglobin; Mb) usually are assumed to be non-allosteric. However, the modulation of the functional properties of monomeric globins by non-covalent (or allosteric) and covalent modifications casts doubts on this general assumption. Here, we report examples referable to these two extreme mechanisms modulating the reactivity of three mammalian monomeric globins. Sperm whale Mb, which acts as a reserve supply of O₂ and facilitates the O₂ flux within a myocyte, displays the allosteric modulation of the O₂ affinity on lactate, an obligatory product of glycolysis under anaerobic conditions, thus facilitating O₂ diffusion to the mitochondria in supporting oxidative phosphorylation. Human neuroglobin (NGB), which appears to protect neurons from hypoxia in vitro and in vivo, undergoes hypoxia-dependent phosphorylation (i.e., covalent modulation) affecting the coordination equilibrium of the heme-Fe atom and, in turn, the heme-protein reactivity. This facilitates heme-Fe-ligand binding and enhances the rate of anaerobic nitrite reduction to form NO, thus contributing to cellular adaptation to hypoxia. The reactivity of human cytoglobin (CYGB), which has been postulated to protect cells against oxidative stress, depends on both non-covalent and covalent mechanisms. In fact, the heme reactivity of CYGB depends on the lipid, such as oleate, binding which stabilizes the penta-coordination geometry of the heme-Fe atom. Lastly, the reactivity of NGB and CYGB is modulated by the redox state of the intramolecular CysCD7/CysD5 and CysB2/CysE9 residue pairs, respectively, affecting the heme-Fe atom coordination state. In conclusion, the modulation of monomeric globins reactivity by non-covalent and covalent modifications appears a very widespread phenomenon, opening new perspectives in cell survival and protection. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

Immunomodulatory and Antibacterial Effects of Cystatin 9 against Francisella tularensis

Cystatin 9 (CST9) is a member of the type 2 cysteine protease inhibitor family, which has been shown to have immunomodulatory effects that restrain inflammation, but its functions against bacterial infections are unknown. Here, we report that purified human recombinant (r)CST9 protects against the deadly bacterium Francisella tularensis (Ft) in vitro and in vivo. Macrophages infected with the Ft human pathogen Schu 4 (S4), then given 50 pg of rCST9 exhibited significantly decreased intracellular bacterial replication and increased killing via preventing the escape of S4 from the phagosome. Further, rCST9 induced autophagy in macrophages via the regulation of the mammalian target of rapamycin (mTOR) signaling pathways. rCST9 promoted the upregulation of macrophage proteins involved in antiinflammation and antiapoptosis, while restraining proinflammatory-associated proteins. Interestingly, the viability and virulence of S4 also was decreased directly by rCST9. In a mouse model of Ft inhalation, rCST9 significantly decreased organ bacterial burden and improved survival, which was not accompanied by excessive cytokine secretion or subsequent immune cell migration. The current report is the first to show the immunomodulatory and antimicrobial functions of rCST9 against Ft. We hypothesize that the attenuation of inflammation by rCST9 may be exploited for therapeutic purposes during infection.     

Ligand- and proton-linked conformational changes of the ferrous 2/2 hemoglobin of Pseudoalteromonas haloplanktis TAC125

The spectroscopic and ligand-binding properties of a 2/2 globin from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 have been studied in the ferrous state. It displays two major conformations characterized by CO-association rates that differ by a factor of 20, with relative fractions that depend on pH. A dynamic equilibrium is found between the two conformations, as indicated by an enhanced slower phase when lower CO levels were used to allow a longer time to facilitate the transition. The deoxy form, in the absence of external ligands, is a mixture of a predominant six-coordinate low spin form and a five-coordinate high-spin state; the proportion of low spin increasing at alkaline pH. In addition, at temperatures above the physiological temperature of 1 °C, an enhanced tendency of the protein to oxidize is observed.