Cobalt-substituted derivatives ofCarcinus hemocyanin

Summary Four derivatives ofCarcinus maenas hemocyanin containing Co(II) in the active site have been prepared under different experimental conditions. Two of them contain one Co(II) ion/active site and most probably represent isomeric forms containing Co(II) either in the fast-reacting or in the slow-reacting position within the active site. A third derivative contains two Co(II) ions active site, which reproduces the metal/protein stoichiometry of native hemocyanin. The fourth derivative is a metal hybrid form containing one Cu(I) ion and one Co(II) ion/active site. The derivatives have been characterized by absorption, circular dichroic and fluorescence spectroscopies. The results indicate that in all derivatives the metal is bound with a low coordination number, in agreement with the presence of three histidine residues/copper ion in the native protein. The two alternative metal-binding positions have different structures as shown by the different spectroscopic properties of the bound Co(II) ions. A marked hyperchromic effect on the optical absorption of Co(II) is observed as a result of the presence of a metal ion in the neighbouring metal-binding position in the active site.     

Spectroscopic studies on Neurospora copper metallothionein

The spectral properties of Neurospora copper metallothionein were investigated and compared with those of the Cu(I)-2-mercaptoethanesulfonic acid complex. In both cases, the absorption spectra are rather similar, showing a characteristic shoulder at approximately 250 nm. However, marked differences were observed in their emissive properties. Thus, only metallothionein emits detectable luminescence in solution, but both the copper protein and the Cu(I) complex are luminescent at 77 K. The circular dichroism spectrum of Neurospora copper metallothionein shows several Cotton extrema attributable to asymmetry in metal coordination. The influence of HgCl2 and p-(chloromercuri)benzoate on the spectral properties of metallothionein was also investigated. The two mercurials exerted a pronounced effect on the electronic absorption, chiroptical, and emissive properties of the protein. Spectroscopic titrations followed by gel filtration experiments indicate that two mercurials can be bound per metallothionein molecule without loss of copper. This binding is responsible for the disappearance of the emissive properties of metallothionein and for the distinct changes in its electronic absorption and circular dichroism spectra. From these data, it is suggested that the Cu(I) ions are coordinated to the cysteinyl residues in the form of a single metal cluster.     

The aromatic circular dichroism spectrum as a probe for conformational changes in the active site environment of hemocyanins.

The binding of various ligand molecules to the binuclear Cu(I) site of deoxy-hemocyanin has been investigated through the changes produced in the aromatic region of the circular dichroism spectrum of the protein, where a cluster of tryptophan residues located in the vicinity of copper site undergo conformational reorientations in the presence of exogenous ligands coordinated to the metal. In agreement with expectations, the binuclear site of arthropod hemocyanin is severely hindered to the access of exogenous ligands except for very small molecules like CO, O2 or CN- while for mollusc proteins ligands such as thiourea and 2-mercaptoethanol bind easily to the Cu(I) sites. However, the access of the ligand becomes progressively hindered and eventually prevented as the size of substituents on the ligand increases.     

An oxygenation-sensitive dye binding to Carcinus maenas hemocyanin

The interaction of 4',6-diamidino-2-phenylindole (DAPI) with Carcinus maenas hemocyanin has been investigated by steady state fluorescence, dynamic fluorescence and circular dichroism measurements. The dye binds to apohemocyanin (without copper) as well as to oxygenated hemocyanin and to deoxygenated hemocyanin with very similar affinities (kd approximately equal to 1 microM ) and number of binding sites (one per subunit). In contrast, the fluorescence quantum yield enhancement of DAPI bound to oxygenated hemocyanin is nearly 60% lower than that observed for deoxygenated and apo forms. The decrease of fluorescence of the dye bound to deoxygenated hemocyanin is a sigmoidal function of the oxygen partial pressure, specular to that observed by following the absorbance of the copper-oxygen charge transfer band at 340 nm. This result provides preliminary evidence that DAPI may be used as a functional probe to monitor the cooperative binding of oxygen to the protein. The higher fluorescence quantum yield of DAPI bound to either apohemocyanin or deoxygenated protein is characterized by a single fluorescence decay with lifetime of about 3 ns, while with the oxygenated protein two components of about 1 ns and 3.0 ns are observed. This result is interpreted assuming the existence of two rotamers of DAPI in solution (Szabo et al. Photochem. Photobiol. 44 (1986) 143-150) both able to interact with oxygenated hemocyanin but only one to deoxygenated and apo forms. We conclude that the different fluorescence behaviour of the dye induced by the presence of oxygen bound to the protein is probably due to a structural change of hemocyanin in cooperative interaction with oxygen. Furthermore, the interaction is confirmed by the induced negative ellipticity of DAPI bound to apohemocyanin and deoxy-hemocyanin and by the increase of fluorescence anisotropy of DAPI bound to all forms of protein investigated.     

Low temperature optical spectroscopy of cobalt-substituted hemocyanin from Carcinus maenas

In this work we report the optical absorption spectra of three cobalt-substituted derivatives of hemocyanin (He) from Carcinus maenas, in the temperature range 300–20 K. The derivatives studied are the mononuclear (Co²⁺)-He with a single cobalt ion in the Cu_A site, the binuclear (Co²⁺)₂-He and the binuclear mixed metal (Co²⁺-Cu¹⁺)-He. At low temperature three main bands are clearly resolved; the temperature dependence of their zeroth, first and second moments sheds light on the stereodynamic properties in the surroundings of the chromophore. Within the limits of the reported analysis, in the binuclear derivatives the motions coupled to the chromophore appear to be essentially harmonic in the whole temperature range investigated; moreover the data are consistent with the presence of an exogenous ligand strongly bound to the two metal ions. For the mononuclear derivative an essentially harmonic behavior is evident only up to 200 K where the data are consistent with the presence of an exogenous ligand much less strongly bound, while at higher temperatures the behavior of the spectra indicates the onset of very large anharmonic contributions to motions, that plausibly involve the above exogenous ligand and, quite likely, the entire active site.Abbreviations He Hemocyanin - M₀ zeroth moment - M₁ first moment - M₂ second moment - (Co^2–)₂-He binuclear bicobalt hemocyanin derivative - (Co²⁺)-He mononuclear monocobalt hemocyanin derivative - (Co²⁺-Cu¹⁺)-He binuclear mixed metals hemocyanin derivative - LFT ligand field theory - CT charge transfer - EPR electronic paramagnetic resonance - XANES X-ray absorption near edge structure Correspondence to: L. Cordone     

Genetic diversity of Puccinia kuehnii,the causal agent of orange rust of sugarcane,from Brazil

The use of resistant genotypes is the preferred method to control orange rust of sugarcane (Saccharum spp) caused by Puccinia kuehnii. This approach has been adopted in Brazil but outbreaks of the disease on previously resistant varieties showed that the efficacy of this method is limited and requires a better understanding of pathogen diversity. Nevertheless, adequate molecular markers for examining pathogen diversity at population level are not available, which limits the success of orange rust control by genetic resistance. Therefore, two independent investigations were conducted to examine genetic diversity of P. kuehnii from São Paulo state, the most important sugarcane growing state of Brazil. First, simple-sequence repeat (SSR) markers were developed in the present work and genotypic diversity of orange rust isolates from different locations investigated. Second, phenotypic diversity was examined by the single-pustule inoculation technique on P. kuehnii isolates retrieved from three susceptible commercial sugarcane cultivars. A total of 96 SSR markers were generated and tested for this species. Subsequently, 29 isolates of P. kuehnii were fingerprinted with nine SSR markers to estimate the genotypic diversity by neighbour-joining and 3D principal coordinates. The 29 isolates of the pathogen clustered into four main groups, which were identified by three SSR markers (NPRL_PK_108a, NPRL_PK_162_spka and NPRL_PK_221_spka). Phenotypic data at 21 days after the single-pustule inoculation showed that P. kuehnii from highly susceptible commercial cultivars harboured a small proportion of variants capable of causing disease on resistant cultivars. A differential reaction was demonstrated for the most virulent variant in a repeated experiment confirming the existence of races within P. kuehnii in Brazil.     

Detection of Mycobacterium leprae DNA in clinical and environmental samples using serological analysis and PCR

Molecular Biology Reports - Leprosy is a chronic infectious disease caused by Mycobacterium leprae and persists as a serious public health problem in Brazil. This microorganism is inculturable,...     

Habitat and host associations of the fish‐burrowing parasite Artystone minima (Cymothoidae: Isopoda) in eastern Amazonia

Cymothoid fish parasites settle on hosts in ways that may impact fish health and energetics. High abundances of Artystone minima observed in Nannostomus beckfordi from the Jeju River in eastern Amazonia were investigated to answer the following questions: (a) What factors are associated with the high prevalence at this locality?; (b) Is high abundance associated with co‐infestation of alternative hosts?; and (c) Is parasite presence associated with host species growth and/or reproduction? Fish assemblages were sampled quarterly (August 2017–May 2018) from five habitats along with environmental data. Parasitic indices were calculated, and parasite presence used to evaluate differences in growth of hosts using analysis of covariance considering host sex and sampling season (wet vs. dry). Parasites were only abundant in one of the habitats, a large, shallow backwater bay with macrophytes. Abiotic environmental factors (flow and depth) likely impact parasite transmission and are, therefore, particularly important in producing these local patterns. Two secondary hosts, Hyphessobrycon cf. rosaceus and Moenkhausia collettii, were found in the wet season. Based on host biology compared to other fish in the habitat, parasite infestation is inferred to be depth associated and long‐term infestation is apparently limited in alternative hosts. Parasite presence was significantly associated with reduced weight (standardized for length) of female Nannostomus beckfordi in the wet season. Furthermore, ovaries of non‐parasitized females from the wet season presented a range of maturation stages, while parasitized females were all immature, indicating a significant association of parasites with host reproductive capacity. Abstract in Portuguese is available with online material     

Invasive potential of golden and zebra mussels in present and future climatic scenarios in the new world

Hydrobiologia - Biological invasions and climate change are important drivers of biodiversity loss. In freshwater ecosystems, golden and zebra mussels are two highly aggressive invasive species...