Characterization and functional analysis of the β-1,3-glucanosyltransferase 3 of the human pathogenic fungus Paracoccidioides brasiliensis

The fungus Paracoccidioides brasiliensis causes paracoccidioidomycosis, a systemic granulomatous mycosis prevalent in Latin America. In an effort to elucidate the molecular mechanisms involved in fungus cell wall assembly and morphogenesis, β-1,3-glucanosyltransferase 3 ( Pb Gel3p) is presented here. Pb Gel3p presented functional similarity to the glucan-elongating/glycophospholipid-anchored surface/pH-regulated /essential for pseudohyphal development protein families, which are involved in fungal cell wall biosynthesis and morphogenesis. The full-length cDNA and gene were obtained. Southern blot and in silico analysis suggested that there is one copy of the gene in P. brasiliensis . The recombinant Pb Gel3p was overexpressed in Escherichia coli , and a polyclonal antibody was obtained. The PbGEL3 mRNA, as well as the protein, was detected at the highest level in the mycelium phase. The protein was immunolocalized at the surface in both the mycelium and the yeast phases. We addressed the potential role of Pb Gel3p in cell wall biosynthesis and morphogenesis by assessing its ability to rescue the phenotype of the Saccharomyces cerevisiae gas1 Δ mutant. The results indicated that Pb Gel3p is a cell wall-associated protein that probably works as a β-1,3-glucan elongase capable of mediating fungal cell wall integrity.     

Improving the production of the denatured recombinant N-terminal domain of rhoptry-associated protein 2 from a Plasmodium falciparum target in the pathology of anemia in falciparum malaria

Rhoptry-associated protein 2 (RAP2) is known to be discharged from rhoptry onto the membrane surface of infected and uninfected erythrocytes (UEs) ex vivo and in vitro and this information provides new insights into the understanding of the pathology of severe anemia in falciparum malaria. In this study, a hexahistidine-tagged recombinant protein corresponding to residues 5-190 of the N-terminal of Plasmodium falciparum RAP2 (rN-RAP2) was produced using a new method of solubilization and purification. Expression was induced with D-lactose, a less expensive alternative inducer to the more common isopropyl-beta-D-thio-galactopyranosidase. The recombinant protein was purified using two types of commercially-available affinity columns, iminodiacetic and nitrilotriacetic. rN-RAP2 had immunogenic potential, since it induced high titers of anti-RAP2 antibodies in mice. These antibodies recognized full-length RAP2 prepared from Triton X-100 extracts from two strains of P. falciparum. In fact, the antibody recognized a 29-kDa product of RAP2 cleavage as well as 82 and 70-kDa products of RAP1 cleavage. These results indicate that the two antigens share sequence epitopes. Our expressed protein fragment was shown to contain a functional epitope that is also present in rhoptry-derived ring surface protein 2 which attaches to the surface of both infected and UEs and erythroid precursor cells in the bone marrow of malaria patients. Serum from malaria patients who developed anemia during infection recognized rN-RAP2, suggesting that this protein fragment may be important for epidemiological studies investigating whether immune responses to RAP2 exacerbate hemolysis in falciparum malaria patients.     

The use of phenological data to calculate chilling units in Olea europaea L. in relation to the onset of reproduction

The aim of this study was to develop a practical method to evaluate the effective relationship between the amount of winter chilling and the response expressed as the spring reproductive re-starting dates in the olive (Olea europaea L.). Two olive cultivars growing in a special olive orchard in Umbria (central Italy) were studied over a 3-year period (1998-2000): the cultivar Ascolana, typical of central Italy, and the cultivar Giarraffa, typical of southern Italy. The spring reproductive restarts were assessed using data from detailed phenological observations made on 60 trees of each cultivar in an effort to establish the exact date of reproductive bud swelling. The chilling phenomenon was evaluated by using 341 functions derived from a formula developed by researchers at Utah State University to calculate chilling units. The mathematical functions are defined, and show the very close relationship between the amount of winter chilling and the spring reproductive response in the two cultivars in the orchard studied. The results can be used to define the relationship between local climate and plant development, and the mathematical approach can be used to draw maps that can show the suitability of different cultivars on the basis of local climatic conditions.     

X-chromosome inactivation and mutation pattern in the Bruton's tyrosine kinase gene in patients with X-linked agammaglobulinemia. Italian XLA Collaborative Group

BACKGROUND: The diagnosis of X-linked agammaglobulinemia (XLA) is not always clearcut. Not all XLA conform to the classic phenotype and less than 50% of affected boys have a family history of immunodeficiency. Mutations in the gene for Bruton's tyrosine kinase (BTK) are responsible for the majority of agammaglobulinemia cases. However, a certain proportion of patients may have mutations involving other genes, although they show with an XLA phenotype. We performed BTK gene mutation analysis in 37 males with presumed XLA and analyzed the pattern of X-chromosome inactivation (XCI) in 31 mothers to evaluate the relevance of these approaches to diagnosis and genetic counseling. MATERIALS AND METHODS: Twenty affected males with a sporadic occurrence and 17 familial cases belonging to nine families were enrolled within the framework of the Italian Multicenter Clinical Study on XLA. We used non-isotopic RNase cleavage assay (NIRCA), followed by cDNA sequence determination to screen for BTK mutations and X-chromosome inactivation analysis for carrier detection. RESULTS: Using the cDNA-based approach, the identification of BTK gene abnormalities confirmed the clinical diagnosis of XLA in 31 of 37 affected infants. Missense was the most frequent mutational event and the kinase domain was mostly involved. In addition, nine novel mutations were identified. In sporadic cases, BTK gene abnormalities were identified in 9 out of 10 patients whose mothers had a nonrandom pattern of XCI and in 5 out of 6 patients whose mother had a random pattern of XCI. With the exception of one family, all patients with a familial occurrence and born to mothers with a nonrandom pattern of XCI had mutations of the BTK gene. CONCLUSIONS: Our findings indicate that in sporadic cases BTK gene sequencing is the only reliable tool for a definitive diagnosis of XLA and support XCI as the first diagnostic tool in the mothers of affected males in multiple generations. Furthermore, our molecular analysis confirms that 10-20% of BTK-unaltered patients have disorders caused by defects in other genes.     

The giants of the phylum Brachiopoda: a matter of diet?

The species of the brachiopod Gigantoproductus are giants within the Palaeozoic sedentary benthos. This presents a dilemma as living brachiopods have low‐energy lifestyles. Although brachiopod metabolic rates were probably higher during the Palaeozoic than today, the massive size reached by species of Gigantoproductus is nevertheless unusual. By examining the diet of Gigantoproductus species from the Visean (Mississippian, Carboniferous) of Derbyshire (UK), we seek to understand the mechanisms that enabled those low‐metabolism brachiopod species to become giants. Were they suspension feeders, similar to all other brachiopods, or did endosymbiosis provide a lifestyle that allowed them to have higher metabolic rates and become giants? We suggest that the answer to this conundrum may be solved by the identification of the biogeochemical signatures of symbionts, through combined analyses of the carbon and nitrogen‐isotopic compositions of the occluded organic matrix within their calcite shells. The shells are formed of substructured columnar units that are remarkably long and a few hundreds of microns wide, deemed to be mostly pristine based on multiple analyses (petrography, cathodoluminescence (CL), scanning electron microscopy (SEM), electron backscatter diffraction (EBSD), transmission electron microscopy (TEM)); they contain occluded organic fractions detected by TEM, nuclear magnetic resonance (NMR) and gas chromatography mass spectrometry (GC‐MS) analyses. We conclude that the gigantic size reached by the species of Gigantoproductus is probably the result of a mixotroph lifestyle, by which they could rely on the energy and nutrients derived both from photosymbiotic microbes and from filtered particulate food.     

Diacetylcurcumin: a new photosensitizer for antimicrobial photodynamic therapy in Streptococcus mutans biofilms

This study evaluated the effect of antimicrobial photodynamic therapy (aPDT) on S. mutans using diacetylcurcumin (DAC) and verified DAC toxicity. In vitro, S. mutans biofilms were exposed to curcumin (CUR) and DAC and were light-irradiated. Biofilms were collected, plated and incubated for colony counts. DAC and CUR toxicity assays were conducted with Human Gingival Fibroblast cells (HGF). In vivo, G. mellonella larvae were injected with S. mutans and treated with DAC, CUR and aPDT. The hemolymph was plated and incubated for colony counts. Significant reductions were observed when DAC and CUR alone were used and when aPDT was applied. HGF assays demonstrated no differences in cell viability for most groups. DAC and CUR reduced the S. mutans load in G. mellonella larvae both alone and with aPDT. Systematic toxicity assays on G. mellonella demonstrated no effect of DAC and CUR or aPDT on the survival curve.     

Toward an in vitro test for the diagnosis of allergy to penicillins. Synthesis, characterization, and use of beta-lactam and beta-lactam metabolite poly-L-lysines which recognize human IgE antibodies.

Conjugates of poly-L-lysine (PLL) containing a penicillin or a penicilloyl residue were prepared and characterized by 1H NMR and by size-exclusion (SE) HPLC. These conjugates were used in a radio allergo sorbent tests (RAST) test for the determination of allergy toward beta-lactams. The chemiometric evaluation of the data indicates that allergy to amoxycillin is different from allergy to the other beta-lactams tested. Furthermore, careful chemical characterization of the conjugates appears to be crucial to obtain meaningful information from the RAST data.