Role of phage ϕ1 in two strains of <Emphasis Type="Italic">Salmonella</Emphasis> Rissen,sensitive and resistant to phage ϕ1

Background

The study describes the Salmonella Rissen phage ?1 isolated from the ?1-sensitive Salmonella Rissen strain R^W. The same phage was then used to select the resistant strain R^R?1+, which can harbour or not ?1.

Results

Following this approach, we found that ?1, upon excision from R^W cells with mitomycin, behaves as a temperate phage: lyses host cells and generates phage particles; instead, upon spontaneous excision from R^R?1+ cells, it does not generate phage particles; causes loss of phage resistance; switches the O-antigen from the smooth to the rough phenotype, and favors the transition of Salmonella Rissen from the planktonic to the biofilm growth.The R^W and R^R?1+ strains differ by 10 genes; of these, only two (phosphomannomutase_1 and phosphomannomutase_2; both involved in the mannose synthesis pathway) display significant differences at the expression levels. This result suggests that phage resistance is associated with these two genes.

Conclusions

Phage ?1 displays the unusual property of behaving as template as well as lytic phage. This feature was used by the phage to modulate several phases of Salmonella Rissen lifestyle.

     

Identification of major royal jelly proteins in the brain of the honeybee Apis mellifera

The consumption of royal jelly (RJ) determines the differences between castes and behavioral development in the honeybee Apis mellifera. However, it is not known whether the proteins of RJ are related to these differences, or which proteins are responsible for the changes. To understand the functions of RJ proteins that are present in other tissues of the bee, in addition to hypopharyngeal gland, we used a polyclonal antibody anti-MRJP1 to investigate the presence of this protein in nervous system of honeybee. This study showed the presence of three polypeptides (p57, p70 and p128) in specific tissues of bee brain. Mushroom body, optic lobe and antennal lobe neuropils all contained proteins recognized by anti-MRJP1. Proteomic analysis showed that the three polypeptides are correlated with proteins of the MRJP family. p57 is correlated with MRJP1, p70 with MRJP3, while p128 may be an oligomeric form or a new polypeptide. Immunostaining of the brain and hypopharyngeal gland revealed differential expression of MRJPs in various brain regions and in different honeybee castes and subcastes. The identification and localization of these MRJPs contribute to the elucidation of the biological roles of this protein family.     

Heterogeneous shedding of Brucella abortus in milk and its effect on the control of animal brucellosis

Aims: To ascertain whether in Brucella abortus‐infected water buffalo herds, the number of newly infected animals could be reduced by culling superspreaders (the animals secreting ≥10⁴ CFU per ml of milk). Methods and Results: The number of B. abortus present in the milk (CFU per ml) from 500 water buffaloes was measured by the culture. Each animal was tested three times, at one month intervals. The presence or the absence of B. abortus in each milk sample was confirmed by PCR. A majority of infected animals shed the pathogen at a low level (≤10³ CFU ml^?1). However, a few infected individuals (superspreaders) shed large numbers of B. abortus (≥10⁴ CFU ml^?1). Quantitative PCR of B. abortus positive milk samples gave comparable results to culture. Culling of the superspreaders was sufficient to arrest the spread of infection. Significance and Impact of the Study: The approach described here can reduce significantly the cost of controlling brucellosis. Culture and quantitative PCR tests identify superspreaders and, compared with the serological tests in use to detect brucellosis, provide also a more accurate estimate of the disease incidence.     

Apoptosis during bone-like tissue development in vitro

We present evidence of cell death by apoptosis during the development of bone-like tissue formation in vitro. Fetal rat calvaria-derived osteoblasts differentiate in vitro, progressing through three stages of maturation: a proliferation period, a matrix maturation period when growth is downregulated and expression of the bone cell phenotype is induced, and a third mineralization stage marked by the expression of bone-specific genes. Here we show for the first time that cells differentiating to the mature bone cell phenotype undergo programmed cell death and express genes regulating apoptosis. Culture conditions that modify expression of the osteoblast phenotype simultaneously modify the incidence of apoptosis. Cell death by apoptosis is directly demonstrated by visualization of degraded DNA into oligonucleosomal fragments after gel electrophoresis. Bcl-X_L, an inhibitor of apoptosis, and Bax, which can accelerate apoptosis, are expressed at maximal levels 24 h after initial isolation of the cells and again after day 25 in heavily mineralized bone tissue nodules. Bcl-2 is expressed in a reciprocal manner to its related gene product Bcl-X_L with the highest levels observed during the early post-proliferative stages of osteoblast maturation. Expression of p53, c-fos, and the interferon regulatory factors IRF-1 and IRF-2, but not cdc2 or cdk, were also induced in mineralized bone nodules. The upregulation of Msx-2 in association with apoptosis is consistent with its in vivo expression during embryogenesis in areas that will undergo programmed cell death. We propose that cell death by apoptosis is a fundamental component of osteoblast differentiation that contributes to maintaining tissue organization. J. Cell. Biochem. 68:31–49, 1998. © 1998 Wiley-Liss, Inc.     

Starchy food residue on a potsherd from a late Holocene hunter-gatherer site in Argentine Patagonia: towards the visibility of wild underground storage organs

Vegetation History and Archaeobotany - The aim of this paper is to present recent advances in the microbotanical analyses of an organic residue on a potsherd from a late Holocene hunter-gatherer...     

Synthesis, in vitro antifungal activity and mechanism of action of four sterol hydrazone analogues against the dimorphic fungus Paracoccidioides brasiliensis

The design and synthesis of novel sterol hydrazone analogues (9, 10, 11 and 12) are described, followed by their evaluation as inhibitors of fungal growth, using Paracoccidioides brasiliensis as the biological tester. Compounds 9, 10, 11 and 12 generated a dose-dependent effect in fungal growth, particularly 9, 11 and 12, which were active at nanomolar concentrations (100 nM). When P. brasiliensis in its pathogenic yeast-like phase was treated individually with each of the aforementioned compounds at concentrations that reduced growth rate around 50%, the analysis of sterol composition in the resulting surviving cells demonstrated a 50% reduction of the final sterols brasicasterol and ergosterol, and concomitant increase in the levels of lanosterol. These results indicate that these compounds inhibit the enzyme Δ(24)-sterol methyl transferase (SMT), in a manner dependent on the stereochemical location of the hydrazone group. Compound 12, instead, induced a good antiproliferative activity not associated with blockage of any step in the pathway to sterol biosynthesis, suggesting a different mode of action. The X-ray crystal structure of H1 was determined to obtain information regarding the rings and side chain conformation of the sterol hydrazones. Comparison of the inhibitory effects of sterol hydrazones (9-12) and azasterols (AZA1-AZA3) on SMT with the molecular electrostatic potential, negative isopotential energy surfaces (-10 kcal/mol) and local ionization potential calculated via DFT methods, showed that changes in the electronic moiety introduced by the N and O atoms were not as important as the additional flexibility of the side chain introduced by an extra methylene group.     

Use of molecular markers and flow cytometry to preserve ancient Annurca apple germplasm

The old Annurca apple cultivar (Malus domestica), particularly appreciated for its peculiar flavor and crispy flesh, was studied in order to preserve its ancient germplasm. Twelve clones of Annurca were analyzed using random amplified polymorphic DNA (RAPD) and simple-sequence repeat (SSR) markers. Two out of 30 RAPD primers and nine out of ten SSR primers were able to discriminate all the clones analyzed. Data were confirmed by measuring DNA content using flow cytometry. The results provide a good procedure to improve germplasm field management, in order to removing redundant material in the Annurca collection. This represents an efficient way to create a data bank in order to preserve the genetic variability of the Annurca cultivar. M. Iannaccone, D. Palumbo and I. Ventimiglia contributed equally to this work     

β-sheet to α-helix conversion and thermal stability of β-Galactosidase encapsulated in a nanoporous silica gel

The effect on protein conformation and thermal stability was studied for β-Galactosidase (β-Gal) encapsulated in the nanopores of a silicate matrix (E_β-Gal). Circular dichroism spectra showed that, compared with the enzyme in buffer (S_β-Gal), E_β-Gal exhibited a higher content of α-helix structure. Heating E_β-Gal up to 75?°C caused a decrease in the content of β-sheet structure and additional augments on E_β-Gal components attributed to helical content, instead of the generalized loss of the ellipticity signal observed with S_β-Gal. Steady state fluorescence spectroscopy analysis evidenced an E_β-Gal structure less compact and more accessible to solvent and also less stable against temperature increase. While for S_β-Gal the denaturation midpoint (Tm) was 59?°C, for E_β-Galit was 48?°C. The enzymatic activity assays at increasing temperatures showed that in both conditions, the enzyme lost most of its hydrolytic activity against ONPG at temperatures above 65?°C and E_β-Gal did it even at lower T values. Concluding, confinement in silica nanopores induced conformational changes on the tertiary/cuaternary structure of E_β-Gal leading to the loss of thermal stability and enzymatic activity.