Protist-Type Lysozymes of the Nematode Caenorhabditis elegans Contribute to Resistance against Pathogenic Bacillus thuringiensis

Pathogens represent a universal threat to other living organisms. Most organisms express antimicrobial proteins and peptides, such as lysozymes, as a protection against these challenges. The nematode Caenorhabditis elegans harbours 15 phylogenetically diverse lysozyme genes, belonging to two distinct types, the protist- or Entamoeba-type (lys genes) and the invertebrate-type (ilys genes) lysozymes. In the present study we characterized the role of several protist-type lysozyme genes in defence against a nematocidal strain of the Gram-positive bacterium Bacillus thuringiensis. Based on microarray and subsequent qRT-PCR gene expression analysis, we identified protist-type lysozyme genes as one of the differentially transcribed gene classes after infection. A functional genetic analysis was performed for three of these genes, each belonging to a distinct evolutionary lineage within the protist-type lysozymes (lys-2, lys-5, and lys-7). Their knock-out led to decreased pathogen resistance in all three cases, while an increase in resistance was observed when two out of three tested genes were overexpressed in transgenic lines (lys-5, lys-7, but not lys-2). We conclude that the lysozyme genes lys-5, lys-7, and possibly lys-2 contribute to resistance against B. thuringiensis, thus highlighting the particular role of lysozymes in the nematode's defence against pathogens

A large decrease in heat-shock-induced proteolysis after tryptophan starvation leads to increased expression of phage lambda lysozyme cloned in Escherichia coli.

The R gene coding for phage lambda lysozyme (lambda L), cloned under the control of the PL promoter on a multicopy vector, is expressed in an Escherichia coli strain auxotrophic for tryptophan. Induction by a thermal shift after tryptophan supplementation in a culture initially brought into stationary phase by tryptophan starvation leads to highly increased expression. A thermally unstable mutant protein, difficult to obtain under standard conditions, can be easily produced by post-stationary-phase expression. It is shown that this is due to a drastic decrease in the heat-shock-induced proteolysis normally observed on thermal induction. These data are discussed in relation to our present knowledge of stringent and heat-shock responses

Belgian experience of robot assisted laparoscopic para – aortic lymphadenectomy for staging of locally advanced cervical carcinoma. A multicentric study.

Objectives: FIGO clinical staging, imaging techniques sometimes underestimate extension of locally advanced cervical cancer (LACC). The presence of para-aortic lymph node (LN) metastases in LACC identifies patients with poor prognosis. Laparoscopic para-aortic lymphadenectomy is now proposed as a diagnostic tool. Feasibility and safety of robot assisted laparoscopic para-aortic lymphadenectomy has been reported. We collected data from different Belgian centers to assess its oncological safety and complication rate. Study design: Three centers participated in the study. Thirty seven patients with LACC underwent a pre treatment robot assisted laparoscopic para-aortic lymphadenectomy. Data was prospectively collected. Results: The median number of LN collected was 27.5 (1-54) per patient. Five out of 37 patients had para-aortic node metastasis. The false negative rate for PET CT diagnosing para- aortic node metastasis was 10.8% (4/37). We encountered 2 major intra operative complications (5.4%). Post operative morbidity was low (13.5%). Median follow up was 27 months (95% CI 24–30). Median disease free survival (DFS) was 16 months (95% CI 2.4- 29.6). Patients with negative LN had a median DFS of 24 months (NA) although patients with positive LN had a median DFS of 9 months (95% CI 6.9-11.9). Conclusions: In this series we report that robot assisted laparoscopic para-aortic lymphadencetomy provided the surgeon with useful information to avoid understaging in 10.8% of women with LACC, at the expense of low morbidity (13.5%). Presence of microscopic para-aortic LN metastasis is correlated with shorter DFS. The number of patients with positive LN doesn’t allow us to draw any firm conclusion

Biosynthetic incorporation of 7-azatryptophan into the phage lambda lysozyme: estimation of tryptophan accessibility, effect on enzymatic activity and protein stability.

The phage lambda lysozyme (lambda L) contains four tryptophans. These have been efficiently replaced by 7-azatryptophan (7aW) through biosynthetic incorporation into the overexpressed protein. Comparative analysis of the effect of temperature or pH on the fluorescence of the wild-type lambda L and 7aWs-containing protein (a lambda L) shows that the stability of the protein is only mildly reduced by 7aW incorporation above pH 5 but that it is strongly decreased below pH 4 on protonation of inaccessible 7aWs. The a lambda L fluorescence depends on pH as a consequence of its effect on the denaturation equilibrium, on the state of protonation of accessible 7aWs in the native state and of all 7aWs in the denatured state. The pH dependence of the fluorescence is used to estimate the number of accessible tryptophans in the protein. The result agrees with that derived from tryptophan NH exchange measurements by 1H-NMR. The acid limb of the activity-pH profile is characterized by a sharp drop that might arise from a cooperative acid-induced denaturation. The difference in acid stability of a lambda L versus lambda L is used to rule out this acid denaturation hypothesis as tryptophan replacement does not affect the lytic activity on chloroform-sensitized Escherichia coli cells or its pH profile

Adsorption of succinylated lysozyme on hydroxyapatite

Hen egg white lysozyme has been succinylated, the isoelectric point being shifted thereby from about 11 to 4.5-4.7. Its adsorption on a chromatography grade hydroxyapatite has been investigated at 20 degrees C and in the pH range from 5.9 to 7.4. Adsorption takes place despite an electrical repulsion between the surface and the adsorbate; consequently, it is favored by a decrease of pH and phosphate concentration and an increase of calcium concentration and ionic strength. While adsorption of native lysozyme is driven by electrostatic attraction to the surface, adsorption of succinylated lysozyme is controlled by hydrophobic interactions. This may be attributed to a different structure of modified lysozyme, compared to native lysozyme, or to a greater tendency to undergo conformational changes. (C) 1997 Academic Press

Bivalent cations stabilize yeast alcohol dehydrogenase I

The thermostability of yeast alcohol dehydrogenase (ADH) I is strongly dependent on the presence of NaCl, a salt that is almost neutral on the Hofmeister scale, which suggests that solvent-accessible electrostatic repulsion might play a role in the inactivation of the enzyme. Moreover, CaCl2 and MgCl2 are able to stabilize the enzyme at millimolar concentrations. Ca2+ stabilizes yeast ADH I by preventing the dissociation of the reduced form of the enzyme and by preventing the unfolding of the oxidized form of the enzyme. An analysis of several chimaeric ADHs suggests that Ca2+ is fixed by the Asp-236 and Glu-101 side chains in yeast ADH I, but that Ca2+ can be displaced by replacing Met-168 by an Arg residue, as suggested by a three-dimensional model of the enzyme structure. These results indicate that electrostatic repulsion can cause protein unfolding and/or dissociation. It is proposed that yeast ADH I binds Mg2+ in vivo