Immunohistochemical evidence of lactoferrin in human embryo–fetal bone and cartilage tissues

Lf (lactoferrin) is an 80‐kDa iron‐binding protein, which has been suggested to promote bone growth in murine models. In view of this, we aimed to analyse the immunohistochemical distribution of Lf in human embryonal and fetal bone and cartilaginous tissues at different gestational weeks in order to evaluate whether a role for this protein might be proposed also in human osteogenesis. Bone and cartilaginous specimens were taken at autopsy from 25 fetuses (8–34 weeks of gestation). Ten samples of human adult bone and cartilage were also submitted to the immunohistochemical procedures. Sections, 4‐μm thick, were cut from formalin‐fixed paraffin‐embedded tissue blocks and stained with a monoclonal antibody against human Lf, following antigen retrieval procedures. Lf immunoreactivity was maily localized in the mesenchymal cells forming the periosteum as well as in chondroblasts at the eighth gestational week; a strong Lf immunoexpression in immature osteocytes and osteoblasts was noted up to the 18th gestation week, with a considerable decrease by the 24th week. No Lf expression was found in any bone area after the 30th and up to the 34th week of gestation. Our data seem to suggest an important role for Lf as a bone growth regulator in the early phases of the human endochondral ossification, with an anabolic action similar to that previously reported in cell culture lines and in animal models.     

Immunohistochemical localisation of FMRF-amide-like peptide in the brain of icefish and red-blooded Antarctic Teleosts

The distribution of Phe-Met-Arg-Phe-NH₂ (FMRF-amide) -like immunoreactivity was investigated by indirect immunofluorescence technique using the molluscan FMRF-amide antibody in the brain of icefish (Pagetopsis macropterus and Chionodraco hamatus used as positive control) and red blooded (Trematomus bernacchii, Gymnodraco acuticeps, Histiodraco velifer, Cygnodraco mawsoni) Antarctic Teleosts. Immunoreactive perikarya were localised in the ventral thalamus, in the hypothalamus (preoptic and periventricular regions) and in the intermedioventral rhombencephalon (vagal motor nucleus) as well as in the telencephalon and in the mesencephalon. Positive nerve fibres were seen to project towards the caudal brainstem to reach the rhombencephalon. No differences were observed in the immunopositivity of FMRF-amide new distribution in the Antarctic Teleosts examined. In the icefishes the immunoreaction was stronger than in the hemoglobin-rich Teleosts. The distribution patterns of the FMRF-amide immunostaining suggest that this peptide may play a pivotal role in the cardiovascular regulation in the Antarctic Teleosts.     

Image filtering for two-photon deep imaging of lymphonodes

Non-linear excitation microscopy is considered an ideal spectroscopic method for imaging thick tissues in vivo due to the reduced scattering of infrared radiation. Although imaging has been reported on brain neocortex at 600-800 mum of depth, much less uniform tissues, such as lymphonodes, are characterized by highly anisotropic light scattering that limits the penetration length. We show that the most severe limitation for deep imaging of lymphonodes appears to be the tissue scattering and the diffuse fluorescence emission of labeled cell (lymphocytes) in layers above the focusing plane. We report a study of the penetration depth of the infrared radiation in a model system and in ex vivo lymphonodes and discuss the possibility to apply Fourier filtering to the images in order to improve the observation depth.     

Molecular simulations of lipid-mediated protein-protein interactions

Recent experimental results revealed that lipid-mediated interactions due to hydrophobic forces may be important in determining the protein topology after insertion in the membrane, in regulating the protein activity, in protein aggregation and in signal transduction. To gain insight into the lipid-mediated interactions between two intrinsic membrane proteins, we developed a mesoscopic model of a lipid bilayer with embedded proteins, which we studied with dissipative particle dynamics. Our calculations of the potential of mean force between transmembrane proteins show that hydrophobic forces drive long-range protein-protein interactions and that the nature of these interactions depends on the length of the protein hydrophobic segment, on the three-dimensional structure of the protein and on the properties of the lipid bilayer. To understand the nature of the computed potentials of mean force, the concept of hydrophilic shielding is introduced. The observed protein interactions are interpreted as resulting from the dynamic reorganization of the system to maintain an optimal hydrophilic shielding of the protein and lipid hydrophobic parts, within the constraint of the flexibility of the components. Our results could lead to a better understanding of several membrane processes in which protein interactions are involved.     

Enoate reductases from non conventional yeasts: bioconversion, cloning, and functional expression in Saccharomyces cerevisiae

Old yellow enzymes (OYEs, EC 1.6.99.1) are flavin-dependent oxidoreductases that catalyze the stereoselective trans-hydrogenation of the double bond, representing a promising alternative to metal-based catalysis. Bioconversion of ketoisophorone (KIP) by 28 non-conventional yeasts belonging to 16 different species was investigated. Growing cells of most of the strains reduced KIP via OYE and showed high stereoselectivity, producing R-levodione as major product. Competition by carbonyl reductase (CR) activity was observed in several strains. The best performing yeasts belong to Candida castellii, Kazachstania spencerorum and Kluyveromyces marxianus exhibited yields of levodione ≥77% up to 95% e.e., and. Candida freyschussii, the sole strain lacking the OYE gene, reduced KIP only to unsaturated alcohols via CR. Nine unedited OYE genes were cloned, sequenced, and heterologously expressed in Saccharomyces cerevisiae BY4741ΔOye2, a mutant that showed negligible OYE and CR activities. Compared with the corresponding wild-type yeasts, growing cells of the recombinant strains bioconverted KIP with improved yields of OYE products, minor competition by CR activity, and lower enantioselectivity. In particular, resting cells of recombinant S. cerevisae presented the best performance in KIP bioconversion. Based on the results herein reported, selected strains of non-conventional yeasts and novel OYE genes can be profitably used as innovative biocatalysts in asymmetric reductions.     

Probing protein aggregation by time-resolved fluorescence during beta-lactoglobulin crystal growth

We have used the fluorescence anisotropy (FA) decay of retinol bound to bovine beta-lactoglobulin to monitor the time evolution of protein aggregation during the early stages of crystal growth. With this approach we have followed the formation of aggregates at different concentrations of ammonium sulfate, the precipitant used for crystallization. The average aggregation number is found to depend on precipitant concentration, and to be restricted to small numbers ranging from 2 to 5, also in the presence of visible growing crystals. The effect of particle distribution and of low probe-to-protein saturation on the FA response is also discussed in detail.