Brownian dynamics simulations of fluorescence fluctuation spectroscopy

We have developed a program for the simulation of the fluorescence fluctuations as detected from highly diluted samples of (bio)molecules. The model is applied to translational diffusion and takes into account the hydrodynamic interactions. The solution concentration is kept constant by assuming periodic boundary conditions and spans here the range 0.5< C < 10 nM. We show that the fluorescence correlation functions can be accurately computed on systems of limited size (a few molecules per simulation box) by simulating for a total time approximately 100-300 times the diffusion relaxation time of the fluorescence autocorrelation function. The model is applied also to the simulation of the scanning fluorescence correlation spectroscopy (FCS) and of the photon counting histograms for the confocal collection configuration. Scanning FCS simulations of highly diluted samples (C approximately equals 0.5 nM) show anticorrelation effects in the autocorrelation functions of the fluorescence signal that are less evident for higher concentrations. We suggest here that this effect may be due to the non-uniform occupancy of the scanning area by the fluorophores.     

Robertsonian polymorphism in house mouse<Emphasis Type="Italic">Mus musculus domesticus</Emphasis> from an area of intense seismic activity

This paper presents a case study of the chromosomal complement of 16 miceMus musculus domesticus Linnaeus, 1758 from the region near one of the most recent disastrous earthquakes in Italy (named Umbria-Marche 1997), with the aim of examining chromosomal variability among the mice from the seismically active zones. For the present investigation, the sampling sites were chosen in the vicinity of some active faults, supposedly the main earthquake generators in the area. In the three localities, that lie approximately on the fault lines, mice with a reduced chromosomal number (2n = 36 to 39) were trapped. This reduction is due to the presence of three different Robertsonian metacentrics — Rb(9.14), Rb(10.12) and Rb(15.17) — in both the homozygous and heterozygous states. Mice trapped in four localities more distant from the fault zone only had the standard karyotype (2n = 40). These results increase the need to analyze in more detail the distribution of karyotypes in relation with active faults.     

Method for inducing experimental pneumococcal meningitis in outbred mice

Background  

Streptococcus pneumoniae is the leading cause of bacterial meningitis. Pneumococcal meningitis is associated with the highest mortality among bacterial meningitis and it may also lead to neurological sequelae despite the use of antibiotic therapy. Experimental animal models of pneumococcal meningitis are important to study the pathogenesis of meningitis, the host immune response induced after infection, and the efficacy of novel drugs and vaccines.     

A novel C-terminal sequence from barley polyamine oxidase is a vacuolar sorting signal

Barley contains two different isoforms of flavin-containing polyamine oxidase (BPAO1 and BPAO2). We have previously demonstrated that BPAO2 is a symplastic protein in barley leaves. On the contrary, maize polyamine oxidase (MPAO), the best characterized member of this enzyme class, is apoplastic. Comparison of the derived amino-acid sequences of BPAO2 and MPAO has revealed that both precursor proteins include a cleavable N-terminal signal peptide of 25 amino acid residues, but the barley enzyme shows an extra C-terminal extension of eight amino acids. By means of MPAO engineering with BPAO2 C-terminal tail (MPAO-T) and exploiting transient expression in Nicotiana tabacum protoplasts, we demonstrate that this oligopeptide is a signal for protein sorting to the plant vacuole. The vacuolar sorting of MPAO-T was saturable. Specific mutations of the C-terminal tail were constructed to determine which amino acid residues of this novel propeptide affect proper protein sorting. No consensus sequence or common structural determinant is required for the intracellular retention of the MPAO-T protein, but a gradual lowering of the efficiency was observed as a result of progressive deletion of the C-terminus.     

A procedure for the preparation of GM3 ganglioside from GM1-lactone

A simple procedure is described for preparing GM3 ganglioside, from a few milligrams to grams, from GM1-lactone (Sonnino et al., (1985) Glycoconjugate J 2: 343-54) [1]. The synthesis was carried out under the following optimal conditions: 30 mM GM1-lactone in 0.25 M H2SO4 in DMSO, 30 min, 70 degrees C, nitrogen atmosphere, strong stirring. The yield of GM3 was 55%. The procedure applied to milligram amounts of GD1b-dilactone gave GD3 ganglioside.     

Deletion of the gene encoding c-Cbl alters the ability of osteoclasts to migrate, delaying resorption and ossification of cartilage during the development of long bones

During development of the skeleton, osteoclast (OC) recruitment and migration are required for the vascular invasion of the cartilaginous anlage and the ossification of long bones. c-Cbl lies downstream of the vitronectin receptor and forms a complex with c-Src and Pyk2 in a signaling pathway that is required for normal osteoclast motility. To determine whether the decreased motility we observed in vitro in c-Cbl(-/-) OCs translated into decreased cell migration in vivo, we analyzed the long bones of c-Cbl(-/-) mice during development. Initiation of vascularization and replacement of cartilage by bone were delayed in c-Cbl(-/-) mice, due to decreased osteoclast invasion of the hypertrophic cartilage through the bone collar. Furthermore, c-Cbl(-/-) mice show a delay in the formation of secondary centers of ossification, a thicker hypertrophic zone of the growth plate, and a prolonged presence of cartilaginous remnants in the spongiosa, confirming a decrease in resorption of the calcified cartilage. Thus, the decrease in motility of c-Cbl(-/-) osteoclasts observed in vitro results in a decreased ability of osteoclasts to invade and resorb bone and mineralized cartilage in vivo. These results confirm that c-Cbl plays an important role in osteoclast motility and resorbing activity.     

Characterization of a novel Drosophila melanogaster acylphosphatase

Analysis of the Drosophila melanogaster EST database led to the characterization of a novel acylphosphatase (AcPDro2). This is coded by the CG18505 (Acyp2) gene and is clearly distinct from a previously described AcPDro coded by the CG16870 (Acyp) gene from D. melanogaster. The two proteins show a 60% homology with both vertebrate isoenzymes. All the residues involved in the catalytic mechanism are conserved. AcPDro2 is a stable enzyme with a correct globular folded structure. Its activity on benzoylphosphate shows higher K(cat) but lower K(m) with respect to AcPDro. It is possible that AcPDro and AcPDro2 genes are not the direct ancestor of MT and CT vertebrate isoenzymes.     

Checking cell size in budding yeast: a systems biology approach

The regulation of cell cycle progression via the attainment of a critical cell size is a conserved feature from simpler unicellular organisms to mammalian cells that is obtaining much attention recently. Genome wide analysis of Saccharomyces cerevisiae deletion strains, genetic epistasis, DNA microarray analysis have recently revealed an increasingly complex network of cell size modulation mechanisms. A systems biology-based approach, that is needed to structure the underlying complexity of cell cycle regulatory mechanisms, is described.     

Degumming of silk fabric with several proteases

A crêpe silk fabric was treated with different alkaline (3374-L, GC 897-H), neutral (3273-C), and acid (EC 3.4 23.18) proteases with the aim to study their effectiveness as degumming agents. Proteases were used under optimum conditions of pH and temperature, while enzyme dosage (0.05-2 U/g fabric) and treatment time (5-240 min) were changed in order to study the kinetics of sericin removal. Degumming loss with soap and alkali was 27 wt.%. The maximum amount of sericin removed in 1 h was 17.6, 24, and 19 wt.% for 3374-L (2 U/g fabric), GC 897-H (1U/g fabric), and 3273-C (0.1 U/g fabric), respectively. Under the experimental conditions adopted, EC 3.4 23.18 was almost ineffective as a degumming agent. Degumming loss increased as a function of the treatment time, reaching a value of 25 wt.% with 1 U/g fabric of 3374-L. The morphological analysis showed that sericin was completely removed from the warp yarns of the crêpe fabric, while the highly twisted weft yarns still exhibited the presence of sericin deposits within the most internal parts of the close fibre texture. The chromatographic pattern of soluble sericin peptides changed as a function of the kind of enzyme used, enzyme dosage, and treatment time. A mixture of peptides from 5 to 20 kDa in weight, with a weight-average molecular weight of about 12 kDa was obtained.     

Polyamine oxidase,a hydrogen peroxide-producing enzyme,is up-regulated by light and down-regulated by auxin in the outer tissues of the maize mesocotyl

Exogenously supplied auxin (1-naphthaleneacetic acid) inhibited light-induced activity increase of polyamine oxidase (PAO), a hydrogen peroxide-producing enzyme, in the outer tissues of maize (Zea mays) mesocotyl. The same phenomenon operates at PAO protein and mRNA accumulation levels. The wall-bound to extractable PAO activity ratio was unaffected by auxin treatment, either in the dark or after light exposure. Ethylene treatment did not affect PAO activity, thus excluding an effect of auxin via increased ethylene biosynthesis. The auxin polar transport inhibitors N(1)-naphthylphthalamic acid or 2,3,5-triiodobenzoic acid caused a further increase of PAO expression in outer tissues after light treatment. The small increase of PAO expression, normally occurring in the mesocotyl epidermis during plant development in the dark, was also inhibited by auxin, although to a lesser extent with respect to light-exposed tissue, and was stimulated by N(1)-naphthylphthalamic acid or 2,3,5-triiodobenzoic acid, thus suggesting a complex regulation of PAO expression. Immunogold ultrastructural analysis in epidermal cells revealed the association of PAO with the secretory pathway and the cell walls. The presence of the enzyme in the cell walls of this tissue greatly increased in response to light treatment. Consistent with auxin effects on light-induced PAO expression, the hormone treatment inhibited the increase in immunogold staining both intraprotoplasmically and in the cell wall. These results suggest that both light and auxin finely tune PAO expression during the light-induced differentiation of the cell wall in the maize mesocotyl epidermal tissues.