Locomotor activity patterns of two sympatric species Orchestia montagui and Orchestia gammarellus (Crustacea,Amphipoda)

Freshly adult individuals of two sympatric species, Orchestia gammarellus and Orchestia montagui, collected in spring from the supralittoral zone of Bizerte lagoon (Northern of Tunisia) at Menzel Jemil, were housed in a controlled environment cabinet. Locomotor activity rhythm was recorded in isolated individuals and groups by infrared actograph every 20 min by a data-logger, at a constant temperature of 18 ± 0.5 °C under constant darkness. According to double-plotted actograms and waveform curves, results showed the presence of two different locomotor patterns; in fact, individuals of O. gammarellus concentrated their activity during the hours of subjective night, whereas O. montagui were active during the subjective night and beyond the subjective dawn. Furthermore, whatever the species studied, periodogram analysis indicated a distinct circadian pattern of activity. Moreover, whatever the experiment condition is, the most clearly defined rhythms were found in O. gammarellus. In contrary to O. gammarellus, the group effect on the locomotor rhythm parameters seems to be less marked in O. montagui. On the other hand, a highly inter-individual variability was observed in the activity time for these two species and especially for O. montagui groups.     

Molecular characterization and immunohistochemical localization of tilapia piscidin 3 in response to Aeromonas hydrophila infection in Nile tilapia

The antimicrobial activity of tilapia piscidin 3 (TP3) was determined in vitro against a locally isolated Aeromonas hydrophila. A 388 bp fragment was amplified from the TP3 cDNA and sequenced. The coding sequence (CDS) of TP3 was estimated to be 231 bp codes for 76 amino acids long and stop codon. In silico analysis was performed to detect both the signal peptide and the prodomain cleavage sites to follow the amino acids number 22 and 70, respectively. Based on this, a peptide 23 amino acids long with a remarkably high computed antimicrobial probability was synthesized and used in the subsequent experiments. The antimicrobial activity of TP3 was determined with minimum inhibitory concentration (MIC) and minim um bactericidal concentration (MBC) methods. TP3 exhibited relatively weak antimicrobial activities against the tested bacteria. A challenge experiment was then performed in Nile tilapia with low and high doses of A. hydrophila, followed by timely recognition; after 3, 6, 24 h, and 7 days of the specific TP3 gene expression, immunohistochemical localization was also performed. Histopathological examination revealed provoked inflammatory responses and congestion in the same organs of TP3 expression. Immunohistochemical localization showed that A. hydrophila induced tilapia fish to express TP3 after 24 h within the gills, intestine, hepatopancreas, spleen, and posterior kidney. In quantitative real time (RT)‐polymerase chain reaction analysis, the high dose showed higher mRNA expression levels than the low dose, and its expression levels increased in the A. hydrophila‐infected fish. It was therefore concluded that TP3 plays an essential role in fish immunity.     

Monoclonal auto-antibodies and sera of autoimmune patients react with Plasmodium falciparum and inhibit its in vitro growth

The relationship between autoimmunity and malaria is not well understood. To determine whether autoimmune responses have a protective role during malaria, we studied the pattern of reactivity to plasmodial antigens of sera from 93 patients with 14 different autoimmune diseases (AID) who were not previously exposed to malaria. Sera from patients with 13 different AID reacted against Plasmodium falciparum by indirect fluorescent antibody test with frequencies varying from 33-100%. In addition, sera from 37 AID patients were tested for reactivity against Plasmodium yoelii 17XNL and the asexual blood stage forms of three different P. falciparum strains. In general, the frequency of reactive sera was higher against young trophozoites than schizonts (p < 0.05 for 2 strains), indicating that the antigenic determinants targeted by the tested AID sera might be more highly expressed by the former stage. The ability of monoclonal auto-antibodies (auto-Ab) to inhibit P. falciparum growth in vitro was also tested. Thirteen of the 18 monoclonal auto-Ab tested (72%), but none of the control monoclonal antibodies, inhibited parasite growth, in some cases by greater than 40%. We conclude that autoimmune responses mediated by auto-Ab may present anti-plasmodial activity.     

A thermostable glucose isomerase having a relatively low optimum pH: study of activity and molecular cloning of the corresponding gene

A thermostable glucose isomerase from a newly isolated thermophilic Streptomyces sp. SK strain, had a wide pH range with an optimum of 6 at 60°C and 6.4 at 90°C. It was optimally active at 95°C and completely stable at 80°C for at least 5.5 h with a half-life of 5 h at 90°C. Using E.coli as a host strain and an internal fragment of xylA of S. olivochromogenes as a probe, a 6.5 kb DNA fragment from Streptomyces sp. SK was cloned. This fragment carries the entire xylA gene since the recombinant plasmid complements the E.coli xyl-5 mutant strain HB101. © Rapid Science Ltd. 1998     

A novel multisite confocal system for rapid Ca2+ imaging from submicron structures in brain slices

In brain slices, resolving fast Ca²⁺ fluorescence signals from submicron structures is typically achieved using 2‐photon or confocal scanning microscopy, an approach that limits the number of scanned points. The novel multiplexing confocal system presented here overcomes this limitation. This system is based on a fast spinning disk, a multimode diode laser and a novel high‐resolution CMOS camera. The spinning disk, running at 20 000 rpm, has custom‐designed spiral pattern that maximises light collection, while rejecting out‐of‐focus fluorescence to resolve signals from small neuronal compartments. Using a 60× objective, the camera permits acquisitions of tens of thousands of pixels at resolutions of ~250 nm per pixel in the kHz range with 14 bits of digital depth. The system can resolve physiological Ca²⁺ transients from submicron structures at 20 to 40 μm below the slice surface, using the low‐affinity Ca²⁺ indicator Oregon Green BAPTA‐5N. In particular, signals at 0.25 to 1.25 kHz were resolved in single trials, or through averages of a few recordings, from dendritic spines and small parent dendrites in cerebellar Purkinje neurons. Thanks to an unprecedented combination of temporal and spatial resolution with relatively simple implementation, it is expected that this system will be widely adopted for multisite monitoring of Ca²⁺ signals.      

Altered tubulin assembly dynamics with N-homocysteinylated human 4R/1N tau in vitro

Tau isoforms promote neuronal integrity through binding and stabilization of microtubule proteins (MTP). It has been shown that hyperphosphorylation of tau contributes to Alzheimer’s disease (AD) pathology and related tauopathies. However, other pathogenic modifications of tau have not been well characterized. It is well accepted that elevated level of homocysteine (Hcy) is associated with neurodegenerative diseases such as AD. As a result of N-homocysteinylation of lysine residues, Hcy becomes a component of proteins, as a protein–homocystamide adduct, which affects protein structure and function. Here we demonstrate that N-homocysteinylation of human tau (4R/1N isoform) inhibits its function via impaired tau–tubulin specific binding and MTP assembly dynamics in vitro.     

A genome-wide collection of Mos1 transposon insertion mutants for the C. elegans research community

Methods that use homologous recombination to engineer the genome of C. elegans commonly use strains carrying specific insertions of the heterologous transposon Mos1. A large collection of known Mos1 insertion alleles would therefore be of general interest to the C. elegans research community. We describe here the optimization of a semi-automated methodology for the construction of a substantial collection of Mos1 insertion mutant strains. At peak production, more than 5,000 strains were generated per month. These strains were then subject to molecular analysis, and more than 13,300 Mos1 insertions characterized. In addition to targeting directly more than 4,700 genes, these alleles represent the potential starting point for the engineered deletion of essentially all C. elegans genes and the modification of more than 40% of them. This collection of mutants, generated under the auspices of the European NEMAGENETAG consortium, is publicly available and represents an important research resource.     

Membrane Topology and Biochemical Characterization of the Escherichia coli BacA Undecaprenyl-Pyrophosphate Phosphatase

Several integral membrane proteins exhibiting undecaprenyl-pyrophosphate (C₅₅-PP) phosphatase activity were previously identified in Escherichia coli that belonged to two distinct protein families: the BacA protein, which accounts for 75% of the C₅₅-PP phosphatase activity detected in E. coli cell membranes, and three members of the PAP2 phosphatidic acid phosphatase family, namely PgpB, YbjG and LpxT. This dephosphorylation step is required to provide the C₅₅-P carrier lipid which plays a central role in the biosynthesis of various cell wall polymers. We here report detailed investigations of the biochemical properties and membrane topology of the BacA protein. Optimal activity conditions were determined and a narrow-range substrate specificity with a clear preference for C₅₅-PP was observed for this enzyme. Alignments of BacA protein sequences revealed two particularly well-conserved regions and several invariant residues whose role in enzyme activity was questioned by using a site-directed mutagenesis approach and complementary in vitro and in vivo activity assays. Three essential residues Glu21, Ser27, and Arg174 were identified, allowing us to propose a catalytic mechanism for this enzyme. The membrane topology of the BacA protein determined here experimentally did not validate previous program-based predicted models. It comprises seven transmembrane segments and contains in particular two large periplasmic loops carrying the highly-conserved active site residues. Our data thus provide evidence that all the different E. coli C₅₅-PP phosphatases identified to date (BacA and PAP2) catalyze the dephosphorylation of C₅₅-PP molecules on the same (outer) side of the plasma membrane.