Cchobo, a hobo-related sequence in Ceratitis capitata

A hobo-related sequence, Cchobo, with high similarity to the Drosophila melanogaster HFL1 and hobo₁₀₈ elements was isolated from the medfly. Thirteen PCR-derived clones, which share 97.9–100% DNA identity, were sequenced, seven of which do not show frame-shift or stop codon mutations in their conceptual translations. The consensus sequence has 99.7% DNA identity with the D. melanogaster hobo element HFL1. In a phylogenetic analysis with other hobo-related elements, Cchobo clusters with the HFL1 and hobo₁₀₈ elements from D. melanogaster and hobo-related elements from D. simulans, D. mauritiana and Mamestra brassicae. These elements may have undergone horizontal transfer in the recent past. The genomic distribution of Cchobo was studied by FISH to mitotic and polytene chromosomes, which revealed that Cchobo is distributed within both the heterochromatin and euchromatin. Intra- and interstrain polymorphisms were detected both at euchromatic and heterochromatic sites. These findings suggest that active copies of the element may be present in the medfly genome.     

Group III muscle afferents evoke reflex depressor responses to repetitive muscle contractions in rabbits

Repetitive-twitch contraction of the hindlimb muscles in anesthetized rabbits consistently evokes a reflex depressor response, whereas this type of contraction in anesthetized cats evokes a reflex pressor response in about one-half of the preparations tested. Rapidly conducting group III fibers appear to comprise the afferent arm of the reflex arc, evoking the depressor response to twitch contraction in rabbits because electrical stimulation of their axons reflexly decreases arterial pressure. In contrast, electrical stimulation of the axons of slowly conducting group III and group IV afferents reflexly increases arterial pressure in rabbits. In the present study, we examined the discharge properties of group III and IV muscle afferents and found that the former (i.e., 13 of 20), but not the latter (i.e., 0 of 10), were stimulated by 5 min of repetitive-twitch contraction (1 Hz) of the rabbit triceps surae muscles. Moreover, most of the group III afferents responding to contraction appeared to be mechanically sensitive, discharging in synchrony with the muscle twitch. On average, rapidly conducting group III afferents responded for the 5-min duration of 1-Hz repetitive-twitch contraction, whereas slowly conducting group III afferents responded only for the first 2 min of contraction. We conclude that rapidly conducting group III afferents, which are mechanically sensitive, are primarily responsible for evoking the reflex depressor response to repetitive-twitch contractions in anesthetized rabbits.     

Beta2-microglobulin isoforms display an heterogeneous affinity for type I collagen

It has been claimed that beta2-microglobulin (beta2-m) interacts with type I and type II collagen, and this property has been linked to the tissue specificity of the beta2-m amyloid deposits that target the osteo-articular system. The binding parameters of the interaction between collagen and beta2-m were determined by band shift electrophoresis and surface plasma resonance by using bovine collagen of type I and type II and various isoforms of beta2-m. Wild-type beta2-m binds collagen type I with a Kd of 4.1 x 10(-4) M and type II with 2.3 x 10(-3) M. By the BIAcore system we monitored the binding properties of the conformers of the slow phase of folding of beta2-m. The folding intermediates during the slow phase of folding do not display any significant difference with respect to the binding properties of the fully folded molecule. The affinity of beta2-m truncated at the third N-terminal residue does not differ from that reported for the wild-type protein. Increased affinity for collagen type I is found in the case of N-terminal truncated species lacking of six residues. The Kd of this species is 3.4 x 10 (-5) M at pH 7.4 and its affinity increases to 4.9 x 10(-6) M at pH 6.4. Fluctuations of the affinity caused by beta2-m truncation and pH change can cause modifications of protein concentration in the solvent that surrounds the collagen, and could contribute to generate locally a critical protein concentration able to prime the protein aggregation.     

Low TLR4 expression by liver dendritic cells correlates with reduced capacity to activate allogeneic T cells in response to endotoxin

Signaling via TLRs results in dendritic cell (DC) activation/maturation and plays a critical role in the outcome of primary immune responses. So far, no data exist concerning TLR expression by liver DC, generally regarded as less immunostimulatory than secondary lymphoid tissue DC. Because the liver lies directly downstream from the gut, it is constantly exposed to bacterial LPS, a TLR4 ligand. We examined TLR4 expression by freshly isolated, flow-sorted C57BL/10 mouse liver DC compared with spleen DC. Real-time PCR revealed that liver CD11c+CD8alpha- (myeloid) and CD11c+CD8alpha+ ("lymphoid-related") DC expressed lower TLR4 mRNA compared with their splenic counterparts. Lower TLR4 expression correlated with reduced capacity of LPS (10 ng/ml) but not anti-CD40-stimulated liver DC to induce naive allogeneic (C3H/HeJ) T cell proliferation. By contrast to LPS-stimulated splenic DC, these LPS-activated hepatic DC induced alloantigen-specific T cell hyporesponsiveness in vitro, correlated with deficient Th1 (IFN-gamma) and Th2 (IL-4) responses. When higher LPS concentrations (> or =100 ng/ml) were tested, the capacity of liver DC to induce proliferation of T cells and Th1-type responses was enhanced, but remained inferior to that of splenic DC. Hepatic DC activated by LPS in vivo were inferior allogeneic T cell stimulators compared with splenic DC, whereas adoptive transfer of LPS-stimulated (10 ng/ml) liver DC induced skewing toward Th2 responses. These data suggest that comparatively low expression of TLR4 by liver DC may limit their response to specific ligands, resulting in reduced or altered activation of hepatic adaptive immune responses.     

Localisation of the Vacuolar Proton Pump (V-H+-ATPase) and Carbonic Anhydrase II in the Human Eccrine Sweat Gland

The localisation of the vacuolar proton pump (V-H+ -ATPase) and the enzyme carbonic anhydrase II (CAII) was investigated in the human eccrine sweat gland employing standard immunohistochemical techniques after antigen retrieval using microwave heat treatment and high pressure. The high-pressure antigen retrieval unmasked the presence of V-H+ -ATPase in the clear cells of the secretory coil, with a distribution similar to that previously observed for CAII. However, the dark cells were unreactive to both antibodies. In addition, heat and high-pressure antigen retrieval demonstrated the presence of CAII in the apical zone of luminal cells of the reabsorptive duct, a location not previously reported. The localisation of V-H+ -ATPase and CAII in the secretory coil clear cells suggests that the formation of HCO3- and H+ by carbonic anhydrase II and the transport of H+ by V-H+ -ATPase may play an role in sweat fluid secretion. Their presence at the apex of the duct cells indicates involvement in ductal ion reabsorption.     

In vitro and in silico processes to identify differentially expressed proteins

We present an integrated proteomics platform designed for performing differential analyses. Since reproducible results are essential for comparative studies, we explain how we improved reproducibility at every step of our laboratory processes, e.g. by taking advantage of the powerful laboratory information management system we developed. The differential capacity of our platform is validated by detecting known markers in a real sample and by a spiking experiment. We introduce an innovative two-dimensional (2-D) plot for displaying identification results combined with chromatographic data. This 2-D plot is very convenient for detecting differential proteins. We also adapt standard multivariate statistical techniques to show that peptide identification scores can be used for reliable and sensitive differential studies. The interest of the protein separation approach we generally apply is justified by numerous statistics, complemented by a comparison with a simple shotgun analysis performed on a small volume sample. By introducing an automatic integration step after mass spectrometry data identification, we are able to search numerous databases systematically, including the human genome and expressed sequence tags. Finally, we explain how rigorous data processing can be combined with the work of human experts to set high quality standards, and hence obtain reliable (false positive < 0.35%) and nonredundant protein identifications.     

High sensitivity of human epidermal keratinocytes (HaCaT) to topoisomerase inhibitors

In the panorama of the numerous established cell lines, the human keratinocyte line HaCaT has a very interesting feature, having a close similarity in functional competence to normal keratinocytes. This cell line has been used in many studies as a paradigm for epidermal cells and therefore we selected HaCaT as a cell model for investigating the activity of three antitopoisomerase drugs (Camptothecin, Doxorubicin, Ciprofloxacin) on in vitro cell growth. The effect was evaluated both by a 24-h cytotoxicity test and by a 7-day antiproliferation assay, in which the cell viability was assessed by an MTT (3-(4,5-dimethyl-2-thiazolyl) 2,5-diphenil-2-H-tetrazolium bromide) test. DNA topoisomerase I was also partially purified from a nuclear extract of HaCaT cells, the level of topo I catalytic activity was measured by a pBR322 DNA relaxation assay and then the in vitro effect of antitopoisomerase drugs on the target enzyme was also assessed. The results indicated that the in vitro sensitivity of human epidermal HaCaT cells to antitopoisomerase drugs is comparable to that of many human tumour cell lines. HaCaT cells express a high level of topoisomerase I activity that is significantly inhibited by both Camptothecin and Doxorubicin and to a minor degree by Ciprofloxacin. A high correlation between the cell sensitivity to the antitopoisomerase I drug measured by the MTT test and the in vitro direct inhibition of HaCaT topoisomerase I was observed, suggesting that HaCaT cells can represent a very interesting model both for studying cellular pharmacokinetics of antineoplastic drugs on keratinocytes and for predicting possible secondary effects, exerted by these drugs on cutaneous cells, during treatment with chemotherapy.     

Desiccation,predation, and mussel-barnacle interactions in the northern Gulf of California

Summary Field experiments were conducted in order to determine the potential for desiccation and predation to mediate the effect of mussels (Brachidontes semilaevis) on barnacles (Chthamalus anisopoma) in the highly seasonal northern Gulf of California. We did this by removing both mussels and a common mussel predator (Morula ferruginosa: Gastropoda) and by spraying selected sites with sea water during summertime spring low tides. We also determined the effect of crowding on resistance to desiccation in barnacles, and the effect of barnacles on colonization by mussels. The mussel-barnacle community was not affected by keeping experimental quadrats damp during daytime low tides throughout the summer. Exposure to summertime low tides, however, did affect the survivorship of isolated, but not crowded, barnacles; and barnacle clumps enhanced the recruitment of mussels. Hence crowding in barnacles had a positive effect on both barnacle survivorship and mussel recruitment. Morula had a negative effect on mussel density, and mussels had a negative effect on barnacle density. The effect of Morula on barnacle density was positive, presumably due to its selective removal of mussels. These results suggest an indirect mutualism between barnacles and the gastropod predator, because barnacles attract settlement or enhance the survival of mussels, and the predator reduces the competitive effect of mussels on barnacles.