Cross‐species comparison of mammalian saliva using an LC–MALDI based proteomic approach

Despite the importance of saliva in the regulation of oral cavity homeostasis, few studies have been conducted to quantitatively compare the saliva of different mammal species. Aiming to define a proteome signature of mammals’ saliva, an in‐depth SDS‐PAGE–LC coupled to MS/MS (GeLC–MS/MS) approach was used to characterize the saliva from primates (human), carnivores (dog), glires (rat and rabbit), and ungulates (sheep, cattle, horse). Despite the high variability in the number of distinct proteins identified per species, most protein families were shared by the mammals studied with the exception of cattle and horse. Alpha‐amylase is an example that seems to reflect the natural selection related to digestion efficacy and food recognition. Casein protein family was identified in all species but human, suggesting an alternative to statherin in the protection of hard tissues. Overall, data suggest that different proteins might assure a similar role in the regulation of oral cavity homeostasis, potentially explaining the specific mammals’ salivary proteome signature. Moreover, some protein families were identified for the first time in the saliva of some species, the presence of proline‐rich proteins in rabbit's saliva being a good example.     

Cab45 is required for Ca2+-dependent secretory cargo sorting at the trans-Golgi network

Ca²⁺ import into the lumen of the trans-Golgi network (TGN) by the secretory pathway calcium ATPase1 (SPCA1) is required for the sorting of secretory cargo. How is Ca²⁺ retained in the lumen of the Golgi, and what is its role in cargo sorting? We show here that a soluble, lumenal Golgi resident protein, Cab45, is required for SPCA1-dependent Ca²⁺ import into the TGN; it binds secretory cargo in a Ca²⁺-dependent reaction and is required for its sorting at the TGN.     

Associations between fish assemblages and environmental factors for Mediterranean-type rivers during summer

The structure of summer fish assemblages was examined along longitudinal gradients in 31 Mediterranean-type rivers of the middle Guadiana basin (south-west Iberian Peninsula), using data from 157 sites including small streams to deep rivers. An ordination analysis, based on 16 variables, was applied to species presence, using principal component and canonical correspondence analysis. The results for the habitat data were compared with those for the biological data using a Mantel analysis, and the agreement was highly significant. Spatial structure was considered by partitioning the total variability among the environmental and geographical variables. The fish assemblages showed longitudinal zoning during the summer, with species distributed over gradients of habitat size (depth), water quality (current and physico-chemical variables), and cover (substratum and vegetation), according to their adult size and life history. The size of the habitat that remained available in summer had the greatest biological effect, being the most important factor explaining fish species distribution and assemblage structure during this stressing season. Strictly spatial variation was low, but there was still a high residual variation. Habitat associations and life-history strategies are discussed for native and exotic species.     

In vitro hydroxyapatite adsorbed salivary proteins

In spite of the present knowledge about saliva components and their respective functions, the mechanism(s) of pellicle and dental plaque formation have hitherto remained obscure. This has prompted recent efforts on in vitro studies using hydroxyapatite (HA) as an enamel model. In the present study salivary proteins adsorbed to HA were extracted with TFA and EDTA and resolved by 2D electrophoresis over a pH range between 3 and 10, digested, and then analysed by MALDI-TOF/TOF mass spectrometry and tandem mass spectrometry. Nineteen different proteins were identified using automated MS and MS/MS data acquisition. Among them, cystatins, amylase, carbonic anhydrase, and calgranulin B, were identified.     

Efficacy of reconstituted and stored botulinum toxin type A: an electrophysiologic and visual study in the auricular muscle of the rabbit

Once botulinum toxin type A is reconstituted, the manufacturer recommends that it be used in approximately 4 hours. As a result, a significant amount of this costly drug is often discarded because it is not completely used in the recommended period. The purpose of the present study was to compare fresh versus stored reconstituted botulinum toxin type A for (1) initial potency, (2) duration of action, and (3) bacterial colonization.Using a rabbit model, 20 New Zealand White rabbits were divided into four groups (I to IV). All rabbits had an injection of 2.5 U of reconstituted botulinum toxin into the right anterior auricular muscle. The first group was injected with botulinum toxin type A that was freshly reconstituted and served as the control. The second, third, and fourth groups were injected with botulinum toxin type A that had been reconstituted and stored for 2, 6, and 12 weeks, respectively, in a conventional freezer. Each rabbit had daily visual evaluation of the ear, with the position of auricle being graded from I to III. In addition, each rabbit had a nerve conduction study performed on the right anterior auricular muscle before injection and every 2 weeks after injection. Amplitude was chosen as the principal variable in the data analysis because it is the best predictor of physiologic changes at the muscle motor unit level. The endpoint of the study was defined as the time at which the nerve conduction studies and the visual inspections returned to baseline, preinjection levels. Botulinum toxin type A was also cultured before injection into each group.Overall, the nerve conduction data revealed a trend with a faster recovery (return to baseline) with the stored botulinum toxin. Groups IV and III returned to baseline first, followed by groups II and I. However, there was no significant difference among the groups at 2 and 4 weeks after injection, indicating that initial potency was unchanged. The differences between the groups became significant (p < 0.05) at 6 weeks and onward, suggesting that the duration was affected. Group I (fresh botulinum toxin) and group II (toxin stored for 2 weeks) had comparable outcomes and were not significantly different at any time period. Under visual inspection, the mean recovery time for each group was as follows: group IV, 5.4 weeks; group III, 7.0 weeks; group II, 6.75 weeks; and group I, 7.80 weeks. The results showed significance (p < 0.05) beginning after 3 weeks among some groups. Again, there was an overall quicker trend to return to baseline with the longer storage of the botulinum toxin (groups III and IV). These results support the authors' conduction study data, which suggest that the initial potency is not affected but the duration of action is. Again, groups I and II had comparable results. Microbiology cultures showed no growth of either aerobic or anaerobic bacteria at 7 days.In conclusion, using the rabbit model, it seems that reconstituted and stored botulinum toxin type A has the same initial potency but the duration of action is affected sometime after 2 weeks of storage. No bacterial contamination was associated with storing unpreserved reconstituted botulinum toxin type A for up to 12 weeks.     

Drosophila melanogaster larval hemolymph protein mapping

With the completion of the genome sequence of Drosophila melanogaster the importance of constructing a proteome map is to be considered. Therefore, with the application of recent advances in proteomic analysis approaches, a protein map of D. melanogaster larvae hemolymph proteins was obtained using 2-DE in the range of pH 3-10. After Coomassie colloidal detection of 289 spots, a total of 105 were excised from the gel and digested with trypsin. Identification was done based on a combination of MALDI-TOF/TOF MS and MS/MS spectra. The 99 proteins identified using this approach include a large number of metabolic enzymes, translational apparatus components, and structural proteins. Among these we emphasize the identification of proteins with molecular chaperone properties (heat shock proteins and PPIases) and protein spots involved in defense responses such as antioxidant and immunological defense mechanisms (thioredoxin, prophenoloxidase, and serine proteases), as well as in signal transduction pathways.     

Use of FTIR,FT-Raman and 13C-NMR spectroscopy for identification of some seaweed phycocolloids

Many seaweeds produce phycocolloids, stored in the cell wall. Members of the Rhodophyceae produce polysaccharides the main components of which are galactose (galactans)-agar and carrageenan. In addition, alginic acid is extracted from members of the Phaeophyceae. This is a binary polyuronide made up of mannuronic acid and guluronic acid. The wide uses of these phycocolloids are based on their gelling, viscosifying and emulsifying properties, which generate an increasing commercial and scientific interest. In this work, the FTIR and FT-RAMAN spectra of carrageenan and agar, obtained by alkaline extraction from different seaweeds (e.g. Mastocarpus stellatus, Chondrus crispus, Calliblepharis jubata, Chondracanthus acicularis, Chondracanthus teedei and Gracilaria gracilis), were recorded in order to identify the type of phycocolloid produced. The spectra of commercial carrageenan, alginic acid and agar samples (SIGMA and TAAB laboratories) were used as references. Special emphasis was given to the 500-1500 cm(-1) region, which presents several vibrational modes, sensitive to the type of polysaccharide and to the type of glycosidic linkage. The FT-Raman spectra present a higher resolution than FTIR spectra, this allowing the identification of a larger number of characteristic bands. In some cases, phycocolloids can be identified by FT-Raman spectroscopy alone.     

Serum interleukin-6 in renal osteodystrophy: relationship with serum PTH and bone remodeling markers.

Interleukin-6, synthesized by osteoblasts in response to PTH, stimulates osteoclastogenesis and bone resorption in vitro, and it has been implicated in the pathogenesis of bone loss in several clinical situations. The aim of this study was to evaluate whether serum levels of interleukin-6 were increased in patients with renal osteodystrophy, and to investigate the possible relationships between serum interleukin-6 and PTH levels on one hand, and serum interleukin-6 and bone remodeling markers on the other. Serum interleukin-6 (IL-6), intact PTH, osteocalcin, bone alkaline phosphatase (BAP) and carboxyterminal telopeptide of Type 1 collagen (ICTP) were measured in 86 uremic patients. IL-6 (median [range] 16.5 [1.0-430] pg/ml), PTH (279.8 [11-2004] pg/ml), osteocalcin (143.8 [8-921] ng/ml), BAP (20.9 [6-169] U/I) and ICTP (38.8 [1.5-181.5] microg/l) were higher than normal. IL-6 levels correlated with PTH (r= 0.22, p = 0.04) and with ICTP (r = 0.31, p = 0.004). A stronger correlation was found between PTH and circulating bone remodeling markers (r = 0.66 for osteocalcin, r = 0.56 for BAP, and r = 0.39 for ICTP). The correlation between PTH and IL-6 was stronger in those patients (n = 15) with severe secondary hyperparathyroidism (r= 0,71, p = 0.003). On the other hand, in the group of patients (n = 41) with PTH lower than 250 pg/ml, there was no correlation between IL-6 and PTH, while IL-6 correlated with ICTP (r = 0.44, p = 0.006). Serum IL-6 correlates with ICTP which suggests that it may mediate bone resorption in renal osteodystrophy.     

Proteomics of immune-challenged Drosophila melanogaster larvae hemolymph

In the last decade, the fruit fly Drosophila melanogaster has emerged as a promising invertebrate model for the investigation of innate immunity, in part because of its well characterised genetics. The information provided by the innumerous reports on Drosophila's immune response indicates that a large number of genes, in addition to the well-known antimicrobial peptide genes, are both up- and down-regulated upon immune challenge. Nevertheless, their contribution to fighting off infection has not been seriously addressed. With the application of recent advances in proteomics, the effects of an immune challenge in the overall modification of Drosophila 2-DE protein patterns were investigated. The aim of this study was to investigate hemolymph proteins differentially expressed between control and immunised larvae sets, which could be related solely to the Drosophila immune response. The list of immune-related protein spots included heat shock proteins and other proteins with chaperone properties, serine proteases, phenol oxidase, and Drosophila antioxidant system components, which accounted for 21% of the total of 70 identified proteins, metabolic enzymes implicated in pathways such as cellular respiration, fatty-acid oxidation, protein biosynthesis, and structural proteins.