The structural analysis of three‐dimensional fibrous collagen hydrogels by raman microspectroscopy

To investigate molecular effects of 1‐Ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide (EDC), EDC/N‐hydroxysuccinimide (NHS), glyceraldehyde cross‐linking as well as polymerization temperature and concentration on the three‐dimensional (3D) collagen hydrogels, we analyzed the structures in situ by Raman microspectroscopy. The increased intensity of the 814 and 936 cm^?1 Raman bands corresponding to the C—C stretch of a protein backbone and a shift in the amide III bands from 1241 cm^?1/1268 cm^?1 in controls to 1247 cm^?1/1283 cm^?1 in glyceraldehyde‐treated gels indicated changes to the alignment of the collagen molecules, fibrils/fibers and/or changes to the secondary structure on glyceraldehyde treatment. The increased intensity of 1450 cm^?1 band and the appearance of a strong peak at 1468 cm^?1 reflected a change in the motion of lysine/arginine CH₂ groups. For the EDC‐treated collagen hydrogels, the increased intensity of 823 cm^?1 peak corresponding to the C—C stretch of the protein backbone indicated that EDC also changed the packing of collagen molecules. The 23% decrease in the ratio of 1238 cm^?1 to 1271 cm^?1 amide III band intensities in the EDC‐modified samples compared with the controls indicated changes to the alignment of the collagen molecules/fibrils and/or the secondary structure. A change in the motion of lysine/arginine CH₂ groups was detected as well. The addition of NHS did not induce additional Raman shifts compared to the effect of EDC alone with the exception of a 1416 cm^?1 band corresponding to a COO^? stretch. Overall, the Raman spectra suggest that glyceraldehyde affects the collagen states within 3D hydrogels to a greater extent compared to EDC and the effects of temperature and concentration are minimal and/or not detectable. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 349–356, 2013.     

Identification and functional analysis of the gene encoding methionine-gamma-lyase in Brevibacterium linens

The enzymatic degradation of L-methionine and subsequent formation of volatile sulfur compounds (VSCs) is believed to be essential for flavor development in cheese. L-methionine-gamma-lyase (MGL) can convert L-methionine to methanethiol (MTL), alpha-ketobutyrate, and ammonia. The mgl gene encoding MGL was cloned from the type strain Brevibacterium linens ATCC 9175 known to produce copious amounts of MTL and related VSCs. The disruption of the mgl gene, achieved in strain ATCC 9175, resulted in a 62% decrease in thiol-producing activity and a 97% decrease in total VSC production in the knockout strain. Our work shows that L-methionine degradation via gamma-elimination is a key step in the formation of VSCs in B. linens.     

On the taxonomic status of some Glyptodontidae (Mammalia,Xenarthra, Cingulata) from the Pleistocene of South America

The anatomical and taxonomic knowledge of some groups of Glyptodontidae (Mammalia, Cingulata) is still very poor. In addition, a strict typological/morphological taxonomic criterion was used in recognizing many taxa, especially during the second half of the 19th century and the first of the 20th century. This particular situation resulted in a clear overestimation diversity, mainly with respect to the South American glyptodonts. In this scenario, this paper analyzes the taxonomic status of some Glyptodontinae through a precise comparative study with well characterized taxa. The main results show that the genera Pseudothoracophorus Castellanos (P. depressus) and Chlamydotherium Lund, one species of the genus “Boreostracon” Simpson (B. corondanus), and one species of the genus Glyptodon Owen (G. falkneri), actually represents juvenile specimens referable to Glyptodon Owen; the genus Neothoracophorus Ameghino (N. elevatus) could be related either to Glyptodon Owen or Glyptotherium Osborn. Another species, Heteroglyptodon genuarioi, presents an almost identical morphology compared with the recognized species of Glyptodon (e.g. G. munizi and G. reticulatus). In this sense, the diagnostic characters described for Glyptodon perforatus Ameghino, a taxon with biostratigraphic relevance since it is characteristic of the Lujanian Age/Stage (late Pleistocene-early Holocene), are also present in other species of Glyptodon, especially G. reticulatus and G. clavipes, very frequent taxa in southern South America.     

Expression in E. coli and purification of the nucleoside diphosphate kinase b from Leishmania major

Leishmaniasis is considered by the World Health Organization to be the second most important disease caused by a protozoan parasite. Biochemical and molecular biology studies can help in the understanding of the biology of the Leishmania parasite. All protozoan parasites, including Leishmania, are unable to synthesize purines de novo, and nucleoside diphosphate kinases (NDK) are involved in the salvage pathway by which free purines are converted to nucleosides and subsequently to nucleotides. In this report, we describe the cloning of NDK coding-sequence from Leishmania major, the expression of the enzyme containing a His(6)-tag in Escherichia coli, and purification of the catalytically active native protein by affinity chromatography using Ni-NTA resin.