Penicillin-binding proteins of protoplast and sporoplast membranes of Streptomyces griseus strains

Membrane-bound penicillin-binding proteins (PBPs) of two Streptomyces griseus strains that sporulate well in liquid and solid medium have been investigated during the course of their life-cycle. The PBP patterns were analyzed by sodium dodecylsulphate polyacrylamide-gel electrophoresis and fluorography. One strain (No. 45 H) has only a single band (mol wt: 27,000) in early log phase, and two additional PBPs of higher mol wt (69,000 and 80,000) in the late log phase. The other strain (No. 2682) possessed two bands with mol wts 27,000 and 38,000 which did not change during its vegetative phase. In strain No. 2682, a new PBP with a mol wt of 58,000 appeared in spore membranes while one of those (mol wt 38,000) present in mycelial membranes disappeared. Our results suggest that appearance of the new PBP in the spore may be associated with the sporulation process. The major PBP band (mol wt: 27,000) present in all stages of the life cycle of these strains, may be characteristic of S. griseus while the other PBPs reflect certain stages of the life cycle. A new method was developed for the production of spore protoplasts by consecutive enzymatic treatments.

Secretion by Overexpression and Purification of the Water-Soluble Streptomyces K15 Dd-Transpeptidase/Penicillin-Binding Protein

Though synthesized with a cleavable signal peptide and devoid of membrane anchors, the 262-amino-acid-residue Streptomyces K15 DD-transpeptidase/penicillin-binding protein is membrane-bound. Overexpression in Streptomyces lividans resulted in the export of an appreciable amount of the synthesized protein (4 mg/litre of culture supernatant). The water-soluble enzyme was purified close to protein homogeneity with a yield of 75%. It requires the presence of 0.5 M-NaCl to remain soluble. It is indistinguishable from the detergent-extract wild-type enzyme with respect to molecular mass, thermostability, transpeptidase activity and penicillin-binding capacity

Site-directed mutagenesis of conserved inverted repeat sequences in the xylanase C promoter region from Streptomyces sp EC3

Streptomyces sp. EC3, a strain which was originally isolated from cattle manure compost, was shown to possess a strong xylanolytic activity. One of the genes responsible for this activity, xlnC, encodes a secreted xylanase. In the native strain, as in the heterologous host S. lividans, expression of xlnC was detectable in the presence of xylan but not in the presence of glucose. Induction by xylan was shown to take place at the transcriptional level. The transcriptional start site of xlnC was mapped and likely -35 (5'-TTGACA-3') and -10 (5'-GAGAAC-3') motifs were identified. In order to localise putative conserved regulatory sequences, the promoter regions of xylanase-encoding genes from various Streptomyces species were aligned. This alignment revealed the existence of three sets of quite well conserved palindromic AT rich sequences called boxes 1, 2 and 3. Box 3 (5'-CGAAA N TTTCG-3') is the farthest away from the promoter region (150-200 bp). A shorter version of this palindrome (5'-GAAA NN TTTC-3') or (5'-CGAAA-3') constitutes box 1, which is located just upstream of the putative -35 promoter sequence. Box 2, located 5-7 bp upstream of box 1, comprises a shorter palindrome than box 3, with inverted polarity [5'-(G/C)TTTC (N) GAAA(G/C)-3']. The putative regulatory role of the conserved inverted repeats in boxes 2 and 3 in the promoter region of the xlnC gene from Streptomyces sp. EC3, was assessed. These boxes were modified by site-directed mutagenesis, and the mutant promoter regions, as well as the wild-type promoter region, were separately fused to a beta-lactamase reporter gene. Analysis of the expression patterns of these fusions in cultures grown in the presence of glucose, xylan or both carbon sources demonstrated that these motifs were cis -acting negative regulatory elements, each playing a specific role in the regulation of xlnC expression. Box 3 was shown to be critical for the establishment of repression of xlnC expression by glucose, whereas box 2 was shown to play an important role in the induction of xlnC expression by xylan.Peer reviewe

The peptidoglycan crosslinking enzyme system in Streptomyces R61, K15 and rimosus. Immunological studies

The exocellular DD-carboxypeptidases from Streptomyces R61, K 15, the lysozyme-releasable DD-carboxypeptidases from Streptomyces R61, K15 and rimosus, and the membrane-bound DD-carboxypeptidase of Streptomyces K15 are immunologically related to each other

Regular graphs of large girth and arbitrary degree

For every integer d > 9, we construct infinite families {G_n}_n of d+1-regular graphs which have a large girth > log_d |G_n|, and for d large enough > 1,33 log_d |G_n|. These are Cayley graphs on PGL_2(q) for a special set of d+1 generators whose choice is related to the arithmetic of integral quaternions. These graphs are inspired by the Ramanujan graphs of Lubotzky-Philips-Sarnak and Margulis, with which they coincide when d is prime. When d is not equal to the power of an odd prime, this improves the previous construction of Imrich in 1984 where he obtained infinite families {I_n}_n of d+1-regular graphs, realized as Cayley graphs on SL_2(q), and which are displaying a girth > 0,48 log_d |I_n|. And when d is equal to a power of 2, this improves a construction by Morgenstern in 1994 where certain families {M_n}_n of 2^k+1-regular graphs were shown to have a girth > 2/3 log_d |M_n|.Comment: (15 pages) Accepted at Combinatorica. Title changed following referee's suggestion. Revised version after reviewing proces

The penicillin receptor in Streptomyces

Kinetics and optical studies of Streptomyces DD-carboxypeptidases-transpeptidases led to the conclusion that the donor, acceptor, and penicillin sites on these enzymes are different but not independent and that penicillin acts as a modifier of the conformation of the protein. In the presence of penicillin, the penicillin-sensitive enzymes would be frozen in a conformation that prevents catalytic activity

DIGITAL EARTH OBSERVATION INFRASTRUCTURES AND INITIATIVES: A REVIEW FRAMEWORK BASED ON OPEN PRINCIPLES

Recent years have seen a tremendous increase of digital Earth Observation (EO) infrastructures, which provide web-based environments for accessing and processing data in a highly automated and scalable way. However, the current landscape of EO infrastructures and initiatives is fragmented, with various levels of user on-boarding and uptake success. The current work aims to make sense of this complex landscape by providing two main contributions. First, it offers a classification scheme used to review and analyse more than 150 EO infrastructures and initiatives. Then, adopting a user-centric perspective, the main limitations and obstacles currently faced by users when working with the existing EO platforms are identified. For each of these limitations, we propose a number of good practices that could benefit, from a user point of view, the design and functioning of EO platforms. Some technological enablers, i.e. specific resources (such as software components, standards and data encodings) that emerged from the analysis as holding a great potential for improving the usability of existing EO platforms, are finally listed. The work aims to provide a first scientific insight on how to best design and operate EO platforms to maximise the benefits of their user communities

Template-based Fault Injection Analysis of Block Ciphers

We present the first template-based fault injection analysis of FPGA-based block cipher implementations. While template attacks have been a popular form of side-channel analysis in the cryptographic literature, the use of templates in the context of fault attacks has not yet been explored to the best of our knowledge. Our approach involves two phases. The first phase is a profiling phase where we build templates of the fault behavior of a cryptographic device for different secret key segments under different fault injection intensities. This is followed by a matching phase where we match the observed fault behavior of an identical but black-box device with the pre-built templates to retrieve the secret key. We present a generic treatment of our template-based fault attack approach for SPN block ciphers, and illustrate the same with case studies on a Xilinx Spartan-6 FPGA-based implementation of AES-128

Disability, atrophy and cortical reorganization following spinal cord injury

The impact of traumatic spinal cord injury on structural integrity, cortical reorganization and ensuing disability is variable and may depend on a dynamic interaction between the severity of local damage and the capacity of the brain for plastic reorganization. We investigated trauma-induced anatomical changes in the spinal cord and brain, and explored their relationship to functional changes in sensorimotor cortex. Structural changes were assessed using cross-sectional cord area, voxel-based morphometry and voxel-based cortical thickness of T1-weighted images in 10 subjects with cervical spinal cord injury and 16 controls. Cortical activation in response to right-sided (i) handgrip; and (ii) median and tibial nerve stimulation were assessed using functional magnetic resonance imaging. Regression analyses explored associations between cord area, grey and white matter volume, cortical activations and thickness, and disability. Subjects with spinal cord injury had impaired upper and lower limb function bilaterally, a 30% reduced cord area, smaller white matter volume in the pyramids and left cerebellar peduncle, and smaller grey matter volume and cortical thinning in the leg area of the primary motor and sensory cortex compared with controls. Functional magnetic resonance imaging revealed increased activation in the left primary motor cortex leg area during handgrip and the left primary sensory cortex face area during median nerve stimulation in subjects with spinal cord injury compared with controls, but no increased activation following tibial nerve stimulation. A smaller cervical cord area was associated with impaired upper limb function and increased activations with handgrip and median nerve stimulation, but reduced activations with tibial nerve stimulation. Increased sensory deficits were associated with increased activations in the left primary sensory cortex face area due to median nerve stimulation. In conclusion, spinal cord injury leads to cord atrophy, cortical atrophy of primary motor and sensory cortex, and cortical reorganization of the sensorimotor system. The degree of cortical reorganization is predicted by spinal atrophy and is associated with significant disability

Progressive Purkinje Cell Degeneration in tambaleante Mutant Mice Is a Consequence of a Missense Mutation in HERC1 E3 Ubiquitin Ligase

The HERC gene family encodes proteins with two characteristic domains: HECT and RCC1-like. Proteins with HECT domains have been described to function as ubiquitin ligases, and those that contain RCC1-like domains have been reported to function as GTPases regulators. These two activities are essential in a number of important cellular processes such as cell cycle, cell signaling, and membrane trafficking. Mutations affecting these domains have been found associated with retinitis pigmentosa, amyotrophic lateral sclerosis, and cancer. In humans, six HERC genes have been reported which encode two subgroups of HERC proteins: large (HERC1-2) and small (HERC3-6). The giant HERC1 protein was the first to be identified. It has been involved in membrane trafficking and cell proliferation/growth through its interactions with clathrin, M2-pyruvate kinase, and TSC2 proteins. Mutations affecting other members of the HERC family have been found to be associated with sterility and growth retardation. Here, we report the characterization of a recessive mutation named tambaleante, which causes progressive Purkinje cell degeneration leading to severe ataxia with reduced growth and lifespan in homozygous mice aged over two months. We mapped this mutation in mouse chromosome 9 and then performed positional cloning. We found a G⇔A transition at position 1448, causing a Gly to Glu substitution (Gly483Glu) in the highly conserved N-terminal RCC1-like domain of the HERC1 protein. Successful transgenic rescue, with either a mouse BAC containing the normal copy of Herc1 or with the human HERC1 cDNA, validated our findings. Histological and biochemical studies revealed extensive autophagy associated with an increase of the mutant protein level and a decrease of mTOR activity. Our observations concerning this first mutation in the Herc1 gene contribute to the functional annotation of the encoded E3 ubiquitin ligase and underline the crucial and unexpected role of this protein in Purkinje cell physiology