Metabolic differentiation in the embryonic retina

Unlike healthy adult tissues, cancers produce energy mainly by aerobic glycolysis instead of oxidative phosphorylation. This adaptation, called the Warburg effect, may be a feature of all dividing cells, both normal and cancerous, or it may be specific to cancers. It is not known whether, in a normally growing tissue during development, proliferating and postmitotic cells produce energy in fundamentally different ways. Here we show in the embryonic Xenopus retina in vivo, that dividing progenitor cells depend less on oxidative phosphorylation for ATP production than non-dividing differentiated cells, and instead use glycogen to fuel aerobic glycolysis. The transition from glycolysis to oxidative phosphorylation is connected to the cell differentiation process. Glycolysis is indispensable for progenitor proliferation and biosynthesis, even when it is not used for ATP production. These results suggest that the Warburg effect can be a feature of normal proliferation in vivo, and that the regulation of glycolysis and oxidative phosphorylation is critical for normal development.     

Differential interaction of NMDA receptor subtypes with the post-synaptic density-95 family of membrane associated guanylate kinase proteins

NMDA receptors are a subclass of ionotropic glutamate receptors. They are trafficked and/or clustered at synapses by the post-synaptic density (PSD)-95 membrane associated guanylate kinase (MAGUK) family of scaffolding proteins that associate with NMDA receptor NR2 subunits via their C-terminal glutamate serine (aspartate/glutamate) valine motifs. We have carried out a systematic study investigating in a heterologous expression system, the association of the four major NMDA receptor subtypes with the PSD-95 family of MAGUK proteins, chapsyn-110, PSD-95, synapse associated protein (SAP) 97 and SAP102. We report that although each PSD-95 MAGUK was shown to co-immunoprecipitate with NR1/NR2A, NR1/NR2B, NR1/NR2C and NR1/NR2D receptor subtypes, they elicited differential effects with regard to the enhancement of total NR2 subunit expression which then results in an increased cell surface expression of NMDA receptor subtypes. PSD-95 and chapsyn-110 enhanced NR2A and NR2B total expression which resulted in increased NR1/NR2A and NR1/NR2B receptor cell surface expression whereas SAP97 and SAP102 had no effect on total or cell surface expression of these subtypes. PSD-95, chapsyn-110, SAP97 and SAP102 had no effect on either total NR2C and NR2D subunit expression or cell surface NR1/NR2C and NR1/NR2D expression. A comparison of PSD-95α, PSD-95β and PSD-95α^C3S,C5S showed that PSD-95-enhanced cell surface expression of NR1/NR2A receptors was dependent upon the PSD-95 N-terminal C3,C5 cysteines. These observations support differential interaction of NMDA receptor subtypes with different PSD-95 MAGUK scaffolding proteins. This has implications for the stabilisation, turnover and compartmentalisation of NMDA receptor subtypes in neurones during development and in the mature brain.     

Ability of bone graft substitutes to support the osteoprogenitor cells: An in-vitro study

AIM: To compare seven commercially available bone graft substitutes(BGS) in terms of these properties and without using any additional biological growth factors.METHODS: Porcine osteoprogenitor cells were loaded on seven commercially available BGS and allowed to proliferate for one week followed by osteogenic induction. Staining for live/dead cells as well as scanning electron microscopy(SEM) was carried out to determine viability and cellular binding. Further outcome measures included alkaline phosphatase(ALP) assays with normalisation for DNA content to quantify osteogenic potential. Negative and positive control experiments were carried out in parallel to validate the results.RESULTS: Live/dead and SEM imaging showed higher viability and attachment with β-tricalcium phosphate(β-TCP) than with other BGS(P 0.05). The average ALP activity in nmol/mL(normalised value for DNA content in nmol/μg DNA) per sample was 657.58(132.03) for β-TCP, 36.22(unable to normalise) for calcium sulphate, 19.93(11.39) for the Hydroxyapatite/Tricalcium Phosphate composite, 14.79(18.53) for polygraft, 13.98(8.15) for the highly porous β-Tricalcium Phosphate, 5.56(10.0) for polymers, and 3.82(3.8) for Hydroxyapatite.CONCLUSION: Under the above experimental conditions, β-TCP was able to maintain better the viability of osteoprogenitor cells and allow proliferation and differentiation(P 0.05).     

Archaeal N-terminal protein maturation commonly involves N-terminal acetylation: a large-scale proteomics survey

We present the first large-scale survey of N-terminal protein maturation in archaea based on 873 proteomically identified N-terminal peptides from the two haloarchaea Halobacterium salinarum and Natronomonas pharaonis. The observed protein maturation pattern can be attributed to the combined action of methionine aminopeptidase and N-terminal acetyltransferase and applies to cytosolic proteins as well as to a large fraction of integral membrane proteins. Both N-terminal maturation processes primarily depend on the amino acid in penultimate position, in which serine and threonine residues are over represented. Removal of the initiator methionine occurs in two-thirds of the haloarchaeal proteins and requires a small penultimate residue, indicating that methionine aminopeptidase specificity is conserved across all domains of life. While N-terminal acetylation is rare in bacteria, our proteomic data show that acetylated N termini are common in archaea affecting about 15% of the proteins and revealing a distinct archaeal N-terminal acetylation pattern. Haloarchaeal N-terminal acetyltransferase reveals narrow substrate specificity, which is limited to cleaved N termini starting with serine or alanine residues. A comparative analysis of 140 ortholog pairs with identified N-terminal peptide showed that acetylatable N-terminal residues are predominantly conserved amongst the two haloarchaea. Only few exceptions from the general N-terminal acetylation pattern were observed, which probably represent protein-specific modifications as they were confirmed by ortholog comparison.     

High throughput two-dimensional blue-native electrophoresis: a tool for functional proteomics of cytoplasmatic protein complexes from Chlorobium tepidum

Chl. tepidum is a Gram-negative green-sulfur bacterium, which is strict by anaerobic and grows by utilizing sulfide or thiosulfate as an electron source. Blue native-polyacrylamide gel electrophoresis (BN-PAGE) is widely used for the analysis of oligomeric state and molecular mass non-dissociated protein complexes. In this study, a number of proteomic techniques were used to investigate the oligomeric state enzymes. In particular, the Chl. tepidum-soluble proteome was monitored under native condition by using BN-PAGE. The BN-PAGE protein complexes map was analyzed by MALDI-TOF MS after trypsin treatment and from 42 BN proteins bands, 62 different proteins were identified. Additionally, functional information regarding protein–protein interactions was assembled, by coupling 2-D BN-PAGE with MALDI-TOF MS. One-hundred and seventy gel bands were spotted, out of which 187 different proteins were identified. The identified proteins belong to various functional categories like energy metabolism, protein synthesis, amino acid biosynthesis, central intermediate metabolism, and biosynthesis of cofactors indicating the potential of the method for elucidation of functional proteomes.     

Plyometric exercise increases serum indices of muscle damage and collagen breakdown

The aim of the present study was to examine the effect of acute plyometric exercise on indices of muscle damage and collagen breakdown. Nine untrained men performed an intense bout of plyometric jumping exercises (experimental group) and nine men remained at rest (control group). Seven days before and 24, 48, and 72 hours after plyometric exercise or rest, several physiological and biochemical indices of muscle damage and two biochemical indices of collagen damage were determined. No significant changes in concentric and eccentric peak torque of knee extensors and flexors or flexion and extension range of motion were found after the plyometric exercise. Delayed-onset muscle soreness increased 48 hours after exercise. Creatine kinase increased 48 and 72 hours post exercise, whereas lactate dehydrogenase increased 24, 48, and 72 hours post exercise. Serum hydroxyproline increased 24 hours post exercise, peaked at 48 hours, and remained elevated up to 72 hours post exercise. Hydroxylysine (which was measured only before exercise and at 48 hours) was found increased 48 hours post exercise. No differences were found in any physiological or biochemical index in the control group. Intense plyometric exercise increased muscle damage, delayed-onset muscle soreness, and serum indices of collagen breakdown without a concomitant decrease in the functional capacity of muscles. Hydroxyproline and hydroxylysine levels in serum seem promising measures for describing exercise-induced collagen degradation. Coaches need to keep in mind that by using plyometric activities, despite the increased muscle damage and collagen turnover that follow, it is not necessarily accompanied by decreases in skeletal muscle capacity.     

Rescue from replication stress during mitosis

Genomic instability is a hallmark of cancer and a common feature of human disorders, characterized by growth defects, neurodegeneration, cancer predisposition, and aging. Recent evidence has shown that DNA replication stress is a major driver of genomic instability and tumorigenesis. Cells can undergo mitosis with under-replicated DNA or unresolved DNA structures, and specific pathways are dedicated to resolving these structures during mitosis, suggesting that mitotic rescue from replication stress (MRRS) is a key process influencing genome stability and cellular homeostasis. Deregulation of MRRS following oncogene activation or loss-of-function of caretaker genes may be the cause of chromosomal aberrations that promote cancer initiation and progression. In this review, we discuss the causes and consequences of replication stress, focusing on its persistence in mitosis as well as the mechanisms and factors involved in its resolution, and the potential impact of incomplete replication or aberrant MRRS on tumorigenesis, aging and disease.     

Isolation and Characterization of an Outer Membrane Protein of Chlorobium tepidum

A protein was isolated from membranes of the green sulfur bacterium Chlorobium tepidum. This protein was characterized by gel electrophoresis, gel filtration, analytical ultracentrifugation and amino acid sequencing. The molecular weight of the purified protein was shown to be 26 kDa by SDS-PAGE. HPLC gelfiltration, SDS-PAGE and analytical ultracentrifugation are consistent with the presence of a homogenous protein in the preparations. Amino acid analysis was obtained from the isolated protein after fragmentation with Lys-C, trypsin and cyanogen bromide. The cleavage pattern resulting from these treatments combined with Edman sequencing yield a sequence allowing the identification of an integral membrane agglutinin in Chl. tepidum.