The dorsal reverse adipofascial flap for fingertip reconstruction

Nine patients who presented with fingertip amputations were treated with the dorsal reverse adipofascial flap. The mean age of the patients was 41.3 years and the mean follow-up was 18 months. The flap described here was used only for amputations at the level of the nail fold, from approximately the lunula to the proximal nail matrix. This flap is based on the dorsal arterial branches that originate from the volar digital arteries just distal to the distal interphalangeal joint. The flap uses only the adipofascial tissue over the middle phalanx of the injured finger; it is turned over to cover the fingertip defect and then covered with a split-thickness skin graft. All flaps survived completely, and the patients continue to use their fingertips as before the amputation injury. This one-step operation is easily performed (even in the emergency department), makes no use of the adjacent digits, and provides a pleasing and stable cover for the fingertips.     

Enhancement of cytochrome c catalytic behaviour by affecting the heme environment using functionalized carbon-based nanomaterials

The effect of carbon-based nanomaterials (CBNs), such as multi-wall carbon nanotubes (CNTs) and graphene oxide (GO) nanomaterials functionalized with carboxyl, alkyl and amine groups, on the peroxidase-like activity and structure of cytochrome c (cyt c) was investigated. The catalytic efficiency of cyt c increases up to 78-fold in the presence of graphene oxide and up to 2.5-fold in the presence of other functionalized CBNs. Moreover, the use of functionalized CBNs enhances the thermal stability of the protein as well as its tolerance against hydrogen peroxide up to 2.5-fold. UV–vis and circular dichroism spectroscopy studies suggest that the increase in the peroxidase activity of cyt c in the presence of some functionalized GO nanomaterials, correlates to perturbations of the heme microenvironment, while the secondary structure of the enzyme remains intact. These results indicate that the beneficial effect the functionalized CBNs have on the activity and on the stability of cyt c depends on CBNs geometry and surface functionalization.     

Arabidopsis Sec1/Munc18 Protein SEC11 Is a Competitive and Dynamic Modulator of SNARE Binding and SYP121-Dependent Vesicle Traffic

The Arabidopsis thaliana Qa-SNARE SYP121 (=SYR1/PEN1) drives vesicle traffic at the plasma membrane of cells throughout the vegetative plant. It facilitates responses to drought, to the water stress hormone abscisic acid, and to pathogen attack, and it is essential for recovery from so-called programmed stomatal closure. How SYP121-mediated traffic is regulated is largely unknown, although it is thought to depend on formation of a fusion-competent SNARE core complex with the cognate partners VAMP721 and SNAP33. Like SYP121, the Arabidopsis Sec1/Munc18 protein SEC11 (=KEULE) is expressed throughout the vegetative plant. We find that SEC11 binds directly with SYP121 both in vitro and in vivo to affect secretory traffic. Binding occurs through two distinct modes, one requiring only SEC11 and SYP121 and the second dependent on assembly of a complex with VAMP721 and SNAP33. SEC11 competes dynamically for SYP121 binding with SNAP33 and VAMP721, and this competition is predicated by SEC11 association with the N terminus of SYP121. These and additional data are consistent with a model in which SYP121-mediated vesicle fusion is regulated by an unusual “handshaking” mechanism of concerted SEC11 debinding and rebinding. They also implicate one or more factors that alter or disrupt SEC11 association with the SYP121 N terminus as an early step initiating SNARE complex formation.     

Conjugation with polyamines enhances the antibacterial and anticancer activity of chloramphenicol

Chloramphenicol (CAM) is a broad-spectrum antibiotic, limited to occasional only use in developed countries because of its potential toxicity. To explore the influence of polyamines on the uptake and activity of CAM into cells, a series of polyamine–CAM conjugates were synthesized. Both polyamine architecture and the position of CAM-scaffold substitution were crucial in augmenting the antibacterial and anticancer potency of the synthesized conjugates. Compounds 4 and 5, prepared by replacement of dichloro-acetyl group of CAM with succinic acid attached to N4 and N1 positions of N⁸,N⁸-dibenzylspermidine, respectively, exhibited higher activity than CAM in inhibiting the puromycin reaction in a bacterial cell-free system. Kinetic and footprinting analysis revealed that whereas the CAM-scaffold preserved its role in competing with the binding of aminoacyl-tRNA 3′-terminus to ribosomal A-site, the polyamine-tail could interfere with the rotatory motion of aminoacyl-tRNA 3′-terminus toward the P-site. Compared to CAM, compounds 4 and 5 exhibited comparable or improved antibacterial activity, particularly against CAM-resistant strains. Compound 4 also possessed enhanced toxicity against human cancer cells, and lower toxicity against healthy human cells. Thus, the designed conjugates proved to be suitable tools in investigating the ribosomal catalytic center plasticity and some of them exhibited greater efficacy than CAM itself.     

Mechanisms of programmed cell death during oogenesis in <Emphasis Type="Italic">Drosophila virilis</Emphasis>

We describe the features of programmed cell death occurring in the egg chambers of Drosophila virilis during mid-oogenesis and late oogenesis. During mid-oogenesis, the spontaneously degenerating egg chambers exhibit typical characteristics of apoptotic cell death. As revealed by propidium iodide, rhodamine-conjugated phalloidin staining, and the TUNEL assay, respectively, the nurse cells contain condensed chromatin, altered actin cytoskeleton, and fragmented DNA. In vitro caspase activity assays and immunostaining procedures demonstrate that the atretic egg chambers possess high levels of caspase activity. Features of autophagic cell death are also observed during D. virilis mid-oogenesis, as shown by monodansylcadaverine staining, together with an ultrastructural examination by transmission electron microscopy. During the late stages of oogenesis in D. virilis, once again, the two mechanisms, viz., nurse cell cluster apoptosis and autophagy, operate together, manifesting features of cell death similar to those detailed above. Moreover, an altered form of cytochrome c seems to be released from the mitochondria in the nurse cells proximal to the oocyte. We propose that apoptosis and autophagy function synergistically during oogenesis in D. virilis in order to achieve a more efficient elimination of the degenerated nurse cells and abnormal egg chambers. The present study was co-financed within Op. Education by the European Social Fund and by National Resources via a grant (HRAKLEITOS 70/3/7164) to Professor L.H. Margaritis.     

Hydrogen peroxide inhibits caspase-dependent apoptosis by inactivating procaspase-9 in an iron-dependent manner

Apoptosis represents a physiological form of cell death, the perturbation of which may contribute to the development of several diseases connected with accumulation of unwanted cells or excessive cell loss. We have previously shown that the continuous presence of low concentrations of H2O2 (generated by the action of glucose oxidase) was able to inhibit caspase-mediated apoptosis in Jurkat cells. The main purpose of the present study was to elucidate the exact molecular mechanism(s) underlying this inhibitory action of H2O2. The results presented show that events like outer mitochondrial membrane permeabilization, release of cytochrome c from mitochondria, oligomerization of Apaf-1, and recruitment of procaspase-9 to apoptosomes were taking place normally, but further advancement toward activation of the execution caspases was interrupted when H2O2 was present during the apoptotic process. From the results presented in this work, it emerges that the inhibition of procaspase-9 autoactivation was probably due to the reversible oxidation of sensitive cysteine residues in this molecule. Remarkably, caspase-9 activation and the ensuing caspase cascade proceeded normally in the presence of H2O2 under conditions of iron deprivation, indicating that the inhibition of procaspase-9 activation was an iron-dependent process. Collectively, these results highlighted the potential role of available intracellular iron ions in signaling mechanisms related to apoptotic cell death.     

Association of NADPH oxidase p22phox gene C242T, A640G and −930A/G polymorphisms with primary knee osteoarthritis in the Greek population

Osteoarthritis (OA) is the most common form of arthritis with still unknown pathogenic etiology and considerable contribution of genetic factors. Recently, a new emerging role of oxidative stress in the pathology of OA has been reported, lacking however elucidation of the underlying mechanism. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase being a complex enzyme produced by chondrocytes, presents the major source of reactive oxygen species and main contributor of increased oxidative stress. The present study aims to evaluate the association of NADPH oxidase p22phox gene C242T, A640G and ?A930G polymorphisms with primary knee OA in the Greek population. One hundred fifty five patients with primary symptomatic knee OA participated in the study along with 139 matched controls. Genotypes were determined using polymerase chain reaction and restriction fragment length polymorphism technique. Allelic and genotypic frequencies were compared between both study groups. NADPH p22phox ?A930G polymorphism was significantly associated with knee OA in the crude analysis (P = 0.018). No significant difference was detected for C242T and A640G polymorphisms (P > 0.05). The association between ?A930G polymorphism and knee OA disappeared when the results were adjusted for obesity (P = 0.078, odds ratio 0.54, 95 % CI 0.272–1.071). The interaction between all three polymorphisms was not significant. The present study shows that NADPH oxidase p22phox gene C242T, A640G and ?A930G polymorphisms are not risk factors for knee OA susceptibility in the Greek population. Further studies are needed to give a global view of the importance of this polymorphism in the pathogenesis of OA.     

Isolation and partial characterisation of a putative monoterpene synthase from Melaleuca alternifolia.

Melaleuca alternifolia (Cheel) is an Australia native tree harvested for its monoterpene-rich, essential oil. Monoterpene synthases (E.C. 4.2.3.20) were partially purified from the flush growth of the commercially important, high terpinen-4-ol chemotype of M. alternifolia. The purified fractions produced an acyclic monoterpene, linalool that is not present in the essential oil. To further characterise the monoterpene synthase, a cDNA library was constructed and 500 expressed sequence tags (ESTs) were sequenced to isolate putative terpene synthases. A single clone with similarity to the TspB gene sub-family of angiosperm monoterpene and isoprene synthases was isolated but was truncated at the 5' end. This single clone was used to design a probe for a cDNA library and was applied to isolate a full-length clone. This gene encoded a polypeptide 583 amino acids in length (67 kDa) including a putative transit peptide. Heterologous expression of the gene in Escherichia coli and subsequent assay of the recombinant enzyme did not result in the production of terpinen-4-ol, the major constituent of tea tree oil, or of its precursor sabinene hydrate. Significant quantities of linalool were observed in these assays, and in the assays of monoterpene synthase activity of a native enzyme in vitro, but the racemic nature of the linalool means that it may have a non-enzymatic origin.     

Degradation of Activated K-Ras Orthologue via K-Ras-specific Lysine Residues Is Required for Cytokinesis

Mammalian cells encode three closely related Ras proteins, H-Ras, N-Ras, and K-Ras. Oncogenic K-Ras mutations frequently occur in human cancers, which lead to dysregulated cell proliferation and genomic instability. However, mechanistic role of the Ras isoform regulation have remained largely unknown. Furthermore, the dynamics and function of negative regulation of GTP-loaded K-Ras have not been fully investigated. Here, we demonstrate RasG, the Dictyostelium orthologue of K-Ras, is targeted for degradation by polyubiquitination. Both ubiquitination and degradation of RasG were strictly associated with RasG activity. High resolution tandem mass spectrometry (LC-MS/MS) analysis indicated that RasG ubiquitination occurs at C-terminal lysines equivalent to lysines found in human K-Ras but not in H-Ras and N-Ras homologues. Substitution of these lysine residues with arginines (4KR-RasG) diminished RasG ubiquitination and increased RasG protein stability. Cells expressing 4KR-RasG failed to undergo proper cytokinesis and resulted in multinucleated cells. Ectopically expressed human K-Ras undergoes polyubiquitin-mediated degradation in Dictyostelium, whereas human H-Ras and a Dictyostelium H-Ras homologue (RasC) are refractory to ubiquitination. Our results indicate the existence of GTP-loaded K-Ras orthologue-specific degradation system in Dictyostelium, and further identification of the responsible E3-ligase may provide a novel therapeutic approach against K-Ras-mutated cancers.