c-Myc localization within the nucleus: evidence for association with the PML nuclear body

Definitive localization of c-Myc within the nucleus is important to fully understand the regulation and function of this oncoprotein. Studies of c-Myc distribution, however, have produced conflicting results. To overcome technical challenges inherent in c-Myc cytology, we use here three methods to visualize c-Myc and in addition examine the impact of proteasome inhibition. EYFP or HA-tagged Myc was reintroduced by stable transfection into myc null diploid rat fibroblasts, replacing endogenous Myc with tagged Myc expressed at or near normal levels. This tagged Myc is shown to functionally replace the endogenous Myc by restoration of normal cell morphology and growth rate. We were able to confirm key findings using antibodies to the endogenous c-Myc and/or its partner, Max. Contrary to some published reports, by all three methods the c-Myc protein in rat fibroblasts distributes predominantly throughout the nucleus in a dispersed granular pattern, avoiding the nucleolus. Importantly, however, several findings provide evidence for an unanticipated relationship between c-Myc and PML nuclear bodies, which is enhanced under conditions of proteasome inhibition. Evidence of Max concentration within PML bodies is shown both with and without proteasome inhibition, strengthening the relationship between PML bodies and Myc/Max. Some accumulation of Myc and Max in nucleoli upon proteasome inhibition is also observed, although co-localization of ubiquitin was only seen with PML bodies. This work provides a comprehensive study of c-Myc distribution and also presents the first evidence of a relationship between turnover of this oncoprotein and PML nuclear bodies, known to break down in certain cancers.     

Identification of synaptic plasma membrane proteins co-precipitated with fibrillar beta-amyloid peptide

The beta-amyloid peptide that is overproduced in Alzheimer's disease rapidly forms fibrils, which are able to interact with various molecular partners. This study aimed to identify abundant synaptosomal proteins binding to the fibrillar beta-amyloid (fAbeta) 1-42. Triton X-100-soluble proteins were extracted from the rat synaptic plasma membrane fraction. Interacting proteins were isolated by co-precipitation with fAbeta, or with fibrillar crystallin as a negative control. Protein identification was accomplished (1) by separating the tryptically digested peptides of the protein pellet by one-dimensional reversed-phase HPLC and analysing them using an ion-trap mass spectrometer with electrospray ionization; and (2) by subjecting the precipitated proteins to gel electrophoretic fractionation, in-gel tryptic digestion and to matrix-assisted laser desorption/ionization time-of-flight mass measurements and post-source decay analysis. Six different synaptosomal proteins co-precipitated with fAbeta were identified by both methods: vacuolar proton-pump ATP synthase, glyceraldehyde-3-phosphate dehydrogenase, synapsins I and II, beta-tubulin and 2',3'-cyclic nucleotide 3'-phosphodiesterase. Most of these proteins have already been associated with Alzheimer's disease, and the biological and pathophysiological significance of their interaction with fAbeta is discussed.     

Amino acid preferences for a critical substrate binding subsite of retroviral proteases in type 1 cleavage sites

The specificities of the proteases of 11 retroviruses representing each of the seven genera of the family Retroviridae were studied using a series of oligopeptides with amino acid substitutions in the P2 position of a naturally occurring type 1 cleavage site (Val-Ser-Gln-Asn-Tyr Pro-Ile-Val-Gln; the arrow indicates the site of cleavage) in human immunodeficiency virus type 1 (HIV-1). This position was previously found to be one of the most critical in determining the substrate specificity differences of retroviral proteases. Specificities at this position were compared for HIV-1, HIV-2, equine infectious anemia virus, avian myeloblastosis virus, Mason-Pfizer monkey virus, mouse mammary tumor virus, Moloney murine leukemia virus, human T-cell leukemia virus type 1, bovine leukemia virus, human foamy virus, and walleye dermal sarcoma virus proteases. Three types of P2 preferences were observed: a subgroup of proteases preferred small hydrophobic side chains (Ala and Cys), and another subgroup preferred large hydrophobic residues (Ile and Leu), while the protease of HIV-1 preferred an Asn residue. The specificity distinctions among the proteases correlated well with the phylogenetic tree of retroviruses prepared solely based on the protease sequences. Molecular models for all of the proteases studied were built, and they were used to interpret the results. While size complementarities appear to be the main specificity-determining features of the S2 subsite of retroviral proteases, electrostatic contributions may play a role only in the case of HIV proteases. In most cases the P2 residues of naturally occurring type 1 cleavage site sequences of the studied proteases agreed well with the observed P2 preferences.     

Inp1p is a peroxisomal membrane protein required for peroxisome inheritance in Saccharomyces cerevisiae

Cells have evolved molecular mechanisms for the efficient transmission of organelles during cell division. Little is known about how peroxisomes are inherited. Inp1p is a peripheral membrane protein of peroxisomes of Saccharomyces cerevisiae that affects both the morphology of peroxisomes and their partitioning during cell division. In vivo 4-dimensional video microscopy showed an inability of mother cells to retain a subset of peroxisomes in dividing cells lacking the INP1 gene, whereas cells overexpressing INP1 exhibited immobilized peroxisomes that failed to be partitioned to the bud. Overproduced Inp1p localized to both peroxisomes and the cell cortex, supporting an interaction of Inp1p with specific structures lining the cell periphery. The levels of Inp1p vary with the cell cycle. Inp1p binds Pex25p, Pex30p, and Vps1p, which have been implicated in controlling peroxisome division. Our findings are consistent with Inp1p acting as a factor that retains peroxisomes in cells and controls peroxisome division. Inp1p is the first peroxisomal protein directly implicated in peroxisome inheritance.     

IFITM/Mil/fragilis family proteins IFITM1 and IFITM3 play distinct roles in mouse primordial germ cell homing and repulsion

The family of interferon-induced transmembrane protein (Ifitm/mil/fragilis) genes encodes cell surface proteins that may modulate cell adhesion and influence cell differentiation. Mouse Ifitm1 and -3, which are expressed in primordial germ cells (PGCs), are implicated to have roles in germ cell development, but the specific functions have been unclear. Our results show that Ifitm1 activity is required for PGC transit from the mesoderm into the endoderm, and that it appears to act via a repulsive mechanism, such that PGCs avoid Ifitm1-expressing tissues. In contrast, Ifitm3, which is expressed in migratory PGCs, is sufficient to confer autonomous PGC-like homing properties to somatic cells. These guidance activities are mediated by the N-terminal extracellular domain of the specific IFITM, which cannot be substituted by that of another family member. Complex homo- and/or heterotypic intercellular interactions among various IFITMs in PGCs and neighboring cells may underpin coordinated germ cell guidance in mice.     

TT-232: An Anti-tumour and Anti-inflammatory Peptide Therapeutic in Clinical Development

TT-232 is a structural derivative of the peptide hormone somatostatin with selective anti-proliferative and anti-inflammatory properties. It has a strong anti-tumour activity both in vitro and in vivo on a wide range of tumour models and induces apoptosis. Its anti-tumour activity is mediated through the SSTR1 receptor and by the tumour specific isoform of pyruvate kinase. TT-232 has been shown to be a potent neurogenic inflammation inhibitory, anti-inflammatory and analgesic agent with a broader spectrum than presently available anti-inflammatory/analgesic drugs. In animal models it is effective against neurogenic inflammation and blocks neuropathic hyperalgesia where COX-1 or COX-2 inhibitors (e.g. diclofenac or meloxicam) proved ineffective. TT-232 has passed phase I clinical trials without toxicity and significant side effects. Human phase II efficacy studies are ongoing in melanoma indication. Two more oncological indications and phase II clinical trials in inflammatory diseases, including rheumatoid arthritis and burn injuries are in preparation. This compound has the perspective to become the first drug in molecularly targeted therapy of inflammation where a combined effect of anti-inflammatory, analgesic and neurogenic inflammation inhibiting activity can be achieved.     

A kinetic model for suspended and attached growth of a defined mixed culture

Kinetic experiments were carried out in a semicontinuous wastewater treatment process called self-cycling fermentation (SCF) using a defined mixed culture and various concentrations of synthetic brewery wastewater. The same consortium, which had been previously identified as Acinetobacter sp., Enterobacter sp., and Candida sp., were used in these experiments. The overall rate of substrate removal was attributable to both suspended microbes and the biofilm that formed during the treatment process. A rate expression was developed for the SCF system for a range of synthetic wastewaters containing glucose and various initial concentrations of ethanol and maltose. The data indicated that substrate removal by the suspended cells was directly related to the biomass concentration. However, substrate removal by the biofilm was apparently not affected by the biofilm thickness and was a function of substrate concentration only.     

Catechol Oxidase and SOD Mimicking by Copper(II) Complexes of Multihistidine Peptides

Copper(II) complexes of five peptide ligands containing at least three histidine residues have been tested as catalysts in catechol oxidation and superoxide dismutation. All systems exhibit considerable catechol oxidase-like activity, and the Michaelis–Menten enzyme kinetic model is applicable in all cases. Beside the Michaelis–Menten parameters, the effects of pH, catalyst and dioxygen concentration on the reaction rates are also reported. Considering the rather different sequences, the observed oxidase activity seems to be a general behavior of copper(II) complexes with multihistidine peptides. Interestingly, in all cases {N_im/2N_im,2N^?} coordinated complexes are the pre-active species, the bound amide nitrogens were proposed to be an acid/base site for facilitating substrate binding. The studied copper(II)-peptide complexes are also able to effectively dismutate superoxide radical in the neutral pH range.     

Cardiovascular risk factors differently affect the survival of patients undergoing manual or mechanical resuscitation

Background

Chest compression is a decisive element of cardio-pulmonary resuscitation (CPR). By applying a mechanical CPR device, compression interruptions can be minimised. We examined the efficiency of manual and device-assisted resuscitation as well as the effects of cardiovascular risk factors on the outcome of resuscitation.

Methods

In our retrospective, randomised 3-year study the data of adult patients suffering non-traumatic, out-of-hospital, sudden cardiac death (SCD) were analysed (n?=?287). The data were retrieved by processing case reports, Utstein sheets and acute coronary syndrome sheets. We compared the data of patients undergoing manual (n?=?232) and device-assisted resuscitation (LUCAS-2, n?=?55). The primary endpoint was the on-site restoration of spontaneous circulation (ROSC).

Results and conclusion

In 37% of the cases ROSC happened. With respect to ROSC an insignificantly more favourable tendency was demonstrated in the case of device-assisted resuscitation (p =?0.072). In the Lucas group, a higher success rate occurred even in the case of prolonged resuscitation. We found a better outcome in the Lucas group in the case of CPR started a longer time after the SCD (p?<?0.05). A positive correlation was established between age and unsuccessful resuscitation (p?=?<?0.017; r?=?0.125). An unfavourable correlation was observed between hypertension and the outcome of resuscitation (p?=?0.018; r?=?0.143). According to our results the presence of left ventricular hypertrophy poses 5.1-fold risk of unsuccessful CPR (CI: 4.97–5.29).Advanced age and structural heart diseases can play a role in the genesis of SCD. Importantly, left ventricular hypertrophy and hypertension negatively affect survival.