Cation-exchange high-performance liquid chromatography: separation of highly basic proteins using volatile acidic solvents

The chromatographic behavior of a number of globular proteins was studied on a Bio-Sil TSK CM-2-SW weak cation exchange HPLC column under acidic conditions. A linear gradient of 0-1 M NH4Ac in 1 M HOAc, inducing a convex pH gradient from 2.4-4.8, resulted in an excellent separation of highly basic proteins. For these proteins a linear relationship between isoelectric point and retention time was determined experimentally. The effect of pH and the ion composition of the eluting buffer system on this linear correlation was studied. Although the exact basis for protein separation on the CM-2-SW column at low pH is not clear yet, both the pH-dependent net positive charge per unit surface area and most likely the relative percentage of arginine in the total number of basic residues contribute to this separation. Because of the high resolving power and the high protein recovery obtained in a system using only acidic volatile buffer solutions, the cation exchanger is particularly suitable for the purification of nanogram amounts of acid-stable basic growth factors. The present sterile conditions (1 M HOAc/NH4Ac system, pH less than 4) and the easy removal of salt by lyophilization facilitate the detection of these proteins by biological assays.     

Reduced Context Effects on Retrieval in First-Episode Schizophrenia

Background

A recent modeling study by the authors predicted that contextual information is poorly integrated into episodic representations in schizophrenia, and that this is a main cause of the retrieval deficits seen in schizophrenia.

Methodology/Principal Findings

We have tested this prediction in patients with first-episode schizophrenia and matched controls. The benefit from contextual cues in retrieval was strongly reduced in patients. On the other hand, retrieval based on item cues was spared.

Conclusions/Significance

These results suggest that reduced integration of context information into episodic representations is a core deficit in schizophrenia and one of the main causes of episodic memory impairment.     

z-VAD-fmk-induced non-apoptotic cell death of macrophages: possibilities and limitations for atherosclerotic plaque stabilization

Macrophages play a pivotal role in atherosclerotic plaque destabilization in contrast to smooth muscle cells (SMCs). As a consequence, removal of macrophages from plaques via selective induction of cell death represents a promising approach to stabilize non-obstructive, rupture-prone atherosclerotic lesions. However, the mechanisms to initiate cell death in macrophages but not in other cell types of the plaque, in particular SMCs, are unknown. Recently, we have shown that the pan-caspase inhibitor z-VAD-fmk induces autophagy and necrotic cell death in J774A.1 and RAW264.7 macrophages as well as in IFN-gamma primed primary mouse peritoneal macrophages, but not in vascular SMCs or C2C12 myoblasts. The different sensitivity to z-VAD-fmk is largely based on differential expression of receptor-interacting protein 1 (RIP1). This finding suggests that caspase inhibition activates RIP1 which in turn initiates autophagy, although other explanations should be taken into account. z-VAD-fmk-treated J774A.1 macrophages overexpress and secrete several chemokines and cytokines, including TNFalpha. The combination of z-VAD-fmk and TNFalpha, but not TNFalpha alone, induces SMC necrosis. In this regard, z-VAD-fmk is detrimental and not beneficial for atherosclerotic plaque stability due to stimulation of inflammatory responses and indirect induction of SMC death. Future work is needed to determine the mechanism(s) that selectively trigger non-apoptotic cell death in plaque macrophages without evoking inflammation and SMC death.     

PDGF-like growth factor induces EGF-potentiated phenotypic transformation of normal rat kidney cells in the absence of TGF beta

Using a growth factor defined assay for anchorage-independent growth (van Zoelen, E.J.J., van Oostwaard, Th.M.J., van der Saag, P.T. and de Laat, S.W. (1985) J. Cell. Physiol. 123, 151- 160, we have studied the ability of polypeptide growth factors produced by Neuro-2A neuroblastoma cells to induce anchorage-independent growth of normal rat kidney cells. Neuro-2A cells produce and secrete a PDGF-like growth factor in addition to TGF beta, which can be fully separated from each other by means of reverse-phase HPLC. Using a new, very sensitive technique for detection of TGF beta in growth factor samples based on its additional ability to act as a growth inhibitory factor, it is shown that the PDGF-like growth factor does not contain any detectable TGF beta. Still this neuroblastoma derived PDGF-like growth factor is able to induce anchorage-independent growth of NRK cells, particularly in the additional presence of EGF. It is concluded that under growth factor defined assay conditions TGF beta is not essential for phenotypic transformation of NRK cells.     

Alternative dimerization is required for activity and inhibition of the HEPN ribonuclease RnlA

The rnlAB toxin-antitoxin operon from Escherichia coli functions as an anti-phage defense system. RnlA was identified as a member of the HEPN (Higher Eukaryotes and Prokaryotes Nucleotide-binding domain) superfamily of ribonucleases. The activity of the toxin RnlA requires tight regulation by the antitoxin RnlB, the mechanism of which remains unknown. Here we show that RnlA exists in an equilibrium between two different homodimer states: an inactive resting state and an active canonical HEPN dimer. Mutants interfering with the transition between states show that canonical HEPN dimerization via the highly conserved RX_4-6H motif is required for activity. The antitoxin RnlB binds the canonical HEPN dimer conformation, inhibiting RnlA by blocking access to its active site. Single-alanine substitutions mutants of the highly conserved R255, E258, R318 and H323 show that these residues are involved in catalysis and substrate binding and locate the catalytic site near the dimer interface of the canonical HEPN dimer rather than in a groove located between the HEPN domain and the preceding TBP-like domain. Overall, these findings elucidate the structural basis of the activity and inhibition of RnlA and highlight the crucial role of conformational heterogeneity in protein function.     

Monitoring of cell therapy and assessment of cardiac function using magnetic resonance imaging in a mouse model of myocardial infarction

We have developed a mouse severe combined immunodeficient (SCID) model of myocardial infarction based on permanent coronary artery occlusion that allows long-term functional analysis of engrafted human embryonic stem cell-derived cardiomyocytes, genetically marked with green fluorescent protein (GFP), in the mouse heart. We describe methods for delivery of dissociated cardiomyocytes to the left ventricle that minimize scar formation and visualization and validation of the identity of the engrafted cells using the GFP emission spectrum, and histological techniques compatible with GFP epifluorescence, for monitoring phenotypic changes in the grafts in vivo. In addition, we describe how magnetic resonance imaging can be adapted for use in mice to monitor cardiac function non-invasively and repeatedly. The model can be adapted to include multiple control or other cell populations. The procedure for a cohort of six mice can be completed in a maximum of 13 weeks, depending on follow-up, with 30 h of hands-on time.     

Signatures of selection in the three‐spined stickleback along a small‐scale brackish water – freshwater transition zone

Local adaptation is often obvious when gene flow is impeded, such as observed at large spatial scales and across strong ecological contrasts. However, it becomes less certain at small scales such as between adjacent populations or across weak ecological contrasts, when gene flow is strong. While studies on genomic adaptation tend to focus on the former, less is known about the genomic targets of natural selection in the latter situation. In this study, we investigate genomic adaptation in populations of the three‐spined stickleback Gasterosteus aculeatus L. across a small‐scale ecological transition with salinities ranging from brackish to fresh. Adaptation to salinity has been repeatedly demonstrated in this species. A genome scan based on 87 microsatellite markers revealed only few signatures of selection, likely owing to the constraints that homogenizing gene flow puts on adaptive divergence. However, the detected loci appear repeatedly as targets of selection in similar studies of genomic adaptation in the three‐spined stickleback. We conclude that the signature of genomic selection in the face of strong gene flow is weak, yet detectable. We argue that the range of studies of genomic divergence should be extended to include more systems characterized by limited geographical and ecological isolation, which is often a realistic setting in nature.