6-Methylmercaptopurine Riboside Is a Potent and Selective Inhibitor of Nerve Growth Factor-Activated Protein Kinase N

Protein kinase N (PKN) is a soluble, apparently novel serine protein kinase that is activated by nerve growth factor (NGF) and other agents in PC12 pheochromocytoma cells as well as in several nonneuronal cell lines. Purine analogs, such as 6-thioguanine and 2-aminopurine, have been found to inhibit PKN in vitro. When applied to intact cells, these compounds suppress certain biological responses to NGF, but not others, a findings suggesting the presence of multiple pathways in the NGF mechanism. We report here that 6-methylmercaptopurine riboside (6-MMPR) inhibits NGF-stimulated PKN activity in vitro with an apparent Ki of approximately 5 nM. This is approximately 1,000-fold lower than the Ki of the most potent purine inhibitor of PKN. Compounds similar to 6-MMPR, but lacking the methyl or riboside groups, were much less potent as PKN inhibitors. A survey of six additional purified protein kinases shows no inhibitory effect of 6-MMPR, thus indicating a good degree of specificity of this compound for PKN. In contrast to NGF-stimulated PKN, a PKN-like activity stimulated in PC12 cells in response to activation of cyclic AMP-dependent protein kinase was nearly insensitive to 6-MMPR. Application of 6-MMPR to intact PC12 cells resulted in blockade of several responses to NGF (neurite regeneration and ornithine decarboxylase induction) but not of several others (rapid enhancement of tyrosine hydroxylase phosphorylation and PKN activation). These findings suggest that 6-MMPR is a potent and selective agent for characterizing PKN in vitro and for assessing its potential role in the multiple pathways of the NGF mechanism of action.     

First appearance of Globorotalia truncatulinoides: cladogenesis and immigration

The first appearance datum of the planktic foraminifer Globorotalia truncatulinoides is widely used to identify the base of the Quaternary Period. However, its appearance is not globally isochronous. We have previously shown that G. truncatulinoides evolved gradually from its ancestor Globorotalia crassaformis via the intermediate species Globorotalia tosaensis. This cladogenesis was documented in the southwest Pacific during the Late Pliocene in sympatric or parapatric populations. Based on qualitative observations, similar but younger, gradual transitions have been reported from other areas of the world's oceans. Therefore, this gradual evolutionary branching might have occurred in response to changing environments at different times in different ocean areas. To evaluate the hypothesis of a repeated, environmentally driven gradual, sympatric cladogenesis, we studied the morphological transitions of the three taxa, using image analytical techniques, in several deep-sea sections from various areas. Our study confirms that G. truncatulinoides evolved between 2.8 and 2.3 Ma sympatrically in large populations from its ancestor G. crassaformis in the southwest Pacific. Differentiated morphotypes of G. truncatulinoides subsequently immigrated into the Indian and Atlantic oceans between 2.3 and 1.9 Ma. Our morphometric data show these younger appearances outside the southwest Pacific to be punctuated.We hypothesize that the global cooling of surface waters, coinciding with the northern hemisphere glaciation, led to the formation of oceanographic barriers that could have retarded the expansion of G. truncatulinoides up to 2.3 Ma. At this time, a relative warming and subsequent transgression could have spurred the migration, possibly through the Indonesian passage. A direct link between the speciation and surface water changes linked to the northern hemisphere glaciation has not been proven so far and seems unlikely. In fact, stable isotope data in this lineage indicate that the three species' depth habitat preferences remained unchanged through the speciation and migration of G. truncatulinoides and that all three species were dominantly deep-dwellers, in agreement with their present environmental preferences.     

Fast Room Temperature Very Low Field-Magnetic Resonance Imaging System Compatible with MagnetoEncephaloGraphy Environment

In recent years, ultra-low field (ULF)-MRI is being given more and more attention, due to the possibility of integrating ULF-MRI and Magnetoencephalography (MEG) in the same device. Despite the signal-to-noise ratio (SNR) reduction, there are several advantages to operating at ULF, including increased tissue contrast, reduced cost and weight of the scanners, the potential to image patients that are not compatible with clinical scanners, and the opportunity to integrate different imaging modalities. The majority of ULF-MRI systems are based, until now, on magnetic field pulsed techniques for increasing SNR, using SQUID based detectors with Larmor frequencies in the kHz range. Although promising results were recently obtained with such systems, it is an open question whether similar SNR and reduced acquisition time can be achieved with simpler devices. In this work a room-temperature, MEG-compatible very-low field (VLF)-MRI device working in the range of several hundred kHz without sample pre-polarization is presented. This preserves many advantages of ULF-MRI, but for equivalent imaging conditions and SNR we achieve reduced imaging time based on preliminary results using phantoms and ex-vivo rabbits heads.     

Elements of the C-terminal t peptide of acetylcholinesterase that determine amphiphilicity, homomeric and heteromeric associations, secretion and degradation.

The C-terminal t peptide (40 residues) of vertebrate acetylcholinesterase (AChE) T subunits possesses a series of seven conserved aromatic residues and forms an amphiphilic alpha-helix; it allows the formation of homo-oligomers (monomers, dimers and tetramers) and heteromeric associations with the anchoring proteins, ColQ and PRiMA, which contain a proline-rich motif (PRAD). We analyzed the influence of mutations in the t peptide of Torpedo AChE(T) on oligomerization and secretion. Charged residues influenced the distribution of homo-oligomers but had little effect on the heteromeric association with Q(N), a PRAD-containing N-terminal fragment of ColQ. The formation of homo-tetramers and Q(N)-linked tetramers required a central core of four aromatic residues and a peptide segment extending to residue 31; the last nine residues (32-40) were not necessary, although the formation of disulfide bonds by cysteine C37 stabilized T(4) and T(4)-Q(N) tetramers. The last two residues of the t peptide (EL) induced a partial intracellular retention; replacement of the C-terminal CAEL tetrapeptide by KDEL did not prevent tetramerization and heteromeric association with Q(N), indicating that these associations take place in the endoplasmic reticulum. Mutations that disorganize the alpha-helical structure of the t peptide were found to enhance degradation. Co-expression with Q(N) generally increased secretion, mostly as T(4)-Q(N) complexes, but reduced it for some mutants. Thus, mutations in this small, autonomous interaction domain bring information on the features that determine oligomeric associations of AChE(T) subunits and the choice between secretion and degradation.     

Heparin binding to human plasma low-density lipoproteins: dependence on heparin sulfation degree and chain length.

Binding between low-density lipoproteins (LDL) and fluorescein-labeled heparin was studied quantitatively with a modified form of a published procedure [Cardin, A. D., Randall, C. I., Hirose, N., & Jackson, R. L. (1987) Biochemistry 26, 5513-5518], using fluorescence anisotropy titrations. Assumption of binding site equivalence satisfactorily interpreted experimental data. Accordingly, the apparent total capacity, n, and the average dissociation constant, Kd, were estimated as n approximately 24 disaccharides per LDL particle and Kd approximately 4 microM in 0.05 M HEPES/0.1 M NaCl, pH 7.4, 22 degrees C. Competition experiments with unlabeled heparins were exploited for the quantitative study of Kd as a function of heparin chain length and sulfation degree (ns = sulfate groups per disaccharide). The former parameter was investigated with a series of bovine lung heparin fractions with Mw ranging from 1,800 to 21,000 and constant sulfation degree (ns = 2.8 +/- 0.1). A series of physically fractionated or chemically modified heparins having 1.2 less than ns less than 3.5 were used to explore the dependence on sulfation degree. LDL affinity was found to increase with increasing both ns and Mw: an empirical Mw-1.6 dependence represented very well the chain length data set; a linear dependence was observed for log Kd as a function of ns, after appropriate allowance was made for chain length differences among samples. This regularity confirmed that LDL-heparin binding is mainly driven by electrostatic forces.(ABSTRACT TRUNCATED AT 250 WORDS)     

Improvement in the lipid extraction of staphylococcal cells by lysostaphin treatment

A new method for extracting lipids from Staphylococcus aureus is described in this paper. The extracts of cells treated with lysostaphin are compared with those from untreated cells. The recovery of lipids improved with the enzymatic treatment. Tentative identification of the components of the lipid extracts has been carried out.     

How will fish that evolved at constant sub‐zero temperatures cope with global warming? Notothenioids as a case study

Current climate change has raised concerns over the fate of the stenothermal Antarctic marine fauna (animals that evolved to live in narrow ranges of cold temperatures). The present paper focuses on Notothenioidei, a taxonomic group that dominates Antarctic fish. Notothenioids evolved in the Southern Ocean over the last 20 million years, providing an example of a marine species flock with unique adaptations to the cold at morphological, physiological and biochemical levels. Their phenotypic modifications are often accompanied by ‘irreversible’ genomic losses or gene amplifications. On a micro‐evolutionary scale, relatively ‘shallow’ genetic variation is observed, on account of past fluctuations in population size, and a significant genetic structure is evident, suggesting low population connectivity. These features suggest that Antarctic fish might have relatively little potential to adapt to global warming, at least at a genetic level. The extent of their phenotypic plasticity, which is evident to some degree, awaits further research.