Role of cell shape in determination of the division plane in Schizosaccharomyces pombe: random orientation of septa in spherical cells

The establishment of growth polarity in Schizosaccharomyces pombe cells is a combined function of the cytoplasmic cytoskeleton and the shape of the cell wall inherited from the mother cell. The septum that divides the cylindrical cell into two siblings is formed midway between the growing poles and perpendicularly to the axis that connects them. Since the daughter cells also extend at their ends and form their septa at right angles to the longitudinal axis, their septal (division) planes lie parallel to those of the mother cell. To gain a better understanding of how this regularity is ensured, we investigated septation in spherical cells that do not inherit morphologically predetermined cell ends to establish poles for growth. We studied four mutants (defining four novel genes), over 95% of whose cells displayed a completely spherical morphology and a deficiency in mating and showed a random distribution of cytoplasmic microtubules, Tea1p, and F-actin, indicating that the cytoplasmic cytoskeleton was poorly polarized or apolar. Septum positioning was examined by visualizing septa and division scars by calcofluor staining and by the analysis of electron microscopic images. Freeze-substitution, freeze-etching, and scanning electron microscopy were used. We found that the elongated bipolar shape is not essential for the determination of a division plane that can separate the postmitotic nuclei. However, it seems to be necessary for the maintenance of the parallel orientation of septa over the generations. In the spherical cells, the division scars and septa usually lie at angles to each other on the cell surface. We hypothesize that the shape of the cell indirectly affects the positioning of the septum by directing the extension of the spindle.     

Elevation as a barrier: genetic structure for an Atlantic rain forest tree (Bathysa australis) in the Serra do Mar mountain range,SE Brazil

Distance and discrete geographic barriers play a role in isolating populations, as seed and pollen dispersal become limited. Nearby populations without any geographic barrier between them may also suffer from ecological isolation driven by habitat heterogeneity, which may promote divergence by local adaptation and drift. Likewise, elevation gradients may influence the genetic structure and diversity of populations, particularly those marginally distributed. Bathysa australis (Rubiaceae) is a widespread tree along the elevation gradient of the Serra do Mar, SE Brazil. This self‐compatible species is pollinated by bees and wasps and has autochoric seeds, suggesting restricted gene dispersal. We investigated the distribution of genetic diversity in six B. australis populations at two extreme sites along an elevation gradient: a lowland site (80–216 m) and an upland site (1010–1100 m.a.s.l.). Nine microsatellite loci were used to test for genetic structure and to verify differences in genetic diversity between sites. We found a marked genetic structure on a scale as small as 6 km (F_ST = 0.21), and two distinct clusters were identified, each corresponding to a site. Although B. australis is continuously distributed along the elevation gradient, we have not observed a gene flow between the extreme populations. This might be related to B. australis biological features and creates a potential scenario for adaptation to the different conditions imposed by the elevation gradient. We failed to find an isolation‐by‐distance pattern; although on the fine scale, all populations showed spatial autocorrelation until ~10‐20 m. Elevation difference was a relevant factor though, but we need further sampling effort to check its correlation with genetic distance. The lowland populations had a higher allelic richness and showed higher rare allele counts than the upland ones. The upland site may be more selective, eliminating rare alleles, as we did not find any evidence for bottleneck.     

Synthetic substrates for measuring activity of autophagy proteases: Autophagins (Atg4)

Atg4 cysteine proteases (autophagins) play crucial roles in autophagy by proteolytic activation of Atg8 paralogs for targeting to autophagic vesicles by lipid conjugation, as well as in subsequent deconjugation reactions. However, the means to measure the activity of autophagins is limited. Herein, we describe two novel substrates for autophagins suitable for a diversity of in vitro assays, including (i) fluorogenic tetrapeptide acetyl-Gly-L-Thr-L-Phe-Gly-AFC (Ac-GTFG-AFC) and (ii) a fusion protein comprised of the natural substrate LC3B appended to the N-terminus of phospholipase A₂ (LC3B-PLA₂), which upon cleavage releases active PLA₂ for fluorogenic assay. To generate the synthetic tetrapeptide substrate, the preferred tetrapeptide sequence recognized by autophagin-1/Atg4B was determined using a positional scanning combinatorial fluorogenic tetrapeptide library. With the LC3B-PLA₂ substrate, we show that mutation of the glycine proximal to the scissile bond in LC3B abolishes activity. Both substrates showed high specificity for recombinant purified autophagin-1/Atg4B compared to closely related proteases and the LC3B-PLA₂ substrate afforded substantially higher catalytic rates (k_cat/K_m 5.26 × 10⁵ M⁻¹/sec⁻¹) than Ac-GTFG-AFC peptide (0.92 M⁻¹/sec⁻¹), consistent with substrate-induced activation. Studies of autophagin-1 mutants were also performed, including the protease lacking a predicted autoinhibitory domain at residues 1 to 24 and lacking a regulatory loop at residues 259 to 262. The peptide and fusion protein substrates were also employed for measuring autophagin activity in cell lysates, showing a decrease in cells treated with autophagin-1/Atg4B siRNA or transfected with a plasmid encoding Atg4B (Cys74Ala) dominant-negative. Therefore, the synthetic substrates for autophagins reported here provide new research tools for studying autophagy.Key words: autophagin, fluorogenic assay, tetrapeptide, phospholipase A2, LC3     

Interactions of the NPXY microdomains of the low density lipoprotein receptor‐related protein 1

The low density lipoprotein receptor‐related protein 1 (LRP1) mediates internalization of a large number of proteins and protein–lipid complexes and is widely implicated in Alzheimer's disease. The cytoplasmic domain of LRP1 (LRP1‐CT) can be phosphorylated by activated protein‐tyrosine kinases at two NPXY motifs in LRP1‐CT; Tyr 4507 is readily phosphorylated and must be phosphorylated before phosphorylation of Tyr 4473 occurs. Pull‐down experiments from brain lysate revealed numerous proteins binding to LRP1‐CT, but the results were highly variable. To separate which proteins bind to each NPXY motif and their phosphorylation dependence, each NPXY motif microdomain was prepared in both phosphorylated and non‐phosphorylated forms and used to probe rodent brain extracts for binding proteins. Proteins that bound specifically to the microdomains were identified by LC‐MS/MS, and confirmed by Western blot. Recombinant proteins were then tested for binding to each NPXY motif. The NPXY₄₅₀₇ (membrane distal) was found to interact with a large number of proteins, many of which only bound the tyrosine‐phosphorylated form. This microdomain also bound a significant number of other proteins in the unphosphorylated state. Many of the interactions were later confirmed to be direct with recombinant proteins. The NPXY₄₄₇₃ (membrane proximal) bound many fewer proteins and only to the phosphorylated form.     

The new 2,3-benzodiazepine derivative EGIS-8332 inhibits AMPA/kainate ion channels and cell death

We observed in vitro neuroprotective and AMPA/kainate receptor antagonist effects of the new 2,3-benzodiazepine derivative EGIS-8332 (R,S-1-(4-aminophenyl)-7,8-methylenedioxy-4-cyano-4-methyl-3-N-acetyl-5H-3,4-dihydro-2,3-benzodiazepine) using the lactate dehydrogenase (LDH) release assay and patch clamp recordings on primary cultures of rat embryonic telencephalon neurons exposed to AMPA/kainate receptor agonists. EGIS-8332 potently decreased alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and quisqualate induced LDH release (IC(50)=5.2+/-0.4 and 7.4+/-1.3 microM, respectively) from the cells. Whole-cell patch clamp studies carried out on the ionotropic glutamate receptors N-methyl D-aspartate (NMDA), as well as AMPA (and kainate) in cultured telencephalon neurons verified that EGIS-8332 blocked steady state responses to AMPA and kainate (IC(50)=1.7+/-0.4 and 6.2+/-1.6 microM, respectively), but hardly influenced currents evoked by NMDA. EGIS-8332 also inhibited kainate-evoked response in CHO cells expressing the flop variant of GluR1 receptor and, in cerebellar Purkinje cells at similar efficiency. The stereoselectivity of the inhibitory site is established by the clearly dissimilar inhibitory potency of the enantiomer components of EGIS-8332 differing in the configuration of methyl and cyano substituents on carbon C(4): the R(-) enantiomer was found to be the efficient species. This finding suggests that the inhibitory interaction between the channel protein and drug is promoted by presence of the C(4) methyl group. The inhibition of the AMPA/kainate ion channels by EGIS-8332 is non-competitive, not use dependent, and depends neither on the closed/open state of the channel, nor the membrane potential. These findings suggest an allosteric mechanism for the inhibition. These in vitro observations suggest that the compound might be useful in the treatments of certain acute and chronic neurological syndromes initiated by derangements of ionotropic glutamate receptor function.     

Molecular probes for the A2A adenosine receptor based on a pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine scaffold

Pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine derivatives such as SCH 442416 display high affinity and selectivity as antagonists for the human A_2A adenosine receptor (AR). We extended ether-linked chain substituents at the p-position of the phenyl group using optimized O-alkylation. The conjugates included an ester, carboxylic acid and amines (for amide condensation), an alkyne (for click chemistry), a fluoropropyl group (for ¹⁸F incorporation), and fluorophore reporter groups (e.g., BODIPY conjugate 14, K_i 15 nM). The potent and A_2AAR-selective N-aminoethylacetamide 7 and N-[2-(2-aminoethyl)-aminoethyl]acetamide 8 congeners were coupled to polyamidoamine (PAMAM) G3.5 dendrimers, and the multivalent conjugates displayed high A_2AAR affinity. Theoretical docking of an AlexaFluor conjugate to the receptor X-ray structure highlighted the key interactions between the heterocyclic core and the binding pocket of the A_2AAR as well as the distal anchoring of the fluorophore. In conclusion, we have synthesized a family of high affinity functionalized congeners as pharmacological probes for studying the A_2AAR.     

Regional Distribution and Relative Abundance of Serotonin2c Receptors in Human Brain: Effect of Suicide

Abnormalities in serotonin receptor subtypes have been observed in the postmortem brain of suicide victims. We examined the regional distribution of serotonin (5HT)(2C) receptor mRNA in several areas of the human brain and also compared its protein and mRNA expression in the prefrontal cortex (PFC), hippocampus, and choroid plexus between suicide victims and normal control subjects. 5HT(2C) receptors were found to be distributed in several areas of the human brain (in order of abundance): highly concentrated and richest in choroid plexus; hypothalamus; nucleus accumbens; with the lowest abundance in PFC and cerebellum. Comparison of 5HT(2C) receptors between suicide victims and control subjects showed higher protein levels in the PFC but not the hippocampus or choroid plexus of suicide victims. However, there were no significant differences in mRNA levels between suicide victims and control subjects in these brain areas. These results suggest that 5HT(2C) receptors are richly distributed throughout the brain with the highest level in the choroid plexus and that abnormalities in protein expression of 5HT(2C) receptors in the PFC may be associated with suicide.     

Autophagy occurs upstream or parallel to the apoptosome during histolytic cell death

Histolysis refers to a widespread disintegration of tissues that is morphologically distinct from apoptosis and often associated with the stimulation of autophagy. Here, we establish that a component of the apoptosome, and pivotal regulator of apoptosis, is also required for histolytic cell death. Using in vivo and ex vivo assays, we demonstrate a global apoptogenic requirement for dark, the fly ortholog of Apaf1, and show that a required focus of dark(-) organismal lethality maps to the central nervous system. We further demonstrate that the Dark protein itself is a caspase substrate and find that alterations of this cleavage site produced the first hypermorphic point mutation within the Apaf1/Ced-4 gene family. In a model of ;autophagic cell death', dark was essential for histolysis but dispensable for characteristic features of the autophagic program, indicating that the induction of autophagy occurs upstream or parallel to histolytic cell death. These results demonstrate that stimulation of autophagy per se is not a ;killing event' and, at the same time, establish that common effector pathways, regulated by the apoptosome, can underlie morphologically distinct forms of programmed cell death.     

Tree growth and its association with climate between individual tree-ring series at three mountain ranges in north central China

Individual tree-ring series may show changed growth trends and divergent climate–growth associations even within a site, highlighting the need to examine tree growth and its climate association before building a chronology. We provided a case study for the stratification and temporal variability of tree growth and its climate associations of individual cores for three mountain ranges in north central China. Tree growth is mainly limited by moisture conditions in previous July–September and current June–August. Repeated sampling and field investigations of Picea wilsonii at Xinglong Mountain over a growth year of 2004 suggested that the growing season is from about the end of April to the end of September. It appears that the moisture conditions in previous and current growing seasons are crucial for tree growth in this region. However, a decrease in drought limitation was observed for a few tree-ring series. We thereby built the pooled chronology and sub-site chronologies with only drought-sensitive tree rings similar climate–growth relationships from the three mountain slopes. Growth disturbances of tree-ring series are detected by checking the occurrence of successively low values of the biweight series, which are treated by fitting a flexible curve.