Assessment of structural similarities in chick oviduct progesterone receptor subunits by partial proteolysis of photoaffinity-labeled proteins

Partial proteolytic fragmentation of the two chick oviduct progesterone receptor subunits was used to identify structural features shared by the two proteins. Both subunits can be photoaffinity labeled at their hormone-binding sites (Birnbaumer, M., Schrader, W. T., and O'Malley, B. W. (1983) J. Biol. Chem. 258, 1637-1644) using the radioactive steroid [methyl-3H] 17 alpha, 21-dimethyl-19-nor-pregn-4,9-diene-3,20-dione. Native subunits A (Mr = 79,000) and B (Mr = 108,000) were partially purified, photoaffinity-labeled, and then subjected to various mild proteolytic digestions. Labeled fragments were analyzed by fluorography after electrophoresis of the digests under denaturing conditions. Digestion patterns were characteristic for each protease tested. However, fragments from both A and B were indistinguishable for all peptides of less than Mr = 60,000. Time course studies demonstrated the sequential production of progressively smaller discrete fragments in a manner consistent with a precursor-product relationship among them and established the existence of similar structural domains resistant to proteolysis in both proteins. Autoradiographic peptide maps were obtained by 125I-labeling of pure A and B protein isolated by two-dimensional gel electrophoresis followed by exhaustive tryptic digestion and two-dimensional separation. These studies revealed that a significant proportion of the smaller A protein differs in its primary sequence from that of the B protein which excludes the possibility of their sharing a precursor-product relationship. We conclude that B and A subunits are separate proteins with common structural features in the native state, but with considerable amino acid sequence differences. The simplest hypothesis consistent with these findings is that B and A are the products of two separate genes which have diverged to give rise to two different but related proteins that fold in such a manner as to be almost indistinguishable by proteolytic attack of their native conformation.     

Stress resistance and longevity are not directly linked to levels of enzymatic antioxidants in the ponerine ant Harpegnathos saltator

Background

The molecular mechanisms of variations in individual longevity are not well understood, even though longevity can be increased substantially by means of diverse experimental manipulations. One of the factors supposed to be involved in the increase of longevity is a higher stress resistance. To test this hypothesis in a natural system, eusocial insects such as bees or ants are ideally suited. In contrast to most other eusocial insects, ponerine ants show a peculiar life history that comprises the possibility to switch during adult life from a normal worker to a reproductive gamergate, therewith increasing their life expectancy significantly.

Results

We show that increased resistance against major stressors, such as reactive oxygen species and infection accompanies the switch from a life-history trait with normal lifespan to one with a longer life expectancy. A short period of social isolation was sufficient to enhance stress resistance of workers from the ponerine ant species Harpegnathos saltator significantly. All ant groups with increased stress resistances (reproducing gamergates and socially isolated workers) have lower catalase activities and glutathione levels than normal workers. Therewith, these ants resemble the characteristics of the youngest ants in the colony.

Conclusions

Social insects with their specific life history including a switch from normal workers to reproducing gamergates during adult life are well suited for ageing research. The regulation of stress resistance in gamergates seemed to be modified compared to foraging workers in an economic way. Interestingly, a switch towards more stress resistant animals can also be induced by a brief period of social isolation, which may already be associated with a shift to a reproductive trajectory. In Harpegnathos saltator, stress resistances are differently and potentially more economically regulated in reproductive individuals, highlighting the significance of reproduction for an increase in longevity in social insects. As already shown for other organisms with a long lifespan, this trait is not directly coupled to higher levels of enzymatic and non-enzymatic antioxidants.     

Distinct structural features of caprin-1 mediate its interaction with G3BP-1 and its induction of phosphorylation of eukaryotic translation initiation factor 2alpha, entry to cytoplasmic stress granules, and selective interaction with a subset of mRNAs

Caprin-1 is a ubiquitously expressed, well-conserved cytoplasmic phosphoprotein that is needed for normal progression through the G(1)-S phase of the cell cycle and occurs in postsynaptic granules in dendrites of neurons. We demonstrate that Caprin-1 colocalizes with RasGAP SH3 domain binding protein-1 (G3BP-1) in cytoplasmic RNA granules associated with microtubules and concentrated in the leading and trailing edge of migrating cells. Caprin-1 exhibits a highly conserved motif, F(M/I/L)Q(D/E)Sx(I/L)D that binds to the NTF-2-like domain of G3BP-1. The carboxy-terminal region of Caprin-1 selectively bound mRNA for c-Myc or cyclin D2, this binding being diminished by mutation of the three RGG motifs and abolished by deletion of the RGG-rich region. Overexpression of Caprin-1 induced phosphorylation of eukaryotic translation initiation factor 2alpha (eIF-2alpha) through a mechanism that depended on its ability to bind mRNA, resulting in global inhibition of protein synthesis. However, cells lacking Caprin-1 exhibited no changes in global rates of protein synthesis, suggesting that physiologically, the effects of Caprin-1 on translation were limited to restricted subsets of mRNAs. Overexpression of Caprin-1 induced the formation of cytoplasmic stress granules (SG). Its ability to bind RNA was required to induce SG formation but not necessarily its ability to enter SG. The ability of Caprin-1 or G3BP-1 to induce SG formation or enter them did not depend on their association with each other. The Caprin-1/G3BP-1 complex is likely to regulate the transport and translation of mRNAs of proteins involved with synaptic plasticity in neurons and cellular proliferation and migration in multiple cell types.     

Synthesis and biological activity of 5-methylidene 1,2-dihydrochromeno[3,4-f]quinoline derivatives as progesterone receptor modulators

A series of 5-methylidene 1,2-dihydrochromeno[3,4-f]quinoline derivatives were synthesized and tested in biological assays to evaluate scope and limitations of the nonsteroidal SPRM pharmacophore (3). A number of orally available highly potent nonsteroidal modulators were identified by modification of the substituents at 5-methylidene position.     

Peroxisome morphology in pathology

Peroxisomes are remarkably dynamic and versatile organelles that are essential for human health and development. They respond to physiological changes in the cellular environment by adapting their morphology, number, enzyme content and metabolic functions accordingly. With the discovery of the first key peroxisomal morphology proteins, the investigation of peroxisomal shape, distribution and dynamics has become an exciting new field in cell biology and biomedical sciences because of its relation to organelle functionality and its impact on developmental and physiological processes. In this review, we summarize recent findings on peroxisome biology, dynamics and the modulation of peroxisome morphology, especially in mammals. Furthermore, we discuss the roles of peroxisome dynamics and morphology in cell pathology and present recent examples for alterations in peroxisome morphology under disease conditions. Besides defects in the peroxisomal morphology machinery, we also address peroxisome biogenesis disorders, alterations of peroxisome number during carcinogenesis and liver cirrhosis, and morphological alterations of peroxisomes during viral infection.     

Entirely artificial signal transduction with adrenaline

Multifunctional transmembrane-building blocks with recognition sites for adrenaline on one end and the reaction partners for an SN2 reaction on the opposite end have been embedded in DPPC-liposomes. These doped vesicles can be quantitatively reduced at their disulfide head groups by externally added reducing agents; their composition and chemical processes taking place within can be monitored by NMR spectroscopy and--with limitations--by UV/Vis spectroscopy. Attempted release of thiopyridine as a second messenger into the interior of the liposome on external adrenaline addition could not be proven unambiguously because the detection system does not fulfill the necessary rigorous specificity and sensitivity requirements.     

Reducing arginase activity via dietary manganese deficiency enhances endothelium-dependent vasorelaxation of rat aorta

L-Arginine is a common substrate for the enzymes arginase and nitric oxide synthase (NOS). Acute inhibition of arginase enzyme activity improves endothelium-dependent vasorelaxation, presumably by increasing availability of substrate for NOS. Arginase is activated by manganese (Mn), and the consumption of a Mn-deficient (Mn-) diet can result in low arginase activity. We hypothesize that endothelium-dependent vasorelaxation is greater in rats fed Mn- versus Mn sufficient (Mn+) diets. Newly weaned rats fed Mn+ diets (0.5 microg Mn/g; n = 12) versus Mn+ diets (45 microg Mn/g; n = 12) for 44 +/- 3 days had (i) lower liver and kidney Mn and arginase activity (P < or = 0.05), (ii) higher plasma L-arginine (P < or = 0.05), (iii) similar plasma and urine nitrate + nitrite, and (iv) similar staining for endothelial nitric oxide synthase in thoracic aorta. Vascular reactivity of thoracic aorta (approximately 720 microm i.d.) and small coronary arteries (approximately 110 microm i.d.) was evaluated using wire myographs. Acetylcholine (ACh; 10(-8)-10(-4) M) produced greater (P < or = 0.05) vasorelaxation in thoracic aorta from Mn- rats (e.g., maximal percent relaxation, 79 +/- 7%) versus Mn + rats (e.g., maximal percent relaxation, 54 +/- 9%) at 5 of 7 evaluated doses. Tension produced by NOS inhibition using N(G) monomethyl-L-arginine (L-NMMA; 10(-3) M) and vasorelaxation evoked by (i) arginase inhibition using difluoromethylornithine (DFMO; 10(-7) M), (ii) ACh (10(-8)-10(-4) M) in the presence of DFMO, and (iii) sodium nitroprusside (10(-9)-10(-4) M) were unaffected by diet. No differences existed between groups concerning these responses in small coronary arteries. These findings support our hypothesis that endothelium-dependent vasorelaxation is greater in aortic segments from rats that consume Mn- versus Mn+ diets; however, responses from small coronary arteries were unaffected.     

Rapid Identification of a Natural Knockout Allele of ARMADILLO REPEAT-CONTAINING KINESIN1 That Causes Root Hair Branching by Mapping-By-Sequencing

In Arabidopsis (Arabidopsis thaliana), branched root hairs are an indicator of defects in root hair tip growth. Among 62 accessions, one accession (Heiligkreuztal2 [HKT2.4]) displayed branched root hairs, suggesting that this accession carries a mutation in a gene of importance for tip growth. We determined 200- to 300-kb mapping intervals using a mapping-by-sequencing approach of F2 pools from crossings of HKT2.4 with three different accessions. The intersection of these mapping intervals was 80 kb in size featuring not more than 36 HKT2.4-specific single nucleotide polymorphisms, only two of which changed the coding potential of genes. Among them, we identified the causative single nucleotide polymorphism changing a splicing site in ARMADILLO REPEAT-CONTAINING KINESIN1. The applied strategies have the potential to complement statistical methods in high-throughput phenotyping studies using different natural accessions to identify causative genes for distinct phenotypes represented by only one or a few accessions.Root hairs are tubular tip outgrowths of single root epidermal cells (trichoblasts). They are an excellent genetic system and serve as a model to study the molecular components regulating tip growth (Carol and Dolan, 2002; Samaj et al., 2004; Lee and Yang, 2008). One of the main regulators of tip growth in root hairs is the small G protein RHO OF PLANTS2 (ROP2; Jones et al., 2002; Payne and Grierson, 2009). ROP2 determines the position of root hairs in incipient epidermal root hair cells and remains localized in the emerging tip during root hair tip growth (Molendijk et al., 2001; Jones et al., 2002). In addition, other factors have been identified to be important for growth and its directionality including phosphoinositides, cytoplasmic [Ca²⁺] gradients and their oscillation, reactive oxygen species, the RAB GTPase homolog A4B, and the cytoskeleton (Foreman et al., 2003; Preuss et al., 2004, 2006; Carol et al., 2005; Thole et al., 2008; Heilmann, 2009).Defects in essential processes for the establishment and maintenance of tip growth lead to deviations in root hair morphology such as branching and waviness (Samaj et al., 2004; Lee and Yang, 2008). Both the microtubules (MTs) and actin are important regulators of tip growth with MTs maintaining one growth point (Bibikova et al., 1999; Miller et al., 1999; Baluska et al., 2000). The latter is evident from the finding that artificially induced [Ca²⁺] gradients can induce additional growth tips when MTs are destroyed by drug treatments (Bibikova et al., 1999).Although the genetic and molecular analysis revealed a well-understood working model for root hair growth, little is known about the natural variation of the underlying processes. Which are the adaptive processes of relevance to specific environmental cues and which have already been selected for in natural accessions? One way to address this question is to link genotype and phenotype by association mapping using various Arabidopsis (Arabidopsis thaliana) accessions. This is greatly facilitated by the 1001 Genomes Project (http://1001genomes.org), providing an increasing number of sequenced accessions. In some cases, phenotypes are only found in one or a few accessions. When the minor allele frequency (AF) is low, the identification of such rare causative alleles with genome-wide association mapping studies is challenging because they cannot be discriminated from false positives (e.g. sequencing errors or synthetic associations; Korte and Farlow, 2013), they are not detectable because of chosen thresholds, or they do not support a statistically significant value (Cantor et al., 2010). Other time-consuming approaches with low mapping resolution, such as quantitative trait loci mapping, need to be followed to identify the causative gene. For this, mapping-by-sequencing, which was originally developed for the identification of mutagen-induced changes in model species (Schneeberger et al., 2009b), can help to rapidly identify causal polymorphisms including nonmodel and nonreference strains (Nordström et al., 2013; Takagi et al., 2013). The resolution of mapping-by-sequencing experiments in Arabidopsis mapping populations is typically between multiple hundreds of kilobase pairs up to a few megabase pairs (James et al., 2013). Although intervals of this size allow the identification of causal mutations in forward genetic screens, they are problematic for the analysis of diverse Arabidopsis accessions because the single nucleotide polymorphism (SNP) density is very high; consequently, hundreds of polymorphisms have to be considered.Here we report on a modification of the mapping-by-sequencing strategy providing a shortcut from distinct, monogenic accession-specific phenotypes to the causative SNP. When studying root hair morphology in 62 accessions for which the genome sequences were released by the 1001 Genomes Project (Cao et al., 2011), we found one accession (Heiligkreuztal2 [HKT2.4]) in which almost all root hairs were branched. To identify the causative gene, we used an approach based on mapping-by-sequencing. Instead of one outcross, we used outcrosses with three different accessions. We selected F2 seedlings exhibiting the distinct, monogenic, recessive root hair branching phenotype for sequencing. Combining the intersection of the three resulting mapping intervals with a selection for accession-specific SNPs revealed two primary candidate genes responsible for the root phenotype. We demonstrate that the causative SNP renders a splicing site in ARMADILLO REPEAT-CONTAINING KINESIN1 (ARK1) inactive and therefore leads to a defective ARK1/MORPHOGENESIS OF ROOT HAIR2 (MRH2) protein that is thought to coordinate actin microfilaments and MTs during tip growth of root hairs (Yang et al., 2007; Yoo and Blancaflor, 2013).