Structural variability of sunflower gene for methionine-rich albumin SFA8

Background. The 2S albumins of sunflower and other oilseed plants possess a high nutritional quality, the defense activity against fungi diseases casual gents and also valuable functional properties. The major component of albumin fraction, the SFA8 protein consists of 103 amino acid residues among which methionine constitutes 15 Mole %. In the cultivated sunflower gene pool the SFA8 structural gene is represented by the two alleles the products of which have different isoelectric points and differ by the electrophoretic mobility, however molecular mechanisms of the polymorphism are still unknown. Results. The amplified sequences of the SFA8 gene from seven Helianthus annuus L. accessions and three accessions of wild Helianthus L. species from VIR collection were sequences. The intron of 258-303 bp length depending on the genotype was firstly found in the central part of the gene. The length of the first exon constitutes 99 bp, the second exon is of 210 bp length. The nucleotide and translated amino acid sequences are polymorphic among different genotypes. The line VIR 130 in which the two expressing SFA8 proteins, the normal polypeptide with isoelectric point (pI) approximately 6.0 (normal SFA8) and its allelic variant with pI 6.5 (variant SFA8) have been earlier revealed possesses two types of the SFA8 encoding sequence. In one sequence the substitution 108С—G is present that results in the substitution of the polar uncharged amino acid serine for the positively charged arginine and respectively in alteration of the protein charge and isoelectric point. The intron sequence is also polymorphic and characterized by the presence of indels of approximately 45 bp. The intron sequences of all accessions contain dinucleotides GT at the 5΄ end and AG at the 3΄ end which are characteristic for consensus sequences of splicing sites in the U2-type introns. The variants of the secondary structure of the SFA8 intron sequences of H. argophyllus Torr. & A. Gray and all the analyzed H. annuus genotypes are similar and differ from those of H. petiolaris Nutt. and H. giganteus L. Conclusions. The data on the SFA8 gene sequence polymorphism are important understanding the molecular mechanisms of genotypic differences in biochemical and functional properties of the protein, and he revealed differences in the intron secondary structure can be important for understanding expression patterns of the protein

Magnetic phases and reorientation transitions in antiferromagnetically coupled multilayers

In antiferromagnetically coupled superlattices grown on (001) faces of cubic substrates, e.g. based on materials combinations as Co/Cu, Fe/Si, Co/Cr, or Fe/Cr, the magnetic states evolve under competing influence of bilinear and biquadratic exchange interactions, surface-enhanced four-fold in-plane anisotropy, and specific finite-size effects. Using phenomenological (micromagnetic) theory, a comprehensive survey of the magnetic states and reorientation transitions has been carried out for multilayer systems with even number of ferromagnetic sub-layers and magnetizations in the plane. In two-layer systems (N=2) the phase diagrams in dependence on components of the applied field in the plane include ``swallow-tail'' type regions of (metastable) multistate co-existence and a number of continuous and discontinuous reorientation transitions induced by radial and transversal components of the applied field. In multilayers (N \ge 4) noncollinear states are spatially inhomogeneous with magnetization varying across the multilayer stack. For weak four-fold anisotropy the magnetic states under influence of an applied field evolve by a complex continuous reorientation into the saturated state. At higher anisotropy they transform into various inhomogeneous and asymmetric structures. The discontinuous transitions between the magnetic states in these two-layers and multilayers are characterized by broad ranges of multi-phase coexistence of the (metastable) states and give rise to specific transitional domain structures.Comment: Manuscript 34 pages, 14 figures; submitted for publicatio

The Neuronal EGF-Related Gene Nell2 Interacts with Macf1 and Supports Survival of Retinal Ganglion Cells after Optic Nerve Injury

Nell2 is a neuron-specific protein containing six epidermal growth factor-like domains. We have identified Nell2 as a retinal ganglion cell (RGC)-expressed gene by comparing mRNA profiles of control and RGC-deficient rat retinas. The aim of this study was to analyze Nell2 expression in wild-type and optic nerve axotomized retinas and evaluate its potential role in RGCs. Nell2-positive in situ and immunohistochemical signals were localized to irregularly shaped cells in the ganglion cell layer (GCL) and colocalized with retrogradely-labeled RGCs. No Nell2-positive cells were detected in 2 weeks optic nerve transected (ONT) retinas characterized with approximately 90% RGC loss. RT-PCR analysis showed a dramatic decrease in the Nell2 mRNA level after ONT compared to the controls. Immunoblot analysis of the Nell2 expression in the retina revealed the presence of two proteins with approximate MW of 140 and 90 kDa representing glycosylated and non-glycosylated Nell2, respectively. Both products were almost undetectable in retinal protein extracts two weeks after ONT. Proteome analysis of Nell2-interacting proteins carried out with MALDI-TOF MS (MS) identified microtubule-actin crosslinking factor 1 (Macf1), known to be critical in CNS development. Strong Macf1 expression was observed in the inner plexiform layer and GCL where it was colocalizied with Thy-1 staining. Since Nell2 has been reported to increase neuronal survival of the hippocampus and cerebral cortex, we evaluated the effect of Nell2 overexpression on RGC survival. RGCs in the nasal retina were consistently more efficiently transfected than in other areas (49% vs. 13%; n = 5, p<0.05). In non-transfected or pEGFP-transfected ONT retinas, the loss of RGCs was approximately 90% compared to the untreated control. In the nasal region, Nell2 transfection led to the preservation of approximately 58% more cells damaged by axotomy compared to non-transfected (n = 5, p<0.01) or pEGFP-transfected controls (n = 5, p<0.01)

Microtubule Actin Crosslinking Factor 1 Regulates the Balbiani Body and Animal-Vegetal Polarity of the Zebrafish Oocyte

Although of fundamental importance in developmental biology, the genetic basis for the symmetry breaking events that polarize the vertebrate oocyte and egg are largely unknown. In vertebrates, the first morphological asymmetry in the oocyte is the Balbiani body, a highly conserved, transient structure found in vertebrates and invertebrates including Drosophila, Xenopus, human, and mouse. We report the identification of the zebrafish magellan (mgn) mutant, which exhibits a novel enlarged Balbiani body phenotype and a disruption of oocyte polarity. To determine the molecular identity of the mgn gene, we positionally cloned the gene, employing a novel DNA capture method to target region-specific genomic DNA of 600 kb for massively parallel sequencing. Using this technique, we were able to enrich for the genomic region linked to our mutation within one week and then identify the mutation in mgn using massively parallel sequencing. This is one of the first successful uses of genomic DNA enrichment combined with massively parallel sequencing to determine the molecular identity of a gene associated with a mutant phenotype. We anticipate that the combination of these technologies will have wide applicability for the efficient identification of mutant genes in all organisms. We identified the mutation in mgn as a deletion in the coding sequence of the zebrafish microtubule actin crosslinking factor 1 (macf1) gene. macf1 is a member of the highly conserved spectraplakin family of cytoskeletal linker proteins, which play diverse roles in polarized cells such as neurons, muscle cells, and epithelial cells. In mgn mutants, the oocyte nucleus is mislocalized; and the Balbiani body, localized mRNAs, and organelles are absent from the periphery of the oocyte, consistent with a function for macf1 in nuclear anchoring and cortical localization. These data provide the first evidence for a role for spectraplakins in polarization of the vertebrate oocyte and egg

BPAG1a and b Associate with EB1 and EB3 and Modulate Vesicular Transport, Golgi Apparatus Structure, and Cell Migration in C2.7 Myoblasts

BPAG1a and BPAG1b (BPAG1a/b) constitute two major isoforms encoded by the dystonin (Dst) gene and show homology with MACF1a and MACF1b. These proteins are members of the plakin family, giant multi-modular proteins able to connect the intermediate filament, microtubule and microfilament cytoskeletal networks with each other and to distinct cell membrane sites. They also serve as scaffolds for signaling proteins that modulate cytoskeletal dynamics. To gain better insights into the functions of BPAG1a/b, we further characterized their C-terminal region important for their interaction with microtubules and assessed the role of these isoforms in the cytoskeletal organization of C2.7 myoblast cells. Our results show that alternative splicing does not only occur at the 5′ end of Dst and Macf1 pre-mRNAs, as previously reported, but also at their 3′ end, resulting in expression of additional four mRNA variants of BPAG1 and MACF1. These isoform-specific C-tails were able to bundle microtubules and bound to both EB1 and EB3, two microtubule plus end proteins. In the C2.7 cell line, knockdown of BPAG1a/b had no major effect on the organization of the microtubule and microfilament networks, but negatively affected endocytosis and maintenance of the Golgi apparatus structure, which became dispersed. Finally, knockdown of BPAG1a/b caused a specific decrease in the directness of cell migration, but did not impair initial cell adhesion. These data provide novel insights into the complexity of alternative splicing of Dst pre-mRNAs and into the role of BPAG1a/b in vesicular transport, Golgi apparatus structure as well as in migration in C2.7 myoblasts

Hippocampal pyramidal cells: the reemergence of cortical lamination

The increasing resolution of tract-tracing studies has led to the definition of segments along the transverse axis of the hippocampal pyramidal cell layer, which may represent functionally defined elements. This review will summarize evidence for a morphological and functional differentiation of pyramidal cells along the radial (deep to superficial) axis of the cell layer. In many species, deep and superficial sublayers can be identified histologically throughout large parts of the septotemporal extent of the hippocampus. Neurons in these sublayers are generated during different periods of development. During development, deep and superficial cells express genes (Sox5, SatB2) that also specify the phenotypes of superficial and deep cells in the neocortex. Deep and superficial cells differ neurochemically (e.g. calbindin and zinc) and in their adult gene expression patterns. These markers also distinguish sublayers in the septal hippocampus, where they are not readily apparent histologically in rat or mouse. Deep and superficial pyramidal cells differ in septal, striatal, and neocortical efferent connections. Distributions of deep and superficial pyramidal cell dendrites and studies in reeler or sparsely GFP-expressing mice indicate that this also applies to afferent pathways. Histological, neurochemical, and connective differences between deep and superficial neurons may correlate with (patho-) physiological phenomena specific to pyramidal cells at different radial locations. We feel that an appreciation of radial subdivisions in the pyramidal cell layer reminiscent of lamination in other cortical areas may be critical in the interpretation of studies of hippocampal anatomy and function