Functional characterization of seed coat-specific members of the barley germin gene family

The present project aimed to isolate testa-, pericarp- and epicarp-specific gene promoters for the developing caryopsis of barley (Hordeum vulgare L.). These might be applied in transgenic plants to express antifungal agents or modify metabolic pathways. A testa-specific 379-nucleotide fragment was cloned by differential amplification and used to screen a bacterial artificial chromosome (BAC) library of 6.3 haploid genome equivalents. Fifty-three clones containing genes encoding for proteins of the germin family were found. Characterization of the clones identified a minimum of six seed coat- and eight leaf-specific germin genes. Four seed coat- and one leaf-specific genes were sequenced. The deduced primary structure of the proteins revealed a remarkable conservation of the manganese(II) binding His and Glu residues and β-barrel secondary structure of oxalate oxidase – also in barley, wheat, rice and Arabidopsis germins, for which an enzymatic activity has not yet been identified. The oxalate oxidase and germins of barley and other species are synthesized with a conserved pre-sequence of 23 or 24 amino acids for targeting into the cell wall. β-Glucuronidase expression with the barley germin F gene promoter occurs specifically in the testa and epicarp of the developing barley caryopsis, while expression with the B gene promoter is restricted to the testa. Oxalate oxidase activity is prominent in the epicarp and the root tips of the developing embryo. A family tree based on primary structure homologies of germins distinguishes three groups: oxalate oxidases, leaf-specific germins and seed coat-specific germins.     

Bcl-2-related protein family gene expression during oligodendroglial differentiation

Oligodendroglial lineage cells (OLC) vary in susceptibility to both necrosis and apoptosis depending on their developmental stages, which might be regulated by differential expression of Bcl-2-related genes. As an initial step to test this hypothesis, we examined the expression of 19 Bcl-2-related genes in purified cultures of rat oligodendroglial progenitors, immature and mature oligodendrocytes. All 'multidomain' anti-apoptotic members (Bcl-x, Bcl-2, Mcl-1, Bcl-w and Bcl2l10/Diva/Boo) except Bcl2a1/A1 are expressed in OLC. Semiquantitative and real-time RT-PCR revealed that Bcl-xL and Mcl-1 mRNAs are the dominant anti-apoptotic members and increase four- and twofold, respectively, with maturation. Bcl-2 mRNA is less abundant than Bcl-xL mRNA in progenitors and falls an additional 10-fold during differentiation. Bcl-w mRNA also increases, with significant changes in its splicing pattern, as OLC mature. Transfection studies demonstrated that Bcl-xL overexpression protects against kainate-induced excitotoxicity, whereas Bcl-2 overexpression does not. As for 'multidomain' pro-apoptotic members (Bax, Bad and Bok/Mtd), Bax and Bak are highly expressed throughout differentiation. Among 'BH3 domain-only' members examined (Bim, Biklk, DP5/Hrk, Bad, Bid, Noxa, Puma/Bbc3, Bmf, BNip3 and BNip3L), BNip3 and Bmf mRNAs increase markedly during differentiation. These results provide basic information to guide further studies on the roles for Bcl-2-related family proteins in OLC death.     

Functional characterization of the H-2Dp gene product

A clone containing the D ^p gene was used to transform L cells. The D^p product expressed was identified by two-dimensional gel electrophoreis and flow cytometry. The D^p product expressed by the L cells was recognized by D^P-specific flow cytometry. The D^p product expressed by the L cells was recognized by D^p-specific but not K^p-specific killer T cells. This killing was inhibited by monoclonal antibodies specific for D^p but not K^p or K^k antigens. Similarly, lymphocytic choriomeningitis virus (LCMV) killer T cells from B 10.P mice were able to kill LCMV-infected L12a cells, but not LCMV-infected Ltk⁺. Again only D^p monoclonal antibodies could inhibit this killing.     

The secrets of the Bcl-2 family

The Bcl-2 family of proteins is formed by pro- and antiapoptotic members. Together they regulate the permeabilization of the mitochondrial outer membrane, a key step in apoptosis. Their complex network of interactions both in the cytosol and on mitochondria determines the fate of the cell. In the past 2 decades, the members of the family have been identified and classified according to their function. Several competing models have been proposed to explain how the Blc-2 proteins orchestrate apoptosis signaling. However, basic aspects of the action of these proteins remain elusive. This review is focused on the biophysical mechanisms that are relevant for their action in apoptosis and on the challenging gaps in our knowledge that necessitate further exploration to finally understand how the Bcl-2 family regulates apoptosis.     

Characterization of zebrafish PSD-95 gene family members

The PSD-95 family of membrane- associated guanylate kinases (MAGUKs) are thought to act as molecular scaffolds that regulate the assembly and function of the multiprotein signaling complex found at the postsynaptic density of excitatory synapses. Genetic analysis of PSD-95 family members in the mammalian nervous system has so far been difficult, but the zebrafish is emerging as an ideal vertebrate system for studying the role of particular genes in the developing and mature nervous system. Here we describe the cloning of the zebrafish orthologs of PSD-95, PSD-93, and two isoforms of SAP-97. Using in situ hybridization analysis we show that these zebrafish MAGUKs have overlapping but distinct patterns of expression in the developing nervous system and craniofacial skeleton. Using a pan-MAGUK antibody we show that MAGUK proteins localize to neurons within the developing hindbrain, cerebellum, visual and olfactory systems, and to skin epithelial cells. In the olfactory and visual systems MAGUK proteins are expressed strongly in synaptic regions, and the onset of expression in these areas coincides with periods of synapse formation. These data are consistent with the idea that PSD-95 family members are involved in synapse assembly and function, and provide a platform for future functional studies in vivo in a highly tractable model organism.     

Functional characterization of a WRKY family gene involved in somatic embryogenesis in Panax ginseng

Protoplasma - As a perennial herbaceous species, Panax ginseng is widely cultivated and used as traditional herbal medicine. The root of Panax ginseng commonly remains expensive as conventional...     

Functional divergence in the Arabidopsis LOB-domain gene family

The Arabidopsis LOB-domain (LBD) gene family is composed by 43 members divided in two classes based on amino acid conservation within the LOB-domain. The LOB domain is known to be responsible for DNA binding and protein-protein interactions. There is very little functional information available for most genes in the LBD family and many lbd single mutants do not exhibit conspicuous phenotypes. One plausible explanation for the limited loss-of-function phenotypes observed in this family is that LBD genes exhibit significant functional redundancy. Here we discuss an example of one phylogenetic subgroup of the LBD family, in which genes that are closely related based on phylogeny exhibit distinctly different expression patterns and do not have overlapping functions. We discuss the challenges of using phylogenetic analyses to predict redundancy in gene families.     

Role of Bcl-2 family members in invertebrates

Proteins belonging to the Bcl-2 family function as regulators of 'life-or-death' decisions in response to various intrinsic and extrinsic stimuli. In mammals, cell death is controlled by pro- and anti-apoptotic members of the Bcl-2 family, which function upstream of the caspase cascade. Structural and functional homologues of the Bcl-2 family proteins also exist in lower eukaryotes, such as nematodes and flies. In nematodes, an anti-apoptotic Bcl-2 family protein, CED-9, functions as a potent cell death inhibitor, and a BH3-only protein, EGL-1, acts as an inhibitor of CED-9 to facilitate the spatio-temporal regulation of programmed cell death. On the other hand, the Drosophila genome encodes two Bcl-2 family proteins, Drob-1/Debcl/dBorg-1/dBok and Buffy/dBorg-2, both of which structurally belong to the pro-apoptotic group, despite abundant similarities in the cell death mechanisms between flies and vertebrates. Drob-1 acts as a pro-apoptotic factor in vitro and in vivo, and Buffy/dBorg-2 exhibits a weak anti-apoptotic function. The ancestral role of the Bcl-2 family protein may be pro-apoptotic, and the evolution of the functions of this family of proteins may be closely linked with the contribution of mitochondria to the cell death pathway.     

Bcl-2 family: life-or-death switch

Proteolytic cleavage of the amyloid protein from the amyloid protein precursor (APP) by APP secretases is a key event in Alzheimer's disease (AD) pathogenesis. alpha-Secretases cleave APP within the amyloid sequences, whereas beta- and gamma-secretases cleave on the N- and C-terminal ends respectively. The transmembrane aspartyl protease BACE has been identified as beta-secretase and several proteases (ADAM-10, TACE, PC7) may be alpha-secretases. A number of studies have suggested that presenilins could be gamma-secretases, although this remains to be demonstrated conclusively. Inhibition of beta- and gamma-secretase, or stimulation of alpha-secretase, is a rational strategy for therapeutic intervention in AD.     

Bcl-2 family proteins as ion-channels

The Bcl-2 protein family function(s) as important regulators of cellular decisions to heed or ignore death signals. The three-dimensional structure of the Bcl-2 homolog, Bcl-XL, bears a strong resemblance to some pore-forming bacterial toxins. This similarity suggested that the Bcl-2 family proteins may also possess channel-forming capability. This review summarizes the recent initial studies on the in vitro channel activity of Bcl-2, Bcl-XL and Bax and offers some speculation as to the physiological role that these channels may play in the cell death pathway.     

Bcl-2 family members and disease

Apoptosis plays an important role during development and in the maintenance of multicellular organisms. Bcl-2 family members affect cell death in either a positive or negative fashion. Although some redundancy exists between family members, expression of certain family members is important during development in an organ-specific manner. The founding family member bcl-2 tends to be highly expressed in the embryo and declines postnatally following differentiation and maturation. Altered expression of bcl-2, as well as other family members, has been observed in disease states potentially affecting treatment modalities. Here we examine the distribution and role death repressors bcl-2, bcl-x(L) and bcl-w as well as death effectors bax and bak play regulating apoptosis in a tissue-specific manner. Understanding the normal role of these proteins during embryogenesis and in the mature organ will give us important insight into what goes awry in various disease states.