Analysis of chromosomal and organellar DNA of somatic hybrids between Triticum aestiuvm and Haynaldia villosa Schur

Intergeneric somatic hybridization between wheat (cv. Jinan 177) protoplasts that have 24-28 chromosomes and Haynaldia villosa protoplasts containing 11-14 chromosomes was carried out by the polyethylene glycol (PEG) method. A high frequency of hybrid calli and plants were obtained from the fusion products, as revealed by cytological and biochemical techniques and by PCR analysis of 5S rDNA spacer sequences. GISH (genomic in situ hybridization) analysis confirmed the presence of chromosomes from both parents in the hybrid clones and the common occurrence of translocations between them. The RFLP analysis of the organellar DNA using mitochondrion- and chloroplast-specific probes revealed that mitochondria from both parents existed in the cells of hybrid calli and their recombination, whereas chloroplasts segregated and recombined randomly. The gross morphology of hybrid plants resembled that of wheat, but the gross morphology of their ovaries and anthers were intermediate between those of the two parents. The relationship between hybrid plant regeneration and the balance of genetic materials in hybrid clones is discussed.     

Presenilin-1 exists in both pre- and post-Golgi compartments and recycles via COPI-coated membranes

Presenilin-1 is involved in intramembrane proteolysis of various proteins, but its intracellular site of action has remained elusive. Here, we determined by quantitative immunogold-electron microscopy that presenilin-1 in Chinese hamster ovary cells is present in pre-Golgi compartments as well as at the plasma membrane and endosomes. Notably, a high percentage of presenilin-1 resides in COPI-coated membranes between the endoplasmic reticulum and the Golgi complex, indicating significant recycling to the endoplasmic reticulum. By contrast, the inactive aspartate mutant presenilin-1_D257A is relatively excluded from COPI-coated membranes, concomitant with increased post-Golgi levels. These data provide critical evidence for the scenario that the complex containing presenilin-1 can serve as γ-secretase at the plasma membrane or endosomes and suggest a role for COPI-mediated retrograde transport in regulating post-Golgi levels of presenilin-1.     

Regulation of hematopoietic-specific G-protein Galpha15 and Galpha16 by protein kinase C

Heterotrimeric G proteins mediate cell growth and differentiation by coupling cell surface receptors to intracellular effector enzymes. The G-protein alpha subunit, Galpha(16), and its murine homologue Galpha(15), are expressed specifically in hematopoietic cells and their expression is highly regulated during differentiation of normal and leukemic cells. In this study, we examined the phosphorylation of Galpha(15)/Galpha(16) and its role in receptor and effector coupling. We observed a PMA-stimulated intact cell phosphorylation of Galpha(15) in COS7 cells transfected with Galpha(15) and protein kinase Calpha (PKCalpha), and phosphorylation of endogenous Galpha(16) in HL60 cells. We also showed that peptides derived from the two G-proteins were phosphorylated in vitro using purified brain PKC. Furthermore, we identified the putative phosphorylation site and showed that mutation or deletion of this PKC phosphorylation site inhibited phospholipase C (PLC) activation. The behavior of double mutants with the constitutively active G-protein mutation (QL-mutant) and mutation in the putative phosphorylation site suggests that the phosphorylation site of Galpha(15/16) is essential for receptor-coupled activation of PLC, but not for direct interaction of the G-protein with PLC-beta.     

A Two-Species Test of the Hypothesis That Spatial Isolation Influences Microbial Diversity in Soil

The hypothesis that spatial isolation is a key determinant of microbial community structure in soils was evaluated by examining the competitive dynamics of two species growing on a single resource in a uniform sand matrix under varied moisture content. One species dominated the community under highly connected, saturated treatments, suggesting that these conditions allow competitive interactions to structure the community. As moisture content decreased, however, the less competitive species became established in the community. This effect was most pronounced at a matric water potential of -0.14 MPa where estimates of final population density and species fitness were equal. A second but more closely related species pair exhibited a similar response to decreasing moisture, suggesting that the effects of spatial isolation we observed are not simply a species-pair-specific phenomenon. These findings indicate that spatial isolation, created by low moisture content, plays an important role in structuring soil microbial communities.     

Synthesis of beta-substituted cationic porphyrins and their interactions with DNA

The beta-substituted cationic porphyrins (7, 8 and 10) have been synthesized and their interactions with plasmid DNA investigated. We found that substituents at the beta-position of porphyrins (7 and 8) have apparently affected their interactions with DNA compared with non-beta-substituted porphyrins (10).     

Ultrastructural study of polyspermy during early embryo development in pigs, observed by scanning electron microscope and transmission electron microscope

Polyspermy is generally considered a pathological phenomenon in mammals. Incidence of polyspermy in porcine eggs in vivo is extremely high (30-40%) compared with other species, and polyspermy rate in the in vitro fertilized eggs in pigs can reach 65%. It is still unknown whether polyspermy to a certain degree is a physiological condition in pigs, and whether porcine eggs have any capability with which to remove the accessory sperm in the cytoplasm. The objectives in the present study are to observe the ultrastructural changes of accessory sperm during early embryonic development in pigs. A total of 58 normal, early embryos at one-, two, three-, and four-cell and morular stages were collected from gilts and were studied by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The surface ultrastructure showed that sperm fusion with the zona pellucida was a continuous process during one-, two-, three-, and four-cell and morular stages, as observed by the SEM. Accessory sperm were present in the cytoplasm of cleaved embryos. The sperm heads in the cytoplasm of cleaved embryos did not decondense. TEM revealed the presence of a condensed sperm head within a lysosome (or phagolysosome) in a three-cell embryo. These observations suggest that polyspermy may be a physiological condition in pigs and that early embryos may develop to term if accessory sperm do not interrupt the embryo genome. Furthermore, lysosome activity could be another physiological mechanism for removing accessory sperm in the cytoplasm of fertilized eggs and cleaved embryos after fertilization in pigs.     

The transmembrane aspartates in presenilin 1 and 2 are obligatory for gamma-secretase activity and amyloid beta-protein generation

The discovery that a deficiency of presenilin 1 (PS1) decreases the production of amyloid beta-protein (Abeta) identified the presenilins as important mediators of the gamma-secretase cleavage of beta-amyloid precursor protein (APP). Recently, we found that two conserved transmembrane (TM) aspartates in PS1 are critical for Abeta production, providing evidence that PS1 either functions as a required diaspartyl cofactor for gamma-secretase or is itself gamma-secretase. Presenilin 2 (PS2) shares substantial sequence and possibly functional homology with PS1. Here, we show that the two TM aspartates in PS2 are also critical for gamma-secretase activity, providing further evidence that PS2 is functionally homologous to PS1. Cells stably co-expressing TM Asp --> Ala mutations in both PS1 and PS2 show further accumulation of the APP-derived gamma-secretase substrates, C83 and C99. The production of Abeta is reduced to undetectable levels in the conditioned media of these cells. Furthermore, endoproteolysis of the exogenous Asp mutant PS2 is absent, and endogenous PS1 C-terminal fragments are diminished to undetectable levels. Therefore, the co-expression of PS1 and PS2 TM Asp --> Ala mutants suppresses the formation of any detectable PS1 or PS2 heterodimeric fragments and essentially abolishes the production of Abeta. These results explain the residual Abeta production seen in PS1-deficient cells and demonstrate the absolute requirement of functional presenilins for Abeta generation. We conclude that presenilins, and their TM aspartates in particular, are attractive targets for lowering Abeta therapeutically to prevent Alzheimer's disease.