Production and characterization of somatic hybrids between upland cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>) and wild cotton (<Emphasis Type="Italic">G. klotzschianum</Emphasis> Anderss) via electrofusion

Symmetric somatic hybrid plants between Gossypium hirsutum Coker 201 and G. klotzschianum were obtained through electrofusion. The fusion products were cultured in KM₈P medium supplemented with 2.685 M -naphthaleneacetic acid and 0.465 M kinetin, and the regenerated plants were morphologically, genetically, and cytologically characterized. Nuclear-DNA flow cytometric analysis revealed that the plants tested (31 of 40) had a relative DNA content close to the total DNA contents of the two parents. Subsequent genome DNA analysis using random amplified polymorphic DNA markers revealed 16 of 18 plants were true somatic hybrids. Cytological investigation of the metaphase root-tip cells of seven hybrids revealed there were 72–81 chromosomes in the hybrids, a value close to the expected 78 chromosomes. The morphology of the hybrids was distinct from that of the parents and from that of the regenerants from protoplasts of Coker 201. Somatic hybridization represents a potent and novel tool for transferring genomes of wild cottons containing economically important traits to cultivars in breeding programs. This is the first report on the regeneration of somatic hybrids via protoplast fusion in cotton.     

Direct interaction of focal adhesion kinase with p190RhoGEF

Focal adhesion kinase (FAK) is a protein-tyrosine kinase that associates with multiple cell surface receptors and signaling proteins through which it can modulate the activity of several intracellular signaling pathways. FAK activity can influence the formation of distinct actin cytoskeletal structures such as lamellipodia and stress fibers in part through effects on small Rho GTPases, although the molecular interconnections of these events are not well defined. Here, we report that FAK interacts with p190RhoGEF, a RhoA-specific GDP/GTP exchange factor, in neuronal cells and in brain tissue extracts by co-immunoprecipitation and co-localization analyses. Using a two-hybrid assay and deletion mutagenesis, the binding site of the FAK C-terminal focal adhesion targeting (FAT) domain was identified within the C-terminal coiled-coil domain of p190RhoGEF. Binding was independent of a LD-like binding motif within p190RhoGEF, yet FAK association was disrupted by a mutation (Leu-1034 to Ser) that weakens the helical bundle structure of the FAK FAT domain. Neuro-2a cell binding to laminin increased endogenous FAK and p190RhoGEF tyrosine phosphorylation, and co-transfection of a dominant-negative inhibitor of FAK activity, termed FRNK, inhibited lamininstimulated p190RhoGEF tyrosine phosphorylation and p21 RhoA GTP binding. Overexpression of FAK in Neuro-2a cells increased both endogenous p190RhoGEF tyrosine phosphorylation and RhoA activity, whereas these events were inhibited by FRNK co-expression. Because insulin-like growth factor 1 treatment of Neuro-2a cells increased FAK tyrosine phosphorylation and enhanced p190RhoGEF-mediated activation of RhoA, our results support the conclusion that FAK association with p190RhoGEF functions as a signaling pathway downstream of integrins and growth factor receptors to stimulate Rho activity.     

Simultaneous cytomegalovirus infection and Kaposi's sarcoma of the thyroid diagnosed by fine needle aspiration in an AIDS patient. A case report and first cytologic description of the two entities occurring together

BACKGROUND: Cytomegalovirus (CMV) infection and Kaposi's sarcoma (KS) are common in AIDS patients but rarely involve the thyroid, and coexistence of these two entities in that organ has not yet been described before. CASE: A 41-year-old female AIDS patient presented with a 2 x 1-cm, well-demarcated, rubbery mass in the right side of the thyroid. On fine needle aspiration (FNA), spindle cells were retrieved singly or in small, loose clusters; they had bland, fusiform to cigar-shaped nuclei; inconspicuous nucleoli; delicate cytoplasmic vacuoles; cytoplasmic hyaline drops; and hemosiderin granules. A single endothelial cell showed an enlarged nucleus with a basophilic intranuclear inclusion and periinclusional halo. CONCLUSION: This is the first reported case of an AIDS patient with KS and CMV infection simultaneously involving the thyroid diagnosed by FNA.     

Cell cycle-dependent expression of volume-activated chloride currents in nasopharyngeal carcinoma cells

Patch-clamping and cell imageanalysis techniques were used to study the expression of thevolume-activated Cl current,I_Cl(vol), and regulatory volume decrease (RVD)capacity in the cell cycle in nasopharyngeal carcinoma cells (CNE-2Z). Hypotonic challenge caused CNE-2Z cells to swell and activated aCl current with a linear conductance, negligibletime-dependent inactivation, and a reversal potential close to theCl equilibrium potential. The sequence of anionpermeability was I > Br > Cl > gluconate. The Cl channelblockers tamoxifen, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB),and ATP inhibited I_Cl(vol). Synchronous cultures of cells were obtained by the mitotic shake-off technique and by adouble chemical-block (thymidine and hydroxyurea) technique. Theexpression of I_Cl(vol) was cell cycle dependent,being high in G₁ phase, downregulated in S phase, butincreasing again in M phase. Hypotonic solution activated RVD, whichwas cell cycle dependent and inhibited by the Cl channelblockers NPPB, tamoxifen, and ATP. The expression of I_Cl(vol) was closely correlated with the RVDcapacity in the cell cycle, suggesting a functional relationship.Inhibition of I_Cl(vol) by NPPB (100 µM)arrested cells in G₀/G₁. The data also suggest that expression of I_Cl(vol) and RVD capacity areactively modulated during the cell cycle. The volume-activatedCl current associated with RVD may therefore play animportant role during the cell cycle progress.

     

Mutual dependence of MDM2 and MDMX in their functional inactivation of p53

MDMX, an MDM2-related protein, has emerged as yet another essential negative regulator of p53 tumor suppressor, since loss of MDMX expression results in p53-dependent embryonic lethality in mice. However, it remains unknown why neither homologue can compensate for the loss of the other. In addition, results of biochemical studies have suggested that MDMX inhibits MDM2-mediated p53 degradation, thus contradicting its role as defined in gene knockout experiments. Using cells deficient in either MDM2 or MDMX, we demonstrated that these two p53 inhibitors are in fact functionally dependent on each other. In the absence of MDMX, MDM2 is largely ineffective in down-regulating p53 because of its extremely short half-life. MDMX renders MDM2 protein sufficiently stable to function at its full potential for p53 degradation. On the other hand, MDMX, which is a cytoplasmic protein, depends on MDM2 to redistribute into the nucleus and be able to inactivate p53. We also showed that MDMX, when exceedingly overexpressed, inhibits MDM2-mediated p53 degradation by competing with MDM2 for p53 binding. Our findings therefore provide a molecular basis for the nonoverlapping activities of these two p53 inhibitors previously revealed in genetic studies.     

Cytological study of Tibetia (Fabaceae) in the Hengduan Mountains region,China

The Hengduan Mountains comprise one of the world's most important hot spots of biodiversity. Tibetia (Ali) H.P. Tsui (Fabaceae), which has four or five species in two sections, is one of the genera endemic to the region. This paper describes for the first time the karyotype of three of those species. The chromosome counts of all three are 2 n = 16. The karyotypes of the species examined contain chromosomes of variable karyotypic symmetry with centromeres at median and submedian positions that correlate with the morphological characteristics of the species. Karyotypic variation at the diploid level appears to be the predominant feature of chromosome evolution in the genus and may provide a clue to the study of evolutionary patterns of plants in this region.     

Crystallization and preliminary X-ray diffraction analysis of FKBP12 complexed with a novel neurotrophic ligand

A novel neurotrophic ligand, (3R)-4-(p-Toluenesulfonyl)-1,4-thiazane-3-carboxylic acid-L-Leucine ethyl ester, has been complexed with FKBP12 and crystallized using the hanging-drop vapor-diffusion method. Crystals belong to P2(1) space group, with unit cell parameters a=41.2, b=29.6, c=41.5 A, beta=114.0 degrees. The crystals diffract to 1.8 A resolution limit.     

Molecular variation of Bothriocephalus acheilognathi Yamaguti, 1934 (Cestoda: Pseudophyllidea) in different fish host species based on ITS rDNA sequences

The molecular variation in Bothriocephalus acheilognathi Yamaguti, 1934 from 11 species of freshwater fish collected in Australia, China, the Czech Republic, England and Hawaii was investigated by determining the nucleotide sequences of the internal transcribed spacer region. The length of the first and second internal transcribed spacer sequences of multiple individuals ranged from 553 to 571 bp and 553 to 615 bp, and the G + C content from 53.1 to 53.5%. The percentage sequence divergence varied between 0 and 0.9% in the ITS1 and 0 and 6.6% in the ITS2, respectively, indicating the occurrence of intraspecific variation. It is demonstrated that the fragment length variation resulted primarily from microsatellite polymorphisms present in the ITS region, especially in the ITS2 region. Phylogenetic analyses revealed that B. acheilognathi examined in this study consisted of three closely related genotypes with certain degrees of host-specificity, and the genotype representing isolates from Cyprinus carpio L. was the most common and diverse form within the species B. acheilognathi.     

AGAP1, an endosome-associated,phosphoinositide-dependent ADP-ribosylation factor GTPase-activating protein that affects actin cytoskeleton

We have identified three members of the AGAP subfamily of ASAP family ADP-ribosylation factor GTPase-activating proteins (Arf GAPs). In addition to the Arf GAP domain, these proteins contain GTP-binding protein-like, ankyrin repeat and pleckstrin homology domains. Here, we have characterized the ubiquitously expressed AGAP1/KIAA1099. AGAP1 had Arf GAP activity toward Arf1>Arf5>Arf6. Phosphatidylinositol 4,5-bisphosphate and phosphatidic acid synergistically stimulated GAP activity. As found for other ASAP family Arf GAPs, the pleckstrin homology domain was necessary for activity. Deletion of the GTP-binding protein-like domain affected lipid dependence of Arf GAP activity. In vivo effects of AGAP1 were distinct from other ASAP family Arf GAPs. Overexpressed AGAP1 induced the formation of and was associated with punctate structures containing the endocytic markers transferrin and Rab4. AP1 was redistributed from the trans-Golgi to the punctate structures. Like other ASAP family members, AGAP1 overexpression inhibited the formation of PDGF-induced ruffles. However, distinct from other ASAP family members, AGAP1 also induced the loss of actin stress fibers. Thus, AGAP1 is a phosphoinositide-dependent Arf GAP that impacts both the endocytic compartment and actin.     

Regulatory volume decrease is actively modulated during the cell cycle

Nasopharyngeal carcinoma cells, CNE-2Z, when swollen by 47% hypotonic solution, exhibited a regulatory volume decrease (RVD). The RVD was inhibited by extracellular applications of the chloride channel blockers tamoxifen (30 microM; 61% inhibition), 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 100 microM; 60% inhibition), and ATP (10 mM; 91% inhibition). The level and time constant of RVD varied greatly between cells. Most cells conducted an incomplete RVD, but a few had the ability to recover their volume completely. There was no obvious correlation between cell volume and RVD capacity. Flow cytometric analysis showed that highly synchronous cells were obtained by the mitotic shake-off technique and that the cells progressed through the cell cycle synchronously when incubated in culture medium. Combined application of DNA synthesis inhibitors, thymidine and hydroxyurea arrested cells at the G1/S boundary and 87% of the cells reached S phase 4 h after being released. RVD capacity changed significantly during the cell cycle progression in cells synchronized by shake-off technique. RVD capacity being at its highest in G1 phase and lowest in S phase. The RVD capacity in G1 (shake-off cells sampled after 4 h of incubation), S (obtained by chemical arrest), and M cells (selected under microscope) was 73, 33, and 58%, respectively, and the time constants were 435, 769, and 2,000 sec, respectively. We conclude that RVD capacity is actively modulated in the cell cycle and RVD may play an important role in cell cycle progress.