FISH mapping and molecular organization of the major repetitive sequences of tomato

This paper presents a bird’s-eye view of the major repeats and chromatin types of tomato. Using fluorescence in-situ hybridization (FISH) with Cot-1, Cot-10 and Cot-100 DNA as probes we mapped repetitive sequences of different complexity on pachytene complements. Cot-100 was found to cover all heterochromatin regions, and could be used to identify repeat-rich clones in BAC filter hybridization. Next we established the chromosomal locations of the tandem and dispersed repeats with respect to euchromatin, nucleolar organizer regions (NORs), heterochromatin, and centromeres. The tomato genomic repeats TGRII and TGRIII appeared to be major components of the pericentromeres, whereas the newly discovered TGRIV repeat was found mainly in the structural centromeres. The highly methylated NOR of chromosome 2 is rich in [GACA]₄, a microsatellite that also forms part of the pericentromeres, together with [GA]₈, [GATA]₄ and Ty1-copia. Based on the morphology of pachytene chromosomes and the distribution of repeats studied so far, we now propose six different chromatin classes for tomato: (1) euchromatin, (2) chromomeres, (3) distal heterochromatin and interstitial heterochromatic knobs, (4) pericentromere heterochromatin, (5) functional centromere heterochromatin and (6) nucleolar organizer region.     

Integrin expression and osteopontin regulation in human fetal osteoblastic cells mediated by substratum surface characteristics

Integrin-mediated adhesion of anchorage-dependent cells to scaffolds is a critical component of tissue engineering. We investigated integrin expression by the human fetal osteoblastic cell line, hFOB 1.19 (hFOB), as a function of substratum surface wettability. The influence of surface wettability on bone cell phenotype was also examined. Plasma-treated quartz (PTQ) and glass (PTG) (hydrophilic, contact angles of 0 degrees), octadecyltrichlorosilane-treated quartz (STQ) and glass (STG) (hydrophobic, contact angles above about 100 degrees), and tissue culture polystyrene were used for cell culture. hFOB cells cultured on hydrophilic substrata displayed well-developed actin stress fibers relative to cells on hydrophobic substrata. Western blot analysis revealed that hFOB cells cultured on hydrophobic substrata (STQ or STG) express lower levels of alphav and beta3 integrin subunits than do cells on hydrophilic substrata (PTQ or PTG). This effect was more pronounced in cells on STQ than on STG. These variations in integrin expression were lessened by extended culture time. Double- labeled integrin/actin immunofluorescence confirmed Western blot results, that is, cells cultured on PTQ displayed distinct, large plaques of alphav and beta3 subunits and integrin alphavbeta3, as well as their colocalization with actin stress fiber ends, whereas cells on STQ did not display integrin plaques after 24 h and displayed only minimal plaque formation after 3 days. Vinculin, a focal adhesion protein that mediates binding between the integrin and actin cytoskeleton, appeared in Western blots to mimic the variations of alphav and beta3 expression with respect to surface wettability. Interestingly, real-time RT-PCR analysis showed that hFOB cultured on hydrophobic substrata, which have downregulated alphav and beta3 integrin subunits, displayed greater steady state mRNA levels of osteopontin, an extracellular matrix (ECM) protein containing the Arg-Gly-Asp (RGD) integrin recognition sequence, than did cells cultured on hydrophilic substrata. Our results imply that substratum surface wettability regulates integrin-mediated bone cell adhesion and further influences the expression of bone cell-ECM complexes.     

Role of predicted transmembrane domains for type III translocation, pore formation, and signaling by the Yersinia pseudotuberculosis YopB protein

YopB is a 401-amino-acid protein that is secreted by a plasmid-encoded type III secretion system in pathogenic Yersinia species. YopB is required for Yersinia spp. to translocate across the host plasma membrane a set of secreted effector proteins that function to counteract immune signaling responses and to induce apoptosis. YopB contains two predicted transmembrane helices (residues 166 to 188 and 228 to 250) that are thought to insert into the host plasma membrane during translocation. YopB is also required for pore formation and host-cell-signaling responses to the type III machinery, and these functions of YopB may also require membrane insertion. To elucidate the importance of membrane insertion for YopB function, YopB proteins containing helix-disrupting double consecutive proline substitutions in the center of each transmembrane domain were constructed. Yersinia pseudotuberculosis strains expressing the mutant YopB proteins were used to infect macrophages or epithelial cells. Effector translocation, pore formation, and host-cell-signaling responses were studied. Introduction of helix-disrupting substitutions into the second transmembrane domain of YopB resulted in a nonfunctional protein that was not secreted by the type III machinery. Introduction of helix-disrupting substitutions into the first transmembrane domain of YopB resulted in a protein that was fully functional for secretion and for interaction with YopD, another component of the translocation machinery. However, the YopB protein with helix-disrupting substitutions in the first transmembrane domain was partially defective for translocation, pore formation, and signaling, suggesting that all three functions of YopB involve insertion into host membrane.     

Role of apoptosis in basal cell and squamous cell carcinoma formation

Long-term ultraviolet-light (UV) exposure of human skin epidermis is associated with an increased risk for the development of skin cancers, such as melanoma, basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). UV radiation not only induces DNA damage in epidermal cells, it also interferes with skin homeostasis, which is maintained by a unique distribution pattern of apoptosis-inducing and -preventing molecules. If the DNA damage is not repaired or the damaged cells are not eliminated by apoptosis, the consequence can be cell transformation, uncontrolled proliferation and eventually skin tumor formation. An important "repair" gene is the p53 suppressor gene. Excessive UV exposure can mutate the p53 gene leading to the loss of its repair function and thus apoptosis resistance of the DNA-damaged cell. For BCC formation an additional pathway has been identified. Mutation of genes of the Hedgehog signaling pathway evokes the downregulation of apoptotic genes and upregulation of anti-apoptotic genes preventing the elimination of damaged cells. In addition, BCC and SCC strongly express the apoptosis-inducing Fas-ligand (FasL) which may help the tumor to escape the attack of immune effector cells. Silencing the genes involved in tumor formation by RNA interference might become a promising new approach to treat skin tumors.     

Vaso-occlusive and prothrombotic mechanisms associated with tumor hypoxia, necrosis, and accelerated growth in glioblastoma

Glioblastoma (GBM) has explosive biologic properties with rapid clinical progression leading to death. Its distinguishing pathologic features, necrosis with surrounding pseudopalisades and microvascular hyperplasia, are believed to be instrumental to its accelerated growth. Microvascular hyperplasia arises in response to the secretion of proangiogenic factors by hypoxic pseudopalisades and allows for peripheral neoplastic expansion. Mechanisms underlying necrosis and hypoxia remain obscure, but vaso-occlusive and prothrombotic contributions could be substantial. Recent investigations on the origin of pseudopalisades suggest that this morphologic phenomenon is created by a tumor cell population actively migrating away from a central hypoxic region and that, in at least a significant subset, hypoxia-induced migration appears due to vaso-occlusion caused by intravascular thrombosis. Both vascular endothelial growth factor induced vascular permeability to plasma coagulation factors and the increased neoplastic expression of tissue factor likely contribute to a prothrombotic state favoring intravascular thrombosis. In addition to prothrombotic mechanisms, vaso-occlusion could also result from angiopoietin-2-mediated endothelial cell apoptosis and vascular regression, which follows neoplastic co-option of native vessels in animal models of gliomas. Vaso-occlusive and prothrombotic mechanisms in GBM could readily explain the presence of pseudopalisades and coagulative necrosis in tissue sections, the emergence of central contrast enhancement and its rapid peripheral expansion on neuroimaging, and the dramatic shift to an accelerated rate of clinical progression. Since the hypoxic induction of angiogenesis appears to support further neoplastic growth, therapeutic targeting of the underlying vascular pathology and thrombosis could provide a new means to prolong time to progression.     

The DNA polymerase gene from chlorella viruses PBCV-1 and NY-2A contains an intron with nuclear splicing sequences.

The deduced amino acid sequences of two eukaryotic chlorella virus (PBCV-1 and NY-2A) DNA polymerases are 90% identical and contain amino acid motifs typical of alpha-like (Family B) DNA polymerases. The open reading frames of both PBCV-1 and NY-2A DNA polymerases are interrupted by an identically located, small (101 or 86 nucleotides, respectively) intron that resembles eukaryotic nuclear-spliced messenger RNA introns. This discovery suggests that chlorella virus replication has a nuclear phase.     

High prevalence of mixed genotype infections in hepatitis B virus infected intravenous drug users

The clinical relevance of hepatitis B virus (HBV) genotypes has been documented; however, the prevalence of mixed HBV genotype infections in at-risk groups remains controversial. The HBV genotypes were determined in 325 HBV-infected intravenous drug users (IVDU) who were at a greater risk of multiple exposures to different HBV genotypes by using a newly developed line probe assay. The distribution of HBV genotype was as follows: genotype A alone in 2 (0.6%); genotype B alone in 256 (78.8%); genotype C alone in 10 (3.1%); mixed genotype A and B in 18 (5.5%); genotype B and C in 30 (9.2%); genotype B and D in 1 (0.3%); genotype A and C in 1 (0.3%); and mixed infections of genotype A, B, and C in 3 (0.9%). Clonal analysis confirmed further the existence of mixed genotype infection and recombination between different genotypes. Compared with our previous data, the line probe assay seemed more sensitive than polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assay in identifying HBV genotype (98.8% vs. 65.0%) and detecting mixed genotype infections (16.3% vs. 0%). In conclusion, the prevalence of mixed HBV infections is substantially higher in IVDU in endemic areas, and the line probe assay is a useful method for rapid genotyping of HBV, with particular reference to the detection of mixed genotype infections.     

Multicenter clinical laboratory evaluation of a beta-lactamase disk assay employing a novel chromogenic cephalosporin, S1.

S1, a new chromogenic cephalosporin (International BioClinical, Inc., Portland, Oreg.), was used to detect beta-lactamase production among a variety of commonly encountered bacteria in a four-center collaborative study. Results of an S1 disk assay were compared with those obtained by a nitrocefin-based disk procedure (Cefinase; Becton-Dickinson Microbiology Systems, Cockeysville, Md.), with repetitive testing of five quality control organisms and with individual tests of recent clinical isolates of Neisseria gonorrhoeae (162 strains), Haemophilus influenzae (162 strains), Moraxella catarrhalis (155 strains), Staphylococcus aureus (161 strains), and Bacteroides fragilis (164 strains). The performances of the two beta-lactamase disk assays were comparable for the first three species cited above. However, the S1 assay appeared to be a more sensitive procedure than the Cefinase assay when applied to S. aureus and B. fragilis, with respect to both total numbers of positive results and length of time to a definitive positive endpoint.     

Smad4 overexpression causes germ cell ablation and leydig cell hyperplasia in transgenic mice

Members of the transforming growth factor-beta (TGF-beta) superfamily play a variety of important roles in testicular development and function. The tumor suppressor gene, Smad4, is a common mediator of TGF-beta, activin, and bone morphogenetic protein-mediated signaling pathways. To investigate the role of the Smad4 gene during testicular development and function, transgenic mice were generated using a Flag-tagged Smad4 gene driven by 180-bp fragment of the Mullerian inhibiting substance upstream promoter sequence. Three Smad4 transgenic founders (A, B, and G) were detected by Southern blot analysis; line B showed the highest expression of the Smad4 transgene and was further studied. The fertility in F1 generation (B) and F2 generation (BB) of the Smad4 transgenic mice was not impaired. However, in the F3 generation (B2x) all animals were impacted by the overexpression of the Smad4 transgene and two kinds of phenotypes were observed. In one group animals were completely infertile, while in the other group animals were fertile and sired the normal number of pups/litter. These groups are designated as infertile and fertile in the text. Histological evaluation of the testes from the infertile group showed variable degrees of Leydig cell hyperplasia, apoptosis of germ cells, spermatogenic arrest, seminiferous tubule degeneration, and infertility. In the fertile group, there was no apparent change in the histology of the testis except for a slight increase in the number of Leydig cells. Serum follicle-stimulating hormone levels in the adult animals of both groups of Smad4 transgenic male mice were not significantly different from normal littermates; however, testosterone levels in both groups were significantly (P < 0.05) increased. These results suggest that overexpression of Smad4 leads to testicular abnormalities and infertility supporting the hypothesis that the TGF-beta signaling pathways are carefully orchestrated during testicular development. In the absence of normal levels of Smad4 testicular function is compromised.