Free Edges in Epithelial Cell Sheets Stimulate Epidermal Growth Factor Receptor Signaling

The ability of epithelia to migrate and cover wounds is essential to maintaining their functions as physical barriers. Wounding induces many cues that may affect the transition to motility, including the immediate mechanical perturbation, release of material from broken cells, new interactions with adjacent extracellular matrix, and breakdown of physical separation of ligands from their receptors. Depending on the exact nature of wounds, some cues may be present only transiently or insignificantly. In many epithelia, activation of the epidermal growth factor receptor (EGFR) is a central event in induction of motility, and we find that its continuous activation is required for progression of healing of wounds in sheets of corneal epithelial cells. Here, we examine the hypothesis that edges, which are universally and continuously present in wounds, are a cue. Using a novel culture model we find that their presence is sufficient to cause activation of the EGFR and increased motility of cells in the absence of other cues. Edges that are bordered by agarose do not induce activation of the EGFR, indicating that activation is not due to loss of any specific type of cell–cell interaction but rather due to loss of physical constraints.     

Chromosome distribution of intracisternal A-particle sequences in the Syrian hamster and mouse

Metaphase chromosomes of Syrian hamster and BALB/c mice were hybridized in situ with radiolabeled probes derived from cloned intracisternal A-particle (IAP) genes of the corresponding species. The DNAs of these species are known to contain about 900 and 1,000 copies, respectively, of the retrovirus-like IAP sequence elements per haploid genome. Multiple IAP sequences were found on all chromosomes of both hamster and mouse. In the hamster, more than half of the IAP sequences were located in regions of non-centromeric constitutive heterochromatin, at an average concentration per unit chromosome length 5 times greater than in the euchromatic regions. The other dispersed sequences showed marked local variations in concentration along the chromosome lengths; both discrete foci and large grain clusters were observed as well as regions apparently lacking IAP sequences. Within the resolution of the techniques, IAP sequences appeared to be more evenly distributed over the mouse chromosomes; however, some prominent variations in concentration were seen. The number of potentially active IAP genes in the Syrian hamster, and by extension in the mouse, may be restricted by the preferential location of IAP sequences in genetically inert regions of the genome.     

A survey of toxicity of cyanobacterial blooms in Lake Ladoga and adjacent water bodies

Twentyfive cyanobacterial blooms in Lake Ladoga and adjacent water bodies were studied in the summer of 1990–1992. Toxicity of the water bloom material for mice was detected in 9 cases. The maximal tolerable doses (MTD) of the material extracted from biomass varied within 3–30 mg kg^–1 mouse body weight; 50% lethal doses (LD₅₀) were within 45–125 mg kg^–1. Toxic water blooms were registered in Karelian lakes and in the Neva Bay, Gulf of Finland. Cyanobacterial samples collected on the eastern coast of Lake Ladoga proved to be non-toxic. The species identified in toxic bloom material included Anabaena circinalis, A. flos-aquae, A. lemmermannii, Anabaena sp., Aphanizomenonflos-aquae, Gloeotrichia echinulata, G. pisum, Microcystis aeruginosa and Oscillatoria sp. These data suggest that toxic forms of cyanobacteria are widespread in Karelian lakes belonging to the drainage basin of Lake Ladoga.     

REPRODUCTIVE ISOLATION BY HOST SPECIFICITY IN A PAIR OF PHYTOPHAGOUS LADYBIRD BEETLES

Crossing experiments and food-choice tests show that two sympatric species of phytophagous ladybird beetles, Epilachna niponica and E. yasutomii, are reproductively isolated by host-plant specificity. Adult beetles selected their natural hosts when given choices, though some accepted the host of the other species when no choice was offered. In each species, survival of larvae to the second instar was significantly higher on their own host plant. No evidence for sexual isolation, gametic isolation, hybrid inviability, or reduced hybrid fertility was detected. Reproductive isolation by host specificity is an important prerequisite for certain models of sympatric speciation. Although the present example supports the plausibility of such models, an allopatric origin of host-plant specificity cannot be discounted.     

Identification and characterization of ispA, a Shigella flexneri chromosomal gene essential for normal in vivo cell division and intercellular spreading

The virulent phenotype of Shigella requires loci on the chromosome as well as on the large virulence plasmid, and is regulated via a complex web of interactions amongst various chromosomal and large plasmid genes. To further investigate the role of chromosomal loci in virulence, we performed random Tn 10 mutagenesis in Shigella flexneri YSH6000T, and isolated an avirulent mutant (V3404) incapable of spreading throughout an epithelial cell monolayer. Although V3404 initially spread intercellularly at the same rate as the wild-type, it gradually slowed down and ceased spreading as a result of increasing defects in cell division, leading to the formation of long filamentous bacteria lacking septa, trapped within cells. In addition, the mutation affected the ability of V3404 to polymerize actin, a prerequisite for intra- and inter-cellular spreading ability. Sequencing of Tn 10 -flanking DNA revealed that the mutated gene, designated ispA (intracellular septation), was equivalent to a previously sequenced but uncharacterised gene of Escherichia coli located between trp and tonB . Using E. coli sequence data, we cloned the ispA gene from the YSH6000T chromosome and found that it complemented the V3404 mutation. Nucleotide sequencing and in vitro expression experiments revealed that ispA coded for a small (21 kDa), very hydrophobic protein. These results thus show that ispA is an essential virulence gene affecting several functions of the virulence process.     

Comparison of the DNA content of megakaryocytes identified immunologically with that identified morphologically

 We devised a new microfluorometric method for determining the ploidy of megakaryocytes identified immunologically in bone marrow smears. The smears were immunostained by incubation with mouse monoclonal anti-glycoproteins (GP) IIb antibodies, followed by fluorescein isothiocyanate-conjugated goat anti-mouse IgG antibodies. They were then stained with 4′,6-diamidino-2-phenylindole (DAPI). Megakaryocytes were identified by their GPIIb immunofluorescence using a microfluorometer and, after the filters were changed, their DNA content was assayed by measuring the intensity of DAPI fluorescence. This intensity was shown to be proportional to the DNA content when the aperture of the objective lens was reduced. We compared these results with those obtained when megakaryocytes were identified morphologically, using DAPI staining after Wright-Giemsa destaining. In all 12 normal controls, the ploidy peaks were shown to be 16N by both methods, and the mean ploidy detected by the immunological method was only reduced 0.961 times relative to the estimate from the morphological method. In contrast, in eight myelodysplastic syndrome (MDS) patients, the ploidy peaks were either 8N or 4N and the mean was reduced by 0.906 times (P=0.018). Thus we could immunologically identify small megakaryocytes which we could not identify morphologically. Therefore, this method is useful for measuring megakaryocytic ploidy, especially in the pathological megakaryocytes of MDS patients. Accepted: 29 April 1997     

Mutational analysis of human immunodeficiency virus type 1 (HIV-1) accessory genes: requirement of a site in the nef gene for HIV-1 replication in activated CD4+ T cells in vitro and in vivo.

Human immunodeficiency virus type 1 (HIV-1) accessory genes including nef, vif, and vpr are important factors that determine the replication and pathogenesis of HIV-1. The state of activation is also important for the replication of HIV-1. We evaluated the properties of nef-, vif-, and vpr-minus macrophage-tropic HIV-1(JR) CSF in primary CD4+ Th1- or Th2-like cell cultures which had been activated through CD3 molecules in the presence of interleukin-2 (IL-2) and IL-12 (Th1-like culture) or IL-4 (Th2-like culture), respectively. In activated Th1- or Th2-like cultures, replication of nef-minus HIV-1(JR-CSF) was markedly lower than that of wild-type HIV-1. Subsequent analysis by site-directed mutagenesis showed that (i) the presence of an acidic amino acid-rich domain (amino acid residues 72 to 75) in the Nef protein was critical for the enhancement of viral DNA synthesis, resulting in increased virus growth rate, and (ii) prolines that form part of Src homology 3 binding domain were not essential for viral replication. We also confirmed the importance of sites by using an HIV-1-infected animal model, the hu-PBL-SCID mouse system, representing HIV-1 replication and pathogenesis in activated CD4+ T cells in vivo. These results indicate that Nef accelerates viral replication in activated CD4+ T cells.     

Nucleotide Sequence Relationship Between Intracisternal Type A Particles of Mus musculus and an Endogenous Retrovirus (M432) of Mus cervicolor

Intracisternal type A particles are retrovirus-like structures found in embryonic cells and many tumors of Mus musculus but having no clear relationship with other retroviruses of this mouse species. We have observed a partial nucleotide sequence homology between the high-molecular-weight (32S and 35S) RNA components of intracisternal A-particles from a neuroblastoma cell line and the 70S RNA fraction from M432, a type of retrovirus endogenous to the Asian mouse Mus cervicolor. M432 complementary DNA (cDNA) was hybridized to the extent of 30% by the A-particle RNAs. The hybrids showed a lower thermal stability (DeltaT(m), 7 degrees C) than those formed with homologous RNA. The reaction was commensurate with that found between M432 cDNA and divergent sequences in the M. musculus genome. The capacity to hybridize M432 cDNA was closely correlated with the concentration of A-particle sequences in the cytoplasmic RNA of several M. musculus cell types. The major RNA fraction of M432 virus showed a reciprocal partial reaction with the A-particle cDNA's; the virus, which was grown in NIH/3T3 (M. musculus) cells, also contained a small proportion of apparently authentic A-particle nucleotide sequences. A subset of A-particle sequences seemed to be almost totally lacking in the main M432 RNA. The A-particle cDNA's hybridized extensively with divergent sequences in M. cervicolor cellular DNA, indicating that this mouse species may contain not only the partially homologous M432 virogene, but also a more complete genetic equivalent of the intracisternal A-particle.     

DIROM: an experimental design interactive system for directed mutagenesis and nucleic acids engineering

A computer system DIROM for oligonucleotide-directed mutagenesisand artificial gene design has been designed for better experimentalplanning and control. DIROM permits searching for optimal oligonucleotideswith respect to certain important parameters, namely sufficientenergy of oligonucleotide-target hybridization, the secondarystructure of oligonuc-tide and target DNA, the presence of alternatebinding sites in the target DNA and terminal G/C pairs. It canalso be used to plan polymerase chain reaction experiments,for optimal primer selection, in sequencing, etc. DIROM enablesone to search for both existing and potential restriction sites,to perform vector + target sequence construction. The systemconsists of a set of original algorithms that formalize theempirical knowledge of oligonucleotide action as primers.