Human T-cell leukemia virus type 1 (HTLV-1) infection of mice: proliferation of cell clones with integrated HTLV-1 provirus in lymphoid organs

Human T-cell leukemia virus type 1 (HTLV-1) is suggested to cause adult T-cell leukemia after 40 to 50 years of latency in a small percentage of carriers. However, little is known about the pathophysiology of the latent period and the reservoir organs where polyclonal proliferation of cells harboring integrated provirus occurs. The availability of animal models would be useful to analyze the latent period of HTLV-1 infection. At 18 months after HTLV-1 infection of C3H/HeJ mice inoculated with the MT-2 cell line, which is an HTLV-1-producing human T-cell line, HTLV-1 provirus was detected in spleen DNA from eight of nine mice. No more than around 100 proviruses were found per 10(5) spleen cells. Cellular sequences flanking the 3' long terminal repeat (LTR) and the clonalities of the cells which harbor integrated HTLV-1 provirus were analyzed by linker-mediated PCR. The results showed that the flanking sequences are of mouse genome origin and that polyclonal proliferation of the spleen cells harboring integrated HTLV-1 provirus had occurred in three mice. A sequence flanking the 5' LTR was isolated from one of the mice and revealed the presence of a 6-nucleotide duplication of cellular sequences, consistent with typical retroviral integration. Moreover, PCR was performed on DNA from infected tissues, with LTR primers and primers derived from seven novel flanking sequences of the three mice. Data revealed that the expected PCR products were found from lymphatic tissues of the same mouse, suggesting that the lymphatic tissues were the reservoir organs for the infected and proliferating cell clones. The mouse model described here should be useful for analysis of the carrier state of HTLV-1 infection in humans.     

Genetic and functional analysis of PARP, a DNA strand break-binding enzyme

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme activated by binding to a single- or double-strand break of DNA and is one of the death substrates for caspase-3 in apoptosis. The nuclear function of PARP is well studied and recent PARP-knockout studies indicate that PARP takes part in chromosomal stability. To analyze the effect of PARP overexpression, or loss of function, we have cloned PARP cDNA and the gene from Drosophila melanogaster and studied its function in developmental stages. Organization of exons corresponds to the functional domains of PARP. An alternatively spliced form of PARP lacking exon 5, which encodes the auto-modification domain, is found in Drosophila. Expression of the PARP gene is at high levels in embryos at 0-6h after egg laying and gradually decreased. In situ mRNA hybridization indicates localization of PARP mRNA in cells along the central nervous system at a late stage of embryogenesis. Overexpression of the gene in the developing eye primordia of D. melanogaster is an excellent experimental model to analyze the cell cycle and programmed cell death. We introduced PARP expression vector overexpresses PARP in the eye discs of Drosophila, and established the PARP transgenic flies by P element-mediated germ line transformation. These flies showed mild roughening of the normally smooth ommatidial lattice involving tissue polarity disruption characterized by missrotation and incorrect chirality of ommatidia. Possible mechanisms of involvement of PARP in the development are discussed.     

Isolation and characterization of cDNAs encoding mitochondrial phosphate transporters in soybean,maize, rice,and Arabidopsis

cDNA clones encoding mitochondrial phosphate transporters were isolated from four herbaceous plants. The cDNAs for the soybean, maize and rice transporters contained entire coding regions, whereas the Arabidopsis cDNA lacked the 5 portion. The hydropathy profiles of the deduced amino acid sequences predicted the existence of six membrane-spanning domains which are highly conserved in the mitochondrial transporter family. In soybeans, the mRNA level for the transporter was high in tissues containing dividing cells. It was suggested that there are multiple copies of transporter genes in both dicots and monocots. The soybean transporter was expressed as inclusion bodies in Escherichia coli, solubilized with detergents, and then reconstituted into liposomes. The resulting proteoliposomes exhibited high phosphate transport activity. The activity was inhibited by N-ethylmaleimide, like those of mammalian phosphate transporters.     

Cerebral Blood Flow Threshold and Regional Heterogeneity of Heat Shock Protein 72 Induction Following Transient Forebrain Ischemia in Rats

Heat shock proteins (HSPs) induced by brain ischemia may play an important role in neuroprotection from neuronal degeneration. In this study, we examined the cerebral blood flow (CBF) threshold to produce regional differences in HSP72 induction after transient forebrain ischemia in spontaneously hypertensive rats (SHRs). Female SHRs were subjected to 20 min of cerebral ischemia induced by bilateral carotid artery occlusion. The CBF was measured by laser Doppler flowmetry. At forty-eight hours after cerebral ischemia and reperfusion, the rats were decapitated and the brains were removed. Specific areas (hippocampal CA1, CA2-3, dentate gyrus, dorsolateral and ventromedial striatum, and parietal cortex) were thereafter dissected from the brain. The amounts of HSP72 in these samples were determined using Western blot analysis. In the hippocampus, HSP72 was induced when the CBF decreased to less than 18–25% of the resting level. The mean values of HSP72 produced in the CA1 area, CA2-3 area, and the dentate gyrus following ischemia and reperfusion treatment were 4.44 ± 1.43 (±SD) ng/g prtein, 3.51 ± 0.72 ng/g protein and 3.77 ± 1.05 ng/g protein, respectively. In the parietal cortex, the amount of HSP72 induction was less pronounced (2.55 ± 0.40 ng/g protein), while HSP72 was hardly detected at all in the striatum, even under conditions of very severe CBF reduction and reperfusion. We demonstrated the existence of both a CBF threshold (i.e., approximately 20% of the resting level) for HSP72 induction and regional heterogeneity for the induction of HSP72 protein.     

Transforming growth factor-beta1-dependent activation of Smad2/3 and up-regulation of PAI-1 expression is negatively regulated by Src in SKOV-3 human ovarian cancer cells

The net balance between urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) has been implicated in tumor cell invasion and metastasis. To elucidate the mechanism of the transforming growth factor-beta1 (TGF-beta1)-dependent up-regulation of PAI-1 expression, we investigated which signaling pathway transduced by TGF-beta1 is responsible for this effect. Here, we show (1) nontoxic concentrations of TGF-beta1 up-regulates uPA expression in HRA and SKOV-3 human ovarian cancer cells, (2) TGF-beta1 activates Smads (phosphorylation of Smad2 and nuclear translocation of Smad3) and subsequently up-regulates PAI-1 expression in HRA cells, whereas TGF-beta1 neither activates Smads nor up-regulates PAI-1 in SKOV-3 cells, (3) pharmacological Src inhibitor PP2 or antisense (AS) c-Src oligodeoxynucleotide (ODN) treatment significantly induces TGF-beta1-dependent activation of Smads, leading to PAI-1 synthesis, compared with controls, in SKOV-3 cells, (4) combination of TGF-beta1 and PP2, which activates PAI-1 expression and reduces uPA expression in SKOV-3, results in decreased invasiveness, (5) pharmacological inhibitors for mitogen-activated protein kinase (MAPK) (PD98059) and phosphoinositide-3-kinase (PI3K) (LY294002 and wortmannin) or AS-PI3K ODN transfection do not affect TGF-beta1-induced Smad signaling and up-regulation of PAI-1 expression in SKOV-3 cells pretreated with PP2, and (6) the induction of PAI-1 protein was partially inhibited by an inhibitor of Sp1-DNA binding, mithramycin, implicating, at least in part, Sp1 in the regulation of this gene by TGF-beta1. In conclusion, TGF-beta1-dependent activation of Smad2/3, leading to PAI-1 synthesis, may be negatively regulated by Src, but not its downstream targets MAPK and PI3K in SKOV-3 cells. These data also reflect the complex biological effect of uPA-PAI-1 system.     

Effect of dilution rate on competitive interactions between the cyanobacterium Microcystis novacekii and the green alga Scenedesmus quadricauda in mixed chemostat cultures

We examined the competition between the cyanobacterium Microcystisnovacekii (Kom.) Comp. and the green alga Scenedesmus quadricauda(Turpin) Brébisson using unialgal and mixed chemostatcultures with various supply rates of culture medium where limited algal growth. In unialgal cultures, bothspecies grew at all of the dilution rates examined (0.1, 0.3and 0.8 day^-1): steady-state cell densities were 1 x 10⁴ to8 x 10⁴ cells mL^-1 for M. novacekii and 0.5 x 10⁵ to 2.1 x 10⁵cells mL^-1 for S. quadricauda. Microcystis novacekii was dominantin mixed cultures at a dilution rate of 0.1 day^-1, where thesteady-state cell density was 1 x 10⁴ to 7 x 10⁴ cells mL^-1for M. novacekii and 1 x 10² to 5 x 10² cells mL^-1 for S. quadricauda.Scenedesmus quadricauda was dominant in mixed cultures at thehigher dilution rates (0.3 and 0.8 day^-1), where the final celldensity was 0.5 x 10² to 6.4 x 10² cells mL^-1 for M. novacekiiand 0.2 x 10⁵ to 7 x 10⁵ cells mL^-1 for S. quadricauda. Thisresult indicates that the dilution rate affects the competitiveinteraction. We conclude that it is necessary to consider waterexchange in the study of mechanisms of cyanobacterial blooms.     

Regrowth of the stalk of the sea lily, Metacrinus rotundus (Echinodermata: Crinoidea)

Sea lilies are critical to understanding the evolution of the echinoderm body plan, because they are the only extant group whose adults possess a stalk, a prevalent feature in the radiation of a number of primitive echinoderm lineages. Extensive crown regeneration ability has been reported in Metacrinus rotundus, but the regenerative potential of the stalk has never been determined in any species of sea lilies. In this study, we show that M. rotundus whose stalks have been completely excised are capable of stalk regeneration. The process is similar to the growth of the original stalk, but much slower, and the regenerated stalks are not morphologically identical to the original stalk. Since stalk regeneration, in contrast to well-studied regeneration events, probably requires little additional activation of morphogenetic programs, we refer to the stalk regeneration phenomenon as "stalk regrowth" to distinguish it as a special form of regeneration. Since specimens whose entire stalk below the basal plates had been removed were able to regrow, the basal plates, and probably the aboral nerve center within them, are essential for stalk regrowth. Sea lily stalk regrowth is described in detail, and the evolution of feather stars is discussed in light of the growth pattern of the sea lily stalk.