Structural and Functional Characterization of IS679 and IS66-Family Elements

A new insertion sequence (IS) element, IS679 (2,704 bp in length), has been identified in plasmid pB171 of enteropathogenic Escherichia coli B171. IS679 has imperfect 25-bp terminal inverted repeats (IRs) and three open reading frames (ORFs) (here called tnpA, tnpB, and tnpC). A plasmid carrying a composite transposon (Tn679) with the kanamycin resistance gene flanked by an intact IS679 sequence and an IS679 fragment with only IRR (IR on the right) was constructed to clarify the transposition activity of IS679. A transposition assay done with a mating system showed that Tn679 could transpose at a high frequency to the F plasmid derivative used as the target. On transposition, Tn679 duplicated an 8-bp sequence at the target site. Tn679 derivatives with a deletion in each ORF of IS679 did not transpose, finding indicative that all three IS679 ORFs are essential for transposition. The tnpA and tnpC products appear to have the amino acid sequence motif characteristic of most transposases. A homology search of the databases found that a total of 25 elements homologous to IS679 are present in Agrobacterium, Escherichia, Rhizobium, Pseudomonas, and Vibrio spp., providing evidence that the elements are widespread in gram-negative bacteria. We found that these elements belong to the IS66 family, as do other elements, including nine not previously reported. Almost all of the elements have IRs similar to those in IS679 and, like IS679, most appear to have duplicated an 8-bp sequence at the target site on transposition. These elements have three ORFs corresponding to those in IS679, but many have a mutation(s) in an ORF(s). In almost all of the elements, tnpB is located in the -1 frame relative to tnpA, such that the initiation codon of tnpB overlaps the TGA termination codon of tnpA. In contrast, tnpC, separated from tnpB by a space of ca. 20 bp, is located in any one of three frames relative to tnpB. No common structural features were found around the intergenic regions, indicating that the three ORFs are expressed by translational coupling but not by translational frameshifting.     

Dynamic Behavior of Microtubules during Dynein-dependent Nuclear Migrations of Meiotic Prophase in Fission Yeast

During meiotic prophase in fission yeast, the nucleus migrates back and forth between the two ends of the cell, led by the spindle pole body (SPB). This nuclear oscillation is dependent on astral microtubules radiating from the SPB and a microtubule motor, cytoplasmic dynein. Here we have examined the dynamic behavior of astral microtubules labeled with the green fluorescent protein during meiotic prophase with the use of optical sectioning microscopy. During nuclear migrations, the SPB mostly follows the microtubules that extend toward the cell cortex. SPB migrations start when these microtubules interact with the cortex and stop when they disappear, suggesting that these microtubules drive nuclear migrations. The microtubules that are followed by the SPB often slide along the cortex and are shortened by disassembly at their ends proximal to the cortex. In dynein-mutant cells, where nuclear oscillations are absent, the SPB never migrates by following microtubules, and microtubule assembly/disassembly dynamics is significantly altered. Based on these observations, together with the frequent accumulation of dynein at a cortical site where the directing microtubules interact, we propose a model in which dynein drives nuclear oscillation by mediating cortical microtubule interactions and regulating the dynamics of microtubule disassembly at the cortex.     

Intervention of somatic mutational events in vivo by a germline defect at the adenine phosphoribosyltransferase locus

Both germline and somatic mutations are known to affect phenotypes of human cells in vivo. In previous studies, we cloned mutant peripheral blood T cells from germline heterozygous humans for adenine phosphoribosyltransferase (APRT) (EC 2.4.2.7) deficiency and found that approximately 1.3 × 10^–4 peripheral T cells had undergone in vivo somatic mutations. Loss of heterozygosity (LOH) was the major cause of the mutations at the APRT locus since approximately 80% of the mutant T cell clones exhibited loss of normal alleles. In the present study, we identified three heterozygous individuals for APRT deficiency (representing two separate families), in whom none of the somatic mutant cells exhibited LOH at the APRT locus. The germline mutant APRT alleles of these heterozygotes from two unrelated families had the same gross DNA abnormalities detectable by Southern blotting. None of the germline mutant APRT alleles so far reported had such gross DNA abnormalities. The data suggest that the germline mutation unique to these heterozygous individuals is associated with the abrogation of LOH in somatic cells. The absence of LOH at a different locus has already been reported in vitro in an established cell line but the present study describes the first such event in vivo in human individuals. Received: 10 May 1996     

Amyloid β-Mediated Zn2+ Influx into Dentate Granule Cells Transiently Induces a Short-Term Cognitive Deficit

We examined an idea that short-term cognition is transiently affected by a state of confusion in Zn²⁺ transport system due to a local increase in amyloid-β (Aβ) concentration. A single injection of Aβ (25 pmol) into the dentate gyrus affected dentate gyrus long-term potentiation (LTP) 1 h after the injection, but not 4 h after the injection. Simultaneously, 1-h memory of object recognition was affected when the training was performed 1 h after the injection, but not 4 h after the injection. Aβ-mediated impairments of LTP and memory were rescued in the presence of zinc chelators, suggesting that Zn²⁺ is involved in Aβ action. When Aβ was injected into the dentate gyrus, intracellular Zn²⁺ levels were increased only in the injected area in the dentate gyrus, suggesting that Aβ induces the influx of Zn²⁺ into cells in the injected area. When Aβ was added to hippocampal slices, Aβ did not increase intracellular Zn²⁺ levels in the dentate granule cell layer in ACSF without Zn²⁺, but in ACSF containing Zn²⁺. The increase in intracellular Zn²⁺ levels was inhibited in the presence of CaEDTA, an extracellular zinc chelator, but not in the presence of CNQX, an AMPA receptor antagonist. The present study indicates that Aβ-mediated Zn²⁺ influx into dentate granule cells, which may occur without AMPA receptor activation, transiently induces a short-term cognitive deficit. Extracellular Zn²⁺ may play a key role for transiently Aβ-induced cognition deficits.     

Zebra Stripes through the Eyes of Their Predators,Zebras, and Humans

The century-old idea that stripes make zebras cryptic to large carnivores has never been examined systematically. We evaluated this hypothesis by passing digital images of zebras through species-specific spatial and colour filters to simulate their appearance for the visual systems of zebras’ primary predators and zebras themselves. We also measured stripe widths and luminance contrast to estimate the maximum distances from which lions, spotted hyaenas, and zebras can resolve stripes. We found that beyond ca. 50 m (daylight) and 30 m (twilight) zebra stripes are difficult for the estimated visual systems of large carnivores to resolve, but not humans. On moonless nights, stripes are difficult for all species to resolve beyond ca. 9 m. In open treeless habitats where zebras spend most time, zebras are as clearly identified by the lion visual system as are similar-sized ungulates, suggesting that stripes cannot confer crypsis by disrupting the zebra’s outline. Stripes confer a minor advantage over solid pelage in masking body shape in woodlands, but the effect is stronger for humans than for predators. Zebras appear to be less able than humans to resolve stripes although they are better than their chief predators. In conclusion, compared to the uniform pelage of other sympatric herbivores it appears highly unlikely that stripes are a form of anti-predator camouflage.     

Heat coma and its relationship to ocean dynamics in the oceanic sea skaters of Halobates (Heteroptera: Gerridae) inhabiting Indian and Pacific Oceans

This study was performed to clarify how weather and current dynamics affect the resistance to temperature change in the oceanic sea skaters, Halobates. Heat coma temperature (HCT) was measured for the adults and 5th instar larvae of four Halobates species collected from a fixed sampling location (12°00′N, 135°00′E ) in western tropical Pacific Ocean and from 13 locations in the eastern area of the India Ocean ranging from 08°00′N-06°00′S and 86°00-76°00′E. Both the gap temperature for heat coma (GTHC, mean±SD: 7.83±1.86 °C, n=32) and the heat coma temperature (HCP, 35.03±1.80 °C, n=32) of individuals collected from the Pacific Ocean, during the first half (10 days) of the sampling period at the fixed sampling point, were significantly higher than those during the second half (GTHC: 5.10±2.05 °C, n=63; HCP: 34.03±2.02 °C, n=63). The reduction in heat tolerance shown in the second half of the 20 day period may have been caused by a decrease in air temperature due to rainfall that occurred around the sampling point accompanied with the arrival of Typhoon No. 6.In the study of individuals collected from the Indian Ocean, significantly higher average GTHCs of >8 °C were recorded for the adult H. micans collected at 02°00′S and 06°00′S (89°00′E) than those at 0°00-8°00′N in the eastern Indian Ocean. Dynamic mixture of water from northern and southern currents occurs at 02°00-6°00′S of the Indian Ocean and might relate to such high heat tolerance.Temperature dynamics in the ocean habitat might directly affect the temperature resistance of the oceanic sea skaters.     

Shear Stress-induced Redistribution of Vascular Endothelial-Protein-tyrosine Phosphatase (VE-PTP) in Endothelial Cells and Its Role in Cell Elongation

Vascular endothelial cells (ECs) are continuously exposed to shear stress (SS) generated by blood flow. Such stress plays a key role in regulation of various aspects of EC function including cell proliferation and motility as well as changes in cell morphology. Vascular endothelial-protein-tyrosine phosphatase (VE-PTP) is an R3-subtype PTP that possesses multiple fibronectin type III-like domains in its extracellular region and is expressed specifically in ECs. The role of VE-PTP in EC responses to SS has remained unknown, however. Here we show that VE-PTP is diffusely localized in ECs maintained under static culture conditions, whereas it undergoes rapid accumulation at the downstream edge of the cells relative to the direction of flow in response to SS. This redistribution of VE-PTP triggered by SS was found to require its extracellular and transmembrane regions and was promoted by integrin engagement of extracellular matrix ligands. Inhibition of actin polymerization or of Cdc42, Rab5, or Arf6 activities attenuated the SS-induced redistribution of VE-PTP. VE-PTP also underwent endocytosis in the static and SS conditions. SS induced the polarized distribution of internalized VE-PTP. Such an effect was promoted by integrin engagement of fibronectin but prevented by inhibition of Cdc42 activity or of actin polymerization. In addition, depletion of VE-PTP by RNA interference in human umbilical vein ECs blocked cell elongation in the direction of flow induced by SS. Our results suggest that the polarized redistribution of VE-PTP in response to SS plays an important role in the regulation of EC function by blood flow.     

Phylogeny and mechanisms of shared hierarchical patterns in birdsong

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N-type Calcium Channel in the Pathogenesis of Experimental Autoimmune Encephalomyelitis

One of the family of voltage-gated calcium channels (VGCC), the N-type Ca²⁺ channel, is located predominantly in neurons and is associated with a variety of neuronal responses, including neurodegeneration. A precise mechanism for how the N-type Ca²⁺ channel plays a role in neurodegenerative disease, however, is unknown. In this study, we immunized N-type Ca²⁺ channel α_1B-deficient (α_1B^−/−) mice and their wild type (WT) littermates with myelin oligodendrocyte glycoprotein 35–55 and analyzed the progression of experimental autoimmune encephalomyelitis (EAE). The neurological symptoms of EAE in the α_1B^−/− mice were less severe than in the WT mice. In conjunction with these results, sections of the spinal cord (SC) from α_1B^−/− mice revealed a reduction in both leukocytic infiltration and demyelination compared with WT mice. No differences were observed in the delayed-type hypersensitivity response, spleen cell proliferation, or cytokine production from splenocytes between the two genotypes. On the other hand, Western blot array analysis and RT-PCR revealed that a typical increase in the expression of MCP-1 in the SC showed a good correlation with the infiltration of leukocytes into the SC. Likewise, immunohistochemical analysis showed that the predominant source of MCP-1 was activated microglia. The cytokine-induced production of MCP-1 in primary cultured microglia from WT mice was significantly higher than that from α_1B^−/− mice and was significantly inhibited by a selective N-type Ca²⁺ channel antagonist, ω-conotoxin GVIA or a withdrawal of extracellular Ca²⁺. These results suggest that the N-type Ca²⁺ channel is involved in the pathogenesis of EAE at least in part by regulating MCP-1 production by microglia.     

Insight into early diversification of leucine-rich repeat receptor-like kinases provided by the sequenced moss and hornwort genomes

Plant Molecular Biology - Identification of the subfamily X leucine-rich repeat receptor-like kinases in the recently sequenced moss and hornwort genomes points to their diversification into...