Emergence of gp120 V3 Variants Confers Neutralization Resistance in an R5 Simian-Human Immunodeficiency Virus-Infected Macaque Elite Neutralizer That Targets the N332 Glycan of the Human Immunodeficiency Virus Type 1 Envelope Glycoprotein

Neutralization-resistant simian-human immunodeficiency virus AD8 (SHIV_AD8) variants that emerged in an infected macaque elite neutralizer targeting the human immunodeficiency virus type 1 (HIV-1) gp120 N332 glycan acquired substitutions of critical amino acids in the V3 region rather than losing the N332 glycosylation site. One of these resistant variants, carrying the full complement of gp120 V3 changes, was also resistant to the potent anti-HIV-1 monoclonal neutralizing antibodies PGT121 and 10-1074, both of which are also dependent on the presence of the gp120 N332 glycan.     

Macrophage-tropic simian/human immunodeficiency virus chimeras use CXCR4, not CCR5, for infections of rhesus macaque peripheral blood mononuclear cells and alveolar macrophages

After the nearly complete and irreversible depletion of CD4(+) T lymphocytes induced by highly pathogenic simian/human immunodeficiency virus chimeric viruses (SHIVs) during infections of rhesus monkeys, tissue macrophages are able to sustain high levels (>10(6) viral RNA copies/ml) of plasma viremia for several months. We recently reported that the virus present in the plasma during the late macrophage phase of infection had acquired changes that specifically targeted the V2 region of gp120 (H. Imamichi et al., Proc. Natl. Acad. Sci. USA 99:13813-13818, 2002); some of these SHIV variants were macrophage-tropic (M-tropic). Those findings have been extended by examining the tropic properties, coreceptor usage, and gp120 structure of five independent SHIVs recovered directly from lymph nodes of late-stage animals. All of these tissue-derived SHIV isolates were able to infect alveolar macrophages. These M-tropic SHIVs used CXCR4, not CCR5, for infections of rhesus monkey PBMC and primary alveolar macrophages. Because the starting highly pathogenic T-tropic SHIV inoculum also utilized CXCR4, these results indicate that the acquisition of M-tropism in the SHIV-macaque system is not accompanied by a change in coreceptor usage. Compared to the initial T-tropic SHIV inoculum, tissue-derived M-tropic SHIVs from individual infected animals carry gp120s containing similar changes (specific amino acid deletions, substitutions, and loss of N-linked glycosylation sites), primarily within the V1 and/or V2 regions of gp120.     

Induction of disease by a molecularly cloned highly pathogenic simian immunodeficiency virus/human immunodeficiency virus chimera is multigenic

One of three full-length infectious molecular clones of SHIV(DH12R), designated SHIV(DH12R-CL-7) and obtained from productively infected rhesus monkey peripheral blood mononuclear cells, directed rapid and irreversible loss of CD4+ T cells within 3 weeks of its inoculation into Indian rhesus monkeys. Induction of complete CD4+ T-cell depletion by SHIV(DH12R-CL-7) was found to be dependent on inoculum size. The acquisition of this pathogenic phenotype was accompanied by the introduction of 42 amino acid substitutions into multiple genes of parental nonpathogenic SHIV(DH12). Transfer of the entire SHIV(DH12R-CL-7) env gene into the genetic background of nonpathogenic SHIV(DH12) failed to confer the rapid CD4+ T-lymphocyte-depleting syndrome; similarly, the substitution of gag plus pol sequences from SIV(smE543) for analogous SIV(mac239) genes in SHIV(DH12R-CL-7) attenuated the pathogenic phenotype. Amino acid changes affecting multiple viral genes are necessary, but insufficient by themselves, to confer the prototypically rapid and irreversible CD4+ T-cell-depleting phenotype exhibited by molecularly cloned SHIV(DH12R-CL-7).     

Pollen wall ontogeny in <Emphasis Type="Italic">Polemonium caeruleum</Emphasis> (Polemoniaceae) and suggested underlying mechanisms of development

By a detailed ontogenetic study of Polemonium caeruleum pollen, tracing each stage of development at high TEM resolution, we aim to understand the establishment of the pollen wall and to unravel the mechanisms underlying sporoderm development. The main steps of exine ontogeny in Polemonium caeruleum, observed in the microspore periplasmic space, are spherical units, gradually transforming into columns, then to rod-like units (procolumellae), the appearance of the initial tectum, growth of columellae in height and tectum in thickness and initial sporopollenin accumulation on them, the appearance of the endexine lamellae and of dark-contrasted particles on the tectum, the appearance of a sponge-like layer and of the intine in aperture sites, the appearance of the foot layer on the base of the sponge-like layer and of spinules on the tectum, and massive sporopollenin accumulation. This sequence of developmental events fits well to the sequence of self-assembling micellar mesophases. This gives (together with earlier findings and experimental exine simulations) strong evidence that genome and self-assembly probably share control of exine formation. It is highly probable that self-assembly is an intrinsic instrument of evolution.     

Ontogeny of anastomosed laticifers in the stem apex of <Emphasis Type="Italic">Hancornia speciosa</Emphasis> (Apocynaceae): a topographic approach

Latex is a complex plant secretion with both ecological and economic importance. There is little information currently available on the cytological aspects of the ontogenesis of anastomosed laticifers, the ducts originating through the lysis of adjacent cell walls. Hancornia speciosa is a tree typical of the Cerrado (neotropical savanna) biome. Its latex has medicinal value and is also used to produce rubber. The ontogenesis of its laticifers and the process of latex synthesis are described here. Structural, cytochemical, and ultrastructural analyses of the stem apex and phytochemical analyses of the latex were performed. Laticifer ontogenesis begins early in promeristem cells and subsequently extends through the procambial region. The laticifer precursor cells demonstrate intense metabolic activity, evidenced by starch accumulation and the proliferation of mitochondria, dictyosomes, endoplasmic reticulum, and ribosomes—resulting in the thickening of the cell walls and accumulations of oil droplets in the cytoplasm and fibrous materials in the vacuoles. The ontogenetic process culminates with the partial dissolution of adjacent cell walls and the collapse of the cytoplasm, giving rise to anastomosed laticifers distributed throughout the phloem and adjacent regions of the cortex and medulla. The latex itself is composed of terpenes, mucilage, proteins, alkaloids, and organelle residues that form an emulsion. Laticifer development takes place in three phases: (1) the formation of the emulsion in the promeristem, (2) anastomosis and the collapse of the cytoplasm in the distal region of the procambium, and (3) the maturation of laticifers and latex storage in a central vacuole in the proximal region of the procambium.     

Replication timing of large <Emphasis Type="Italic">Sorex granarius</Emphasis> (<Emphasis Type="Italic">Soricidae</Emphasis>, <Emphasis Type="Italic">Eulipotyphla</Emphasis>) telomeres

Previously, we described the unique feature of telomeric regions in Iberian shrew Sorex granarius: its telomeres have two ranges of size, very small (3.8 kb of telomeric repeats on average) and very large discontinuous telomeres (213 kb) interrupted with 18S rDNA. In this study, we have demonstrated extraordinary replication pattern of S. granarius large telomeres that have not been shown before in other studied mammal. Using the ReD-FISH procedure, we observed prolonged, through S period, large telomere replication. Furthermore, revealed ReD-FISH asymmetric signals were probably caused by partial replication of telomeres within an hour of 5-bromodeoxyuridine treatment due to the large size and special organization. We also found that in contrast to the telomeric halo from primary fibroblasts of bovine, mink, and common shrew, telomere halo of S. granarius consists of multiple loops bundled together, some of which contain rDNA. Here, we suggested several replicons firing possibly stochastic in each large telomere. Finally, we performed the TIF assay to reveal DNA damage responses at the telomeres, and along with TIF in nuclei, we found large bodies of telomeric DNA and ?-H2AX in the cytoplasm and on the surface of fibroblasts. We discuss the possibility of additional origin activation together with recombination-dependent replication pathways, mainly homologous recombination including BIR for replication fork stagnation overcoming and further S. granarius large telomere replication.     

Phospholipase Dδ assists to cortical microtubule recovery after salt stress

The dynamic microtubule cytoskeleton plays fundamental roles in the growth and development of plants including regulation of their responses to environmental stress. Plants exposed to hyper-osmotic stress commonly acclimate, acquiring tolerance to variable stress levels. The underlying cellular mechanisms are largely unknown. Here, we show, for the first time, by in vivo imaging approach that linear patterns of phospholipase Dδ match the localization of microtubules in various biological systems, validating previously predicted connection between phospholipase Dδ and microtubules. Both the microtubule and linear phospholipase Dδ structures were disintegrated in a few minutes after treatment with oryzalin or salt. Moreover, by using immunofluorescence confocal microscopy of the cells in the root elongation zone of Arabidopsis, we have shown that the cortical microtubules rapidly depolymerized within 30 min of treatment with 150 or 200 mM NaCl. Within 5 h of treatment, the density of microtubule arrays was partially restored. A T-DNA insertional mutant lacking phospholipase Dδ showed poor recovery of microtubule arrays following salt exposition. The restoration of microtubules was significantly retarded as well as the rate of root growth, but roots of overexpressor GFP-PLDδ prepared in our lab, have grown slightly better compared to wild-type plants. Our results indicate that phospholipase Dδ is involved in salt stress tolerance, possibly by direct anchoring and stabilization of de novo emerging microtubules to the plasma membrane, providing novel insight into common molecular mechanism during various stress events.     

Ultrastructure of the larval Malpighian tubules in <Emphasis Type="Italic">Terrobittacus implicatus</Emphasis> (Mecoptera: Bittacidae)

The larvae of Bittacidae, a cosmopolitan family in Mecoptera, have an interesting habit of spraying the body surface with soil through the anus after hatching, and each molts. The fine structure of Malpighian tubules, however, remains largely unknown in the larvae of Bittacidae to date. Here, we studied the ultrastructure of the larval Malpighian tubules in the hangingfly Terrobittacus implicatus (Huang & Hua) using scanning and transmission electron microscopy. The larvae of T. implicatus have six elongate Malpighian tubules at the junction of the midgut and hindgut. The tubule comprises a basal lamina, a single-layered epithelium, and a central lumen. The basal plasma membranes of the epithelial cells are conspicuously infolded and generate a labyrinth. The epithelium consists of two types of cells: large principal cells and scattered stellate cells. Mitochondria and cisterns of rough endoplasmic reticulum are numerous in the principal cells but are sparsely distributed in the stellate cells, indicating that the principal cells are active in transport. On the other hand, spherites are only abundant in the principal cells and are likely associated with the soil-spraying habit of the larvae.     

The discovery of plastid-to-nucleus retrograde signaling—a personal perspective

DNA and machinery for gene expression have been discovered in chloroplasts during the 1960s. It was soon evident that the chloroplast genome is relatively small, that most genes for chloroplast-localized proteins reside in the nucleus and that chloroplast membranes, ribosomes, and protein complexes are composed of proteins encoded in both the chloroplast and the nuclear genome. This situation has made the existence of mechanisms highly probable that coordinate the gene expression in plastids and nucleus. In the 1970s, the first evidence for plastid signals controlling nuclear gene expression was provided by studies on plastid ribosome deficient mutants with reduced amounts and/or activities of nuclear-encoded chloroplast proteins including the small subunit of Rubisco, ferredoxin NADP+ reductase, and enzymes of the Calvin cycle. This review describes first models of plastid-to-nucleus signaling and their discovery. Today, many plastid signals are known. They do not only balance gene expression in chloroplasts and nucleus during developmental processes but are also generated in response to environmental changes sensed by the organelles.