Ecdysteroids from the flowers of Aerva javanica

Four new ecdysteroids (1–4), along with three known steroids, β-ecdysone (5), 5-β-2-deoxyintegristerone A (6) and 24-epi-makisterone A (7) (Fig. 1), were isolated from the methanolic extract of the flowers of Aerva javanica by using normal and reverse phase chromatography. The structures of the new compounds (1–4) were determined due to 1D (¹H and ¹³C), 2D NMR (HSQC, HMBC, COSY, NOESY) techniques and high resolution mass spectrometry (HREIMS). The known compounds (5–7) were characterized based on the 1D NMR spectroscopy and mass spectrometry and by comparison with the literature values. All isolates were evaluated for their inhibitory activities against enzymes acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and lipoxygenase (LOX).     

The C-Terminal Domains of NF-H and NF-M Subunits Maintain Axonal Neurofilament Content by Blocking Turnover of the Stationary Neurofilament Network

Newly synthesized neurofilaments or protofilaments are incorporated into a highly stable stationary cytoskeleton network as they are transported along axons. Although the heavily phosphorylated carboxyl-terminal tail domains of the heavy and medium neurofilament (NF) subunits have been proposed to contribute to this process and particularly to stability of this structure, their function is still obscure. Here we show in NF-H/M tail deletion [NF-(H/M)^tailΔ] mice that the deletion of both of these domains selectively lowers NF levels 3–6 fold along optic axons without altering either rates of subunit synthesis or the rate of slow axonal transport of NF. Pulse labeling studies carried out over 90 days revealed a significantly faster rate of disappearance of NF from the stationary NF network of optic axons in NF-(H/M)^tailΔ mice. Faster NF disappearance was accompanied by elevated levels of NF-L proteolytic fragments in NF-(H/M)^tailΔ axons. We conclude that NF-H and NF-M C-terminal domains do not normally regulate NF transport rates as previously proposed, but instead increase the proteolytic resistance of NF, thereby stabilizing the stationary neurofilament cytoskeleton along axons.     

Mutational Analysis of the Human Immunodeficiency Virus Type 1 Vpu Transmembrane Domain That Promotes the Enhanced Release of Virus-Like Particles from the Plasma Membrane of Mammalian Cells

Human immunodeficiency virus type 1 Vpu is a multifunctional phosphoprotein composed of the N-terminal transmembrane (VpuTM) and C-terminal cytoplasmic domains. Each of these domains regulates a distinct function of the protein; the transmembrane domain is critical in virus release, and phosphorylation of the cytoplasmic domain is necessary for CD4 proteolysis. We carried our experiments to identify amino acids in the VpuTM domain that are important in the process of virus-like particle (VLP) release from HeLa cells. VLPs are released from the plasma membrane of HeLa cells at constitutive levels, and Vpu expression enhanced the release of VLPs by a factor of 10 to 15. Deletion of two to five amino acids from both N- and C-terminal ends or the middle of the VpuTM domain generated mutant Vpu proteins that have lost the ability to enhance VLP release. These deletion mutants have not lost the ability to associate with the wild-type or mutant Vpu proteins and formed complexes with equal efficiency. They were also transported normally to the Golgi complex. Furthermore, a Vpu protein having the CD4 transmembrane and Vpu cytoplasmic domains was completely inactive, and Vpu proteins harboring hybrid Vpu-CD4 TM domains were also defective in the ability to enhance the release of VLPs. When tested for functional complementation in cotransfected cells, two inactive proteins were not able to reconstitute Vpu activity that enhances the release of Gag particles. Coexpression of functional CD4/Vpu hybrids or wild-type Vpu with inactive mutant CD4/Vpu proteins revealed that mutations in the VpuTM domain could dominantly interfere with Vpu activity in Gag release. Taken together, these results demonstrated that the structural integrity of the VpuTM domain is critical for Vpu activity in the release of VLPs from the plasma membrane of mammalian cells.     

Cell surface transport, oligomerization, and endocytosis of chimeric type II glycoproteins: role of cytoplasmic and anchor domains.

We investigated the role of cytoplasmic and anchor domains of type II glycoproteins in intracellular transport, oligomerization, and endocytosis by expressing the wild-type and chimeric genes in mammalian cells. Chimeric genes were constructed by exchanging the DNA segments that encode the cytoplasmic and anchor domains between the human influenza virus (A/WSN/33) neuraminidase (NA) and transferrin receptor (TR). The chimeric proteins in which domains were exchanged precisely were productively targeted to the cell surface. However, the proteins appeared to assemble differently in the intracellular compartment. For example, while TR existed predominantly as a dimer, NATR delta 90, containing the cytoplasmic and signal-anchor domains of NA and the ectodomain of TR, was present as a tetramer, a dimer, and a monomer. Similarly, the influenza virus NA existed predominantly as a tetramer but TRNA delta 35, in which the cytoplasmic and signal-anchor domains of TR were joined to the ectodomain of NA, existed predominantly as a dimer, suggesting that the cytoplasmic and anchor domains of type II glycoproteins affect the subunit assembly of heterologous ectodomains. In addition, we analyzed the role of the cytoplasmic domain in endocytosis. NA and NATR delta 90 did not undergo endocytosis, whereas both TR and TRNA delta 35 were internalized efficiently, demonstrating that the NH2 cytoplasmic domain of TR was capable of internalizing a heterologous ectodomain (NA) from the cell surface.     

Caenorhabditis elegans OSR-1 regulates behavioral and physiological responses to hyperosmotic environments

The molecular mechanisms that enable multicellular organisms to sense and modulate their responses to hyperosmotic environments are poorly understood. Here, we employ Caenorhabditis elegans to characterize the response of a multicellular organism to osmotic stress and establish a genetic screen to isolate mutants that are osmotic stress resistant (OSR). In this study, we describe the cloning of a novel gene, osr-1, and demonstrate that it regulates osmosensation, adaptation, and survival in hyperosmotic environments. Whereas wild-type animals exposed to hyperosmotic conditions rapidly lose body volume, motility, and viability, osr-1(rm1) mutant animals maintain normal body volume, motility, and viability even upon chronic exposures to high osmolarity environments. In addition, osr-1(rm1) animals are specifically resistant to osmotic stress and are distinct from previously characterized osmotic avoidance defective (OSM) and general stress resistance age-1(hx546) mutants. OSR-1 is expressed in the hypodermis and intestine, and expression of OSR-1 in hypodermal cells rescues the osr-1(rm1) phenotypes. Genetic epistasis analysis indicates that OSR-1 regulates survival under osmotic stress via CaMKII and a conserved p38 MAP kinase signaling cascade and regulates osmotic avoidance and resistance to acute dehydration likely by distinct mechanisms. We suggest that OSR-1 plays a central role in integrating stress detection and adaptation responses by invoking multiple signaling pathways to promote survival under hyperosmotic environments.     

Delineation of Cohen syndrome following a large-scale genotype-phenotype screen

Cohen syndrome is an autosomal recessive condition associated with developmental delay, facial dysmorphism, pigmentary retinopathy, and neutropenia. The pleiotropic phenotype, combined with insufficient clinical data, often leads to an erroneous diagnosis and has led to confusion in the literature. Here, we report the results of a comprehensive genotype-phenotype study on the largest cohort of patients with Cohen syndrome assembled to date. We found 22 different COH1 mutations, of which 19 are novel, in probands identified by our diagnostic criteria. In addition, we identified another three novel mutations in patients with incomplete clinical data. By contrast, no COH1 mutations were found in patients with a provisional diagnosis of Cohen syndrome who did not fulfill the diagnostic criteria ("Cohen-like" syndrome). This study provides a molecular confirmation of the clinical phenotype associated with Cohen syndrome and provides a basis for laboratory screening that will be valuable in its diagnosis.     

Modifying and Fine Controlling of Silver Nanoparticle Nucleation Sites and SERS Performance by Double Silicon Etching Process

Different forms of modified and well-controlled plasmonic silver nanoparticles (AgNPs) were synthesized by silver ion reduction process of porous silicon (PS). Fine control of PS surface morphology was accomplished by employing two etching processes: light-induced etching (LIE) and photo electrochemical etching (PECE). The idea was to prepare excellent and reproducible surface-enhanced Raman scattering (SERS) substrates with high enhancement performance. PS surface modification was employed to create efficient and nearly uniformly distributed AgNP hotspot regions with very high specific surface areas. Reproducibility deviation of no more than 5% and enhancement factor of 1.2 × 10¹⁴ were obtained by SERS measurements at very low, rhodamine 6G (R6G) dye, concentration 10^?15 M. The PS morphology SERS substrate was well discussed and analyzed using field emission scanning electron microscopy (FE-SEM), X-ray diffraction spectroscopy (XRD), and Raman measurements.     

New species of <Emphasis Type="Italic">Cloacina</Emphasis> von Linstow, 1898 (Nematoda: Strongyloidea) parasitic in the stomachs of wallaroos, <Emphasis Type="Italic">Osphranter</Emphasis> spp. (Marsupialia: Macropodidae) from northern Australia

Three new species of the parasitic nematode genus Cloacina von Linstow, 1898 (Strongyloidea: Cloacininae) are described from the stomachs of wallaroos, Osphranter spp. (Marsupialia: Macropodidae), from northern Australia. Cloacina spearei n. sp. is described from O. robustus woodwardi (Thomas) and O. antilopinus (Gould) and is distinguished from congeners by the shape of the cephalic papillae, the shallow buccal capsule, the presence of an oesophageal denticle and the convoluted but non-recurrent vagina in the female. Cloacina longibursata n. sp. also from O. robustus woodwardi and O. antilopinus is distinguished from congeners by the elongate dorsal lobe of the bursa, with the origin of the lateral branchlets posterior to the principal bifurcation, in the features of the spicule tip, the lack of bosses lining the oesophagus and the absence of an oesophageal denticle. Cloacina crassicaudata n. sp., from the same two host species was formerly identified as C. cornuta (Davey & Wood, 1938). Differences in the cephalic cuticle (inflation lacking in the new species), the shape of the cephalic papillae, the dorsal oesophageal tooth and the spicule tips, as well as differences in the sequences of the internal transcribed spacers of the nuclear ribosomal DNA, indicate that this is an independent species. The geographical distribution of this species is disjunct with populations in both the Northern Territory and Queensland. Possible reasons for the disjunct distribution are discussed.     

Localisation of three host-protective oncospheral antigens of Taenia ovis

Immunohistochemistry, confocal immunofluorescence and immunogold labelling were used to determine the localisation of the host-protective antigens To16, To18 and To45W in Taenia ovis oncospheres. During maturation of the adult tapeworm the antigens were initially seen as diffuse staining in the developing oncospheres but in mature oncospheres four distinct cells stained positively for the antigens. Confocal fluorescence microscopy using different fluorophores revealed that each of the antigens co-localises within the same cells in the oncosphere. No surface localisation was seen in non-activated or recently activated parasites. Immunogold labelling of non-activated oncosphere sections viewed in transmission electron microscopy revealed labelling of bilateral cells, however the identities of these cells was unclear due to deficiencies in the current level of understanding of oncosphere ultrastructure. Localisation of all the antigens changed dramatically after oncospheres were activated in vitro with each of the antigens being dispersed more generally throughout the parasite parenchyma. During development of the parasites in in vitro culture, surface localisation of the proteins was seen in parasites after 3 or more days in culture. All three antigens were found to be completely absent in parasites by 15 days of culture. The location of the host-protective antigens suggests that initially the invading oncospheres are not susceptible to vaccine-induced antibody and complement mediated attack, but that as the parasites mature, the host-protective antigens come to be associated with the parasite’s surface, rendering them susceptible to immune attack.