Morphology and Small Subunit rDNA Gene Sequence of Pseudoamphisiella quadrinucleata n. sp. (Ciliophora,Urostylida) from the South China Sea

ABSTRACT. The urosylid genus Pseudoamphisiella was established by Song (1996) with hitherto only two known congeners. In the present work, the morphology and infraciliature of a new member, Pseudoamphisiella quadrinucleata n. sp., a form with conspicuous alveolar layer and four macronuclear nodules isolated from the coastal waters both near Hong Kong and near Guangzhou, South China were investigated using living observation and protargol silver impregnation methods. Pseudoamphisiella quadrinucleata differs from other two known forms mainly by the number of macronuclear nodules: constantly four vs. two in Pseudoamphisiella alveolata and 24–57 in Pseudoamphisiella lacazei. To support this, the sequence of the small subunit rDNA of P. quadrinucleata n. sp. showed 14 and 74 nucleotides in comparison with that of the two known congeners, respectively, which hence firmly supports the validity of the new species.     

Cloning, structure and expression of a cDNA encoding the human androgen receptor

A cDNA clone has been isolated from a library prepared of mRNA of human breast cancer T47D cells with an oligonucleotide probe homologous to part of the region encoding the DNA-binding domain of steroid receptors. The clone has a size of 1505 bp and sequence analysis revealed an open reading frame of 1356 bp. The deduced amino acid sequence displays two highly conserved regions identified as the putative DNA-binding and hormone binding domains respectively of steroid receptors. Expression of this cDNA clone in COS cells produces a nuclear protein with all the binding characteristics of the human androgen receptor (hAR). The gene encoding the cDNA is assigned to the human X-chromosome. High levels of three hybridizing mRNA species of 11, 8.5 and 4.7 kb respectively are found in the human prostate cancer cell line (LNCaP), which contains elevated levels of hAR. The present data provide evidence that we have isolated a cDNA that encodes a major part of the human androgen receptor.     

Interactions of hydrophobic peptides with lipid bilayers: Monte Carlo simulations with M2delta

We introduce here a novel Monte Carlo simulation method for studying the interactions of hydrophobic peptides with lipid membranes. Each of the peptide's amino acids is represented as two interaction sites: one corresponding to the backbone alpha-carbon and the other to the side chain, with the membrane represented as a hydrophobic profile. Peptide conformations and locations in the membrane and changes in the membrane width are sampled using the Metropolis criterion, taking into account the underlying energetics. Using this method we investigate the interactions between the hydrophobic peptide M2delta and a model membrane. The simulations show that starting from an extended conformation in the aqueous phase, the peptide first adsorbs onto the membrane surface, while acquiring an ordered helical structure. This is followed by formation of a helical-hairpin and insertion into the membrane. The observed path is in agreement with contemporary understanding of peptide insertion into biological membranes. Two stable orientations of membrane-associated M2delta were obtained: transmembrane (TM) and surface, and the value of the water-to-membrane transfer free energy of each of them is in agreement with calculations and measurements on similar cases. M2delta is most stable in the TM orientation, where it assumes a helical conformation with a tilt of 14 degrees between the helix principal axis and the membrane normal. The peptide conformation agrees well with the experimental data; average root-mean-square deviations of 2.1 A compared to nuclear magnetic resonance structures obtained in detergent micelles and supported lipid bilayers. The average orientation of the peptide in the membrane in the most stable configurations reported here, and in particular the value of the tilt angle, are in excellent agreement with the ones calculated using the continuum-solvent model and the ones observed in the nuclear magnetic resonance studies. This suggests that the method may be used to predict the three-dimensional structure of TM peptides.     

Complex chromosome 17p rearrangements associated with low-copy repeats in two patients with congenital anomalies

Recent molecular cytogenetic data have shown that the constitution of complex chromosome rearrangements (CCRs) may be more complicated than previously thought. The complicated nature of these rearrangements challenges the accurate delineation of the chromosomal breakpoints and mechanisms involved. Here, we report a molecular cytogenetic analysis of two patients with congenital anomalies and unbalanced de novo CCRs involving chromosome 17p using high-resolution array-based comparative genomic hybridization (array CGH) and fluorescent in situ hybridization (FISH). In the first patient, a 4-month-old boy with developmental delay, hypotonia, growth retardation, coronal synostosis, mild hypertelorism, and bilateral club feet, we found a duplication of the Charcot-Marie–Tooth disease type 1A and Smith-Magenis syndrome (SMS) chromosome regions, inverted insertion of the Miller-Dieker lissencephaly syndrome region into the SMS region, and two microdeletions including a terminal deletion of 17p. The latter, together with a duplication of 21q22.3-qter detected by array CGH, are likely the unbalanced product of a translocation t(17;21)(p13.3;q22.3). In the second patient, an 8-year-old girl with mental retardation, short stature, microcephaly and mild dysmorphic features, we identified four submicroscopic interspersed 17p duplications. All 17 breakpoints were examined in detail by FISH analysis. We found that four of the breakpoints mapped within known low-copy repeats (LCRs), including LCR17pA, middle SMS-REP/LCR17pB block, and LCR17pC. Our findings suggest that the LCR burden in proximal 17p may have stimulated the formation of these CCRs and, thus, that genome architectural features such as LCRs may have been instrumental in the generation of these CCRs.     

A new staphylococcal anti-inflammatory protein that antagonizes the formyl peptide receptor-like 1

Bacteria have developed mechanisms to escape the first line of host defense, which is constituted by the recruitment of phagocytes to the sites of bacterial invasion. We previously described the chemotaxis inhibitory protein of Staphylococcus aureus, a protein that blocks the activation of neutrophils via the formyl peptide receptor (FPR) and C5aR. We now describe a new protein from S. aureus that impaired the neutrophil responses to FPR-like1 (FPRL1) agonists. FPRL1 inhibitory protein (FLIPr) inhibited the calcium mobilization in neutrophils stimulated with MMK-1, WKYMVM, prion-protein fragment PrP(106-126), and amyloid beta(1-42). Stimulation with low concentrations of fMLP was partly inhibited. Directed migration was also completely prevented toward MMK-1 and partly toward fMLP. Fluorescence-labeled FLIPr efficiently bound to neutrophils, monocytes, B cells, and NK cells. HEK293 cells transfected with human C5aR, FPR, FPRL1, and FPRL2 clearly showed that FLIPr directly bound to FPRL1 and, at higher concentrations, also to FPR but not to C5aR and FPRL2. FLIPr can reveal unknown inflammatory ligands crucial during S. aureus infections. As a novel described FPRL1 antagonist, it might lead to the development of therapeutic agents in FPRL1-mediated inflammatory components of diseases such as systemic amyloidosis, Alzheimer's, and prion disease.     

Soil 13C–15N dynamics in an N2-fixing clover system under long-term exposure to elevated atmospheric CO2

Reduced soil N availability under elevated CO₂ may limit the plant's capacity to increase photosynthesis and thus the potential for increased soil C input. Plant productivity and soil C input should be less constrained by available soil N in an N₂‐fixing system. We studied the effects of Trifolium repens (an N₂‐fixing legume) and Lolium perenne on soil N and C sequestration in response to 9 years of elevated CO₂ under FACE conditions. ¹⁵N‐labeled fertilizer was applied at a rate of 140 and 560 kg N ha^?1 yr^?1 and the CO₂ concentration was increased to 60 Pa pCO₂ using ¹³C‐depleted CO₂. The total soil C content was unaffected by elevated CO₂, species and rate of ¹⁵N fertilization. However, under elevated CO₂, the total amount of newly sequestered soil C was significantly higher under T. repens than under L. perenne. The fraction of fertilizer‐N (f_N) of the total soil N pool was significantly lower under T. repens than under L. perenne. The rate of N fertilization, but not elevated CO₂, had a significant effect on f_N values of the total soil N pool. The fractions of newly sequestered C (f_C) differed strongly among intra‐aggregate soil organic matter fractions, but were unaffected by plant species and the rate of N fertilization. Under elevated CO₂, the ratio of fertilizer‐N per unit of new C decreased under T. repens compared with L. perenne. The L. perenne system sequestered more ¹⁵N fertilizer than T. repens: 179 vs. 101 kg N ha^?1 for the low rate of N fertilization and 393 vs. 319 kg N ha^?1 for the high N‐fertilization rate. As the loss of fertilizer‐¹⁵N contributed to the ¹⁵N‐isotope dilution under T. repens, the input of fixed N into the soil could not be estimated. Although N₂ fixation was an important source of N in the T. repens system, there was no significant increase in total soil C compared with a non‐N₂‐fixing L. perenne system. This suggests that N₂ fixation and the availability of N are not the main factors controlling soil C sequestration in a T. repens system.     

Localization of a gene involved in complementation of the defect in xeroderma pigmentosum group A cells on human chromosome 1

Human, Chinese hamster or Chinese hamster/human hybrid cytoplasts were fused with UV-irradiated xeroderma pigmentosum group A (XP-A) cells. Unscheduled DNA synthesis (UDS) of the XP-A nucleus was measured 0-2 and 2-4 h after seeding of the fused population. Human cytoplasts did correct the defect in the XP-A nucleus immediately after fusion, whereas the chinese hamster cytoplasts did not show this rapid increase in excision repair. The results obtained after fusion of cytoplasts isolated from a panel of 26 Chinese hamster-human hybrids showed that chromosome 1 bears genetic information that is necessary for the rapid correction of the XP-A defect. Furthermore, this genetic information was regionally assigned to 1q42-qter by analysing hybrid cell lines having retained various segments of chromosome 1. Cytoplasts from a Chinese hamster/XP-A hybrid containing chromosome 1 of XP-A origin corrected also the defect with fast kinetics. This result indicate that the correcting factor consists of human and Chinese hamster components. As a consequence, the gene mapped on chromosome 1 may not be the gene which is mutated in XP-A cells.     

Phagocytosis Escape by a Staphylococcus aureus Protein That Connects Complement and Coagulation Proteins at the Bacterial Surface

Upon contact with human plasma, bacteria are rapidly recognized by the complement system that labels their surface for uptake and clearance by phagocytic cells. Staphylococcus aureus secretes the 16 kD Extracellular fibrinogen binding protein (Efb) that binds two different plasma proteins using separate domains: the Efb N-terminus binds to fibrinogen, while the C-terminus binds complement C3. In this study, we show that Efb blocks phagocytosis of S. aureus by human neutrophils. In vitro, we demonstrate that Efb blocks phagocytosis in plasma and in human whole blood. Using a mouse peritonitis model we show that Efb effectively blocks phagocytosis in vivo, either as a purified protein or when produced endogenously by S. aureus. Mutational analysis revealed that Efb requires both its fibrinogen and complement binding residues for phagocytic escape. Using confocal and transmission electron microscopy we show that Efb attracts fibrinogen to the surface of complement-labeled S. aureus generating a ‘capsule’-like shield. This thick layer of fibrinogen shields both surface-bound C3b and antibodies from recognition by phagocytic receptors. This information is critical for future vaccination attempts, since opsonizing antibodies may not function in the presence of Efb. Altogether we discover that Efb from S. aureus uniquely escapes phagocytosis by forming a bridge between a complement and coagulation protein.     

Inhibition of histone acetylation and deacetylation enzymes affects longevity,development, and fecundity in the pea aphid (Acyrthosiphon pisum)

Histone acetylation is an evolutionarily conserved epigenetic mechanism of eukaryotic gene regulation which is tightly controlled by the opposing activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs). In insects, life-history traits such as longevity and fecundity are severely affected by the suppression of HAT/HDAC activity, which can be achieved by RNA-mediated gene silencing or the application of chemical inhibitors. We used both experimental approaches to investigate the effect of HAT/HDAC inhibition in the pea aphid (Acyrthosiphon pisum) a model insect often used to study complex life-history traits. The silencing of HAT genes (kat6b, kat7, and kat14) promoted survival or increased the number of offspring, whereas targeting rpd3 (HDAC) reduced the number of viviparous offspring but increased the number of premature nymphs, suggesting a role in embryogenesis and eclosion. Specific chemical inhibitors of HATs/HDACs showed a remarkably severe impact on life-history traits, reducing survival, delaying development, and limiting the number of offspring. The selective inhibition of HATs and HDACs also had opposing effects on aphid body weight. The suppression of HAT/HDAC activity in aphids by RNA interference or chemical inhibition revealed similarities and differences compared to the reported role of these enzymes in other insects. Our data suggest that gene expression in A. pisum is regulated by multiple HATs/HDACs, as indicated by the fitness costs triggered by inhibitors that suppress several of these enzymes simultaneously. Targeting multiple HATs or HDACs with combined effects on gene regulation could, therefore, be a promising approach to discover novel targets for the management of aphid pests.