Replication of an Autonomous Human Parvovirus in Non-dividing Human Airway Epithelium Is Facilitated through the DNA Damage and Repair Pathways

Human bocavirus 1 (HBoV1) belongs to the genus Bocaparvovirus of the Parvoviridae family, and is an emerging human pathogenic respiratory virus. In vitro, HBoV1 infects well-differentiated/polarized primary human airway epithelium (HAE) cultured at an air-liquid interface (HAE-ALI). Although it is well known that autonomous parvovirus replication depends on the S phase of the host cells, we demonstrate here that the HBoV1 genome amplifies efficiently in mitotically quiescent airway epithelial cells of HAE-ALI cultures. Analysis of HBoV1 DNA in infected HAE-ALI revealed that HBoV1 amplifies its ssDNA genome following a typical parvovirus rolling-hairpin DNA replication mechanism. Notably, HBoV1 infection of HAE-ALI initiates a DNA damage response (DDR) with activation of all three phosphatidylinositol 3-kinase–related kinases (PI3KKs). We found that the activation of the three PI3KKs is required for HBoV1 genome amplification; and, more importantly, we identified that two Y-family DNA polymerases, Pol η and Pol κ, are involved in HBoV1 genome amplification. Overall, we have provided an example of de novo DNA synthesis (genome amplification) of an autonomous parvovirus in non-dividing cells, which is dependent on the cellular DNA damage and repair pathways.     

Chromosomal aberrations in patients with primary biliary cirrhosis

Summary Chromosomal aberrations in untreated lymphocyte cultures, bleomycin (BLM)-induced aberrations and sister chromatid exchanges (SCE) in the peripheral blood lymphocytes of 11 patients suffering from primary biliary cirrhosis (PBC) and 14 matched control individuals were analysed. The lymphocytes of the PBC patients had on average a lower mitotic index (2.3) compared with controls (3.5) in the untreated cultures. The mean baseline rate of aberrations of the cultured lymphocytes of the patients was 5.3 aberrations per 100 metaphases (%); this was significantly different (P=0.0291) from that of the controls with a mean of 2.3%. In lymphocytes of the patients and controls, most of the aberrations observed took the form of gaps; there was an almost equal breakage rate in both groups (0.5% and 0.4%, respectively). The average number of mitoses with aberrations in the PBC patients studied was double that of the controls (4.9% and 2.3% respectively, P=0.0323). The mean number of the BLM-induced aberrations was 54.0% and 27.7% for the lymphocytes of the patients and controls, respectively. The mean number of the aberrant mitoses in the BLM cultures was 6 times higher than that of the untreated cultures for both groups, 25.7% and 14.6% respectively (P=0.018). The chromosomal distribution of baseline and induced aberrations was not random. The PBC patients had a mean number of 8.7 SCE per mitosis, which was significantly higher than the SCEs in the controls (6.3 SCE per mitosis; P=0.0156). The evidence suggests that the chromosomes of the lymphocytes of PBC patients may be less stable than those of the control individuals in this study.     

Der Abbau von Phenylamiden durch Bacillus sphaericus

Zusammenfassung Die Acylanilide Monalide, Carboxin, Oxycarboxin, 2,5-Dimethylfurancarbonsäureanilid, Pyracarbolid, 2-Methyl- und 2-Chlorbenzoesäureanilid, der N-Phenylcarbaminsäureester Propham und die N-Phenylharnstoff-derivate Monolinuron, Linuron, Metobromuron und Maloran wurden durch B. sphaericus an der Amidbindung zu den entsprechenden Anilinen und Säuren gespalten.Der qualitative Nachweis der Zwischenprodukte gelang dünnschicht- und gaschromatographisch mit Hilfe von Vergleichssubstanzen.Für den Abbau der Phenylamide durch B. sphaericus wurde ein Abbauschema aufgestellt.

---

The degradation of phenylamides by Bacillus sphaericus

Summary B. sphaericus is degrading the acylamides monalide, carboxin, oxycarboxin, 2,5-dimethylfurancarboxylic acid anilide, pyracarbolid, 2-methyl-and 2-chloro-benzoic acid anilide, the N-phenylcarbamate propham and the N-methoxyphenylurea compounds monolinuron, linuron, metabromuron and maloran by cleaving the amide linkage, forming the corresponding anilines and acids.The metabolites were determined qualitatively by the means of co-thinlayer- and co-gaschromatography.A pathway for degradation of the phenylamides by B. sphaericus is proposed.

     

Two-dimensional crystallization of a bacterial surface protein on lipid vesicles under controlled conditions.

The solubilized surface protein of the Gram-negative bacterium Comamonas acidovorans was reconstituted on lipid vesicles by means of controlled dialysis. To this end, a multichamber dialysis apparatus was built which allows one to control the temperature and the dialysis rate, to apply various temperatures or buffer systems and sample conditions in a single experiment, and to monitor the turbidity of the sample by means of light scattering. The reconstitution conditions were optimized such that the surface protein formed two-dimensional crystals suitable for electron crystallography. The recrystallized surface protein arrays gave a resolution of approximately 1.3 nm in projection after correlation averaging of negatively stained preparations. The surface protein assembled into partially self-contained two-dimensional crystals which possess a strong shape-determining effect and formed cylinders and various cone-shaped vesicles. The development of the various vesicle forms is described in a model.     

Endocytosis of human IgG:Fc receptor complexes by transfected BHK cells

We have analyzed the mode of uptake of human beta FcRII molecules expressed in BHK cells (clone 2/14). When challenged with aggregated human IgG (ahIgG), these cells bind the ligand at 4 degrees C and endocytose the IgG: receptor complexes rapidly upon warming to 37 degrees C, as seen by fluorescence microscopy with antibodies directed against human IgG. Using 125I-labeled ahIgG, we found that 40% of the bound ligand was internalized within 15 min, and approximately 60% within 2 h. Surface replication and thin sectioning combined with immunogold labeling revealed that the ligand was taken up by coated vesicles and was transferred to the endosomal/lysosomal compartment. This was confirmed by confocal laser microscopy of cells double labeled for clathrin and ahIgG. After modulation of the coated vesicle pattern by hypertonic medium, ahIgG transport was impaired. These data show that a single isoform of human FcRII, expressed in an animal cell negative for Fc receptors, can use the coated vesicle based endocytic pathway of the host cell. Reincubation of cycloheximide-treated cells with a second batch of ligand showed that approximately 20% of the beta FcRII was recycled. This finding is in apparent contrast to the fate of the endogenous Fc receptors expressed on mouse macrophages.     

Three-dimensional structure of an open form of the surface layer from the fish pathogen Aeromonas salmonicida.

Cell-free culture supernatants of a lipopolysaccharide (LPS) O-polysaccharide-deficient, single-insertion transposon mutant of the tetragonal surface protein array (S layer)-containing fish pathogen Aeromonas salmonicida were examined by electron microscopy. Negative staining showed that the S layer was released as sheets of tetragonal material, indicating that although surface retention of assembled S layer requires the presence of wild-type LPS oligosaccharides, initial assembly of S-layer subunits into sheets does not require the presence of O-polysaccharide chains. The three-dimensional structure of the S layer was reconstructed from tilted micrographs of the released sheets. Horizontal sections through this reconstruction showed that the released sheets were composed of two identical S layers that were perfectly in register. The reconstructed layer had a lattice constant of 12.5 nm. At a resolution of 1.6 nm, the layer consisted of a major tetragon at one fourfold axis of symmetry and a minor tetragon at the second fourfold axis of symmetry. The core, composed of four of the major domains, contained a large depression and was located toward the inside of the layer. The minor tetragon provided connectivity within the layer and was located toward the outer surface of the layer. Projections through the double layer gave a type I (closed) pattern (M. Stewart, T. J. Beveridge, and T. J. Trust, J. Bacteriol. 166:120-127, 1986), yet projections through the single layer indicated that the type II (open) pattern was present. This open pattern was indistinguishable from that seen in S layer released from the surfaces of wild-type cells.     

Pattern of motility and flow of digesta in the forestomach of the Llama (Lama guanacoe f.glama)

Summary A study of the pattern of stomach motility was made in five conscious llamas. Motility was recorded continuously by X-radiography, electromyography and/or balloons with pressure transducers, and correlated with outflow of fore-stomach contents measured by a thermistor. The llama forestomach showed a rhythmic pattern of contractions (motility cycle). A single cycle lasted about 82 s. Evidence is presented that each cycle comprises a single A- and numerous B-contraction sequences. Each motility cycle started with an A-sequence which began with a contraction of the canal between compartment 2 (C2) and compartment 3 (C3). A vigorous contraction of C2 followed, as the pressure in the canal fell below the baseline, then a contraction of the caudal sac of compartment 1 (C1) and another contraction of the canal occurred. The B-sequence of contractions began with a contraction of the cranial sac of C1 at the same time as the second canal contraction, followed by a weak contraction of C2 and finally a contraction of the caudal sac of C1. This B-sequence was repeated a number of times in each motility cycle. Passage of contents from C2 to C3 only occurred as the canal relaxed during the A-contraction. Continuous aborally-directed segmention and peristaltic-like movements were recorded in C3. These contractions occurred at 10 min⁻¹ in the proximal part, and at 5 min⁻¹ in the distal part where they were strongest. In the hind stomach mixing movements could be seen. At the border between C3 and the hind stomach, within a length of 10 cm the pH fell from 6 to less than 2. No reflux of contents was observed from the hind stomach to C3.     

Antisense RNA decreases AP33 gene expression and cytoadherence by <Emphasis Type="Italic">T. vaginalis</Emphasis>

Background  

Host parasitism by Trichomonas vaginalis is complex. Adherence to vaginal epithelial cells (VECs) is mediated by surface proteins. We showed before that antisense down-regulation of expression of adhesin AP65 decreased amounts of protein, which lowered levels of T. vaginalis adherence to VECs. We now perform antisense down-regulation of expression of the ap33 gene to evaluate and confirm a role for AP33 in adherence by T. vaginalis. We also used an established transfection system for heterologous expression of AP33 in T. foetus as an additional confirmatory approach.