XR-C1, a new CHO cell mutant which is defective in DNA-PKcs, is impaired in both V(D)J coding and signal joint formation

DNA-dependent protein kinase (DNA-PK) plays an important role in DNA double-strand break (DSB) repair and V(D)J recombination. We have isolated a new X-ray-sensitive CHO cell line, XR-C1, which is impaired in DSB repair and which was assigned to complementation group 7, the group that is defective in the XRCC7 / SCID ( Prkdc ) gene encoding the catalytic subunit of DNA-PK (DNA-PKcs). Consistent with this complementation analysis, XR-C1 cells lackeddetectable DNA-PKcs protein, did not display DNA-PK catalytic activity and were complemented by the introduction of a single human chromosome 8 (providing the Prkdc gene). The impact of the XR-C1 mutation on V(D)J recombination was quite different from that found in most rodent cells defective in DNA-PKcs, which are preferentially blocked in coding joint formation, whereas XR-C1 cells were defective in forming both coding and signal joints. These results suggest that DNA-PKcs is required for both coding and signal joint formation during V(D)J recombination and that the XR-C1 mutant cell line may prove to be a useful tool in understanding this pathway.     

Somatic antigens of pseudomonads: structure of the O-specific polysaccharide of Pseudomonas fluorescens biovar B, strain IMV 247

The O-specific polysaccharide of Pseudomonas fluorescens biovar B, strain IMV 247, was studied by acid hydrolysis, GLC-MS and 1H and 13C NMR spectroscopy, including 1D and 2D NOE, 2D hybrid TOCSY and ROESY (TORO), and 2D H-detected heteronuclear multiple-bond correlation (HMBC) experiments. The polysaccharide was found to contain L-rhamnose, 3.6-dideoxy-3-[(S)-3-hydroxybutyramido]-D-glucose (D-Qui3NHb), 2-acetamido- 2,4,6-trideoxy-4-[(S)-3-hydroxybutyramido-D-glucose (D-QuiNAc4NHb) and 2-acetamido-2- deoxy-D-galacturonic acid (D-GalNAcA). Partial acid hydrolysis of the polysaccharide resulted in a non-reducing GalNAcA-->QuiNAc4NHb disaccharide with the 3-hydroxybutyryl group glycosylated intramolecularly by the QuiN4N residue. The following structure of the tetrasaccharide repeating unit of the polysaccharide was established:-->4) -alpha-D-GalpNAcA-(1-->3)- alpha-D-QuipNAc4NHb-(1-->2)-beta-D-Quip3NHb-(1-->2)-alpha-L- Rhap(1-->.     

Toxicity of the styrene metabolite, phenylglyoxylic acid, in rats after three months' oral dosing

Male Wistar rats were dosed with 0, 1250, 3750 or 5000 mg/l of phenylglyoxylic acid (PGA) (CAS no. 611-73-4) in the drinking water ad libitum for 3 months. During the entire treatment period, there were no gross signs of toxicity related to PGA. No changes in neurobehavior were found after using a functional observational battery or radial arm maze. An increased relative kidney weight was seen in the highest dose-group (Controls: 0.504 +/- 0.031 g/100 g b.wt.; 5000 mg PGA/l: 0.579 +/- 0.033 g/100 g b.wt.; p<0.01). No other organ weights were affected. Histopathology revealed no change in kidney structure. No changes in clinical biochemistry. In the highest dose-group three animals out of ten showed reduction in peripheral nerve myelin sheath thickness. No such changes were seen in the control group. The study revealed no changes in auditory brain stem response but minor changes in electroretinography. The noradrenaline (NA) concentration decreased in pons and thalamus whereas it increased in medulla oblongata and whole brain. The dopamine (DA) concentration increased in cerebellum, hippocampus, pons, and whole brain. The most marked DA increase was seen in hippocampus (Controls: 0.56 +/- 0.10 nmol/g tissue; 5000 mg/l: 1.04 +/- 0.11 nmol/g tissue; p<0.001). The 5-hydroxytryptamine (5-HT) concentration decreased in cerebellum, cerebral cortex, hippocampus, and medulla oblongata, whereas it increased in thalamus. The yield of synaptosomal protein, synaptosomal NA, DA, and 5-HT concentrations, and DA uptake rate were not affected. When dosed males were mated with naive females, there were no differences between groups in the pregnancy rate, number of corpora luteae, implantations, live or dead fetuses, resorptions, preimplantation loss, or postimplantation loss. It is concluded that a part of the effects on kidney, peripheral nerves, and vision, which have previously been reported after exposure to styrene, might be induced by the styrene metabolite, PGA. If PGA has ototoxic effects in rats, the dosing in the present study is not sufficient to induce the necessary ototoxic concentration in blood. Alternatively, the ototoxicity of styrene, like toluene, may be caused the parent compound itself and not by a metabolite like PGA.     

Role of endoproteolytic dibasic proprotein processing in maturation of secretory proteins in Trichoderma reesei

Cell extracts of Trichoderma reesei exhibited dibasic endopeptidase activity toward the carboxylic side of KR, RR, and PR sequences. This activity was stimulated by the presence of Ca2+ ions and localized in vesicles of low bouyant density; it therefore exhibited some similarity to yeast Kex2. Analytical chromatofocusing revealed a single peak of activity. The dibasic endopeptidase activity was strongly and irreversibly inhibited in vitro as well as in vivo by 1 mM p-amidinophenylmethylsulfonyl fluoride (pAPMSF) but not by PMSF at concentrations up to 5 mM. We therefore used pAPMSF to study the role of the dibasic endopeptidase in the secretion of protein by T. reesei. Secretion of xylanase I (proprotein processing sequence -R-R- downward arrow-R- downward arrow-A-) and xylanase II (-K-R- downward arrow-Q-) was strongly inhibited by 1 mM pAPMSF, and a larger, unprocessed enzyme form was detected intracellularly under these conditions. Secretion of cellobiohydrolase II (CBH II; -E-R- downward arrow-Q-) was only slightly inhibited by pAPMSF, and no accumulation of unprocessed precursors was detected. In contrast, secretion of CBH I (-R-A- downward arrow-Q-) was stimulated by pAPMSF addition, and a simultaneous decrease in the concentration of intracellular CBH I was detected. Similar experiments were also carried out with a single heterologous protein, ShBLE, the phleomycin-binding protein from Streptoalloteichus hindustanus, fused to a series of model proprotein-processing sequences downstream of the expression signals of the Aspergillus nidulans gpdA promoter. Consistent with the results obtained with homologous proteins, pAPMSF inhibited the secretion of ShBLE with fusions containing dibasic (RK and KR) target sequences, but it even stimulated secretion in fusions to LR, NHA, and EHA target sequences. Addition of 5 mM PMSF, a nonspecific inhibitor of serine protease, nonspecifically inhibited the secretion of heterologous proteins from fusions bearing the NHA and LR targets. These data point to the existence of different endoproteolytic proprotein processing enzymes in T. reesei and demonstrate that dibasic processing is obligatory for the secretion of the proproteins containing this target.     

Ultrastructural features of diffuse malignant mesotheliomas

The distinction of malignant mesothelioma from tumors metastatic to the serosal membranes can often be made based on the results of histochemical or immunohistochemical studies. However, in some cases, these techniques are inadequate to make a firm diagnosis. In these instances, electron microscopic studies with the observation of a constellation of characteristic ultrastructural findings may permit an unequivocal diagnosis of mesothelioma.     

Results of transplantation for acute and chronic hepatic allograft rejection

B-chronic lymphocytic leukemia (CLL) is characterized by an accumulation of long-lived, resting B cells expressing the Bcl-2 protein. However, less than 10% of the CLL patients shows bcl-2 gene rearrangement in blood cells, using traditional Southern blotting analysis. In the present study, rearrangement of the bcl-2 gene in CLL cells was studied by pulsed-field gel electrophoresis (PFGE). With this method, large DNA fragments (> 50-10,000 kb) could be analyzed. Blood CLL cells from 9 of 9 patients and 2 of 2 CLL cell lines showed rearranged bcl-2 gene. In comparison, healthy blood B cells and lymphoblastoid cell lines (LCLs) established from normal peripheral blood lymphocytes of the patients showed only germ line configuration. Thus, the possibility of restriction fragment length polymorphisms (RFLPs) in this gene could be excluded. The primary cell involved in CLL might be a progenitor B cell that has accidentally rearranged the bcl-2 gene. As a consequence, such cells express stable amount of Bcl-2 protein and do not enter apoptosis. During prolonged survival, such cells may acquire secondary changes including chromosomal translocations and mutations.