The association of nonsense mutation with exon-skipping in hprt mRNA of Chinese hamster ovary cells results from an artifact of RT-PCR

RT-PCR of RNA from CHO cells with nonsense mutations in the hprt gene frequently detects minor hprt mRNA species lacking one or more exons. Many nonsense mutants also contain greatly reduced concentrations of the major, normally spliced hprt mRNA. In this study, we examined the hypothesis that exon-deleted mRNAs are normal constituents of CHO cells, but are not detected in wild-type parental cells and most missense mutants because their amplification is suppressed by relatively high concentrations of normally spliced hprt mRNA. A protocol designed to specifically detect exon-deleted mRNAs was conducted using RNA from parental cells and identified all the exon-deleted species typical of nonsense mutants. Quantitative analysis of parental cell RNA measured these exon-deleted mRNAs at < or = 0.7% of the abundance of the full-sized species. Nonsense and missense mutants had comparable amounts of exon-deleted mRNAs, which varied both above and below parental concentrations. The relative concentrations of particular exon-deleted species could be explained by the location of nonsense mutations remaining in the mRNA or by structural effects of mutations on splicing. Exon-deleted mRNAs were detected by RT-PCR when the concentration of the most abundant exon-deleted species was > or = 2% of the full-length mRNA. This occurred for mutants with nonsense mutations in internal exons. RT-PCR conditions were shown to suppress the amplification of exon-deleted species 40-fold when full-length mRNA was abundant, which occurred for parental lines and missense mutants. Our results verify that RT-PCR conditions can produce an artifactual association between nonsense mutation and exon-skipping when minor, exon-deleted mRNA is relatively enriched.     

Factor VIII inhibitors in mild and moderate-severity haemophilia A

Inhibitors are an uncommon complication of mild haemophilia, occurring in 3-13% of patients and usually arising during adulthood. The risk of inhibitor development in this group appears to be associated with relatively few high-risk factor VIII genotypes clustered in the A2 and C2 domains, especially the Arg593-Cys and the Trp2229-Cys mutations. Kindreds with these mutations have an inhibitor incidence of up to 40%. These mutations may induce a stable conformational change in the factor VIII molecule rendering it antigenically distinct from wild-type factor VIII. Inhibitors in mild haemophilia usually cross-react with endogenous factor VIII reducing the basal VIIIC to < 0.01 IU/ml, and causing spontaneous bleeding. This bleeding is sometimes severe and life-threatening, two-thirds of patients developing a pattern of soft tissue, gastrointestinal (GI) and urinogenital bleeding reminiscent of acquired haemophilia. Bleeding has been treated with human and porcine factor VIII, bypass therapy and DDAVP. Recombinant factor VIIa and DDAVP have the advantage that they do not induce an anamnestic rise in inhibitor titre. About 60% of these inhibitors disappear in the remainder over a median of 9 months. Few of these inhibitors recur, suggesting that most such patients have become tolerant. The inhibitors persist long-term and remain troublesome in about 40% of patients. The limited data available on immune tolerance induction in this group indicate a generally poor response to this approach. Two of nine achieved tolerance, with a partial response in a further four. Inhibitors are an uncommon but life-threatening complication of haemophilia. This complication should be considered when selecting the treatment modality for patients with a family history of inhibitors, and DDAVP used whenever possible.     

Effects of consecutive thiouracil exposures in the juvenile and adult single comb White Leghorn chicken on body weight and reproductive performance

To develop a novel potent radical-scavenging antioxidant, the ideal structure of a phenolic compound was designed considering the factors that determine antioxidant potency. 2,3-Dihydro-5-hydroxy-2,2-dipentyl-4, 6-di-tert-butylbenzofuran (BO-653) was thus synthesized and its antioxidant activity was evaluated against lipid peroxidations in vitro. The electron spin resonance study showed that the phenoxyl radical derived from BO-653 was more stable than alpha-tocopheroxyl radical. BO-653 reduced alpha-tocopheroxyl radical rapidly, but alpha-tocopherol did not reduce the phenoxyl radical derived from BO-653. However, the chemical reactivity of BO-653 toward peroxyl radical was smaller than that of alpha-tocopherol. This was interpreted as the steric effect of bulky tert-butyl groups at both ortho positions which hindered the access of peroxyl radical to the phenolic hydrogen. However, the tertbutyl substituents increased the stability of BO-653 radical and also lipophilicity, and its antioxidant potency against lipid peroxidation in phosphatidylcholine liposomal membranes was superior to that of alpha-tocopherol. Ascorbic acid reduced the phenoxyl radical derived from BO-653 and spared BO-653 during the oxidation of lipid in the homogeneous solution. On the other hand, ascorbic acid did not spare BO-653 in the oxidation of liposomal membranes. It was concluded that BO-653 is a potent novel radical-scavenging antioxidant.     

Mechanobiological adaptation of subchondral bone as a function of joint incongruity and loading

Computed tomography (CT) has been employed to determine non-invasively the distribution of subchondral bone density in joints and to evaluate their dominant loading pattern. The objective of this study was to investigate the relationship between subchondral bone adaptation, joint incongruity and loading, in order to determine to what extent the loading conditions and/or geometric configuration can be inferred from the distribution of subchondral density. Finite element models of joints with various degrees of incongruity were designed and a current remodeling theory implemented using the node-based approach. Appropriate combinations of joint incongruity and loading yielded subchondral bone density patterns consistent with experimental findings, specifically a bicentric distribution in the humero-ulnar joint and a monocentric distribution in the humero-radial joint. However, other combinations of incongruity and loading produced similar subchondral density patterns. Both the geometric joint configuration and the loading conditions influence the distribution of subchondral density in such a way that one of these factors must be known a priori to estimate the other. Since subchondral density can be assessed by CT and joint geometry by magnetic resonance imaging, the dominant loading pattern of joints may be potentially derived in the living using these non-invasive imaging methods.     

Reduction of fecal shedding and egg contamination of Salmonella enteritidis by increasing the number of heterophil adaptations

Serial passage of Salmonella enteritidis (SE) in chicken heterophils resulted in heterophil-adapted SE (HASE). We now report that an additional five heterophil passages have further reduced the number and frequency of fecal shedding of HASE. Eleven-times HASE (11 x HASE) given to 12 laying hens for three consecutive days reduced fecal shedding of 11 x HASE to three isolations from fecal samples during the 70-day postexposure observation period. Hens were exposed to challenge SE 74 days after treatment with 11 x HASE. Three of 12 11 x HASE-treated hens were positive for challenge SE (11/396 fecal samples, or 2.8%) between days 5 and 40 postchallenge, whereas all 12 challenge control birds were positive (118/420 fecal samples, or 28.1%) for SE. None of 12 11 x HASE-treated hens was fecal positive from day 9 postchallenge, whereas 10 of 12 challenge control hens (82/372 fecal samples, or 22.0%) remained positive until day 40, the termination of the experiment. None of 525 eggs and eggshells cultured after 11 x HASE exposure was positive for Salmonella, and none of 422 eggs and eggshells cultured after challenge SE exposure was positive for Salmonella. Eggs or eggshells from challenge control hens were positive for Salmonella in 12/479 (2.5%) cases after challenge SE exposure.     

The peroxisome proliferators are hepatocyte mitogens in chemically-defined media: glucocorticoid-induced PPAR alpha is linked to peroxisome proliferator mitogenesis

OBJECTIVES: To investigate the induction of apoptosis by methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC) therapy as well as p53 alterations in a rat model of bladder cancer induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). METHODS: At 20 and 28 weeks after starting the administration of BBN, 60 rats (30 at 20 weeks, and 30 at 28 weeks) were divided into an MVAC treatment group (20 rats) and a control group of untreated rats (10 rats). After one intraperitoneal injection of MVAC, the rat bladder was obtained on day 1 or 7 for evaluation of apoptosis by biohistochemical and electron microscopic observations, and of p53 alterations immunohistochemically. RESULTS: All tumors were noninvasive transitional cell carcinoma (TCC) at 20 weeks and invasive TCC at 28 weeks. In comparison with untreated tumors, a threefold increase of apoptotic indexes (Als) was noted in invasive TCC and a twofold increase in noninvasive TCC on day 1 after MVAC therapy. Both decreased Als and a frequent occurrence of apoptotic necrosis were observed on day 7. Occurrence of tumor necrosis was not affected by MVAC therapy, and the extent of necrosis was not related to apoptosis. Detection of p53 alterations, 45% and 40% in MVAC treated and untreated tumors, respectively, did not correlate with Als. CONCLUSIONS: MVAC therapy may act through the induction of apoptosis, and invasive TCC cells may be much more sensitive to MVAC therapy in the rat model of bladder cancer. Neither spontaneous nor MVAC-induced apoptosis may be related to p53 alterations in the rat model of bladder cancer.     

Identification of elicitor-induced cytochrome P450s of soybean (Glycine max L.) using differential display of mRNA

Magnesium (Mg) absorption across the intestinal epithelium is crucial for Mg homeostasis in all vertebrates. Besides paracellular transport, it involves a cell-mediated component, and this predicts the presence of specific Mg carriers at both the apical and basolateral pole of the epithelial cells. Although the mechanism of transmembrane Mg transport in enterocytes, as in most cell types, has remained an elusive topic for many years, recent studies have provided promising new insights. We here recapitulate the progress that has been made in this field, and advance evidence for membrane carriers that are involved in transcellular Mg transport in the intestine.