Mammalian Fbh1 is important to restore normal mitotic progression following decatenation stress

We have addressed the role of the F-box helicase 1 (Fbh1) protein during genome maintenance in mammalian cells. For this, we generated two mouse embryonic stem cell lines deficient for Fbh1: one with a homozygous deletion of the N-terminal F-box domain (Fbh1^f/f), and the other with a homozygous disruption (Fbh1^?/?). Consistent with previous reports of Fbh1-deficiency in vertebrate cells, we found that Fbh1^?/? cells show a moderate increase in Rad51 localization to DNA damage, but no clear defect in chromosome break repair. In contrast, we found that Fbh1^f/f cells show a decrease in Rad51 localization to DNA damage and increased cytoplasmic localization of Rad51. However, these Fbh1^f/f cells show no clear defects in chromosome break repair. Since some Rad51 partners and F-box-associated proteins (Skp1-Cul1) have been implicated in progression through mitosis, we considered whether Fbh1 might play a role in this process. To test this hypothesis, we disrupted mitosis using catalytic topoisomerase II inhibitors (bisdioxopiperazines), which inhibit chromosome decatenation. We found that both Fbh1^f/f and Fbh1^?/? cells show hypersensitivity to topoisomerase II catalytic inhibitors, even though the degree of decatenation stress was not affected. Furthermore, following topoisomerase II catalytic inhibition, both Fbh1-deficient cell lines show substantial defects in anaphase separation of chromosomes. These results indicate that Fbh1 is important for restoration of normal mitotic progression following decatenation stress.     

An antisense RNA regulates the bidirectional silencing property of the Kcnq1 imprinting control region

The Kcnq1 imprinting control region (ICR) located in intron 10 of the Kcnq1 gene is unmethylated on the paternal chromosome and methylated on the maternal chromosome and has been implicated in the manifestation of parent-of-origin-specific expression of six neighboring genes. The unmethylated Kcnq1 ICR harbors bidirectional silencer activity and drives expression of an antisense RNA, Kcnq1ot1, which overlaps the Kcnq1 coding region. To elucidate whether the Kcnq1ot1 RNA plays a role in the bidirectional silencing activity of the Kcnq1 ICR, we have characterized factor binding sites by genomic footprinting and tested the functional consequence of various deletions of these binding sites in an episome-based system. Deletion of the elements necessary for Kcnq1ot1 promoter function resulted in the loss of silencing activity. Furthermore, interruption of Kcnq1ot1 RNA production by the insertion of a polyadenylation sequence downstream of the promoter also caused a loss of both silencing activity and methylation spreading. Thus, the antisense RNA plays a key role in the silencing function of the ICR. Double-stranded RNA (dsRNA)-mediated RNA interference is unlikely to be involved, as the ICR is active irrespective of the simultaneous production of dsRNA from the genes it silences.     

The in vitro and in vivo effects of anti-galactose antibodies on endothelial cell activation and xenograft rejection

We have previously produced a series of antigalactose (anti-Gal) hybridomas and characterized their heavy chain gene usage. Here we have quantified the affinity of these Abs for the alpha-Gal epitope and characterized their in vitro effects on endothelial cell activation and apoptosis. We report that anti-Gal mAbs derived from Gal(-/-) mice show a range of affinity for the alpha-Gal epitope, and that affinity was generally increased as the V(H) gene usage transitioned from germline sequences to sequences exhibiting somatic maturation. Despite an 85-fold range in affinity, all the anti-Gal mAbs examined induced alpha-Gal-specific endothelial cell activation, and after prolonged exposure induced endothelial cell apoptosis in a complement-independent manner. Only murine anti-Gal mAbs of the IgM or IgG3 subclass, but not IgG1, were effective at initiating complement-dependent cell lysis. Using a novel rat to mouse xenograft model, we examined the in vivo ability of these mAbs to induce xenograft rejection and characterized the rejection using histology and immunohistochemistry. Infusion of complement-fixing IgG3 mAbs resulted in either hyperacute rejection or acute vascular rejection of the xenograft. Surprisingly, infusion of an equal amount of a high affinity anti-Gal IgG1 mAb, that fixed complement poorly also induced a rapid xenograft rejection, which we have labeled very acute rejection. These studies emphasize the importance of in vivo assays, in addition to in vitro assays, in understanding the role of anti-Gal IgG-mediated tissue injury and xenograft rejection.     

New class of HIV-1 integrase (IN) inhibitors with a dual mode of action

tert-Butoxy-(4-phenyl-quinolin-3-yl)-acetic acids (tBPQA) are a new class of HIV-1 integrase (IN) inhibitors that are structurally distinct from IN strand transfer inhibitors but analogous to LEDGINs. LEDGINs are a class of potent antiviral compounds that interacts with the lens epithelium-derived growth factor (LEDGF) binding pocket on IN and were identified through competition binding against LEDGF. LEDGF tethers IN to the host chromatin and enables targeted integration of viral DNA. The prevailing understanding of the antiviral mechanism of LEDGINs is that they inhibit LEDGF binding to IN, which prevents targeted integration of HIV-1. We showed that in addition to the properties already known for LEDGINs, the binding of tBPQAs to the IN dimer interface inhibits IN enzymatic activity in a LEDGF-independent manner. Using the analysis of two long terminal repeat junctions in HIV-infected cells, we showed that the inhibition by tBPQAs occurs at or prior to the viral DNA 3'-processing step. Biochemical studies revealed that this inhibition operates by compound-induced conformational changes in the IN dimer that prevent proper assembly of IN onto viral DNA. For the first time, tBPQAs were demonstrated to be allosteric inhibitors of HIV-1 IN displaying a dual mode of action: inhibition of IN-viral DNA assembly and inhibition of IN-LEDGF interaction.     

Detection of methandienone (methandrostenolone) and metabolites in horse urine by gas chromatography—mass spectrometry

The metabolic transformation of methandienone (I) in the horse was investigated. After administration of a commercial drug preparation to a female horse (0.5 mg/kg), urine samples were collected up to 96 h and processed without enzymic hydrolysis. Extraction was performed by a series of solid—liquid and liquid—liquid extractions, thus avoiding laborious purification techniques. For analysis by gas chromatography—mass spectrometry, the extracts were trimethylsilylated. Besides the parent compound I and its C-17 epimer II, three monohydroxylated metabolites were identified: 6β-hydroxymethandienone (III), its C-17 epimer (IV) and 16β-hydroxy-methandienone (V). In addition, three isomers of 6β,16-dihydroxymethandienone (VIa–c) were discovered. Apparently, reduction of the δ⁴ double bond of 16β-hyroxymethandienone (V) in the horse yields 16β,17β-dihydroxy-17α-methyl-5β-androst-1-en-3-one (VII). Reduction of the isomers VIa–c results in the corresponding 6β,16,17-trihydroxy-17-methyl-5β-androst-1-en-3-ones (VIIIa–c). The data presented here suggest that screening for the isomers of VI and VIII, applying the selected-ion monitoring technique, will be the most successful way of proving methandienone administration to a horse.     

How Do Medical Students Respond to Emotional Cues and Concerns Expressed by Simulated Patients during OSCE Consultations? – A Multilevel Study

Objectives

How medical students handle negative emotions expressed by simulated patients during Objective Structured Clinical Examinations (OSCE) has not been fully investigated. We aim to explore (i) whether medical students respond differently to different types of patients’ emotional cues; and (2) possible effects of patients’ progressive disclosure of emotional cues on students’ responses.

Methods

Forty OSCE consultations were video recorded and coded for patients’ expressions of emotional distress and students’ responses using a validated behavioural coding scheme (the Verona Coding Definitions of Emotional Sequence). Logistic multilevel regression was adopted to model the probability of the occurrence of student reduce space response behaviour as a function of the number of patients’ expressions of emotional cues.

Results

We found that medical students offered responses that differed to emotional cue types expressed by simulated patients. Students appeared to provide space to emotional cues when expressed in vague and unspecific words and reduce space to cues emphasizing physiological or cognitive correlates. We also found that medical students were less likely to explore patients’ emotional distress nearer the end of the consultation and when the duration of a patient speech turn got larger. Cumulative frequency of patients’ emotional cues also predicted students’ reduce space behaviour.

Practical Implications

Understanding how medical students manage negative emotions has significant implications for training programme development focusing on emotion recognition skills and patient-centred communication approach. In addition, the statistical approaches adopted by this study will encourage researchers in healthcare communication to search for appropriate analytical techniques to test theoretical propositions.     

VEGF,HIF-1α Expression and MVD as an Angiogenic Network in Familial Breast Cancer

Angiogenesis, which plays an important role in tumor growth and progression of breast cancer, is regulated by a balance between pro- and anti-angiogenic factors. Expression of vascular endothelial growth factor (VEGF) is up-regulated during hypoxia by hypoxia-inducible factor-1α (HIF-1α). It is known that there is an interaction between HIF-1α and BRCA1 carrier cancers, but little has been reported about angiogenesis in BRCA1-2 carrier and BRCAX breast cancers. In this study, we investigated the expression of VEGF and HIF-1α and microvessel density (MVD) in 26 BRCA1-2 carriers and 58 BRCAX compared to 77 sporadic breast cancers, by immunohistochemistry. VEGF expression in BRCA1-2 carriers was higher than in BRCAX cancer tissues (p = 0.0001). Furthermore, VEGF expression was higher in both BRCA1-2 carriers and BRCAX than the sporadic group (p<0.0001). VEGF immunoreactivity was correlated with poor tumor grade (p = 0.0074), hormone receptors negativity (p = 0.0206, p = 0.0002 respectively), and MIB-1-labeling index (p = 0.0044) in familial cancers (BRCA1-2 and BRCAX). The percentage of nuclear HIF-1α expression was higher in the BRCA1-2 carriers than in BRCAX cancers (p<0.05), and in all familial than in sporadic tumor tissues (p = 0.0045). A higher MVD was observed in BRCA1-2 carrier than in BRCAX and sporadic cancer tissues (p = 0.002, p = 0.0001 respectively), and in all familial tumors than in sporadic tumors (p = 0.01). MVD was positively related to HIF-1α expression in BRCA1-2 carriers (r = 0.521, p = 0.006), and, in particular, we observed a highly significant correlation in the familial group (r = 0.421, p<0.0001). Our findings suggest that angiogenesis plays a crucial role in BRCA1-2 carrier breast cancers. Prospective studies in larger BRCA1-2 carrier series are needed to improve the best therapeutic strategies for this subgroup of breast cancer patients.     

Casein-derived bioactive phosphopeptides: role of phosphorylation and primary structure in promoting calcium uptake by HT-29 tumor cells

Casein phosphopeptides beta-CN(1-25)4P and alpha(s1)-CN(59-79)5P, from beta- and alpha(s1)-casein, respectively, both carrying the characteristic 'acidic motif' Ser(P)-Ser(P)-Ser(P)-Glu-Glu, were chemically synthesized and administered to HT-29 cells differentiated in culture, which are a used model of intestinal epithelium for absorption studies. Both casein phosphopeptides caused an increase of [Ca(2+)](i) due to influx of extracellular Ca(2+). The response was quantitatively higher with beta-CN(1-25)4P than alpha(s1)-CN(59-79)5P. The synthetic peptide corresponding to the 'acidic motif' was ineffective and the dephosphorylated form of beta-CN(1-25)4P almost inactive. The lack of the N-terminally located five amino acids, or sequence modifications within the N-terminal segment of beta-CN(1-25)4P, caused a total loss of activity, whereas the lack of the C-terminal segment preserved activity. In conclusion, the influx of calcium into HT-29 cells caused by beta-CN(1-25)4P appears to depend on the phosphorylated 'acidic motif' and the preceding N-terminal region.