The BET inhibitor JQ1 selectively impairs tumour response to hypoxia and downregulates CA9 and angiogenesis in triple negative breast cancer.

The availability of bromodomain and extra-terminal inhibitors (BETi) has enabled translational epigenetic studies in cancer. BET proteins regulate transcription by selectively recognizing acetylated lysine residues on chromatin. BETi compete with this process leading to both downregulation and upregulation of gene expression. Hypoxia enables progression of triple negative breast cancer (TNBC), the most aggressive form of breast cancer, partly by driving metabolic adaptation, angiogenesis and metastasis through upregulation of hypoxia-regulated genes (for example, carbonic anhydrase 9 (CA9) and vascular endothelial growth factor A (VEGF-A). Responses to hypoxia can be mediated epigenetically, thus we investigated whether BETi JQ1 could impair the TNBC response induced by hypoxia and exert anti-tumour effects. JQ1 significantly modulated 44% of hypoxia-induced genes, of which two-thirds were downregulated including CA9 and VEGF-A. JQ1 prevented HIF binding to the hypoxia response element in CA9 promoter, but did not alter HIF expression or activity, suggesting some HIF targets are BET-dependent. JQ1 reduced TNBC growth in vitro and in vivo and inhibited xenograft vascularization. These findings identify that BETi dually targets angiogenesis and the hypoxic response, an effective combination at reducing tumour growth in preclinical studies.

The contextual change paradox is still unresolved: comment on Bouton, Nelson, and Rosas (1999)

According to the contextual change theory of memory loss, spontaneous forgetting reflects a retrieval impairment due to subtle and unprogrammed shifts in environmental cues over a retention interval. However, Riccio, Richardson, and Ebner (1984) noted an apparent paradox in this model; specifically, laboratory studies inducing explicit shifts in contextual cues found less disruption of performance as retention intervals increased. Bouton, Nelson, and Rosas (1999) critiqued several of the claims made by Riccio et al. and concluded that the contextual cue theory is still a valid account of spontaneous forgetting. In this comment, the authors address the 3 major criticisms offered by Bouton et al., point out an inconsistency in their argument, and conclude that the original paradox still poses problems for the contextual change theory of forgetting.

Use of topical NSAIDs in patients receiving systemic NSAID treatment: a pharmacy-based study in Germany.

Topical non-steroidal anti-inflammatory drugs NSAID account for two-thirds of the most frequently prescribed NSAIDs in Germany. Nevertheless, the extent of use and the benefit of local NSAIDs are viewed critically. We described the use of topical NSAIDs in people with systemic NSAID therapy. The study population consisted of 526 people with an average age of 57 years; two-thirds of the patients were women. We observed that the elderly (60 years and older) and patients with osteo- and/or rheumatoid arthritis had an increased likelihood of being treated with both topical and systemic NSAIDs. We suggest that the frequent use of topical NSAIDs in these patients is based on their supportive therapeutic effect rather than on their efficacy. Further research is needed in order to investigate the potential beneficial effects of topical NSAIDs in NSAID therapy.

Lack of covalent binding to DNA of di-n-octyltin dichloride (DOTC) in vivo and in vitro.

[14C]Di-n-octyltin dichloride ([14C]DOTC) was administered by oral gavage to male and female rats. After 96 h hepatic and thymic DNA was isolated. All DNA fractions were radioactive, but analysis of DNA hydrolysates by HPLC revealed that the radioactivity was incorporated via biosynthesis and was not due to adduct formation. The limit of detection for adduct formation, expressed in units of the covalent binding index (CBI = mumol chemical bound per mol nucleotides/mmol chemical applied per kg body wt.) was approximately 0.2 for liver DNA and about 0.7 for thymus DNA. This maximum possible DNA-binding ability is about 30,000 times lower than the corresponding value for the strong carcinogen, aflatoxin B1. In addition, [14C]DOTC did not bind covalently to calf thymus DNA in the presence or absence of rat liver S9 or to DNA of V79 Chinese hamster cells. This study therefore gives no indication for genotoxic activity of DOTC mediated by DNA binding.

Detection of antibodies to avian reverse transcriptase by indirect ELISA.

An improved indirect ELISA test for detection of antibodies to the reverse transcriptase (revertase) is described. The sensitivity of the revertase ELISA test was compared to that of the revertase inhibition test. Serum samples of various origin and sera of specific pathogen free (SPF) hens were examined for the revertase antibodies. The presence of these antibodies in the sera of SPF chickens is discussed.

Identification of the coronavirus MHV-JHM mRNA 4 product.

A bacterial expression vector was constructed to encode a fusion protein which had, at its carboxy terminus, a polypeptide encoded within the 5' proximal open reading frame of the coronavirus MHV-JHM mRNA 4. This polypeptide was isolated and used to produce an antiserum. The antiserum reacted specifically with a 15,000 Mr polypeptide synthesized in MHV-JHM-infected cells, or in vitro translations of infected cell poly(A) RNA enriched for mRNA 4. These results demonstrate the translational activity of mRNA 4 during infection, identify conclusively the translation product and provide a means to investigate the synthesis and function of this protein.

Coronavirus MHV-JHM mRNA 5 has a sequence arrangement which potentially allows translation of a second, downstream open reading frame.

The sequence of a 5'-proximal region of mRNA 5 of coronavirus MHV-JHM was determined by chain-terminator sequencing of cDNA subcloned in M13. The sequence contained two long open reading frames of 321 bases and 264 bases, overlapping by five bases but in different frames. Both open reading frames are initiated by AUG codons in sequence contexts that are relatively infrequently used as initiator codons. The smaller, downstream open reading frame encoded a neutral protein (mol. wt. 10200) with a hydrophobic amino terminus. The larger, 5'-proximal open reading frame encoded a basic protein (mol. wt. 12400) which lacks internal methionine residues. With the exception of the AUG codon initiating the downstream open reading frame, no internal AUG codons were found within the sequence covered by the upstream open reading frame. These results suggest that the MHV-JHM mRNA 5 is translated to produce two proteins by a mechanism involving internal initiation of protein synthesis. Preliminary evidence is presented showing that the downstream open reading frame is functional in vivo.

Functional and molecular analysis of mitochondria in thyroid oncocytoma.

We report a functional and molecular analysis of nine oncocytic tumors of the human thyroid. In all the abundance of mitochondria observed ultrastructurally was accompanied by an increase in enzymatic activities of respiratory complexes 1 (NADH dehydrogenase), 11 (succinate dehydrogenase) IV (cytochrome c oxidase), and V (ATPase). Western blot analysis failed to detect uncoupling protein in the tumors. The elevated respiratory enzyme activities were paralleled by an increase in the mitochondrial DNA content. Restriction analysis of mitochondrial DNA gave no indication of heteroplasmy or other gross alterations. We conclude that the mitochondrial proliferation in oncocytic tumors is probably not the result of a compensatory mechanism for the deficiency in enzyme complexes of the mitochondrial respiratory chain.

The Rorschach and the assessment of impulsivity.

The validity of the Rorschach as an instrument to assess impulsivity was examined in a sample of 55 adolescent psychiatric inpatients. The Rorschach variables considered to be related to impulsivity (D, Adjusted D, M, Afr, X + %, FC:CF+C, and L) were used to predict performance on the Gordon Diagnostic System (GDS). Only the discriminant function for the GDS Delay Task (which assesses the ability to formulate response strategies and to benefit from feedback) was statistically significant, with a 76.36% correct classification of subjects. The Rorschach variables with the highest correlations within the discriminant function were D, FC:CF+C, and M. The results of this study appeared to provide some support for the utility of the Rorschach in the assessment of impulsivity.

[Serologic and virologic investigations on the presence of BLV infection in a dairy herd in Syria]. / Serologische und virologische Untersuchungen über das Vorkommen der BLV-Infektion in einer Milchviehherde in Syrien.

237 cattle of a dairy herd in Syria were tested for anti-BLV antibody by the ELISA. 194 animals were additionally examined by the agar gel immunodiffusions test (AGID) on BLV antibodies and 100 by polymerase chain reaction (PCR) for BLV provirus. BLV specific antibodies were determined by means of AGID and ELISA at 62.9% and 69.2% of the examined animals, respectively. Using the PCR method the BLV provirus was detected in 89% of the investigated cattle. Only one ELISA seropositive animal was negative for BLV provirus. The results show the high BLV contamination of this herd and lead to the presumption of wide spread enzootic bovine leukosis in Syria. In the case of the diagnosis of BLV-infection, the PCR-technique compared to the serological tests proved to be much more sensitive. By the detection of BLV antibody, the ELISA showed a higher sensitivity than the AGID and in this way, is advisable as a method of choice for screening investigations. Restriction enzyme and sequence analysis of PCR-amplificates demonstrate that different BLV provirus variants (A, B and C) in the examined herd occur, where the variant C which a high similarity to an Australian BLV provirus isolates showed, occurred most frequently at 92.5%.

A study of the longitudinal utilization and switching-patterns of non-steroidal anti-inflammatory drugs using a pharmacy based approach.

The extent of the heterogeneity of drug utilization among NSAID users has not been extensively studied. We studied the longitudinal prescribing and switching patterns of NSAID users in a 1-year follow-up study in four German pharmacies. The study population consisted of 526 persons with an average age of 57 years. We observed that the legend duration of prescription increased with age; 14.3 days for patients aged 44 or younger to 25.1 days for persons 75 years or older, and was dependent on disease chronicity; 16.0 days for acutely ill persons compared to 23.9 days for chronic patients. The average legend duration also varied between different types of NSAIDs, from 18.0 days for ibuprofen to 29.1 days for tiaprofen. Switching from one type of NSAID to another proved to be related to the legend duration of prescription and patient characteristics such as compliance with NSAID therapy, duration of the disease and the effectiveness (poor tolerability or insufficient effect) of the NSAID therapy reported by the patients 4 weeks after recruitment. We conclude that NSAID users cannot be viewed as an homogeneous group of patients with respect to exposure time to the drug. This heterogeneity should be considered in the exposure definition, the 'time-window' design of observational studies dealing with risk comparisons in particular.

Direct use of cell lysates in PCR-based diagnosis of bovine leukemia virus infection.

Polymerase chain reaction (PCR) has been used for direct detection of bovine leukemia virus (BLV) proviral DNA in cattle, but it is still mainly used for experimental research. One bottleneck for routine diagnosis of BLV by PCR has always been the isolation and purification of DNA. We compare the use of not purificated with highly-purified DNA in the PCR-based diagnosis of BLV infection. DNA extracted from whole blood by chloroform extraction (CP-DNA) and DNA prepared only by osmotic shock, washing, heating and freezing procedures (RPoS-DNA), were utilized. Fifteen cattle well characterized serologically were investigated for BLV-provirus with PCR using this different DNA preparations. With both methods all but one investigated animal were correctly identified. It was estimated that in case of CP-DNA PCR 10 BLV-provirus copies were sufficient to obtain a positive result. The sensitivity of RPoS-DNA PCR was similar. Because of the greater practicability of the latter technique we used it in a small field study with ten cattle. All serologically positive animals were correctly identified by the PCR. In addition one seronegative animal was found to carry BLV-provirus. Therefore RPoS-DNA PCR might be a good tool for the routine diagnosis of BLV-infected cattle.

Evaluation of polymerase chain reaction (PCR) application in diagnosis of bovine leukaemia virus (BLV) infection in naturally infected cattle.

The practical application of polymerase chain reaction (PCR) for the diagnosis of bovine leukaemia virus (BLV) infections in naturally infected cattle was evaluated. Compared to serological tests the PCR was definitely found to be a more sensitive method, yielding the highest number of positive results (10% more compared to enzyme-linked immunosorbent assay, (ELISA), and 17.7% more compared to agar-gel immunodiffusion, (AGID)). In testing cattle from herds with BLV incidence under 5%, out of 52 provirus positive cattle only 43 were correctly identified by ELISA. When compared to AGID only 37 of the 52 PCR positive animals were correctly identified. Of 18 cattle imported from the Slovak Republic and kept in a quarantine stable, four were found to be BLV provirus positive by PCR, while serological tests indicated one animal positive and three negative. Therefore, it is impossible to prevent the spread of the infection from one country to another by serological testing only. Moreover, it is feasible to identify animals with changing antibody titres correctly by PCR. Using PCR we were also able to distinguish BLV infected from uninfected calves that were serologically positive due to colostral antibodies. Higher sensitivity of BLV provirus detection by PCR was achieved using env gene rather than tax gene specific primers. Negative results by PCR in cases of positive serological reactions are still possible, as shown in case of one adult animal. These findings indicate that PCR is a highly sensitive method and might be successfully used and economically advantageous for different practical applications in detection of BLV infection in naturally infected cattle.

[Comparison of standard methods for the preparation of egg yolk antibodies]. / Vergleich von Standardmethoden zur Präparation von Dotterantikörpern.

Chicken egg yolk antibodies (lgY) play an increasing role as alternative to mammalian polyclonal antibodies. They are widely used in biomedical research, for diagnostics, prophylaxis, and therapy of diseases. The extraction steps of IgY from egg yolk must be simple, with high output of purified antibodies. The aim of the present study was a comparison of different purification methods of egg yolk antibodies. The results of eight extraction methods of IgY and method combinations were investigated by PAGE and densitometric analysis. It has been demonstrated, that the IgY preparation with dextran sulfate is very effective, quick and simple to perform. It is well-suited in combination with other methods, e.g. ammonium sulfate precipitation.

[Polymerase chain reaction (PCR) for detection of BLV provirus-- a practical complement for BLV diagnosis?]. / Polymerase Kettenreaktion (PCR) zum Nachweis von BLV-Provirus--praktikable Ergänzung der BLV-Diagnostik?

A typical infection with bovine leukemia virus (BLV) induces a permanent antibody (Ab) response with high titers against BLV-antigens. In the last few years atypical courses of infection with low or transient BLV-Ab-titers or even lack of any detectable BLV-Ab-titers in animals with BLV-provirus integration have been described. This makes it difficult to eliminate BLV infection from herds using serological assays only. Whether or not polymerase chain reaction (PCR) is a useful tool to complement serological Ab-assays in BLV-eradication in herds was clarified in three ways: (i) different DNA-quick-preparations of blood were examined in nested PCR, (ii) cows of a BLV infected herd that was involved in a national eradication program were investigated for 6 months und (iii) BLV-provirus-variants occurring in this herd were differentiated. The results show, that even by using PCR it was not possible to detect all infected animals all the time and that eradication of BLV from this herd was not completed in this short time. The PCR is useful for the investigation of herds and more sensitive than ELISA. PCR using LTR-primers (34 positive cattle) was more sensitive than PCR with env-primers (30 positive cattle). Using PCR 34 BLV infected cattle were detected of which only 21 reacted in ELISA. Restriction enzyme analysis or sequence analysis of PCR-amplificates allowed the detection of virus variants and conclusions about the way of infection. PCR should be used for BLV-eradication in cattle herds with low BLV-incidence, for the investigation of new outbreaks or tumor cases in long term BLV free herds and for investigation of breeding cattle.