bluuebunny/crossref_metadata_2025_split huggingface dataset:
Zebrafish: an emerging technology for in vivo pharmacological assessment to identify potential safety liabilities in early drug discovery
Authors: Barros T P , Alderton W K , Reynolds H M , Roach A G , Berghmans S 
Published: June 2008

The zebrafish is a well‐established model organism used in developmental biology. In the last decade, this technology has been extended to the generation of high‐value knowledge on safety risks of novel drugs. Indeed, the larval zebrafish appear to combine advantages of whole organism phenotypic assays and those (rapid production of results with minimal resource engagement) of in vitro high‐throughput screening techniques. Thus, if appropriately evaluated, it can offer undeniable advantages in drug discovery for identification of target and off‐target effects. Here, we review some applications of zebrafish to identify potential safety liabilities, particularly before lead/candidate selection. For instance, zebrafish cardiovascular system can be used to reveal decreases in heart rate and atrial–ventricular dissociation, which may signal human ether‐a‐go‐go‐related gene (hERG) channel blockade. Another main area of interest is the CNS, where zebrafish behavioural assays have been and are further being developed into screening platforms for assessment of locomotor activity, convulsant and proconvulsant liability, cognitive impairment, drug dependence potential and impaired visual and auditory functions. Zebrafish also offer interesting possibilities for evaluating effects on bone density and gastrointestinal function. Furthermore, available knowledge of the renal system in larval zebrafish can allow identification of potential safety issues of drug candidates on this often neglected area in early development platforms. Although additional validation is certainly needed, the zebrafish is emerging as a versatile in vivo animal model to identify off‐target effects that need investigation and further clarification early in the drug discovery process to reduce the current, high degree of attrition in development. British Journal of Pharmacology (2008) 154, 1400–1413; doi:10.1038/bjp.2008.249; published online 16 June 2008
The Importance of Patents for Scientists
Authors: Scott Sue 
Published: June 2008

AbstractPatents have become a hot topic and the subject of much media attention. If your main source of information about patents is the newspapers, you could be forgiven for having an extremely distorted view of what patents are, what they do, and what benefits and problems are associated with them. All scientists want to be able to improve the world in some way, whether by contributing in a fundamental way to the fund of human knowledge or by delivering new and valuable technology. If you are a scientist making inventions, it is increasingly important that you should have an understanding of the importance of patents in the modern world, and should have sufficient understanding of the patent system to make sure that your aims for your inventions are achievable. This talk sets out some basic facts about patents and also de-bunks some the most common misapprehensions about patents.
Biotransformation of p‐methoxyphenylacetonitrile into p‐methoxyphenylacetic acid by resting cells of Bacillus subtilis
Authors: Chen Jing , Zheng Yu‐Guo , Shen Yin‐Chu 
Published: June 2008

Resting cells of Bacillus subtilis ZJB‐063 were used for the direct transformation of MOPAN (p‐methoxyphenylacetonitrile) to MOPAA (p‐methoxyphenylacetic acid), which is an important pharmaceutical intermediate. The B. subtilis ZJB‐063 culture conditions for the production of nitrilase and the reaction conditions for this nitrilase‐mediated conversion were optimized. The maximum production of nitrilase was achieved when glucose and a combination of ammonium sulfate and yeast powder were added as carbon and nitrogen sources respectively. Previously reported inducers were found to be unnecessary for the production of nitrilase from B. subtilis ZJB‐063, which indicated that this nitrilase appeared to be constitutive. However, when ε‐caprolactam (6‐hexanolactam) was added as the inducer, B. subtilis ZJB‐063 exhibited nitrile hydratase and amidase activity. The maximum conversion of MOPAN into MOPAA (specific activity 17.03 units·g−1DCW; DCW is dry cell weight) was observed in a solution containing 50 mM phosphate buffer (pH 7.0), 10 mM MOPAN, 2.7 mg DCW·ml−1 wet resting cells and 5% (v/v) DMSO for 4 h at 32 °C. MOPAN (10 mM) was completely converted into MOPAA (9.65 mM) in 5 h in shake flasks without the formation of p‐methoxyphenylacetamide. The small deviation of MOPAA (9.65 mM) from the theoretical amount (10 mM) may be due to partial consumption of the products by B. subtilis ZJB‐063. Both MOPAN and MOPAA inhibited the hydrolysis at concentrations above 15 mM. Scale up of the reaction to 200 ml in a bubble bioreactor shortened the reaction time compared with the reactions performed in shake flasks.
Bioengineering functional human aortic vascular smooth‐muscle strips in vitro
Authors: Hecker Louise , Khait Luda , Welsh Michael J. , Birla Ravi 
Published: June 2008

The contraction and relaxation of VSM (vascular smooth muscle) are responsible for the maintenance of vascular tone, which is a major determinant of blood pressure. However, the molecular events leading to the contraction and relaxation of VSM are poorly understood. The development of three‐dimensional bioengineered tissues provides an opportunity to investigate the molecular events controlling vascular tone in vitro. In the present study we used fibrin‐gel casting to bioengineer functional VSM strips from primary human aortic VSM cells. Our bioengineered VSM strips are functionally similar to VSM in vivo and remained viable in culture for up to 5 weeks. VSM strips demonstrate spontaneous basal tone and can generate an active force (contraction) of up to 85.2 μN on stimulation with phenylephrine. Bioengineered VSM strips exhibited Ca2+‐dependent contraction and calcium‐independent relaxation. The development of functional bioengineered VSM tissue provides a new in vitro model system that can be used to investigate the molecular events controlling vascular tone.
Development of transgenic Artemisia annua (Chinese wormwood) plants with an enhanced content of artemisinin, an effective anti‐malarial drug, by hairpin‐RNA‐mediated gene silencing
Authors: Zhang Ling , Jing Fuyuan , Li Fupeng , Li Meiya , Wang Yuliang , Wang Guofeng , Sun Xiaofen , Tang Kexuan 
Published: June 2008

Artemisinin is an effective anti‐malarial drug isolated from Artemisia annua L. (Chinese wormwood), but the content of artemisinin in A. annua is low. In the present study we explored the possibility of using genetic engineering to increase the artemisinin content of A. annua by suppressing the expression of SQS (squalene synthase), a key enzyme of sterol pathway (a pathway competitive with that of artemisinin biosynthesis) by means of a hairpin‐RNA‐mediated RNAi (RNA interference) technique. A total of 23 independent transgenic A. annua plants were obtained through Agrobacterium tumefaciens‐mediated transformation, which was confirmed by PCR and Southern‐blot analyses. HPLC‐evaporative light‐scattering detection analysis showed that the artemisinin content of some transgenic plants was significantly increased, with the highest values reaching 31.4 mg/g dry weight, which is about 3.14‐fold the content observed in untransformed control plants. Real‐time reverse transcription–PCR analysis demonstrated that the expression of SQS was suppressed significantly, and GC–MS analysis showed that sterol was efficiently decreased in the transgenic plants. The present study demonstrated that genetic‐engineering strategy of RNAi is an effective means of increasing artemisinin content in plants.
Knockdown of Pdcd4 results in induction of proprotein convertase 1/3 and potent secretion of chromogranin A and secretogranin II in a neuroendocrine cell line
Authors: Lankat‐Buttgereit Brigitte , Müller Sabine , Schmidt Harald , Parhofer Klaus G. , Gress Thomas M. , Göke Rüdiger 
Published: June 2008

Background information. Pdcd4 (programmed cell death 4) is up‐regulated during apoptosis and seems to play an important role as a tumour suppressor. To gain further insights into its biological functions, we suppressed Pdcd4 expression in the neuroendocrine cell line Bon‐1 via siRNA (small interfering RNA) technology.Results. Using this cell line, we found that suppression of Pdcd4 resulted in an increased release of CgA (chromogranin A) and Sg II (secretogranin II), and was accompanied by an up‐regulation of intracellular PC1 (proprotein convertase 1/3). The enhanced secretion of CgA and Sg II seemed to be mediated by an activation of protein kinase Akt via PI3K (phosphoinositide 3‐kinase). In accordance with this, inhibition of PI3K activity and, thereby, reduced phosphorylation of Akt was shown to enhance Pdcd4 expression. Neither the PKC (protein kinase C) signal transduction cascade nor the MAPK (mitogen‐activated protein kinase) pathway seemed to play a role in the regulation of CgA and Sg II secretion by Pdcd4.Conclusions. CgA is considered to be a marker for neuroendocrine tumours, and up‐regulation of PC1 has been reported in various types of cancers. The repression of PC1 by Pdcd4 may represent a novel mechanism for the function of Pdcd4 as a tumour suppressor. Our results are of particular interest, as we observed that pioglitazone, an oral medication used in the treatment of Type 2 diabetes, decreased Pdcd4 levels, activated Akt, increased CgA and Sg II secretion and augmented PC1 protein in Bon‐1 cells. Enhanced PC1 levels, leading to improved processing of proinsulin and proglucagon, may contribute to the benefits of pioglitazone therapy. The in vivo relevance of our findings was highlighted by data indicating elevated CgA amounts in the sera of patients treated with pioglitazone. This is the first study connecting Pdcd4 levels, secretion behaviour of neuroendocrine cells and regulation of PI3K activity.
How do surfactants and DTT affect the size, dynamics, activity and growth of soluble lysozyme aggregates?
Authors: Kumar Satish , Ravi Vijay Kumar , Swaminathan Rajaram 
Published: June 2008

The early intermediates in the protein aggregation pathway, the elusive soluble aggregates, play a pivotal role in growth and maturation of ordered aggregates such as amyloid fibrils. Blocking the growth of soluble oligomers is an effective strategy to inhibit aggregation. To decipher the molecular mechanisms and develop better strategies to arrest aggregation, it is imperative to understand how the size, molecular dynamics, activity and growth kinetics of soluble aggregates are affected when aggregation is inhibited. With this objective, in the present study we have investigated the influence of additives such as SDS, CTAB (cetyltrimethylammonium bromide) and DTT (dithiothreitol) on the slow aggregation of HEWL (hen eggwhite lysozyme) at pH 12.2. For this purpose, techniques such as steady-state and time-resolved fluorescence anisotropy of covalently labelled dansyl probe, gel-filtration chromatography, estimation of free thiol groups, thioflavin T and ANS (8-anilinonaphthalene-1-sulfonic acid) fluorescence, CD and atomic-force microscopy were employed to monitor the soluble oligomers over a period spanning 30 days. The results of the present study reveal that: (i) the spontaneous formation of soluble aggregates is irreversible and abolishes activity; (ii) the initial growth of aggregates (0–24 h) is promoted by a gradual increase in the exposure of hydrophobic surfaces; (iii) subsequently intermolecular disulfide bonds are critical for the assembly and stability of aggregates; (iv) the tight molecular packing inside large aggregates which contributed to slow (∼5 ns) and restricted segmental motion of dansyl probe was clearly loosened up in the presence of additives, enabling fast (1–2 ns) and free motion (unlike DTT, the size of lysozyme complexes with surfactants, was large, due to a conglomeration of proteins and surfactants); (v) the aggregates show reduced helical content compared with native lysozyme, except in the presence of SDS; and (vi) DTT was more potent than SDS/CTAB in arresting the growth of aggregates.
Evidence of multiple retrotransposons in two litopenaeid species
Authors: Hizer S. E. , Tamulis W. G. , Robertson L. M. , Garcia D. K. 
Published: June 2008

SummaryRetrotransposons encompass a specific class of mobile genetic elements that are widespread across eukaryotic genomes. The impact of the varied types of retrotransposons on these genomes is just beginning to be deciphered. In a step towards understanding their role in litopenaeid shrimp, we have herein identified nine non‐LTR retrotransposons, among which several appear to exist outside the standard defined clades. Two Litopenaeus stylirostris elements were discovered through degenerate PCR amplification using previously defined non‐LTR degenerate primers, and through primers designed from a RAPD‐derived sequence. A third genomic L. stylirostris element was identified using specific priming from an amplification protocol. These three PCR‐derived sequences showed conserved domains of the non‐LTR reverse transcriptase gene. In silico searching of genome databases and subsequent contig construction yielded six non‐LTR retrotransposons (both genomic and expressed) in the Litopenaeus vannamei genome that also exhibited the highly conserved domains found in our PCR‐derived sequences. Phylogenetic placement among representatives from all non‐LTR clades showed a possibly novel monophyletic group that included five of our nine sequences. This group, which included elements from both L. stylirostris and L. vannamei, appeared most closely related to the highly active RTE clade. Our remaining four sequences placed in the CR1 and I clades of retrotransposons, with one showing strong similarity to ancient Penelope elements. This research describes three newly discovered retrotransposons in the L. stylirostris genome. Phylogenetic analysis clusters these in a monophyletic grouping with retrotransposons previously described from two closely related species, L. vannamei and Penaeus monodon.
Molecular variation in pigmentation genes contributing to coat colour in native Korean Hanwoo cattle
Authors: Mohanty T. R. , Seo K. S. , Park K. M. , Choi T. J. , Choe H. S. , Baik D. H. , Hwang I. H. 
Published: June 2008

SummaryPigmentation genes such as TYR (tyrosinase), TYRP1 (tyrosinase‐related protein 1), DCT (previously TYRP2, or tyrosinase‐related protein 2), ASIP (agouti) and MC1R (melanocortin receptor 1) play a major role in cattle coat colour. To understand the genotypic profile underlying coat colour in native Korean Hanwoo cattle and Angus black cattle, portions of the above‐mentioned genes were amplified. Sequence analysis revealed variation in the TYRP1 (exon 5) and MC1R genes. Restriction enzyme analysis of these two genes could distinguish between different colours of Hanwoo cattle. Quantitative estimates of melanin and eumelanin in hair from three different‐coloured Hanwoo phenotypes and Angus black showed significant differences at the breed and phenotypic levels. Finally, sequence variants in MC1R were associated with total melanin and eumelanin in breeds as well as in Hanwoo phenotypes.
A proline‐to‐histidine mutation in POU1F1 is associated with production traits in dairy cattle
Authors: Huang W. , Maltecca C. , Khatib H. 
Published: June 2008

SummaryPOU class 1 homeobox 1 (POU1F1) is a member of the tissue‐specific POU‐containing transcription factor family. The expression of POU1F1 in mammalian pituitary gland controls the transcription of the genes encoding growth hormone, prolactin (PRL) and the subunits of thyroid‐stimulating hormone. In addition, some genes in the JAK/STAT signalling pathway downstream of POU1F1 have been shown to be associated with different production traits in dairy cattle. To investigate whether the POU1F1 gene is associated with economically important traits in dairy cattle, a pooled DNA sequencing approach was used to identify single nucleotide polymorphisms (SNPs) in the gene. An SNP in exon 3 of POU1F1 that changes a proline to a histidine was identified. A total of 2141 individuals from two North American Holstein cattle resource populations were genotyped for this SNP using a modified PCR‐RFLP method. Statistical analyses revealed significant association of POU1F1 variants with milk yield and productive life, which makes POU1F1 a possible candidate for marker‐assisted selection in dairy cattle breeding programmes.
Two variable active site residues modulate response regulator phosphoryl group stability
Authors: Thomas Stephanie A. , Brewster Jocelyn A. , Bourret Robert B. 
Published: June 2008

SummaryMany signal transduction networks control their output by switching regulatory elements on or off. To synchronize biological response with environmental stimulus, switching kinetics must be faster than changes in input. Two‐component regulatory systems (used for signal transduction by bacteria, archaea and eukaryotes) switch via phosphorylation or dephosphorylation of the receiver domain in response regulator proteins. Although receiver domains share conserved active site residues and similar three‐dimensional structures, rates of self‐catalysed dephosphorylation span a ≥ 40 000‐fold range in response regulators that control diverse biological processes. For example, autodephosphorylation of the chemotaxis response regulator CheY is 640‐fold faster than Spo0F, which controls sporulation. Here we demonstrate that substitutions at two variable active site positions decreased CheY autodephosphorylation up to 40‐fold and increased the Spo0F rate up to 110‐fold. Particular amino acids had qualitatively similar effects in different response regulators. However, mutant proteins matched to other response regulators at the two key variable positions did not always exhibit similar autodephosphorylation kinetics. Therefore, unknown factors also influence absolute rates. Understanding the effects that particular active site amino acid compositions have on autodephosphorylation rate may allow manipulation of phosphoryl group stability for useful purposes, as well as prediction of signal transduction kinetics from amino acid sequence.
Chemical and genetic validation of dihydrofolate reductase–thymidylate synthase as a drug target in African trypanosomes
Authors: Sienkiewicz Natasha , Jarosławski Szymon , Wyllie Susan , Fairlamb Alan H. 
Published: June 2008

SummaryThe phenotypes of single‐ (SKO) and double‐knockout (DKO) lines of dihydrofolate reductase–thymidylate synthase (DHFR–TS) of bloodstream Trypanosoma brucei were evaluated in vitro and in vivo. Growth of SKO in vitro is identical to wild‐type (WT) cells, whereas DKO has an absolute requirement for thymidine. Removal of thymidine from the medium triggers growth arrest in S phase, associated with gross morphological changes, followed by cell death after 60 h. DKO is unable to infect mice, whereas the virulence of SKO is similar to WT. Normal growth and virulence could be restored by transfection of DKO with T. brucei DHFR–TS, but not with Escherichia coli TS. As pteridine reductase (PTR1) levels are unchanged in SKO and DKO cells, PTR1 is not able to compensate for loss of DHFR activity. Drugs such as raltitrexed or methotrexate with structural similarity to folic acid are up to 300‐fold more potent inhibitors of WT cultured in a novel low‐folate medium, unlike hydrophobic antifols such as trimetrexate or pyrimethamine. DKO trypanosomes show reduced sensitivity to these inhibitors ranging from twofold for trimetrexate to >10 000‐fold for raltitrexed. These data demonstrate that DHFR–TS is essential for parasite survival and represents a promising target for drug discovery.
Rust severity in bioenergy willow plantations treated with additional nutrients
Authors: Toome M. , Heinsoo K. , Ramstedt M. , Luik A. 
Published: June 2008

SummaryA 3‐year field study was carried out to determine the effect of wastewater irrigation and previous differences in mineral fertilization on the occurrence of willow leaf rust (Melampsora epitea). The experiment was conducted in two energy forest plantations: one designed for wastewater purification and the other as a mineral fertilization experiment. The severity of leaf rust on different clones and sites with different treatments was assessed by counting the number of uredinia per leaf unit area. Generally, plants irrigated with wastewater consistently had more leaf rust, irrespective of the study years or willow clones. Previous mineral fertilization had mixed effects on different clones 2 years after the last application. Three years after the last fertilizer application, however, no impact of the treatment on rust disease development was detected. In general, the rust levels differed from year to year probably due to climate. In this study, no correlation was detected between shoot age and rust severity, whereas climate and treatments strongly influenced leaf rust levels on some willow clones.
Dendritic cell therapy in combination with interferon‐α for the treatment of metastatic renal cell carcinoma
Authors: Tatsugami Katsunori , Eto Masatoshi , Harano Masahiko , Hamaguchi Masumitsu , Miyamoto Toshihiro , Morisaki Takashi , Furue Masutaka , Akashi Koichi , Naito Seiji 
Published: June 2008

Objectives:  To evaluate the safety and efficacy of dendritic cell (DC) therapy in combination with interferon‐α (IFN‐α) in patients with advanced renal cell carcinoma.Methods:  Seven patients, with progressive disease following IFN‐α and interleukin (IL)‐2 treatment, were treated with monocyte‐derived DC (Mo‐DC) and IFN‐α between February 2004 and September 2006. They received Mo‐DC once a week for 5 weeks and then every 2 weeks either intradermally or intratumorally. IFN‐α (5–6 million U) was subcutaneously administered three times a week. Tumor size was evaluated by computed tomography scans before and after the 5th and 10th DC vaccination. A delayed‐type hypersensitivity test was performed after the 4th and 5th DC administration for immunological monitoring.Results:  Five patients had stable disease while the remaining two patients had progressive disease following 4 months of vaccination. In six patients the time to progression was prolonged in comparison with the previous cytokine treatment. Six patients showed delayed‐type hypersensitivity after the 4th or 5th immunization. Three patients developed high fever following DC immunization. Treatment was associated with transient flu‐like symptoms.Conclusions:  Our data indicate that DC therapy combined with IFN‐α is safe and has the potential for prolonging the time to progression in patients with advanced renal cell carcinoma.
Prepuce‐Sparing hypospadias repair with tubularized incised plate urethroplasty
Authors: Shimada Kenji , Matsumoto Fumi , Matsui Futoshi , Takano Syuichi 
Published: June 2008

Objectives:  Modifications in surgical methods of hypospadias repair have been influenced by social considerations. Most Japanese parents wish their children to retain their foreskin during hypospadias repair. We report on short‐term results of foreskin reconstruction associated with hypospadias repair.Methods:  Of 44 tubularized incised plate urethroplasty‐patients, 42 (95%) underwent foreskin reconstruction at the time of urethroplasty during the last 2 years. After the urethroplasty was completed, the foreskin was re‐approximated in three layers.Results:  Complications related to urethroplasty consisted of fistula in two patients with penoscrotal hypospadia, and mild stenosis of the glandular urethra in one patient. Complications related to foreskin reconstruction included dehiscence of the ventral foreskin in two patients. In all cases parents were well satisfied with the reconstructed prepuce.Conclusions:  Pre‐operative information about preputial reconstruction should be given to parents if they would prefer their son to be uncircumcised.