Toxicity Potential of Aqueous Kava Extracts on Liver Function Tests in Rats

Kava, prepared as the traditional aqueous infusion, was tested in the rat for possible effects on liver function tests. Extracts were administered in dosages containing 200 or 500 mg of the active kavalactones per kg for two or four weeks. Sera were assayed for four enzymes that are markers of liver toxicity and liver homogenates for malondiadehyde formation that indicates changes in lipid peroxidation. The data showed that none of the enzymes, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and lactate dehydrogenase, nor malondialdehyde levels were elevated, in fact in some cases they were significantly reduced, suggesting the lack of a toxic effect on the liver by kava

A scalable lysyl hydroxylase 2 expression system and luciferase-based enzymatic activity assay

Hydroxylysine aldehyde-derived collagen cross-links (HLCCs) accumulate in fibrotic tissues and certain types of cancer and are thought to drive the progression of these diseases. HLCC formation is initiated by lysyl hydroxylase 2 (LH2), an Fe(II) and α-ketoglutarate (αKG)-dependent oxygenase that hydroxylates telopeptidyl lysine residues on collagen. Development of LH2 antagonists for the treatment of these diseases will require a reliable source of recombinant LH2 protein and a non-radioactive LH2 enzymatic activity assay that is amenable to high throughput screens of small molecule libraries. However, LH2 protein generated previously using E coli– or insect-based expression systems was either insoluble or enzymatically unstable, and LH2 enzymatic activity assays have measured radioactive CO2 released from 14C-labeled αKG during its conversion to succinate. To address these deficiencies, we have developed a scalable process to purify human LH2 protein from Chinese hamster ovary cell-derived conditioned media samples and a luciferase-based assay that quantifies LH2-dependent conversion of αKG to succinate. These methodologies may be applicable to other Fe(II) and αKG-dependent oxygenase systems

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Calcium/Calmodulin Stimulates the Autophosphorylation of Elongation Factor 2 Kinase on Thr-348 and Ser-500 To Regulate Its Activity and Calcium Dependence

Eukaryotic elongation factor 2 kinase (eEF-2K) is an atypical protein kinase regulated by Ca²⁺ and calmodulin (CaM). Its only known substrate is eukaryotic elongation factor 2 (eEF-2), whose phosphorylation by eEF-2K impedes global protein synthesis. To date, the mechanism of eEF-2K autophosphorylation has not been fully elucidated. To investigate the mechanism of autophosphorylation, human eEF-2K was coexpressed with λ-phosphatase and purified from bacteria in a three-step protocol using a CaM affinity column. Purified eEF-2K was induced to autophosphorylate by incubation with Ca²⁺/CaM in the presence of MgATP. Analyzing tryptic or chymotryptic peptides by mass spectrometry monitored the autophosphorylation over 0–180 min. The following five major autophosphorylation sites were identified: Thr-348, Thr-353, Ser-445, Ser-474, and Ser-500. In the presence of Ca²⁺/CaM, robust phosphorylation of Thr-348 occurs within seconds of addition of MgATP. Mutagenesis studies suggest that phosphorylation of Thr-348 is required for substrate (eEF-2 or a peptide substrate) phosphorylation, but not self-phosphorylation. Phosphorylation of Ser-500 lags behind the phosphorylation of Thr-348 and is associated with the Ca²⁺-independent activity of eEF-2K. Mutation of Ser-500 to Asp, but not Ala, renders eEF-2K Ca²⁺-independent. Surprisingly, this Ca²⁺-independent activity requires the presence of CaM