Development and validation of a generic fluorescent methyltransferase activity assay based on the transcreener AMP/GMP assay

Methylation is a ubiquitous covalent modification used to control the function of diverse biomolecules including hormones, neurotransmitters, xenobiotics, proteins, nucleic acids, and lipids. Histone methyltransferases (HMTs) are currently of high interest as drug targets because of their role in epigenetic regulation; however, most HMT assay methods are either not amenable to a high-throughput screening (HTS) environment or are applicable to a limited number of enzymes. The authors developed a generic methyltransferase assay method using fluorescent immunodetection of adenosine monophosphate (AMP), which is formed from the MT reaction product S-adenosylhomocysteine in a dual-enzyme coupling step. The detection range of the assay; its suitability for HTS, including stability of reagents following dispensing and after addition to reactions; and the potential for interference from drug-like molecules was investigated. In addition, the use of the assay for measuring inhibitor potencies with peptide or intact protein substrates was examined through pilot screening with selected reference enzymes including HMT G9a. By combining a novel enzymatic coupling step with the well-characterized Transcreener AMP/GMP assay, the authors have developed a robust HTS assay for HMTs that should be broadly applicable to other types of methyltransferases as well.     

Establishment and characterization of bovine mammary epithelial cell lines

Summary One bovine mammary epithelial cell clone, designated PS-BME-C1, and two bovine mammary epithelial cell lines, designated PS-BME-L6 and PS-BME-L7, were derived from mammary tissue of a pregnant (270 day) Holstein cow. The cells exhibit the distinctive morphologic characteristics of mammary epithelial cells and express the milk fat globule membrane protein, PAS-III. They form domes when cultured on plastic substrata and acinilike aggregates when cultured on a collagen matrix. These cells are capable of synthesizing and secretingα-lactalbumin andα-s₁-casein when cultured on a collagen matrix in the presence of insulin, cortisol, and prolactin. The cells have a near-normal diploid number and do not grow in suspension culture. When transplanted to the cleared mammary fat pads of female athymic nude mice, the cells readily proliferate forming noninvasive palpable spherical cellular masses within 8 wk after inoculation. The cells may become a useful tool to study the regulation of ruminant mammary epithelial cell growth and differentation. This work was supported by the Pennsylvania State University Experiment Station. The PS-BME cells are the property of The Pennsylvania Research Corporation. Scientists interested in obtaining the PS-BME clone or cell lines for their research may request them from the corresponding author.     

Synthesis of monoterpene hydrocarbons from [1-3H]linalyl pyrophosphate by carbocyclase from Citrus limonum

A partially purified enzyme preparation from the flavedo of Citrus limonum utilized [1-³H]linalyl pyrophosphate as a substrate for cyclic terpene hydrocarbon formation more efficiently than the pyrophosphates of nerol and geraniol. The products formed from all three substrates are α-pinene, β-pinene, limonene, and γ-terpinene. Neryl and geranyl pyrophosphate inhibit the formation of these products from linalyl pyrophosphate. No free linalyl pyrophosphate could be detected during the enzymatic formation of cyclic terpene hydrocarbons from geranyl pyrophosphate. Mn²⁺ catalyzes the nonenzymatic solvolysis of linalyl pyrophosphate, forming myrcene and ocymenes and no bicyclic hydrocarbons. Linalyl pyrophosphate is a sterically plausible precursor of cyclic hydrocarbons, but the present data support only its role as an alternative substrate and not as an obligatory free intermediate in terpene biosynthesis.     

Biochemical Characterization of Recombinant Human Nerve Growth Factor

Recombinant human nerve growth factor (rhNGF) was expressed and secreted by Chinese hamster ovary cells and purified to homogeneity using ion-exchange and reversed-phase (RP) chromatography. The isolated product was shown to be consistent with a 120-amino-acid residue polypeptide chain by amino acid composition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), RP-HPLC, and mass spectrometry and with an N-terminal sequence consistent with that expected from the cDNA for human nerve growth factor. By size-exclusion chromatography, rhNGF behaves like a noncovalent dimer. Limited enzymatic digests of the 120-residue monomer produced additional species of 118 (trypsin, removal of the C-terminal Arg119-Ala120 sequence) and 117 (trypsin plus carboxypeptidase B, removal of the C-terminal Arg118-Arg119-Ala120 sequence) residues. Each of these species was isolated by high-performance ion-exchange chromatography and characterized by amino acid and N-terminal sequence analyses, SDS-PAGE, RP-HPLC, and mass spectrometry. All three species were present in the digests as both homodimeric and heterodimeric combinations and found to be equipotent in both the chick dorsal root ganglion cell survival and rat pheochromocytoma neurite extension assays.     

Evaluation of disruptive camouflage of avian cup‐nests

Parent birds employ various strategies to protect their offspring against nest predators. Two well‐researched anti‐nest‐predation strategies involve visual concealment of the nest by way of parental camouflage and egg camouflage. By contrast, camouflage of nest structures is relatively under‐researched, particularly in the case of cup‐nests in trees and bushes. We explored how birds camouflage cup‐nests in nature. Specifically, we tested Hansell’s hypothesis that birds use externally applied pale and white objects such as spider cocoons and lichens to achieve cup‐nest camouflage. To test Hansell’s hypothesis, three complementary experiments were performed: (1) an in situ nest predation experiment; (2) a photo‐based visual search experiment; and (3) contrast analyses using PAT‐GEOM software in IMAGEJ. White paper and chalk spots were used to mimic white objects used by birds in nature. Whereas predation rates in Experiment 1 were not affected by white spots, location rates in Experiment 2 were lower for natural nests with white spots than without white spots. Experiment 3 demonstrated that white spots significantly increased the contrast between different visual elements of nests. It was concluded that white objects can potentially camouflage nests against some nest predators, and that any improved camouflage was probably achieved via disruptive camouflage.     

Bayesian Reconstruction of Disease Outbreaks by Combining Epidemiologic and Genomic Data

Recent years have seen progress in the development of statistically rigorous frameworks to infer outbreak transmission trees (“who infected whom”) from epidemiological and genetic data. Making use of pathogen genome sequences in such analyses remains a challenge, however, with a variety of heuristic approaches having been explored to date. We introduce a statistical method exploiting both pathogen sequences and collection dates to unravel the dynamics of densely sampled outbreaks. Our approach identifies likely transmission events and infers dates of infections, unobserved cases and separate introductions of the disease. It also proves useful for inferring numbers of secondary infections and identifying heterogeneous infectivity and super-spreaders. After testing our approach using simulations, we illustrate the method with the analysis of the beginning of the 2003 Singaporean outbreak of Severe Acute Respiratory Syndrome (SARS), providing new insights into the early stage of this epidemic. Our approach is the first tool for disease outbreak reconstruction from genetic data widely available as free software, the R package outbreaker. It is applicable to various densely sampled epidemics, and improves previous approaches by detecting unobserved and imported cases, as well as allowing multiple introductions of the pathogen. Because of its generality, we believe this method will become a tool of choice for the analysis of densely sampled disease outbreaks, and will form a rigorous framework for subsequent methodological developments.     

Eighteen years of heart transplantation--a single center experience

The best option for the treatment of a failing heart is heart transplantation. The transplantation program at the University Hospital Center Rebro Zagreb started in 1988. To the best of our knowledge this is the first retrospective study on cardiac transplantation in Croatia looking into survival following heart transplantation. Between 1988 and 2006, we performed 81 heart transplantations at the University Hospital Center Rebro Zagreb. Our study focused on the last ten years after establishment of the Department of cardiac surgery as a separate institution. There were thirteen different hospitals throughout Croatia, which contributed to the donor network. Average age of the heart recipient was 48+/-11.8 years (range 14-72), and average age of the heart donor was 34+/-10.7 years (range 14-56). There were more women among the heart donors (34%) then among the heart recipients (18%). During the first ten years, from 1988-1998, the average number of cardiac transplantations was 3 per year In the period from 1998-2006, average number of cardiac transplantations increased to 6 per year. The average thirty-day mortality for the last nine years was 27%. It declined from 30% and 40% in 1998 and 1999, respectively down to 0% in the last two years. Average age of the patients who died was 50+/-6.5years (range 44-62) and did not significantly differ from those who survived. The donor network has grown up to fourteen different hospitals throughout Croatia. The limiting factor in cardiac transplant surgery is the number of available donors. Therefore in attempt to form a good transplant program it is crucial to form an efficient donor network. The number of performed cardiac transplantations is expected to rise until it reaches the number of available donors. With advances in operative technique and postoperative management--immunosuppressive therapy we have observed a remarkable drop in the early operative mortality in the studied period.