Chemical genetics suggests a critical role for lysyl oxidase in zebrafish notochord morphogenesis

As a result of a chemical genetic screen for modulators of metalloprotease activity, we report that 2-mercaptopyridine-N-oxide induces a conspicuous undulating notochord defect in zebrafish embryos, a phenocopy of the leviathan mutant. The location of the chemically-induced wavy notochord correlated with the timing of application, thus defining a narrow chemical sensitivity window during segmentation stages. Microscopic observations revealed that notochord undulations appeared during the phase of notochord cell vacuolation and notochord elongation. Notochord cells become swollen as well as disorganized, while electron microscopy revealed disrupted organization of collagen fibrils in the surrounding sheath. We demonstrate by assay in zebrafish extracts that 2-mercaptopyridine-N-oxide inhibits lysyl oxidase. Thus, we provide insight into notochord morphogenesis and reveal novel compounds for lysyl oxidase inhibition. Taken together, these data underline the utility of small molecules for elucidating the dynamic mechanisms of early morphogenesis and provide a potential explanation for the recently established role of copper in zebrafish notochord formation.     

Calmodulin kinase II inhibition enhances ischemic preconditioning by augmenting ATP-sensitive K+ current

Mice with genetic inhibition (AC3-I) of the multifunctional Ca(2+)/calmodulin dependent protein kinase II (CaMKII) have improved cardiomyocyte survival after ischemia. Some K(+) currents are up-regulated in AC3-I hearts, but it is unknown if CaMKII inhibition increases the ATP sensitive K(+) current (I(KATP)) that underlies ischemic preconditioning (IP) and confers resistance to ischemia. We hypothesized increased I(KATP) was part of the mechanism for improved ventricular myocyte survival during ischemia in AC3-I mice. AC3-I hearts were protected against global ischemia due to enhanced IP compared to wild type (WT) and transgenic control (AC3-C) hearts. IKATP was significantly increased, while the negative regulatory dose-dependence of ATP was unchanged in AC3-I compared to WT and AC3-C ventricular myocytes, suggesting that CaMKII inhibition increased the number of functional I(KATP) channels available for IP. We measured increased sarcolemmal Kir6.2, a pore-forming I(KATP) subunit, but not a change in total Kir6.2 in cell lysates or single channel I(KATP) opening probability from AC3-I compared to WT and AC3-C ventricles, showing CaMKII inhibition increased sarcolemmal I(KATP) channel expression. There were no differences in mRNA for genes encoding I(KATP) channel subunits in AC3-I, WT and AC3-C ventricles. The I(KATP) opener pinacidil (100 microM) reduced MI area in WT to match AC3-I hearts, while the I(KATP) antagonist HMR1098 (30 microM) increased MI area to an equivalent level in all groups, indicating that increased I(KATP) and augmented IP are important for reduced ischemic cell death in AC3-I hearts. Our study results show CaMKII inhibition enhances beneficial effects of IP by increasing I(KATP).     

A Review of the Salivary Proteome and Peptidome and Saliva-derived Peptide Therapeutics

Saliva is a glandular secretion that is vital in the maintenance of healthy oral tissues. In this review we outline the high abundance salivary proteins, summarise the status of the salivary proteome and peptidome, the genetic origin and recognised functions of these proteins, the diseases associated with salivary disorders, and the emerging saliva-derived peptide therapeutics. Different proteomic approaches have reported the identification of over 1,300 proteins in saliva. However there are fewer than 100 high abundance proteins, identified by multiple methods including, two-dimensional polyacrylamide gel electrophoresis and HPLC combined with mass spectrometry. Analysis of the genes coding for the salivary proteins demonstrated a non-uniform chromosomal distribution with chromosome 4 having the largest proportion of genes expressed in salivary glands. Several diseases are associated with salivary disorders including Sjögren’s syndrome, Prader-Willi syndrome, dental caries and stress related disorders. Saliva as a diagnostic medium for various biochemical tests has provided a non-invasive and accessibility advantage over other more regularly tested body fluids such as blood and urine. To-date the emerging saliva-based therapeutics include artificial salivas and antimicrobial agents based on histatins and mucins.     

Discovering robust protein biomarkers for disease from relative expression reversals in 2-D DIGE data

This study assesses the ability of a novel family of machine learning algorithms to identify changes in relative protein expression levels, measured using 2-D DIGE data, which support accurate class prediction. The analysis was done using a training set of 36 total cellular lysates comprised of six normal and three cancer biological replicates (the remaining are technical replicates) and a validation set of four normal and two cancer samples. Protein samples were separated by 2-D DIGE and expression was quantified using DeCyder-2D Differential Analysis Software. The relative expression reversal (RER) classifier correctly classified 9/9 training biological samples (p<0.022) as estimated using a modified version of leave one out cross validation and 6/6 validation samples. The classification rule involved comparison of expression levels for a single pair of protein spots, tropomyosin isoforms and alpha-enolase, both of which have prior association as potential biomarkers in cancer. The data was also analyzed using algorithms similar to those found in the extended data analysis package of DeCyder software. We propose that by accounting for sources of within- and between-gel variation, RER classifiers applied to 2-D DIGE data provide a useful approach for identifying biomarkers that discriminate among protein samples of interest.     

Characterization of a novel gene for strain typing reveals substructuring of Aspergillus fumigatus across North America

Fifty-five epidemiologically linked Aspergillus fumigatus isolates obtained from six nosocomial outbreaks of invasive aspergillosis were subtyped by sequencing the polymorphic region of the gene encoding a putative cell surface protein, Afu3g08990 (denoted as CSP). Comparative sequence analysis showed that genetic diversity was generated in the coding region of this gene by both tandem repeats and point mutations. Each unique sequence in an outbreak cluster was assigned an arbitrary number or CSP sequence type. The CSP typing method was able to identify "clonal" and genotypically distinct A. fumigatus isolates, and the results of this method were concordant with those of another discriminatory genotyping technique, the Afut1 restriction fragment length polymorphism typing method. The novel single-locus sequence typing (CSP typing) strategy appears to be a simple, rapid, discriminatory tool that can be readily shared across laboratories. In addition, we found that A. fumigatus isolates substructured into multiple clades; interestingly, one clade consisted of isolates predominantly representing invasive clinical isolates recovered from cardiac transplant patients from two different outbreak situations. We also found that the A. fumigatus isolate Af293, whose genome has been sequenced, possesses a CSP gene structure that is substantially different from those of the other A. fumigatus strains studied here, highlighting the need for further taxonomic study.     

Perinatal physiology in cloned and normal calves: hematologic and biochemical profiles

Although a majority of clones are born normal and apparently healthy, mortality rates of nearly 30% are described in many reports. Such losses are a major limitation of cloning technology and represent substantial economic investment as well as justifiable animal health and welfare concerns. Prospective, controlled studies are needed to understand fully the causes of neonatal mortality in clones and to develop preventive and therapeutic strategies to minimize losses. We report here the findings of studies on the hematologic and biochemical profiles of cloned and control calves in the immediate 48-h postpartum period. Cloned calves were similar to control calves for a majority of parameters studied including blood gases, concentrations of plasma proteins, minerals and electrolytes, and white blood cell, neutrophil, lymphocyte, and platelet counts. The most notable differences between clones and controls in this study were reduced red- and white-blood cell counts in clones at birth and 1 h of age. As a group, plasma electrolyte concentrations were more variable in clones, and the variability tended to be shifted either higher (sodium, chloride) or lower (potassium, bicarbonate) than in controls. Previously, we noted differences in carbohydrate parameters, the length of time required for clones to make the neonatal adaptation to life ex utero, and morphology of the cloned placenta. Taken together, our findings suggest that cloned calves experience greater difficulty adjusting to life ex utero and that further research is warranted to determine the nature of the relationship between the physiological differences noted here in clones at birth and concomitant abnormal placental morphology.     

Experimental Test of Connector Rotation during DNA Packaging into Bacteriophage ϕ29 Capsids

The bacteriophage ϕ29 generates large forces to compact its double-stranded DNA genome into a protein capsid by means of a portal motor complex. Several mechanical models for the generation of these high forces by the motor complex predict coupling of DNA translocation to rotation of the head-tail connector dodecamer. Putative connector rotation is investigated here by combining the methods of single-molecule force spectroscopy with polarization-sensitive single-molecule fluorescence. In our experiment, we observe motor function in several packaging complexes in parallel using video microscopy of bead position in a magnetic trap. At the same time, we follow the orientation of single fluorophores attached to the portal motor connector. From our data, we can exclude connector rotation with greater than 99% probability and therefore answer a long-standing mechanistic question.     

A model system for analyzing somatic mutations in Drosophila melanogaster

Presently there are no good assays for comparing somatic mutation frequencies and spectra between different vertebrate and invertebrate organisms. Here we describe a new lacZ mutation reporter system in D. melanogaster, which complements existing systems in the mouse. The results obtained with the new model indicate two-to threefold higher frequencies of spontaneous mutations than in the mouse, with most of the mutations characterized as large genome rearrangements.     

Fly photoreceptors demonstrate energy-information trade-offs in neural coding

Trade-offs between energy consumption and neuronal performance must shape the design and evolution of nervous systems, but we lack empirical data showing how neuronal energy costs vary according to performance. Using intracellular recordings from the intact retinas of four flies, Drosophila melanogaster, D. virilis, Calliphora vicina, and Sarcophaga carnaria, we measured the rates at which homologous R1–6 photoreceptors of these species transmit information from the same stimuli and estimated the energy they consumed. In all species, both information rate and energy consumption increase with light intensity. Energy consumption rises from a baseline, the energy required to maintain the dark resting potential. This substantial fixed cost, ∼20% of a photoreceptor's maximum consumption, causes the unit cost of information (ATP molecules hydrolysed per bit) to fall as information rate increases. The highest information rates, achieved at bright daylight levels, differed according to species, from ∼200 bits s⁻¹ in D. melanogaster to ∼1,000 bits s⁻¹ in S. carnaria. Comparing species, the fixed cost, the total cost of signalling, and the unit cost (cost per bit) all increase with a photoreceptor's highest information rate to make information more expensive in higher performance cells. This law of diminishing returns promotes the evolution of economical structures by severely penalising overcapacity. Similar relationships could influence the function and design of many neurons because they are subject to similar biophysical constraints on information throughput.     

Protein misfolding and aggregation

Interest in the problem of protein misfolding and aggregation has exploded in recent years for two reasons: (1) the sharp rise in the number and volume of therapeutic proteins produced commercially and (2) the recognition of the central role of protein aggregates in degenerative diseases. The systematic study of protein aggregation presents major challenges to both the experimentalist and the theoretician. Much of the work retains an empirical flavor due to the experimental complexities; the sensitivity of protein aggregation to the slightest change in protein amino acid composition, solvent properties, or protein concentration; and the lack of robust theoretical models of misfolding and aggregation. Novel experimental and computational approaches are being developed, and we anticipate substantial progress will be made in the near future. Several presentations describing the latest advances in protein misfolding and aggregation were given at the American Chemical Society meeting (BIOT division) held in September, 2006 in San Francisco.