Molecular weight differences among potato peroxidases

The isoenzyme of potato peroxidase A5 has MW 105 000; C3—94 000; C4 and C5—56 500 C6—48 500. The isoenzymes retain activity on SDS-gels thereby allowing direct measurement of monomeric MW, even in crude extracts. One of the isoperoxidases showed anomalous behaviour on SDS-electrophoresis.     

Sufficient Penetration of Peracetic Acid into Drilled Human Femoral Heads

Chemical sterilisation methods for musculoskeletal transplants have the problem of penetration into all tissue strata. The present study examined if a peracetic acid/ethanol solution penetrated to a sufficient extent into specifically prepared femoral heads. To this effect, 10 femoral heads have been provided with drillings (diameter 2 mm, depth 10 mm) at a distance of 15 mm (series B) and placed in a diffusion chamber with sterilisation solution. From an additional central drilling at the femoral neck junction, the sample drawing was made after 30 min each over a period of 4 h for the iodometric determination of peracetic acid (PAA) concentration. Ten femoral heads, which did contain only the central drilling, served as controls (series A). In 9 of the examined femoral heads of series A the defined minimum concentration of PAA of 0.2% (inactivation of bacteria, spores, fungi) has been clearly exceeded over the complete period of measurement. About 0.8% PAA (inactivation of viruses) was achieved within 4 h only with six femoral heads. Nine out of the 10 examined femoral heads in series B show a clearly improved penetration behaviour which was expressed in smaller standard deviations, a faster increase in concentration, as well as in higher starting and final concentrations (approx. 0.9%) of PAA. Previous drying in air leads to a faster penetration into the centre of the bone. Standardised drilling of decartilaged femoral heads creates favourable conditions for the penetration of the PAA sterilisation solution into the whole tissue and guarantees a sufficient inactivation of microorganisms.     

Assessment of sublethal effects of methoxyfenozide on oriental fruit Moth (Lepidoptera: Tortricidae)

Sublethal effects of the insect growth regulator methoxyfenozide were examined in oriental fruit moth, Grapholita molesta (Busck), in laboratory and field studies. In laboratory studies, oriental fruit moth larvae reared on diet amended with 0.1 ppm methoxyfenozide developed at the same rate as larvae reared on untreated diet, and paired moths reared as larvae from the same treated or untreated diets exhibited similar fecundity and fertility. Population growth differences over multiple generations were used to examine sublethal effects of methoxyfenozide on population dynamics in the field. Multiple single-tree cages were placed over apple (Malus spp.) trees treated with two applications of methoxyfenozide (70 g [AI] /ha) and nontreated trees. Cages were infested at a single time point with virgin male and female oriental fruit moth adults, and population growth was evaluated by egg counts, shoot infestation, fruit damage, and larval counts over a 12-wk period. Significantly fewer eggs, larvae, and damaged fruit were found on methoxyfenozide-treated compared with nontreated trees in 2001. Observed population differences may have been a result of direct mortality to eggs and larvae of the first generation rather than sublethal effects. In 2002, no differences were observed between treatments, but a heavy rain event shortly after the early infestation impacted the experiment. A late moth release treatment was tested in 2002 to examine the effects of residual methoxyfenozide 55 d after initial application. Significantly fewer eggs were found in the methoxyfenozide treatment compared with the control, but no differences existed among treatments in shoot infestation, percentage of damaged fruit, or larval populations. It was concluded direct mortality of eggs and larvae exposed to methoxyfenozide rather than sublethal effects were most important in reduction of subsequent generations.     

Further study on the role of HSP70 on Ca2+ homeostasis in rat ventricular myocytes subjected to simulated ischemia

We hypothesized that activation of heat shock protein 70 (HSP70) by preconditioning, which is known to confer delayed cardioprotection, attenuates the impaired handling of Ca²⁺ at multiple sites. To test the hypothesis, we determined how the ryanodine receptor (RyR), sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA), and Na⁺/Ca²⁺ exchanger (NCX) handled Ca²⁺ in rat ventricular myocytes preconditioned with a -opioid receptor agonist, U50488H (UP), followed by blockade of HSP70 with a selective antisense oligonucleotide and subsequently subjected to simulated ischemia. We determined the following: 1) the Ca²⁺ transients induced by electrical stimulation and caffeine, which provide the overall picture of Ca²⁺ homeostasis; 2) expression of RyR, SERCA, and NCX; and 3) Ca²⁺ fluxes via NCX by the use of ⁴⁵Ca²⁺ in the rat ventricular myocyte. We found that UP increased the activity of RyR, SERCA, and NCX and the expression of RyR and SERCA. These effects led to increases in the release of Ca²⁺ from the sarcoplasmic reticulum via RyR and in the removal of Ca²⁺ from the cytoplasm by reuptake of Ca²⁺ to the SR via SERCA and by extrusion of Ca²⁺ out of the cell via NCX. UP also reduced mitochondrial Ca²⁺ accumulation. All of the effects of UP were either abolished or significantly attenuated by blockade of HSP70 synthesis with a selective antisense oligonucleotide. The results are evidence that activation of HSP70 by preconditioning improves the ischemia-impaired Ca²⁺ homeostasis at multiple sites in the heart, which may be responsible, at least partly, for attenuated Ca²⁺ overload, improved recovery in contractile function, and cardioprotection. intracellular Ca²⁺, -opioid receptor; Na⁺/Ca²⁺ exchanger; ryanodine receptor; sarco(endo)plasmic reticulum Ca²⁺-ATPase     

Seed fate of Marah macrocarpus (Cucurbitaceae) following fire: do seedlings recruit from rodent burrows?

For numerous species in fire-prone ecosystems, the passage of fire triggers the release of large quantities of seeds within 2 years of the fire. This special case of masting has been described for species in an array of floras, but few studies have followed the fates of seeds liberated into the postfire environment. In this study, I followed the fates of 990, magnet-bearing Marah macrocarpus seeds sown at three seed densities in a large, high-intensity burn area. Seeds disappeared over 6.6 months and removal at all three densities became negatively density-dependent in late summer until all seeds disappeared in mid winter. I recovered only 5% of the magnets, mostly from rodent tunnels. Based on the recovery of magnets, I estimate rodents moved ~91% of the seeds belowground. Seed burial trials showed that seedlings established from seeds buried up to 16 cm whereas seeds sown on the surface did not germinate. Seedlings also readily established from burial in soils with 50% rock fragments as well as from artificial tunnels in both pot and field experiments. Excavation of 50 natural seedlings suggested up to 20% established in rodent burrows. Findings of this study suggest that ecologists may have underestimated the role of burrows as locations for recruitment of large-seeded species.     

Zebrafish transgenic Enhancer TRAP line database (ZETRAP)

Background  

The zebrafish, Danio rerio, is used as a model organism to study vertebrate genetics and development. An effective enhancer trap (ET) in zebrafish using the Tol2 transposon has been demonstrated. This approach could be used to study embryogenesis of a vertebrate species in real time and with high resolution.     

A novel biotinylated suicide inhibitor for directed molecular evolution of lipolytic enzymes

A bifunctional activity label (8) for directed molecular evolution of lipolytic enzymes has been designed and synthesized. The structure is composed of a 4-nitrophenyl activated phosphonate, that is, a suicide substrate of lipases/esterases, connected to a biotin moiety through a spacer containing a disulfide bridge. The phosphonate (3) was prepared by Michaelis-Arbuzov reaction of trimethylsilyl-protected 11-bromoundecanol (2) with triethyl phosphite. The deprotected omega-hydroxyalkylphosphonate (4) was transformed into an active N-hydroxysuccinimide carbonate (5) followed by 4-nitrophenyl activation of the phosphonate using standard procedures. The biotinylated phosphonate inhibitor (8) was then synthesised by coupling the phosphonate inhibitor (6) to the epsilon-amino-caproic acid and cystamine containing biotinyl spacer (7). The function of all relevant groups of the final activity label (8) (biotin-label, cleavable disulfide bridge, phosphonate-inhibitor) have been successfully tested with the commercial lipase Lipolase (Novo Nordisk). Hence, a tool for directed molecular evolution of lipolytic enzymes has been developed.     

Cannabidiol inhibits lung cancer cell invasion and metastasis via intercellular adhesion molecule-1

Cannabinoids inhibit cancer cell invasion via increasing tissue inhibitor of matrix metalloproteinases-1 (TIMP-1). This study investigates the role of intercellular adhesion molecule-1 (ICAM-1) within this action. In the lung cancer cell lines A549, H358, and H460, cannabidiol (CBD; 0.001-3 μM) elicited concentration-dependent ICAM-1 up-regulation compared to vehicle via cannabinoid receptors, transient receptor potential vanilloid 1, and p42/44 mitogen-activated protein kinase. Up-regulation of ICAM-1 mRNA by CBD in A549 was 4-fold at 3 μM, with significant effects already evident at 0.01 μM. ICAM-1 induction became significant after 2 h, whereas significant TIMP-1 mRNA increases were observed only after 48 h. Inhibition of ICAM-1 by antibody or siRNA approaches reversed the anti-invasive and TIMP-1-upregulating action of CBD and the likewise ICAM-1-inducing cannabinoids Δ(9)-tetrahydrocannabinol and R(+)-methanandamide when compared to isotype or nonsilencing siRNA controls. ICAM-1-dependent anti-invasive cannabinoid effects were confirmed in primary tumor cells from a lung cancer patient. In athymic nude mice, CBD elicited a 2.6- and 3.0-fold increase of ICAM-1 and TIMP-1 protein in A549 xenografts, as compared to vehicle-treated animals, and an antimetastatic effect that was fully reversed by a neutralizing antibody against ICAM-1 [% metastatic lung nodules vs. isotype control (100%): 47.7% for CBD + isotype antibody and 106.6% for CBD + ICAM-1 antibody]. Overall, our data indicate that cannabinoids induce ICAM-1, thereby conferring TIMP-1 induction and subsequent decreased cancer cell invasiveness.     

Toward Fast Determination of Protein Stability Maps: Experimental and Theoretical Analysis of Mutants of a Nocardiopsis prasina Serine Protease

The stability of serine proteases is of major importance for their application in industrial processes. Here we study the determinants of the stability of a Nocardiopsis prasina serine protease using fast residual activity assays, a feature classification algorithm, and structure-based energy calculation algorithms for 121 micropurified mutant enzyme clones containing multiple point mutations. Using a multivariate regression analysis, we deconvolute the data for the mutant clones and find that mutations of residues Asn47 and Pro124 are deleterious to the stability of the enzyme. Both of these residues are situated in loops that are known to be important for the stability of the highly homologous α-lytic protease. Structure-based energy calculations with PEATSA give a good general agreement with the trend of experimentally measured values but also identify a number of clones that the algorithm fails to predict correctly. We discuss the significance of the results in relation to the structure and function of closely related proteases, comment on the optimal experimental design when performing high-throughput experiments for characterizing the determinants of protein stability, and discuss the performance of structure-based energy calculations with complex data sets such as the one presented here.