Antimicrobial peptides from the skin of the Tsushima brown frog Rana tsushimensis

The Tsushima brown frog Rana tsushimensis Stejneger, 1907 exists in reproductive isolation on the island of Tsushima, Japan. Six peptides with antimicrobial activity were isolated in pure form from an extract of the skin of this species and their amino acid sequences identified them as members of the brevinin-1 (one peptide), brevinin-2 (one peptide) and temporin (four peptides) families. The C-terminally alpha-amidated brevinin-1 peptide (FLGSIVGALASALPSLISKIRN.NH2) lacks the cyclic heptapeptide domain Cys18-(Xaa)4-Lys-Cys24 at the COOH-terminus of the molecule that characterizes other members of that family. A structurally similar brevinin-1 peptide, also lacking the cyclic domain, was previously isolated from the skin of the Ryukyu brown frog Rana okinavana, indicative of a close phylogenetic relationship between the species. Brevinin-2TSa (GIMSLFKGVLKTAGKHVAGSLVDQLKCKITGGC) showed broad-spectrum growth inhibitory activity against a range of Gram-negative and Gram-positive bacteria (including methicillin-resistant Staphylococcus aureus) (minimum inhibitory concentrations< or =25 microM) and relatively low hemolytic activity against human erythrocytes (LD50=100 microM). The peptide therefore represents a candidate for drug development.     

Screening for Tannin Degradation by Rumen and Faecal Samples of Wild and Domestic Animals in Ethiopia

Summary Tannins limit the use of fodder trees as feed for ruminants. Removal of the effects of tannins would thus improve the nutritional quality of these trees. This prompted the study to evaluate the effect of rumen or faecal mixed cultures from different animals on tannin degradation. Tannin extracts, tannic acid and gallic acid were used to enrich media to assess if rumen or faecal mixed cultures could degrade the phenolic compounds. Rumen fluid of Acacia-adapted sheep, sheep fed on wheat bran, bush duikers (Sylvicapra grimmia) and goats fed on Leucaena pallida and Sesbania goetzei were separately inoculated into Growth Study Medium (GSM) and incubated for 5-15 days. Faecal samples from dikdik (Madoqua guentheri), camel (Camelus dromedarius), zebra (Equus quagga), Grant’s gazelle (Gazella granti) and hartebeest (Alcelaphus buselaphus) were also separately inoculated into GSM media and incubated from 3-5 days. TLC results showed that mixed cultures from rumen fluids of Acacia-adapted sheep, sheep on wheat bran, goats on Leucaena pallida and Sesbania goetzei partially hydrolysed tannic acid to pyrogallol. Complete degradation of the heterocyclic ring in tannic acid and gallic acid was achieved by the mixed cultures from the faecal samples of dikdik and this was confirmed by HPLC. Mixed cultures from faecal samples of camel hydrolysed gallic acid to phloroglucinol. This study has demonstrated that faecal microorganisms of Ethiopian dikdik could completely degrade hydrolysable tannin.     

The inhibitory activity of natural products on plant p-hydroxyphenylpyruvate dioxygenase

The inhibitory activity of 34 natural products of various structural classes on hydroxyphenylpyruvate dioxygenase (HPPD), the target site for triketone herbicides, and the mode of interaction of selected natural products were investigated. Recombinant HPPD from arabidopsis is sensitive to several classes of natural compounds including, in decreasing order of sensitivity, triketones, benzoquinones, naphthoquinones and anthraquinones. The triketone natural products acted as competitive tight-binding inhibitors, whereas the benzoquinones and naphthoquinones did not appear to bind tightly to HPPD. While these natural products may not have optimal structural features required for in vivo herbicidal activity, the differences in their kinetic behavior suggest that novel classes of HPPD inhibitors may be developed based on their structural backbones.     

Human DNA polymerase mu (Pol mu) exhibits an unusual replication slippage ability at AAF lesion

We analyzed the ability of various cell extracts to extend a radiolabeled primer past an N-2-acetylaminofluorene (AAF) adduct located on a primed single-stranded template. When the 3′ end of the primer is located opposite the lesion, partially fractionated human primary fibroblast extracts efficiently catalyzed primer-terminus extension by adding a ladder of about 15 dGMPs, in an apparently non-templated reaction. This activity was not detected in SV40-transformed fibroblasts or in HeLa cell extracts unless purified human DNA polymerase mu (Pol µ) was added. In contrast, purified human Pol µ alone could only add three dGMPs as predicted from the sequence of the template. These results suggest that a cofactor(s) present in cellular extracts modifies Pol µ activity. The production of the dGMP ladder at the primer terminus located opposite the AAF adduct reveals an unusual ability of Pol µ (in conjunction with its cofactor) to perform DNA synthesis from a slipped intermediate containing several unpaired bases.     

Oxidation and nitrosylation of oxyhemoglobin by S-nitrosoglutathione via nitroxyl anion

The reaction between low molecular weight S-nitrosothiols and hemoglobin is often used to synthesize S-nitrosohemoglobin, a form of hemoglobin suggested to be involved in the regulation of vascular oxygen delivery. However, this reaction has not been studied in detail, and several groups have reported a variable co-formation of oxidized methemoglobin (metHb) during synthesis. This study examines the mechanism of metHb formation and shows that nitrosylhemoglobin (HbNO) can also be formed. Generation of metHb and HbNO is largely dependent on the presence of protein thiol groups. We present evidence for a mechanism for the formation of metHb and HbNO involving the intermediacy of nitroxyl anion. Specifically, the reaction of nitroxyl with S-nitrosothiols to liberate nitric oxide and reduced thiol is proposed to be central to the reaction mechanism.     

Novel derivatives of 9,10-anthraquinone are selective algicides against the musty-odor cyanobacterium Oscillatoria perornata

Musty "off-flavor" in pond-cultured channel catfish (Ictalurus punctatus) costs the catfish production industry in the United States at least 30 million US dollars annually. The cyanobacterium Oscillatoria perornata (Skuja) is credited with being the major cause of musty off-flavor in farm-raised catfish in Mississippi. The herbicides diuron and copper sulfate, currently used by catfish producers as algicides to help mitigate musty off-flavor problems, have several drawbacks, including broad-spectrum toxicity towards the entire phytoplankton community that can lead to water quality deterioration and subsequent fish death. By use of microtiter plate bioassays, a novel group of compounds derived from the natural compound 9,10-anthraquinone have been found to be much more selectively toxic towards O. perornata than diuron and copper sulfate. In efficacy studies using limnocorrals placed in catfish production ponds, application rates of 0.3 micro M (125 micro g/liter) of the most promising anthraquinone derivative, 2-[methylamino-N-(1'-methylethyl)]-9,10-anthraquinone monophosphate (anthraquinone-59), dramatically reduced the abundance of O. perornata and levels of 2-methylisoborneol, the musty compound produced by O. perornata. The abundance of green algae and diatoms increased dramatically 2 days after application of a 0.3 micro M concentration of anthraquinone-59 to pond water within the limnocorrals. The half-life of anthraquinone-59 in pond water was determined to be 19 h, making it much less persistent than diuron. Anthraquinone-59 appears to be promising for use as a selective algicide in catfish aquaculture.     

Activity-dependent Protein Dynamics Define Interconnected Cores of Co-regulated Postsynaptic Proteins

Synapses are highly dynamic structures that mediate cell–cell communication in the central nervous system. Their molecular composition is altered in an activity-dependent fashion, which modulates the efficacy of subsequent synaptic transmission events. Whereas activity-dependent trafficking of individual key synaptic proteins into and out of the synapse has been characterized previously, global activity-dependent changes in the synaptic proteome have not been studied.To test the feasibility of carrying out an unbiased large-scale approach, we investigated alterations in the molecular composition of synaptic spines following mass stimulation of the central nervous system induced by pilocarpine. We observed widespread changes in relative synaptic abundances encompassing essentially all proteins, supporting the view that the molecular composition of the postsynaptic density is tightly regulated. In most cases, we observed that members of gene families displayed coordinate regulation even when they were not known to physically interact.Analysis of correlated synaptic localization revealed a tightly co-regulated cluster of proteins, consisting of mainly glutamate receptors and their adaptors. This cluster constitutes a functional core of the postsynaptic machinery, and changes in its size affect synaptic strength and synaptic size. Our data show that the unbiased investigation of activity-dependent signaling of the postsynaptic density proteome can offer valuable new information on synaptic plasticity.Excitatory synaptic transmission is the primary mode of cell–cell communication in the central nervous system. The efficacy of synaptic transmission is highly regulated, and alterations in the strength of synaptic signaling within networks of neurons provide a mechanism for learning and memory storage, as well as for overall network stability. Modulation of synapse efficacy can occur through alterations in the structure and composition of the postsynaptic spine. The synaptic abundance of several molecules has been shown to be regulated in response to activity (1).The levels of individual proteins at postsynaptic spines are regulated through multiple processes. Active transport mechanisms exist and have been well characterized for AMPA-type glutamate receptors (AMPA-Rs)¹ via either insertion into the synapse or tighter association with the postsynaptic density (PSD) following lateral diffusion within the cell membrane (2). In addition to AMPA-Rs, other proteins known to be subject to activity-dependent regulation include calcium calmodulin-dependent protein kinase II alpha and beta, NMDA-type glutamate receptors (NMDA-Rs), and proteosome subunits (3–5). Synaptic protein content is dysregulated in a number of neuropsychiatric and neurodegenerative diseases, including Alzheimer''s disease and fragile X mental retardation (6–8).Most studies reported thus far have focused on a small number of selected molecules in individual experiments using a subset of synapses. Whereas learning and memory rely on the differential response of individual synapses to their specific input patterns, overall network excitability has to be maintained by homeostatic means. This homeostasis is governed by multiple pathways, and very little is known about the principles that regulate synaptic protein content across large numbers of synapses and neurons. The contributions of individual pathways and the interactions among them are largely unknown.In order to explore synaptic dynamics with a global view, we took advantage of a chemically induced mass stimulation protocol to stimulate synapses broadly throughout the central nervous system. We employed mass spectrometry and isotopically encoded isobaric peptide tagging with the iTRAQ reagent to quantify changes in the abundance of 893 proteins (9). We then analyzed changes in the relative abundance of these proteins at 0, 10, 20, and 60 min after the onset of stimulation.We observed evidence of the coordinated activation of synaptic protein groups, thereby identifying functional core complexes within the PSD. We demonstrate that adopting a quantitative systems biology approach provides insight allowing for a new level of analysis of synaptic function.     

Developmental regulation of dUTPase in Drosophila melanogaster

dUTPase prevents uracil incorporation into DNA by strict regulation of the cellular dUTP:dTTP ratio. Lack of the enzyme initiates thymineless cell death, prompting studies on enzyme regulation. We investigated expression pattern and localization of Drosophila dUTPase. Similarly to human, two isoforms of the fly enzyme were identified at both mRNA and protein levels. During larval stages, a drastic decrease of dUTPase expression was demonstrated at the protein level. In contrast, dUTPase mRNAs display constitutive character throughout development. A putative nuclear localization signal was identified in one of the two isoforms. However, immunohistochemistry of ovaries and embryos did not show a clear correlation between the presence of this signal and subcellular localization of the protein, suggesting that the latter may be perturbed by additional factors. Results are in agreement with a multilevel regulation of dUTPase in the Drosophila proteome, possibly involving several interacting protein partners of the enzyme. Using independent approaches, the existence of such macromolecular partners was verified.     

Socioeconomic and Sociodemographic Factors Associated with Asthma Related Outcomes in Early Childhood: The Generation R Study

Rationale

Few studies have analyzed the association of socioeconomic and sociodemographic factors with asthma related outcomes in early childhood, including Fraction of exhaled Nitric Oxide (FeNO) and airway resistance (Rint). We examined the association of socioeconomic and sociodemographic factors with wheezing, asthma, FeNO and Rint at age 6 years. Additionally, the role of potential mediating factors was studied.

Methods

The study included 6717 children participating in The Generation R Study, a prospective population-based cohort study. Data on socioeconomic and sociodemographic factors, wheezing and asthma were obtained by questionnaires. FeNO and Rint were measured at the research center. Statistical analyses were performed using logistic and linear regression models.

Results

At age 6 years, 9% (456/5084) of the children had wheezing symptoms and 7% (328/4953) had asthma. Children from parents with financial difficulties had an increased risk of wheezing (adjusted Odds Ratio (aOR) = 1.63, 95% Confidence Interval (CI):1.18–2.24). Parental low education, paternal unemployment and child''s male sex were associated with asthma, independent of other socioeconomic or sociodemographic factors (aOR = 1.63, 95% CI:1.24–2.15, aOR = 1.85, 95% CI:1.11–3.09, aOR = 1.58, 95% CI:1.24–2.01, respectively). No socioeconomic or gender differences in FeNO were found. The risks of wheezing, asthma, FeNO and Rint measurements differed between ethnic groups (p<0.05). Associations between paternal unemployment, child''s sex, ethnicity and asthma related outcomes remained largely unexplained.

Conclusions

This study showed differences between the socioeconomic and sociodemographic correlates of wheezing and asthma compared to the correlates of FeNO and Rint at age 6 years. Several socioeconomic and sociodemographic factors were independently associated with wheezing and asthma. Child''s ethnicity was the only factor independently associated with FeNO. We encourage further studies on underlying pathways and public health intervention programs, focusing on reducing socioeconomic or sociodemographic inequalities in asthma.     

Phosphatidylcholine could be the source of 1,2-DAG which activates protein kinase C in EGF-stimulated colon carcinoma cells (HT29)

In our previous study (A. Balogh et al, Cell. Signalling 5 (6), 795-802, 1993.), we have shown that epidermal growth factor (EGF) increased protein kinase C (PKC) activities in colon carcinoma cell line (HT29), possibly through the increased 1,2-diacylglycerol (1,2-DAG) production via phosphatidylcholine (PC). Here we investigate the effect of the well-known PKC activator 12-O-tetradecanoyl-2 phorbol-13-acetate (TPA), on the levels of ³²P incorporation into EGF induced phosphatidylinositols (PI, PI4P, PI4, 5P₂) and different phospholipids (PC, PA, PS) as well as on induced tyrosine kinase activity. TPA significantly decreased the effects of EGF and it had the biggest inhibitory effect on EGF induced PC level. These data support our contention that PC plays an important role in the activation of PKC via 1,2-DAG production in the EGF stimulated pathway.