Antitumour activities of levans produced by Zymomonas mobilis strains

Levans produced by four Zymomonas mobilis strains showed antitumour activity against sarcoma 180 and Ehrlich carcinoma in Swiss albino mice. Levans from two strains (ZAP and CP4) had the highest effects. NMR analysis showed that the polymers were composed only of fructose units. The results suggested that the antineoplasic effect is associated to the polysaccharide molecular weight and that a particular molecular weight range may be responsible for this effect.     

Mammalian-heart adenylate deaminase: Cross-species immunoanalysis of tissue distribution with a cardiac-directed antibody

A sheep antiserum against purified rabbit-heart adenylate deaminase (EC 3.5.4.6) (AMPD) was developed and validated as an immunologic probe to assess the cross-species tissue distribution of the mammalian cardiac AMPD isoform. The antiserum and the antibodies purified therefrom recognized both native and denatured rabbit-heart AMPD in immunoprecipitation and immunoblot experiments, respectively, and antibody binding did not affect native enzyme activity. The immunoprecipitation experiments further demonstrated a high antiserum titer. Immunoblot analysis of either crude rabbit-heart extracts or purified rabbit-heart AMPD revealed a major immunoreactive band with the molecular mass (81 kDa) of the soluble rabbit-heart AMPD subunit. AMPD in heart extracts from mammalian species other than rabbit (including human) was equally immunoreactive with this antiserum by quantitative immunoblot criteria. Although generally held to be in the same isoform class as heart AMPD, erythrocyte AMPD was not immunoreactive either within or across species. Nor was AMPD from most other tissues [e.g., white (gastrocnemius) muscle, lung, kidney] immunoreactive with the cardiac-directed antibody. Limited immunoreactivity was evidenced by mammalian liver, red (soleus) muscle, and brain extracts across species, indicating the presence of a minor cardiac(-like) AMPD isoform in these tissues. The results of this study characterize the tissue distribution of the cardiac AMPD isoform using a molecular approach with the first polyclonal antibodies prepared against homogeneous cardiac AMPD. This immunologic probe should prove useful at the tissue level for AMPD immunohistochemistry.     

Effects of long-term fixation on histological quality of undecalcified murine bones embedded in methylmethacrylate

While long-term fixation and storage of specimens is common and useful for many research projects, it is particularly important for space flight investigations where samples may not be returned to Earth for several months (International Space Station) or years (manned mission to Mars). We examined two critical challenges of space flight experimentation: the effect of long-term fixation on the quality of mouse bone preservation and the preservation of antigens and enzymes for both histochemical and immunohistochemical analyses, and how the animal/sample processing affects the preservation. We show that long-term fixation minimally affects standard histological staining, but that enzyme histochemistry and immunolabeling are greatly compromised. Further, we demonstrate that whole animal preservation is not as suitable as whole leg or stripped leg preservation for long-term fixation and all histological analyses. Overall, we recommend whole leg processing for long-term storage of bone specimens in fixative prior to embedding in plastic for histological examination.     

Factors influencing in the response of <i>Schizolobium parahybum</i> (Vell) Blake to <i>Ceratocystis paradoxa</i> and <i>C. moniliformis</i>

In the Ecuadorian coast one of the most destructive diseases of the pachaco is vascular wilt or stem rot caused by Ceratocystis complex, so the aim of this study was to determine the factors that affect the efficiency of the reaction of bark pachaco to this disease. This research was conducted under laboratory conditions, using trees pachaco S38, S41, S98, AE-1, AE-2 and AE-3, and pathogenic species Ceratocystis paradoxa and C. moniliformis. The method utilized was tissue stem bark,with bark sections with 4.5 cm², and a suspension of 3x10⁴ units infection and remained in a humid chamber for 96 hours at 25 ± 5 °C. Were determined grades of resistance/ susceptibility using a scale from 0 to 4, depending on the amount of mycelia and peritecio in each plant sample. Three factors were used: four colonies obtained by several transfers from each fungal specie, four ages of colonies of each fungal specie and four volumes of inoculum applied (units of infection), using for each experiment separately Completely Randomized Design with 4 replications factorial arrangement. For comparison between treatment means was used Tukey test at 5% probability of error. For future trials using this technique, you could use 30-day colonies for C. paradoxa and 40 days for C. moniliformis, and an application volume of 100 μL/cm², it would improve the level of response for the formation of perithecium and mycelia in samples cortex.     

The MAP kinase ERK2 inhibits the cyclic AMP-specific phosphodiesterase HSPDE4D3 by phosphorylating it at Ser579

The extracellular receptor stimulated kinase ERK2 (p42(MAPK))-phosphorylated human cAMP-specific phosphodiesterase PDE4D3 at Ser579 and profoundly reduced ( approximately 75%) its activity. These effects could be reversed by the action of protein phosphatase PP1. The inhibitory state of PDE4D3, engendered by ERK2 phosphorylation, was mimicked by the Ser579-->Asp mutant form of PDE4D3. In COS1 cells transfected to express PDE4D3, challenge with epidermal growth factor (EGF) caused the phosphorylation and inhibition of PDE4D3. This effect was blocked by the MEK inhibitor PD98059 and was not apparent using the Ser579-->Ala mutant form of PDE4D3. Challenge of HEK293 and F442A cells with EGF led to the PD98059-ablatable inhibition of endogenous PDE4D3 and PDE4D5 activities. EGF challenge of COS1 cells transfected to express PDE4D3 increased cAMP levels through a process ablated by PD98059. The activity of the Ser579-->Asp mutant form of PDE4D3 was increased by PKA phosphorylation. The transient form of the EGF-induced inhibition of PDE4D3 is thus suggested to be due to feedback regulation by PKA causing the ablation of the ERK2-induced inhibition of PDE4D3. We identify a novel means of cross-talk between the cAMP and ERK signalling pathways whereby cell stimuli that lead to ERK2 activation may modulate cAMP signalling.     

Dauer Larvae of Caenorhabditis briggsae in Axenic Culture

The free-living hermaphroditic nematode, Caenorhabditis briggsae, enters a dauer stage under certain conditions in axenic culture. Dauer larvae differ from directly-developing third-stage larvae in internal structure, size at time of second molt, morphology of second and third cuticles, separation zone of cuticular caps, and survival at 4 C and 37 C, temperatures fatal to other stages. Males, which occur rarely in liquid medium, may mature under conditions which cause most of the hermaphrodites to go into the dauer stage, resulting in a culture with increased male-to-hermaphrodite ratio.     

Isocitrate dehydrogenase 1 R132C mutation occurs exclusively in microsatellite stable colorectal cancers with the CpG island methylator phenotype

The CpG Island Methylator Phenotype (CIMP) is fundamental to an important subset of colorectal cancer; however, its cause is unknown. CIMP is associated with microsatellite instability but is also found in BRAF mutant microsatellite stable cancers that are associated with poor prognosis. The isocitrate dehydrogenase 1 (IDH1) gene causes CIMP in glioma due to an activating mutation that produces the 2-hydroxyglutarate oncometabolite. We therefore examined IDH1 alteration as a potential cause of CIMP in colorectal cancer. The IDH1 mutational hotspot was screened in 86 CIMP-positive and 80 CIMP-negative cancers. The entire coding sequence was examined in 81 CIMP-positive colorectal cancers. Forty-seven cancers varying by CIMP-status and IDH1 mutation status were examined using Illumina 450K DNA methylation microarrays. The R132C IDH1 mutation was detected in 4/166 cancers. All IDH1 mutations were in CIMP cancers that were BRAF mutant and microsatellite stable (4/45, 8.9%). Unsupervised hierarchical cluster analysis identified an IDH1 mutation-like methylation signature in approximately half of the CIMP-positive cancers. IDH1 mutation appears to cause CIMP in a small proportion of BRAF mutant, microsatellite stable colorectal cancers. This study provides a precedent that a single gene mutation may cause CIMP in colorectal cancer, and that this will be associated with a specific epigenetic signature and clinicopathological features.     

In addition to the SH3 binding region, multiple regions within the N-terminal noncatalytic portion of the cAMP-specific phosphodiesterase, PDE4A5, contribute to its intracellular targeting

The long cyclic AMP (cAMP)-specific phosphodiesterase isoform, PDE4A5 (PDE4A subfamily isoform variant 5), when transiently expressed in COS-7 cells, was shown in subcellular fractionation studies to be associated with both membrane and cytosol fractions, with immunofluorescence analyses identifying PDE4A5 as associated both with ruffles at the cell margin and also at a distinct perinuclear localisation. Deletion of the first nine amino acids of PDE4A5 (1) ablated its ability to interact with the SH3 domain of the tyrosyl kinase, LYN; (2) reduced, but did not ablate, membrane association; and (3) disrupted the focus of PDE4A5 localisation within ruffles at the cell margin. This deleted region contained a Class I SH3 binding motif of similar sequence to those identified by screening a phage display library with the LYN-SH3 domain. Truncation to remove the PDE4A5 isoform-specific N-terminal region caused a further reduction in membrane association and ablated localisation at the cell margin. Progressive truncation to delete the PDE4A long isoform common region and then the long isoform-specific UCR1 did not cause any further change in membrane association or intracellular distribution. However, deletion up to the super-short form splice junction generated an entirely soluble 'core' PDE4A species. We propose that multiple sites in the N-terminal noncatalytic portion of PDE4A5 have the potential to associate with intracellular structures and thus define its intracellular localisation. At least two such sites lie within the PDE4A5 isoform-specific N-terminal region and these appear to be primarily responsible for targeting PDE4A5 to, and organising it within, the cell margin; one is an SH3 binding motif able to interact with LYN kinase and the other lies within the C-terminal portion of the PDE4A5 unique region. A third membrane association region is located within the N-terminal portion of UCR2 and appears to be primarily responsible for targeting to the perinuclear region. Progressive N-terminal truncation, to delete defined regions of PDE4A5, identified activity changes occurring upon deletion of the SH3 binding site region and then upon deletion of the membrane association site region located within UCR2. This suggests that certain of these anchor sites may not only determine intracellular targeting but may also transduce regulatory effects on PDE4A5 activity.     

QMEANclust: estimation of protein model quality by combining a composite scoring function with structural density information

Background  

The selection of the most accurate protein model from a set of alternatives is a crucial step in protein structure prediction both in template-based and ab initio approaches. Scoring functions have been developed which can either return a quality estimate for a single model or derive a score from the information contained in the ensemble of models for a given sequence. Local structural features occurring more frequently in the ensemble have a greater probability of being correct. Within the context of the CASP experiment, these so called consensus methods have been shown to perform considerably better in selecting good candidate models, but tend to fail if the best models are far from the dominant structural cluster. In this paper we show that model selection can be improved if both approaches are combined by pre-filtering the models used during the calculation of the structural consensus.     

The pharmacophore of the human C5a anaphylatoxin.

We have determined which amino acids contribute to the pharmacophore of human C5a, a potent inflammatory mediator. A systematic mutational analysis of this 74-amino acid protein was performed and the effects on the potency of receptor binding and of C5a-induced intracellular calcium ion mobilization were measured. This analysis included the construction of hybrids between C5a and the homologous but unreactive C3a protein and site-directed mutagenesis. Ten noncontiguous amino acids from the structurally well-defined 4-helix core domain (amino acids 1-63) and the C-terminal arginine-containing tripeptide were found to contribute to the pharmacophore of human C5a. The 10 mostly charged amino acids from the core domain generally made small incremental contributions toward binding affinity, some of which were independent. Substitutions of the C-terminal amino acid Arg 74 produced the largest single effect. We also found the connection between these 2 important regions to be unconstrained.