Disruption of Trichoderma reesei gene encoding protein O-mannosyltransferase I results in a decrease of the enzyme activity and alteration of cell wall composition

In fungi transfer of the first mannosyl residue to proteins during their O-glycosylation is catalyzed by protein O-mannosyltransferases encoded by pmt genes. Disruption of the pmt1 gene in Trichoderma caused a significant decrease in the total activity of protein O-mannosyltransferases. Moreover, disruption of the pmt1 gene also led to osmotic sensitivity of the strain, indicating an essential role of the PMTI protein activity for cell wall synthesis. At the same time, the strain was defective in septa formation, producing only half the number of septa per unit length of hypha compared with the wild type. Disruption of the pmt1 gene decreased protein secretion but had no effect on glycosylation of secreted proteins, which suggests that PMTI protein O-mannosyltranferase does not take part in glycosylation of these proteins.     

Control of phenylalanine ammonia-lyase gene promoters from pea by UV radiation

The gene fusion system was used to study UV light-control of PS PAL1 and PS PAL2 genes encoding phenylalanine ammonia-lyase of pea. The induction of pea PAL promoters was analysed in transgenic tobacco plants. Binary plasmids (derivatives of pBI101.2 vector) containing 5′ regulatory fragments of PS PAL1 and PS PAL2 linked to reporter genes (GUS,LUC) were constructed. The analyses were performed with the use of single constructs (containing one variant of PS PAL promoter and one reporter gene) and dual constructs (containing both PS PAL1 and PS PAL2 promoters connected with different reporter genes). The use of dual constructs enabled the evaluation of both PS PAL promoters activity in the same plant. The analyses of in vitro grown plants have shown that both PAL promoters are strongly induced in leaves subjected to UV radiation. In some cases, the UV-stimulated expression exceeded the exposed areas. This phenomenon was observed more often in the leaves of plants containing the PS PAL1::GUS than PS PAL2::GUS construct. Removal of boxes 2, 4, 5 from PS PAL1 promoter and deletion of its 5′ end region (-339 to -1394) decreases the level of gene expression but does not eliminate its responsiveness to UV.     

Rats fed a high sucrose diet have altered heart antioxidant enzyme activity and gene expression

Several studies in human and animal models have shown that consumption of fructose facilitates oxidative damage but the mechanisms involved are unclear. In this study, the effects of two weeks of high sucrose on both oxidative stress parameters and stress-related gene expression, using a cDNA array, were investigated in rat heart. Both increased TBARS and lower Cu-Zn-SOD activity were found in heart from high sucrose fed rats compared to rats on a starch diet. Higher plasma NO level was also found in the high sucrose group, corroborating the pro-oxidant effect of fructose. The Cu-Zn-SOD mRNA level was also greater in the high sucrose group; the Mn-SOD, GPX and catalase were not different between the two groups. Increased HSP70 and decreased COMT genes expression were observed, underlying the hypertensive effect of dietary fructose. These findings confirm the pro-oxidant effect of high sucrose feeding to rats and highlight the NO/O(2)(*-) balance importance in oxidative homeostasis.     

Comparison of the Ames test and a newly developed assay for detection of mutagenic pollution of marine environments

Mismatch repair (MMR) genes, such as Msh2, are classified as "mutator" genes, responsible for the microsatellite instability identified in many tumors. In the current study, the mutation frequency and mutational spectrum in thymic lymphoma arising in Msh2 deficient mice are investigated. Thymic lymphoma developed in Msh2-/- background displayed an eight to nine-fold increase in mutation frequency compared to the normal thymi in Msh2 deficient animals. Sequencing demonstrated significantly different mutational spectra between normal thymus tissue and thymic lymphomas in Msh2-/- mice (P=0.02). The tumor mutational spectrum is characterized by an increase in base substitutions occurring at A:T sites, and multiple mutations, as well as a minor increase in -1 frameshifts. We analyzed mutations in different parts of the tumors, and different regional hotspots could be identified. Several hotspot mutations that are a rare event in normal tissues were identified in the tumor tissues. We conclude that thymic lymphomas arising in Msh2 deficient genetic background are hypermutable and the altered mutational spectrum might be an indication of infidelity of DNA replication during tumorigenesis.     

Biological evaluation of analogues of an insect neuropeptide proctolin

Continuing our studies on proctolin (Arg-Tyr-Leu-Pro-Thr) we performed the synthesis and biological evaluation of 52 analogues substituted in position 2, 3, 4, and 5 of the peptide chain. The peptides were bioassayed for cardiotropic activity in vitro on Tenebrio molitor and myotropic activity on foregut of Schistocerca gregaria. Twenty analogues retained 20-80% of proctolin activity.     

Pharmacological blockade of CXCR3 by (±)-NBI-74330 reduces neuropathic pain and enhances opioid effectiveness - Evidence from in vivo and in vitro studies

It has been suggested that CXCR3 is important for nociception. Our experiments were conducted to evaluate involvement of CXCR3 and its ligands (CXCL4, CXCL9, CXCL10, CXCL11/CCL21) in neuropathic pain. Our studies give new evidence that intrathecal administration of each CXCR3 ligand induces pain-like behaviour in naive mice that occurs shortly after injection due to its location of neurons, which is confirmed by immunofluorescent staining. Moreover, intrathecal administrations of CXCL9, CXCL10, CCL21 neutralizing antibodies diminished pain-related behaviour. RT-PCR/Western blot analysis unprecedentedly showed spinal elevated levels of CXCR3 after chronic constriction injury of the sciatic nerve in rats in parallel with different time-course changes of its endogenous ligands. Initially, on day 2 we observed spinal increased levels of CXCL10 and CXCL11 indicating that these chemokines have important roles in triggering neuropathy. Then, on day 7, we observed increased levels of CXCL4, CXCL9, CXCL10. Interestingly, changes in CXCL9 level persisted until day 28, suggesting that these chemokines are responsible for long-term, persistent neuropathy. Additionally, in DRG the CXCL4, CXCL9 were elevated. The results obtained from primary glial cultures, suggests that all CXCR3 ligands can be produced in microglia, but also, except for CXCL4, in astrocytes. We provide the first evidence that in neuropathy chronic intrathecal administration of CXCR3 antagonist, (±)-NBI-74330, attenuates hypersensitivity with concomitant occurrence of microglial and some of CXCR3 ligands activation observed in the spinal cord and/or DRG level. This paper underlies the significance of CXCR3 in neuropathic pain and shows therapeutic potential of its blockade for enhancement of morphine analgesia as the major novelty of this work.     

Structural and thermodynamic properties of rice starches with different genetic background Part 2. Defectiveness of different supramolecular structures in starch granules

High-sensitivity differential scanning microcalorimetry (HSDSC), small-angle X-ray scattering (SAXS), light (LM) and scanning electronic (SEM) microscopy techniques were used to study the defectiveness of different supramolecular structures in starches extracted from 11 Thai cultivars of rice differing in level of amylose and amylopectin defects in starch crystalline lamellae. Despite differences in chain-length distribution of amylopectin macromolecules and amylose level in starches, the invariance in the sizes of crystalline lamellae, amylopectin clusters and granules was established. The combined analysis of DSC, SAXS, LM and SEM data for native starches, as well as the comparison of the thermodynamic data for native and annealed starches, allowed to determine the structure of defects and the localization of amylose chains in crystalline and amorphous lamellae, defectiveness of lamellae, clusters and granules. It was shown that amylose “tie chains”, amylose–lipid complexes located in crystalline lamellae, defective ends of double helical chains dangling from crystallites inside amorphous lamellae (“dangling” chains), as well as amylopectin chains with DP 6–12 and 25–36 could be considered as defects. Their accumulation can lead to a formation of remnant granules. The changes observed in the structure of amylopectin chains and amylose content in starches are reflected in the interconnected alterations of structural organization on the lamellar, cluster and granule levels.     

Application of Two‐Way Hierarchical Cluster Analysis for the Identification of Similarities between the Individual Lipid Fractions of Lucilia sericata

The composition of the cuticular and internal lipids of larvae and pupae of Lucilia sericata was studied using chromatographic techniques. The lipids from both stages of L. sericata had similar free fatty acid (FFA) profiles and also contained alcohols and cholesterol. The range of the number of C‐atoms detected for these classes of compounds was to some extent similar in larvae and pupae, but the relative amounts of each class differed between stages. Saturated as well as unsaturated FFAs with even and odd numbered C‐atom chains were present in both cuticular and internal lipids. The alcohol fractions of L. sericata were represented by free, straight‐chain primary alcohols containing an even number of C‐atoms. The lipid composition of male and female L. sericata adults and the hydrocarbon composition of all stages of L. sericata had previously been analyzed. To have a full overview of the lipid composition and to identify similarities or dissimilarities between the individual lipid fractions in this insect species, two‐way hierarchical cluster analysis (HCA) was performed using also the data from these previous publications. The content of FFA 18 : 1 (n‐9) was noticed to be very high in the cuticular fractions of larvae and pupae as well as in all internal fractions (male, female, larvae, and pupae) and low in the cuticular fractions of male and female imago. The contents of FFAs 16 : 0 and 16 : 1 (n‐9), cholesterol, and the n‐alkanes n‐C₃₁, n‐C₂₉, n‐C₂₇, n‐C₂₅, and n‐C₂₃ varied between particular fractions, whereas the amounts of other compounds were similar in all fractions.