Savignin, a potent thrombin inhibitor isolated from the salivary glands of the tick Ornithodoros savignyi (Acari: Argasidae).

A thrombin (E.C. 3.4.21.5) inhibitor, savignin, was isolated from the salivary glands of Ornithodoros savignyi by a combination of size exclusion, anion-exchange, and reversed-phase chromatography. The inhibitor has a molecular mass of 12,430.4 Da as determined by electrospray mass spectrometry. The behavior of savignin during anion-exchange chromatography indicated that it has an acidic pI. The available N-terminal sequence (residues 1-11) differed from that of ornithodorin with only one residue. Savignin inhibits thrombin-induced platelet aggregation, but has no effect on ADP- or collagen-induced aggregation. Kinetic studies indicated that savignin is a competitive, slow-, tight-binding inhibitor of alpha-thrombin (K(i) = 4.89 +/- 1.39 pM). Tight-binding kinetics showed that the inhibitor has a lower affinity for gamma-thrombin (K(i) = 22.3 +/- 5.9 nM). Plasmin, factor Xa, and trypsin are not inhibited by savignin.     

Involvement of putrescine in the inductive rooting phase of poplar shoots raised in vitro

Micropropagated poplar shoots rooted 100% on a rooting medium (A) containing NAA, but they did not root in the absence of auxin (NA). Putrescine, but not spermidine and spermine, promoted rooting up to 42% when added to the NA medium. Cyclohexylamine (CHA), an inhibitor of spermine synthase, also promoted (up to 36%) rooting in the absence of auxin. The inhibitors of polyamine biosynthesis DFMA (α-difluoromethylarginine) and DFMO (α-difluoromethylomithine), aminoguanidine (AG) and methylglyoxal-bis-guanylhydrazone (MGBG), inhibited rooting when applied in the presence of auxin and had no effect in its absence.
The rooting inductive phase (in the presence of auxin) was determined by periodical transfer of shoots from A to NA medium, and by changes in peroxidase activity, to be 7 h. Putrescine (not spermidine and spermine) accumulated to a maximum during the inductive phase. Both putrescine and CHA promoted rooting on NA medium when applied during the first 7 h. In contrast DFMA and AG inhibited rooting during this period. The results point to the involvement of putrescine and its Δ¹-pyrroline pathway, in the inductive phase of rooting in poplar shoots.     

The formation of titan cells in Cryptococcus neoformans depends on the mouse strain and correlates with induction of Th2‐type responses

Cryptococcus neoformans is a pathogenic yeast that can form titan cells in the lungs, which are fungal cells of abnormal enlarged size. Little is known about the factors that trigger titan cells. In particular, it is not known how the host environment influences this transition. In this work, we describe the formation of titan cells in two mouse strains, CD1 and C57BL/6J. We found that the proportion of C. neoformans titan cells was significantly higher in C57BL/6J mice than in CD1. This higher proportion of titan cells was associated with a higher dissemination of the yeasts to the brain. Histology sections demonstrated eosinophilia in infected animals, although it was significantly lower in the CD1 mice which presented infiltration of lymphocytes. Both mouse strains presented infiltration of granulocytes, but the amount of eosinophils was higher in C57BL/6J. CD1 mice showed a significant accumulation of IFN‐γ, TNF‐α and IL17, while C57BL/BL mice had an increase in the anti‐inflammatory cytokine IL‐4. IgM antibodies to the polysaccharide capsule and total IgE were more abundant in the sera from C57BL/6J, confirming that these animals present a Th2‐type response. We conclude that titan cell formation in C. neoformans depends, not only on microbe factors, but also on the host environment.     

Disturbed nitrogen metabolism associated with the hyperhydric status of fully habituated callus of sugarbeet

The content of polyamines and proline was much lower in a normal (N) callus of Beta vulgaris L. than in a fully habituated hyperhydric (H) callus. The H callus also contained more glutamate and had a higher glutamate dehydrogenase activity. The excess of glutamate, in this chlorophyll-deficient callus, was linked to accumulation of proline and polyamines. Experiments with α-difluoromethylornithine (DFMO) and α-difluoromethylarginine (DFMA) showed that both ornithine decarboxylase and arginine decarboxylase participated in the synthesis of polyamines (especially spermidine and putrescine) and removal of ammonia. It is hypothesized that the H callus was subjected to ammonia stress from the start of the culture. Experiments with gabaculine, an inhibitor of ornithine aminotransferase, showed that this enzyme linked proline degradation to polyamine synthesis through the production of ornithine. This disturbed nitrogen metabolism appeared to be characteristic of the fully habituated callus and might explain the low growth of this hyperhydric tissue.     

Biosynthesis of 5-aminolevulinic Acid via the Shemin Pathway in a Green Sugar Beet Callus

5-Aminolevulinic acid synthase (ALAS) has been detected in a normal (auxin- and cytokinin-dependent) green sugar beet callus under light and under darkness. ALAS activity was lower when the callus was grown under light. The supply of precursors of the Shemin pathway (glycine and succinate) to dark-grown callus enhanced considerably the capacity of the 5-aminolevulinic acid (ALA) formation. Glutamate, -aminobutyrate or -ketoglutarate also increased ALA accumulation. Such an accumulation was also obtained after inhibition of polyamine synthesis. The results show that glutamate or its derivatives might feed the Shemin pathway in conditions preventing glutamate to be used through the Beale pathway.     

Effect of ankle joint position and electrode placement on the estimation of the antagonistic moment during maximal plantarflexion.

During maximal efforts, antagonistic activity can significantly influence the joint moment. During maximal voluntary "isometric" contractions, certain joint rotation can not be avoided. This can influence the estimation of the antagonistic moment from the EMG activity. Our study aimed to quantify the influence on the calculated agonistic moment produced during maximal voluntary isometric plantarflexions (a) when estimating antagonistic moments at different ankle angles and (b) when placing the EMG electrodes at different portions over the m. tibialis anterior. Ten subjects performed maximal voluntary isometric plantarflexions at 90 degrees ankle angle. In order to estimate the antagonistic moment, submaximal isometric dorsiflexions were performed at various ankle angles. Moment and EMG signals from mm. triceps surae and tibialis anterior were measured. The RMS differences between plantarflexors moment calculated considering the antagonistic cocontraction estimated at the same ankle angle at which the maximal plantarflexion moment was achieved and at different ankle angles ranged from 0.10 to 2.94 Nm. The location of the electrodes led to greater RMS differences (2.35-5.18 Nm). In conclusion, an angle 10 degrees greater than the initial plantarflexion angle is enough to minimize the effect of the change in length of the m. tibialis anterior during the plantarflexion on the estimation of the plantarflexors moment. The localisation of the electrodes over the m. tibialis anterior can influence the estimation of its cocontraction during maximal plantarflexion efforts.     

Preapoptotic chromatin changes induced by ultraviolet B irradiation in human erythroleukemia K562 cells

Exponentially growing human erythroleukemia K562 cells were permeabilized and the dose dependent decrease of DNA synthesis rate was measured after ultraviolet (UV B, 290 nm) irradiation. Cells were able to overcome 2 and 5 J/m2 UV doses, partial recovery was observed at 15 J/m2, while at high (25 J/m2) UV dose replicative DNA synthesis remained suppressed. K562 cells were subjected to synchronization prior to and after UV irradiation (24 J/m2) and 18 fractions were collected by centrifugal elutriation. Cell cycle analysis by flow cytometry did not show early apoptotic cells after UV irradiation. The gradual increase in DNA content typical for non-irradiated cells was contrasted by an early S phase block between 2.2 and 2.4 C-values after UV irradiation. Cell cycle dependent chromatin changes after ultraviolet irradiation were seen as a fine fibrillary network covering the mainly fibrous chromatin structures and incompletely folded primitive chromosomes. Based on observations after UV irradiation and on earlier results with cadmium treatment and gamma irradiation, we confirm that typical chromatin changes characteristic to genotoxic agents can be recognized and classified.     

The effect of falling height on muscle activity and foot motion during landings

The aims of this study were: (a) to examine the effect of falling height on the kinematics of the tibiotalar, talonavicular and calcaneocuboid joints and (b) to study the influence of falling height on the muscle activity of the leg during landings. Six female gymnasts (height: 1.63±0.04 m, weight: 58.21±3.46 kg) participated in this study. All six gymnasts carried out barefoot landings, falling from 1.0, 1.5 and 2.0 m height onto a mat. Three genlocked digital high speed video cameras (250 Hz) captured the motion of the left shank and foot. Surface electromyography (EMG) was used to measure muscle activity (1000 Hz) from five muscles (gastrocnemius medialis, tibialis anterior, peroneus longus, vastus lateralis and hamstrings) of the left leg. The kinematics of the tibiotalar, talonavicular and calcaneocuboid joints were studied. The lower-leg and the foot were modelled by means of a multi-body system, comprising seven rigid bodies. The falling height does not show any influence on the kinematics neither of the tibiotalar nor of the talonavicular joints during landing. The eversion at the calcaneocuboid joint increases with increasing falling height. When augmenting falling height, the myoelectric activity of the muscles of the lower limb increases as well during the pre-activation phase as during the landing itself. The muscles of the lower extremities are capable of stabilizing the tibiotalar and the talonavicular joints actively, restricting their maximal motion by means of a higher activation before and after touchdown. Maximal eversion at the calcaneocuboid joint increases about 52% when landing from 2.0 m.