Gas chromatographic–mass spectrometric investigation of metabolites from the needles and roots of pine seedlings at early stages of pathogenic fungi <Emphasis Type="Italic">Armillaria ostoyae</Emphasis> attack

An investigation was carried out of the composition of metabolites in pine seedlings tissues at the initial stages of the infectious process caused by pathogenic fungi Armillaria ostoyae, which causes a root rot of trees and degradation of forest resources. With the help of successive extraction with organic solvents of different polarity, more than 190 metabolites were extracted from the needles and roots of the seedlings and then identified by GC–MS method. The composition of the extracts from control plants and those inoculated with Armillaria ostoyae were compared. It was established that part of secondary metabolites (glucosamines and free amino acids, carbohydrates raffinose and trehalose) were present only in the tissues of inoculated plants. Possible roles of some of these compounds appearing in the roots of seedlings infected with the fungus are also discussed in the paper.     

Bone-marrow-derived stem cells — our key to longevity?

Bone marrow (BM) was for many years primarily regarded as the source of hematopoietic stem cells. In this review we discuss current views of the BM stem cell compartment and present data showing that BM contains not only hematopoietic but also heterogeneous non-hematopoietic stem cells. It is likely that similar or overlapping populations of primitive non-hematopoietic stem cells in BM were detected by different investigators using different experimental strategies and hence were assigned different names (e.g., mesenchymal stem cells, multipotent adult progenitor cells, or marrow-isolated adult multilineage inducible cells). However, the search still continues for true pluripotent stem cells in adult BM, which would fulfill the required criteria (e.g. complementation of blastocyst development). Recently our group has identified in BM a population of very small embryonic-like stem cells (VSELs), which express several markers characteristic for pluripotent stem cells and are found during early embryogenesis in the epiblast of the cylinder-stage embryo.     

Jack bean urease: the effect of active-site binding inhibitors on the reactivity of enzyme thiol groups

In view of the complexity of the role of the active site flap cysteine in the urease catalysis, in this work we studied how the presence of typical active-site binding inhibitors of urease, phenylphosphorodiamidate (PPD), acetohydroxamic acid (AHA), boric acid and fluoride, affects the reactivity of enzyme thiol groups, the active site flap thiol in particular. For that the inhibitor-urease complexes were prepared with excess inhibitors and had their thiol groups titrated with DTNB. The effects observed were analyzed in terms of the structures of the inhibitor-urease complexes reported in the literature. We found that the effectiveness in preventing the active site cysteine from the modification by disulfides, varied among the inhibitors studied, even though they all bind to the active site. The variations were accounted for by different extents of geometrical distortion in the active site that the inhibitors introduced upon binding, leaving the flap either open in AHA-, boric acid- and fluoride-inhibited urease, like in the native enzyme or closed in PPD-inhibited urease. Among the inhibitors, only PPD was found to be able to thoroughly protect the flap cysteines from the further reaction with disulfides, this apparently resulting from the closed conformation of the flap. Accordingly, in practical terms PPD may be regarded as the most suitable inhibitor for active-site protection experiments in inhibition studies of urease.     

Yeast holoenzyme of protein kinase CK2 requires both β and β′ regulatory subunits for its activity

Protein kinase CK2 is a highly conserved Ser/Thr protein kinase that is ubiquitous among eucaryotic organisms and appears to play an important role in many cellular functions. This enzyme in yeast has a tetrameric structure composed of two catalytic (α and/or α′) subunits and two regulatory β and β′ subunits. Previously, we have reported isolation from yeast cells four active forms of CK2, composed of αα′ββ′, α₂ββ′, α′₂ββ′ and a free α′-catalytic subunit. Now, we report that in Saccharomyces cerevisiae CK2 holoenzyme regulatory β subunit cannot substitute other β′ subunit and only both of them can form fully active enzymatic unit. We have examined the subunit composition of tetrameric complexes of yeast CK2 by transformation of yeast strains containing single deletion of the β or β′ regulatory subunits with vectors carrying lacking CKB1 or CKB2 genes. CK2 holoenzyme activity was restored only in cases when both of them were present in the cell. Additional, co-immunoprecypitation experiments show that polyadenylation factor Fip1 interacts with catalytic α subunits of CK2 and interaction with beta subunits in the holoenzyme decreases CK2 activity towards this protein substrate. These data may help to elucidate the role of yeast protein kinase CK2β/β′ subunits in the regulation of holoenzyme assembly and phosphotransferase activity.     

tRNA residues that have coevolved with their anticodon to ensure uniform and accurate codon recognition

The structure, phylogeny and in vivo function of the base pair formed between nucleotides 32 and 38 of the tRNA anticodon loop are reviewed. The A32-U38 pair, which is highly conserved in tRNA2(Ala) and sometimes observed in tRNA2(Pro), was recently found to decrease the affinity of tRNAs to the ribosomal A site relative to other 32-38 combinations. This suggests that the role of 32-38 pair is to tune the tRNA affinity in the A site to a uniform value. New experiments presented here show that the U32C mutation in tRNA1(Gly) increases its affinity to the cognate codon and to codons with third position mismatches in the A site. This suggests that one reason for uniform tRNA binding to evolve was to avoid incorrect codon recognition.     

Predator-induced diapause in Daphnia magna may require two chemical cues

The production of diapausing eggs by Daphnia magna stimulated by fish exudates can be explained as an anti-predator defence ensuring genome protection in periods of high risk from fish predation. The combined effects on the induction of D. magna diapause of an “alarm” chemical originating from injured conspecific prey and fish kairomones were tested. The results of the experiment showed that the cues when present together promote both the production of ephippial eggs and male formation, indicating their role in the synchronization of the entire mode of Daphnia sexual reproduction. Ephippial eggs were only produced in the presence of both fish kairomone and conspecific alarm chemicals, while male offspring occurred in the treatments where both, one or none of the cues were present. However, production of males was the highest when both cues were provided. D. magna responded similarly to the tested cues whether or not the hypothetical alarm substance associated with predator odour came from Daphnia specimens actually eaten by fish or from crushed conspecific individuals. However, chemicals from crushed chironomid larvae combined with fish kairomones did not induce a similar response in D. magna. The relative advantage of utilization of alarm cues or predator kairomones in the induction of defence responses in prey organisms is discussed. Received: 8 June 1998 / Accepted: 11 January 1999     

A novel c.581C>T transition localized in a highly conserved homeobox sequence ofMSX1: is it responsible for oligodontia?

Even though selective tooth agenesis is the most common developmental anomaly of human dentition, its genetic background still remains poorly understood. To date, familial as well as sporadic forms of both hypodontia and oligodontia have been associated with mutations or polymorphisms of MSX1, PAX9, AXIN2 and TGFa, whose protein products play a crucial role in odontogenesis. In the present report we described a novel mutation of MSX1, which might be responsible for the lack of 14 permanent teeth in our proband. However, this c.581C>T transition, localized in a highly conserved homeobox sequence of MSX1, was identified also in 2 healthy individuals from the proband's family. Our finding suggests that this transition might be the first described mutation of MSX1 that might be responsible for oligodontia and showing incomplete penetrance. It may also support the view that this common anomaly of human dentition might be an oligogenic trait caused by simultaneous mutations of different genes.     

The molecular and biochemical characteristics of proline iminopeptidase from rye seedlings (Secale cereale L.)

A proline iminopeptidase (EC. 3.4.11.5) was isolated from shoots of 3 day old seedlings. The purification procedure consisted of 5 steps: acid precipitation, gel filtration on Sephadex G-200, ion-exchange chromatography on Sepharose CL 6B, twice repeated hydrophoic chromatography on Phenyl-Sepharose HP. The enzyme was purified 404.8-fold, with the specific activity of 8.5 units mg⁻¹ of protein with recovery yield of 3%. The purified enzyme had a molecular mass of 225 kDa estimated by gel filtration and 55.4 kDa by SDS PAGE. This indicates that native enzyme is composed of four subunits. The enzyme was specific for proline β-naphtylamide among various amino acid β-naphtylamides. An optimal activity was observed at 37 °C at pH 7.75. The enzyme was thermostable up to 37 °C for 30 min. The enzyme was strongly inhibited by pHMB, E-64, heavy metal ions and partially by PMSF, DFP. The results suggest that cysteine and serine residues may participate in the enzyme activity.