Interdependency of sequence and positional specificities for cysteine proteases of the papain family

The specificity of cysteine proteases is characterized by the nature of the amino acid sequence recognized by the enzymes (sequence specificity) as well as by the position of the scissile peptide bond (positional specificity, i.e., endopeptidase, aminopeptidase, or carboxypeptidase). In this paper, the interdependency of sequence and positional specificities for selected members of this class of enzymes has been investigated using fluorogenic substrates where both the position of the cleavable peptide bond and the nature of the sequence of residues in P2-P1 are varied. The results show that cathepsins K and L and papain, typically considered to act strictly as endopeptidases, can also display dipeptidyl carboxypeptidase activity against the substrate Abz-FRF(4NO2)A and dipeptidyl aminopeptidase activity against FR-MCA. In some cases the activity is even equal to or greater than that observed with cathepsin B and DPP-I (dipeptidyl peptidase I), which have been characterized previously as exopeptidases. In contrast, the exopeptidase activities of cathepsins K and L and papain are extremely low when the P2-P1 residues are A-A, indicating that, as observed for the normal endopeptidase activity, the exopeptidase activities rely heavily on interactions in subsite S2 (and possibly S1). However, cathepsin B and DPP-I are able to hydrolyze substrates through the exopeptidase route even in absence of preferred interactions in subsites S2 and S1. This is attributed to the presence in cathepsin B and DPP-I of specific structural elements which serve as an anchor for the C- or N-terminus of a substrate, thereby allowing favorable enzyme-substrate interaction independently of the P2-P1 sequence. As a consequence, the nature of the residue at position P2 of a substrate, which is usually the main factor determining the specificity for cysteine proteases of the papain family, does not have the same contribution for the exopeptidase activities of cathepsin B and DPP-I.     

Membrane skeleton detachment in spherical and cylindrical microexovesicles

We observed that amphiphile-induced microexovesicles may be spherical or cylindrical, depending on the species of the added amphiphile. The spherical microexovesicle corresponds to an extreme local difference between the two monolayer areas of the membrane segment with a fixed area, while the cylindrical microexovesicle corresponds to an extreme local area difference if the area of the budding segment is increased due to lateral influx of anisotropic membrane constituents. Protein analysis showed that both types of vesicles are highly depleted in the membrane skeleton. It is suggested that a partial detachment of the skeleton in the budding region is favoured due to accumulated skeleton shear deformations in this region.     

Ferrioxamine-mediated Iron(III) utilization by Salmonella enterica

Utilization of ferrioxamines as sole sources of iron distinguishes Salmonella enterica serotypes Typhimurium and Enteritidis from a number of related species, including Escherichia coli. Ferrioxamine supplements have therefore been used in preenrichment and selection media to increase the bacterial growth rate while selectivity is maintained. We characterized the determinants involved in utilization of ferrioxamines B, E, and G by S. enterica serotype Typhimurium by performing siderophore cross-feeding bioassays. Transport of all three ferric siderophores across the outer membrane was dependent on the FoxA receptor encoded by the Fur-repressible foxA gene. However, only the transport of ferrioxamine G was dependent on the energy-transducing protein TonB, since growth stimulation of a tonB strain by ferrioxamines B and E was observed, albeit at lower efficiencies than in the parental strain. Transport across the inner membrane was dependent on the periplasmic binding protein-dependent ABC transporter complex comprising FhuBCD, as has been reported for other hydroxamate siderophores of enteric bacteria. The distribution of the foxA gene in the genus Salmonella, as indicated by DNA hybridization studies and correlated with the ability to utilize ferrioxamine E, was restricted to subspecies I, II, and IIIb, and this gene was absent from subspecies IIIa, IV, VI, and VII (formerly subspecies IV) and Salmonella bongori (formerly subspecies V). S. enterica serotype Typhimurium mutants with either a transposon insertion or a defined nonpolar frameshift (+2) mutation in the foxA gene were not able to utilize any of the three ferrioxamines tested. A strain carrying the nonpolar foxA mutation exhibited a significantly reduced ability to colonize rabbit ileal loops compared to the foxA+ parent. In addition, a foxA mutant was markedly attenuated in mice inoculated by either the intragastric or intravenous route. Mice inoculated with the foxA mutant were protected against subsequent challenge by the foxA+ parent strain.     

A Novel Rieske Iron-Sulfur Protein from the Hyperthermophilic Crenarchaeon Pyrobaculum aerophilum: Sequencing of the Gene, Expression in E. coli and Characterization of the Protein

The crenarchaeon Pyrobaculum aerophilum is with an optimalgrowth temperature of 100 °C one of the most thermophilic organisms knownto possess an aerobic respiratory chain. The analysis of DNA sequences fromthe Pyrobaculum genome project lead to the identification of an openreading frame potentially coding for a Rieske iron-sulfur protein. Thecomplete gene (named parR) was cloned and sequenced. The deducedamino acid sequence displays unusual amino acid exchanges and a so farunknown sequence insertion. The N-terminus shows similarities to bacterialsignal sequences. Several forms of the gene were expressed in E.coli in order to verify the classification as a Rieske protein and tofacilitate biophysical studies. Soluble, thermo-stable proteins withcorrectly inserted iron-sulfur clusters were expressed from two versions ofthe gene. The 1–23 truncated holo-protein is redox active. Itdisplays the typical spectroscopic properties of a Rieske protein. The redoxpotential was determined to be +215 mV at pH 6.5 and is pH dependentabove pH 7.5 revealing the influence of two protonation equilibria with pKavalues of 8.1 and 9.8. Phylogenetic analysis demonstrates that the parRprotein clusters together with the two other available archaeal Rieskesequences from Sulfolobus on a separate branch of the phylogenetictree apart from the proteins from thermophilic bacteria like Aquifexand Thermus.     

Translocation of ornithine decarboxylase to the surface membrane during cell activation and transformation

Ornithine decarboxylase (ODC) is highly up-regulated in proliferating and transforming cells. Here we show that upon induction, an initial cytosolic increase of ODC is followed by translocation of a fraction of the enzyme to the surface membrane. ODC membrane translocation is mediated by a p47(phox) membrane-targeting motif-related sequence, as indicated by reduced ODC activity in the membrane fraction of cells treated with a competing, ODC-derived (amino acids 165-172) peptide, RLSVKFGA, which is homologous to the p47(phox) membrane-targeting sequence. p47(phox) membrane translocation is known to be dependent on the phosphorylation of the targeting motif. Analogously, overexpressed ODC.S167A, a mutant ODC lacking the putative phosphorylation site Ser67, is unable to move to the surface membrane. Cells blocked with the RLSVKFGA peptide showed defective transformation, indicating that the motif-mediated translocation of ODC is prerequisite to its biological function. Constitutive targeting of ODC to the membrane using a plasmid encoding the chimeric protein, wild-type ODC with C-terminal linkage to the farnesylation motif of K-ras, caused impaired cytokinesis with an accumulation of polykaryotic cells. Impaired cytokinesis confirms that ODC is involved in mitotic cytoskeletal rearrangement events and pinpoints the importance of relevant membrane targeting to its physiological function.     

Evolution of heterospecific attraction: using other species as cues in habitat selection

We analyzed the ecological conditions that may favor a habitat selection process in which later arriving individuals (colonists) use the presence of earlier established species (residents) as a cue to profitable breeding sites (heterospecific attraction). In our model, colonists assessing potential breeding patches could select between high-quality source and low-quality sink patches. A proportion of the source patches were occupied by residents. Colonists could either directly sample the relative quality of the patches (termed samplers) or, alternatively, they could also use residents as a cue of patch quality (cue-users). Cue-users gained benefit from lowered costs when assessing occupied source patches. The cue-using strategy is an efficient way to choose the best possible patch not only when interspecific competition is intense, but also when benefits from social aggregation exceed the effects of competition. High relative cost of sampling empty patches increases the fitness of the cue-using strategy relative to samplers. The strongest attraction to heterospecifics was predicted when the benefit from aggregating with residents exceeded the effects of competition, and approximately half of the landscape consisted of occupied, high-quality source patches.     

Liquid chromatography “on-flow” 1H nuclear magnetic resonance on native glycosphingolipid mixtures together with gas chromatography/mass spectrometry on the released oligosaccharides for screening and characterisation of carbohydrate-based antigens from pig lungs

Glycosphingolipids were prepared from pig lung and pooled into two fractions with (i) 3 sugar residues, and (ii) 3 sugar residues. Oligosaccharides were prepared and used for gas chromatography, gas chromatography-mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry. The glycolipid fractions i and ii were further characterised and purified using a novel method based on high performance liquid chromatography on-flow proton nuclear magnetic resonance. The LC on-flow NMR technique showed good chromatographic separation and gave NMR spectral information which could be used as guidance for pooling of the separated mixture glycolipids. Conventional ¹H NMR, thin layer immunostaining, gas chromatography, gas chromatography/mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry were used to characterise the glycolipids and to validate LC-NMR spectral data.     

Comparison between low- and standard-dose ACTH tests in premature infants at risk for chronic lung disease

In order to find a sensitive method to evaluate adrenocortical function in premature infants, we compared low- (0.5 microg/1.73 m(2)) and standard-dose (250 microg/1.73 m(2)) adrenocorticotropin tests (LD- and SD-ACTH) in 12 very-low-birth-weight infants before and 2 days after the end of dexamethasone therapy (duration 9-14 days) for chronic lung disease. Basal serum cortisol levels were inappropriately low in several infants already before dexamethasone therapy (median 190, range 60-357 nmol/l). The 95% confidence intervals of mean serum cortisol levels at 20 min were equal in LD- and SD-ACTH, while at 60 min, the low-dose gave a clearly lower response than the standard-dose test. The LD-ACTH can be used in premature infants as in older children and adults, but the criteria for adrenocortical insufficiency need to be defined.     

Effect of exhausting stretch-shortening cycle exercise on the time course of mechanical behaviour in the drop jump: possible role of muscle damage

The purpose of the present study was to investigate the effect of stretch-shortening-cycle-induced muscle damage on the time course of mechanical behaviour in the drop jump. Ten healthy male subjects performed submaximal stretch-shortening cycle (SSC) exercise on a special sledge apparatus. Exhaustion occurred on average within 3 min. A drop jump (DJ) test from a 50-cm height was performed before and immediately after the sledge exercise as well as 2 h, 2 days and 4 days later. The fatigue exercise showed relatively high blood lactate concentration [12.5 (SD 2.6) mmol x l(-1)] and an increase of serum creatine kinase (CK) activity delayed by 2 days [540 (SD 407) U x l(-1)]. The initial decline in the jump performance (before - immediately after) was related negatively to the early recovery in performance (immediately after 2 h) (P < 0.05). The early recovery of the knee joint moment at the end of stretch showed a negative correlation to the delayed decrease in DJ performance (2 h 2 days) (P < 0.01). Thus, the DJ performance showed an initial decline followed by an early recovery and a secondary decline. Both the initial decline and early recovery in the knee joint moment at the end of stretch were related to the corresponding initial (after 2 h) (P < 0.05) and secondary increases (2 h - 2 days) (P < 0.01) in CK. It is suggested that the early recovery as well as the initial decline in the knee joint function could depend on the degree of muscle damage. Delayed decrease in initial stiffness (2 h - 2 days) was negatively related to the corresponding changes in the knee joint angle at touch down in DJ (P < 0.001). These interactions would imply that the decrease in the stiffness regulation and the modulation of the prelanding motor control might be attributable to secondary muscle damage during 2 days after the SSC exercise. Therefore, it may be suggested that the changes in the DJ performance after the exhausting SSC exercise accompany the progress of muscle damage observed by the corresponding increase in serum CK concentration and the corresponding deterioration of stiffness regulation and motor control in DJ.