Telomere stability and telomerase in mesenchymal stem cells

Telomeres are repetitive genetic material that cap and thereby protect the ends of chromosomes. Each time a cell divides, telomeres get shorter. Telomere length is mainly maintained by telomerase. This enzyme is present in high concentrations in the embryonic stem cells and in fast growing embryonic cells, and declines with age. It is still unclear to what extent there is telomerase in adult stem cells, but since these are the founder cells of cells of all the tissues in the body, understanding the telomere dynamics and expression of telomerase in adult stem cells is very important. In the present communication we focus on telomere expression and telomere length in stem cells, with a special focus on mesenchymal stem cells. We consider different mechanisms by which stem cells can maintain telomeres and also focus on the dynamics of telomere length in mesenchymal stem cells, both the overall telomere length and the telomere length of individual chromosomes.     

Cold adaptation of enzymes: structural, kinetic and microcalorimetric characterizations of an aminopeptidase from the Arctic psychrophile Colwellia psychrerythraea and of human leukotriene A(4) hydrolase

The relationships between structure, activity, stability and flexibility of a cold-adapted aminopeptidase produced by a psychrophilic marine bacterium have been investigated in comparison with a mesophilic structural and functional human homolog. Differential scanning calorimetry, fluorescence monitoring of thermal- and guanidine hydrochloride-induced unfolding and fluorescence quenching were used to show that the cold-adapted enzyme is characterized by a high activity at low temperatures, a low structural stability versus thermal and chemical denaturants and a greater structural permeability to a quenching agent relative to the mesophilic homolog. These findings support the hypothesis that cold-adapted enzymes maintain their activity at low temperatures as a result of increased global or local structural flexibility, which results in low stability. Analysis of the thermodynamic parameters of irreversible thermal unfolding suggests that entropy-driven factors are responsible for the fast unfolding rate of the cold-adapted aminopeptidase. A reduced number of proline residues, a lower degree of hydrophobic residue burial and a decreased surface accessibility of charged residues may be responsible for this effect. On the other hand, the reduction in enthalpy-driven interactions is the primary determinant of the weak conformational stability.     

Verocytotoxin-Producing Escherichia coli in Wild Birds and Rodents in Close Proximity to Farms

Wild animals living close to cattle and pig farms (four each) were examined for verocytotoxin-producing Escherichia coli (VTEC; also known as Shiga toxin-producing E. coli). The prevalence of VTEC among the 260 samples from wild animals was generally low. However, VTEC isolates from a starling (Sturnus vulgaris) and a Norway rat (Rattus norvegicus) were identical to cattle isolates from the corresponding farms with respect to serotype, virulence profile, and pulsed-field gel electrophoresis type. This study shows that wild birds and rodents may become infected from farm animals or vice versa, suggesting a possible role in VTEC transmission.     

Lipid dynamics in fast-tumbling bicelles with varying bilayer thickness: Effect of model transmembrane peptides

The morphology of q = 0.5 fast-tumbling bicelles prepared with three different acyl chain lengths has been investigated by NMR. It is shown that bicelles prepared with DLPC (12 C) and DHPC are on average larger than those containing DMPC or DPPC (14 and 16 C) and DHPC, which may be due to a higher degree of mixing between DLPC and DHPC. The fast internal mobility of the lipids was determined from natural abundance carbon-13 relaxation. A similar dynamical behaviour of the phospholipids in the three different bicelles was observed, although the DPPC lipid acyl chain displayed a somewhat lower degree of mobility, as evidenced by higher generalized order parameters throughout the acyl chain. Carbon-13 relaxation was also used to determine the effect of different model transmembrane peptides, with flanking Lys residues, on the lipid dynamics in the three different bicelles. All peptides had the effect of increasing the order parameters for the DLPC lipid, while no effect was observed on the longer lipid chains. This effect may be explained by a mismatch between the hydrophobic length of the peptides and the DLPC lipid acyl chain.     

Expression of recombinant murine pregnancy-associated plasma protein-A (PAPP-A) and a novel variant (PAPP-Ai) with differential proteolytic activity.

Murine pregnancy-associated plasma protein-A (PAPP-A) cDNA encoding a 1545 amino-acid protein has been cloned. We have also identified and cloned cDNA that encodes a novel variant of PAPP-A, PAPP-Ai, carrying a 29-residue highly basic insert. The point of insertion corresponds to a junction between two exons in the human PAPP-A gene. The human intron flanked by these exons does not encode a homologous corresponding insert, which is unique to the mouse. The overall sequence identity between murine and human PAPP-A is 91%, and murine PAPP-A contains sequence motifs previously described in the sequence of human PAPP-A. Through expression in mammalian cells, we show that murine PAPP-A and PAPP-Ai are active metalloproteinases, both capable of cleaving insulin-like growth factor binding protein (IGFBP)-4 and -5. Cleavage of IGFBP-4 is dramatically enhanced by the addition of IGF, whereas cleavage of IGFBP-5 is slightly inhibited by IGF, as previously established with human PAPP-A. Surprisingly, however, quantitative analyses demonstrate that the murine PAPP-Ai cleaves IGFBP-4 very slowly compared to PAPP-A, even though its ability to cleave IGFBP-5 is unaffected by the presence of the insert. By RT-PCR analysis, we find that both variants are expressed in several tissues. The level of mRNA in the murine placenta does not exceed the levels of other tissues analyzed. Furthermore, the IGFBP-4-proteolytic activity of murine pregnancy serum is not elevated. This is in striking contrast to the increase seen in human pregnancy serum, and the expression of PAPP-A in the human placenta, which exceeds other tissues at least 250-fold. Interestingly, the position of the insert of PAPP-Ai, within the proteolytic domain, lies in close proximity to the cysteine residue, which in human PAPP-A forms a disulfide bond with the proform of eosinophil major basic protein (proMBP). ProMBP functions as a proteinase inhibitor in the PAPP-A-proMBP complex, but whether any mechanistic parallel on regulation of proteolytic activity can be drawn between the insert of PAPP-Ai and the linkage to proMBP is not known. Importantly, these data support the development of the mouse as a model organism for the study of PAPP-A, which must take into account the differences between the mouse and the human.     

Leukotriene A4 hydrolase/aminopeptidase. Glutamate 271 is a catalytic residue with specific roles in two distinct enzyme mechanisms.

Leukotriene A(4) hydrolase/aminopeptidase is a bifunctional zinc metalloenzyme that converts the fatty acid epoxide leukotriene A(4) into leukotriene B(4), a potent chemoattractant and immune-modulating lipid mediator. Recently, the structure of leukotriene A(4) hydrolase revealed that Glu-271, which belongs to a conserved GXMEN motif in the M1 family of zinc peptidases, and Gln-136 are located at the active site. Here we report that mutagenetic replacements of Glu-271, but not Gln-136, abrogate both catalytic activities of leukotriene A(4) hydrolase. Furthermore, the 2.1 A crystal structure of [E271Q]leukotriene A(4) hydrolase revealed minimal conformational changes that could not explain the loss of enzyme function. We propose that the carboxylate of Glu-271 participates in an acid-induced opening of the epoxide moiety of leukotriene A(4) and formation of a carbocation intermediate. Moreover, Glu-271 appears to act as an N-terminal recognition site and may potentially stabilize the transition-state during turnover of peptides, a property that most likely pertains to all members of the M1 family of zinc aminopeptidases. Hence, Glu-271 is a unique example of an amino acid, which has dual and separate functions in two different catalytic reactions, involving lipid and peptide substrates, respectively.     

Recessive Resistance in Pisum sativum and Potyvirus Pathotype Resolved in a Gene-for-Cistron Correspondence between Host and Virus

Pea seed-borne mosaic potyvirus (PSbMV) isolates are divided into pathotypes P-1, P-2, and P-4 according to their infection profile on a panel of Pisum sativum lines. P. sativum PI 269818 is resistant to P-1 and P-2 isolates and is susceptible to P-4 isolates. Resistance to P-1 is inherited as a single recessive gene, denoted sbm-1, and the pathogenicity determinant has previously been mapped to the virus-coded protein VPg. In the cultivar Bonneville, a second recessive gene, sbm-2, confers specific resistance to P-2. By exchanging cistrons between a P-2 and a P-4 isolate, the P3-6k1 cistron was identified as the PSbMV host-specific pathogenicity determinant on Bonneville. Exchange of P3-6k1 did not affect infection on PI 269818, and infection of Bonneville was not altered by substitution of the VPg cistron, indicating that P3-6k1 and VPg are independent determinants of pathotype-specific infectivity. On PI 269818 the pathogenicity determinant of both P-1 and P-2 mapped to the N terminus of VPg. This suggests that VPg from the P-1 and P-2 isolates are functionally similar on this host and that resistance to P-1 and P-2 in PI 269818 may operate by the same mechanism. Identification of VPg-sbm-1 and P3-6k1-sbm-2 as independent pairs of genetic interactors between PSbMV and P. sativum provides a simple explanation of the three known pathotypes of PSbMV. Furthermore, analysis of beta-glucuronidase-tagged P-2 virus indicated that sbm-2 resistance affected an early step in infection, implying that the P3-6k1 region plays a critical role in potyvirus replication or cell-to-cell movement.     

Lactobacillus coryniformis subsp. coryniformis Strain Si3 Produces a Broad-Spectrum Proteinaceous Antifungal Compound

The antifungal activity spectrum of Lactobacillus coryniformis subsp. coryniformis strain Si3 was investigated. The strain had strong inhibitory activity in dual-culture agar plate assays against the molds Aspergillus fumigatus, A. nidulans, Penicillium roqueforti, Mucor hiemalis, Talaromyces flavus, Fusarium poae, F. graminearum, F. culmorum, and F. sporotrichoides. A weaker activity was observed against the yeasts Debaryomyces hansenii, Kluyveromyces marxianus, and Saccharomyces cerevisiae. The yeasts Rhodotorula glutinis, Sporobolomyces roseus, and Pichia anomala were not inhibited. In liquid culture the antifungal activity paralleled growth, with maximum mold inhibition early in the stationary growth phase, but with a rapid decline in antifungal activity after 48 h. The addition of ethanol to the growth medium prevented the decline and gave an increased antifungal activity. The activity was stable during heat treatment and was retained even after autoclaving at 121°C for 15 min. Maximum activity was observed at pH values of between 3.0 and 4.5, but it decreased rapidly when pH was adjusted to a level between 4.5 and 6.0 and was lost at higher pH values. The antifungal activity was fully regained after readjustment of the pH to the initial value (pH 3.6). The activity was irreversibly lost after treatment with proteolytic enzymes (proteinase K, trypsin, and pepsin). The antifungal activity was partially purified using ion-exchange chromatography and (NH₄)₂SO₄ precipitation, followed by gel filtration chromatography. The active compound(s) was estimated to have a molecular mass of approximately 3 kDa. This is the first report of the production of a proteinaceous antifungal compound(s) from L. coryniformis subsp. coryniformis.     

Critical parameters in the MCF-7 cell proliferation bioassay (E-Screen)

The MCF-7 cell proliferation bioassay has grown in popularity as a rapid test for detecting potentially oestrogenic compounds. Several MCF-7 cell sublines with different sensitivities to oestrogens are currently used, with maximal proliferation responses ranging from two- to 10-fold above those of hormone-free controls. In the highly responsive MCF-7 BUS cell line, we evaluated critical assay parameters for test performance, including growth conditions, initial seeding densities and differences in growth stimulation in medium containing human serum or fetal calf serum as well as appropriate solvents for oestrogen-mimicking compounds. Modifications significantly reduced the labour-intensive steps and overall assay costs without affecting the sensitivity of the assay. Using this optimized test regimen, the responsiveness of treated MCF-7 BUS cells was consistently increased up to 11-fold over hormone-free controls. The specificity was characterized by examining the effects of oestradiol-17 β, the anti-oestrogen ICI 182,780, and dieldrin, a recognized xeno-oestrogen. The improved proliferation bioassay will be a useful tool in identifying potential xeno-oestrogens.