Structural and Functional Studies on the Overproduced L11 Protein from Thermus thermophilus

The L11 ribosomal protein from Thermus thermophilus (TthL11) has been overproduced and purified to homogeneity using a two-step purification protocol. The overproduced protein carries a similar methylation pattern at Lys-3 as does its homolog from Escherichia coli. Chymotrypsin digested only a small part of the TthL11 protein and did not cleave TthL11 into two peptides, as in the case of EcoL11, but produced only a single N-terminal peptide. Tryptic digestion of TthL11 also produced an N-terminal peptide, in contrast to the C-terminal peptide obtained with L11 from Bacillus stearothermophilus. The recombinant protein forms a specific complex with a 55-nt 23S rRNA fragment known to interact with members of the L11 family from several organisms. Cooperative binding of TthL11 and thiostrepton to 23S rRNA leads to an increased protection of TthL11 from tryptic digestion. The similar structural and biochemical properties as well as the significant homology between L11 from E. coli and B. stearothermophilus with the corresponding protein from Thermus thermophilus indicate an evolutionarily conserved protein important for ribosome function.     

Effect of sibutramine on regional fat pads and leptin levels in rats fed with three isocaloric diets

AIM: The aim of the study was to investigate: a) the differential effect of the three main macronutrients on food intake, fat depots and serum leptin levels and b) the impact of sibutramine on the above parameters in rats fed ad libitum with three isocaloric diets. METHODS: Three groups of male Wistar rats (n = 63) were fed with a high fat diet (HFD), a high carbohydrate diet (HCD) or a high protein diet (HPD) for 13 weeks. In the last three weeks, each group was divided into three subgroups and received sibutramine (S) either at 5 mg/kg or 10 mg/kg, or vehicle. Food intake was measured daily during the last week of the experiment; perirenal and epididymal fat and fat/lean ratio were calculated and serum leptin was assayed. RESULTS: HFD-fed rats demonstrated elevated food intake and higher regional fat depots. S at 10 mg/kg decreased food intake in the HFD and epididymal fat in the HCD group. S also reduced perirenal fat in the HCD and HPD groups. Leptin levels were higher in rats fed with either the HFD or the HPD compared to those fed with the HCD. Moreover, S at 10 mg/kg decreased serum leptin levels in the HPD group. CONCLUSIONS: Results suggest a preferential effect of S on perirenal visceral fat and support the view that body fat loss is greater when its administration is accompanied by a HCD diet. No effect of S on leptin levels was found, besides that expected as a result of the decrease in body fat.     

A conditional export system provides new insights into protein export in Plasmodium falciparum-infected erythrocytes

The human malarial parasite Plasmodium falciparum exports determinants of virulence and pathology to destinations within its host erythrocyte, including the cytoplasm, the plasma membrane and membrane profiles of parasite origin termed Maurer's clefts. While there is some information regarding the signals that allot proteins for export, the trafficking route itself has remained largely obscure, partly due to technical limitations in following protein trafficking with time. To overcome these shortcomings, we have established a conditional protein export system in P. falciparum, based on the previously described conditional aggregation domain (CAD domain) that self-aggregates in the endoplasmic reticulum in a manner that is reversible by the addition of a small molecule. By fusing the CAD domain to the first 80 amino acids of STEVOR and full-length PfSBP1, we were able to control export of a soluble and a transmembrane protein to the erythrocyte cytosol and the Maurer's clefts respectively. The conditional export system allowed us to study the temporal sequence of events of protein export and identify intermediate steps. We further explored the potential of the conditional export system in identifying factors that interact with exported proteins en route. Our data provide evidence for a physical interaction of exported proteins with the molecular chaperone PfBiP during early export steps.     

Ability of <Emphasis Type="Italic">Lactobacillus fermentum</Emphasis> to overcome host α-galactosidase deficiency,as evidenced by reduction of hydrogen excretion in rats consuming soya α-galacto-oligosaccharides

Background  

Soya and its derivatives represent nutritionally high quality food products whose major drawback is their high content of α-galacto-oligosaccharides. These are not digested in the small intestine due to the natural absence of tissular α-galactosidase in mammals. The passage of these carbohydrates to the large intestine makes them available for fermentation by gas-producing bacteria leading to intestinal flatulence. The aim of the work reported here was to assess the ability of α-galactosidase-producing lactobacilli to improve the digestibility of α-galacto-oligosaccharides in situ.     

Marked defects in the expression and glycosylation of alpha2-HS glycoprotein/fetuin-A in plasma from neonates with intrauterine growth restriction: proteomics screening and potential clinical implications

Intrauterine growth restriction (IUGR) has been associated with increased perinatal morbidity and mortality and increased morbidity and metabolic abnormalities later in life. IUGR is characterized as the failure of a fetus to achieve his or her genetic growth potential in utero. Altered protein expression profiles associated with IUGR may be informative on the pathologic mechanisms of this condition and might reveal potential markers for postnatal complications. The aim of this study was to compare protein profiles of umbilical cord plasma from IUGR and appropriate for gestational age full-term neonates. Blood samples from doubly clamped umbilical cord at delivery from 10 IUGR and 10 appropriate for gestational age full-term neonates were analyzed by two-dimensional electrophoresis and MS. Prominent changes of the alpha2-HS glycoprotein/fetuin-A were observed in IUGR cases. Specifically we showed that these changes occur primarily at the level of post-translational modifications of the protein. Using a combination of mass spectrometry and classical biochemical assays, single and heavy chain forms of fetuin-A were found to lack the normally present O-linked sialic acids in IUGR neonates. Fetuin A is a glycoprotein that has been associated with promotion of in vitro cell replication, fetal growth and osteogenesis, and protection from Gram-negative bacterial endotoxins. Prominent defects in glycosylation/sialylation of fetuin-A revealed by our study might be responsible for impaired function of fetuin-A, leading to deficient fetal growth, especially osteogenesis, and/or to the development of complications frequently seen later in the lives of IUGR neonates.     

Invasive plant integration into native plant–pollinator networks across Europe

The structure of plant–pollinator networks has been claimed to be resilient to changes in species composition due to the weak degree of dependence among mutualistic partners. However, detailed empirical investigations of the consequences of introducing an alien plant species into mutualistic networks are lacking. We present the first cross-European analysis by using a standardized protocol to assess the degree to which a particular alien plant species (i.e. Carpobrotus affine acinaciformis, Impatiens glandulifera, Opuntia stricta, Rhododendron ponticum and Solanum elaeagnifolium) becomes integrated into existing native plant–pollinator networks, and how this translates to changes in network structure.Alien species were visited by almost half of the pollinator species present, accounting on average for 42 per cent of the visits and 24 per cent of the network interactions. Furthermore, in general, pollinators depended upon alien plants more than on native plants. However, despite the fact that invaded communities received more visits than uninvaded communities, the dominant role of alien species over natives did not translate into overall changes in network connectance, plant linkage level and nestedness. Our results imply that although supergeneralist alien plants can play a central role in the networks, the structure of the networks appears to be very permeable and robust to the introduction of invasive alien species into the network.     

Importance of Proteins Controlling Initiation of DNA Replication in the Growth of the High-Pressure-Loving Bacterium Photobacterium profundum SS9

The molecular mechanism(s) by which deep-sea bacteria grow optimally under high hydrostatic pressure at low temperatures is poorly understood. To gain further insight into the mechanism(s), a previous study screened transposon mutant libraries of the deep-sea bacterium Photobacterium profundum SS9 and identified mutants which exhibited alterations in growth at high pressure relative to that of the parent strain. Two of these mutants, FL23 (PBPRA3229::mini-Tn10) and FL28 (PBPRA1039::mini-Tn10), were found to have high-pressure sensitivity and enhanced-growth phenotypes, respectively. The PBPRA3229 and PBPRA1039 genes encode proteins which are highly similar to Escherichia coli DiaA, a positive regulator, and SeqA, a negative regulator, respectively, of the initiation of DNA replication. In this study, we investigated the hypothesis that PBPRA3229 and PBPRA1039 encode DiaA and SeqA homologs, respectively. Consistent with this, we determined that the plasmid-carried PBPRA3229 and PBPRA1039 genes restored synchrony to the initiation of DNA replication in E. coli mutants lacking DiaA and SeqA, respectively. Additionally, PBPRA3229 restored the cold sensitivity phenotype of an E. coli dnaA(Cs) diaA double mutant whereas PBPRA1039 suppressed the cold sensitivity phenotype of an E. coli dnaA(Cs) single mutant. Taken together, these findings show that the genes disrupted in FL23 and FL28 encode DiaA and SeqA homologs, respectively. Consequently, our findings add support to a model whereby high pressure affects the initiation of DNA replication in P. profundum SS9 and either the presence of a positive regulator (DiaA) or the removal of a negative regulator (SeqA) promotes growth under these conditions.Despite the fact that more than 70% of the earth''s surface is covered by oceans, which have an average temperature of 3°C and exert an average hydrostatic pressure of 38 MPa (atmospheric pressure is ∼0.1 MPa), little is understood about the molecular basis of cold- and high-pressure-adapted deep-ocean life. The discovery and isolation of the pyschrotolerant facultative piezophile (high-pressure-loving organism) Photobacterium profundum SS9 (8) have made it possible to more readily address the mechanisms of piezophilic growth at cold temperatures (for a recent review, see reference 3). P. profundum SS9 is a gammaproteobacterium originally isolated from an amphipod homogenate obtained from the Sulu Sea in the Philippines at a depth of 2.5 km and a temperature of 9°C (8). Although it grows optimally at 28 MPa and 15°C, P. profundum SS9 can also grow over a wide range of pressures (0.1 to 90 MPa) and temperatures (2 to 20°C). The ability to grow at atmospheric pressure has made P. profundum SS9 more amenable to genetic manipulation than obligate piezophiles. The P. profundum SS9 genome has been sequenced and annotated (26) and consists of two chromosomes and an 80-kb plasmid. It was determined that the 80-kb plasmid is nonessential for the piezophilic growth of P. profundum SS9 (26).To gain insights into the genetic basis of high-pressure-adapted growth, transposon mutant libraries of P. profundum SS9R (a rifampin [rifampicin]-resistant derivative of SS9) were screened in liquid culture for mutants with defects in the ability to grow at high pressure (45 MPa, 15°C) (19). One of the putative high-pressure-sensitive mutants (FL23) isolated from these screens had a mini-Tn10 insertion in the gene PBPRA3229, which encodes a protein with 75% identity (85% similarity) to Escherichia coli DiaA (DnaA initiator-associating factor) (14). Although FL23 shows growth defects at 0.1 MPa (15°C) relative to the parent strain, the ratio of growth at 45 MPa to growth at 0.1 MPa and 15°C is substantially reduced compared to that of the parent strain, confirming that disruption of PBPRA3229 results in a high-pressure sensitivity growth phenotype (19).In E. coli, DiaA is necessary to ensure the timely initiation of DNA replication (14). DiaA forms a tetramer and binds to multiple molecules of DnaA, promoting (i) the binding of DnaA to the origin of replication in E. coli (known as oriC), (ii) ATP-DnaA-specific conformational changes in the oriC complex, and (iii) the unwinding of oriC DNA (17). Consequently, E. coli DiaA acts as a positive regulator of the initiation of DNA replication. In the absence of DiaA, initiation of DNA replication is delayed and in E. coli cells with two oriC copies, it only occurs from one of these, resulting in cells with three copies of their chromosome (14). In contrast, this is an extremely rare occurrence in wild-type E. coli cells. Although disruption of diaA in E. coli results in an asynchronous DNA replication phenotype, it does not appear to affect growth or morphology at atmospheric pressure at 37°C in a genetic background with a wild-type dnaA gene. However, disruption of the diaA gene suppresses the cold sensitivity phenotype of an E. coli dnaA(Cs) mutant at 30°C.Even though PBPRA3229 is highly similar to E. coli DiaA, it also shows 45% identity (65% similarity) to a phosphoheptose isomerase in E. coli known as GmhA (4). GmhA is involved in lipopolysaccharide (LPS) biosynthesis and catalyzes the isomerization of d-sedoheptulose 7-phosphate into d-glycero-d-manno-heptose 7-phosphate, which is the first step in the biosynthesis of ADP-glycero-manno-heptose, a subunit of the LPS inner core. The LPS forms the outermost leaflet of the outer membrane of gram-negative bacterial cells, and in E. coli K-12 strains, the LPS is composed of inner and outer sugar cores and lipid A (25). E. coli K-12 mutants lacking GmhA produce truncated LPS species relative to that of the parent strain due to the absence of the inner core, which can be easily visualized by gel electrophoresis followed by silver staining (4). Due to the high degree of sequence similarity between PBPRA3229 and GmhA, it is also possible that FL23 has an alteration in its LPS relative to that of the parent strain.In contrast to DiaA, SeqA is a negative regulator of the initiation of DNA replication in E. coli (20). E. coli SeqA binds to hemimethylated oriC and prevents the binding of ATP-DnaA. Disruption of seqA in E. coli also results in an asynchronous-replication phenotype. However, the effect of DiaA on the timing of DNA replication initiation appears to be SeqA independent (14). Interestingly, a putative P. profundum SS9R seqA transposon insertion mutant (PBPRA1039::Tn10) was identified as having high-pressure-enhanced growth at 45 MPa and 15°C relative to its growth at atmospheric pressure (19). Therefore, this preliminary finding suggests that the removal of a negative regulator of the initiation of DNA replication could promote the growth of P. profundum SS9R at high pressure.In this study, we investigated the hypothesis that proteins that regulate the initiation of DNA replication play a key role in the piezophilic growth of P. profundum SS9. We determined that PBPRA3229 and PBPRA1039 encode functional DiaA and SeqA homologs, respectively, and we propose a model whereby the initiation of DNA replication is sensitive to high pressure and either the production of a positive regulator (DiaA) or the removal of a negative regulator (SeqA) can promote growth under these conditions.     

The impact of <Emphasis Type="Italic">Solanum elaeagnifolium</Emphasis>, an invasive plant in the Mediterranean,on the flower visitation and seed set of the native co-flowering species <Emphasis Type="Italic">Glaucium flavum</Emphasis>

The sprouting response types of 1,151 cork oak (Quercus suber) trees one and half years after a wildfire in southern Portugal were characterised. It was hypothesised that different response types should occur according to the following conceptual model: an increased level of damage (fire severity) on a sprouting tree that suffered a crown fire was expected to be reflected in a sequence of four alternative events, namely (a) resprouting exclusively from crown, (b) simultaneous resprouting from crown and base, (c) resprouting exclusively from base and (d) plant death. To assess whether the level of expected damage was influenced by the level of protection from disturbance, we explored the relationships between response types and tree size, bark thickness and cork stripping, using an information-theoretic approach. The more common response type was crown resprouting (68.8% of the trees), followed by plant death (15.8%), simultaneous resprouting from crown and base (10.1%) and basal resprouting (5.3%). In agreement with the conceptual model, trees which probably suffered a higher level of damage by fire (larger trees with thinner bark; exploited for cork) died or resprouted exclusively from base. On the other hand, trees that were well protected (smaller trees with thicker bark not exploited for cork) were able to rebuild their canopy through crown resprouting. Simultaneous resprouting from the crown and base was determined mainly by tree size, and it was more common in smaller trees.