A multipurpose vector system for the screening of libraries in bacteria, insect and mammalian cells and expression in vivo

We have constructed a novel tetra-promoter vector (pBVboostFG) system that enables screening of gene/cDNA libraries for functional genomic studies. The vector enables an all-in-one strategy for gene expression in mammalian, bacterial and insect cells and is also suitable for direct use in vivo. Virus preparation is based on an improved mini Tn7 transpositional system allowing easy and fast production of recombinant baculoviruses with high diversity and negligible background. Cloning of the desired DNA fragments or libraries is based on the recombination system of bacteriophage lambda. As an example of the utility of the vector, genes or cDNAs of 18 different proteins were cloned into pBVboostFG and expressed in different hosts. As a proof-of-principle of using the vector for library screening, a chromophoric Thr⁶⁵-Tyr-Gly⁶⁷-stretch of enhanced green fluorescent protein was destroyed and subsequently restored by novel PCR strategy and library screening. The pBVboostFG enables screening of genome-wide libraries, thus making it an efficient new platform technology for functional genomics.     

Neisserial pilin genes display extensive interspecies diversity

All Neisseria live in association with host cells, however, little is known about the genetic potential of nonpathogenic Neisseria species to express attachment factors such as pili. In this study, we demonstrate that type IV pilin-encoding genes are present in a wide range of Neisseria species. N. sicca, N. subflava, and N. elongata each contain two putative pilE genes arranged in tandem, while single genes were identified in N. polysaccharea, N. mucosa, and N. denitrificans. Neisserial pilE genes are highly diverse and display features consistent with a history of horizontal gene transfer.     

OSBP-Related Proteins (ORPs) in Human Adipose Depots and Cultured Adipocytes: Evidence for Impacts on the Adipocyte Phenotype

Oxysterol-binding protein (OSBP) homologues, ORPs, are implicated in lipid homeostatic control, vesicle transport, and cell signaling. We analyzed here the quantity of ORP mRNAs in human subcutaneous (s.c.) and visceral adipose depots, as well as in the Simpson-Golabi-Behmel syndrome (SGBS) adipocyte cell model. All of the ORP mRNAs were present in the s.c and visceral adipose tissues, and the two depots shared an almost identical ORP mRNA expression pattern. SGBS adipocytes displayed a similar pattern, suggesting that the adipose tissue ORP expression pattern mainly derives from adipocytes. During SGBS cell adipogenic differentiation, ORP2, ORP3, ORP4, ORP7, and ORP8 mRNAs were down-regulated, while ORP11 was induced. To assess the impacts of ORPs on adipocyte differentiation, ORP3 and ORP8, proteins down-regulated during adipogenesis, were overexpressed in differentiating SGBS adipocytes, while ORP11, a protein induced during adipogenesis, was silenced. ORP8 overexpression resulted in reduced expression of the aP2 mRNA, while down-regulation of adiponectin and aP2 was observed in ORP11 silenced cells. Furthermore, ORP8 overexpression or silencing of ORP11 markedly decreased cellular triglyceride storage. These data identify the patterns of ORP expression in human adipose depots and SGBS adipocytes, and provide the first evidence for a functional impact of ORPs on the adipocyte phenotype.     

Genetics of the neuronal ceroid lipofuscinoses

The neuronal ceroid lipofuscinoses (NCLs) are an intriguing group of inherited neurodegenerative disorders characterized by blindness, progressive psychomotor deterioration and death of neocortical neurons. Clinically, four major NCL groups have been identified: infantile, late infantile, juvenile and adult. In recent years, our understanding of the molecular basis of different NCLs has advanced significantly. The accumulation of autofluorescent material in patients' tissues has been shown to be caused by defects in either lysosomal enzymes or in novel membrane proteins of unknown function. Although the accumulated material is biochemically well defined and some of the causative mutations are known, a unifying hypothesis for the molecular basis of the NCLs remains elusive. Further work will be required to characterize the interactiving molecules and metabolic pathways involved in the pathogenesis of NCLs.     

TGF-beta type I receptor Alk5 regulates tooth initiation and mandible patterning in a type II receptor-independent manner

TGF-beta superfamily members signal through a heteromeric receptor complex to regulate craniofacial development. TGF-beta type II receptor appears to bind only TGF-beta, whereas TGF-beta type I receptor (ALK5) also binds to ligands in addition to TGF-beta. Our previous work has shown that conditional inactivation of Tgfbr2 in the neural crest cells of mice leads to severe craniofacial bone defects. In this study, we examine and compare the defects of TGF-beta type II receptor (Wnt1-Cre;Tgfbr2(fl/fl)) and TGF-beta type I receptor/Alk5 (Wnt1-Cre;Alk5(fl)(/fl)) conditional knockout mice. Loss of Alk5 in the neural crest tissue resulted in phenotypes not seen in the Tgfbr2 mutant, including delayed tooth initiation and development, defects in early mandible patterning and altered expression of key patterning genes including Msx1, Bmp4, Bmp2, Pax9, Alx4, Lhx6/7 and Gsc. Alk5 controls the survival of CNC cells by regulating expression of Gsc and other genes in the proximal aboral region of the developing mandible. We conclude that ALK5 regulates tooth initiation and early mandible patterning through a pathway independent of Tgfbr2. There is an intrinsic requirement for Alk5 signal in regulating the fate of CNC cells during tooth and mandible development.     

Binding of LL-37 to model biomembranes: insight into target vs host cell recognition

Pursuing the molecular mechanisms of the concentration dependent cytotoxic and hemolytic effects of the human antimicrobial peptide LL-37 on cells, we investigated the interactions of this peptide with lipids using different model membranes, together with fluorescence spectroscopy for the Trp-containing mutant LL-37(F27W). Minimum concentrations inhibiting bacterial growth and lipid interactions assessed by dynamic light scattering and monolayer penetration revealed the mutant to retain the characteristics of native LL-37. Although both LL-37 and the mutant intercalated effectively into zwitterionic phosphatidylcholine membranes the presence of acidic phospholipids caused augmented membrane binding. Interestingly, strongly attenuated intercalation of LL-37 into membranes containing both cholesterol and sphingomyelin (both at X=0.3) was observed. Accordingly, the distinction between target and host cells by LL-37 is likely to derive from i) acidic phospholipids causing enhanced association with the former cells as well as ii) from attenuated interactions with the outer surface of the plasma membrane of the peptide secreting host, imposed by its high content of cholesterol and sphingomyelin. Our results further suggest that LL-37 may exert its antimicrobial effects by compromising the membrane barrier properties of the target microbes by a mechanism involving cytotoxic oligomers, similarly to other peptides forming amyloid-like fibers in the presence of acidic phospholipids.     

Microplate screening assay for binding of ligands to bovine or reindeer beta-lactoglobulins

Several analytical methods have been used to determine whether ligands bind to bovine beta-lactoglobulin (betaLG). The most common methods are based on fluorescence quenching. We have miniaturised this method from a quartz cell to a 96-well plate. The miniaturisation was evaluated using retinol. The binding constants between the two methods demonstrated a good correlation. The 96-well plate method is much faster and allows many references to be used in the same analysis. The miniaturised method was used to study the binding of three different ligands (4-HPR, arotinoid, warfarinyl palmitate) modelled to bind to betaLG. The binding data showed that all of these ligands bound to betaLG. The method was further used to demonstrate that reindeer betaLG could also bind the four ligands in the same way as bovine betaLG. Because one aim is to use bovine and reindeer betaLG as a binder molecule for aliments in e.g. functional food or for drugs, the influence of pH was also studied and demonstrated that short-term acidic conditions had only a slight effect on the binding properties.     

Symbiotic nitrogen fixation in eight Acacia senegal provenances in dryland clays of the Blue Nile Sudan estimated by the 15N natural abundance method

The symbiotic biological N₂fixation by Acacia senegal was estimated using the ¹⁵N natural abundance (δ ¹⁵N) procedure on eight provenances collected from different environments and soil types grown in a clay soil in the Blue Nile region, Sudan. Balanites aegyptiaca (a non-legume) was used as a non-N₂-fixing reference plant to allow ¹⁵N-based estimates of the proportion of the Acacia N derived from atmospheric N₂ (N_dfa) to be calculated. Results show variation in leaf δ ¹⁵N between A. senegal and the reference plant and among years. The relative δ ¹⁵N values (‰) were higher in B. aegyptiaca than in the N₂-fixing acacia provenances. Provenances originally collected from clay soils fixed little N in the first year, but the amount fixed increased as the trees aged. All provenances showed a decrease in δ ¹⁵N with age. The N_dfa varied between 24% (Mazmoom provenance) and 61% (Rahad provenance) 4 years after planting. There was no significant difference in δ ¹⁵N between provenance groups based on soil type or rainfall at original growing site. The amount of N_dfa increased significantly with age in all provenances. The above-ground contribution of fixed N to foliage growth in a 4-year-old A. senegal was highest in the Rahad sand–soil provenance (46.7 kg N ha⁻¹) and lowest in the Mazmoom clay-soil provenance (28.7 kg N ha⁻¹). Our study represents the first use of the δ ¹⁵N method for estimating the N input by A. senegal to the clay plain soils of the gum belt in the Sudan.     

Young flying squirrels (Pteromys volans) dispersing in fragmented forests

Dispersal is a key determinant of the population dynamics ofspecies. Thus, a better understanding of how dispersal is affectedby the landscape structure and how animals make decisions aboutmoving across different landscapes is needed. We studied thedispersal of 60 radio-collared juvenile Siberian flying squirrels(Pteromys volans) in southern Finland. The effect of landscapestructure on selected dispersal direction, dispersal distance,and straightness of dispersal path was studied. Flying squirrelswere capable of dispersing over long distances in fragmentedforest landscapes. The patches used as temporary roosting sitesduring dispersal were of a lower quality than were those usedas finally occupied patches. The patches used were larger thanwere patches on average in the study areas. There was a veryclear directional bias in the dispersal path (i.e., it was nearlya straight line), which remained over a large scale, but wide-openareas obstructed the straightness of the path. As the distancesbetween crossed patches increased, short-distance disperserswere found further away from their natal home range. However,there were no differences in the landscape that could explainthe differences between individuals in decisions to remain philopatricor to become short- or long-distance dispersers. In addition,whereas short-distance dispersers dispersed in random directions,long-distance dispersers started to disperse in directions dominatedby preferred habitat. Thus, there were behavioral differencesbetween dispersers. Our results supported the hypotheses statingthat individuals decide to disperse long or short distancesbefore the onset of dispersal.