A new locus for coeliac disease mapped to chromosome 15 in a population isolate

Coeliac disease is a common multifactorial disease with a strong genetic component, which is not entirely explained by the HLA association. Four previous whole-genome screens have produced somewhat inconsistent results suggesting genetic heterogeneity. We attempted to overcome this problem by performing a genome-wide scan in a Finnish sub-population, expected to be more homogeneous than the general population of Finland. The families in our study originate from the northeastern part of Finland, the Koilliskaira region, which has been relatively isolated since its founding in the 16th century. Genealogical studies have confirmed that the families share a common ancestor in the 16th century. Nine families with altogether 23 patients were genotyped for 399 microsatellite markers and the data were analysed with parametric linkage analysis using two dominant and one recessive model. A region on chromosome 15q11-q13 was implicated with a LOD score of 3.14 using a highly penetrant dominant model. Addition of more markers and one more sib-pair increased the LOD score to 3.74. This result gives preliminary evidence for existence of a susceptibility factor in this chromosomal region.     

Refuge sites of voles under owl predation risk: priority of dominant individuals?

In an aviary experiment, we studied whether body size or habitatfamiliarity of field voles (Microtus agrestis) affected predationrisk by Tengmalm's owls (Aegolius funereus). In the field, wecompared the body size of field voles snap-trapped in good (covered)and poor (open) habitats in 1992 and 1994 to determine whetherthere were habitat-related differences in the body size of voles.In the aviary, large individuals occupied the good habitat significantlymore than small individuals both in the control (owl not present)and experimental treatments (owl present). Furthermore, habitat-familiarvoles inhabited the good habitat more than habitat-unfamiliarvoles did when an owl was present Our field data were consistentwith our aviary data: larger field voles were more frequentlyfound in good habitats than in poor habitats. In the aviary,Tengmalm's owl predation risk was higher for small and habitat-unfamiliarvoles. This suggests that large field voles may have priorityto sheltered habitats. Furthermore, habitat familiarity mayplay a central role in avoiding risky habitats.     

Neuromuscular function during drop jumps in young and elderly males

The Hoffman reflex (H-reflex), indicating alpha-motoneuron pool activity, has been shown to be task – and in resting conditions – age dependent. How aging affects H-reflex activity during explosive movements is not clear at present. The purpose of this study was to examine the effects of aging on H-reflexes during drop jumps, and its possible role in drop jump performance. Ten young (26.8 ± 2.7 years) and twenty elderly (64.2 ± 2.7 years) subjects participated in the study. Maximal drop jump performance and soleus H-reflex response (H/M jump) 20 ms after ground contact were measured in a sledge ergometer. Maximal H-reflex, maximal M-wave, Hmax/Mmax-ratio and H-reflex excitability curves were measured during standing rest. Although in young the H-reflex response (Hmax/Mmax) was 6.5% higher during relaxed standing and 19.7% higher during drop jumps (H jump/M jump) than in the elderly group, these differences were not statistically significant. In drop jumps, the elderly subjects had lower jumping height (30.4%, p < 0.001), longer braking time (32.4%, p < 0.01), lower push-off force (18.0%, p < 0.05) and longer push-off time (31.0% p < 0.01). H jump/M jump correlated with the average push-off force (r = 0.833, p < 0.05) and with push-off time (r = ?0.857, p < 0.01) in young but not in the elderly. Correlations between H-reflex response and jumping parameters in young may indicate different jumping and activation strategies in drop jumps. However, it does not fully explain age related differences in jumping performance, since age related differences in H-reflex activity were non-significant.     

DNA family shuffling within the chicken avidin protein family - A shortcut to more powerful protein tools

Avidins represent an interesting group of proteins showing high structural similarity and ligand-binding properties but low similarity in primary structure. In this study, we show that it is possible to create functional chimeric proteins from the avidin protein family when applying DNA family shuffling to the genes of the avidin protein family: avidin, avidin related gene 2 and biotin-binding protein A. The novel chimeric proteins were selected by phage display biopanning against biotin, and the selected enriched proteins were characterized, displaying diverse features distinct from the parental genes, including binding to cysteine.     

The Wnt pool of glycogen synthase kinase 3beta is critical for trophic-deprivation-induced neuronal death

Glycogen synthase kinase 3 (GSK-3) is implicated in neuronal death through a causal role, and precise mechanisms have not been unambiguously defined. We show that short hairpin RNA (shRNA) knockdown of GSK-3β, but not GSK-3α, protects cerebellar granule neurons from trophic-deprivation-induced death. Using compartment-targeted inhibitors of the Wnt-regulated GSK-3 pool, NLS-FRAT1, NES-FRAT1, and axin-GSK-3-interacting domain (axin-GID), we locate proapoptotic GSK-3 action to the cytosol and regulation of Bim protein turnover despite constitutive cycling of GSK-3 between the cytosol and nucleus, revealed by leptomycin B. We examine the importance of Ser21/9 (GSK-3α/β) phosphorylation on proapoptotic GSK-3 function. Neurons isolated from GSK-3α/β^S21A/S9A knock-in mice survive normally and are fully sensitive to trophic-deprivation-induced death. Nonetheless, inhibition of GSK-3 catalytic activity with lithium or SB216763 protects GSK-3α/β^S21A/S9A neurons from death. This indicates that dephosphorylation of GSK-3β/Ser9 and GSK-3α/Ser21 is insufficient for GSK-3 proapoptotic function and that another level of regulation is required. Gel filtration reveals a stress-induced loss of neuronal GSK-3β from a high-molecular-mass complex with a concomitant decrease in axin-bound GSK-3β. These data imply that Wnt-regulated GSK-3β plays a nonredundant role in trophic-deprivation-induced death of neurons.     

Cardiac outflow tract defects in mice lacking ALK2 in neural crest cells

Cardiac neural crest cells are multipotent migratory cells that contribute to the formation of the cardiac outflow tract and pharyngeal arch arteries. Neural crest-related developmental defects account for a large proportion of congenital heart disorders. Recently, the genetic bases for some of these disorders have been elucidated, and signaling pathways required for induction, migration and differentiation of cardiac neural crest have emerged. Bone morphogenetic proteins comprise a family of secreted ligands implicated in numerous aspects of organogenesis, including heart and neural crest development. However, it has remained generally unclear whether BMP ligands act directly on neural crest or cardiac myocytes during cardiac morphogenesis, or function indirectly by activating other cell types. Studies on BMP receptor signaling during organogenesis have been hampered by the fact that receptor knockouts often lead to early embryonic lethality. We have used a Cre/loxP system for neural crest-specific deletion of the type I receptor, ALK2, in mouse embryos. Mutant mice display cardiovascular defects, including persistent truncus arteriosus, and abnormal maturation of the aortic arch reminiscent of common forms of human congenital heart disease. Migration of mutant neural crest cells to the outflow tract is impaired, and differentiation to smooth muscle around aortic arch arteries is deficient. Moreover, in Alk2 mutants, the distal outflow tract fails to express Msx1, one of the major effectors of BMP signaling. Thus, the type I BMP receptor ALK2 plays an essential cell-autonomous role in the development of the cardiac outflow tract and aortic arch derivatives.     

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.     

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.