Developmental mechanisms and experimental models to understand forebrain malformative diseases

The development of the central nervous system can be divided into a number of phases, each of which can be subject of genetic or epigenetic alterations that may originate particular developmental disorders. In recent years, much progress has been made in elucidating the molecular and cellular mechanisms by which the vertebrate forebrain develops. Therefore, our understanding of major developmental brain disorders such as cortical malformations and neuronal migration disorders has significantly increased. In this review, we will describe the major stages in forebrain morphogenesis and regionalization, with special emphasis on developmental molecular mechanisms derailing telencephalic development with subsequent damage to cortical function. Because animal models, mainly mouse, have been fundamental for this progress, we will also describe some characteristic mouse models that have been capital to explore these molecular mechanisms of malformative diseases of the human brain. Although most of the genes involved in the regulation of basic developmental processes are conserved among vertebrates, the extrapolation of mouse data to corresponding gene expression and function in humans needs careful individual analysis in each functional system.     

Determination of glibenclamide in human plasma by solid-phase extraction and high-performance liquid chromatography

A sensitive high-performance liquid chromatographic method for determination of intact glibenclamide in human plasma has been developed. Sample clean-up prior to chromatographic analysis was accomplished by extraction of the drug using a solid-phase RP-8 or RP-18 cartridge instead of the conventional liquid-liquid extraction methods described. For the separation of the drug from the endogenous components a reversed-phase column (LiChrosorb RP-8) of 5 μm particle size and 250×4 mm I.D., together with a mobile phase consisting of acetonitrile-12 μM perchloric acid (47:53) was selected. The method employs progesterone as an internal standard, and a reversed-phase column combined with UV detection of the drug at 230 nm. The detector response was linear up to the concentration of 400 ng/ml and the average recovery was 100.36%. The sensitivity of the method was 5 ng/ml.     

Reassessment of the Systematic Status of Miamira Bergh, 1875 and Orodoris Bergh, 1875 (Nudibranchia: Chromodorididae) in Light of Phylogenetic Analysis

The external morphology and anatomy of the opisthobranch gastropodsMiamira sinuata (van Hasselt, 1824) and Orodoris miamiranaBergh, 1875, the type species of the genera Miamira Bergh, 1875and Orodoris Bergh, 1875, and their phylogenetic relationshipsare studied. The phylogeny obtained supports the placement ofM. sinuata and O. miamirana in the genus Ceratosoma J. E. Gray, 1850.Therefore, Miamira and Orodoris become synonyms of the seniorvalid name Ceratosoma. In addition, the family name MiamiridaeBergh, 1891, based on Miamira, is newly recognized as a synonymof Chromodorididae Bergh, 1891. Ceratosoma sinuata and C. miamirana are more closely relatedto the highly derived Ceratosoma alleni than to other membersof the genus. C. miamirana appears to present reversal to theplesiomorphic state in the body shape and has secondarily lostits mantle glands. (Received 5 January 1998; accepted 23 April 1998)     

Quaternary climate changes explain diversity among reptiles and amphibians

It is widely believed that contemporary climate determines large-scale patterns of species richness. An alternative view proposes that species richness reflects biotic responses to historic climate changes. These competing "contemporary climate" vs "historic climate" hypotheses have been vigorously debated without reaching consensus. Here, we test the proposition that European species richness of reptiles and amphibians is driven by climate changes in the Quaternary. We find that climate stability between the Last Glacial Maximum (LGM) and the present day is a better predictor of species richness than contemporary climate; and that the 0°C isotherm of the LGM delimits the distributions of narrow-ranging species, whereas the current 0°C isotherm limits the distributions of wide-ranging species. Our analyses contradict previous studies of large-scale species richness patterns and support the view that "historic climate" can contribute to current species richness independently of and at least as much as contemporary climate.     

Generation of a histidine-tagged antibotulinum toxin antibody fragment in E. coli: effects of post-induction temperature on yield and IMAC binding-affinity

Recombinant E. coli clones expressing a 50-kDa poly-histidine tail tagged antibody fragment against botulinum toxin (bt-Fab) were initially screened for yield and binding affinity. One clone was selected for bioprocess development. The selected bt-Fab vector was induced by addition of IPTG and the protein was targeted to the periplasm by inclusion of a pelB leader sequence. A histidine₆ affinity ligand at the heavy chain C-terminus facilitated single-step purification by immobilized metal-affinity chromatography (IMAC). Notably, the effects of post-induction temperature on bt-Fab expression and downstream purification were evaluated. Our results demonstrated that fermentation conditions interfered with purification on the IMAC column at 37°C. Protease analysis by gelatin polyacrylamide gel electrophoresis (GPAGE) indicated the presence of a membrane-bound ∼39 kDa protease activity shortly after induction. The appearance of the protease activity was inversely correlated with the bt-Fab yield. The protease was purified and was shown to degrade bt-Fab. A simple kinetic model was developed describing temporal regulation of protease and bt-Fab degradation. Partially degraded bt-Fab was unrecoverable by IMAC, presumably due to the loss of the His₆ affinity ligand. The amount of purified bt-Fab obtained per liter of fermentation broth was typically ∼1 mg. Received 18 August 1997/ Accepted in revised form 4 October 1998     

CUP-1 is a novel protein involved in dietary cholesterol uptake in Caenorhabditis elegans

Sterols transport and distribution are essential processes in all multicellular organisms. Survival of the nematode Caenorhabditis elegans depends on dietary absorption of sterols present in the environment. However the general mechanisms associated to sterol uptake in nematodes are poorly understood. In the present work we provide evidence showing that a previously uncharacterized transmembrane protein, designated Cholesterol Uptake Protein-1 (CUP-1), is involved in dietary cholesterol uptake in C. elegans. Animals lacking CUP-1 showed hypersensitivity to cholesterol limitation and were unable to uptake cholesterol. A CUP-1-GFP fusion protein colocalized with cholesterol-rich vesicles, endosomes and lysosomes as well as the plasma membrane. Additionally, by FRET imaging, a direct interaction was found between the cholesterol analog DHE and the transmembrane "cholesterol recognition/interaction amino acid consensus" (CRAC) motif present in C. elegans CUP-1. In-silico analysis identified two mammalian homologues of CUP-1. Most interestingly, CRAC motifs are conserved in mammalian CUP-1 homologous. Our results suggest a role of CUP-1 in cholesterol uptake in C. elegans and open up the possibility for the existence of a new class of proteins involved in sterol absorption in mammals.     

G Protein–Coupled Receptor-Type G Proteins Are Required for Light-Dependent Seedling Growth and Fertility in Arabidopsis

G protein–coupled receptor-type G proteins (GTGs) are highly conserved membrane proteins in plants, animals, and fungi that have eight to nine predicted transmembrane domains. They have been classified as G protein–coupled receptor-type G proteins that function as abscisic acid (ABA) receptors in Arabidopsis thaliana. We cloned Arabidopsis GTG1 and GTG2 and isolated new T-DNA insertion alleles of GTG1 and GTG2 in both Wassilewskija and Columbia backgrounds. These gtg1 gtg2 double mutants show defects in fertility, hypocotyl and root growth, and responses to light and sugars. Histological studies of shoot tissue reveal cellular distortions that are particularly evident in the epidermal layer. Stable expression of GTG1_pro:GTG1-GFP (for green fluorescent protein) in Arabidopsis and transient expression in tobacco (Nicotiana tabacum) indicate that GTG1 is localized primarily to Golgi bodies and to the endoplasmic reticulum. Microarray analysis comparing gene expression profiles in the wild type and double mutant revealed differences in expression of genes important for cell wall function, hormone response, and amino acid metabolism. The double mutants isolated here respond normally to ABA in seed germination assays, root growth inhibition, and gene expression analysis. These results are inconsistent with their proposed role as ABA receptors but demonstrate that GTGs are fundamentally important for plant growth and development.     

A twin study of mitochondrial DNA polymorphisms shows that heteroplasmy at multiple sites is associated with mtDNA variant 16093 but not with zygosity

The mitochondrial theory of ageing proposes that damage to mitochondria and diminished mitochondrial DNA (mtDNA) repair are major contributors to cellular dysfunction and age-related diseases. We investigate the prevalence of heteroplasmy in the mtDNA control region in buccal swab and blood derived samples for 178 women from the TwinsUK cohort (41 DZ pair 39 MZ pairs, 18 singletons, mean age 57.5 range 28–82) and its relationship to age, BMI and fasting insulin and glucose serum levels. The overall estimated prevalence of heteroplasmy for both tissues in the control region measured for 37 sites was 17%. The prevalence of heteroplasmy was higher among the older half of the study subjects than in the younger half (23% vs 10% p<0.03), primarily reflecting the increase in the prevalence of a heteroplasmic dinucleotide CA repeat in variable region II (VRII) with age. The VRII 523–524 heteroplasmic site (heteroplasmic in 25 subjects) was also associated with a decrease in BMI. In addition, concordance rates for common heteroplasmy were observed to be near complete for both dizygotic (DZ = 94%) and monozygotic twin pairs (MZ = 100%), consistent with previous reports that suggest variation in heteroplasmy rates between generations are determined by bottlenecks in maternal transmission of mitochondria. Differences in the prevalence of heteroplasmy were observed overall between samples derived from buccal swabs (19%) and blood (15%, p<0.04). These were particularly marked at position 16093 of hypervariable region I (HVI, 7% vs 0%, respectively, p<4×10⁻¹¹). The presence of the C allele at position 16093 in blood was associated with the presence of heteroplasmy in buccal swabs at this position (p = 3.5×10⁻¹⁴) and also at VRII (p = 2×10⁻⁴) suggesting a possible predisposing role for this site in the accumulation of heteroplasmy. Our data indicate that BMI is potentially associated with control region heteroplasmy.     

Glycosylation of Immunoglobulin G: Role of Genetic and Epigenetic Influences

Objective

To determine the extent to which genetic and epigenetic factors contribute to variations in glycosylation of immunoglobulin G (IgG) in humans.

Methods

76  N-glycan traits in circulating IgG were analyzed by UPLC in 220 monozygotic and 310 dizygotic twin pairs from TwinsUK. A classical twin study design was used to derive the additive genetic, common and unique environmental components defining the variance in these traits. Epigenome-wide association analysis was performed using the Illumina 27k chip.

Results

51 of the 76 glycan traits studied have an additive genetic component (heritability, h ²)≥  0.5. In contrast, 12 glycan traits had a low genetic contribution (h²<0.35). We then tested for association between methylation levels and glycan levels (P<2 x10^-6). Among glycan traits with low heritability probe cg08392591 maps to a CpG island 5’ from the ANKRD11 gene, a p53 activator on chromosome 16. Probe cg26991199 maps to the SRSF10 gene involved in regulation of RNA splicing and particularly in regulation of splicing of mRNA precursors upon heat shock. Among those with high heritability we found cg13782134 (mapping to the NRN1L gene) and cg16029957 mapping near the QPCT gene to be array-wide significant. The proportion of array-wide epigenetic associations was significantly larger (P<0.005) among glycans with low heritability (42%) than in those with high heritability (6.2%).

Conclusions

Glycome analyses might provide a useful integration of genetic and non-genetic factors to further our understanding of the role of glycosylation in both normal physiology and disease.