Mutations in BICD2, which Encodes a Golgin and Important Motor Adaptor,Cause Congenital Autosomal-Dominant Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is a heterogeneous group of neuromuscular disorders caused by degeneration of lower motor neurons. Although functional loss of SMN1 is associated with autosomal-recessive childhood SMA, the genetic cause for most families affected by dominantly inherited SMA is unknown. Here, we identified pathogenic variants in bicaudal D homolog 2 (Drosophila) (BICD2) in three families afflicted with autosomal-dominant SMA. Affected individuals displayed congenital slowly progressive muscle weakness mainly of the lower limbs and congenital contractures. In a large Dutch family, linkage analysis identified a 9q22.3 locus in which exome sequencing uncovered c.320C>T (p.Ser107Leu) in BICD2. Sequencing of 23 additional families affected by dominant SMA led to the identification of pathogenic variants in one family from Canada (c.2108C>T [p.Thr703Met]) and one from the Netherlands (c.563A>C [p.Asn188Thr]). BICD2 is a golgin and motor-adaptor protein involved in Golgi dynamics and vesicular and mRNA transport. Transient transfection of HeLa cells with all three mutant BICD2 cDNAs caused massive Golgi fragmentation. This observation was even more prominent in primary fibroblasts from an individual harboring c.2108C>T (p.Thr703Met) (affecting the C-terminal coiled-coil domain) and slightly less evident in individuals with c.563A>C (p.Asn188Thr) (affecting the N-terminal coiled-coil domain). Furthermore, BICD2 levels were reduced in affected individuals and trapped within the fragmented Golgi. Previous studies have shown that Drosophila mutant BicD causes reduced larvae locomotion by impaired clathrin-mediated synaptic endocytosis in neuromuscular junctions. These data emphasize the relevance of BICD2 in synaptic-vesicle recycling and support the conclusion that BICD2 mutations cause congenital slowly progressive dominant SMA.     

Glucocorticoids regulate transendothelial fluid flow resistance and formation of intercellular junctions

The regulation of transendothelial fluid flow by glucocorticoidswas studied in vitro with use of human endothelial cells cultured fromSchlemm's canal (SCE) and the trabecular meshwork (TM) in conjunctionwith computer-linked flowmeters. After 2-7 wk of 500 nMdexamethasone (Dex) treatment, the following physiological, morphometric, and biochemical alterations were observed: a 3- to 5-foldincrease in fluid flow resistance, a 2-fold increase in therepresentation of tight junctions, a 10- to 30-fold reduction in themean area occupied by interendothelial "gaps" or preferential flow channels, and a 3- to 5-fold increase in the expression of thejunction-associated protein ZO-1. The more resistive SCE cells expressed two isoforms of ZO-1; TM cells expressed only one. To investigate the role of ZO-1 in the aforementioned Dex effects, itsexpression was inhibited using antisense phosphorothioate oligonucleotides, and the response was compared with that observed withthe use of sense and nonsense phosphorothioate oligonucleotides. Inhibition of ZO-1 expression abolished the Dex-induced increase inresistance and the accompanying alterations in cell junctions and gaps.These results support the hypothesis that intercellular junctions arenecessary for the development and maintenance of transendothelial flowresistance in cultured SCE and TM cells and are likely involved in themechanism of increased resistance associated with glucocorticoid exposure.

     

Choline head groups stabilize the matrix loop regions of the ATP/ADP carrier ScAAC2

ATP/ADP carriers (AACs) are essential to the cell as they exchange ATP produced in mitochondria for cytosolic ADP. Monoclonal antibodies against the isoform 2 of Saccharomyces cerevisiae AAC (ScAAC2) were used to probe the accessibility of the matrix loops 1 and 3 depending on the environment of the carrier. In mitochondrial membranes ScAAC2 was not recognized, whereas in dodecylmaltoside the antibodies bound to the carrier, suggesting that the epitopes are hidden in the native environment. Exposure of the epitopes by detergents was reversed by reconstitution of the carrier in phospholipids or by exchanging with detergents having a choline or a trimethylammonium head group. Circular dichroism spectroscopy on peptides representing the C-terminal regions of all three matrix loops showed that only phosphocholine detergents induced a structural reorganization. Since in addition phosphatidylcholine was found to be tightly associated with the purified carrier, the matrix loop regions are likely to be associated to the membrane by phosphatidylcholine.     

Bioavailability of anthocyanidin-3-glucosides following consumption of red wine and red grape juice

Pharmacokinetic parameters and the bioavailability of several dietary anthocyanins following consumption of red wine and red grape juice were compared in nine healthy volunteers. They were given a single oral dose of either 400 mL of red wine (279.6 mg total anthocyanins) or 400 mL of red grape juice (283.5 mg total anthocyanins). Within 7 h, the urinary excretion of total anthocyanins was 0.23 and 0.18% of the administered dose following red grape juice and red wine ingestion, respectively. Pharmacokinetic parameters derived from plasma and urine concentrations exhibited higher variability after ingestion of red grape juice. Compared to red grape juice anthocyanins, the relative bioavailability of red wine anthocyanins was calculated to be 65.7, 61.3, 61.9, 291.5, 57.1, and 76.3% for the glucosides of cyanidin, delphinidin, malvidin, peonidin, petunidin, and its sum (referred to as total anthocyanins), respectively. Bioequivalence was established for none of the anthocyanins. On a low level, urinary excretion of anthocyanins was fast, and the excretion rates seem to exhibit monoexponential characteristics over time after ingestion of both red grape juice and red wine. Due to low bioavailability, any significant contribution of anthocyanins to health protecting properties of red wine or red grape juice seems questionable, but the clinical relevance of these findings awaits further investigation.     

Functional reconstitution of purified metabotropic glutamate receptor expressed in the fly eye

G-protein-coupled receptors (GPCRs) form one of the largest superfamilies of membrane proteins. Obtaining high yields of GPCRs remains one of the major factors limiting a detailed understanding of their structure and function. Photoreceptor cells (PRCs) contain extensive stacks of specialized membranes where high levels of rhodopsins are naturally present, which makes them ideal for the overexpression of GPCRs. We have generated transgenic flies expressing a number of GPCRs in the PRCs. Drosophila melanogaster metabotropic glutamate receptor (DmGluRA) expressed by this novel strategy was purified to homogeneity, giving at least 3-fold higher yields than conventional baculovirus expression systems due to the higher membrane content of the PRCs. Pure DmGluRA was then reconstituted into liposomes of varying composition. Interestingly, glutamate binding was strictly dependent on the presence of ergosterol.     

Epoxysuccinyl peptide-derived cathepsin B inhibitors: modulating membrane permeability by conjugation with the C-terminal heptapeptide segment of penetratin

Besides its physiological role in lysosomal protein breakdown, extralysosomal cathepsin B has recently been implicated in apoptotic cell death. Highly specific irreversible cathepsin B inhibitors that are readily cell-permeant should be useful tools to elucidate the effects of cathepsin B in the cytosol. We have covalently functionalised the poorly cell-permeant epoxysuccinyl-based cathepsin B inhibitor [R-Gly-Gly-Leu-(2S,3S)-tEps-Leu-Pro-OH; R=OMe] with the C-terminal heptapeptide segment of penetratin (R=epsilonAhx-Arg-Arg-Nle-Lys-Trp-Lys-Lys-NH2). The high inhibitory potency and selectivity for cathepsin B versus cathepsin L of the parent compound was not affected by the conjugation with the penetratin heptapeptide. The conjugate was shown to efficiently penetrate into MCF-7 cells as an active inhibitor, thereby circumventing an intracellular activation step that is required by other inhibitors, such as the prodrug-like epoxysuccinyl peptides E64d and CA074Me.     

The ramification of Apinagia riedelii: A key to the understanding of the plant architecture of Podostemaceae,subfamily Podostemoideae

The study of the ramification pattern of Apinagia riedelii results in a new concept of the architecture of this species, with general implications to members of subfamily Podostemoideae with dithecous leaves. The presence of a subtending leaf below the floriferous shoot proves axillary branching also for species with dithecous leaves. Previous opinions of an unusual ramification mode by subfoliar or non-axillary branching or stem bifurcation in combination with dithecous leaves hitherto pleaded for Podostemoideae is refuted. Moreover, the view of the so-called dithecous leaves with one sheath (theca) at the ventral and one at the dorsal side of the leaf, previously regarded as initially connected with branching, has to be changed. The dithecous leaf arises from the branch and not from the mother shoot axis – as previously believed – and represents the addorsed hypsophyll, i.e., the first leaf (prophyll) of the floriferous branch. This finding leads to the conclusion that the lower sheath of the dithecous leaf is the ventral (not dorsal) sheath pointing to the branch and surrounding its flower bud with a ligule or an ochrea and a hood upon the bud. In this way, the branch and its flower bud become seemingly sunk in the leaf base. At the fusion of leaf basis and shoot results this enigmatic common tissue. The wings of the dorsal (upper) sheath of the dithecous leaf point to the mother shoot axis of the branch. Successive floriferous branches along the main stem disclose the shoot axis of A. riedelii as a monopodium (not sympodium) that develops an anthocladial (foliated) inflorescence in the form of a botrys or a compound botrys, respectively. Since it is generally difficult to define cymose or racemose inflorescences if subtending leaves are absent – which occur in most other species of subfamily Podostemoideae with dithecous leaves – the nature of these inflorescences is discussed anew. The findings on A. riedelii have consequences on our comprehension of the shoot architecture of Podostemoideae.