Lipid Binding Defects and Perturbed Synaptogenic Activity of a Collybistin R290H Mutant That Causes Epilepsy and Intellectual Disability

Signaling at nerve cell synapses is a key determinant of proper brain function, and synaptic defects—or synaptopathies—are at the basis of many neurological and psychiatric disorders. In key areas of the mammalian brain, such as the hippocampus or the basolateral amygdala, the clustering of the scaffolding protein Gephyrin and of γ-aminobutyric acid type A receptors at inhibitory neuronal synapses is critically dependent upon the brain-specific guanine nucleotide exchange factor Collybistin (Cb). Accordingly, it was discovered recently that an R290H missense mutation in the diffuse B-cell lymphoma homology domain of Cb, which carries the guanine nucleotide exchange factor activity, leads to epilepsy and intellectual disability in human patients. In the present study, we determined the mechanism by which the Cb^R290H mutation perturbs inhibitory synapse formation and causes brain dysfunction. Based on a combination of biochemical, cell biological, and molecular dynamics simulation approaches, we demonstrate that the R290H mutation alters the strength of intramolecular interactions between the diffuse B-cell lymphoma homology domain and the pleckstrin homology domain of Cb. This defect reduces the phosphatidylinositol 3-phosphate binding affinity of Cb, which limits its normal synaptogenic activity. Our data indicate that impairment of the membrane lipid binding activity of Cb and a consequent defect in inhibitory synapse maturation represent a likely molecular pathomechanism of epilepsy and mental retardation in humans.     

Genetic variability and association of ISSR markers with some biochemical traits in mulberry (Morus spp.) genetic resources available in India

Utilizing intersimple sequence repeat (ISSR) markers, 18 mulberry (Morus spp.) germplasm collections were studied for genetic variability, phylogenetic relationship, and association with protein and sugar content. The genetic polymorphism exhibited by ISSR primers was 100%, and the genetic diversity recorded among the mulberry accessions had an average of 0.263 ± 0.094. Dendrogram (unweighted pair group method analysis) clustered the mulberry accessions into two major groups, one comprised the accessions collected from north or northeast regions of India, and the other comprised three subclusters and one isolate, i.e., Assamjati, a collection from Assam. Another subcluster contained accessions collected from Kerala, which belong to Morus indica. These accessions of M. indica from Kerala were found to be genetically diverse from north and northeast India. Multidimensional scaling of the ISSR data clearly separated the mulberry accessions according to their genetic diversity and protein content. Mulberry accessions were arbitrarily grouped into three classes viz. very low, moderate, and high in terms of protein and sugar content using standard statistical programs. Stepwise multiple regression analysis identified four ISSR markers (835_1,600, 835_5,600, 822_2,500, and 807_2,500) associated with protein content with highly positive correlation (p < 0.001) with linear curves with high F values (18.055 to 48.674; p < 0.001). In case of sugar content, four ISSR markers viz. 812₉₀₀, 817_1,500, 826_1,500, and 810_8,000 showed negative correlation. Hence, DNA markers for proteins seem promising and may be used in marker-assisted breeding program.     

High resolution genetic mapping uncovers chitin synthase-1 as the target-site of the structurally diverse mite growth inhibitors clofentezine,hexythiazox and etoxazole in Tetranychus urticae

The acaricides clofentezine, hexythiazox and etoxazole are commonly referred to as ‘mite growth inhibitors’, and clofentezine and hexythiazox have been used successfully for the integrated control of plant mite pests for decades. Although they are still important today, their mode of action has remained elusive. Recently, a mutation in chitin synthase 1 (CHS1) was linked to etoxazole resistance. In this study, we identified and investigated a Tetranychus urticae strain (HexR) harboring recessive, monogenic resistance to each of hexythiazox, clofentezine, and etoxazole. To elucidate if there is a common genetic basis for the observed cross-resistance, we adapted a previously developed bulk segregant analysis method to map with high resolution a single, shared resistance locus for all three compounds. This finding indicates that the underlying molecular basis for resistance to all three compounds is identical. This locus is centered on the CHS1 gene, and as supported by additional genetic and biochemical studies, a non-synonymous variant (I1017F) in CHS1 associates with resistance to each of the tested acaricides in HexR. Our findings thus demonstrate a shared molecular mode of action for the chemically diverse mite growth inhibitors clofentezine, hexythiazox and etoxazole as inhibitors of an essential, non-catalytic activity of CHS1. Given the previously documented cross-resistance between clofentezine, hexythiazox and the benzyolphenylurea (BPU) compounds flufenoxuron and cycloxuron, CHS1 should be also considered as a potential target-site of insecticidal BPUs.     

Phytoplankton composition and spatial distribution of copper and zinc in the Fal Estuary (Cornwall,UK)

In the estuaries near Falmouth (Cornwall, UK) levels of dissolved copper and zinc are high, due to drainage of copper and tin mines. Phytoplankton species composition in the autumn of 1989 deviated in the metal-contaminated Restronguet Creek from that in other estuarine branches,viz. Fal, Tresillian and Percuil. In the riverine part of Restronguet Creek (Carnon River)Euglena mutabilis, known as an acidophilic (pH 3) metal-resistant flagellate, occurred at micromolar Cu and Zn, whereas in the clean riversChlamydomonas sp. andOocystis sp. occurred at nanomolar Cu and Zn. An ordination analysis revealed the following patterns in Cu, Zn and phytoplankton species composition in the poly- and euhaline waters. Seston-bound Zn and dissolved Zn were in equilibrium,Katodinium rotundatum is Zn-tolerant, andSkeletonema costatum occurred in water with high contents of Cu in seston. However, none of the variables in these patterns correlated at a significant level (p>0.05). The results show that algae-metal interactions are complicated, and that statistical correlations foundin situ need experimental verification.Communication no. 542 of the Delta Institute for Hydrobiological Research.     

Effects of amiprilose hydrochloride on the components of human skin equivalents

Summary Amiprilose hydrochloride has been shown to inhibit the proliferation of a number of hyperproliferative cell types including psoriatic skin cells. In the present study, the effects of amiprilose hydrochloride on human tissue equivalents were examined by incubating a) dermal equivalents, b) skin equivalents in the process of epidermalization, and c) mature skin equivalents, with varying concentrations of the drug. In all three models amiprilose hydrochloride concentrations of 0.1% (wt/vol) and lower were not toxic to fibroblasts and keratinocytes and did not interfere with the differentiation of the skin equivalent and the developing skin equivalent. When tested in dermal equivalents, concentrations of amiprilose hydrochloride between 0.1 and 0.5% resulted in changes in fibroblast morphology with development of large intracellular vacuoles, and concentrations greater than 5% were toxic. In mature skin equivalents, in addition to changes in fibroblast morphology, amiprilose hydrochloride in concentrations of 1 to 10% affected the epidermis. When 0.5% amiprilose hydrochloride was present in the developing skin equivalent during differentiation, the epidermal keratinocytes were also affected. Thus the morphology of basal keratinocytes was modified, the differentiation was incomplete, and the dermalepidermal attachment was compromised. These studies suggest the possibility of an extracellular mechanism of action of amiprilose hydrochloride and delineate acceptable dosage ranges for the potential drug. Supported in part by research grant AG01274 from the National Institutes of Health, Bethesda, MD, The R. A. Welch Foundation (B0502), The Texas Advanced Technology and Research Program (Wound Healing and Aging no. 2147), and Greenwich Pharmaceuticals, Inc. R. W. G. is the recipient of a MERIT award from the National Institute on Aging, Bethesda, MD.     

Low mtDNA Cytb diversity and shallow population structure of Eleutheronema tetradactylum in the East China Sea and the South China Sea

Eleutheronema tetradactylum is an economically important fish species in China water. To investigate the genetic diversity and describe population structure of it, an 1151 base pair (bp) fragment of the mitochondrial DNA Cytb sequence was analyzed in 120 individuals from four populations in the East China Sea and the South China Sea. A total of 16 haplotypes were defined by 24 variable nucleotide sites. High level of haplotype diversity and low nucleotide diversity were observed in all populations. The results of AMOVA detected that 89.44% of the genetic variation occurred within populations. Significant genetic differentiations were detected among populations (0.05097, P < 0.05), but no large-scale regional differences were detected. Analysis of neutral evolution and mismatch distribution suggested no recent population expansion happened. The present results provided new information for genetic assessment, fishery management and conservation of this species.     

Mitochondrial toxicity and caspase activation in HIV pregnant women

To assess the impact of HIV‐infection and highly active anti‐retroviral treatment in mitochondria and apoptotic activation of caspases during pregnancy and their association with adverse perinatal outcome. Changes of mitochondrial parameters and apoptotic caspase activation in maternal peripheral blood mononuclear cells were compared at first trimester of pregnancy and delivery in 27 HIV‐infected and ‐treated pregnant women versus 24 uninfected pregnant controls. We correlated immunovirological, therapeutic and perinatal outcome with experimental findings: mitochondrial DNA (mtDNA) content, mitochondrial protein synthesis, mitochondrial function and apoptotic caspase activation. The HIV pregnancies showed increased adverse perinatal outcome (OR: 4.81 [1.14–20.16]; P < 0.05) and decreased mtDNA content (42.66 ± 5.94%, P < 0.01) compared to controls, even higher in naïve participants. This depletion caused a correlated decrease in mitochondrial protein synthesis (12.82 ± 5.73%, P < 0.01) and function (20.50 ± 10.14%, P < 0.001), not observed in controls. Along pregnancy, apoptotic caspase‐3 activation increased 63.64 ± 45.45% in controls (P < 0.001) and 100.00 ± 47.37% in HIV‐pregnancies (P < 0.001), in correlation with longer exposure to nucleoside analogues. HIV‐infected women showed increased obstetric problems and declined genetic and functional mitochondrial parameters during pregnancy, especially those firstly exposed to anti‐retrovirals. The apoptotic activation of caspases along pregnancy is emphasized in HIV pregnancies promoted by nucleoside analogues. However, we could not demonstrate direct mitochondrial or apoptotic implication in adverse obstetric outcome probably because of the reduced sample size.     

SSR markers for a critically endangered species Euryodendron excelsum and a distantly related species Ternstroemia gymnanthera (Ternstroemiaceae)

Euryodendron excelsum is a critically endangered tertiary relict plant endemic to China. It has only one population remaining in Ba Jia Zhen of Yangchun, Guangdong. In this study, we discovered 25 microsatellite markers from E. excelsumusing a Fast Isolation by Amplified Fragment Length Polymorphism of Sequences COntaining Repeats (FIASCO) protocol. Thirteen loci demonstrated polymorphisms, with the number of alleles per locus ranging from 2 to 13. Values for observed and expected heterozygosity ranged from 0.176 to 1.000 and from 0.261 to 0.889, respectively. Three loci (ZXM-17, ZXM-54, and ZXM-92) were found to significantly deviate from Hardy-Weinberg equilibrium. In addition, five of 13 loci could be successfully cross-amplified in Ternstroemia gymnanthera. These microsatellite loci may help to further survey the adaptive evolution and genetic variation of E. excelsum for guiding its conservation.