Characterization of events preceding the release of malaria parasite from the host red blood cell

The process of merozoite release involves proteolysis of both the parasitophorous vacuole membrane (PVM) and red blood cell membrane (RBCM), but the precise temporal sequence remains controversial. Using immunofluorescence microscopy and Western blotting of parasite-infected RBCs, we observed that the intraerythrocytic parasite was enclosed in a continuous ring of PVM at early stages of parasite development while at the segmented schizont stage, the PVM appeared to be integrated in the cluster of newly formed merozoites. Subsequently, such clusters were detected extraerythrocytically together with single merozoites devoid of the PVM at low frequency, suggesting a primary rupture of RBCM, followed by PVM rupture and release of invasive merozoites. Secondly, since cysteine proteases are implicated in the process of parasite release, antimalarial effects of 4 cysteine protease inhibitors (leupeptin, E64, E64d, and MDL) were tested at the late schizont stage and correlated with the integrity of PVM and RBCM. We observed that leupeptin and E64 treatment produced extraerythrocytic clusters of merozoites associated with PVM suggesting inhibition of PVM lysis but not RBCM lysis. Merozoites in these clusters developed into rings upon removal of the inhibitors. In contrast, E64d and MDL caused an irreversible parasite death blocking further development. Future characterization of the mechanism(s) of inhibition may facilitate the design of novel antimalarial inhibitors.     

A novel missense mutation in MSX1 underlies autosomal recessive oligodontia with associated dental anomalies in Pakistani families

Tooth agenesis constitutes the most common anomaly of dental development in humans. In the majority of familial cases of hypodontia alone or in association with other anomalies, the mode of inheritance is autosomal dominant. In the present study, we have identified two distantly related consanguineous Pakistani kindreds with an autosomal recessive form of oligodontia with associated dental anomalies. Locus in this case has been mapped on chromosome 4p16.1–p16.3. The maximum two-point LOD score of 2.85 (θ=0.0) was obtained at markers D4S2925 and D4S2285. A maximum multipoint LOD score exceeding 4 was obtained at the same markers. Recombination events observed in affected individuals localized the disease locus between markers D4S412 and D4S2935, spanning a 9.24-cM region on chromosome 4p16.1–p16.3. Sequence analysis of candidate gene MSX1 revealed a novel recessive missense mutation resulting in substitution of alanine to threonine amino acid (p. A219T), located in the MSX1 homeodomain, which is important for DNA binding and protein–protein interaction. The mutation, p. A219T, is the first recessive mutation identified in MSX1.     

Glutamate receptors localize postsynaptically at neuromuscular junctions in mice

Dlg (Discs Large) is a multidomain protein that interacts with glutamate receptors and potassium channels at Drosophila neuromuscular junctions (NMJs) and at mammalian central nervous system synapses. Dlg also localizes postsynaptically at cholinergic mammalian NMJs. We show here that α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐proprionate (AMPA) receptor subunits, together with glutamate, are present at the mammalian NMJ. Both AMPA and NMDA (N‐methyl‐D‐aspartate) glutamate receptor subunits display overlapping postsynaptic localization patterns with Dlg at all NMJs examined in normal mice. Kir2 potassium channels also localize with Dlg and glutamate receptors at this synapse. Localization of the components of a glutamatergic system suggests novel mechanisms at mammalian neuromuscular synapses. Muscle Nerve 39: 343–349, 2009     

Seasonal changes in infrapopulations of Diplozoon kashmirensis Kaw, 1950 (Monogenea: Diplozoidae) along a eutrophic gradient

Seasonal population dynamics of the monogenean, Diplozoon kashmirensis Kaw, on the gills of two cyprinid fish species, Schizothorax niger Heckel and Carassius carassius (Linnaeus), was investigated in three limnologically distinct trophic habitats located along the flood plain of River Jhelum in Kashmir from June 2006 to May 2008. The parasite infrapopulations exhibited a marked seasonal regime in infestation pattern as the infection indices increased to a much higher plateau during summer season at all the lakes, while the lows were recorded in winter. The heterogeneity in infection pattern indicates that water temperature is an important determinant of the seasonality of infrapopulations at all the localities. Furthermore, the results of our work clearly indicate that the parasite infrapopulations increased proportionally with eutrophication level and, as such, the highly eutrophic habitat, Anchar Lake, was significantly more favourable for parasite infrapopulations than the less eutrophic ones. However, the lakes presented no significant interlake differences in water temperature. Therefore, we could argue that interlake differences in the infestation pattern of parasite can be safely attributed to respective water quality in the lakes rather than water temperature. We propose that infrapopulations of the diplozoid studied herein do respond to differences in water quality of lakes and, thus, could qualify as simple and reliable indicator species in short-term comparative assays by lake managers.     

Object recognition memory and cholinergic parameters in mice expressing human presenilin 1 transgenes

Most autosomal dominant forms of Alzheimer disease (AD) are related to missense mutations in the human presenilin (PS) 1 gene. Although the underlying mechanisms associated with pathophysiology of AD have yet to be clearly established, pathogenic mutations in the PS1 gene influence the processing of beta-amyloid precursor protein, leading to increased production and deposition of highly fibrillogenic amyloid beta(1-42) peptide in the brains of AD patients. As cognitive dysfunction in AD is associated with a dramatic loss of cholinergic innervation particularly in the hippocampus and neocortex, we investigated learning and cholinergic neurochemistry in transgenic mice expressing pathogenic mutant L286V or wild-type(wt) human PS1 transgenes. Relative to wt, the L286V PS1 transgenic mice exhibited reduced sensorimotor activity and marked deterioration of object memory between 3 and 5 h after the first encounter. Activity of the biosynthetic enzyme choline acetyltransferase was not altered in the hippocampus, frontoparietal cortex, or striatum of mutant transgenic mice relative to wt transgenic or littermate nontransgenic controls. No differences in the densities of M1/[3H]pirenzepine, M2/[3H]AF-DX 384, or alpha(7) nicotinic/125I-alpha-bungarotoxin receptor binding sites were evident in any brain regions among L286V PS1 transgenic, wt PS1 transgenic, and littermate nontransgenic controls. These results suggest that overexpression of a mutated PS1 gene induces a subtle alteration in object memory without affecting cholinergic neurochemistry.     

Headpiece domain of dematin is required for the stability of the erythrocyte membrane

Dematin is an actin-binding and bundling protein of the erythrocyte membrane skeleton. Dematin is localized to the spectrin-actin junctions, and its actin-bundling activity is regulated by phosphorylation of cAMP-dependent protein kinase. The carboxyl terminus of dematin is homologous to the "headpiece" domain of villin, an actin-bundling protein of the microvillus cytoskeleton. The headpiece domain contains an actin-binding site, a cAMP-kinase phosphorylation site, plays an essential role in dematin self-assembly, and bundles F-actin in vitro. By using homologous recombination in mouse embryonic stem cells, the headpiece domain of dematin was deleted to evaluate its function in vivo. Dematin headpiece null mice were viable and born at the expected Mendelian ratio. Hematological evaluation revealed evidence of compensated anemia and spherocytosis in the dematin headpiece null mice. The headpiece null erythrocytes were osmotically fragile, and ektacytometry/micropore filtration measurements demonstrated reduced deformability and filterability. In vitro membrane stability measurements indicated significantly greater membrane fragmentation of the dematin headpiece null erythrocytes. Finally, biochemical characterization, including the vesicle/cytoskeleton dissociation, spectrin self-association, and chemical crosslinking measurements, revealed a weakened membrane skeleton evidenced by reduced association of spectrin and actin to the plasma membrane. Together, these results provide evidence for the physiological significance of dematin and demonstrate a role for the headpiece domain in the maintenance of structural integrity and mechanical properties of erythrocytes in vivo.     

Previously described sequence variant in <Emphasis Type="Italic">CDK5RAP2</Emphasis>gene in a Pakistani family with autosomal recessive primary microcephaly

Background  

Autosomal Recessive Primary Microcephaly (MCPH) is a disorder of neurogenic mitosis. MCPH leads to reduced cerebral cortical volume and hence, reduced head circumference associated with mental retardation of variable degree. Genetic heterogeneity is well documented in patients with MCPH with six loci known, while pathogenic sequence variants in four respective genes have been identified so far. Mutations in CDK5RAP2 gene at MCPH3 locus have been least involved in causing MCPH phenotype.