Surface Area Loss and Increased Sphericity Account for the Splenic Entrapment of Subpopulations of Plasmodium falciparum Ring-Infected Erythrocytes

Ex vivo perfusion of human spleens revealed innate retention of numerous cultured Plasmodium falciparum ring-infected red blood cells (ring-iRBCs). Ring-iRBC retention was confirmed by a microsphiltration device, a microbead-based technology that mimics the mechanical filtering function of the human spleen. However, the cellular alterations underpinning this retention remain unclear. Here, we use ImageStream technology to analyze infected RBCs’ morphology and cell dimensions before and after fractionation with microsphiltration. Compared to fresh normal RBCs, the mean cell membrane surface area loss of trophozoite-iRBCs, ring-iRBCs and uninfected co-cultured RBCs (uRBCs) was 14.2% (range: 8.3–21.9%), 9.6% (7.3–12.2%) and 3.7% (0–8.4), respectively. Microsphilters retained 100%, ∼50% and 4% of trophozoite-iRBCs, ring-iRBCs and uRBCs, respectively. Retained ring-iRBCs display reduced surface area values (estimated mean, range: 17%, 15–18%), similar to the previously shown threshold of surface-deficient RBCs retention in the human spleen (surface area loss: >18%). By contrast, ring-iRBCs that successfully traversed microsphilters had minimal surface area loss and normal sphericity, suggesting that these parameters are determinants of their retention. To confirm this hypothesis, fresh normal RBCs were exposed to lysophosphatidylcholine to induce a controlled loss of surface area. This resulted in a dose-dependent retention in microsphilters, with complete retention occurring for RBCs displaying >14% surface area loss. Taken together, these data demonstrate that surface area loss and resultant increased sphericity drive ring-iRBC retention in microsphilters, and contribute to splenic entrapment of a subpopulation of ring-iRBCs. These findings trigger more interest in malaria research fields, including modeling of infection kinetics, estimation of parasite load, and analysis of risk factors for severe clinical forms. The determination of the threshold of splenic retention of ring-iRBCs has significant implications for diagnosis (spleen functionality) and drug treatment (screening of adjuvant therapy targeting ring-iRBCs).     

Influence of carbon and nitrogen sources on antifungal metabolite production by bacterium associated with entomopathogenic nematode against Penicillium expansum

A specific symbiotic Bacillus sp. isolated from a rhabditid entomopathogenic nematode, Rhabditis (Oscheius) sp. was found to produce large number of bioactive compounds. The present study was conducted to determine the effect of carbon and nitrogen sources for the production of antimicrobial substances by Bacillus sp. The yield of the crude antimicrobial substances and antimicrobial activity against the test micro-organism also differed significantly when carbon and nitrogen sources in the fermentation media were changed. The antifungal activity was significantly high in yeast extract plus fructose (46.5?±?2.12?mm) followed by yeast extract plus maltose, beef extract plus fructose and meat infusion plus glucose. High pressure liquid chromatography analysis of the crude antimicrobial substances revealed different peaks with different retention time indicating that they produced different compounds. When the carbon source was not included in the fermentation media, the antimicrobial production was substantially reduced. The results indicate that carbon source in the fermentation media plays a vital role in the production of antifungal substances. It is concluded that yeast extract and fructose as nitrogen and carbon sources produced maximum activity, which can effectively control the blue mould caused by Penicillium expansum in apples and pears.     

Human and rat adult Schwann cell cultures: fast and efficient enrichment and highly effective non-viral transfection protocol

We present a fast protocol that can be used to obtain highly purified cultures of proliferating adult human and rat Schwann cells accessible for non-viral transfection methods. The use of enriched genetically modified adult Schwann cells is of interest in the context of autologous cell transplantation within nerve transplants for peripheral nerve repair. Cell preparation from pre-degenerated adult peripheral nerves is described, together with the use of melanocyte growth medium plus forskolin, fibroblast growth factor-2 (FGF-2), pituitary extract and heregulin as a selective, serum-free culture medium and a subsequent cell enrichment step (cold jet). Proliferating adult Schwann cells can be efficiently genetically modified using optimized, non-viral electroporation protocols. The protocol results in Schwann cell cultures that are more than 90-95% pure, and transfection efficiencies vary depending on the initial cell constitution from 20 to 40%. The procedure takes up to 21 d, depending on the length of the pre-degeneration period.     

Hypoxia-induced acute lung injury in murine models of sickle cell disease

Vaso-occlusive events are the major source of morbidity and mortality in sickle cell disease (SCD); however, the pathogenic mechanisms driving these events remain unclear. Using hypoxia to induce pulmonary injury, we investigated mechanisms by which sickle hemoglobin increases susceptibility to lung injury in a murine model of SCD, where mice either exclusively express the human alpha/sickle beta-globin (halphabetaS) transgene (SCD mice) or are heterozygous for the normal murine beta-globin gene and express the halphabetaS transgene (mbeta+/-, halphabetaS+/-; heterozygote SCD mice). Under normoxia, lungs from the SCD mice contained higher levels of xanthine oxidase (XO), nitrotyrosine, and cGMP than controls (C57BL/6 mice). Hypoxia increased XO and nitrotyrosine and decreased cGMP content in the lungs of all mice. After hypoxia, vascular congestion was increased in lungs with a greater content of XO and nitrotyrosine. Under normoxia, the association of heat shock protein 90 (HSP90) with endothelial nitric oxide synthase (eNOS) in lungs of SCD and heterozygote SCD mice was decreased compared with the levels of association in lungs of controls. Hypoxia further decreased association of HSP90 with eNOS in lungs of SCD and heterozygote SCD mice, but not in the control lungs. Pretreatment of rat pulmonary microvascular endothelial cells in vitro with xanthine/XO decreased A-23187-stimulated nitrite + nitrate production and HSP90 interactions with eNOS. These data support the hypotheses that hypoxia increases XO release from ischemic tissues and that the local increase in XO-induced oxidative stress can then inhibit HSP90 interactions with eNOS, decreasing *NO generation and predisposing the lung to vaso-occlusion.     

Genetic association analysis of KCNQ3 and juvenile myoclonic epilepsy in a South Indian population

Juvenile myoclonic epilepsy (JME) is a common subtype of idiopathic generalized epilepsy that shows a complex pattern of inheritance. We have tested the association between JME phenotype and an intragenic marker in KCNQ3 by using the transmission disequilibrium test in 119 probands and their parents. Mutations in KCNQ3 are known to cause benign familial neonatal convulsions and are involved in the physiologically important M current in neurons. Our results provide suggestive evidence of allelic association between JME and KCNQ3 (P-value=0.008) and raise an interesting possibility of a genetic contribution to JME, viz., of a gene that causes a monogenic form of human epilepsy.     

A long range restriction map of the distal human X chromosome short arm around the steroid sulfatase locus.

The distal short arm of the human X chromosome is of interest because it contains genes which escape X chromosome inactivation and because it is subject to frequent deletions in human patients. The steroid sulfatase gene has been particularly well studied as an example of a gene which escapes X inactivation and which is included in a number of these deletion events. For these reasons a physical map of the region around the STS gene would be of interest. We have constructed a rare cutting enzyme map of this area and have determined the position of several nearby markers with respect to STS. We have also oriented the 5' and 3' ends of the STS gene on this map and have determined the centromeric and telomeric portions of the region. Finally, we have shown that this map can be used to locate deletion breakpoints in STS deficient patients.     

The human Hb (mu) class glutathione S-transferases are encoded by a dispersed gene family

The human glutathione S-transferases are products of a gene superfamily which consists of at least four gene families. The various glutathione S-transferase genes are located on different human chromosomes, and new gene(s) are still being added to the gene superfamily. We have characterized a cDNA in pGTH4 encoding human glutathione S-transferase subunit 4 (GST mu) and mapped its gene (or a homologous family member) on chromosome 1 at p31 by in situ hybridization. Genomic Southern analysis with the 3' noncoding region of the cDNA revealed at least four human DNA fragments with highly homologous sequences. Using a panel of DNAs from mouse-human somatic cell hybrids in genomic DNA hybridization we show that the Hb (or B) genes of human glutathione S-transferases are on three separate chromosomes: 1, 6, and 13. Therefore, the glutathione S-transferase B gene family, which encodes the Hb (mu) class subunits, is a dispersed gene family. The GST mu (psi) gene, whose expression is polymorphic in the human population, is probably located on chromosome 13. We propose that the GST mu (psi) gene was created by a transposition or recombination event during evolution. The null phenotype may have resulted from a lack of DNA transposition just as much as from the deletion of an inserted gene.     

Two distinct domains of protein 4.1 critical for assembly of functional nuclei in vitro

Protein 4.1R, a multifunctional structural protein, acts as an adaptor in mature red cell membrane skeletons linking spectrin-actin complexes to plasma membrane-associated proteins. In nucleated cells protein 4.1 is not associated exclusively with plasma membrane but is also detected at several important subcellular locations crucial for cell division. To identify 4.1 domains having critical functions in nuclear assembly, 4.1 domain peptides were added to Xenopus egg extract nuclear reconstitution reactions. Morphologically disorganized, replication deficient nuclei assembled when spectrin-actin-binding domain or NuMA-binding C-terminal domain peptides were present. However, control variant spectrin-actin-binding domain peptides incapable of binding actin or mutant C-terminal domain peptides with reduced NuMA binding had no deleterious effects on nuclear reconstitution. To test whether 4.1 is required for proper nuclear assembly, 4.1 isoforms were depleted with spectrin-actin binding or C-terminal domain-specific antibodies. Nuclei assembled in the depleted extracts were deranged. However, nuclear assembly could be rescued by the addition of recombinant 4.1R. Our data establish that protein 4.1 is essential for nuclear assembly and identify two distinct 4.1 domains, initially characterized in cytoskeletal interactions, that have crucial and versatile functions in nuclear assembly.     

Protein 4.1R core domain structure and insights into regulation of cytoskeletal organization

The crystal structure of the core domain (N-terminal 30 kDa domain) of cytoskeletal protein 4.1R has been determined and shows a cloverleaf-like architecture. Each lobe of the cloverleaf contains a specific binding site for either band 3, glycophorin C/D or p55. At a central region of the molecule near where the three lobes are joined are two separate calmodulin (CaM) binding regions. One of these is composed primarily of an alpha-helix and is Ca 2+ insensitive; the other takes the form of an extended structure and its binding with CaM is dramatically enhanced by the presence of Ca 2+, resulting in the weakening of protein 4.1R binding to its target proteins. This novel architecture, in which the three lobes bind with three membrane associated proteins, and the location of calmodulin binding sites provide insight into how the protein 4.1R core domain interacts with membrane proteins and dynamically regulates cell shape in response to changes in intracellular Ca2+ levels.