Attenuation of experimental allergic encephalomyelitis in complement component 6-deficient rats is associated with reduced complement C9 deposition, P-selectin expression, and cellular infiltrate in spinal cords

The role of Ab deposition and complement activation, especially the membrane attack complex (MAC), in the mediation of injury in experimental allergic encephalomyelitis (EAE) is not resolved. The course of active EAE in normal PVG rats was compared with that in PVG rats deficient in the C6 component of complement (PVG/C6(-)) that are unable to form MAC. Following immunization with myelin basic protein, PVG/C6(-) rats developed significantly milder EAE than PVG/C rats. The anti-myelin basic protein response was similar in both strains, as was deposition of C3 in spinal cord. C9 was detected in PVG/C rats but not in PVG/C6(-), consistent with their lack of C6 and inability to form MAC. In PVG/C6(-) rats, the T cell and macrophage infiltrate in the spinal cord was also significantly less than in normal PVG/C rats. There was also reduced expression of P-selectin on endothelial cells, which may have contributed to the reduced cellular infiltrate by limiting migration from the circulation. Assay of cytokine mRNA by RT-PCR in the spinal cords showed no differences in the profile of Th1 or Th2 cytokines between PVG/C and PVG/C6(-) rats. PVG/C rats also had a greater increase in peripheral blood white blood cell, neutrophil, and basophil counts than was observed in the PVG/C6(-). These findings suggest that the MAC may have a role in the pathogenesis of EAE, not only by Ig-activated MAC injury but also via induction of P-selectin on vascular endothelium to promote infiltration of T cells and macrophages into the spinal cord.     

Altering the expression kinetics of VP5 results in altered virulence and pathogenesis of herpes simplex virus type 1 in mice

While many herpes simplex virus (HSV) structural proteins are expressed with strict-late kinetics, the HSV virion protein 5 (VP5) is expressed as a "leaky-late" protein, such that appreciable amounts of VP5 are made prior to DNA replication. Our goal has been to determine if leaky-late expression of VP5 is a requirement for a normal HSV infection. It had been shown previously that recombinant viruses in which the VP5 promoter was replaced with promoters of other kinetic classes (including a strict late promoter) exhibited no alterations in replication kinetics or virus yields in vitro. In contrast, here we report that alterations in pathogenesis were observed when these recombinants were analyzed by experimental infection of mice. Following intracranial inoculation, a recombinant expressing VP5 from a strict-late promoter (U(L)38) exhibited an increased 50% lethal dose and a 10-fold decrease in virus yields in the central nervous system, while a recombinant expressing VP5 from an early (dUTPase) or another leaky-late (VP16) promoter exhibited wild-type neurovirulence. Moreover, following infection of the footpad, changing the expression kinetics of VP5 from leaky-late to strict-late resulted in 100-fold-less virus in the spinal ganglia during the acute infection than produced by either the parent virus or the rescued virus. These data indicate that the precise timing of appearance of the major capsid protein plays a role in the pathogenesis of HSV infections and that changing the expression kinetics has different effects in different cell types and tissues.     

Glyphosate-resistant goosegrass. Identification of a mutation in the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase

The spontaneous occurrence of resistance to the herbicide glyphosate in weed species has been an extremely infrequent event, despite over 20 years of extensive use. Recently, a glyphosate-resistant biotype of goosegrass (Eleusine indica) was identified in Malaysia exhibiting an LD(50) value approximately 2- to 4-fold greater than the sensitive biotype collected from the same region. A comparison of the inhibition of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) activity by glyphosate in extracts prepared from the resistant (R) and sensitive (S) biotypes revealed an approximately 5-fold higher IC(50)(glyphosate) for the (R) biotype. Sequence comparisons of the predicted EPSPS mature protein coding regions from both biotypes revealed four single-nucleotide differences, two of which result in amino acid changes. One of these changes, a proline to serine substitution at position 106 in the (R) biotype, corresponds to a substitution previously identified in a glyphosate-insensitive EPSPS enzyme from Salmonella typhimurium. Kinetic data generated for the recombinant enzymes suggests that the second substitution identified in the (R) EPSPS does not contribute significantly to its reduced glyphosate sensitivity. Escherichia coli aroA- (EPSPS deficient) strains expressing the mature EPSPS enzyme from the (R) biotype exhibited an approximately 3-fold increase in glyphosate tolerance relative to strains expressing the mature EPSPS from the (S) biotype. These results provide the first evidence for an altered EPSPS enzyme as an underlying component of evolved glyphosate resistance in any plant species.     

Human erythroid cells produced ex vivo at large scale differentiate into red blood cells in vivo

New sources of red blood cells (RBCs) would improve the transfusion capacity of blood centers. Our objective was to generate cells for transfusion by inducing a massive proliferation of hematopoietic stem and progenitor cells, followed by terminal erythroid differentiation. We describe here a procedure for amplifying hematopoietic stem cells (HSCs) from human cord blood (CB) by the sequential application of specific combinations of growth factors in a serum-free culture medium. The procedure allowed the ex vivo expansion of CD34+ progenitor and stem cells into a pure erythroid precursor population. When injected into nonobese diabetic, severe combined immunodeficient (NOD/SCID) mice, the erythroid cells were capable of proliferation and terminal differentiation into mature enucleated RBCs. The approach may eventually be useful in clinical transfusion applications.     

Effects of acylation on the structure,lipid binding,and transfer activity of wheat lipid transfer protein

Study of the effect of protein chemical acylation on their functional properties or activity often brings valuable information regarding structure-function relationships. We performed such work on wheat lipid transfer protein, LTP1, to investigate the role of grafted acyl chains on the lipid binding and transfer properties. LTP1 was acylated by using anhydride derivatives of various chain lengths from C2 to C6. Only the chemical modifications with hexanoic acid yielded a marked effect on the tertiary structure and a slight change in the secondary structure. The affinity of the modified proteins for myristoyl-lysophosphatidylcholine was similar to that of the native protein accompanied by a slight decrease in stoichiometry. Interestingly, the acylation of LTP1 enhanced the lipid transfer activity by at least a factor of 10 for hexanoic chain length. Finally, the grafting of acyl chains was investigated by means of molecular modelling, and an attempt is made to correlate with our experimental data.     

The biosynthetic incorporation of short-chain linear saturated fatty acids by Acholeplasma laidlawii B may suppress cell growth by perturbing membrane lipid polar headgroup distribution

Acholeplasma laidlawii B cells made fatty acid auxotrophic by growth in the presence of the biotin-binding agent avidin grow increasingly poorly at 37 degrees C when supplemented with single exogenous linear saturated fatty acids of decreasing hydrocarbon chain length. Interestingly, this progressive decrease in growth yields with decreasing hydrocarbon chain length is not observed when cells are cultured in the presence of other classes of exogenous fatty acids. Moreover, normal growth is observed is other types of fatty acids with equivalent or shorter hydrocarbon chain lengths, indicating that poor growth in the presence of short-chain linear saturated fatty acids cannot be due to a decrease in membrane lipid bilayer thickness per se. To understand the molecular basis of such growth inhibition, we determined the growth yields, membrane lipid fatty acid and polar headgroups compositions, and phase state and fluidity of the membrane lipids in cells progressively biosynthetically enriched in tridecanoic acid (13:0) or dodecanoic acid (12:0). The growth of fatty acid auxotrophic A. laidlawii B cells grown in the presence of binary combinations of an exogenous fatty acid which supports normal growth on its own and 13:0 or 12:0 revealed that growth inhibition is not observed until 13:0 and 12:0 biosynthetic incorporation levels reach about 90 and 60 mol %, respectively, after which growth is markedly inhibited. Differential scanning calorimetric analyses of membranes from cells maximally enriched in 13:0 indicate that the lipid gel/liquid-crystalline phase transition temperature is unexpectedly high but that at the growth temperature of 37 degrees C, the membrane lipid bilayer is almost exclusively in the liquid-crystalline state but is certainly not excessively fluid. However, high levels of 13:0 incorporation produce a greatly elevated level of the high melting, reversed nonlamellar phase-preferring lipid component monoglucosyl diacylglycerol, and greatly reduced levels of all other membrane lipid components. This marked elevation of monoglucosyl diacylglycerol levels can be rationalized as a regulatory response which maintains the lamellar/nonlamellar phase-forming propensity of the total membrane lipid mixture relatively constant in the face of the biosynthetic incorporation of increasing quantities of short-chain saturated fatty acids, which favor the lamellar phase. However, this lipid biosynthetic response produces a marked decline in the levels of anionic phospholipid and phosphoglycolipid which are probably required to maintain the minimal negative surface charge density of the lipid bilayer, which we suggest is responsible for the observed growth inhibition. This work shows that the lipid biosynthetic regulatory mechanisms present in this organism may sometimes operate at cross purposes such that it is not possible to simultaneously optimize all of the biologically relevant physical properties of the membrane lipid bilayer.     

A selective sweep driven by pyrimethamine treatment in southeast asian malaria parasites

Malaria parasites (Plasmodium falciparum) provide an excellent system in which to study the genomic effects of strong selection in a recombining eukaryote because the rapid spread of resistance to multiple drugs during the last the past 50 years has been well documented, the full genome sequence and a microsatellite map are now available, and haplotype data can be easily generated. We examined microsatellite variation around the dihydrofolate reductase (dhfr) gene on chromosome 4 of P. falciparum. Point mutations in dhfr are known to be responsible for resistance to the antimalarial drug pyrimethamine, and resistance to this drug has spread rapidly in Southeast (SE) Asia after its introduction in 1970s. We genotyped 33 microsatellite markers distributed across chromosome 4 in 61 parasites from a location on the Thailand/Myanmar border. We observed minimal microsatellite length variation in a 12-kb (0.7-cM) region flanking the dhfr gene and diminished variation for approximately 100 kb (6 cM), indicative of a single origin of resistant alleles. Furthermore, we found the same or similar microsatellite haplotypes flanked resistant dhfr alleles sampled from 11 parasite populations in five SE Asian countries indicating recent invasion of a single lineage of resistant dhfr alleles in locations 2000 km apart. Three features of these data are of especially interest. (1). Pyrimethamine resistance is generally assumed to have evolved multiple times because the genetic basis is simple and resistance can be selected easily in the laboratory. Yet our data clearly indicate a single origin of resistant dhfr alleles sampled over a large region of SE Asia. (2). The wide valley ( approximately 6 cM) of reduced variation around dhfr provides "proof-of-principle" that genome-wide association may be an effective way to locate genes under strong recent selection. (3). The width of the selective valley is consistent with predictions based on independent measures of recombination, mutation, and selection intensity, suggesting that we have reasonable estimates of these parameters. We conclude that scanning the malaria parasite genome for evidence of recent selection may prove an extremely effective way to locate genes underlying recently evolved traits such as drug resistance, as well as providing an opportunity to study the dynamics of selective events that have occurred recently or are currently in progress.     

Domains of estrogen receptor alpha (ERalpha) required for ERalpha/Sp1-mediated activation of GC-rich promoters by estrogens and antiestrogens in breast cancer cells

Estrogen receptor alpha (ERalpha)/Sp1 activation of GC-rich gene promoters in breast cancer cells is dependent, in part, on activation function 1 (AF1) of ERalpha, and this study investigates contributions of the DNA binding domain (C) and AF2 (DEF) regions of ERalpha on activation of ERalpha/Sp1. 17Beta-estradiol (E2) and the antiestrogens 4-hydroxytamoxifen and ICI 182,780 induced reporter gene activity in MCF-7 and MDA-MB-231 cells cotransfected with human or mouse ERalpha (hERalpha or MOR), but not ERbeta and GC-rich constructs containing three tandem Sp1 binding sites (pSp13) or other E2-responsive GC-rich promoters. Estrogen and antiestrogen activation of hERalpha/Sp1 was dependent on overlapping and different regions of the C, D, E, and F domains of ERalpha. Antiestrogen-induced activation of hERalpha/Sp1 was lost using hERalpha mutants deleted in zinc finger 1 [amino acids (aa) 185-205], zinc finger 2 (aa 218-245), and the hinge/helix 1 (aa 265-330) domains. In contrast with antiestrogens, E2-dependent activation of hERalpha/Sp1 required the C-terminal F domain (aa 579-595), which contains a beta-strand structural motif. Moreover, in peptide competition experiments overexpression of a C-terminal (aa 575-595) F domain peptide specifically blocked E2-dependent activation of hERalpha/Sp1, suggesting that F domain interactions with nuclear cofactors are required for ERalpha/Sp1 action.