kD Protein Research Articles

Investigate the Effect of Nd-Yag Laser Beam on Soybean (Glycin max) Leaves at the Protein Level

The effect of pre-sowing laser treatment on soybean seeds was investigated, using neodymium-doped yttrium aluminium garnet (Nd-Yag) laser source at wave length ʎ = 532nm. Soybean uniform seeds (Glycin max L. cv. Giza 111) were exposed to laser irradiation for 5, 10, 30, 60 and 120 minutes. The growth potential of 4 - weekold seedlings was progressively enhanced with the increase in time durations of laser treatment. In this study, protein banding patterns of the leaf pools of the irradiated and non-irradiated control plants were investigated to elucidate the role of laser at the molecular level. These patterns showed proportional increased levels of a ~ 55 KD protein in alliance with the pre - sowing exposure time to laser irradiation. This protein was gel eluted and identified using de novo protein sequencing analysis of Liquid Chromatography - Mass Spectrometry and Tandem Mass Spectrometry (LC/ MS/MS) technique. The database search of the results led to identification of this protein as the large subunit of the enzyme 1,5 ribulose bisphosphate carboxylase/ oxygenase (RubisCO). The concomitantly enhanced growth potential of soybean seedlings with the elevation of the level of RubisCO will be discussed from different points of views.

Comparative Analysis of the 15.5kD Box C/D snoRNP Core Protein in the Primitive Eukaryote Giardia lamblia Reveals Unique Structural and Functional Features

Box C/D ribonucleoproteins (RNP) guide the 2'-O-methylation of targeted nucleotides in archaeal and eukaryotic rRNAs. The archaeal L7Ae and eukaryotic 15.5kD box C/D RNP core protein homologues initiate RNP assembly by recognizing kink-turn (K-turn) motifs. The crystal structure of the 15.5kD core protein from the primitive eukaryote Giardia lamblia is described here to a resolution of 1.8 Å. The Giardia 15.5kD protein exhibits the typical α-β-α sandwich fold exhibited by both archaeal L7Ae and eukaryotic 15.5kD proteins. Characteristic of eukaryotic homologues, the Giardia 15.5kD protein binds the K-turn motif but not the variant K-loop motif. The highly conserved residues of loop 9, critical for RNA binding, also exhibit conformations similar to those of the human 15.5kD protein when bound to the K-turn motif. However, comparative sequence analysis indicated a distinct evolutionary position between Archaea and Eukarya. Indeed, assessment of the Giardia 15.5kD protein in denaturing experiments demonstrated an intermediate stability in protein structure when compared with that of the eukaryotic mouse 15.5kD and archaeal Methanocaldococcus jannaschii L7Ae proteins. Most notable was the ability of the Giardia 15.5kD protein to assemble in vitro a catalytically active chimeric box C/D RNP utilizing the archaeal M. jannaschii Nop56/58 and fibrillarin core proteins. In contrast, a catalytically competent chimeric RNP could not be assembled using the mouse 15.5kD protein. Collectively, these analyses suggest that the G. lamblia 15.5kD protein occupies a unique position in the evolution of this box C/D RNP core protein retaining structural and functional features characteristic of both archaeal L7Ae and higher eukaryotic 15.5kD homologues.

Isolation, characterization, and function of EBAF/LEFTY B: role in infertility

Human endometrium exhibits cyclic stromal and glandular remodeling in preparation of embryo implantation. We identified EBAF/LEFTY B as a soluble cytokine of the TGF-β superfamily that is expressed at a low level in human endometrium during the receptivity period, while it is maximally expressed during perimenstrual and menstrual phases. Transfection of cells with EBAF/LEFTY B resulted in expression of a 42 kD protein that was proteolytically processed to release two polypeptides of 34 and 28 kD. EBAF/LEFTY B causes tissue remodeling by induction of collagenolysis by matrix metalloproteases. In a subset of infertile patients, the expression EBAF/LEFTY B was prematurely increased during the implantation window. We showed that induced overexpression of EBAF/LEFTY B in transgenic mice impairs implantation. EBAF/LEFTY B inhibits the expression of key decidual proteins, IGFBP-1 and PRL, through regulation of transcription factors FOXO1 and ETS1. Together, these findings show that during embryonic development, EBAF/LEFTY B plays important roles in decidualization and embryo implantation.

Isthmin is a novel secreted angiogenesis inhibitor that inhibits tumour growth in mice

Anti-angiogenesis represents a promising therapeutic strategy for the treatment of various malignancies. Isthmin (ISM) is a gene highly expressed in the isthmus of the midbrain–hindbrain organizer in Xenopus with no known functions. It encodes a secreted 60 kD protein containing a thrombospondin type 1 repeat domain in the central region and an adhesion-associated domain in MUC4 and other proteins (AMOP) domain at the C-terminal. In this work, we demonstrate that ISM is a novel angiogenesis inhibitor. Recombinant mouse ISM inhibited endothelial cell (EC) capillary network formation on Matrigel through its C-terminal AMOP domain. It also suppressed vascular endothelial growth factor (VEGF)-basic fibroblast growth factor (bFGF) induced in vivo angiogenesis in mouse. It mitigated VEGF-stimulated EC proliferation without affecting EC migration. Furthermore, ISM induced EC apoptosis in the presence of VEGF through a caspase-dependent pathway. ISM binds to αvβ5 integrin on EC surface and supports EC adhesion. Overexpression of ISM significantly suppressed mouse B16 melanoma tumour growth through inhibition of tumour angiogenesis without affecting tumour cell proliferation. Knockdown of isthmin in zebrafish embryos using morpholino antisense oligonucleotides led to disorganized intersegmen-tal vessels in the trunk. Our results demonstrate that ISM is a novel endogenous angiogenesis inhibitor with functions likely in physiological as well as pathological angiogenesis.

Genetic Polymorphism Exon 9-11 at the Leptin Gene Receptor in Breeder Hens of Mazandaran Native Fowls

Problem statement: Leptin a 16 kD protein synthesized by white adipose tissue and involved in regulation of feed intake, energy balance, fertility and immune function. In order to evaluate the leptin gene receptor polymorphism, we have used a Restriction Fragment Length Polymorphism (RFLP) method. Approach: Blood samples were collected from randomly chosen 100 native fowls breeding station of Mazandaran. The DNA extraction was based on salting out method and used amplified polymerase chain reaction technique. Exon and intron 9-11 of the fowl leptin gene receptor was amplified to produce a 382 bp fragment. The PCR products were electrophoresed on 1% agarose gel and stained by etidium bromide. Results: Then, they were digested of amplicons with Tsp509I and revealed two alleles A and B. Data were analysed using PopGene 32 package. In this population, AA, AB, BB genotype have been identified with the 69.14, 30.16, 0.7% frequencies. A and B alleles frequencies were 0.84, 0.16, respectively. x2 test didn’t signify Hardy-Weinberg equilibrium in this population (p<0.05). Conclusion: The main objectives of the current strategy in commercial broiler breeding programs aim to increased growth rate, increased breast muscle yield, decreased abdominal fat pad content, increased feed efficiency and increased overall fitness. The obtained results in the present study indicated that the A allele frequency for leptin gene receptor loci was the dominant allele.

Stable High Level Coagulation Factor VIII Expression In Vivo Following Gene Transfer Using a Novel Expression Cassette Encoding a More Potent FVIII Variant

AbstractAbstract 250Hemophilia A (HA, or clotting factor VIII deficiency), the commonest inherited bleeding disorder, is a good model for early proof of concept gene therapy studies. This is primarily because its clinical manifestations are entirely attributable to the lack of a single gene product (FVIII) that circulates in minute amounts (200ng/ml) in the plasma. Furthermore, a modest increase in the level of FVIII (>1% of normal) can ameliorate the severe phenotype. Several different gene transfer strategies for FVIII replacement have been evaluated but these have been hampered by the fact that hFVIII protein expression is highly inefficient. In addition the relatively large size of the hFVIII cDNA, (≂f7.0 kb) far exceeds the normal packaging capacity of adeno-associated viral vectors (AAV), which are currently the vectors of choice for the correction of genetic disorders such as hemophilia A. We have begun to address some of these limitations through the development of a 5.7kb AAV expression cassette (rAAV-HLP-codop-hFVIII-N6) which consists of a novel more potent hFVIII (codop-hFVIII-N6) which contains a short 226 amino-acid B-domain spacer, rich in asparagine-linked oligosaccharides, which is currently the most efficiently expressed hFVIII variant. This variant is under the control of a small ≂f200bp liver specific promoter. This expression cassette can be efficiently packaged into a single AAV vector, without significantly compromising vector yields. Transient transfection of this rAAV expression cassette into the HuH7 liver cell-line resulted in hFVIII expression that was between 4 and 8 (0.05±0.02IU/ml/24h) fold higher than that achieved with the B domain deleted (BDD-hFVIII) and N6-hFVIII variants respectively. Tail vein injection of serotype 5 or 8 pseudotyped vector rAAV-HLP-codop-hFVIII-N6 in C57Bl/6 mice resulted in detectable hFVIII within two weeks of gene transfer, reaching steady state levels of 23±6 IU/ml and 54±12 IU/ml respectively by 10 weeks. This level of expression is at least 400-fold greater than required for therapeutic efficacy (0.05IU/ml) and at least 10 fold higher than achieved in mice transduced with a comparable dose of rAAV encoding either the BDD or N6 variant of hFVIII. Southern blot analysis of DNA extracted from the liver of rAAV-HLP-codop-hFVIII-N6 transduced mice revealed head-to-tail and head-to-head concatemer fragments of ≂f5kb and ≂f10kb respectively in the expected ratio of 3:1. Western blot analysis showed that the rAAV-HLP-codop-hFVIII-N6 cassette mediated the synthesis and secretion of a single chain 210kd protein. To confirm correction of the bleeding phenotype, either 4×1011 (low-dose cohort, n=3) or 4×1012 (high-dose cohort, n=3) rAAV5-HLP-codop-hFVIII-N6 vector genomes were injected into the tail vein of haemophilia A knockout mice. Peak hFVIII levels, as determined by a one-stage clotting assay, were 137±27% and 374±18% of normal levels in the low and high-dose cohorts of F8-/- mice respectively. These levels were sufficient to arrest bleeding in a modified tail clip assay. Anti-hFVIII antibodies were not detected in the rAAV treated HA mice at any stage. Therefore, the higher potency of our novel codop-hFVIII-N6 construct and the ability to package this FVIII variant within AAV virions has substantially improved the prospects of effective gene transfer for Hemophilia A. Disclosures:No relevant conflicts of interest to declare.

Statin Treatment Inhibits FLT3 Signaling by Preventing Glycosylation of the Mature Receptor and Leads to Cell Death In Mutant FLT3 Expressing Cells

AbstractAbstract 779FLT3 is a tyrosine kinase receptor expressed on the surface of hematopoietic stem cells/progenitors and contributes to leukemogenesis as evidenced by its frequent mutation in acute myeloid leukemia (AML). FLT3 normally undergoes N-linked glycosylation to produce an immature 130 Kd protein in the endoplasmic reticulum followed by further processing in the Golgi to form the final 160 Kd mature glycoprotein that translocates to the surface. At the surface, FLT3 binds to FLT3 ligand (FL) resulting in phosphorylation and activation of multiple signaling cascades. FLT3 activating mutations primarily occur as internal tandem duplications (ITD) within the juxtamembrane domain or as point mutations in the kinase domain and have become the target of numerous small molecule tyrosine kinase inhibitors (TKI), several of which have entered clinical trials for AML. However, clinical trials to date have shown limited success due to a variety of reasons including the emergence of resistance mediated by alternative pathway activation, the acquisition of point mutations, expression of increased FLT3 ligand levels and poor inhibition of FLT3 kinase activity for a number of reasons. Statins are a well-defined class of drugs that have been safely used for treatment of hypercholesterolemia. Here, we show for the first time that statins can inhibit constitutive activation of both kinase domain and ITD mutants. Western blotting demonstrates that fluvastatin inhibits FLT3 phosphorylation by preventing receptor glycosylation, initially leading to loss of the mature receptor followed by instability of the receptor and subsequent degradation of FLT3 protein and ultimately, cell death. MTT analysis indicates that BaF3 cells transfected with FLT3/ITD are killed in a dose-dependent fashion by fluvastatin with an IC50 of <1 uM, a level which has been achieved in clinical trials. Annexin V binding confirms that BaF3/ITD cells undergo apoptosis when treated with Fluvastatin. Fluvastatin treatment causes a reduction in surface FLT3 expression as measured by flow cytometry that parallels induction of apoptosis. This finding is consistent with results obtained by immunofluoresence microsopy showing increased intracellular retention of FLT3/ITD. Additionally, fluvastatin eliminates the 2–3 fold shift in the dose-response to TKI seen when FLT3/ITD expressing cells are stimulated by FL. Also, a number of mutations that confer high level resistance to TKI are overcome by fluvastatin. These results demonstrate that statins, a class of drugs already clinically approved, might be useful in the management of AML cases in which FLT3 is over expressed, mutated or resistant to other clinically available inhibitors, either alone or more likely in combination with FLT3 TKI. Disclosures:Levis:Ambit Biosciences, Inc: Consultancy.

874 Serological Diagnosis of Lyme Neuroborreliosis in Children with Various Neurological Disorders - Comparison of Two Elisa Tests

Aim: We wanted to compare two ELISA kits for detection of IgG and IgM antibodies against Borrelia in samples taken from children with suspected neuroborreliosis. Methods: Between 01.04.2009 and 31.03.2010 serum and cerebrospinal fluid from children with various neurological symptoms were analyzed using Biomedica kits. Positive samples were confirmed with MIKROGEN Diagnostik Immunoblot assay and tested with DRG Diagnostics kits. The Biomedica IgG kit contains recombinant antigens: OspC, p18, p100 and VIsE ; the IgM Biomedica kit contains recombinant antigens: OspC (B.afzelii, B.garinii), p41/I, VIsE. The DRG IgG kit contains recombinant VIsE antigen and lysate antigens from B.burgdorferi s.s., B.afzelii and B.garinii; the DRG IgM kit contains recombinant 14 kD protein and native OspC. Interpretation of the ELISA and Immunoblot was done according to the manufacturer's instructions. Results: A total of 379 samples (353 sera and 26 CSF) were analyzed for Borrelia antibodies. Antibodies against B.burgdorferi were detected by Biomedica kit in 55 samples from which 29 (52%) were confirmed with Immunoblot. Based on the Immunoblot analysis 29 / 379 (7,3 %) samples were considered as “true positive”. Among two ELISA tests, 26 sera were false-positive with the Biomedica test and 13 sera were false-positive with the DRG Diagnostics test. However, DRG Diagnostics test failed to detect two sera that were positive with the Biomedica test and Immunoblot. Conclusions: We observed significant differences between the two ELISA tests. Biomedica test showed higher sensitivity but it produced more false-positive results. DRG Diagnostics test was more specific, but less sensitive.

Circatidal rhythms of locomotion in the American horseshoe crab Limulus polyphemus: Underlying mechanisms and cues that influence them

Abstract While eye sensitivity in the American horseshoe crab Limulus polyphemus has long been known to be under the control of an endogenous circadian clock, only recently has horseshoe crab locomotion been shown to be controlled by a separate clock system. In the laboratory, this system drives clear activity rhythms throughout much of the year, not just during the mating season when horseshoe crabs express clear tidal rhythms in the field. Water temperature is a key factor influencing the expression of these rhythms: at 17̊C tidal rhythms are expressed by most animals, while at 11̊C expression of circatidal rhythms is rarely seen, and at 4̊C rhythms are suppressed. Neither long (16:8 Light:Dark) nor short (8:16) photoperiods modify this behavior at any of these temperatures. Synchronization of these circatidal rhythms can be most readily effected by water pressure cycles both in situ and in the lab, while temperature and current cycles play lesser, but possibly contributory, roles. Interestingly, Light:Dark cycles appear to have synchronizing as well as “masking” effects in some individuals. Evidence that each of two daily bouts of activity are independent suggests that the Limulus circatidal rhythm of locomotion is driven by two (circalunidian) clocks, each with a period of 24.8h. While the anatomical locations of either the circadian clock, that drives fluctuations in visual sensitivity, or the circatidal clock, that controls tidal rhythms of locomotion, are currently unknown, preliminary molecular analyses have shown that a 71 kD protein that reacts with antibodies directed against the Drosophila PERIOD (PER) protein is found in both the protocerebrum and the subesophageal ganglion.

Dietary Phenethyl Isothiocyanate Alters Gene Expression in Human Breast Cancer Cells

Phenethyl isothiocyanate (PEITC), a component in cruciferous vegetables, can block chemical carcinogenesis in animal models. Our objective was to determine the effect of treatment with PEITC on gene expression changes in MCF-7 human breast cancer cells in order to evaluate potential mechanisms involved in its chemopreventive effects. MCF-7 cells were treated for 48 hours with either PEITC (3 μM) or the vehicle. Total RNA was extracted from cell membrane preparations, and labeled cDNA's representing the mRNA pool were reverse-transcribed directly from total RNA isolated for use in the microarray hybridizations. Two specific human GE Array Kits (Superarray Inc.) that both contain 23 marker genes, related to signal transduction pathways or cancer/tumor suppression, plus 2 housekeeping genes (β-actin and GAPDH), were utilized. Arrays from treated and control cells (n = 4 per group) were evaluated using a Student's t-test. Gene expression was significantly induced for tumor protein p53 (p53), cyclin-dependent kinase inhibitor 1C (p57 Kip2), breast cancer Type 2 early onset (BRCA2), cAMP responsive element binding protein 2 (ATF-2), interleukin 2 (IL-2), heat shock 27 KD protein (hsp27), and CYP19 (aromatase). Induction of p57 Kip2, p53, BRCA2, IL-2, and ATF-2 would be expected to decrease cellular proliferation and increase tumor suppression and/or apoptosis. PEITC treatment produced significant alterations in some genes involved in tumor suppression and cellular proliferation/apoptosis that may be important in explaining the chemopreventive effects of PEITC.

Identification of α-tubulin as an autoantigen recognized by sera from patients with neuropsychiatric systemic lupus erythematosus

In a previous study we found in 50% of patients with neuropsychiatric manifestations of systemic lupus erythematosus (NP-SLE) organ specific antibodies to 45–56 kD proteins in a 100,000 g supernatant (SN) from bovine brain mitochondria. Aim of the present study was to identify the corresponding target antigen. A 100,000 g SN from bovine brain mitochondria was applied to SDS-gel electrophoresis. A 50 kD band recognized by sera from patients with NP-SLE in the Western blot (WB) was excised from the gels and applied to mass spectrometry. The identified protein was expressed in Escherichia coli and retested against sera from eleven patients with NP-SLE (severe symptoms n = 6, mild symptoms n = 5), 26 SLE-patients without NP manifestations and 53 controls (patients with multiple sclerosis, epilepsy, healthy blood donors). Mass spectrometry of the 50 kD band revealed the presence of α-tubulin. Applying the recombinant α-tubulin in the WB, four of the eleven NP-SLE patients (36%), one of the 26 patients with SLE without NP manifestations (4%) and none of the 53 controls reacted with α-tubulin. The antibodies were more frequently found in patients with severe (50%) than with mild NP-SLE (20%). α-tubulin may be a novel marker autoantigen for a neuropsychiatric manifestation at least in a subgroup of patients with SLE. Whether anti-α-tubulin antibodies are of pathogenetic relevance has still to be clarified.

Rescue of a Dystrophin-like Protein by Exon Skipping In Vivo Restores GABAA-receptor Clustering in the Hippocampus of the mdx Mouse

Dystrophin, the cytoskeletal protein whose defect is responsible for Duchenne muscular dystrophy (DMD), is normally expressed in both muscles and brain. Genetic loss of brain dystrophin in the mdx mouse model of DMD reduces the capacity for type A gamma-aminobutyric acid (GABA(A))-receptor clustering in central inhibitory synapses, which is thought to be a main molecular defect leading to brain and cognitive alterations in this syndrome. U7 small nuclear RNAs modified to encode antisense sequences and expressed from recombinant adeno-associated viral (rAAV) vectors have proven efficient after intramuscular injection to induce skipping of the mutated exon 23 and rescue expression of a functional dystrophin-like product in muscle tissues of mdx mice in vivo. Here, we report that intrahippocampal injection of a single dose of rAAV2/1-U7 can rescue substantial levels of brain dystrophin expression (15-25%) in mdx mice for months. This is sufficient to completely restore GABA(A)-receptor clustering in pyramidal and dendritic layers of CA1 hippocampus, suggesting exon-skipping strategies offer the prospect to investigate and correct both brain and muscle alterations in DMD. This provides new evidence that in the adult brain dystrophin is critical for the control of GABA(A)-receptor clustering, which may have an important role in activity-dependent synaptic plasticity in hippocampal circuits.

OP17 Clinical effect of albumin dialysis in patients with intractable pruritus correlates closely with changes in autotaxin activity but not bile salt levels

IntroductionAbout 5% of patients with severe cholestatic liver disease have intractable pruritus. Albumin dialysis is an effective treatment for these patients but the mechanism through which it reduces the severity...

A MAP for Bundling Microtubules

Microtubules assemble into arrays of bundled filaments that are critical for multiple steps in cell division, including anaphase and cytokinesis. Recent structural and functional studies, including two papers in this issue of Cell (Bieling et al., 2010; Subramanian et al., 2010), demonstrate how the MAP65 protein PRC1 crosslinks microtubules and cooperates with kinesin motors to control the dynamics and size of bundled regions.

The Meningococcal Vaccine Candidate Neisserial Surface Protein A (NspA) Binds to Factor H and Enhances Meningococcal Resistance to Complement

Complement forms an important arm of innate immunity against invasive meningococcal infections. Binding of the alternative complement pathway inhibitor factor H (fH) to fH-binding protein (fHbp) is one mechanism meningococci employ to limit complement activation on the bacterial surface. fHbp is a leading vaccine candidate against group B Neisseria meningitidis. Novel mechanisms that meningococci employ to bind fH could undermine the efficacy of fHbp-based vaccines. We observed that fHbp deletion mutants of some meningococcal strains showed residual fH binding suggesting the presence of a second receptor for fH. Ligand overlay immunoblotting using membrane fractions from one such strain showed that fH bound to a ∼17 kD protein, identified by MALDI-TOF analysis as Neisserial surface protein A (NspA), a meningococcal vaccine candidate whose function has not been defined. Deleting nspA, in the background of fHbp deletion mutants, abrogated fH binding and mAbs against NspA blocked fH binding, confirming NspA as a fH binding molecule on intact bacteria. NspA expression levels vary among strains and expression correlated with the level of fH binding; over-expressing NspA enhanced fH binding to bacteria. Progressive truncation of the heptose (Hep) I chain of lipooligosaccharide (LOS), or sialylation of lacto-N-neotetraose LOS both increased fH binding to NspA-expressing meningococci, while expression of capsule reduced fH binding to the strains tested. Similar to fHbp, binding of NspA to fH was human-specific and occurred through fH domains 6–7. Consistent with its ability to bind fH, deleting NspA increased C3 deposition and resulted in increased complement-dependent killing. Collectively, these data identify a key complement evasion mechanism with important implications for ongoing efforts to develop meningococcal vaccines that employ fHbp as one of its components.

Comparable effect of conventional ventilation versus early high-frequency oscillation on serum CC16 and IL-6 levels in preterm neonates

Clara cell 16 kD protein (CC16) and interleukin (IL)-6 have been used as peripheral blood biomarkers of alveolar leakage and inflammation, respectively. Thus, their measurement in the bloodstream could be used to assess ventilator-induced lung injury. The objective of this study was to evaluate the effect of optimized synchronized intermittent mandatory ventilation (SIMV) and high-frequency oscillatory ventilation (HFOV) on circulating CC16 and IL-6 levels when used as the initial ventilation modes in preterm neonates. Single center, prospective, randomized clinical study in preterm neonates (gestational age 30 weeks) requiring mechanical ventilation within the first 2 h of life. Serum CC16 and IL-6 were measured on establishment of the assigned ventilation mode after admission, at days 3 and 14 of life as well as at 36 weeks postmenstrual age. Demographic-perinatal data and clinical parameters were also recorded. Of the 30 neonates studied, 24 (gestational age 27.1±1.7 weeks, birth weight 942±214 g) were finally analyzed, equally assigned into the SIMV and HFOV groups. Both groups had comparable demographic-perinatal characteristics and clinical parameters. Serum CC16 and IL-6 altered significantly over time (repeated-measures analysis of variance, both P<0.001). However, changes were not affected by the ventilation mode. Post hoc analysis showed a significant decrease in CC16 and IL-6 from birth up to 36 weeks postmenstrual age in both groups. In preterm neonates, SIMV and HFOV are associated with comparable circulating CC16 and IL-6 levels. These findings suggest a similar alveolar leakage and systemic inflammation with any of the ventilation modes evaluated when their usage is optimized.

The meningococcal vaccine candidate Neisserial surface protein A (NspA) binds to Factor H and enhances meningococcal resistance to complement

Complement forms an important arm of innate immunity against invasive meningococcal infections. Binding of the alternative complement pathway inhibitor factor H (fH) to fH-binding protein (fHbp) is one mechanism meningococci employ to limit complement activation on the bacterial surface. fHbp is a leading vaccine candidate against group B Neisseria meningitidis. Novel mechanisms that meningococci employ to bind fH could undermine the efficacy of fHbp-based vaccines. We observed that fHbp deletion mutants of some meningococcal strains showed residual fH binding suggesting the presence of a second receptor for fH. Ligand overlay immunoblotting using membrane fractions from one such strain showed that fH bound to a ∼17 kD protein, identified by MALDI-TOF analysis as Neisserial surface protein A (NspA), a meningococcal vaccine candidate whose function has not been defined. Deleting nspA, in the background of fHbp deletion mutants, abrogated fH binding and mAbs against NspA blocked fH binding, confirming NspA as a fH binding molecule on intact bacteria. NspA expression levels vary among strains and expression correlated with the level of fH binding; over-expressing NspA enhanced fH binding to bacteria. Progressive truncation of the heptose (Hep) I chain of lipooligosaccharide (LOS), or sialylation of lacto-N-neotetraose LOS both increased fH binding to NspA-expressing meningococci, while expression of capsule reduced fH binding to the strains tested. Similar to fHbp, binding of NspA to fH was human-specific and occurred through fH domains 6–7. Consistent with its ability to bind fH, deleting NspA increased C3 deposition and resulted in increased complement-dependent killing. Collectively, these data identify a key complement evasion mechanism with important implications for ongoing efforts to develop meningococcal vaccines that employ fHbp as one of its components.

Methamphetamine Preconditioning Causes Differential Changes in Striatal Transcriptional Responses to Large Doses of the Drug

Methamphetamine (METH) is a toxic drug of abuse, which can cause significant decreases in the levels of monoamines in various brain regions. However, animals treated with progressively increasing doses of METH over several weeks are protected against the toxic effects of the drug. In the present study, we tested the possibility that this pattern of METH injections might be associated with transcriptional changes in the rat striatum, an area of the brain which is known to be very sensitive to METH toxicity and which is protected by METH preconditioning. We found that the presence and absence of preconditioning followed by injection of large doses of METH caused differential expression in different sets of striatal genes. Quantitative PCR confirmed METH-induced changes in some genes of interest. These include small heat shock 27 kD proteins 1 and 2 (HspB1 and HspB2), brain derived neurotrophic factor (BDNF), and heme oxygenase-1 (Hmox-1). Our observations are consistent with previous studies which have reported that ischemic or pharmacological preconditioning can cause reprogramming of gene expression after lethal ischemic insults. These studies add to the growing literature on the effects of preconditioning on the brain transcriptome.

Mixed Lineage Kinase-3 Stabilizes and Functionally Cooperates with TRIBBLES-3 to Compromise Mitochondrial Integrity in Cytokine-induced Death of Pancreatic Beta Cells

Mixed lineage kinases (MLKs) have been implicated in cytokine signaling as well as in cell death pathways. Our studies show that MLK3 is activated in leukocyte-infiltrated islets of non-obese diabetic mice and that MLK3 activation compromises mitochondrial integrity and induces apoptosis of beta cells. Using an ex vivo model of islet-splenocyte co-culture, we show that MLK3 mediates its effects via the pseudokinase TRB3, a mammalian homolog of Drosophila Tribbles. TRB3 expression strongly coincided with conformational change and mitochondrial translocation of BAX. Mechanistically, MLK3 directly interacted with and stabilized TRB3, resulting in inhibition of Akt, a strong suppressor of BAX translocation and mitochondrial membrane permeabilization. Accordingly, attenuation of MLK3 or TRB3 expression each prevented cytokine-induced BAX conformational change and attenuated the progression to apoptosis. We conclude that MLKs compromise mitochondrial integrity and suppress cellular survival mechanisms via TRB3-dependent inhibition of Akt.

Pillars Article: Blimp-1, a Novel Zinc Finger-Containing Protein That Can Drive the Maturation of B Lymphocytes into Immunoglobulin-Secreting Cells. Cell. 1994. 77: 297–306

We describe a novel gene, Blimp-1 (for B lymphocyte-induced maturation protein), transcripts of which are rapidly induced during the differentiation of B lymphocytes into immunoglobulin secretory cells and whose expression is characteristic of late B and plasma cell lines. The 856 amino acid open reading frame contains five Kruppel-type zlnc finger motifs and proline-rich and acidic regions similar to those of known transcription factors. Serological studies show an ∼ 100 kd protein that localizes to the nucleus. Stable or transient transfection of Blimp-1 into B cell lymphoma lines leads to the expression of many of the phenotypic changes associated with B cell differentiation into an early plasma cell stage, including induction of J chain message and immunoglobulin secretion, up-regulation of Syndecan-1, and increased cell size and granularity. Thus, Blimp-1 appears to be a pleiotropic regulatory factor capable of at least partially driving the terminal differentiation of B cells.