Insertion mutations at the maizeOpaque2 locus induced by transposable element familiesAc, En/Spm andBg

Eight independently isolated unstable alleles of theOpaque2 (O2) locus were analysed genetically and at the DNA level. The whole series of mutations was isolated from a maize strain carrying a wild-typeO2 allele and the transposable elementActivator (Ac) at thewx-m7 allele. Previous work with another unstable allele of the same series has shown that it was indeed caused by the insertion of anAc element. Unexpectedly, the remaining eight mutations were not caused by the designatedAc element, but by other insertions that are structurally similar or identical to one of two different autonomous transposable elements. Six mutations were caused by the insertion of a transposable element of theEnhancer/Suppressor-Mutator (En/Spm) family. Two mutations were the result of the insertion of a transposable element of theBergamo (Bg) family. Genetic tests carried out with plants carrying the unstable mutations demonstrated that all were caused by the insertion of an autonomous transposable element.     

Bacterial beta-peptidyl aminopeptidases with unique substrate specificities for beta-oligopeptides and mixed beta,alpha-oligopeptides

We previously discovered that BapA, a bacterial beta-peptidyl aminopeptidase, is able to hydrolyze two otherwise metabolically inert beta-peptides [Geueke B, Namoto K, Seebach D and Kohler H-PE (2005) J Bacteriol 187, 5910-5917]. Here, we describe the purification and characterization of two distinct bacterial beta-peptidyl aminopeptidases that originated from different environmental isolates. Both bapA genes encode a preprotein with a signal sequence and were flanked by ORFs that code for enzymes with similar predicted functions. To form the active enzymes, which had an (alphabeta)(4) quaternary structure, the preproteins needed to be cleaved into two subunits. The two beta-peptidyl aminopeptidases had 86% amino acid sequence identity, hydrolyzed a variety of beta-peptides and mixed beta/alpha-peptides, and exhibited unique substrate specificities. The prerequisite for peptides being accepted as substrates was the presence of a beta-amino acid at the N-terminus; peptide substrates with an N-terminal alpha-amino acid were not hydrolyzed at all. Both enzymes cleaved the peptide bond between the N-terminal beta-amino acid and the amino acid at the second position of tripeptidic substrates of the general structure H-betahXaa-Ile-betahTyr-OH according to the following preferences with regard to the side chain of the N-terminal beta-amino acid: aliphatic and aromatic > OH-containing > hydrogen, basic and polar. Experiments with the tripeptides H-d-betahVal-Ile-betahTyr-OH and H-betahVal-Ile-betahTyr-OH demonstrated that the two BapA enzymes preferred the peptide with the l-configuration of the N-terminal beta-homovaline residue as a substrate.     

Proliferation-Linked Apoptosis of Adoptively Transferred T Cells after IL-15 Administration in Macaques

The adoptive transfer of antigen-specific effector T cells is being used to treat human infections and malignancy. T cell persistence is a prerequisite for therapeutic efficacy, but reliably establishing a high-level and durable T cell response by transferring cultured CD8⁺ T cells remains challenging. Thus, strategies that promote a transferred high-level T cell response may improve the efficacy of T cell therapy. Lymphodepletion enhances persistence of transferred T cells in mice in part by reducing competition for IL-15, a common γ-chain cytokine that promotes T cell memory, but lymphodepleting regimens have toxicity. IL-15 can be safely administered and has minimal effects on CD4⁺ regulatory T cells at low doses, making it an attractive adjunct in adoptive T cell therapy. Here, we show in lymphoreplete macaca nemestrina, that proliferation of adoptively transferred central memory-derived CD8⁺ effector T (T_CM/E) cells is enhanced in vivo by administering IL-15. T_CM/E cells migrated to memory niches, persisted, and acquired both central memory and effector memory phenotypes regardless of the cytokine treatment. Unexpectedly, despite maintaining T cell proliferation, IL-15 did not augment the magnitude of the transferred T cell response in blood, bone marrow, or lymph nodes. T cells induced to proliferate by IL-15 displayed increased apoptosis demonstrating that enhanced cycling was balanced by cell death. These results suggest that homeostatic mechanisms that regulate T cell numbers may interfere with strategies to augment a high-level T cell response by adoptive transfer of CD8⁺ T_CM/E cells in lymphoreplete hosts.     

Efficacy of sulfadoxine-pyrimethamine in Tanzania after two years as first-line drug for uncomplicated malaria: assessment protocol and implication for treatment policy strategies

Background

Early diagnosis and effective treatment with an appropriate drug form the main components of the World Health Organization's strategy to reduce malaria related mortality. The few available drugs might be safeguarded if combined with artesunate. The addition of artesunate to a standard antimalarial treatment substantially reduces treatment failure, recrudescence and gametocyte carriage.

Methods

During late 2004, the efficacy of artesunate (4 mg/kg. day, on days 0–2) plus sulfadoxine-pyrimethamine (25 mg/kg, on day 0) for the treatment of uncomplicated Plasmodium falciparum malaria was investigated in four sentinel areas in Sudan, with different malaria transmission (Damazin, Kassala, Kosti, and Malakal).

Results

Two hundreds and sixty-nine patients completed the 28-day follow-up. On day one, 60 (22.3%) patients were febrile and 15 (5.5%) patients were parasitaemic. On day three, all the patients were afebrile and aparasitaemic. While two patients (0.7%, Kassala) showed late Clinical and Parasitological Failures, the rest (99.3%) of the patients demonstrated Adequate Clinical and Parasitological Response. A gametocytaemia were detected during the follow-up in one patient (0.37%, Kassala). Adverse drug effects were detected in 32 (11.9%) patients

Conclusion

The study showed that AS plus SP is an effective, safe drug in the treatment of uncomplicated P. falciparum malaria in Sudan.     

Analysis of the chloroplast protein complexes by blue-native polyacrylamide gel electrophoresis (BN-PAGE)

Blue-native polyacrylamide gel electrophoresis (BN-PAGE) is a powerful procedure for the separation and characterization of the protein complexes from mitochondria. Membrane proteins are solubilized in the presence of aminocaproic acid and n-dodecylmaltoside and Coomassie-dyes are utilized before electrophoresis to introduce a charge shift on proteins. Here, we report a modification of the procedure for the analysis of chloroplast protein complexes. The two photosystems, the light-harvesting complexes, the ATP synthase, the cytochrome b ₆ f complex and the ribulose-bisphosphate carboxylase/oxygenase are well resolved. Analysis of the protein complexes on a second gel dimension under denaturing conditions allows separation of more than 50 different proteins which are part of chloroplast multi-subunit enzymes. The resolution capacity of the blue-native gels is very high if compared to 'native green gel systems' published previously. N-terminal amino acid sequences of single subunits can be directly determined by cyclic Edman degradation as demonstrated for eight proteins. Analysis of chloroplast protein complexes by blue-native gel electrophoresis will allow the generation of 'protein maps' from different species, tissues and developmental stages or from mutant organelles. Further applications of blue-native gel electrophoresis are discussed.     

Mapping of quantitative trait loci associated with protein expression variation in barley grains

Barley (Hordeum vulgare) is an important cereal crop grown for both the feed and malting industries. Hence, there is great interest to gain deeper insight into the determinants of grain nutritional quality in order to improve the assessment of new traits. Two-dimensional gel electrophoresis was employed for the characterization of the grain proteome of doubled-haploid introgression lines (IL) representing a wild barley genome (Hordeum spontaneum Hs213) within a modern cultivar background (H. vulgare cv. Brenda). Proteome maps were subjected to differential cluster analysis and revealed ILs with similar or different protein expression patterns compared to the Brenda parent. A total of 51 quantitative trait loci for protein expression (pQTL) were detected, and proteins underlying these pQTL were further examined by mass spectrometry. Identification was successful for 49 of the segregating spots and functional annotation of proteins revealed that most proteins are involved in metabolism and disease/defence-related processes. Among those, multigene families of glyceraldehyde-3-phosphate dehydrogenases, heat shock proteins, peroxidases, and serpins were identified. Overall, eight pQTL signals were discovered in two independently grown sets of plants. The mapped spots included protein disulfide isomerase, α-amylase inhibitor BDAI, NADP malic enzyme, adenosine kinase and peroxidase BP1. Specific marker information of proteins involved in developmental events and protein storage as well as in disease- and defence-related processes now allows for targeted breeding approaches to improve the grain quality in barley.     

Mzabimyces algeriensis gen. nov., sp. nov., a halophilic filamentous actinobacterium isolated from a Saharan soil,and proposal of Mzabimycetaceae fam. nov.

Three halophilic mycelium-forming actinobacteria, strains H195^T, H150 and H151, were isolated from a Saharan soil sample collected from Béni-isguen in the Mzab region (Ghardaïa, South of Algeria) and subjected to a polyphasic taxonomic characterisation. These strains were observed to show an aerial mycelium differentiated into coccoid spore chains and fragmented substrate mycelium. Comparative analysis of the 16S rRNA gene sequences revealed that the highest sequence similarities were to Saccharopolyspora qijiaojingensis YIM 91168^T (92.02 % to H195^T). Phylogenetic analyses showed that the strains H195^T, H150 and H151 represent a distinct phylogenetic lineage. The cell-wall hydrolysate was found to contain meso-diaminopimelic acid, and the diagnostic whole-cell sugars were identified as arabinose and galactose. The major cellular fatty acids were identified as iso-C_15:0, iso-C_16:0, iso-C_17:0 and anteiso-C_17:0. The diagnostic phospholipid detected was phosphatidylcholine and MK-9 (H₄) was found to be the predominant menaquinone. The genomic DNA G+C content of strain H195^T was 68.2 mol%. On the basis of its phenotypic features and phylogenetic position, we propose that strain H195^T represents a novel genus and species, Mzabimyces algeriensis gen. nov., sp. nov., within a new family, Mzabimycetaceae fam. nov. The type strain of M. algeriensis is strain H195^T (=DSM 46680^T = MTCC 12101^T).     

Systemic Treatment with Erythropoietin Protects the Neurovascular Unit in a Rat Model of Retinal Neurodegeneration

Rats expressing a transgenic polycystic kidney disease (PKD) gene develop photoreceptor degeneration and subsequent vasoregression, as well as activation of retinal microglia and macroglia. To target the whole neuroglialvascular unit, neuro- and vasoprotective Erythropoietin (EPO) was intraperitoneally injected into four –week old male heterozygous PKD rats three times a week at a dose of 256 IU/kg body weight. For comparison EPO-like peptide, lacking unwanted side effects of EPO treatment, was given five times a week at a dose of 10 µg/kg body weight. Matched EPO treated Sprague Dawley and water-injected PKD rats were held as controls. After four weeks of treatment the animals were sacrificed and analysis of the neurovascular morphology, glial cell activity and pAkt localization was performed. The number of endothelial cells and pericytes did not change after treatment with EPO or EPO-like peptide. There was a nonsignificant reduction of migrating pericytes by 23% and 49%, respectively. Formation of acellular capillaries was significantly reduced by 49% (p<0.001) or 40% (p<0.05). EPO-treatment protected against thinning of the central retina by 10% (p<0.05), a composite of an increase of the outer nuclear layer by 12% (p<0.01) and in the outer segments of photoreceptors by 26% (p<0.001). Quantification of cell nuclei revealed no difference. Microglial activity, shown by gene expression of CD74, decreased by 67% (p<0.01) after EPO and 36% (n.s.) after EPO-like peptide treatment. In conclusion, EPO safeguards the neuroglialvascular unit in a model of retinal neurodegeneration and secondary vasoregression. This finding strengthens EPO in its protective capability for the whole neuroglialvascular unit.     

Eye Movement Deficits Are Consistent with a Staging Model of pTDP-43 Pathology in Amyotrophic Lateral Sclerosis

Background

The neuropathological process underlying amyotrophic lateral sclerosis (ALS) can be traced as a four-stage progression scheme of sequential corticofugal axonal spread. The examination of eye movement control gains deep insights into brain network pathology and provides the opportunity to detect both disturbance of the brainstem oculomotor circuitry as well as executive deficits of oculomotor function associated with higher brain networks.

Objective

To study systematically oculomotor characteristics in ALS and its underlying network pathology in order to determine whether eye movement deterioration can be categorized within a staging system of oculomotor decline that corresponds to the neuropathological model.

Methods

Sixty-eight ALS patients and 31 controls underwent video-oculographic, clinical and neuropsychological assessments.

Results

Oculomotor examinations revealed increased anti- and delayed saccades’ errors, gaze-palsy and a cerebellary type of smooth pursuit disturbance. The oculomotor disturbances occurred in a sequential manner: Stage 1, only executive control of eye movements was affected. Stage 2 indicates disturbed executive control plus ‘genuine’ oculomotor dysfunctions such as gaze-paly. We found high correlations (p<0.001) between the oculomotor stages and both, the clinical presentation as assessed by the ALS Functional Rating Scale (ALSFRS) score, and cognitive scores from the Edinburgh Cognitive and Behavioral ALS Screen (ECAS).

Conclusions

Dysfunction of eye movement control in ALS can be characterized by a two-staged sequential pattern comprising executive deficits in Stage 1 and additional impaired infratentorial oculomotor control pathways in Stage 2. This pattern parallels the neuropathological staging of ALS and may serve as a technical marker of the neuropathological spreading.