Comparison of the heme structures of horseradish peroxidase compounds X and II by resonance Raman spectroscopy

Horseradish peroxidase will catalyze the chlorination of certain substrates by sodium chlorite through an intermediate known as compound X. A chlorite-derived chlorine atom is known to be retained by compound X and has been proposed to be located at the heme active site. Although several heme structures have been proposed for compound X, including an Fe(IV)-OCl group, preliminary data previously reported by our laboratory suggested that compound X contained a heme Fe(IV) = O group, based on the similarity of a compound X resonance Raman band at 788 cm-1 to resonance Raman Fe(IV) = O stretching vibrations recently identified for horseradish peroxidase compound II and ferryl myoglobin. Isotopic studies now confirm that the 788 cm-1 resonance Raman band of compound X is, in fact, due to a heme Fe(IV) = O group, with the oxygen atom derived from chlorite. The Fe(IV) = O frequency of compound X, of horseradish peroxidase isoenzymes B and C, undergoes a pH-induced frequency shift, with behavior which appears to be the same as that previously reported for compound II, formed from the same isoenzymes. These observations strongly suggest that compounds II and X have very similar, if not identical, heme structures. The chlorine atom thus appears not to be heme-bound and may rather be located on an amino acid residue. The studies on compound X reported here were done in a pH region above pH 8, where compound X is moderately stable. The present results do not necessarily apply to compound X below pH 8.     

Zur Cytogenetik vonMedicago falcata,L.

Ohne ZusammenfassungMit 1 Textabbildung.     

Multi-species territoriality and dynamic of neotropical forest understorey bird flocks

1. Eleven contiguous mixed-species bird flocks, with colour-banded individuals, were monitored continuously during 3 years in a 132-ha study area of primary rainforest in French Guiana.
2. Flock members were divided into six categories according to their flocking propensity and occurrence: 10 core or permanent species and 56 regular, occasional or incidental species. Each core species was represented by a single breeding pair with their fledglings and extra 'floaters' (unmated subadults and adults).
3. Flock home ranges overlapped slightly, but were communally defended by all core species in areas of overlap. Their size varied from 3·2 to 14·3 ha and was inversely correlated with vegetation density, but not flock size or species composition.
4. Flock number, size and composition, as well as boundaries were highly stable between seasons and years. Each flock had a single permanent gathering site and bathing site in late afternoon, the latter sometimes shared by 2–3 flocks.
5. Core species produced 0·18–0·73 fledglings per pair per year, which stayed in their natal flock for 200 to over 421 days. Then, these individuals usually moved between two and six different flocks, sometimes for up to 3 years, before finding a mate and a flock where they could settle and breed. Once breeding, they probably remained for life in the same flock. The mean annual survival rate was at least 0·75.
6. This highly evolved and stable organization, associated with a low breeding success and high survival rate was a critical factor maintaining low species density, delayed reproduction and a proportion of floating individuals buffering population fluctuations.
7. These social groups with their multi-species territoriality and co-evolved roles of flock members were similar to those described elsewhere in South America. They seem to be a general phenomenon in neotropical lowland rainforests.     

Cloning and characterization of MS5 from Arabidopsis : a gene critical in male meiosis

In this paper, we describe the cloning of the MS5 gene, a gene essential for male fertility in Arabidopsis . We previously defined the MS5 locus by characterizing an EMS-induced allele, ms5–1 . We identified a new allele of MS5 ( ms5–2 ) that was T-DNA-generated and used the T-DNA tag to clone the gene. Sequencing of mutant and wild-type alleles together with complementation of the ms5–1 mutant phenotype with a wild-type genomic clone confirmed the identity of the gene. Differences between the phenotypes of the two mutant alleles could be attributed to differences in mutant gene structure. The semi-dominant and dominant negative phenotypes of the ms5–2 mutant probably result from production of a truncated polypeptide. An unknown locus in Landsberg erecta can counteract the dominant negative phenotype of ms5–2 . Mutations in MS5 cause the formation of ‘polyads’– tetrads with more than four pools of chromosomes after male meiosis. Similarities between the MS5 sequence and that of a number of proteins were found; two that may be significant were with a synaptonemal complex protein and with a regulatory subunit of a cyclin-dependent kinase. The MS5 gene is a member of a small gene family highly conserved amongst plant species.     

Evidence for a human-specific Escherichia coli clone

Escherichia coli is a widespread commensal of the vertebrate intestinal tract. Until recently, no strong association between a particular clone and a given host species has been found. However, members of the B2 subgroup VIII clone with an O81 serotype appear to be human host specific. To determine the degree of host specificity exhibited by this clone, a PCR-based assay was used to screen 723 faecal and clinical isolates from humans, and 904 faecal isolates from animals. This clone was not detected among the animal isolates, but was discovered in people living in Africa, Europe and South America. The clone is rarely isolated from people suffering from intestinal or extraintestinal disease and is avirulent in a mouse model of extraintestinal infection. Fine-scale epidemiological analysis suggests that this clone is competitively dominant relative to other members of the B2 phylogenetic group and that it has increased in frequency over the past 20 years. This clone appears to be a good candidate for use as a probiotic, and may be suitable as an indicator of human faecal contamination in microbial source tracking studies.     

A mutation (R826W) in nucleotide-binding domain 1 of ABCC8 reduces ATPase activity and causes transient neonatal diabetes

Activating mutations in the pore-forming Kir6.2 (KCNJ11) and regulatory sulphonylurea receptor SUR1 (ABCC8) subunits of the K(ATP) channel are a common cause of transient neonatal diabetes mellitus (TNDM). We identified a new TNDM mutation (R826W) in the first nucleotide-binding domain (NBD1) of SUR1. The mutation was found in a region that heterodimerizes with NBD2 to form catalytic site 2. Functional analysis showed that this mutation decreases MgATP hydrolysis by purified maltose-binding protein MBP-NBD1 fusion proteins. Inhibition of ATP hydrolysis by MgADP or BeF was not changed. The results indicate that the ATPase cycle lingers in the post-hydrolytic MgADP.P(i)-bound state, which is associated with channel activation. The extent of MgADP-dependent activation of K(ATP) channel activity was unaffected by the R826W mutation, but the time course of deactivation was slowed. Channel inhibition by MgATP was reduced, leading to an increase in resting whole-cell currents. In pancreatic beta cells, this would lead to less insulin secretion and thereby diabetes.     

Both Functional LTβ Receptor and TNF Receptor 2 Are Required for the Development of Experimental Cerebral Malaria

Background

TNF-related lymphotoxin α (LTα) is essential for the development of Plasmodium berghei ANKA (PbA)-induced experimental cerebral malaria (ECM). The pathway involved has been attributed to TNFR2. Here we show a second arm of LTα-signaling essential for ECM development through LTβ-R, receptor of LTα1β2 heterotrimer.

Methodology/Principal Findings

LTβR deficient mice did not develop the neurological signs seen in PbA induced ECM but died at three weeks with high parasitaemia and severe anemia like LTαβ deficient mice. Resistance of LTαβ or LTβR deficient mice correlated with unaltered cerebral microcirculation and absence of ischemia, as documented by magnetic resonance imaging and angiography, associated with lack of microvascular obstruction, while wild-type mice developed distinct microvascular pathology. Recruitment and activation of perforin⁺ CD8⁺ T cells, and their ICAM-1 expression were clearly attenuated in the brain of resistant mice. An essential contribution of LIGHT, another LTβR ligand, could be excluded, as LIGHT deficient mice rapidly succumbed to ECM.

Conclusions/Significance

LTβR expressed on radioresistant resident stromal, probably endothelial cells, rather than hematopoietic cells, are essential for the development of ECM, as assessed by hematopoietic reconstitution experiment. Therefore, the data suggest that both functional LTβR and TNFR2 signaling are required and non-redundant for the development of microvascular pathology resulting in fatal ECM.     

Genebank biodiversity assessments regarding optimal sample size and seed harvesting techniques for the regeneration of carrot accessions

Small, finite populations are particularly vulnerable to diversity loss during regeneration. The regeneration of a highly outbreeding open-pollinated variety relies on estimated effective population size, via the measurement of temporal change in allele frequencies. Using appropriate estimators for dominant gene markers, effective sizes were calculated for five sizes of a mating population and two seed harvesting procedures. We have shown that, in the case of carrot regeneration, 70 equally harvested plants should provide an effective size (N _e) of at least 50 plants. This value seems sufficient to limit genetic drift and to preserve an efficient level of genetic diversity within the collection. The efficiency of balanced samples (made of an equal number of seeds per plant) is compared to that of bulk samples (seeds randomly chosen among the total seed lot coming from all the plants).     

SHOC1, an XPF endonuclease-related protein, is essential for the formation of class I meiotic crossovers

Crossovers (COs) are essential for the completion of meiosis in most species and lead to new allelic combinations in gametes. Two pathways of meiotic crossover formation have been distinguished. Class I COs, which are the major class of CO in budding yeast, mammals, Caenorhabditis elegans, and Arabidopsis, depend on a group of proteins called ZMM and rely on specific DNA structure intermediates that are processed to form COs. We identified a novel gene, SHOC1, involved in meiosis in Arabidopsis. Shoc1 mutants showed a striking reduction in the number of COs produced, a similar phenotype to the previously described Arabidopsis zmm mutants. The early steps of recombination, revealed by DMC1 foci, and completion of synapsis are not affected in shoc1 mutants. Double mutant analysis showed that SHOC1 acts in the same pathway as AtMSH5, a conserved member of the ZMM group. SHOC1 is thus a novel gene required for class I CO formation in Arabidopsis. Sequence similarity studies detected putative SHOC1 homologs in a large range of eukaryotes including human. SHOC1 appears to be related to the XPF endonuclease protein family, which suggests that it is directly involved in the maturation of DNA intermediates that lead to COs.