Breakdown rates and macroinvertebrate colonisation of alder (Alnus glutinosa) leaves in an acid lake (Lake Orta, N Italy), before, during and after a liming intervention

To test the effectiveness of the liming intervention on Lake Orta, the speed of leaves decay and of colonisation processes by macrobenthonic fauna were studied on alder leaves (Alnus glutinosa) placed on the bottom of the lake and recovered after appropriate time intervals. Experiments were performed at two sites (North and South) and two depths (-3 and –18 m), during three successive winters: 1988-1989 (pre-liming), 1989-1990 (liming), 1990-1991 (post-liming). Two main results emerged: 1) alder leaves, which are known to have a medium to high decaying speed in a number of aquatic environments, behave in Lake Orta as a low speed species. Decaying processes in the three years are significantly different only in station N3, where the mean breakdown rate in 1988- 1989 is more than twice that measured in the two subsequent winters. 2) The species richness of colonising benthic fauna is low: the community is made up almost exclusively of Chironomidae, which form 70 to 100% of the whole population; among them, the genus Phenopsectra is always present, while Tanytarsus was collected only during the first year and in the less deep sampling sites. The mean population abundances were higher before liming

Neutron cryo-crystallography captures the protonation state of ferryl heme in a peroxidase

Heme enzymes activate oxygen through formation of transient iron-oxo (ferryl) intermediates of the heme iron. A long-standing question has been the nature of the iron-oxygen bond and, in particular, the protonation state. We present neutron structures of the ferric derivative of cytochrome c peroxidase and its ferryl intermediate; these allow direct visualization of protonation states. We demonstrate that the ferryl heme is an Fe(IV)=O species and is not protonated. Comparison of the structures shows that the distal histidine becomes protonated on formation of the ferryl intermediate, which has implications for the understanding of O–O bond cleavage in heme enzymes. The structures highlight the advantages of neutron cryo-crystallography in probing reaction mechanisms and visualizing protonation states in enzyme intermediates

Mutation spectrum of MLL2 in a cohort of kabuki syndrome patients

ABSTRACT: BACKGROUND: Kabuki syndrome (Niikawa-Kuroki syndrome) is a rare, multiple congenital anomalies/mental retardation syndrome characterized by a peculiar face, short stature, skeletal, visceral and dermatoglyphic abnormalities, cardiac anomalies, and immunological defects. Recently mutations in the histone methyl transferase MLL2 gene have been identified as its underlying cause. METHODS: Genomic DNAs were extracted from 62 index patients clinically diagnosed as affected by Kabuki syndrome. Sanger sequencing was performed to analyze the whole coding region of the MLL2 gene including intron-exon junctions. The putative causal and possible functional effect of each nucleotide variant identified was estimated by in silico prediction tools. RESULTS: We identified 45 patients with MLL2 nucleotide variants. 38 out of the 42 variants were never described before. Consistently with previous reports, the majority are nonsense or frameshift mutations predicted to generate a truncated polypeptide. We also identified 3 indel, 7 missense and 3 splice site. CONCLUSIONS: This study emphasizes the relevance of mutational screening of the MLL2 gene among patients diagnosed with Kabuki syndrome. The identification of a large spectrum of MLL2 mutations possibly offers the opportunity to improve the actual knowledge on the clinical basis of this multiple congenital anomalies/mental retardation syndrome, design functional studies to understand the molecular mechanisms underlying this disease, establish genotype-phenotype correlations and improve clinical management

Reduced BRCA1 expression due to promoter hypermethylation in therapy-related acute myeloid leukaemia

BRCA1 plays a pivotal role in the repair of DNA damage, especially following chemotherapy and ionising radiation. We were interested in the regulation of BRCA1 expression in acute myeloid leukaemia (AML), in particular in therapy-related forms (t-AML). Using real-time PCR and Western blot, we found that BRCA1 mRNA was expressed at barely detectable levels by normal peripheral blood granulocytes, monocytes and lymphocytes, whereas control BM-mononuclear cells and selected CD34+ progenitor cells displayed significantly higher BRCA1 expression (P=0.0003). Acute myeloid leukaemia samples showed heterogeneous BRCA1 mRNA levels, which were lower than those of normal bone marrows (P=0.0001). We found a high frequency of hypermethylation of the BRCA1 promoter region in AML (51/133 samples, 38%), in particular in patients with karyotypic aberrations (P=0.026), and in t-AML, as compared to de novo AML (76 vs 31%, P=0.0002). Examining eight primary tumour samples from hypermethylated t-AML patients, BRCA1 was hypermethylated in three of four breast cancer samples, whereas it was unmethylated in the other four tumours. BRCA1 hypermethylation correlated to reduced BRCA1 mRNA (P=0.0004), and to increased DNA methyltransferase DNMT3A (P=0.003) expression. Our data show that reduced BRCA1 expression owing to promoter hypermethylation is frequent in t-AML and that this could contribute to secondary leukaemogenesis

Structural and spectroscopic characterisation of a heme peroxidase from sorghum

A cationic class III peroxidase from Sorghum bicolor was purified to homogeneity. The enzyme contains a high-spin heme, as evidenced by UV-visible spectroscopy and EPR. Steady state oxidation of guaiacol was demonstrated and the enzyme was shown to have higher activity in the presence of calcium ions. A Fe(III)/Fe(II) reduction potential of -266 mV vs NHE was determined. Stopped-flow experiments with H2O2 showed formation of a typical peroxidase Compound I species, which converts to Compound II in the presence of calcium. A crystal structure of the enzyme is reported, the first for a sorghum peroxidase. The structure reveals an active site that is analogous to those for other class I heme peroxidase, and a substrate binding site (assigned as arising from binding of indole-3-acetic acid) at the γ-heme edge. Metal binding sites are observed in the structure on the distal (assigned as a Na(+) ion) and proximal (assigned as a Ca(2+)) sides of the heme, which is consistent with the Ca(2+)-dependence of the steady state and pre-steady state kinetics. It is probably the case that the structural integrity (and, thus, the catalytic activity) of the sorghum enzyme is dependent on metal ion incorporation at these positions