The Mycobacterium avium-intracellulare complex dnaB locus and protein intein splicing

Intein is a protein sequence mebedded in-frame within a precursor protein and is posttranslationally excised by a self-catalytic protein splicing process. Protein splicing is believed to follow a pathway requiring Cys, Ser, or Thr residues at the intein N-terminus and substitutions other than Cys, Ser, or Thr residues prevent splicing. We show that the dnaB locus in some strains of M. avium-intracellulare complex (MAC) contains intein and that the intein N-terminal amino acid is Ala [Ala-type]. We demonstrate that the M. avium DnaB precursor protein undergoes posttranslational proteolytic processing producing proteins corresponding to the sizes of the DnaB and intein. Further, by Western analysis we detect a protein corresponding to the size of the spliced DnaB protein in MAC cell extracts. Together, these results indicate that the Ala-type MAC DnaB inteins can splice and provide another example that points to an interesting alternative splicing mechanism (Southworth, M. W., Benner, J., and Perler, F. B., EMBO J. 19, 5019-5026, 2000).     

High cytokine levels at admission are associated with fatal outcome in patients with necrotizing fasciitis

We evaluated in a blinded fashion the cytokine profiles of patients with suspected necrotizing fasciitis. In 15 out of 20 patients, the diagnosis of necrotizing fasciitis was established; five patients had cellulitis. Eighteen of the 20 patients were i.v. drug users. Five of the 15 patients with necrotizing fasciitis died (33%). On admission, serum levels for interleukin-1beta (IL-1beta), IL-1-receptor antagonist (IL-1Ra), IL-18 and interferon-gamma (IFNgamma) as well as white blood cells (WBC) were significantly elevated in patients with fatal outcome compared to survivors with necrotizing fasciitis. IL-1Ra and WBC levels were also higher than in patients with cellulitis. No differences were observed between patients groups for IL-6 and IL-8. In summary, significantly elevated levels of proinflammatory cytokines and particularly IL-1Ra are associated with fatal outcome in patients with necrotizing fasciitis. The measurement of proinflammatory cytokines and IL-1Ra may help to establish early diagnosis of life-threatening necrotizing fasciitis and thus to initiate aggressive treatment.     

A unique set of 11,008 onion expressed sequence tags reveals expressed sequence and genomic differences between the monocot orders Asparagales and Poales

Enormous genomic resources have been developed for plants in the monocot order Poales; however, it is not clear how representative the Poales are for the monocots as a whole. The Asparagales are a monophyletic order sister to the lineage carrying the Poales and possess economically important plants such as asparagus, garlic, and onion. To assess the genomic differences between the Asparagales and Poales, we generated 11,008 unique ESTs from a normalized cDNA library of onion. Sequence analyses of these ESTs revealed microsatellite markers, single nucleotide polymorphisms, and homologs of transposable elements. Mean nucleotide similarity between rice and the Asparagales was 78% across coding regions. Expressed sequence and genomic comparisons revealed strong differences between the Asparagales and Poales for codon usage and mean GC content, GC distribution, and relative GC content at each codon position, indicating that genomic characteristics are not uniform across the monocots. The Asparagales were more similar to eudicots than to the Poales for these genomic characteristics.     

The DNA sequence of chromosome I of an African trypanosome: gene content,chromosome organisation,recombination and polymorphism

The African trypanosome, Trypanosoma brucei, causes sleeping sickness in humans in sub-Saharan Africa. Here we report the sequence and analysis of the 1.1 Mb chromosome I, which encodes approximately 400 predicted genes organised into directional clusters, of which more than 100 are located in the largest cluster of 250 kb. A 160-kb region consists primarily of three gene families of unknown function, one of which contains a hotspot for retroelement insertion. We also identify five novel gene families. Indeed, almost 20% of predicted genes are members of families. In some cases, tandemly arrayed genes are 99–100% identical, suggesting an active process of amplification and gene conversion. One end of the chromosome consists of a putative bloodstream-form variant surface glycoprotein (VSG) gene expression site that appears truncated and degenerate. The other chromosome end carries VSG and expression site-associated genes and pseudogenes over 50 kb of subtelomeric sequence where, unusually, the telomere-proximal VSG gene is oriented away from the telomere. Our analysis includes the cataloguing of minor genetic variations between the chromosome I homologues and an estimate of crossing-over frequency during genetic exchange. Genetic polymorphisms are exceptionally rare in sequences located within and around the strand-switches between several gene clusters.     

Interaction of the estrogen receptors with the Fas ligand promoter in human monocytes

The predominance of autoimmune diseases among women suggests that estrogen may modulate immune function. Monocytes and macrophages are important in initiating, maintaining, and resolving inflammatory responses through cell-signaling molecules, which control immune cell survival. One important mechanism of cell survival is mediated by the Fas/Fas ligand (FasL) system. In this study, the link between estrogen, monocytes/macrophages, and the Fas/FasL system was investigated. Estrogen treatment increased FasL expression in monocytes through the binding of the estrogen receptors (ER) to the estrogen recognizing elements and AP-1 motifs present at the FasL promoter. Furthermore, estrogen induced apoptosis in monocytes expressing ERbeta, but not in monocyte-differentiated macrophages expressing ERalpha. The expression of either ERalpha or ERbeta and their response to estrogen in monocytes was found to be dependent on the their stage of cell differentiation. Previously, we have shown that estrogen replacement therapy in postmenopausal women decreased the number of circulating monocytes. In this study, we have characterized the molecular mechanism by which estrogen regulates monocytes homeostasis. These findings indicate that estrogen may regulate immune cell survival through the Fas/FasL system. There is biological relevance to these findings in view of studies showing that accumulation of activated monocytes is involved in the pathogenesis of conditions such as vasculititis, arteriosclerosis, and rheumatoid arthritis.     

p53 functional assays: detecting p53 mutations in both the germline and in sporadic tumours

The tumour suppressor gene p53 is the gene most often reported to be mutated in clinical cancers with something like half of all tumours harbouring mutations. Further, many studies have suggested that p53 mutations have prognostic importance and sometimes are a significant factor in determining the response of tumours to therapy. The value of knowing the p53 status of individual tumours will increase if currently researched strategies aimed at developing p53-based treatment protocols come to fruition. There are quite a number of techniques used to detect p53 defects in both tumours and in the germline of cancer-prone families, although some of these methods are indirect and each has certain drawbacks. In this brief review we will discuss the value of two assays of p53 function as a means of detecting and partly characterizing p53 mutations. The two assays are the apoptotic assay, which measures the response of peripheral blood lymphocytes to radiation-induced DNA damage and the FASAY, a yeast based assay which assesses the ability of a given p53 protein to transactivate p53 target genes. Both of these assays are rapid, yielding results within 5 days. Further, they not only offer the possibility of detecting p53 mutations but also of characterizing a given mutation in terms of two of p53's most important functions, namely the induction of apoptosis and the transactivation of target genes.     

Photosystem II and photosynthetic oxidation of water: an overview

Conceptually, photosystem II, the oxygen-evolving enzyme, can be divided into two parts: the photochemical part and the catalytic part. The photochemical part contains the ultra-fast and ultra-efficient light-induced charge separation and stabilization steps that occur when light is absorbed by chlorophyll. The catalytic part, where water is oxidized, involves a cluster of Mn ions close to a redox-active tyrosine residue. Our current understanding of the catalytic mechanism is mainly based on spectroscopic studies. Here, we present an overview of the current state of knowledge of photosystem II, attempting to delineate the open questions and the directions of current research.     

Orientation selection in photosynthetic PS I multilayers: structural investigation of the charge separated state P(700)(+z.rad;)A(1)(-z.rad;) by high-field/high-frequency time-resolved EPR at 3.4 T/95 GHz

The radical-pair state of the primary electron donor and the secondary electron acceptor (P(700)(+z.rad;)A(1)(-z.rad;)) of the photosynthetic reaction center (RC) photosystem I (PS I) of Synechocystis PCC 6803 was studied by time-resolved electron paramagnetic resonance (TREPR) at high field/high frequency (3.4 T/95 GHz) using orientation selection in multilayers. The goal of the present article is to work out the basis for future studies, in which the improved resolution of such multilayers may be used to detect mutation-induced structural changes of PS I in membrane preparations. This approach is particularly interesting for systems that cannot be prepared as single crystals. However, in order to use such multilayers for structural investigations of protein complexes, it is necessary to know their orientation distribution. PS I was chosen as a test example because the wild type was recently crystallized and its X-ray structure determined to 2.5 A resolution [Nature 411 (2001) 909]. On the basis of our experimental results we determined the orientation distribution. Furthermore, a simulation model for the general case in which the orientation distribution is not axially symmetric about the C(2) symmetry axis of the RC is developed and discussed. Spectra simulations show that changes in the TREPR spectra of PS I are much more significant for these oriented multilayers than for disordered samples. In this way the use of oriented multilayers, in conjunction with multifrequency TREPR measurements on oriented as well as on disordered samples, is a promising approach for studies of structural changes of PS I systems that are induced by point mutations.