The lack of chromosomal protein Hmg1 does not disrupt cell growth but causes lethal hypoglycaemia in newborn mice.

High mobility group 1 (HMG1) protein is an abundant component of all mammalian nuclei, and related proteins exist in all eukaryotes. HMG1 binds linear DNA with moderate affinity and no sequence specificity, but bends the double helix significantly on binding through the minor groove. It binds with high affinity to DNA that is already sharply bent, such as linker DNA at the entry and exit of nucleosomes; thus, it is considered a structural protein of chromatin. HMG1 is also recruited to DNA by interactions with proteins required for basal and regulated transcriptions and V(D)J recombination. Here we generate mice harbouring deleted Hmg1. Hmg1-/- pups are born alive, but die within 24 hours due to hypoglycaemia. Hmg1-deficient mice survive for several days if given glucose parenterally, then waste away with pleiotropic defects (but no alteration in the immune repertoire). Cell lines lacking Hmg1 grow normally, but the activation of gene expression by the glucocorticoid receptor (GR, encoded by the gene Grl1) is impaired. Thus, Hmg1 is not essential for the overall organization of chromatin in the cell nucleus, but is critical for proper transcriptional control by specific transcription factors.

Cepacia syndrome in cystic fibrosis: A systematic review of the literature and possible new perspectives in treatment.

BACKGROUND: Cepacia syndrome (CS) is an acute, necrotizing pneumonia with a high mortality rate, occurring in patients with cystic fibrosis (CF) infected with Burkholderia cepacia complex (BCC). Due to its low incidence, data on this condition are limited. METHODS: We conducted a systematic review of the reported cases of CS by searching MEDLINE, Embase and the Cochrane Library to improve knowledge of this rare but potentially lethal condition. RESULTS: We included 15 eligible articles, describing 18 cases (9 females) of CS. Median age at onset was 22 years (range: 10-60 years); median time to CS after first infection by BCC was 5 years (range: 1-26 years). Burkholderia cenocepacia was the most frequently reported causative agent. All patients received intravenous antibiotic treatment (most frequently including cotrimoxazole), while inhaled antibiotics were used in five patients (27.8%). Immunosuppressant agents were the most commonly prescribed supportive treatment (n = 7, 38.9%). Half of the patients died (9/18, 50%). CONCLUSIONS: This study describes epidemiological, clinical characteristics, and prognosis of CS cases reported over the last 24 years. CS is a rare yet severe complication of BCC infection in patients with CF, which occurs several years after BCC colonization and has a negative outcome in 50% of the patients. Data are too scanty to identify the most effective therapeutic approach.

Cytokine storm syndrome in a young patient with cystic fibrosis.

We report the case of a patient with cystic fibrosis (CF) presenting with a full-blown cytokine storm syndrome probably triggered by infection. This condition is rare and the diagnosis can be particularly difficult in patients with a complex chronic disease such as CF. However, timely recognition and appropriate treatment in the early stages are key to avoiding a potentially fatal course.

High mobility group 1 protein is not stably associated with the chromosomes of somatic cells.

High mobility group 1 (HMG1) protein is an abundant and conserved component of vertebrate nuclei and has been proposed to play a structural role in chromatin organization, possibly similar to that of histone H1. However, a high abundance of HMG1 had also been reported in the cytoplasm and on the surface of mammalian cells. We conclusively show that HMG1 is a nuclear protein, since several different anti-HMG1 antibodies stain the nucleoplasm of cultured cells, and epitope-tagged HMG1 is localized in the nucleus only. The protein is excluded from nucleoli and is not associated to specific nuclear structures but rather appears to be uniformly distributed. HMG1 can bind in vitro to reconstituted core nucleosomes but is not stably associated to chromatin in live cells. At metaphase, HMG1 is detached from condensed chromosomes, contrary to histone H1. During interphase, HMG1 readily diffuses out of nuclei after permeabilization of the nuclear membranes with detergents, whereas histone H1 remains associated to chromatin. These properties exclude a shared function for HMG1 and H1 in differentiated cells, in spite of their similar biochemical properties. HMG1 may be stably associated only to a very minor population of nucleosomes or may interact transiently with nucleosomes during dynamic processes of chromatin remodeling.

Targeting Prostate Cancer with a Combination of WNT Inhibitors and a Bi-functional Peptide.

BACKGROUND/AIM: Prostate cancer is the most common cancer in the Western world. A bi-functional peptide was combined with wingless-related integration site (WNT) inhibitors to determine if there is an additive therapeutic effect when they are used against prostate cancer, since their efficacy has already been proven when used alone. MATERIALS AND METHODS: A bi-functional peptide (TP-LYT) was designed with a target domain (LTVSPWY) and a lytic domain (KLAKLAK)2, and a second peptide with the same lytic domain but a random sequence instead of the target domain was used as a negative control. Two different WNT inhibitors were used, ethacrynic acid and ciclopiroxolamine. They were tested on prostate cancer cells using the WST-8 assay. RESULTS: A synergistic effect of peptides and WNT inhibitors was demonstrated, increasing the toxicity against cancer cells. CONCLUSION: Our findings potentially allow safer treatment since lower concentrations of WNT inhibitors can be used in combination with this bi-functional peptide.

In vivo recombination and the production of hybrid genes.

In vivo recombination between homologous genes is increasingly being favoured as a means of generating proteins with altered and novel specificities. The typical procedure requires the cloning of two related genes on a single replicative plasmid of Escherichia coli and the selection or screening of recombinants. Up to now the recombination process between the cloned genes was generally thought to involve the recA function and the availability of free ends in the DNA molecule to be recombined. Our results show that neither is necessary. Recombinants are obtained by simply growing the bacteria that host the plasmid carrying the two cloned genes.

Extreme endovascular revascularization for limb salvage in critical limb ischemia.

AIM: Distal bypass has been considered as a primary choice for the treatment of critical limb ischemia (CLI). When bypass failed with limb threatening ischemia, the amputation rate is high in patients with increased surgical risks and lack of conduit. Percutaneous transluminal angioplasty (PTA) has been shown to be effective and safe in the setting of CLI even in patients with failed bypass graft. The aim of this study was to review our experience and results of extreme endovascular revascularization in patients with CLI following occluded lower limb bypass graft. METHODS: Retrospective review from January 2005 to June 2008 of patients with CLI following occluded bypass graft who underwent PTA was performed. All patients were studied by Duplex scanning and dual-energy computed tomographic angiography (DE-CTA) bone removal technique. Stents were used in cases of residual stenosis or dissection. Technical success was defined as a residual stenosis less than 30%. Demographics, comorbidities, functional status, details of the procedure information were recorded. Descriptive, logistic regression and life-table analyses performed. RESULTS: Thirty-six patients with occluded bypass grafts were treated. The mean age was 69 years (range 56-89), 44% were older than 80 years, 83% had diabetes mellitus, 88% of limbs treated had multiple lesions included Tasc C and D lesions. Technical success was achieved in 91%. Mean follow-up was 24 months. At follow-up, there were 19 PTA failures which were followed by subsequent procedures: redo PTA in 16 limbs, redo bypass in 2, amputation in 5. Cumulative primary patency was 60% (±0.08 SE) and 24% (±0.07 SE). Secondary patency was 96% (±0.03 SE) and 83% (±0.08 SE). Limb salvage was 84% (±0.06 SE) and 70% (±0.10 SE). Freedom from surgical revision was 78% (±0.07 SE) and 54% (±0.11 SE). Overall survival was 89% (±0.05 SE) and 58% (±0.11 SE) at 12 and 24 months, respectively. CONCLUSION: Endovascular revascularization of patients with CLI and occluded bypass graft is a safe and feasible procedure with reasonable technical and clinical success and limb salvage. PTA may be the only alternative to amputation in these patients with extensive comorbidities and limited life expectancy.

The mouse gene coding for high mobility group 1 protein (HMG1).

We have isolated an active gene encoding the mouse HMG1 protein among a multitude of cross-hybridizing sequences, which most likely are retrotransposed pseudogenes. The hmg1 gene contains five exons, of which the first is not translated, and the last contains a long 3'-untranslated sequence and three alternative polyadenylation sites. We found no evidence for a sequence encoding a membrane localization signal in the hmg1 gene, despite the presence of HMG1 protein on the surface of several cell types. The hmg1 promoter coincides with a CpG island, contains no TATA sequence, and derives the expression of reporter genes placed under its control. The hmg1 gene may be a member of a family of closely related genes but appears to be the major or the only active gene coding for HMG1 protein.

Functional analysis of the outB gene of Bacillus subtilis.

Three additional alleles of the outB gene of Bacillus subtilis, whose activity is required for spore outgrowth, were identified. The nucleotide sequence of three mutant genes was determined. Analyses of dominance-recessivity showed that the wild-type allele is dominant over the mutant ones. When the outB gene was placed under the control of the inducible spac-1 promoter, the presence of IPTG was necessary to obtain normal growth. The results suggested that the outB gene is required for growth of B. subtilis. Expression of outB from the sporulation promoter spoIID negatively affected subsequent spore outgrowth, without altering vegetative growth and sporulation.

RocR, a novel regulatory protein controlling arginine utilization in Bacillus subtilis, belongs to the NtrC/NifA family of transcriptional activators.

Bacillus subtilis can use ammonium and various amino acids as sole nitrogen sources. The utilization of arginine or ornithine is abolished in a sigma L-deficient strain of B. subtilis, indicating that one or several genes involved in this pathway are transcribed by a sigma L-RNA polymerase holoenzyme. Three B. subtilis genes, called rocA, rocB, and rocC, which seem to form an operon, were found near the sacTPA locus (P. Glaser, F. Kunst, M. Arnaud, M.-P. Coudart, W. Gonzales, M.-F. Hullo, M. Ionescu, B. Lubochinsky, L. Marcelino, I. Moszer, E. Presecan, M. Santana, E. Schneider, J. Schweizer, A. Vertes, G. Rapport, and A. Danchin, Mol. Microbiol. 10:371-384, 1993). The expression of this putative operon is induced by arginine and is sigma L dependent. Mutants impaired in the transcription of rocA were obtained. One of these mutants was used as recipient to clone and sequence a new regulatory gene, called rocR. This gene encodes a polypeptide of 52 kDa which belongs to the NtrC/NifA family of transcriptional activators. Upstream activating sequences highly similar to those of NtrC in Escherichia coli were also identified upstream from the rocABC genes. A B. subtilis strain containing a rocR null mutation is unable to use arginine as the sole nitrogen source, indicating that RocR is a positive regulator of arginine catabolism. After LevR, RocR is the second example of an activator stimulating sigma 54-dependent promoters in gram-positive bacteria.

Expression of a cloned Bacillus thuringiensis delta-endotoxin gene in Bacillus subtilis.

The entire coding region of the Bacillus thurigiensis HD73 crystal protein gene was subcloned from plasmid pJWK20 into the integration vector pUG2-15. This plasmid expresses chloramphenicol resistance when integrated into the Bacillus subtilis chromosome in the outH locus near the recE region. The correct molecular organization of the integrated plasmid was verified by hybridization to Southern blots of chromosomal DNA digests. Production of the toxic crystal protein was monitored at different time points during the life cycle of B. subtilis. Toxicity assays against Anagasta (Ephestia) larvae, direct electron microscopy crystal detection, and immunoblotting assays proved that the expression of the gene in B. subtilis is time regulated and restricted mainly to the sporulation stage. RNase protection experiments defined the transcription initiation start point and the transcription timing. All tests were made in a strain containing one to three copies of the integrated plasmid and in a strain subjected to an amplification regimen.

Expression, purification, crystallization, and preliminary X-ray diffraction analysis of the homodimeric bacterial hemoglobin from Vitreoscilla stercoraria.

The recombinant homodimeric hemoglobin from the strictly aerobe gram-negative bacterium Vitreoscilla stercoraria has been expressed in Escherichia coli, purified to homogeneity, and crystallized by vapor diffusion techniques, using ammonium sulfate as precipitant. The crystals belong to the monoclinic space group P2(1) and diffract to HIGH resolution. The unit cell parameters are alpha = 62.9, b = 42.5, c = 63.2 A, beta = 106.6 degrees; the asymmetric unit contains the homodimeric hemoglobin, with a volume solvent content of 42%.