IS<Emphasis Type="Italic">Rm31</Emphasis>, a new insertion sequence of the IS<Emphasis Type="Italic">66</Emphasis> family in<Emphasis Type="Italic"> Sinorhizobium meliloti</Emphasis>

Sinorhizobium meliloti natural populations show a high level of genetic polymorphism possibly due to the presence of mobile genetic elements such as insertion sequences (IS), transposons, and bacterial mobile introns. The analysis of the DNA sequence polymorphism of the nod region of S. meliloti pSymA megaplasmid in an Italian isolate led to the discovery of a new insertion sequence, ISRm31. ISRm31 is 2,803 bp long and has 22-bp-long terminal inverted repeat sequences, 8-bp direct repeat sequences generated by transposition, and three ORFs (A, B, C) coding for proteins of 124, 115, and 541 amino acids, respectively. ORF A and ORF C are significantly similar to members of the transposase family. Amino acid and nucleotide sequences indicate that ISRm31 is a member of the IS66 family. ISRm31 sequences were found in 30.5% of the Italian strains analyzed, and were also present in several collection strains of the Rhizobiaceae family, including S. meliloti strain 1021. Alignment of targets sites in the genome of strains carrying ISRm31 suggested that ISRm31 inserts randomly into S. meliloti genomes. Moreover, analysis of ISRm31 insertion sites revealed DNA sequences not present in the recently sequenced S. meliloti strain 1021 genome. In fact, ISRm31 was in some cases linked to DNA fragments homologous to sequences found in other rhizobia species.     

Infarct-induced chronic heart failure increases bidirectional protein movement across the alveolocapillary barrier

Chronic heart failure (CHF) is associated with adaptive structural changes at the alveolocapillary barrier that may be associated with altered protein permeability. Bidirectional protein movement across the barrier was studied in anesthetized rats with infarct-induced CHF by following (125)I-labeled albumin ((125)I-albumin) flux into the alveoli and the leakage of surfactant protein (SP)-B from the alveoli into the circulation. Three groups were studied: controls [0% left ventricular (LV) infarction], moderate infarct (25-45% LV infarction), and large infarct (>46% LV infarction). Wet and dry lung weights increased in the large infarct group (both P < 0.001), consistent with increased lung water and solid lung tissue. (125)I-albumin flux increased across the endothelial (P < 0.001) and epithelial (P < 0.01) components of the alveolocapillary barrier in the large infarct group. Plasma SP-B increased 23% with moderate infarcts (P < 0.05) and 97% with large infarcts (P < 0.001), independent of alveolar levels. Lavage fluid immune cells (P < 0.01) and myeloperoxidase activity (P < 0.05) increased in the large infarct group, consistent with inflammation. Bidirectional protein movement across the alveolocapillary barrier is increased in CHF, and alveolar inflammation may contribute to this pathophysiological defect.     

Serum levels of soluble CD30 in adult patients affected by atopic dermatitis and its relation to age,duration of disease and Scoring Atopic Dermatitis index

The value of CD30 and the soluble circulating fragment of CD30 (sCD30) for atopic dermatitis (AD) remains unclear. In particular, little is known about the effects of age, duration of disease and Scoring Atopic Dermatitis index (SCORAD) on the levels of serum sCD30 in patients affected by AD. In the present study, we have analysed serum sCD30 levels of adult patients affected by AD. The study's population includes 18 non-smoking outpatients, with a diagnosis of AD. As a control group we studied 18 non-atopic subjects from laboratory staff, matched for sex and age. These subjects had no history of AD, urticaria or seasonal or perennial rhinitis or asthma, and had negative skin prick test to a panel of allergens. The sCD30 serum levels were clearly higher in patients affected by AD (14.2+/-9.0 IU/ml) than in healthy subjects (1.2+/-0.8 IU/ml) (p<0.001). No differences were observed between males and females affected by atopic dermatitis, regarding age, duration of disease and SCORAD. Significant correlations were found between serum levels of sCD30 levels and age (r=-0.55; 95% confidence interval (CI) for r (Fisher's z transformed)=-0.81 to -0.12; p=0.01), duration of the disease (months) (r=-0.64; 95% CI for r (Fisher's z transformed)=-0.85 to -0.24; p=0.004) and SCORAD (r=-0.74; 95% CI for r (Fisher's z transformed)=-0.89 to -0.42; p=0.004). As demonstrated by the close correlation with age, duration of disease and SCORAD, serum levels of sCD30 appear to be an additional marker for the follow-up of AD.     

Productive infection maintains a dynamic steady state of residual viremia in human immunodeficiency virus type 1-infected persons treated with suppressive antiretroviral therapy for five years

To provide insight into the dynamics and source of residual viremia in human immunodeficiency virus (HIV) patients successfully treated with antiretroviral therapy, 14 intensely monitored patients treated with indinavir and efavirenz sustaining HIV RNA at <50 copies/ml for >5 years were studied. Abacavir was added to the regimen of eight patients at year 5. After the first 9 months of therapy, HIV RNA levels had reached a plateau ("residual viremia") that persisted for over 5 years. Levels of residual viremia differed among patients and ranged from 3.2 to 23 HIV RNA copies/ml. Baseline HIV DNA was the only significant pretreatment predictor of residual viremia in regression models including baseline HIV RNA, CD4 count, and patient age. In the four of five patients with detectable viremia who added abacavir to their regimen after 5 years, HIV RNA levels declined rapidly. The estimated half-life of infected cells was 6.7 days. Decrease in activated memory cells and a reduction in gamma interferon production to HIV Gag and p24 antigen in ELISpot assays were observed, consistent with a decrease in HIV replication. Thus, in patients treated with efavirenz plus indinavir, levels of residual viremia were established by 9 months, were predicted by baseline proviral DNA, and remained constant for 5 years. Even after years of highly suppressive therapy, HIV RNA levels declined rapidly after the addition of abacavir, suggesting that productive infection contributes to residual ongoing viremia and can be inhibited with therapy intensification.     

Missense,nonsense, and neutral mutations define juxtaposed regulatory elements of splicing in cystic fibrosis transmembrane regulator exon 9

Exonic sequence variations may induce exon inclusion or exclusion from the mature mRNA by disrupting exonic regulatory elements and/or by affecting a nuclear reading frame scanning mechanism. We have carried out a systematic study of the effect on cystic fibrosis transmembrane regulator exon 9 splicing of natural and site-directed sequence mutations. We have observed that changes in the splicing pattern were not related to the creation of premature termination codons, a fact that indicates the lack of a significant nuclear check of the reading frame in this system. In addition, the splice pattern could not be predicted by available Ser/Arg protein matrices score analysis. An extensive site-directed mutagenesis of the 3' portion of the exon has identified two juxtaposed splicing enhancer and silencer elements. The study of double mutants at these regulatory elements showed a complex regulatory activity. For example, one natural mutation (146C) enhances exon inclusion and overrides all of the downstream silencing mutations except for a C to G transversion (155G). This unusual effect is explained by the creation of a specific binding site for the inhibitory splicing factor hnRNPH. In fact, on the double mutant 146C-155G, the silencing effect is dominant. These results indicate a strict dependence between the two juxtaposed enhancer and silencer sequences and show that many point mutations in these elements cause changes in splicing efficiency by different mechanisms.     

The need of specific standards for head dimensions of urban Sardinian boys

In the 1997-1998 and 1998-1999 school years we measured head dimensions of 1600 boys from 6 to 13 years attending elementary and middle schools in towns of the Cagliari area (Sardinia, Italy). For each age, we compared the mean values for circumference, length and width of the head with Canadian standards, widely used by Sardinian pediatricians. The t-test shows that the means of the three variables are significantly lower in the Cagliari boys than in their Canadian contemporaries, with the exception of head circumference in 6 and 7 year-olds, and of head width in 10 year-olds. Therefore, it is necessary to produce specific growth charts for circumference, length and width of the head of Sardinian children rather than evaluate their growth using standards of populations with different ethnic, geographical and socio-economic backgrounds.     

Role of the adaptor protein CIKS in the activation of the IKK complex

Nuclear factor kappaB (NF-kappaB) plays a pivotal role in numerous cellular processes, including stress response, inflammation, and protection from apoptosis. Therefore, the activity of NF-kappaB needs to be tightly regulated. We have previously identified a novel gene, named CIKS (connection to IkappaB-kinase and SAPK), able to bind the regulatory sub-unit NEMO/IKKgamma and to activate NF-kappaB. Here, we demonstrate that CIKS forms homo-oligomers, interacts with NEMO/IKKgamma, and is recruited to the IKK-complex upon cell stimulation. In addition, we identified the regions of CIKS responsible for these functions. We found that the ability of CIKS to oligomerize, and to be recruited to the IKK-complex is not sufficient to activate the NF-kappaB. In fact, a deletion mutant of CIKS able to oligomerize, to interact with NEMO/IKKgamma, and to be recruited to the IKK-complex does not activate NF-kappaB, suggesting that CIKS needs a second level of regulation to efficiently activate NF-kappaB.     

Cell shape provides global control of focal adhesion assembly

Cell spreading was controlled independently of the amount and density of immobilized integrin ligand by culturing cells on single adhesive islands of different sizes (100-2500 microm(2)) and shapes (squares, circles, and lines) or on many smaller (3-5 microm diameter) circular islands that were coated with a saturating density of fibronectin and separated by non-adhesive regions. The amount of focal adhesions (FAs) containing vinculin and phosphotyrosine increased in direct proportion to cell spreading under all conditions. FAs localized asymmetrically along the periphery of the small islands that experienced highest tensional stress, and FA staining increased when cytoskeletal tension was stimulated with thrombin, whereas inhibitors of contractility promoted FA disassembly. Thus, these findings demonstrate the existence of an "inside-out" mechanism whereby global cell distortion produces increases in cytoskeletal tension that feed back to drive local changes in FA assembly. This complex interplay between cell morphology, mechanics, and adhesion may be critical to how cells integrate from and function in living tissues.