Isolation of BM mesenchymal stem cells by plastic adhesion or negative selection: phenotype, proliferation kinetics and differentiation potential

BACKGROUND: BM mesenchymal stem cells (MSC) have the capacity for renewal and the potential to differentiate into multiple tissues. In this study, we compared different enrichment methods to obtain MSC from BM. METHODS: Three different methods were compared with a view to obtaining MSC more rapidly from BM: negative selection (RosetteSep and MACS) and plastic adhesion. The three cell fractions were grown in complete alpha-minimum essential medium in order to evaluate their proliferative capacity, their phenotype during culture and their potential to differentiate into adipocytes, osteocytes and chondrocytes. Identification of MSC was performed by immunofluorescence with putative mesenchymal markers SH2 and SH3 but also with hematopoietic markers. RESULTS: After negative selection, only 1+/-0.2% and 2.9+/-0.8% of cells were recovered from BM with the RosetteSep and MACS methods, respectively. However, negative depletion permitted a homogeneous population of MSC, with more than 90% SH2+ and SH3+ cells, to be obtained rapidly and in large quantities after 10 days of culture. Similar homogeneity was observed after three passages if the plastic adhesion was used as selection method and after an average of 25-30 days of culture. Different levels of MSC maturity were also suggested by the variable level expression of Stro-1. DISCUSSION: Depleting selection by RosetteSep may represent an easy method of obtaining MSC rapidly from BM with the aim of potential therapeutic use.     

Aquaglyceroporins,one channel for two molecules

In the light of the recently published structure of GlpF and AQP1, we have analysed the nature of the residues which could be involved in the formation of the selectivity filter of aquaporins, glycerol facilitators and aquaglyceroporins. We demonstrate that the functional specificity for major intrinsic protein (MIP) channels can be explained on one side by analysing the polar environment of the residues that form the selective filter. On the other side, we show that the channel selectivity could be associated with the oligomeric state of the membrane protein. We conclude that a non-polar environment in the vicinity of the top of helix 5 could allow aquaglyceroporins and GlpF to exist as monomers within the hydrophobic environment of the membrane.     

Membrane topology of the Streptomyces lividans type I signal peptidases

Most bacterial membranes contain one or two type I signal peptidases (SPases) for the removal of signal peptides from export proteins. For Streptomyces lividans, four different type I SPases (denoted SipW, SipX, SipY, and SipZ) were previously described. In this communication, we report the experimental determination of the membrane topology of these SPases. A protease protection assay of SPase tendamistat fusions confirmed the presence of the N- as well as the C-terminal transmembrane anchor for SipY. SipX and SipZ have a predicted topology similar to that of SipY. These three S. lividans SPases are currently the only known prokaryotic-type type I SPases of gram-positive bacteria with a C-terminal transmembrane anchor, thereby establishing a new subclass of type I SPases. In contrast, S. lividans SipW contains only the N-terminal transmembrane segment, similar to most type I SPases of gram-positive bacteria. Functional analysis showed that the C-terminal transmembrane anchor of SipY is important to enhance the processing activity, both in vitro as well as in vivo. Moreover, for the S. lividans SPases, a relation seems to exist between the presence or absence of the C-terminal anchor and the relative contributions to the total SPase processing activity in the cell. SipY and SipZ, two SPases with a C-terminal anchor, were shown to be of major importance to the cell. Accordingly, for SipW, missing the C-terminal anchor, a minor role in preprotein processing was found.     

Structural and functional analysis of a bipolar replication terminus. Implications for the origin of polarity of fork arrest

We have delineated the amino acid to nucleotide contacts made by two interacting dimers of the replication terminator protein (RTP) of Bacillus subtilis with a novel naturally occurring bipolar replication terminus by converting RTP to a site-directed chemical nuclease and mapping its cleavage sites on the terminus. The data show a relatively symmetrical arrangement of the amino acid to base contacts, and a comparison of the bipolar contacts with that of a normal unipolar terminus suggests that the DNA-protein contacts play an important determinative role in generating polarity from structurally symmetrical RTP dimers. The amino acid to nucleotide contacts provided distance constraints that enabled us to build a three-dimensional model of the protein-DNA complex. The model is consistent with features of the bipolar Ter.RTP complex derived from mutational and cross-linking data. The bipolar terminus arrested Escherichia coli DNA replication and DnaB helicase and T7 RNA polymerase in vitro in both orientations. RTP arrested the unwinding of duplex DNA on the bipolar Ter DNA substrate regardless of the length of the duplex DNA. The latter result suggested further that the terminus arrested authentic DNA unwinding by the helicase rather than just translocation of helicase on DNA.     

Evidence-based de-implementation for contradicted, unproven, and aspiring healthcare practices

Abandoning ineffective medical practices and mitigating the risks of untested practices are important for improving patient health and containing healthcare costs. Historically, this process has relied on the evidence base, societal values, cultural tensions, and political sway, but not necessarily in that order. We propose a conceptual framework to guide and prioritize this process, shifting emphasis toward the principles of evidence-based medicine, acknowledging that evidence may still be misinterpreted or distorted by recalcitrant proponents of entrenched practices and other biases.     

Associations between COPD related manifestations: a cross-sectional study

Background

Cardiovascular disease, osteoporosis and emphysema are associated with COPD. Associations between these factors and whether they predict all-cause mortality in COPD patients are not well understood. Therefore, we examined associations between markers of cardiovascular disease (coronary artery calcification [CAC], thoracic aortic calcification [TAC] and arterial stiffness), bone density (bone attenuation of the thoracic vertebrae), emphysema (PI-950 and 15th percentile) and all-cause mortality in a COPD cohort.

Methods

We assessed CAC, TAC, bone attenuation of the thoracic vertebrae, PI-950 and 15th percentile on low-dose chest computed tomography in COPD subjects. We measured arterial stiffness as carotid-radial pulse wave velocity (PWV), and identified deaths from the national register.

Results

We studied 119 COPD subjects; aged 67.8 ±7.3, 66% were males and mean FEV₁% predicted was 46.0 ±17.5. Subjects were classified into three pre-specificed groups: CAC = 0 (n = 14), 0 < CAC ≤ 400 (n = 41) and CAC > 400 (n = 64). Subjects with higher CAC were more likely to be older (p < 0.001) and male (p = 0.03), and more likely to have higher systolic blood pressure (p = 0.001) and a history of hypertension (p = 0.002) or ischemic heart disease (p = 0.003). Higher CAC was associated with higher PWV (OR 1.62, p = 0.04) and lower bone attenuation (OR 0.32, p = 0.02), but not with 15th percentile, after adjustment for age, sex and pack-years of smoking. In a Cox proportional hazards model, CAC, TAC and 15th percentile predicted all-cause mortality (HR 2.01, 2.09 and 0.66, respectively).

Conclusions

Increased CAC was associated with increased arterial stiffness and lower bone density in a COPD cohort. In addition, CAC, TAC and extent of emphysema predicted all-cause mortality.

Trial registration

Lothian NHS Board, Lothian Research Ethics Committee, LREC/2003/8/28.