Polyplex-embedding in polyelectrolyte multilayers for gene delivery

In this work, incorporation of plasmid DNA, pre-complexed with PEI, into polyelectrolyte multilayers has been studied to further develop platforms for local gene delivery. Polyplex embedding in synthetic and naturally degradable architectures was efficient for transfection of human hepato-cellular carcinoma cells.     

Extracellular release of colicin A is non-specific.

The possible involvement of topogenic export sequences within the colicin A polypeptide chain has been investigated. Different constructs have been made using various techniques to introduce deletions in the central and NH2-terminal regions of colicin A. Together, these deletions span the region from amino acid 15 to the end of the protein. None of these regions was found to be required for extracellular release or had any effect on the efficiency of this process. By inserting a termination codon, a Shine-Dalgarno sequence and an initiation codon into the gene for colicin A, the NH2-terminal and central plus COOH-terminal domains could be demonstrated to be released to the same extent when produced as separate polypeptides as when produced as linked ones. The introduction into the COOH-terminal domain of mutations promoting cytoplasmic aggregation had no effect on the secretion of the NH2-terminal polypeptide. These results demonstrated that no specific interaction between the NH2- and COOH-terminal regions of the colicin A polypeptide chain is involved in the release of colicin A. We are led to conclude that there is no topogenic export signal in the polypeptide chain of colicin A involved in the release mechanism. Thus the process is non-specific with respect to the colicin itself and depends solely on the expression of the colicin A lysis protein (Cavard et al., 1985, 1987). The expression of the protein causes the release of not only the colicin but also many other cellular proteins, including beta-lactamase, EF-Tu, and chloramphenicol acetyltransferase.     

Lipoprotein nature of the colicin A lysis protein: effect of amino acid substitutions at the site of modification and processing.

The colicin A lysis protein (Cal) is required for the release of colicin A to the medium by producing bacteria. This protein is produced in a precursor form that contains a cysteine at the cleavage site (-Leu-Ala-Ala-Cys). The precursor must be modified by the addition of lipid before it can be processed. The maturation is prevented by globomycin, an inhibitor of signal peptidase II. Using oligonucleotide-directed mutagenesis, the alanine and cystein residues in the -1 and +1 positions of the cleavage site were replaced by proline and threonine residues, respectively, in two different constructs. Both substitutions prevented the normal modification and cleavage of the protein. The marked activation of the outer membrane detergent-resistant phospholipase A observed with wild-type Cal was not observed with the Cal mutants. Both Cal mutants were also defective for the secretion of colicin A. In one mutant, the signal peptide appeared to be cleaved off by an alternative pathway involving signal peptidase I. Electron microscope studies with immunogold labeling of colicin A on cryosections of pldA and cal mutant cells indicated that the colicin remains in the cytoplasm and is not transferred to the periplasmic space. These results demonstrate that Cal must be modified and processed to activate the detergent-resistant phospholipase A and to promote release of colicin A.     

Sib pair linkage analysis of renin gene haplotypes in human essential hypertension

Summary Although essential arterial hypertension is believed to have a strong genetic predisposition, the gene(s) responsible are unknown. The mechanisms underlying the regulation of blood pressure and experimental studies place the renin gene among the main candidate genes that need to be tested in humans. We tested the hypothesis of a linkage between the renin gene and essential hypertension using the affected sib pair method. Siblings (133 subjects, 52.1±10.9 years) from 57 families were selected for sustained hypertension (160.7 ± 22.9/99.5 ± 12.8 mmHg with 80% of patients under antihypertensive treatment), of early onset (40.7 ± 12.0 years), in the absence of obesity, diabetes mellitus, and secondary hypertension. Eight renin haplotypes were generated from three diallelic renin restriction fragment length polymorphisms (RFLPs) (TaqI, Hinfi, HindIII) located throughout the renin gene. The allelic concordance between the sib pairs was analyzed by identity by state relationships for 98 sib pairs (41 for 41 couples, 39 for 13 trios, 18 for 3 quartets). Allelic frequencies in the 57 hypertensive probands were similar to those observed among 102 hypertensive subjects studied previously. Six of eight possible haplotypes were observed, the informativity of the marker corresponded to 70% of heterozygosity. Allelic concordance for all sib pairs according to sibship size was not significantly different from that expected under the hypothesis of no linkage (t = 0.52, P = 0.15) reflecting only a small excess of renin alleles shared by the hypertensive sibs (1.44 ± 0.6 vs 1.36 ± 0.6). Likewise the linkage hypothesis was unsupported by weighted estimates to correct for possible bias due to large sibship size. Thus, the sib pair analysis suggests that the renin gene does not have a frequent role in the pathogenesis of essential hypertension; further more powerful linkage studies or other approaches will be needed to detect contributions at the renin locus to the heritability of essential hypertension.     

Modulation of the urokinase receptor in human colon cell lines by N,N-dimethylformamide

The present study documents the effect of the planar, polar differentiation promoter N,N-dimethylformamide (DMF) on urokinase binding to colon carcinoma cells. Exposure of the colon carcinoma cell lines to the agent resulted in enhanced specific binding of radioactive urokinase to all cells tested. Insulin binding to the cells was, however, unaffected by DMF. A DMF exposure period of 45 h was required to observe maximum urokinase binding to two representative cell lines FET and RKO. Optimal stimulation of both cell lines occurred with 0.8% DMF. Scatchard analysis revealed the dissociation constants to be unchanged by the agent with the increased binding of radioactive plasminogen activator reflecting an up-regulation of binding sites. In this regard, the cell line RKO upon exposure to DMF, displayed approx. 700,000 receptors/cell, the highest value published, to date, for any cell line.     

A molecular, genetic and immunological approach to the functioning of colicin A, a pore-forming protein

We have constructed, by recombinant DNA techniques, one hybrid protein, colicin A-beta-lactamase (P24), and two modified colicin As, one (P44) lacking a large central domain and the other (PX-345) with a different C-terminal region. The regulation of synthesis, the release into the medium and the properties of these proteins were studied. Only P44 was released into the medium. This suggests that both ends of the colicin A polypeptide chain might be required for colicin release. None of the three proteins was active on sensitive cells in an assay in vivo. However, P44 was able to form voltage-dependent channels in phospholipid planar bilayers. Its lack of activity in vivo is therefore probably caused by the inability to bind to the receptor in the outer membrane. PX-345 is a colicin in which the last 43 amino acids of colicin A have been replaced by 27 amino acids encoded by another reading frame in the same region of the colicin A structural gene; it was totally unable to form pores in planar bilayers at neutral pH but showed a very slight activity at acidic pH. These results confirm that the C-terminal domain of colicin A is involved in pore formation and indicate that at least the 43 C-terminal amino acid residues of this domain play a significant role in pore formation or pore function. Fifteen monoclonal antibodies directed against colicin A have been isolated by using conventional techniques. Five out of the 15 monoclonal antibodies could preferentially recognize wild-type colicin A. In addition, the altered forms of the colicin A polypeptide were used to map the epitopes of ten monoclonal antibodies reacting specifically with colicin A. Some of the antibodies did not bind to colicin A when it was pre-incubated at acidic pH suggesting that colicin A undergoes conformational change at about pH 4. The effects of monoclonal antibodies on activity in vivo of colicin A were investigated. The degree of inhibition observed was related to the location of the epitopes, with monoclonal antibodies reacting with the N terminus giving greater inhibition. The monoclonal antibodies directed against the C-terminal region promoted an apparent activation of colicin activity in vivo.     

Procaine, a local anesthetic interacting with the cell membrane, inhibits the processing of precursor forms of periplasmic proteins in Escherichia coli.

Treatment of Escherichia coli cells with procaine (0.55%, w/v) results in the accumulation of precursor in addition to mature forms of two periplasmic proteins, alkaline phosphatase and glutamine-binding protein. The precursor form of alkaline phosphatase has a higher molecular weight than the mature form by about 2600. An experimental technique is described to isolate and purify precursor forms of any presumably exported protein. After the membrane solubilization step in the presence of nonionic detergent, a peptidase is stimulated, resulting in partial cleavage of the precursors. The products of this cleavage have been identified as the mature protein and presumably the signal peptide in the case of alkaline phosphatase. The amino acid composition of this peptide, which is comprised of 25 residues, has been determined. Procaine (0.55%, w/v) causes an increase in molecular packing of lipid molecules in the membrane which might result in an alteration of membrane fluidity sufficient for selective inhibition of processing of precursors of exported proteins.     

Selective pericentromeric heterochromatin dismantling caused by TP53 activation during senescence

Cellular senescence triggers various types of heterochromatin remodeling that contribute to aging. However, the age-related mechanisms that lead to these epigenetic alterations remain elusive. Here, we asked how two key aging hallmarks, telomere shortening and constitutive heterochromatin loss, are mechanistically connected during senescence. We show that, at the onset of senescence, pericentromeric heterochromatin is specifically dismantled consisting of chromatin decondensation, accumulation of DNA breakages, illegitimate recombination and loss of DNA. This process is caused by telomere shortening or genotoxic stress by a sequence of events starting from TP53-dependent downregulation of the telomere protective protein TRF2. The resulting loss of TRF2 at pericentromeres triggers DNA breaks activating ATM, which in turn leads to heterochromatin decondensation by releasing KAP1 and Lamin B1, recombination and satellite DNA excision found in the cytosol associated with cGAS. This TP53–TRF2 axis activates the interferon response and the formation of chromosome rearrangements when the cells escape the senescent growth arrest. Overall, these results reveal the role of TP53 as pericentromeric disassembler and define the basic principles of how a TP53-dependent senescence inducer hierarchically leads to selective pericentromeric dismantling through the downregulation of TRF2.     

Pseudomonas aeruginosa polysaccharide Psl supports airway microbial community development

Pseudomonas aeruginosa dominates the complex polymicrobial cystic fibrosis (CF) airway and is a leading cause of death in persons with CF. Oral streptococcal colonization has been associated with stable CF lung function. However, no studies have demonstrated how Streptococcus salivarius, the most abundant streptococcal species found in individuals with stable CF lung disease, potentially improves lung function or becomes incorporated into the CF airway biofilm. By utilizing a two-species biofilm model to probe interactions between S. salivarius and P. aeruginosa, we discovered that the P. aeruginosa exopolysaccharide Psl promoted S. salivarius biofilm formation. Further, we identified a S. salivarius maltose-binding protein (MalE) that is required for promotion of biofilm formation both in vitro and in a Drosophila melanogaster co-infection model. Finally, we demonstrate that promotion of dual biofilm formation with S. salivarius is common among environmental and clinical P. aeruginosa isolates. Overall, our data supports a model in which S. salivarius uses a sugar-binding protein to interact with P. aeruginosa exopolysaccharide, which may be a strategy by which S. salivarius establishes itself within the CF airway microbial community.Subject terms: Bacteriology, Biofilms, Microbiome, Clinical microbiology