Dual benefit of reduced Cx43 on atherosclerosis in LDL receptor-deficient mice

Paracrine cell-to-cell interactions are crucial events during atherogenesis, however, little is known on the role of gap junctional communication during this process. We recently demonstrated increased expression of Cx43 in intimal smooth muscle cells and in a subset of endothelial cells covering the shoulder of atherosclerotic plaques. The purpose of this study was to examine the role of Cx43 in the development of atherosclerosis in vivo. Atherosclerosis-susceptible LDL receptor-deficient (LDLR(-/-)) mice were intercrossed with mice heterozygous for Cx43 (Cx43(+/-) mice). Male mice with normal (Cx43(+/+)LDLR(-/-)) or reduced (Cx43(+/-)LDLR(-/-)) Cx43 level of 10 weeks old were fed a cholesterol-rich diet (1.25%) for 14 weeks. Both groups of mice showed similar increases in serum lipids and body weight. Interestingly, the progression of atherosclerosis was reduced by 50% (P < 0.01) in the thoraco-abdominal aorta and in the aortic roots of Cx43(+/-)LDLR(-/-) mice compared with Cx43(+/+)LDLR(-/-) littermate controls. In addition, atheroma in Cx43(+/-)LDLR(-/-) mice contained fewer inflammatory cells and exhibited thicker fibrous caps with more collagen and smooth muscle cells, important features associated, in human, with stable atherosclerotic lesions. Thus, reducing Cx43 expression in mice provides beneficial effects on both the progression and composition of the atherosclerotic lesions.     

Interaction of the bullous pemphigoid antigen 1 (BP230) and desmoplakin with intermediate filaments is mediated by distinct sequences within their COOH terminus

The bullous pemphigoid antigen 1 (BP230) and desmoplakin (DP) are members of the plakin protein family of cytolinkers. Despite their homology, their COOH termini selectively bind distinct intermediate filaments (IFs). We studied sequences within their COOH termini required for their interaction with the epidermal keratins K5/K14, the simple epithelial keratins K8/K18, and type III IF vimentin by yeast three-hybrid, cell transfection, and overlay assays. The results indicate that BP230 interacts with K5/K14 but not with K8/K18 or vimentin via a region encompassing both the B and C subdomains and the COOH extremity, including a COOH-terminal eight-amino-acid stretch. In contrast, the C subdomain with the COOH-terminal extremity of DP interacts with K5/K14 and K8/K18, and its linker region is able to associate with K8/K18 and vimentin. Furthermore, the potential of DP to interact with IF proteins in yeast seems to be regulated by phosphorylation of Ser 2849 within its COOH terminus. Strikingly, BP230 and DP interacted with cytokeratins only when both type I and type II keratins were present. The head and tail domains of K5/K14 keratins were dispensable for their interaction with BP230 or DP. On the basis of our findings, we postulate that (1) the binding specificity of plakins for various IF proteins depends on their linker region between the highly homologous B and C subdomains and their COOH extremity and (2) the association of DP and BP230 with both epidermal and simple keratins is critically affected by the tertiary structure induced by heterodimerization and involves recognition sites located primarily in the rod domain of these keratins.     

A new mouse limb mutation identifies a <Emphasis Type="Italic">Twist</Emphasis> allele that requires interacting loci on Chromosome 4 for its phenotypic expression

Pluridigite (Pdt) is a semi-dominant mutation obtained after a mutagenesis experiment with ethyl-nitroso-urea (ENU). The mutant exhibits abnormal skeletal pattern formation characterized by the formation of extra digits (polydactyly) in the preaxial (anterior) part of the hindlimbs. The phenotype shows incomplete penetrance, depending on the genetic background. In an F₂ cross with C57BL/6, the phenotype could not be associated with a single locus. Strong linkage was observed with markers located on Chromosome (Chr) 12, in a 2-cM interval between D12Mit136 and D12Mit153. This region contains the Twist gene, and we show that the [Pdt] phenotype is dependent upon a new allele of Twist. We further identified that the whole Chr 4 is associated with the [Pdt] phenotype. The Pluridigite phenotype thus results from the combination of a Twist mutant allele and at least two additional loci.     

Amperometric cytochrome c3-based biosensor for chromate determination

The chromate reductase activity of cytochrome c(3) (Cyt c(3), M(r) 13000), isolated from the sulfate-reducing bacterium Desulfomicrobium norvegicum, was used to develop an amperometric biosensor to measure chromate (CrO(4)(2-)) bioavailability. The performance of various biosensor configurations for qualitative and quantitative determination of Cr(VI) was studied. Biosensor properties depend on the technique used to immobilize the enzyme on the electrode (glassy carbon electrode). Immobilization of Cyt c(3) by entrapment in poly 3,4-ethylenedioxythiophene films denatured the enzyme, while application of an adsorption technique did not affect enzyme activity but the detection range was limited. The best results were obtained with dialysis membranes, which allowed the determination of Cr(VI) from 0.20 to 6.84 mg l(-1) (3.85-132 microM) with a sensitivity of 35 nA mg(-1) l (1.82 nA microM(-1)). No interference was observed with As(V), As(III) and Fe(III). Only a small amount of Cyt c(3) (372 ng of protein) was needed for this biosensor.     

The NFP locus of Medicago truncatula controls an early step of Nod factor signal transduction upstream of a rapid calcium flux and root hair deformation

Establishment of the Rhizobium-legume symbiosis depends on a molecular dialogue, in which rhizobial nodulation (Nod) factors act as symbiotic signals, playing a key role in the control of specificity of infection and nodule formation. Using nodulation-defective (Nod-) mutants of Medicago truncatula to study the mechanisms controlling Nod factor perception and signalling, we have previously identified five genes that control components of a Nod factor-activated signal transduction pathway. Characterisation of a new M. truncatula Nod- mutant led to the identification of the Nod Factor Perception (NFP) locus. The nfp mutant has a novel phenotype among Nod- mutants of M. truncatula, as it does not respond to Nod factors by any of the responses tested. The nfp mutant thus shows no rapid calcium flux, the earliest detectable Nod factor response of wild-type plants, and no root hair deformation. The nfp mutant is also deficient in Nod factor-induced calcium spiking and early nodulin gene expression. While certain genes controlling Nod factor signal transduction also control the establishment of an arbuscular mycorrhizal symbiosis, the nfp mutant shows a wild-type mycorrhizal phenotype. These data indicate that the NFP locus controls an early step of Nod factor signal transduction, upstream of previously identified genes and specific to nodulation.     

Potentially active copies of the gypsy retroelement are confined to the y chromosome of some strains of drosophila melanogaster possibly as the result of the female-specific effect of the flamenco gene

Gypsy is an endogenous retrovirus present in the genome of Drosophila melanogaster. This element is mobilized only in the progeny of females which contain active gypsy elements and which are homozygous for permissive alleles of a host gene called flamenco (flam). Some data strongly suggest that gypsy elements bearing a diagnostic HindIII site in the central region of the retrovirus body represent a subfamily that appears to be much more active than elements devoid of this site. We have taken advantage of this structural difference to assess by the Southern blotting technique the genomic distribution of active gypsy elements. In some of the laboratory Drosophila stocks tested, active gypsy elements were found to be restricted to the Y chromosome. Further analyses of 14 strains tested for the permissive vs. restrictive status of their flamenco alleles suggest that the presence of permissive alleles of flam in a stock tends to be associated with the confinement of active gypsy elements to the Y chromosome. This might be the result of the female-specific effect of flamenco on gypsy activity. Received: 13 June 1997 / Accepted: 27 August 1997     

A QTL with major effect on milk yield and composition maps to bovine Chromosome 14

A whole genome scan was undertaken in a granddaughter design comprising 1158 progeny-tested bulls in order to map QTL influencing milk yield and composition. In this paper we report the identification of a locus on the centromeric end of bovine Chromosome (Chr) 14, with major effect on fat and protein percentage as well as milk yield. The genuine nature of this QTL was verified using the grand²-daughter design, that is, by tracing the segregating QTL alleles from heterozygous grandsires to their maternal grandsons and confirming the predicted QTL allele substitution effect. Received: 30 December 1997 / Accepted: 21 February 1998     

Evaluation of 111In-Labelled Exendin-4 Derivatives Containing Different Meprin β-Specific Cleavable Linkers

Background

Cleavable linkers, which are specifically cleaved by defined conditions or enzymes, are powerful tools that can be used for various purposes. Amongst other things, they have been successfully used to deliver toxic payloads as prodrugs into target tissues. In this work novel linker sequences targeting meprin β, a metalloprotease expressed in the kidney brush-border membrane, were designed and included in the sequence of three radiolabelled exendin-4 derivatives. As radiolabelled exendin-4 derivatives strongly accumulate in the kidneys, we hypothesised that specific cleavage of the radiolabelled moiety at the kidney brush-border membrane would allow easier excretion of the activity into the urine and therefore improve the pharmacological properties of the peptide.

Results

The insertion of a cleavable linker did not negatively influence the in vitro properties of the peptides. They showed a good affinity to the GLP-1 receptor expressed in CHL cells, a high internalisation and sufficiently high stability in fresh human blood plasma. In vitro digestion with recombinant meprin β rapidly metabolised the corresponding linker sequences. After 60 min the majority of the corresponding peptides were digested and at the same time the anticipated fragments were formed. The peptides were also quickly metabolised in CD1 nu/nu mouse kidney homogenates. Immunofluorescence staining of meprin β in kidney sections confirmed the expression of the protease in the kidney brush-border membrane. Biodistribution in GLP-1 receptor positive tumour-xenograft bearing mice revealed high specific uptake of the ¹¹¹In-labelled tracers in receptor positive tissue. Accumulation in the kidneys, however, was still high and comparable to the lead compound ¹¹¹In-Ex4NOD40.

Conclusion

In conclusion, we show that the concept of cleavable linkers specific for meprin β is feasible, as the peptides are rapidly cleaved by the enzyme while retaining their biological properties.