Isolation and properties in culture of human adrenal capillary endothelial cells

The isolation of human adrenal capillary endothelial (HACE) cells without resort to fluorescence activated cell sorting is described, together with their properties in culture. HACE cells were isolated by plating collagenase digests at high dilution in the presence of endothelial cell growth supplement, followed by clonal selection of endothelial colonies. HACE cells exhibit a typical endothelial 'cobblestone' morphology at confluence and formed 'tubes' when seeded onto 'Matrigel'. They are positive for human MHC1, and the endothelial markers ENDOCAM (CD31) and weakly CD34, they also take up dil-acetyl low density lipoprotein but are negative for Factor VIII. Their growth is strongly stimulated by FGF and inhibited by TGF-beta I. Like their much studied bovine counterparts they are robust in culture, retaining the properties described up to senescence. HACE cells provide a readily available alternative to human umbilical vein endothelial cells in that they are easily isolated pure and in quantity. They should be particularly useful in studies where human capillary, as opposed to large vessel endothelium, is required.     

A Bird’s Eye View of Discard Reforms: Bird-Borne Cameras Reveal Seabird/Fishery Interactions

Commercial capture fisheries produce huge quantities of offal, as well as undersized and unwanted catch in the form of discards. Declines in global catches and legislation to ban discarding will significantly reduce discards, but this subsidy supports a large scavenger community. Understanding the potential impact of declining discards for scavengers should feature in an eco-system based approach to fisheries management, but requires greater knowledge of scavenger/fishery interactions. Here we use bird-borne cameras, in tandem with GPS loggers, to provide a unique view of seabird/fishery interactions. 20,643 digital images (one min⁻¹) from ten bird-borne cameras deployed on central place northern gannets Morus bassanus revealed that all birds photographed fishing vessels. These were large (>15 m) boats, with no small-scale vessels. Virtually all vessels were trawlers, and gannets were almost always accompanied by other scavenging birds. All individuals exhibited an Area-Restricted Search (ARS) during foraging, but only 42% of ARS were associated with fishing vessels, indicating much ‘natural’ foraging. The proportion of ARS behaviours associated with fishing boats were higher for males (81%) than females (30%), although the reasons for this are currently unclear. Our study illustrates that fisheries form a very important component of the prey-landscape for foraging gannets and that a discard ban, such as that proposed under reforms of the EU Common Fisheries Policy, may have a significant impact on gannet behaviour, particularly males. However, a continued reliance on ‘natural’ foraging suggests the ability to switch away from scavenging, but only if there is sufficient food to meet their needs in the absence of a discard subsidy.     

Responses of pertussis toxin-treated microvascular endothelial cells to transforming growth factor beta 1. No evidence for pertussis-sensitive G-protein involvement in TGF-beta signal transduction.

Responses of bovine adrenal capillary endothelial cells (BACE) on treatment with transforming growth factor beta 1 (TGF-beta 1) have been characterized and tested for sensitivity to inactivation of pertussis toxin-sensitive G-proteins. TGF-beta 1 elicited growth inhibition, monolayer remodeling, elevation of steady state mRNA levels for collagen type 1 (alpha 1(1) and alpha 2(1)) and TGF-beta 1, and inhibition of p34cdc2 histone H1 kinase activity in BACE cells. Pertussis toxin treatment enhanced both inhibition of BACE cell [3H]methylthymidine uptake and remodeling of BACE monolayers by TGF-beta 1. These findings contrast with studies of mink lung epithelial cells, in which TGF-beta 1 growth inhibition has been shown to be pertussis-sensitive. Further investigation revealed that pertussis toxin treatment of BACE cells had no effect on TGF-beta 1-stimulated elevation of steady state mRNA levels for collagen type 1 (alpha 1(1) or alpha 2(1)) or for TGF-beta 1. Analysis of p34cdc2 activity in BACE cells revealed potent inhibition of p34cdc2 histone H1 kinase activity by TGF-beta 1. Pertussis toxin treatment also abolished the increase in p34cdc2 activity, however, precluding the determination of the pertussis toxin sensitivity of this response to TGF-beta 1. Consistent with suppression of p34cdc2 activation, pertussis toxin also caused substantial inhibition of mitogen-stimulated BACE cell [3H]methylthymidine uptake. It is concluded that TGF-beta 1 signal transduction in this cell type does not involve G-proteins of the pertussis toxin-sensitive class and that, in view of its potent effects on DNA synthesis and p34cdc2 activation, the use of pertussis toxin to determine G-protein involvement in cytokine signalling pathways should be approached with caution.     

Radiometric assay for carboxypeptidase H (EC 3.4.17.10) and other carboxypeptidase B-like enzymes

Carboxypeptidase H, EC 3.4.17.10, also known as enkephalin convertase, carboxypeptidase E, and crino carboxypeptidase B, is an important enzyme involved in the biosynthesis of bioactive peptides. To assay the enzyme, tissues are homogenized in at least 20 vol (ml/g) of 0.025 M Tris-HCl buffer, pH 8, with 5 mg/ml of bovine serum albumin. After centrifugation, the supernatant is brought to pH 5.6 and centrifuged again. Following a 20-min preincubation in 2 mM CoCl2, the supernatant is incubated with 0.1 mM (final concentration) of the radioactive substrate [3H]benzoyl-Phe-Ala-Arg. The 100-microliters assay is stopped by the addition of 680 microliters of acetonitrile/0.25 M HCl (0.7/1). The 1.5-ml tube is transferred into a scintillation vial and is flushed with 4 ml of Econofluor, a water-immiscible scintillation fluid. The product, [3H]benzoyl-Phe-Ala, recovered in the organic phase, is counted directly with no interference from the substrate remaining in the aqueous phase. The blank is below 1%. Expressed in nanomoles per minute per milligram of tissue, the activity of the soluble enzyme in rat is 0.34 for striatum, 21.0 for pancreatic islet, 16.6 for anterior pituitary, 46.0 for intermediate pituitary, and 10.9 for neural pituitary. In every case 25 microM guanidinoethylmercaptosuccinic acid, an active site-directed inhibitor of carboxypeptidase H, completely inhibits the activity.     

The pH-dependence of class B and class C beta-lactamases.

The classification by structure allots beta-lactamases to (at present) three classes, A, B and C. The pH-dependence of the kinetic parameters for class B and class C have been determined. They differ from each other and from class A beta-lactamases. The class B enzyme was beta-lactamase II from Bacillus cereus 569/H/9. The plots of kcat against pH for the hydrolysis of benzylpenicillin by Zn(II)-requiring beta-lactamase II and Co(II)-requiring beta-lactamase II were not symmetrical, but those of kcat/Km were. A similar feature was observed for the hydrolysis of both benzylpenicillin and cephalosporin C by a class C beta-lactamase from Pseudomonas aeruginosa. The results have been interpreted by a scheme in which two ionic forms of an intermediate can give product, but do so at differing rates.     

Micropropagation of Hieracium aurantiacum

Hieracium aurantiacum L. is being investigated as a model system for studying the genetic basis of gametophytic apomixis. Micropropagation methods were developed for the multiplication and maintenance of unique genotypes, and to facilitate the development of an Agrobacterium-mediated transformation procedure. A basal medium containing Murashige & Skoog macro- and micro-salts, B5 vitamins and 3% sucrose was used throughout. Shoot regeneration from leaf disks was optimized on a medium supplemented with 2.5 M indole-3-butyric acid (IBA) and 2.2 M 6-benzyladenine (BA). Shoot proliferation was optimized using 2.2 M BA, without additional IBA, giving 5-fold shoot-tip multiplication rates every 4 weeks. Shoots readily formed roots on the basal medium and could be transplanted to potting medium 3 weeks later. Micropropagation was found to have no effect on the expression of apomixis in this species.Abbreviations Ba 6-benzyladenine - iba indole-3-butyric acid - lsd least significant difference     

Selection for β2-microglobulin mutation in mismatch repair-defective colorectal carcinomas

Novel peptide antigens complexed with human leukocyte antigen (HLA) and β₂-microglobulin (β2M) molecules are presented at the cell surface to cytotoxic T lymphocytes (CTLs), provoking lysis of the antigen-presenting cell [1]. In tumour cells, genetically altered or abnormally expressed proteins provide a source of peptides that can be presented to CTLs; the resulting anti-tumour CTL responses may provide part of the body's defence against cancer. Disabling mutations in the HLA and β2M proteins required for peptide presentation allow a tumour cell to escape destruction by CTLs. Cells with deficient DNA mismatch repair have high spontaneous mutation rates [2] and produce many altered proteins that are a potential source of numerous unique peptides. Mutator tumour cells might therefore be particularly vulnerable to immune surveillance and CTL attack. Mutator phenotypes [3] and [4] and loss of β2M (or HLA) expression [5] and [6] are both relatively common among sporadic colorectal tumours. We have compared the frequency of β2M mutations in sporadic colorectal and other tumours with and without a mutator phenotype. Mutations were more frequent among colorectal tumours with the microsatellite instability indicative of a defect in DNA mismatch repair. The inactivating β2M mutations were predominantly frameshifts, which is consistent with the underlying mismatch repair defects. Evasion of immune surveillance by acquiring β2M mutations therefore occurs at high frequency in tumour cells with a mutator phenotype due to defective DNA mismatch repair.