Structural organization of the beta-globin locus of B-haplotype sheep

Goats and some sheep synthesize a juvenile hemoglobin, Hb C (alpha 2 beta C2), at birth and produce this hemoglobin exclusively during severe anemia. Sheep that synthesize this juvenile hemoglobin are of the A haplotype. Other sheep, belonging to a separate group, the B haplotype, do not synthesize hemoglobin C and during anemia continue to produce their adult hemoglobin. To understand the basis for this difference we have determined the structural organization of the beta- globin locus of B-type sheep by constructing and isolating overlapping genomic clones. These clones have allowed us to establish the linkage map 5' epsilon I-epsilon II-psi beta I-beta B-epsilon III-epsilon IV- psi beta II-beta F3' in this haplotype. Thus, B sheep lack four genes, including the BC gene, and have only eight genes, compared with the 12 found in the goat globin locus. The goat beta-globin locus is as follows: 5' epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A-epsilon V-epsilon VI-psi beta Y-beta F3'. Southern blot analysis of A-type sheep reveals that these animals have a beta- globin locus similar to that of goat, i.e., 12 globin genes. Thus, the beta-globin locus of B-haplotype sheep resembles that of cows and may have retained the duplicated locus of the ancestor of cows and sheep. Alternatively, the B-sheep locus arrangement may be the result of a deletion of a four-gene set from the triplicated locus.      

Mode of action of LciA, the lactococcin A immunity protein

Monoclonal antibodies were raised against a fusion between the Escherichia coli maltose-binding protein and LciA, the immunity protein that protects Lactococcus lactis against the effects of the bacteriocin lactococcin A. One of the antibodies directed against the LciA moiety of the fusion protein was used to locate the immunity protein in the L. lactis producer cell. LciA was present in the cytosolic. the membrane-associated, and the membrane fractions in roughly equal amounts, irrespective of the production by the cells of lactococcin A. The monoclonal antibody specifically reacted with right-side-out vesicles obtained from a strain producing the immunity protein. It did not react with inside-out vesicles of the same strain, or with right-side-out vesicles obtained from a strain producing both LciA and lactococcin A. Also, externally added lactococcin A blocked the interaction between the antibody and right-side-out vesicles obtained from a strain producing only LciA. The epitope in LciA was localized between amino acid residues 60 and 80. As the epitope could be removed from right-side-out vesicles by proteinase K, it is located at the outside of the cell. The immunity protein contains a putative a-amphiphilic helix from residue 29 to 47. A model is proposed in which this helix is thought to traverse the membrane in such a way that the C-terminal part of the protein, containing the epitope, is on the outside of the cell. Vesicle-fusion studies together with leucine-uptake experiments suggest that the immunity protein interacts with the putative receptor for lactococcin A, thus preventing pore formation by the bacteriocin.     

Lactococcal plasmid pWV01 as an integration vector for lactococci.

A Bacillus subtilis strain was constructed that contained the repA gene of the lactococcal plasmid pWVO1 in its chromosome. This strain was used to construct the pWVO1-based integration vector pINT1, which lacked the repA gene. The 3.6-kb plasmid pINT1 was not able to replicate in Lactococcus lactis MG1363 but integrated into the chromosome via a Campbell-like mechanism when a lactococcal chromosomal DNA fragment was incorporated in the plasmid. Transformants were obtained that carried between one and four plasmid copies, in stable tandem arrangement on the chromosome. The results indicate that pWVO1 can be used for the development of a Campbell-like integration system fully derived of lactococcal DNA, with which stable multiple copies of any gene of interest can be generated in the lactococcal chromosome.     

Selective inhibition of prostaglandin biosynthesis by gold salts and phenylbutazone

Gold salts and phenylbutazone selectively inhibit the synthesis of PGF_2α and PGE₂ respectively. Lowered production of one prostaglandin species is accompanied by an increased production of the other. Selective inhibition by these drugs was observed in the presence of adrenaline, reduced glutathione and copper sulphate under conditions when most anti-inflammatory compounds inhibited PGE₂ and PGF_2α syntheses equally. It is postulated that selective inhibitors may have a different mode of action and beneficial effects may be related to the endogenous ratio of PGE to PGF required for normal function.     

Two silicone nontoxic fouling release coatings: Hydrosilation cured PDMS and CaCO3 filled,ethoxysiloxane cured RTV11

Two silicone coatings have been evaluated for barnacle adhesion. One coating is an unfilled hydrosilation cured polydimethylsiloxane (PDMS) network, while the other is a room temperature vulcanized (RTV), filled, ethoxysiloxane cured PDMS elastomer, RTV11^?. The adhesion strength of one species of barnacle, Balanus eburneus, to the hydrosilation coatings is in the range of 0.37–0.60 kg cm^‐2 while the corresponding range for RTV11 is 0.64–0.90 kg cm^‐2. The easier release of B. eburneus from the hydrosilation cured network compared to RTV11 is discussed in relationship to differences in bulk and surface properties. Preliminary results suggest bulk modulus may be the most important parameter in determining barnacle adhesion strength. In light or mechanical property analysis, a re‐evaluation of surface properties and chemical stability is presented.     

Two-mutation models for bone cancer due to radium, strontium and plutonium

Data from beagle experiments and radium dial painters were used to derive two-mutation carcinogenesis models for bone cancer induced by the bone-seeking radionuclides radium, strontium and plutonium. For all data, the model fits indicate that at low doses both mutation rates depend linearly and equally strongly on dose rate. For the high-LET alpha-particle emitters, a cell killing term reduces the second mutation rate at high dose rates. In all cases, the combined effect of both mutation rates is a linear-quadratic dose-effect relationship for cancer at low doses. This behavior may lead to experimental data that could be mistaken as showing a threshold below which no cancers are induced. Derived parameters such as toxicity ratios and tumor growth times compare well with values reported in the literature. Furthermore, results for plutonium indicate that rapid burial of the nuclide in the growing bones of juvenile beagles leads to a significant reduction of its toxicity, as was suggested previously. The results for radium in beagles compare well with those for humans and suggest that the models derived for strontium and plutonium in beagles may be translated to humans. The significant model parameters for the accurate animal data could then also be used to fit human epidemiological data.     

Cre-loxP recombination system for large genome rearrangements in Lactococcus lactis

We have used a new genetic strategy based on the Cre-loxP recombination system to generate large chromosomal rearrangements in Lactococcus lactis. Two loxP sites were sequentially integrated in inverse order into the chromosome either at random locations by transposition or at fixed points by homologous recombination. The recombination between the two chromosomal loxP sites was highly efficient (approximately 1 x 10(-1)/cell) when the Cre recombinase was provided in trans, and parental- or inverted-type chromosomal structures were isolated after removal of the Cre recombinase. The usefulness of this approach was demonstrated by creating three large inversions of 500, 1,115, and 1,160 kb in size that modified the lactococcal genome organization to different extents. The Cre-loxP recombination system described can potentially be used for other gram-positive bacteria without further modification.     

Mucosal vaccine delivery of antigens tightly bound to an adjuvant particle made from food-grade bacteria

Mucosal immunization with subunit vaccines requires new types of antigen delivery vehicles and adjuvants for optimal immune responses. We have developed a non-living and non-genetically modified gram-positive bacterial delivery particle (GEM) that has built-in adjuvant activity and a high loading capacity for externally added heterologous antigens that are fused to a high affinity binding domain. This binding domain, the protein anchor (PA), is derived from the Lactococcus lactis AcmA cell-wall hydrolase, and contains three repeats of a LysM-type cell-wall binding motif. Antigens are produced as antigen-PA fusions by recombinant expression systems that secrete the hybrid proteins into the culture growth medium. GEM particles are then used as affinity beads to isolate the antigen-PA fusions from the complex growth media in a one step procedure after removal of the recombinant producer cells. This procedure is also highly suitable for making multivalent vaccines. The resulting vaccines are stable at room temperature, lack recombinant DNA, and mimic pathogens by their bacterial size, surface display of antigens and adjuvant activity of the bacterial components in the GEM particles. The GEM-based vaccines do not require additional adjuvant for eliciting high levels of specific antibodies in mucosal and systemic compartments.     

Novel Surface Display System for Proteins on Non-Genetically Modified Gram-Positive Bacteria

A novel display system is described that allows highly efficient immobilization of heterologous proteins on bacterial surfaces in applications for which the use of genetically modified bacteria is less desirable. This system is based on nonliving and non-genetically modified gram-positive bacterial cells, designated gram-positive enhancer matrix (GEM) particles, which are used as substrates to bind externally added heterologous proteins by means of a high-affinity binding domain. This binding domain, the protein anchor (PA), was derived from the Lactococcus lactis peptidoglycan hydrolase AcmA. GEM particles were typically prepared from the innocuous bacterium L. lactis, and various parameters for the optimal preparation of GEM particles and binding of PA fusion proteins were determined. The versatility and flexibility of the display and delivery technology were demonstrated by investigating enzyme immobilization and nasal vaccine applications.