Nitrite reduction in Paracoccus sp. is affected by a novel plasmid pYR1

Two relatively low-copy plasmids of 9 and 16 kb were found to comprise the extrachromosomal DNA of a Paracoccus strain. Reduction of nitrate by plasmid-cured cells resulted in a significant intermediate nitrite accumulation as compared to wild-type cells. By examining nitrate reduction by transformants containing one of the two plasmids, it was found that nitrite accumulation was influenced by the 9.0-kb plasmid, designated as pYR1. Subcloning analysis showed that a 1.8-kb fragment of this plasmid affected nitrite accumulation. Sequence analysis of this fragment revealed the presence of five open reading frames. One of the six deduced proteins showed a strong homology to ABC transporters.     

Analyzing the role of the putative inositol 1,3,4,5-tetrakisphosphate receptor GAP1IP4BP in intracellular Ca2+ homeostasis

Inositol 1,3,4,5-tetrakisphosphate (IP(4)) has been linked to a potential role in the regulation of intracellular free Ca(2+) concentration ([Ca(2+)](i)) following cellular stimulation with agonists that activate phosphoinositide-specific phospholipase C. However, despite many studies, the function of IP(4) remains unclear and indeed there is still some debate over whether it has a function at all. Here we have used various molecular approaches to address whether manipulation of the potential IP(4) receptor, GAP1(IP4BP), affects [Ca(2+)](i) following cellular stimulation. Using single cell imaging, we show that the overexpression of a constitutively active and a potential dominant negative form of GAP1(IP4BP) appear to have no effect on Ca(2+) mobilization or Ca(2+) entry following stimulation of HeLa cells with histamine. In addition, through the use of small interfering RNA duplexes, we have examined the effect of suppressing endogenous GAP1(IP4BP) production on [Ca(2+)](i). In HeLa cells in which the endogenous level of GAP1(IP4BP) has been suppressed by approximately 95%, we failed to observe any effect on Ca(2+) mobilization or Ca(2+) entry following histamine stimulation. Thus, using various approaches to manipulate the function of endogenous GAP1(IP4BP) in intact HeLa cells, we have been unable to observe any detectable effect of GAP1(IP4BP) on [Ca(2+)](i).     

Modification of a tumor-derived peptide at an HLA-A2 anchor residue can alter the conformation of the MHC-peptide complex: probing with TCR-like recombinant antibodies

A common assumption about peptide binding to the class I MHC complex is that each residue in the peptide binds independently. Based on this assumption, modifications in class I MHC anchor positions were used to improve the binding properties of low-affinity peptides (termed altered peptide ligands), especially in the case when tumor-associated peptides are used for immunotherapy. Using a new molecular tool in the form of recombinant Abs endowed with Ag-specific MHC-restricted specificity of T cells, we show that changes in the identity of anchor residues may have significant effects, such as altering the conformation of the peptide-MHC complex, and as a consequence, may affect the TCR-contacting residues. We herein demonstrate that the binding of TCR-like recombinant Abs, specific for the melanoma differentiation Ag gp100 T cell epitope G9-209, is entirely dependent on the identity of a single peptide anchor residue at position 2. An example is shown in which TCR-like Abs can recognize the specific complex only when a modified peptide, G9-209-2 M, with improved affinity to HLA-A2 was used, but not with the unmodified natural peptide. Importantly, these results demonstrate, using a novel molecular tool, that modifications at anchor residues can dramatically influence the conformation of the MHC peptide groove and thus may have a profound effect on TCR interactions. Moreover, these results may have important implications in designing modifications in peptides for cancer immunotherapy, because most such peptides studied are of low affinity.     

Preferential Th1 immune response in invariant chain-deficient mice

MHC class II molecules associate with the invariant chain (Ii) molecule during biosynthesis. Ii facilitates the folding of class II molecules, interferes with their peptide association, and is involved in MHC class II transport. In this study, we have investigated the in vitro and in vivo immune response of Ii-deficient mice (Ii(-/-)). Our results have demonstrated that CD4(+) T cells from Ii(-/-) mice proliferate normally in vitro after in vivo immunization with protein Ags. However, cytokine secretion profiles of Ag-primed CD4(+) T cells from Ii(-/-) mice differ from CD4(+) T cells from wild-type mice. Whereas cells from wild-type mice secrete IFN-gamma and IL-4, cells from Ii(-/-) mice secrete mostly IFN-gamma. Moreover, Ii(-/-) mice exhibit a normal Th1 response in the delayed-type hypersensitivity and trinitrobenzene sulfonic acid colitis models; however, these mice lack an in vivo Th2 response, as demonstrated in the asthma model. Therefore, we suggest that defective Ag presentation in Ii(-/-) mice leads selectively to a Th1 effector response.     

Investigation of Four Classes of Non-nodulating White Sweetclover (Melilotus alba annua Desr.) Mutants and Their Responses to Arbuscular-Mycorrhizal Fungi

The nitrogen-fixing symbiosis between Rhizobiaceae and legumes is one of the best-studied interactions established between prokaryotes and eukaryotes. The plant develops root nodules in which the bacteria are housed, and atmospheric nitrogen is fixed into ammonia by the rhizobia and made available to the plant in exchange for carbon compounds. It has been hypothesized that this symbiosis evolved from the more ancient arbuscular mycorrhizal (AM) symbiosis, in which the fungus associates with roots and aids the plant in the absorption of mineral nutrients, particularly phosphate. Support comes from several fronts: 1) legume mutants where Nod(-) and Myc(-) co-segregate, and 2) the fact that various early nodulin (ENOD) genes are expressed in legume AM. Both strongly argue for the idea that the signal transduction pathways between the two symbioses are conserved. We have analyzed the responses of four classes of non-nodulating Melilotus alba (white sweetclover) mutants to Glomus intraradices (the mycorrhizal symbiont) to investigate how Nod(-) mutations affect the establishment of this symbiosis. We also re-examined the root hair responses of the non-nodulating mutants to Sinorhizobium meliloti (the nitrogen-fixing symbiont). Of the four classes, several sweetclover sym mutants are both Nod(-) and Myc(-). In an attempt to decipher the relationship between nodulation and mycorrhiza formation, we also performed co-inoculation experiments with mutant rhizobia and Glomus intraradices on Medicago sativa, a close relative of M. alba. Even though sulfated Nod factor was supplied by some of the bacterial mutants, the fungus did not complement symbiotically defective rhizobia for nodulation.     

How to move with no rigid skeleton? The octopus has the answers

The octopus is amazingly flexible and shows exceptional control and coordination in all its movements. It seems remarkable to us skeletal creatures that the octopus achieves all this without a single bone.     

Proliferation and competition in discrete biological systems

We study the emergence of collective spatio-temporal objects in biological systems by representing individually the elementary interactions between their microscopic components. We use the immune system as a prototype for such interactions. The results of this detailed explicit analysis are compared with the traditional procedure of representing the collective dynamics in terms of densities that obey partial differential equations. The simulations show even for very simple elementary reactions the spontaneous emergence of localized complex structures, from microscopic noise. In turn the effective dynamics of these structures affects the average behaviour of the system in a very decisive way: systems which would according to the differential equations approximation die, display in reality a very lively behaviour. As the optimal modelling method we propose a mixture of microscopic simulation systems describing each reaction separately, and continuous methods describing the average behaviour of the agents.     

The Salmonella SpiC protein targets the mammalian Hook3 protein function to alter cellular trafficking

The Salmonella SpiC protein is secreted into the cytosol of macrophages via a unique type III secretion system that functions intracellularly to translocate proteins across the phagosomal membrane. The SpiC protein is required for survival within macrophages and inhibition of phagosome-lysosome fusion in vivo, and it is sufficient to inhibit endosome-endosome fusion in vitro. Here, we establish that SpiC targets the function of Hook3, a mammalian protein implicated in cellular trafficking. Purified GST-SpiC pulled down Hook3 from murine macrophages, and anti-Hook3 antibodies precipitated SpiC from the cytosol of Salmonella-infected macrophages. Expression of the spiC gene disrupted Golgi morphology in Vero cells and altered the distribution of lysosomes in macrophages, mimicking the phenotype of cells expressing a hook3 dominant-negative mutant. By inactivating Hook3 function, the SpiC protein may alter the lysosome network and prevent phagosome-lysosome fusion.     

Cloning and functional expression of alkaline alpha-galactosidase from melon fruit: similarity to plant SIP proteins uncovers a novel family of plant glycosyl hydrolases

Raffinose and stachyose are ubiquitous galactosyl-sucrose oligosaccharides in the plant kingdom which play major roles, second only to sucrose, in photoassimilate translocation and seed carbohydrate storage. These sugars are initially metabolised by alpha-galactosidases (alpha-gal). We report the cloning and functional expression of the first genes, CmAGA1 and CmAGA2, encoding for plant alpha-gals with alkaline pH optima from melon fruit (Cucumis melo L.), a raffinose and stachyose translocating species. The alkaline alpha-gal genes show very high sequence homology with a family of undefined 'seed imbibition proteins' (SIPs) which are present in a wide range of plant families. In order to confirm the function of SIP proteins, a representative SIP gene, from tomato, was expressed and shown to have alkaline alpha-gal activity. Phylogenetic analysis based on amino acid sequences shows that the family of alkaline alpha-gals shares little homology with the known prokaryotic and eukaryotic alpha-gals of glycosyl hydrolase families 27 and 36, with the exception of two cross-family conserved sequences containing aspartates which probably function in the catalytic step. This previously uncharacterised, plant-specific alpha-gal family of glycosyl hydrolases, with optimal activity at neutral-alkaline pH likely functions in key processes of galactosyl-oligosaccharide metabolism, such as during seed germination and translocation of RFO photosynthate.     

Enhanced levels of free and protein-bound threonine in transgenic alfalfa (Medicago sativa L.) expressing a bacterial feedback-insensitive aspartate kinase gene

Threonine, lysine, methionine, and tryptophan are essential amino acids for humans and monogastric animals. Many of the commonly used diet formulations, particularly for pigs and poultry, contain limiting amounts of these amino acids. One approach for raising the level of essential amino acids is based on altering the regulation of their biosynthetic pathways in transgenic plants. Here we describe the first production of a transgenic forage plant, alfalfa (Medicago sativa L.) with modified regulation of the aspartate-family amino acid biosynthetic pathway. This was achieved by over-expressing the Escherichia coli feedback-insensitive aspartate kinase (AK) in transgenic plants. These plants showed enhanced levels of both free and protein-bound threonine. In many transgenic plants the rise in free threonine was accompanied by a significant reduction both in aspartate and in glutamate. Our data suggest that in alfalfa, AK might not be the only limiting factor for threonine biosynthesis, and that the free threonine pool in this plant limits its incorporation into plant proteins.