The thrH gene product of Pseudomonas aeruginosa is a dual activity enzyme with a novel phosphoserine:homoserine phosphotransferase activity

The thrH gene product of Pseudomonas aeruginosa has been shown to complement both homoserine kinase (thrB gene product) and phosphoserine phosphatase (serB gene product) activities in vivo. Sequence comparison has revealed that ThrH is related to phosphoserine phosphatases (PSP, EC 3.1.3.3) and belongs to the l-2-haloacid dehalogenase-like protein superfamily. We have solved the crystal structures of ThrH in the apoform and in complex with a bound product phosphate. The structure confirms an overall fold similar to that of PSP. Most of the catalytic residues of PSP are also conserved in ThrH, suggesting that similar catalytic mechanisms are used by both enzymes. Spectrophotometry-based in vitro assays show that ThrH is indeed a phosphoserine phosphatase with a K(m) of 0.207 mm and k(cat) of 13.4 min(-1), comparable with those of other PSPs. More interestingly, using high pressure liquid chromatography-based assays, we have demonstrated that ThrH is able to further transfer the phosphoryl group to homoserine using phosphoserine as the phosphoryl group donor, indicating that ThrH has a novel phosphoserine:homoserine phosphotransferase activity.     

On the impact of correlation between collaterally consanguineous cells on lymphocyte population dynamics

During an adaptive immune response, lymphocytes proliferate for five to twenty-five cell divisions, then stop and die over a period of weeks. Based on extensive flow cytometry data, Hawkins et al. (Proc Natl Acad Sci USA 104:5032–5037, 2007) introduced a cell-level stochastic model of lymphocyte population dynamics, called the Cyton Model, that accurately captures mean lymphocyte population size as a function of time. In Subramanian et al. (J Math Biol 56(6):861–892, 2008), we performed a branching process analysis of the Cyton Model and deduced from parameterizations for in vitro and in vivo data that the immune response is predictable despite each cell’s fate being highly variable. One drawback of flow cytometry data is that individual cells cannot be tracked, so that it is not possible to investigate dependencies in the fate of cells within family trees. In the absence of this information, while the Cyton Model abandons one of the usual assumptions of branching processes (the independence of lifetime and progeny number), it adopts another of the standard branching processes hypotheses: that the fates of progeny are stochastically independent. However, new experimental observations of lymphocytes show that the fates of cells in the same family tree are not stochastically independent. Hawkins et al. (2008, submitted) report on ciné lapse photography experiments where every founding cell’s family tree is recorded for a system of proliferating lymphocytes responding to a mitogenic stimulus. Data from these experiments demonstrate that the death-or-division fates of collaterally consanguineous cells (those in the same generation within a founding cell’s family tree) are strongly correlated, while there is little correlation between cells of distinct generations and between cells in distinct family trees. As this finding contrasts with one of the assumptions of the Cyton Model, in this paper we introduce three variants of the Cyton Model with increasing levels of collaterally consanguineous correlation structure to incorporate these new found dependencies. We investigate their impact on the predicted expected variability of cell population size. Mathematically we conclude that while the introduction of correlation structure leaves the mean population size unchanged from the Cyton Model, the variance of the population size distribution is typically larger. Biologically, through comparison of model predictions for Cyton Model parameterizations determined by in vitro and in vivo experiments, we deduce that if collaterally consanguineous correlation extends beyond cousins, then the immune response is less predictable than would be concluded from the original Cyton Model. That is, some of the variability seen in data that we previously attributed to experimental error could be due to intrinsic variability in the cell population size dynamics.      

The amino and carboxyl termini of perilipin a facilitate the storage of triacylglycerols

Perilipin A is the most abundant lipid droplet-associated protein in adipocytes and serves important functions in regulating triacylglycerol levels by reducing rates of basal lipolysis and facilitating hormonally stimulated lipolysis. We have previously shown that the central region of perilipin A targets and anchors it to lipid droplets, at least in part via three moderately hydrophobic sequences that embed the protein into the hydrophobic core of the droplet. The current study examines the roles of the amino and carboxyl termini of perilipin A in facilitating triacylglycerol storage. Amino- and carboxyl-terminal truncation mutations of mouse perilipin A were stably expressed in 3T3-L1 preadipocytes, which lack perilipins. Triacylglycerol content of the cells was quantified as a measure of perilipin function and was compared with that of cells expressing full-length perilipin A or control cells lacking perilipins. The amino-terminal sequence between amino acids 122 and 222, including four 10-11-amino acid sequences predicted to form amphipathic beta-strands and a consensus site for cAMP-dependent protein kinase, and the carboxyl terminus of 112 amino acids that is unique to perilipin A were critical to facilitate triacylglycerol storage. The precocious expression of full-length perilipin A in 3T3-L1 preadipocytes aided more rapid storage of triacylglycerol during adipose differentiation. By contrast, the expression of highly truncated amino- or carboxyl-terminal mutations of perilipin failed to serve a dominant negative function in lowering triacylglycerol storage during adipose differentiation. We conclude that the amino and carboxyl termini are critical to the function of perilipin A in facilitating triacylglycerol storage.     

Monomeric recombinant TCR ligand reduces relapse rate and severity of experimental autoimmune encephalomyelitis in SJL/J mice through cytokine switch

Our previous studies demonstrated that oligomeric recombinant TCR ligands (RTL) can treat clinical signs of experimental autoimmune encephalomyelitis (EAE) and induce long-term T cell tolerance against encephalitogenic peptides. In the current study, we produced a monomeric I-A(s)/PLP 139-151 peptide construct (RTL401) suitable for use in SJL/J mice that develop relapsing disease after injection of PLP 139-151 peptide in CFA. RTL401 given i.v. or s.c. but not empty RTL400 or free PLP 139-151 peptide prevented relapses and significantly reduced clinical severity of EAE induced by PLP 139-151 peptide in SJL/J or (C57BL/6 x SJL)F(1) mice, but did not inhibit EAE induced by PLP 178-191 or MBP 84-104 peptides in SJL/J mice, or MOG 35-55 peptide in (C57BL/6 x SJL/J)F(1) mice. RTL treatment of EAE caused stable or enhanced T cell proliferation and secretion of IL-10 in the periphery, but reduced secretion of inflammatory cytokines and chemokines. In CNS, there was a modest reduction of inflammatory cells, reduced expression of very late activation Ag-4, lymphocyte function-associated Ag-1, and inflammatory cytokines, chemokines, and chemokine receptors, but enhanced expression of Th2-related factors, IL-10, TGF-beta3, and CCR3. These results suggest that monomeric RTL therapy induces a cytokine switch that curbs the encephalitogenic potential of PLP 139-151-specific T cells without fully preventing their entry into CNS, wherein they reduce the severity of inflammation. This mechanism differs from that observed using oligomeric RTL therapy in other EAE models. These results strongly support the clinical application of this novel class of peptide/MHC class II constructs in patients with multiple sclerosis who have focused T cell responses to known encephalitogenic myelin peptides.     

N-Phthaloyl gamma-aminobutyric acid affects biochemical circadian rhythms in Wistar rats

N-Phthaloyl gamma-aminobutyric acid (P-GABA) was administered to Wistar rats and 24 hr rhythms of glucose, cholesterol, total protein and lactic acid levels in blood were studied under semi-natural light dark conditions. P-GABA administration caused desynchronisation of the rhythms; while glucose and lactic acid rhythms were advanced, cholesterol and total protein rhythms were delayed. Since GABA is being involved in conveying dark information to the clock, exogenous administration of P-GABA may reduce the photic information received by the clock. The results could be explained by slightly less than 1 hr daily delays (or) advances respectively which would bring the peak times to the points 21 days after the start of administration.     

T lymphocytes promote the development of bone marrow-derived APC in the central nervous system

Certain cells within the CNS, microglial cells and perivascular macrophages, develop from hemopoietic myelomonocytic lineage progenitors in the bone marrow (BM). Such BM-derived cells function as CNS APC during the development of T cell-mediated paralytic inflammation in diseases such as experimental autoimmune encephalomyelitis and multiple sclerosis. We used a novel, interspecies, rat-into-mouse T cell and/or BM cell-transfer method to examine the development and function of BM-derived APC in the CNS. Activated rat T cells, specific for either myelin or nonmyelin Ag, entered the SCID mouse CNS within 3-5 days of cell transfer and caused an accelerated recruitment of BM-derived APC into the CNS. Rat APC in the mouse CNS developed from transferred rat BM within an 8-day period and were entirely sufficient for induction of CNS inflammation and paralysis mediated by myelin-specific rat T cells. The results demonstrate that T cells modulate the development of BM-derived CNS APC in an Ag-independent fashion. This previously unrecognized regulatory pathway, governing the presence of functional APC in the CNS, may be relevant to pathogenesis in experimental autoimmune encephalomyelitis, multiple sclerosis, and/or other CNS diseases involving myelomonocytic lineage cells.     

A correlation between membrane cholesterol level, cell adhesion and tumourigenicity of polyoma virus transformed cells

We have investigated the implications of the rise in membrane cholesterol levels on several in vitro and in vivo properties of polyoma virus transformed rat fibroblasts (PyF), with a special emphasis on alpha5beta1 integrin functions. We show that increased membrane cholesterol causes the PyF cells to change their shape and become more bipolar in appearance. These cells also show significantly higher adhesion to the cell-binding domain of fibronectin, increased localization of alpha5beta1 integrin and talin molecules in focal adhesions and a more robust actin cytoskeleton organization. PyF cells with increased membrane cholesterol show reduced growth in vitro and tumours caused by these cells in nude mice are slow growing. These changes in the growth properties of PyF cells are reversible when the cholesterol levels of PyF cells become normal. Our results suggest that changes in membrane cholesterol levels influence the growth and morphological properties of transformed cells, which can be exploited in controlling the growth of tumours in vivo.     

Immunocontraceptive potential of recombinantly expressed minimized chicken riboflavin carrier protein (mini-RCP) in rodents

Chicken riboflavin carrier protein (RCP; 219 AA) harbours four linear epitopes, constituted by the peptide residues 3-23, 64-83, 130-147 and 200-219. Antibodies to these sequences bioneutralize maternal RCP and provide protection from pregnancy in rodents. In order to overcome the major histocompatibility complex-dependent variability in immune response often encountered with use of single peptides for vaccination in genetically outbred populations, we have assembled a novel synthetic gene, incorporating in tandem the nucleotide sequences coding for all the four neutralizing epitopes of chicken RCP and expressed in Escherichia coli. The gene product, mini-RCP has been characterized for its immunogenic properties and contraceptive potential in rodents. Immunization of rabbits and rats led to generation of antibodies against individual peptide components, as determined by enzyme-linked-immunosorbent assay (ELISA). However, immunized rats carried pregnancy to term and delivered healthy offsprings. Antisera from these rats exhibited decreased affinity of binding to the native protein. These findings suggest that the prospects of covalently-linked epitope peptides need to be cautiously evaluated during the design and development of peptide-based vaccines.     

Tackling the plant proteome: practical approaches, hurdles and experimental tools

The study of complex biological questions through comparative proteomics is becoming increasingly attractive to plant biologists as the rapidly expanding plant genomic and expressed sequence tag databases provide improved opportunities for protein identification. This review focuses on practical issues associated with comparative proteomic analysis, including the challenges of effective protein extraction and separation from plant tissues, the pros and cons of two-dimensional gel-based analysis and the problems of identifying proteins from species that are not recognized models for functional genomic studies. Specific points are illustrated using data from an ongoing study of the tomato and pepper fruit proteomes.