Isotopic Fractionation of Hydrogen in Plants

The naturally-occurring stable isotopes deuterium and hydrogen are fractionated by a number of physical and biological processes. Deuterium has a tendency to precipitate out first from a moist air mass. Thus ground water will become isotopically lighter with an increase in latitude, altitude, or distance inland. Water taken up by the plant from the soil undergoes little change until evapotranspiration results in leaf water becoming isotopically heavier. Thus hydrogen isotopes in plants can reveal something of geography (groundwater) and climate. Hydrogen isotopes undergo little fractionation by passage through the food chain, although plant parasites tend to be enriched in D as compared to their hosts, possibly due to higher rates of transpiration in the parasitic plants. The splitting of water in photosynthesis results in the lighter isotope being incorporated into organic matter. An even larger isotopic fractionation results during lipid synthesis and other processes involving the pyruvate dehydrogenase complex. Differences in metabolic pathway between species can be detected by D/H ratios. Hydrogen isotopic differences can be detected between CAM, C₄, and C₃ species. Within C₄ plants, the NADP-ME plants are isotopically distinguishable from NAD-ME and PEP-CK plants.     

Targeted gene delivery to CD117‐expressing cells in vivo with lentiviral vectors co‐displaying stem cell factor and a fusogenic molecule

The development of a lentiviral system to deliver genes to specific cell types could improve the safety and the efficacy of gene delivery. Previously, we have developed an efficient method to target lentivectors to specific cells via an antibody–antigen interaction in vitro and in vivo. We report herein a targeted lentivector that harnesses the natural ligand–receptor recognition mechanism for targeted modification of c‐KIT receptor‐expressing cells. For targeting, we incorporate membrane‐bound human stem cell factor (hSCF), and for fusion, a Sindbis virus‐derived fusogenic molecule (FM) onto the lentiviral surface. These engineered vectors can recognize cells expressing surface CD117, resulting in efficient targeted transduction of cells in an SCF‐receptor dependent manner in vitro, and in vivo in xenografted mouse models. This study expands the ability of targeting lentivectors beyond antibody targets to include cell‐specific surface receptors. Development of a high titer lentivector to receptor‐specific cells is an attractive approach to restrict gene expression and could potentially ensure therapeutic effects in the desired cells while limiting side effects caused by gene expression in non‐target cells. Biotechnol. Bioeng. 2009; 104: 206–215 © 2009 Wiley Periodicals, Inc.     

Structural Basis for T Cell Alloreactivity among Three HLA-B14 and HLA-B27 Antigens

The existence of cytotoxic T cells (CTL) cross-reacting with the human major histocompatibility antigens HLA-B14 and HLA-B27 suggests that their alloreactivity could be due to presentation of shared peptides in similar binding modes by these molecules. We therefore determined the crystal structures of the subtypes HLA-B*1402, HLA-B*2705, and HLA-B*2709 in complex with a proven self-ligand, pCatA (peptide with the sequence IRAAPPPLF derived from cathepsin A (residues 2–10)), and of HLA-B*1402 in complex with a viral peptide, pLMP2 (RRRWRRLTV, derived from latent membrane protein 2 (residues 236–244) of Epstein-Barr virus). Despite the exchange of 18 residues within the binding grooves of HLA-B*1402 and HLA-B*2705 or HLA-B*2709, the pCatA peptide is presented in nearly identical conformations. However, pLMP2 is displayed by HLA-B*1402 in a conformation distinct from those previously found in the two HLA-B27 subtypes. In addition, the complexes of HLA-B*1402 with the two peptides reveal a nonstandard, tetragonal mode of the peptide N terminus anchoring in the binding groove because of the exchange of the common Tyr-171 by His-171 of the HLA-B*1402 heavy chain. This exchange appears also responsible for reduced stability of HLA-B14-peptide complexes in vivo and slow assembly in vitro. The studies with the pCatA peptide uncover that CTL cross-reactive between HLA-B14 and HLA-B27 might primarily recognize the common structural features of the bound peptide, thus neglecting amino acid replacements within the rim of the binding grooves. In contrast, structural alterations between the three complexes with the pLMP2 peptide indicate how heavy chain polymorphisms can influence peptide display and prevent CTL cross-reactivity between HLA-B14 and HLA-B27 antigens.T cells possessing the ability to recognize major histocompatibility complex (MHC)² molecules from another individual of the same species, also termed alloreactive T cells, may constitute up to 10% of the T cell pool of an individual, and their precursor frequency can be 100–1,000-fold higher than that of self-restricted T cells directed against a foreign peptide (1, 2). The ability of alloreactive T cells to cross-react with nonself-MHC molecules is a major obstacle preventing successful organ transplantations (3–5). Two mechanisms, direct or indirect allorecognition, can be responsible for the rejection of a transplant by alloreactive T cells (6). In the first case, donor cells expressing MHC molecules are directly recognized by host T cells (7), whereas indirect allorecognition involves the presentation of peptides derived from donor proteins by MHC molecules of the host, followed by the detection of the complexes by the host T cells (8). However, although alloreactive T cells are very common and of great clinical importance, neither the primary basis for their existence nor the reasons underlying their cross-reactivity are sufficiently understood to draw general conclusions (9–11). Only very few studies have addressed the structural basis for the recognition of distinct MHC antigens by cross-reactive T cells (12–18). One of the most important questions regards the individual contribution of the bound peptide and binding groove residues of the heavy chain (HC) of MHC class I antigens to the interaction with T cell receptors (TCR).Here we analyze an HLA-B14 subtype, HLA-B*1402 (named B*1402), as well as two HLA-B27 subtypes, HLA-B*2705 and HLA-B*2709 (named B*2705 and B*2709), to shed light on the structural basis of peptide presentation and T cell alloreactivity among these HLA-B molecules. The amino acid sequences of B*1402 and B*2705 HC differ from each other at 18 positions, all of which are part of the peptide-binding groove (Fig. 1). These amino acid exchanges result in different repertoires of bound peptides; B*1402 and B*2705 share only about 4% of their peptides (19), whereas this value rises to 88% for the B*2705 and B*2709 subtypes (20), which are distinguished only by a single residue at the floor of the binding groove (B*2705, Asp-116; B*2709, His-116). The structural similarities between the two HLA-B27 subtypes (21–27) permit extensive cross-reactivity (up to 90%) of cytotoxic T cells (CTL) (28), whereas CTL alloreactivity between B*1402 and B*2705 is drastically reduced (to about 3%) (19), in line with the very limited overlap of their peptide repertoires.Open in a separate windowFIGURE 1.Amino acid sequence differences among B*1402 and B*2705 HC. The 18 residues distinguishing the two subtypes are all located in or in the immediate vicinity of the peptide-binding groove. B*2705 differs from B*2709 only by a D116H exchange (not shown). The residues are indicated by spheres with volumes roughly proportional to the volumes of the respective amino acid side chain in solution (77). The spheres are colored according to the biochemical properties of the respective amino acids, as indicated at the bottom of the image.The HLA-B14 and HLA-B27 subtypes are distinguished from most other HLA class I molecules in their requirement for an arginine at anchor position 2 of the bound peptide (p2) (20, 29, 30). This preference is nearly absolute in B*2705 and B*2709 (31), whereas B*1402 tolerates also glutamine, glutamate, and proline as p2 anchors (19, 29). Statistically significant differences between B*1402 and B*2705 are also found at several other peptide positions (19). Previous structural and cellular studies of the HLA-B27 subtypes have suggested that molecular mimicry between the viral peptide pLMP2 (RRRWRRLTV, derived from Epstein-Barr virus latent membrane protein 2, residues 236–244) and the self-peptide pVIPR (RRKWRRWHL, derived from vasoactive intestinal peptide type 1 receptor, residues 400–408), when bound to B*2705, serves as an example of how a cellular immune response could be triggered that might contribute to the onset of ankylosing spondylitis (AS) through an autoimmune mechanism (22, 24). CTL that recognize the B*2705 and the B*2709 subtypes in complex with the self-peptide pVIPR (22) exemplify alloreactivity in this system, although the D116H micropolymorphism is deeply buried and not directly accessible to a TCR.Alloreactive T cells are known to recognize a very diverse array of alloantigen-bound peptides (32, 33), so that virtually each T cell clone can be assumed to be specific for a distinct peptide. For this reason, the substantial correlation found in previous studies between peptide and the alloreactive T cell epitope sharing among HLA-B27 (reviewed in Ref. 34) or HLA-B14 subtypes (only 28.4% partial or full cross-reactivity, similar to peptide overlapping between the subtypes B*1402 and B*1403, see Ref. 19) supports a prominent role of peptides in determining alloreactive T cell cross-reaction, and it suggests that many shared ligands adopt antigenically similar conformations when bound to distinct HLA-B molecules. On the other hand, the results reported by Merino et al. (19) also demonstrate that the few CTL that cross-react with B*1402 and B*2705 did not exhibit cross-reactivity with B*1403, which is distinguished from B*1402 only by a single amino acid exchange in the α2-helix. Furthermore, they show that alloreactive CTL from various donors directed against B*2705 did not lyse cells expressing either B*1402 or B*1403, although the number of CTL tested might not have been high enough to detect a presumably low degree of cross-reactivity. Without structural data from HLA-B14 subtypes, however, these results are difficult to interpret.The pCatA peptide (IRAAPPPLF, derived from the signal sequence of cathepsin A, residues 2–10) is among the very few known common ligands of B*1402, B*2705 (19), and B*2709³ and can thus serve to study how a very different (B*1402) and two very similar subtypes (B*2705 and B*2709) handle a common ligand. On the other hand, the pLMP2 peptide is a proven natural ligand only of B*2705, whose possible presentation in vivo by B*2709 and HLA-B14 is not yet known, although this peptide can be complexed in vitro with B*2709 (24) and also with B*1402 (35). From previous crystallographic studies, it was known that pLMP2 is presented by the two HLA-B27 antigens in very different conformations (24). We expected that the pronounced sequence differences between B*1402 and the HLA-B27 alloantigens (Fig. 1) might even enhance the conformational dissimilarities that are observed when two very closely related subtypes such as B*2705 and B*2709 are compared. Discrepancies in peptide display could reasonably be expected to prevent CTL cross-reaction, so that pLMP2 might be considered as a representative of the vast majority of HLA-B14- and HLA-B27-presented ligands that must be responsible for the low degree of CTL cross-reactivity between these alloantigens. Despite these presumed differences between pCatA and pLMP2, both peptides may be seen as examples of ligands that could principally allow direct allorecognition.Here we report the crystal structures of B*1402·pCatA, B*2705·pCatA, B*2709·pCatA, and B*1402·pLMP2, and we compare them with each other and with the previously reported structures of B*2705·pLMP2 and B*2709·pLMP2 (24).     

Noise-Induced Coherence in Multicellular Circadian Clocks

In higher organisms, circadian rhythms are generated by a multicellular genetic clock that is entrained very efficiently to the 24-h light-dark cycle. Most studies done so far of these circadian oscillators have considered a perfectly periodic driving by light, in the form of either a square wave or a sinusoidal modulation. However, in natural conditions, organisms are subject to nonnegligible fluctuations in the light level all through the daily cycle. In this article, we investigate how the interplay between light fluctuations and intercellular coupling affects the dynamics of the collective rhythm in a large ensemble of nonidentical, globally coupled cellular clocks modeled as Goodwin oscillators. On the basis of experimental considerations, we assume an inverse dependence of the cell-cell coupling strength on the light intensity, in such a way that the larger the light intensity, the weaker the coupling. Our results show a noise-induced rhythm generation for constant light intensities at which the clock is arrhythmic in the noise-free case. Importantly, the rhythm shows a resonancelike phenomenon as a function of the noise intensity. Such improved coherence can be only observed at the level of the overt rhythm and not at the level of the individual oscillators, thus suggesting a cooperative effect of noise, coupling, and the emerging synchronization between the oscillators.     

Light-induced degradation of storage starch in turions of Spirodela polyrhiza depends on nitrate

Light induces both the germination of turions of the duckweed Spirodela polyrhiza and the degradation of the reserve starch stored in the turions. The germination photoresponse requires nitrate, and we show here that nitrate is also needed for the light-induced degradation of the turion starch. Ammonium cannot substitute for nitrate in this regard, and nitrate thus acts specifically as signal to promote starch degradation in the turions. Irradiation with continuous red light leads to starch degradation via auto-phosphorylation of starch-associated glucan, water dikinase (GWD), phosphorylation of the turion starch and enhanced binding of alpha-amylase to starch granules. The present study shows that all of these processes require the presence of nitrate, and that nitrate exerts its effect on starch degradation at a point between the absorption of light by phytochrome and the auto-phosphorylation of the GWD. Nitrate acts to coordinate carbon and nitrogen metabolism in germinating turions: starch will only be broken down when sufficient nitrogen is present to ensure appropriate utilization of the released carbohydrate. These data constitute the first report of control over the initiation of reserve starch degradation by nitrate.     

Distribution of RNA polymerase on Drosophila polytene chromosomes as studied by indirect immunofluorescence

Using indirect immunofluorescence visualization techniques we investigated the in situ distribution of RNA polymerase B on Drosophila melanogaster polytene chromosomes. The enzyme was found at many sites distributed throughout the genome in a pattern clearly distinct from that observed for histone H1, but it was especially concentrated in puffs induced by heat shock.     

Lipopolysaccharide responsiveness is an important factor in the generation of optimal antigen-specific T cell responses during infection with gram-negative bacteria

We previously have found that the endotoxin (LPS) of Gram-negative bacteria is a major determinant of macrophage Ia induction during infection with these organisms. Specifically, i.p. injection of Gram-negative bacteria elicits a striking macrophage Ia response in LPS-responder mice but virtually no response in LPS-low-responder mice. As an extension of these findings, in this report we have tested the hypothesis that the inability of LPS-low responder mice to mount an Ia response during Gram-negative infection may in turn impair their capacity for generation of appropriate antibacterial T cell responses. Our results demonstrate that for a variety Gram-negative organisms (Salmonella typhimurium, Salmonella minnesota, and Escherichia coli), both macrophage Ia induction and the generation of Ag-specific T cell responses are controlled by the lps gene. We also have asked whether the expression of additional toxins (other than LPS) by infecting Gram-negative organisms can "override" this lps gene control of macrophage and T cell responses. We have found that infection of LPS-low-responder mice with an E. coli strain that expresses a hemolytic exotoxin (Hly) leads to the induction of macrophage Ia expression as well as the generation of T cell responses to both the Hly molecule and to other E. coli-associated Ag, whereas no responses are generated during infection with a Hly- strain. This result suggests that LPS-low responder mice have no inherent defect in T cell responsiveness to Gram-negative bacterial Ag but rather that these mice fail to receive an LPS-mediated signal required for the induction of Ia expression and subsequent generation of peritoneal T cell immunity. These findings, when taken together with results presented in the accompanying paper, strengthen the argument that bacterial toxin production (and the ability of the host to respond to the toxin) can represent a critical determinant of the induction of macrophage Ia expression and in turn, of Ag-specific T cell responses during bacterial infection.     

Role of bacterial hemolysin production in induction of macrophage Ia expression during infection with Listeria monocytogenes

The production of a hemolytic exotoxin (Hly) termed listeriolysin O (LLO) is a major determinant of the virulence of the Gram-positive bacterium Listeria monocytogenes. As determined by lethal inoculum size, LLO- strains of L. monocytogenes generally are several orders of magnitude less virulent than their LLO+ counterparts. The generation of protective anti-Listeria T cell immunity also has been shown to depend on the LLO phenotype of the bacteria present during primary infection, although the cellular basis of this observation is not known. The experiments described here address the role of LLO in regulation of the expression of class II MHC (Ia) molecules by murine macrophages. Because Ia expression by macrophages and other APC is thought to be a central factor in the generation of T cells specific for bacterial Ag, we have tested the hypothesis that the failure of LLO- strains to elicit anti-Listeria T cell responses might be secondary to an inability of these strains to stimulate increases in macrophage Ia levels. Our results show that the macrophage Ia response after i.p. injection of L. monocytogenes correlates strongly with the LLO phenotype of the bacteria. The presence of LLO+ organisms, even at very small numbers (as few as 10), elicits a striking increase in Ia expression by peritoneal macrophages. In contrast, even at very high numbers (up to 10(6) per mouse), LLO- bacteria fail to stimulate a strong Ia response. We also have analyzed macrophage Ia expression after injection of lysates of Escherichia coli expressing recombinant LLO protein. Similar to the results obtained with LLO+ and LLO- L. monocytogenes, we have observed Ia induction only with LLO+ lysates. Ia induction by this crude recombinant LLO preparation can be inhibited by cholesterol or heat. Furthermore, supernatants derived from cultures of LLO+ (but not LLO-) L. monocytogenes can cause Ia induction when administered via i.p. injection. Taken together, these findings suggest that the failure of macrophages to respond to LLO- organisms with an increase in Ia expression may be a major underlying cause of the failure of these bacteria to induce Listeria-specific protective T cell immunity. Furthermore, we propose that the induction of macrophage Ia expression in response to bacterial toxins such as Hly may represent one component of a set of early, innate immune mechanisms, and that this induction may provide a critical "bridge" to later, acquired, Ag-specific immune processes.     

Controlled unfolding and refolding of a single sodium-proton antiporter using atomic force microscopy

Single-molecule force-spectroscopy was employed to unfold and refold single sodium-proton antiporters (NhaA) of Escherichia coli from membrane patches. Although transmembrane alpha-helices and extracellular polypeptide loops exhibited sufficient stability to individually establish potential barriers against unfolding, two helices predominantly unfolded pairwise, thereby acting as one structural unit. Many of the potential barriers were detected unfolding NhaA either from the C-terminal or the N-terminal end. It was found that some molecular interactions stabilizing secondary structural elements were directional, while others were not. Additionally, some interactions appeared to occur between the secondary structural elements. After unfolding ten of the 12 helices, the extracted polypeptide was allowed to refold back into the membrane. After five seconds, the refolded polypeptide established all secondary structure elements of the native protein. One helical pair showed a characteristic spring like "snap in" into its folded conformation, while the refolding process of other helices was not detected in particular. Additionally, individual helices required characteristic periods of time to fold. Correlating these results with the primary structure of NhaA allowed us to obtain the first insights into how potential barriers establish and determine the folding kinetics of the secondary structure elements.