A study of methods for in situ X-ray energy dispersive analysis of polyphosphate bodies in Plectonema boryanum

A variety of preparative methods for in situ X-ray energy dispersive analysis were tested to determine their effects on the elemental composition of polyphosphate bodies in P. boryanum. The bodies were found to contain large amounts of P and K and small amounts of Ca and Mg. Air drying, freeze-drying and freeze-drying from a liquid nitrogen slush all gave similar results. Fixation of the cells in glutaraldehyde and/or OsO₄ resulted in loss of the K and enhancement of the Ca peak. Magnesium was lost during embedding in epoxy.     

A proton magnetic resonance study of water in human stratum corneum

Proton magnetic resonance spectroscopy has been used to study the nature of water in human stratum corneum. For a single planar sheet of stratum corneum mounted at a specific orientation to the applied magnetic field, three distinct absorptions may be seen having different chemical shifts and spin-lattice relaxation times (T₁). All T₁ values for these resonances are smaller than that for normal liquid water. One absorption is unusual in that the resonance position is dependent upon the orientation of the sample within the field.     

Dissecting the telomere region of barley chromosome 5HL using rice genomic sequences as references: new markers for tracking a complex region in breeding

The terminal region of barley chromosome 5HL controls malt extract, diastatic power, free amino acid nitrogen, alpha-amylase activity, seed dormancy and pre-harvest sprouting. Comparative analysis of the barley and rice maps has established that the terminal region of barley chromosome 5HL is syntenic to rice chromosome 3L near the telomere end. The rice BAC (Bacterial Artificial Chromosome) sequences covering the region of chromosome 3L were used to search barley expressed sequenced tags database. Thirty-three genes were amplified by PCR (polymerase chain reaction) with the primers designed from barley ESTs (expressed sequence tag). Comparison of the sequences of the PCR generated DNA fragments revealed polymorphisms including single nucleotide polymorphism (SNP), insertions or deletions between the barley varieties. Seven new PCR based molecular markers were developed and mapped within 10 cM in three doubled haploid barley populations (Stirling × Harrington, Baudin × AC Metcalfe and Chebec × Harrington). The mapped genes maintain the micro-syntenic relationship between barley and rice. These gene specific markers provide simple and efficient tools for germplasm characterization and marker-assisted selection for barley malting quality, and ultimately lead to isolation and identification of the major gene(s) controlling multiple quality traits on barley chromosome 5HL.     

Impaired carotenogenesis can affect organization and functionality of etioplast membranes

The effects of impaired carotenogenesis on plastid membrane organization, functionality and stability were studied in etiolated barley plants grown at 20 and 30°C. The plants were treated with norflurazon or amitrole, two herbicides affecting phytoene desaturation and lycopene cyclization, respectively. At 20°C, the amitrole-treated etioplasts, which accumulated lycopene in their inner membranes, exhibited disorganized prolamellar bodies, containing a prevalent form of non-phototransformable protochlorophyllide (Pchlide). They also showed a certain difficulty in reducing the phototransformable pigment to chlorophyllide when exposed to light, and were unable to reform the active ternary complex [protochlorophyllide–oxidoreductase (POR)–Pchlide–NADPH] when placed back in darkness. No ultrastructural alterations were found in norflurazon-treated etioplasts, with carotenogenesis inhibited at the phytoene desaturation step. In these latter organelles, Pchlide, whose forms were comparable with those of the control etioplasts, was photoreduced quickly after illumination and the ternary complex was reformed during a subsequent dark period. Thus, the impaired carotenogenesis leading to the accumulation of lycopene showed greater interference with the etioplast membrane arrangement and functionality than did the earlier interruption of the biosynthetic pathway at the phytoene level. This might be due to the different interactions of the distinct carotenoid precursors with other membrane components. However, in etioplasts of norflurazon-treated plants, a rise in growth temperature caused a partial demolition of prolamellar bodies, showing a lowered thermostability of the carotenoid-deficient membranes. This latter effect strengthens the concept that a correct and complete carotenogenesis pathway, leading to the synthesis of polar carotenoids (i.e. xanthophylls), is required for the maintenance of stable plastid membranes.     

Distinct Biology of Kaposi’s Sarcoma-Associated Herpesvirus from Primary Lesions and Body Cavity Lymphomas

The DNA sequence for Kaposi’s sarcoma-associated herpesvirus was originally detected in Kaposi’s sarcoma biopsy specimens. Since its discovery, it has been possible to detect virus in cell lines established from AIDS-associated body cavity-based B-cell lymphoma and to propagate virus from primary Kaposi’s sarcoma lesions in a human renal embryonic cell line, 293. In this study, we analyzed the infectivity of Kaposi’s sarcoma-associated herpesvirus produced from these two sources. Viral isolates from cultured cutaneous primary KS cells was transmitted to an Epstein-Barr virus-negative Burkitt’s B-lymphoma cell line, Louckes, and compared to virus induced from a body cavity-based B-cell lymphoma cell line. While propagation of body cavity-based B-cell lymphoma-derived virus was not observed in 293 cell cultures, infection with viral isolates obtained from primary Kaposi’s sarcoma lesions induced injury in 293 cells typical of herpesvirus infection and was associated with apoptotic cell death. Interestingly, transient overexpression of the Kaposi’s sarcoma-associated herpesvirus v-Bcl-2 homolog delayed the process of apoptosis and prolonged the survival of infected 293 cells. In contrast, the broad-spectrum caspase inhibitors Z-VAD-fmk and Z-DEVD-fmk failed to protect infected cell cultures, suggesting that Kaposi’s sarcoma-associated herpesvirus-induced apoptosis occurs through a Bcl-2-dependent pathway. Kaposi’s sarcoma-associated herpesvirus isolates from primary Kaposi’s sarcoma lesions and body cavity-based lymphomas therefore may differ and are likely to have distinct contributions to the pathophysiology of Kaposi’s sarcoma.     

The HOX-5 and surfeit gene clusters are linked in the proximal portion of mouse chromosome 2

Using an interspecies backcross, we have mapped the HOX-5 and surfeit (surf) gene clusters within the proximal portion of mouse chromosome 2. While the HOX-5 cluster of homeobox-containing genes has been localized to chromosome 2, bands C3-E1, by in situ hybridization, its more precise position relative to the genes and cloned markers of chromosome 2 was not known. Surfeit, a tight cluster of at least six highly conserved “housekeeping” genes, has not been previously mapped in mouse, but has been localized to human chromosome 9q, a region of the human genome with strong homology to proximal mouse chromosome 2. The data presented here place HOX-5 in the vicinity of the closely linked set of developmental mutations rachiterata, lethargic, and fidget and place surf close to the proto-oncogene Abl, near the centromere of chromosome 2.     

Targeted tandem affinity purification of PSD‐95 recovers core postsynaptic complexes and schizophrenia susceptibility proteins

The molecular complexity of mammalian proteomes demands new methods for mapping the organization of multiprotein complexes. Here, we combine mouse genetics and proteomics to characterize synapse protein complexes and interaction networks. New tandem affinity purification (TAP) tags were fused to the carboxyl terminus of PSD‐95 using gene targeting in mice. Homozygous mice showed no detectable abnormalities in PSD‐95 expression, subcellular localization or synaptic electrophysiological function. Analysis of multiprotein complexes purified under native conditions by mass spectrometry defined known and new interactors: 118 proteins comprising crucial functional components of synapses, including glutamate receptors, K⁺ channels, scaffolding and signaling proteins, were recovered. Network clustering of protein interactions generated five connected clusters, with two clusters containing all the major ionotropic glutamate receptors and one cluster with voltage‐dependent K⁺ channels. Annotation of clusters with human disease associations revealed that multiple disorders map to the network, with a significant correlation of schizophrenia within the glutamate receptor clusters. This targeted TAP tagging strategy is generally applicable to mammalian proteomics and systems biology approaches to disease.     

New pathways for ammonia conversion in soil and aquatic systems

Ammonia conversion processes are essential for most soil and aquatic systems. Under natural conditions, the many possible reactions are difficult to analyze. For example, nitrification and denitrification have long been regarded as separate phenomena performed by different groups of bacteria in segregated areas of soils, sediments or aquatic systems sequentially in time. It has now been established that strict segregation in place and time of the two processes is not necessary and that both denitrifiers and nitrifiers have versatile metabolisms. However, the rates described for aerobic denitrifiers are very low compared to the rates observed under anoxic conditions. Also the rates of nitrifier denitrification are quite low, indicating that these conversions may not play an important role under natural conditions. In addition, these processes often result in the emission of quite large amounts of undesirable products, NO and N₂O. Heterotrophic nitrification might be of relevance for systems, that contain a high carbon to nitrogen ratio. Recently, a novel process (Anammox) has been discovered in which ammonium serves as the electron donor for denitrification of nitrite into dinitrogen gas. ¹⁵N labeling studies showed that hydrazine and hydroxylamine were important intermediates in this process. Enrichment cultures on ammonium, nitrite and bicarbonate resulted in the dominance of one morphotypical microorganism. The growth rate of the cultures is extremely low (doubling time 11 days), but the affinity for ammonium and nitrite and the conversion rates (9.2 10^–4 mol kg^–1 s^–1) are quite high. Some of the reported high nitrogen losses in soil and aquatic systems might be attributed to anaerobic ammonium oxidation. In addition, this conversion offers new opportunities for nitrogen removal, when it is combined with recently developed processes for partial nitrification.     

Reproducibility of protein identification of selected cell types in Barrett's esophagus analyzed by combining laser-capture microdissection and mass spectrometry

Barrett's esophagus (BE) is associated with increased risk of esophageal adenocarcinoma (EAC) and characterized by replacement of normal esophageal squamous epithelium by columnar epithelium. These alterations are also reflected in changes in the protein-expression profiles of the cell types involved. To separately investigate the proteomes of selected cell-types we combined laser-capture microdissection (LCM) and liquid chromatography-mass spectrometry (LC-MS). Aims were to determine the sensitivity, specificity, and technical reproducibility of the sampling method, and the biological variability within and between biopsies and patients. Frozen biopsies were cryo-sectioned, samples of around 2000 epithelial or stroma cells microdissected, digested and measured by Orbitrap LC-MS. Proteins were then identified by MS/MS database search and quantified by label-free analysis. An average of 366 protein-groups were identified per sample, and more protein-groups were found in epithelial samples than in stromal samples (442 vs 301, p < 0.0001). Altogether, 1254 distinct protein-groups were found, 289 and 88 of them significantly more often in epithelial and stroma samples, respectively. We assessed five different types of reproducibilities (run-to-run, intrabiopsy, biopsy-to-biopsy, experiment-to-experiment, and patient-to-patient) for protein identification and protein quantification. Reproducibility of protein identification ranged from 78 to 57%, and standard deviation of protein quantification was on patient-to-patient level four times higher than for run-to-run. We conclude that sampling around 2000 cells requires groups of 32 samples to detect significant, over 10-fold differences in protein abundances and thus creates a successful compromise between throughput and quality of results. We therefore believe that this method is suitable for investigating protein-expression profiles during carcinogenesis.