Induction of type C virions from normal rat kidney cells by 2-deoxy-D-glucose.

The sugar 2-deoxy-D-glucose (2-DG) induced the release of type C virions from an established line of normal rat kidney (NRK) cells. Within 20 h after the addition of 5 mg of 2-DG per ml to exponentially growing NRK clutures, more than 80% of the cells expressed the mammalian type C virus interspecies-specific antigen (p30) as determined by indirect cytoplasmic immunofluorescence. Maximal virion release occurred 1 to 2 days after 2-DG was added for 24 h to the growth medium although a low level of virion production was detected as early as 2.5 h after 2-DG treatment. Studies with inhibitors of RNA synthesis indicated a requirement for de novo RNA synthesis after the addition of 2-DG. Sensitivity of NRK cells to type C virion induction was limited to a relatively short period of in vitro growth and preceded spontaneous virion release by 8 to 10 subculture generations. A model is presented for the sequential derepression of latent type C virus information in serially propagated NRK cells.     

Impact of antigen presentation on TCR modulation and cytokine release: implications for detection and sorting of antigen-specific CD8+ T cells using HLA-A2 wild-type or HLA-A2 mutant tetrameric complexes

Soluble MHC class I molecules loaded with antigenic peptides are available either to detect and to enumerate or, alternatively, to sort and expand MHC class I-restricted and peptide-reactive T cells. A defined number of MHC class I/peptide complexes can now be implemented to measure T cell responses induced upon Ag-specific stimulation, including CD3/CD8/zeta-chain down-regulation, pattern, and quantity of cytokine secretion. As a paradigm, we analyzed the reactivity of a Melan-A/MART-1-specific and HLA-A2-restricted CD8(+) T cell clone to either soluble or solid-phase presented peptides, including the naturally processed and presented Melan-A/MART-1 peptide AAGIGILTV or the peptide analog ELAGIGILTV presented either by the HLA-A2 wild-type (wt) or mutant (alanineright arrowvaline aa 245) MHC class I molecule, which reduces engagement of the CD8 molecule with the HLA-A2 heavy chain. Soluble MHC class I complexes were used as either monomeric or tetrameric complexes. Soluble monomeric MHC class I complexes, loaded with the Melan-A/MART-1 peptide, resulted in CD3/CD8 and TCR zeta-chain down-regulation, but did not induce measurable cytokine release. In general, differences pertaining to CD3/CD8/zeta-chain regulation and cytokine release, including IL-2, IFN-gamma, and GM-CSF, were associated with 1) the format of Ag presentation (monomeric vs tetrameric MHC class I complexes), 2) wt vs mutant HLA-A2 molecules, and 3) the target Ag (wt vs analog peptide). These differences are to be considered if T cells are exposed to recombinant MHC class I Ags loaded with peptides implemented for detection, activation, or sorting of Ag-specific T cells.     

Genome-inferred spatio-temporal resolution of an uncultivated Roizmanbacterium reveals its ecological preferences in groundwater

Subsurface ecosystems like groundwater harbour diverse microbial communities, including small-sized, putatively symbiotic organisms of the Candidate Phyla Radiation, yet little is known about their ecological preferences and potential microbial partners. Here, we investigated a member of the superphylum Microgenomates (Cand. Roizmanbacterium ADI133) from oligotrophic groundwater using mini-metagenomics and monitored its spatio-temporal distribution using 16S rRNA gene analyses. A Roizmanbacteria-specific quantitative PCR assay allowed us to track its abundance over the course of 1 year within eight groundwater wells along a 5.4 km hillslope transect, where Roizmanbacteria reached maximum relative abundances of 2.3%. In-depth genomic analyses suggested that Cand. Roizmanbacterium ADI133 is a lactic acid fermenter, potentially able to utilize a range of complex carbon substrates, including cellulose. We hypothesize that it attaches to host cells using a trimeric autotransporter adhesin and inhibits their cell wall biosynthesis using a toxin–antitoxin system. Network analyses based on correlating Cand. Roizmanbacterium ADI133 abundances with amplicon sequencing-derived microbial community profiles suggested one potential host organism, classified as a member of the class Thermodesulfovibrionia (Nitrospirae). By providing lactate as an electron donor Cand. Roizmanbacterium ADI133 potentially mediates the transfer of carbon to other microorganisms and thereby is an important connector in the microbial community.     

Pool sequencing of natural HLA-DR,DQ, and DP ligands reveals detailed peptide motifs,constraints of processing,and general rules

We have approached the problem of MHC class II ligand motifs by pool sequencing natural peptides eluted from HLA-DR, DQ, and DP molecules. The results indicate surprisingly clear patterns, although not quite as clear as with natural class I ligands. The most striking feature is a highly dominant Proline at position 2. We interpret this to be a consequence of aminopeptidase N-like activity in processing. Another general aspect is the existence of three to four hydrophobic or aromatic anchors, whereby the first and the last are separated by five to eight residues. The peptide motifs for HLA-DR1, DR5, DQ7, and DPw4 are allele-specific and differ by spacing and occupancy of anchors. The anchors tend to be flanked by clusters of charged residues, and small residues, especially Ala, are frequent in the motif centers. These detailed motifs allow one to interpret most previous (DR-) motifs as fitting one or more of the anchors or conserved clusters. The relative motif symmetry suggests the possibility of bidirectional binding of peptides in the class II groove.     

Correlated evolution between hearing sensitivity and social calls in bats

Echolocating bats are auditory specialists, with exquisite hearing that spans several octaves. In the ultrasonic range, bat audiograms typically show highest sensitivity in the spectral region of their species-specific echolocation calls. Well-developed hearing in the audible range has been commonly attributed to a need to detect sounds produced by prey. However, bat pups often emit isolation calls with low-frequency components that facilitate mother-young reunions. In this study, we examine whether low-frequency hearing in bats exhibits correlated evolution with (i) body size; (ii) high-frequency hearing sensitivity or (iii) pup isolation call frequency. Using published audiograms, we found that low-frequency hearing sensitivity is not dependent on body size but is related to high-frequency hearing. After controlling for high-frequency hearing, we found that low-frequency hearing exhibits correlated evolution with isolation call frequency. We infer that detection and discrimination of isolation calls have favoured enhanced low-frequency hearing because accurate parental investment is critical: bats have low reproductive rates, non-volant altricial young and must often identify their pups within large crèches.     

A novel Pseudomonas putida strain with high levels of hydantoin-converting activity, producing -amino acids

Optically pure chiral amino acids and their derivatives can be efficiently synthesised by the biocatalytic conversion of 5-substituted hydantoins in reactions catalysed by stereo-selective microbial enzymes: initially a hydantoinase catalyses the cleavage of the hydantoin producing an N-carbamyl amino acid. In certain bacteria where an N-carbamyl amino acid amidohydrolase (NCAAH) is present, the N-carbamyl amino acid intermediate is further converted to amino acid, ammonia and CO₂. In this study we report on a novel Pseudomonas putida strain which exhibits high levels of hydantoin-converting activity, yielding -amino acid products including alanine, valine, and norleucine, with bioconversion yields between 60% and 100%. The preferred substrates are generally aliphatic, but not necessarily short chain, 5-alkylhydantoins. In characterizing the enzymes from this microorganism, we have found that the NCAAH has -selectivity, while the hydantoinase is non-stereoselective. In addition, resting cell reactions under varying conditions showed that the hydantoinase is highly active, and is not subject to substrate inhibition, or product inhibition by ammonia. The rate-limiting reaction appears to be the NCAAH-catalysed conversion of the intermediate. Metal-dependence studies suggest that the hydantoinase is dependent on the presence of magnesium and cobalt ions, and is strongly inhibited by the presence of copper ions. The relative paucity of -selective hydantoin-hydrolysing enzyme systems, together with the high level of hydantoinase activity and the unusual substrate selectivity of this P. putida isolate, suggest that is has significant potential in industrial applications.     

Targeting the Tumour Vasculature: Exploitation of Low Oxygenation and Sensitivity to NOS Inhibition by Treatment with a Hypoxic Cytotoxin

Many cancer research efforts focus on exploiting genetic-level features that may be targeted for therapy. Tissue-level features of the tumour microenvironment also represent useful therapeutic targets. Here we investigate the presence of low oxygen tension and sensitivity to NOS inhibition of tumour vasculature as potential tumour-specific features that may be targeted by hypoxic cytotoxins, a class of therapeutics currently under investigation. We have previously demonstrated that tirapazamine (TPZ) mediates central vascular dysfunction in tumours. TPZ is a hypoxic cytotoxin that is also a competitive inhibitor of NOS. Here we further investigated the vascular-targeting activity of TPZ by combining it with NOS inhibitor L-NNA, or with low oxygen content gas breathing. Tumours were analyzed via multiplex immunohistochemical staining that revealed irreversible loss of perfusion and enhanced tumour cell death when TPZ was combined with either low oxygen or a NOS inhibitor. Tumour growth rate was reduced by TPZ + NOS inhibition, and tumours previously resistant to TPZ-mediated vascular dysfunction were sensitized by low oxygen breathing. Additional mapping analysis suggests that tumours with reduced vascular-associated stroma may have greater sensitivity to these effects. These results indicate that poorly oxygenated tumour vessels, also being abnormally organized and with inadequate smooth muscle, may be successfully targeted for significant anti-cancer effects by inhibition of NOS and hypoxia-activated prodrug toxicity. This strategy illustrates a novel use of hypoxia-activated cytotoxic prodrugs as vascular targeting agents, and also represents a novel mechanism for targeting tumour vessels.     

Variability in water temperature affects trait-mediated survival of a newly settled coral reef fish

As animals with complex life cycles metamorphose from one stage to the next, carry-over effects from earlier stages can affect future mortality. To examine the relationship between early life history traits and survival, seven monthly cohorts of newly-settled bluehead wrasse Thalassoma bifasciatum were collected immediately after settlement and over sequential 3-day periods. Otolith analysis was used to quantify mean larval and juvenile growth rates, pelagic larval duration (PLD), and settlement size and condition of different age classes to identify the traits most important for survival. Overall, survivors tended to have shorter PLDs, to settle at smaller sizes and higher condition levels, and to exhibit faster early juvenile growth. Water temperature contributed to among-cohort variability in traits as warmer water led to faster larval and juvenile growth and shorter PLDs. Trait-specific fitness functions demonstrated that temperature can influence fitness by changing the nature of selection on each trait. Estimates of selection intensity revealed that settlement condition contributed the most to variation in fitness across cohorts, followed by juvenile growth. Frequent loss of low settlement condition individuals and occasional loss of the very highest condition fish suggest that particularly high settlement condition during the warmest temperatures may be detrimental. Not only does the quality of settlers vary over time, but selective loss of individuals with particular phenotypic traits is not pervasive and can vary with environmental conditions such as temperature.     

Zooplankton in the arctic Laptev Sea--feeding ecology as indicated by fatty acid composition

The fatty acid and alcohol composition of the mesozooplanktonspecies analysed indicated a diatom-based diet in late summer.For the majority of organisms investigated, an opportunisticfeeding behaviour is assumed.