Exploring the role of structure and dynamics in the function of chymotrypsin inhibitor 2

Increasing awareness of the possible role of internal dynamics in protein function has led to the development of new methods for experimentally characterizing protein dynamics across multiple time scales, especially using NMR spectroscopy. A few analyses of the conformational dynamics of proteins ranging from nonallosteric single domains to multidomain allosteric enzymes are now available; however, demonstrating a connection between dynamics and function remains difficult on account of the comparative lack of studies examining both changes in dynamics and changes in function in response to the same perturbations. In previous work, we characterized changes in structure and dynamics on the ps–ns time scale resulting from hydrophobic core mutations in chymotrypsin inhibitor 2 and found that there are moderate, persistent global changes in dynamics in the absence of gross structural changes (Whitley et al., Biochemistry 2008;47:8566–8576). Here, we assay those and additional mutants for inhibitory ability toward the serine proteases elastase and chymotrypsin to determine the effects of mutation on function. Results indicate that core mutation has only a subtle effect on CI2 function. Using chemical shifts, we also studied the effect of complex formation on CI2 structure and found that perturbations are greatest at the complex interface but also propagate toward CI2's hydrophobic core. The structure–dynamics–function data set completed here suggests that dynamics plays a limited role in the function of this small model system, although we do observe a correlation between nanosecond-scale reactive loop motions and inhibitory ability for mutations at one key position in the hydrophobic core.     

Production of Bioactive Soluble Interleukin-15 in Complex with Interleukin-15 Receptor Alpha from a Conditionally-Replicating Oncolytic HSV-1

Oncolytic type-1 herpes simplex viruses (oHSVs) lacking the γ₁34.5 neurovirulence gene are being evaluated for treatment of a variety of malignancies. oHSVs replicate within and directly kill permissive cancer cells. To augment their anti-tumor activity, oHSVs have been engineered to express immunostimulatory molecules, including cytokines, to elicit tumor-specific immune responses. Interleukin-15 (IL-15) holds potential as an immunotherapeutic cytokine because it has been demonstrated to promote both natural killer (NK) cell-mediated and CD8⁺ T cell-mediated cytotoxicity against cancer cells. The purpose of these studies was to engineer an oHSV producing bioactive IL-15. Two oHSVs were constructed encoding murine (m)IL-15 alone (J100) or with the mIL-15 receptor α (mIL-15Rα, J100D) to determine whether co-expression of these proteins is required for production of bioactive mIL-15 from oHSV. The following were demonstrated: i) both oHSVs retain replication competence and cytotoxicity in permissive tumor cell lines. ii) Enhanced production of mIL-15 was detected in cell lysates of neuro-2a cells following J100D infection as compared to J100 infection, suggesting that mIL-15Rα improved mIL-15 production. iii) Soluble mIL-15 in complex with mIL-15Rα was detected in supernates from J100D-infected, but not J100-infected, neuro-2a, GL261, and CT-2A cells. These cell lines vary in permissiveness to oHSV replication and cytotoxicity, demonstrating soluble mIL-15/IL-15Rα complex production from J100D was independent of direct oHSV effects. iv) The soluble mIL-15/IL-15Rα complex produced by J100D was bioactive, stimulating NK cells to proliferate and reduce the viability of syngeneic GL261 and CT-2A cells. v) J100 and J100D were aneurovirulent inasmuch as no neuropathologic effects were documented following direct inoculation into brains of CBA/J mice at up to 1x10⁷ plaque forming units. The production of mIL-15/mIL-15Rα from multiple tumor lines, as well as the lack of neurovirulence, renders J100D suitable for investigating the combined effects of oHSV and mIL-15/IL-15Rα in various cancer models.     

Effect of Breadmaking Process on In Vitro Gut Microbiota Parameters in Irritable Bowel Syndrome

A variety of foods have been implicated in symptoms of patients with Irritable Bowel Syndrome (IBS) but wheat products are most frequently cited by patients as a trigger. Our aim was to investigate the effects of breads, which were fermented for different lengths of time, on the colonic microbiota using in vitro batch culture experiments. A set of in vitro anaerobic culture systems were run over a period of 24 h using faeces from 3 different IBS donors (Rome Criteria–mainly constipated) and 3 healthy donors. Changes in gut microbiota during a time course were identified by fluorescence in situ hybridisation (FISH), whilst the small -molecular weight metabolomic profile was determined by NMR analysis. Gas production was separately investigated in non pH-controlled, 36 h batch culture experiments. Numbers of bifidobacteria were higher in healthy subjects compared to IBS donors. In addition, the healthy donors showed a significant increase in bifidobacteria (P<0.005) after 8 h of fermentation of a bread produced using a sourdough process (type C) compared to breads produced with commercial yeasted dough (type B) and no time fermentation (Chorleywood Breadmaking process) (type A). A significant decrease of δ-Proteobacteria and most Gemmatimonadetes species was observed after 24 h fermentation of type C bread in both IBS and healthy donors. In general, IBS donors showed higher rates of gas production compared to healthy donors. Rates of gas production for type A and conventional long fermentation (type B) breads were almost identical in IBS and healthy donors. Sourdough bread produced significantly lower cumulative gas after 15 h fermentation as compared to type A and B breads in IBS donors but not in the healthy controls. In conclusion, breads fermented by the traditional long fermentation and sourdough are less likely to lead to IBS symptoms compared to bread made using the Chorleywood Breadmaking Process.     

Dietary constraints of phytosaurian reptiles revealed by dental microwear textural analysis

Phytosaurs are a group of large, semi‐aquatic archosaurian reptiles from the Middle–Late Triassic. They have often been interpreted as carnivorous or piscivorous due to their large size, morphological similarity to extant crocodilians and preservation in fluvial, lacustrine and coastal deposits. However, these dietary hypotheses are difficult to test, meaning that phytosaur ecologies and their roles in Triassic food webs remain incompletely constrained. Here, we apply dental microwear textural analysis to the three‐dimensional sub‐micrometre scale tooth surface textures that form during food consumption to provide the first quantitative dietary constraints for five species of phytosaur. We furthermore explore the impacts of tooth position and cranial robustness on phytosaur microwear textures. We find subtle systematic texture differences between teeth from different positions along phytosaur tooth rows, which we interpret to be the result of different loading pressures experienced during food consumption, rather than functional partitioning of food processing along tooth rows. We find rougher microwear textures in morphologically robust taxa. This may be the result of seizing and processing larger prey items compared to those captured by gracile taxa, rather than dietary differences per se. We reveal relatively low dietary diversity between our study phytosaurs and that individual species show a lack of dietary specialization. Species are predominantly carnivorous and/or piscivorous, with two taxa exhibiting slight preferences for ‘harder’ invertebrates. Our results provide strong evidence for higher degrees of ecological convergence between phytosaurs and extant crocodilians than previously appreciated, furthering our understanding of the functioning and evolution of Triassic ecosystems.     

Atlas of vertebrate decay: a visual and taphonomic guide to fossil interpretation

Like many other important evolutionary transitions, our knowledge of the origin of vertebrates is limited to windows of exceptional preservation of soft‐bodied fossils. Unfortunately, these fossils are rare and have been subjected to complex taphonomic filters including decay, collapse and distortion. To maximize our ability to utilize these crucial fossils to reconstruct the timing and sequence of evolutionary events, we are in the need of a robust taphonomic framework with in which to interpret them. Here, we report the results of a series of experiments designed to examine patterns of transformation and loss during decay of important anatomical characters of chordates and primitive vertebrates (ammocoete, adult lamprey, hagfish, juvenile chondrichthyans and a non‐vertebrate chordate, Branchiostoma). Complex and repeated patterns of transformation during decay are identified and figured for informative character complexes including eyes, feeding apparatus, skull and brain, muscles, branchial apparatus, axial structures, viscera, heart and fins. The resulting data regarding character decay and relative loss serve as a guide to recognition and interpretation of the anatomy of non‐biomineralized fossil vertebrates. The methods and techniques outlined are eminently applicable to other soft‐bodied groups and present a new way to interpret the exceptionally preserved fossil record.     

Apparatus Composition, Growth, And Survivorship Of The Lower Ordovician Conodont paracordylodus Gracilis Lindström, 1955

Analysis of numerous conodont element clusters from the Lower Ordovician cherts of the Burubaital Formation in central Kazakhstan reveals that the apparatus of Paracordylodus gracilis Lindstro¨m, 1955 consisted of 15 elements: two M elements, nine S elements (including 1 S₀), and four P elements (2 P₁, 2 P₂). The clusters probably originated as faecal pellets, but the best preserved indicate that the architecture of the apparatus of P. gracilis was comparable to that of ozarkodinid conodonts, providing strong support for the hypothesis that the 15-element 2M-9S-4P apparatus plan was plesiomorphic for conodonts with morphologically complex elements. All the elements within the P. gracilis clusters appear to be at a similar stage of ontogeny, and there is no evidence for late addition or replacement of elements. Analysis of element growth suggests that the relative dimensions of some elements changed during ontogeny, but the available data support the hypothesis that the growth of the apparatus as a whole was isometric. The size distribution of P. gracilis in the Burubaital Formation suggests that individuals in a particular size range were preferentially selected for consumption by predators. The identity of these predators is unknown, but they may have included other P. gracilis .     

T cells in cryptopatch aggregates share TCR gamma variable region junctional sequences with gamma delta T cells in the small intestinal epithelium of mice

The role of cryptopatch aggregates in the development of intestinal intraepithelial lymphocytes (IEL) is a matter of controversy. Therefore, an important question is whether T cells in cryptopatch aggregates are lineally related to IEL. We hypothesized that if gammadelta+ IEL derive from T cells in cryptopatch aggregates, then a clonal relationship would exist between the two populations. To test this hypothesis, we compared the sequence of rearranged TCR gamma variable region 5 genes in gammadelta+ IEL and cryptopatch cells. We purified IEL by FACS and cryptopatch cells were isolated from frozen sections of the intestine by laser-assisted microdissection. PCR showed that TCR gamma variable region 5 was rearranged in gammadelta+ IEL and in CD3+ cryptopatch cells, but not in CD3- cryptopatch cells. DNA sequence analysis showed that the frequency of in-frame junctions in cryptopatch aggregates was at a level consistent with positive selection in both wild-type and athymic nude mice. In addition, the predicted amino acid sequences of V-J junctions present in gammadelta+ IEL and cryptopatch cells were encoded by identical nucleotide sequences. By contrast, the frequency of in-frame joints was significantly reduced in cryptopatch cells isolated from TCR delta-deficient mice, indicating that the enrichment of in-frame joints in cryptopatch cells must normally depend on expression of surface gammadelta TCR. Our results are consistent with the hypothesis that a subset of gammadelta+ IEL are related to T cells in cryptopatch aggregates. The precise role of cryptopatch aggregates in intestinal gammadelta+ T cell homeostasis still needs to be determined.