Lipidic phase membrane protein serial femtosecond crystallography

X-ray free electron laser (X-FEL)-based serial femtosecond crystallography is an emerging method with potential to rapidly advance the challenging field of membrane protein structural biology. Here we recorded interpretable diffraction data from micrometer-sized lipidic sponge phase crystals of the Blastochloris viridis photosynthetic reaction center delivered into an X-FEL beam using a sponge phase micro-jet.     

Matrilin-4 is processed by ADAMTS-5 in late Golgi vesicles present in growth plate chondrocytes of defined differentiation state

The two aggrecanases ADAMTS-4 and ADAMTS-5 have been shown to not only play roles in the breakdown of cartilage extracellular matrix in osteoarthritis, but also mediate processing of matrilins in the secretory pathway. The matrilins are adaptor proteins with a function in connecting fibrillar and network-like components in the cartilage extracellular matrix. Cleavage resulting in processed matrilins with fewer ligand-binding subunits could make these less efficient in providing matrix cohesion. In this study, the processing and degradation of matrilin-4 during cartilage remodeling in the growth plate of the developing mouse long bones were studied in greater detail. We show that ADAMTS-5 and a matrilin-4 neoepitope, revealed upon ADAMTS cleavage, colocalize in prehypertrophic/hypertrophic chondrocytes while they are not detected in proliferating chondrocytes of the growth plate. ADAMTS-5 and the cleaved matrilin-4 are preferentially detected in vesicles derived from the Golgi apparatus. The matrilin-4 neoepitope was not observed in the growth plate of ADAMTS-5 deficient mice. We propose that in the growth plate ADAMTS-5, and not ADAMTS-4, has a physiological function in the intracellular processing of matrilins and potentially of other extracellular matrix proteins.     

Brain size predicts behavioural plasticity in guppies (Poecilia reticulata): An experiment

Understanding how animal personality (consistent between‐individual behavioural differences) arises has become a central topic in behavioural sciences. This endeavour is complicated by the fact that not only the mean behaviour of individuals (behavioural type) but also the strength of their reaction to environmental change (behavioural plasticity) varies consistently. Personality and cognitive abilities are linked, and we suggest that behavioural plasticity could also be explained by differences in brain size (a proxy for cognitive abilities), since accurate decisions are likely essential to make behavioural plasticity beneficial. We test this idea in guppies (Poecilia reticulata), artificially selected for large and small brain size, which show clear cognitive differences between selection lines. To test whether those lines differed in behavioural plasticity, we reared them in groups in structurally enriched environments and then placed adults individually into empty tanks, where we presented them daily with visual predator cues and monitored their behaviour for 20 days with video‐aided motion tracking. We found that individuals differed consistently in activity and risk‐taking, as well as in behavioural plasticity. In activity, only the large‐brained lines demonstrated habituation (increased activity) to the new environment, whereas in risk‐taking, we found sensitization (decreased risk‐taking) in both brain size lines. We conclude that brain size, potentially via increasing cognitive abilities, may increase behavioural plasticity, which in turn can improve habituation to novel environments. However, the effects seem to be behaviour‐specific. Our results suggest that brain size likely explains some of the variation in behavioural plasticity found at the intraspecific level.     

Marginal Zone Macrophage Receptor MARCO Is Trapped in Conduits Formed by Follicular Dendritic Cells in the Spleen

The marginal zone (MZ) region of the spleen plays an important role in leukocyte traffic and the removal of blood-borne pathogens by resident macrophages. Macrophage receptor with a collagenous structure (MARCO), expressed by MZ macrophages, recognizes several microbial ligands and is also involved in the retention of MZ B cells. Here, we report that MARCO is also associated with follicular dendritic cells (FDCs) in the spleen. In its FDC-associated form MARCO is arranged in 0.3–0.5-μm diameter granular-fibrillar structures with an appearance similar to the white pulp conduit system formed by fibroblastic reticular cells (FRCs), but with different compartment preference. The follicular display of MARCO resists irradiation and requires the presence of both MZ macrophages and differentiated FDCs. The follicular delivery of MARCO is independent from the shuffling of marginal zone B cells, and it persists after clodronate liposome-mediated depletion of MZ macrophages. Our findings thus indicate that MARCO is distributed to both MZ and follicles within the spleen into conduit-like structures, where FDC-bound MARCO may mediate communication between the stromal microenvironments of MZ and follicles.     

Stabilization by a disulfide bond of the N-terminal domain of a mutant troponin C (TnC48/82)

The regulatory activity of troponin C is reversibly inhibited by a disulfide bridge between cysteine residues introduced by site-directed mutagenesis in positions 48 and 82 (TnC48/82) in the N-terminal domain of rabbit skeletal troponin C (sTnC; Grabarek, Z., Tan, R.-Y., Tao, T., and Gergely, J. (1990) Nature 345, 132-135). In the present work we have investigated the effects of the disulfide on structural properties of TnC48/82 monitored by CD spectroscopy and limited trypsinolysis. The CD spectra of the mutant protein in the oxidized form (oxTnC48/82) with and without Ca2+ are similar to the corresponding ones of the reduced and carboxamidomethylated form (CAMTnC48/82), indicating that the disulfide has essentially no effect on the overall secondary structure. The N-terminal domain of oxTnC48/82 is resistant to thermal unfolding, but that of CAMTnC48/82 is only slightly more stable than the corresponding domain of sTnC. In the presence of Ca2+ oxTnC48/82 is more resistant to trypsinolysis than sTnC whereas the rate of tryptic digestion of CAMTnC48/82 is the same as that of sTnC, indicating that peptide bonds adjacent to lysine residues at position 84 and 88, the sites of tryptic attack, are protected by the disulfide. The disulfide cross-linked N-terminal peptide of TnC48/82 does not bind TnI, unlike its reduced or carboxamidomethylated forms. Our data indicate that the disulfide between Cys48 and Cys82 stabilizes the structure of the N-terminal domain of TnC and blocks its ability to interact with TnI. The effects of the disulfide appear to be restricted to the N-terminal domain of TnC.     

Heavy metal cations permeate the TRPV6 epithelial cation channel

TRPV6 belongs to the vanilloid family of the transient receptor potential channel (TRP) superfamily. This calcium-selective channel is highly expressed in the duodenum and the placenta, being responsible for calcium absorption in the body and fetus. Previous observations have suggested that TRPV6 is not only permeable to calcium but also to other divalent cations in epithelial tissues. In this study, we tested whether TRPV6 is indeed also permeable to cations such as zinc and cadmium. We found that the basal intracellular calcium concentration was higher in HEK293 cells transfected with hTRPV6 than in non-transfected cells, and that this difference almost disappeared in nominally calcium-free solution. Live cell imaging experiments with Fura-2 and NewPort Green DCF showed that overexpression of human TRPV6 increased the permeability for Ca(2+), Ba(2+), Sr(2+), Mn(2+), Zn(2+), Cd(2+), and interestingly also for La(3+) and Gd(3+). These results were confirmed using the patch clamp technique. (45)Ca uptake experiments showed that cadmium, lanthanum and gadolinium were also highly efficient inhibitors of TRPV6-mediated calcium influx at higher micromolar concentrations. Our results suggest that TRPV6 is not only involved in calcium transport but also in the transport of other divalent cations, including heavy metal ions, which may have toxicological implications.     

Blood Parasite Infection Intensity Covaries with Risk‐Taking Personality in Male Carpetan Rock Lizards (Iberolacerta cyreni)

Identifying evolutionary and developmental mechanisms underlying consistent between‐individual differences in behaviour is the main goal in ‘animal personality studies’. Here, we explored whether activity and risk‐taking varied consistently between individuals and correlated to various – potentially fitness linked – male traits in Carpetan rock lizards (Iberolacerta cyreni). Lizards showed significant consistency within both behaviours, implying the presence of activity and risk‐taking personalities. However, there were no correlation between activity and risk‐taking, neither on the between‐ nor on the within‐individual levels, implying the absence of a behavioural syndrome. We found a strong link between the intensity of blood parasite (Haemogregarinidae) infection and risk‐taking: lizards with higher infection intensity took more risk. While we cannot distinguish cause from causative in the parasite intensity – risk‐taking correlation – our results are in line with the asset protection hypothesis predicting that individuals with lower future reproductive value should focus on the current reproductive event and take higher risk.     

Location of SH-1 and SH-2 in the heavy chain segment of heavy meromyosin.

The two essential thiol groups of myosin, SH-1 and SH-2, have been localized in an ~ 20K segment of the heavy chain by analysis of the distribution of radioactivity after tryptic digestion of tryptic heavy meromyosin (HMM) or papain-HMM subfragment-1, both labeled at SH-1 and SH-2 with [¹⁴C]iodoacetamide and [¹⁴C]N-ethyl maleimide, respectively. The results are discussed in the framework of earlier work (Bálint, M., Sréter, F. A., Wolf, I., Nagy, B., and Gergely, J. (1975) J. Biol. Chem. 250, 6168–6177) on the tryptic fragmentation of myosin heavy chain and in the light of more recent work on the location of a fragment that reacts with a photoaffinity analog of ATP (Szilágyi, L., Bálint, M., Sréter, F. A., and Gergely, J. (1978) Fed. Proc. 37, 1695) and of suggestions concerning the binding of ATP in the region containing the SH-1 and SH-2 (Elzinga, M., and Collins, J. H. (1977) Proc. Nat. Acad. Sci. USA74, 4281–4284).     

Effect of cross-reinnervation on physiological parameters and on properties of myosin and sarcoplasmic reticulum of fast and slow muscles of the rabbit

Cross-reinnvervation of fast (extensor digitorum longus) and slow (soleus) twitch muscles of the rabbit showed essentially complete fast to slow and slow to fast conversion, respectively, 11-12 mo after surgery with respect to a number of physiological parameters including intrinsic shortening, velocity, and isometric twitch time to peak. There was pronounced bu incomplete biochemical conversion as judged by Ca2+ uptake by sarcoplasmic reticulum, myosin ATPase, alkali lability, and light chain complement. The question of trophic substances of neural origin is discussed in light of the fact that chronic stimulation for 15 wk of a fast muscle produces complete biochemical and physiological conversion to the slow type.     

Thymic Atrophy and Apoptosis of CD4+CD8+ Thymocytes in the Cuprizone Model of Multiple Sclerosis

Previous studies on the degenerative animal model of multiple sclerosis suggested that the copper-chelator cuprizone might directly suppress T-cell functions. Peripheral T-cell function in the cuprizone model has already been explored; therefore, in the present study, we investigated, for the first time, how cuprizone feeding affects the thymus, the organ of T-cell maturation and selection. We found that even one week of cuprizone treatment induced significant thymic atrophy, affecting the cortex over the medulla. Fluorescent microscopy and flow-cytometric analyses of thymi from cuprizone- and vehicle-treated mice indicated that eradication of the cluster of the differentiation-4 (CD4)-CD8 double-positive T-cell subset was behind the substantial cell loss. This result was confirmed with CD3-CD4-CD8 triple-staining experiments. Ultrastructurally, we observed degraded as well as enlarged mitochondria, myelin-bodies, large lipid droplets, and large lysosomes in the thymi of cuprizone-treated mice. Some of these features were similar to those in physiological and steroid-induced accelerated aging. According to our results, apoptosis was mainly of mitochondrial origin mediated by both caspase-3- and apoptosis inducing factor-mediated mechanisms. Additionally, mitogen activated protein kinase activation and increased pro-apoptotic B cell lymphoma-2 family protein expression were the major underlying processes. Our results do not indicate a functional relationship between cuprizone-induced thymus involution and the absence of inflammatory responses or the selective demyelination observed in the cuprizone model. On the other hand, due to the reversible nature of cuprizone’s deleterious effects, the cuprizone model could be valuable in studying thymus regeneration as well as remyelination processes.