Investigating Models of Protein Function and Allostery With a Widespread Mutational Analysis of a Light-Activated Protein

To investigate the relationship between a protein’s sequence and its biophysical properties, we studied the effects of more than 100 mutations in Avena sativa light-oxygen-voltage domain 2, a model protein of the Per-Arnt-Sim family. The A. sativa light–oxygen–voltage domain 2 undergoes a photocycle with a conformational change involving the unfolding of the terminal helices. Whereas selection studies typically search for winners in a large population and fail to characterize many sites, we characterized the biophysical consequences of mutations throughout the protein using NMR, circular dichroism, and ultraviolet/visible spectroscopy. Despite our intention to introduce highly disruptive substitutions, most had modest or no effect on function, and many could even be considered to be more photoactive. Substitutions at evolutionarily conserved sites can have minimal effect, whereas those at nonconserved positions can have large effects, contrary to the view that the effects of mutations, especially at conserved positions, are predictable. Using predictive models, we found that the effects of mutations on biophysical function and allostery reflect a complex mixture of multiple characteristics including location, character, electrostatics, and chemistry.     

A method for culturing chick melanocytes: The effect of BRL-3A cell conditioning and related additives

Summary A method for growing chick embryo melanocytes is described that utilizes medium conditioned by Buffalo Rat liver (BRL-3A) cells. The dissected trunk region of each 72 h (Stages 14 to 19) embryo produces approximately 200,000 melanocytes (purity, 80%) when processed and cultured for 8 d. Thus, a typical experiment involving 20 embryos would produce a total of 4 × 10⁶ melanocytes. Choice of serum, serum concentration, and cell density were determined experimentally. Partially purified multiplication stimulating activity (MSA) from BRL-3A cells and insulin were also tested as medium additives. MSA was not stimulatory, whereas insulin gave a positive response in 2% but not 10 or 0% serum. The final protocol used a modified F12 medium with 10% bovine calf serum conditioned by BRL-3A cells. Cultures were fed every other day. Small colonies of cells became evident by culture Day 3 and increased rapidly to Day 5 when pigmentation became obvious. Colony size continued to increase but more slowly from Days 5 to 8, whereas pigmentation increased rapidly and maximized on Day 8. There is a factor, or factors, present in BRL-3A conditioned medium that stimulates embryonic chick melanocytes to divide preferentially over contaminating cell types. This results in cultures that can provide adequate numbers and purity for biochemical studies. This work was supported by National Institutes of Health grants GM18969 to J. B. and CA17620 to G. S.     

Inferring phylogenetic patterns of land snails of the genus Albinaria on the island of Dia (Crete,Greece)

Albinaria (Gastropoda, Clausiliidae) is a pulmonate genus distributed around the north-eastern coasts of the Mediterranean. It is the most 'speciose' genus within the family of Clausiliidae, exhibiting a high degree of morphological and genetic differentiation, and serving as a model for several ecological, systematics and evolutionary studies. Nevertheless, many aspects remain uncertain mainly due to the large number of taxa whose classification has not yet been evaluated with solid synapomorphic characters. Thirty-one morphological species are currently recognized on the island of Crete and its satellite islets. Four of them (A. retusa, A. torticollis, A. jaeckeli, and A. teres) are distributed on the island of Dia (north of Crete); the first three are island endemics. Here, we combined mitochondrial and nuclear DNA information and Bayesian and Maximum Likelihood approaches to evaluate the phylogenetic relationships, and assess the genetic distinctiveness and cohesiveness of all described species of Dia Island. The produced phylogeny was not congruent with the morphological species, demonstrating a more complex pattern of speciation and diversification. Although each island endemic constitutes a monophyletic lineage, the number of island endemic species could be greater than the three currently recognized species so far (A. retusa, A. torticollis, and A. jaeckeli), since a newly discovered lineage (north-western part of the island), that morphologically differs from the populations of A. torticollis in the eastern part, and genetically is more closely related to A. jaeckeli, could be elevated to the species level. Considering the fourth species found on the island of Dia, A. teres is genetically highly variable, showing low geographic structuring due to either long-distance gene flow or retained ancestral polymorphisms, or a combination of both. Further work (analysis of more specimens and DNA data) both from Dia and Crete is indispensable in order to shed light on aspects of the evolutionary history of the genus in Dia.     

Effect of nitrogen-rich cell culture surfaces on type X collagen expression by bovine growth plate chondrocytes

Background  

Recent evidence indicates that osteoarthritis (OA) may be a systemic disease since mesenchymal stem cells (MSCs) from OA patients express type X collagen, a marker of late stage chondrocyte hypertrophy (associated with endochondral ossification). We recently showed that the expression of type X collagen was suppressed when MSCs from OA patients were cultured on nitrogen (N)-rich plasma polymer layers, which we call "PPE:N" (N-doped plasma-polymerized ethylene, containing up to 36 atomic percentage (at.%) of N.     

The efficacy of Link N as a mediator of repair in a rabbit model of intervertebral disc degeneration

Introduction  

Intervertebral disc (IVD) degeneration is associated with proteolytic degradation of the extracellular matrix, and its repair requires both the production of extracellular matrix and the downregulation of proteinase activity. These properties are associated with several growth factors. However, the use of growth factors in clinical practice is limited by their high cost. This cost can be circumvented using synthetic peptides, such as Link N, which can stimulate the synthesis of proteoglycan and collagen by IVD cells in vitro. The purpose of the present study was to evaluate the effect of Link N in vivo in a rabbit model of IVD degeneration.     

Polymorphisms and haplotypes in MyD88 are associated with the development of sarcoidosis: a candidate-gene association study

Sarcoidosis is considered as a disorder of protracted immune response to an as yet unidentified causative agent that leads to granuloma formation. Material from M. tuberculosis and P. acne has been repeatedly detected in the sarcoidosis lesions, implying the involvement of the Toll-like receptor2 (TLR2) gene that responds to these intracellular pathogens. Since TLR2 association studies have produced controversial results, we sought to investigate whether the downstream signalling molecule MyD88 could be linked to disease susceptibility. We analyzed a total of 93 cases with sarcoidosis and of 89 controls for the most common MyD88 SNPs: ?938C>A (rs4988453) and 1944C>G (rs4988457). There is evidence that the genotype distributions of both variants are associated with the development of sarcoidosis (p = 0.038 for ?938C>A and p = 0.026 for 1944C>G). In particular, ?938A and 1944G carriers were associated with risk of sarcoidosis [OR = 2.48 (1.23–5.02) and OR = 0.33 (0.14–0.76)], respectively, indicating dominance of the mutant alleles; however, the adjustment of the effect size for age and sex diminished the significance. The haplotype analysis showed association for the ?938A/1944G haplotype (p < 0.001). Since genetic association studies have linked MyD88 to Hodgkin’s lymphoma it is tempting to speculate that MyD88 may contribute to the granuloma formation that characterizes sarcoidosis.     

Major histocompatibility complex class I-ERp57-tapasin interactions within the peptide-loading complex

The endoplasmic reticulum-located multimolecular peptide-loading complex functions to load optimal peptides onto major histocompatibility complex (MHC) class I molecules for presentation to CD8(+) T lymphocytes. Two oxidoreductases, ERp57 and protein-disulfide isomerase, are known to be components of the peptide-loading complex. Within the peptide-loading complex ERp57 is normally found disulfide-linked to tapasin, through one of its two thioredoxin-like redox motifs. We describe here a novel trimeric complex that disulfide links together MHC class I heavy chain, ERp57 and tapasin, and that is found in association with the transporter associated with antigen processing peptide transporter. The trimeric complex normally represents a small subset of the total ERp57-tapasin pool but can be significantly increased by altering intracellular oxidizing conditions. Direct mutation of a conserved structural cysteine residue implicates an interaction between ERp57 and the MHC class I peptide-binding groove. Taken together, our studies demonstrate for the first time that ERp57 directly interacts with MHC class I molecules within the peptide-loading complex and suggest that ERp57 and protein-disulfide isomerase act in concert to regulate the redox status of MHC class I during antigen presentation.     

Effects of Anesthetic Ketamine on Anxiety-Like Behaviour in Rats

There is scarce information regarding the effects of anesthetic doses of the non-competitive N-methyl-d-aspartate receptor antagonist ketamine on anxiety. The current study evaluated the acute effects of intraperitoneally (i.p.) administered anesthetic ketamine (100 mg/kg) i.p. on anxiety in rats. For this purpose, the light/dark and the open field tests were utilized. The effects of anesthetic ketamine on motility were also examined using a motility cage. In the light/dark test, anesthetic ketamine, administered 24 h before testing reduced the number of transitions between the light and dark compartments and the time spent in the light compartment in the rats compared with their control cohorts. In addition, ketamine was found to exert a depressive effect on rats’ motility. In the open field test, animals treated with anesthetic ketamine 24 h before testing spent essentially no time in the central area of the apparatus, decreased horizontal ambulatory activity, and preserved to a certain extent their exploratory behaviour compared to their control counterparts. The results suggest that, in spite of its hypokinetic effect, a single anesthetic ketamine administration apparently induces an anxiety-like state, while largely preserving exploratory behaviour in the rat. These effects were time-dependent they since they were extinguished when testing was carried out 48 h after anesthetic ketamine administration.

     

MiR‐134‐dependent regulation of Pumilio‐2 is necessary for homeostatic synaptic depression

Neurons employ a set of homeostatic plasticity mechanisms to counterbalance altered levels of network activity. The molecular mechanisms underlying homeostatic plasticity in response to increased network excitability are still poorly understood. Here, we describe a sequential homeostatic synaptic depression mechanism in primary hippocampal neurons involving miRNA‐dependent translational regulation. This mechanism consists of an initial phase of synapse elimination followed by a reinforcing phase of synaptic downscaling. The activity‐regulated microRNA miR‐134 is necessary for both synapse elimination and the structural rearrangements leading to synaptic downscaling. Results from miR‐134 inhibition further uncover a differential requirement for GluA1/2 subunits for the functional expression of homeostatic synaptic depression. Downregulation of the miR‐134 target Pumilio‐2 in response to chronic activity, which selectively occurs in the synapto‐dendritic compartment, is required for miR‐134‐mediated homeostatic synaptic depression. We further identified polo‐like kinase 2 (Plk2) as a novel target of Pumilio‐2 involved in the control of GluA2 surface expression. In summary, we have described a novel pathway of homeostatic plasticity that stabilizes neuronal circuits in response to increased network activity.