Elucidating the principles of the molecular organization of heteropolymeric tight junction strands

Paracellular barrier properties of tissues are mainly determined by the composition of claudin heteropolymers. To analyze the molecular organization of tight junctions (TJ), we investigated the ability of claudins (Cld) to form homo- and heteromers. Cld1, -2, -3, -5, and -12 expressed in cerebral barriers were investigated. TJ-strands were reconstituted by claudin-transfection of HEK293-cells. cis-Interactions and/or spatial proximity were analyzed by fluorescence resonance energy transfer inside and outside of strands and ranked: Cld5/Cld5?>?Cld5/Cld1?>?Cld3/Cld1?>?Cld3/Cld3?>?Cld3/Cld5, no Cld3/Cld2. Classic Cld1, -3, and -5 but not non-classic Cld12 showed homophilic trans-interaction. Freeze-fracture electron microscopy revealed that, in contrast to classic claudins, YFP-tagged Cld12 does not form homopolymers. Heterophilic trans-interactions were analyzed in cocultures of differently monotransfected cells. trans-Interaction of Cld3/Cld5 was less pronounced than that of Cld3/Cld1, Cld5/Cld1, Cld5/Cld5 or Cld3/Cld3. The barrier function of reconstituted TJ-strands was demonstrated by a novel imaging assay. A model of the molecular organization of TJ was generated.     

A degradation-sensitive anionic trypsinogen (PRSS2) variant protects against chronic pancreatitis

Chronic pancreatitis is a common inflammatory disease of the pancreas. Mutations in the genes encoding cationic trypsinogen (PRSS1) and the pancreatic secretory trypsin inhibitor (SPINK1) are associated with chronic pancreatitis. Because increased proteolytic activity owing to mutated PRSS1 enhances the risk for chronic pancreatitis, mutations in the gene encoding anionic trypsinogen (PRSS2) may also predispose to disease. Here we analyzed PRSS2 in individuals with chronic pancreatitis and controls and found, to our surprise, that a variant of codon 191 (G191R) is overrepresented in control subjects: G191R was present in 220/6,459 (3.4%) controls but in only 32/2,466 (1.3%) affected individuals (odds ratio 0.37; P = 1.1 x 10(-8)). Upon activation by enterokinase or trypsin, purified recombinant G191R protein showed a complete loss of trypsin activity owing to the introduction of a new tryptic cleavage site that renders the enzyme hypersensitive to autocatalytic proteolysis. In conclusion, the G191R variant of PRSS2 mitigates intrapancreatic trypsin activity and thereby protects against chronic pancreatitis.     

Mapping autism risk loci using genetic linkage and chromosomal rearrangements

Autism spectrum disorders (ASDs) are common, heritable neurodevelopmental conditions. The genetic architecture of ASDs is complex, requiring large samples to overcome heterogeneity. Here we broaden coverage and sample size relative to other studies of ASDs by using Affymetrix 10K SNP arrays and 1,181 [corrected] families with at least two affected individuals, performing the largest linkage scan to date while also analyzing copy number variation in these families. Linkage and copy number variation analyses implicate chromosome 11p12-p13 and neurexins, respectively, among other candidate loci. Neurexins team with previously implicated neuroligins for glutamatergic synaptogenesis, highlighting glutamate-related genes as promising candidates for contributing to ASDs.     

Topology of signal recognition particle receptor in endoplasmic reticulum membrane

The signal recognition particle (SRP) receptor is an integral membrane protein of the endoplasmic reticulum which, in conjunction with SRP, ensures the correct targeting of nascent secretory proteins to this membrane system. From the complementary DNA sequence we have deduced the complete primary structure of the SRP receptor and established that its amino-terminal region is anchored in the membrane. The anchor fragment and the cytoplasmic fragment contribute jointly to a functionally important region which is highly charged and may function in nucleic acid binding.     

Vocalization during copulation behavior in greater horseshoe bats, Rhinolophus ferrumequinum

Vocal communication plays an important role for individual recognition and male-female interaction during mating in greater horseshoe bats,especially in respect to mate fidelity,which ensures that the bats can maintain a stable social organization.Few studies,however,have addressed the calling behavior during copulating in bats.Here,we initially report the copulation vocalizations and behaviors of both male and female greater horseshoe bats.During copulation,the male assumed a dorsal position and arched his back,arming around the female using his feet and thumbs.The male repeatedly produced very short constant frequency(SCF) syllables with high intensity and repetition rate(male 1:16.48±4.8 ms,male 2:17.79±4.03 ms) when he tried to insert the penis into the female,and then long syllables(male 1:42.08±12.67 ms,male 2:43.02±11.44 ms) after penile insertion.The female bats sometime refused the male bats in the early phase of copulations as emitting noise bursts and broad-band vocalizations,but kept silence during actual copulation.We also found that the SCF copulation calls of one male remained stable peak frequencies on different copulation days although its echolocation call frequency varied each day.Moreover,different male individuals maintained their own "private frequency" in the SCF copulation calls.Therefore,we predicted that the SCF copulation calls may serve as an indicator for female greater horseshoe bats to recognize the mating males in order to maintain mate fidelity because horseshoe bats exhibit sexual segregation before mating.Our results stipulate further studies on mating system and copulation strategies in polygynous bats.Such work may also aid in promoting the preservation of greater horseshoe bats.     

N2O binding at a [4Cu:2S] copper-sulphur cluster in nitrous oxide reductase

Nitrous oxide (N(2)O) is generated by natural and anthropogenic processes and has a critical role in environmental chemistry. It has an ozone-depleting potential similar to that of hydrochlorofluorocarbons as well as a global warming potential exceeding that of CO(2) 300-fold. In bacterial denitrification, N(2)O is reduced to N(2) by the copper-dependent nitrous oxide reductase (N(2)OR). This enzyme carries the mixed-valent Cu(A) centre and the unique, tetranuclear Cu(Z) site. Previous structural data were obtained with enzyme isolated in the presence of air that is catalytically inactive without prior reduction. Its Cu(Z) site was described as a [4Cu:S] centre, and the substrate-binding mode and reduction mechanism remained elusive. Here we report the structure of purple N(2)OR from Pseudomonas stutzeri, handled under the exclusion of dioxygen, and locate the substrate in N(2)O-pressurized crystals. The active Cu(Z) cluster contains two sulphur atoms, yielding a [4Cu:2S] stoichiometry; and N(2)O bound side-on at Cu(Z), in close proximity to Cu(A). With the substrate located between the two clusters, electrons are transferred directly from Cu(A) to N(2)O, which is activated by side-on binding in a specific binding pocket on the face of the [4Cu:2S] centre. These results reconcile a multitude of available biochemical data on N(2)OR that could not be explained by earlier structures, and outline a mechanistic pathway in which both metal centres and the intervening protein act in concert to achieve catalysis. This structure represents the first direct observation, to our knowledge, of N(2)O bound to its reductase, and sheds light on the functionality of metalloenzymes that activate inert small-molecule substrates. The principle of using distinct clusters for substrate activation and for reduction may be relevant for similar systems, in particular nitrogen-fixing nitrogenase.