Overshooting tipping point thresholds in a changing climate.

Palaeorecords suggest that the climate system has tipping points, where small changes in forcing cause substantial and irreversible alteration to Earth system components called tipping elements. As atmospheric greenhouse gas concentrations continue to rise as a result of fossil fuel burning, human activity could also trigger tipping, and the impacts would be difficult to adapt to. Previous studies report low global warming thresholds above pre-industrial conditions for key tipping elements such as ice-sheet melt. If so, high contemporary rates of warming imply that exceeding these thresholds is almost inevitable, which is widely assumed to mean that we are now committed to suffering these tipping events. Here we show that this assumption may be flawed, especially for slow-onset tipping elements (such as the collapse of the Atlantic Meridional Overturning Circulation) in our rapidly changing climate. Recently developed theory indicates that a threshold may be temporarily exceeded without prompting a change of system state, if the overshoot time is short compared to the effective timescale of the tipping element. To demonstrate this, we consider transparently simple models of tipping elements with prescribed thresholds, driven by global warming trajectories that peak before returning to stabilize at a global warming level of 1.5 degrees Celsius above the pre-industrial level. These results highlight the importance of accounting for timescales when assessing risks associated with overshooting tipping point thresholds.

Abrupt changes in Great Britain vegetation carbon projected under climate change.

Past abrupt 'regime shifts' have been observed in a range of ecosystems due to various forcing factors. Large-scale abrupt shifts are projected for some terrestrial ecosystems under climate change, particularly in tropical and high-latitude regions. However, there is very little high-resolution modelling of smaller-scale future projected abrupt shifts in ecosystems, and relatively less focus on the potential for abrupt shifts in temperate terrestrial ecosystems. Here, we show that numerous climate-driven abrupt shifts in vegetation carbon are projected in a high-resolution model of Great Britain's land surface driven by two different climate change scenarios. In each scenario, the effects of climate and CO2 combined are isolated from the effects of climate change alone. We use a new algorithm to detect and classify abrupt shifts in model time series, assessing the sign and strength of the non-linear responses. The abrupt ecosystem changes projected are non-linear responses to climate change, not simply driven by abrupt shifts in climate. Depending on the scenario, 374-1,144 grid cells of 1.5 km × 1.5 km each, comprising 0.5%-1.5% of Great Britain's land area show abrupt shifts in vegetation carbon. We find that abrupt ecosystem shifts associated with increases (rather than decreases) in vegetation carbon, show the greatest potential for early warning signals (rising autocorrelation and variance beforehand). In one scenario, 89% of abrupt increases in vegetation carbon show increasing autocorrelation and variance beforehand. Across the scenarios, 81% of abrupt increases in vegetation carbon have increasing autocorrelation and 74% increasing variance beforehand, whereas for decreases in vegetation carbon these figures are 56% and 47% respectively. Our results should not be taken as specific spatial or temporal predictions of abrupt ecosystem change. However, they serve to illustrate that numerous abrupt shifts in temperate terrestrial ecosystems could occur in a changing climate, with some early warning signals detectable beforehand.

Exacerbation action plans for patients with COPD and comorbidities: a randomised controlled trial.

This international randomised controlled trial evaluated whether COPD patients with comorbidities, trained in using patient-tailored multidisease exacerbation action plans, had fewer COPD exacerbation days than usual care (UC).COPD patients (Global Initiative for Chronic Obstructive Lung Disease (GOLD) classification II-IV) with ≥1 comorbidity (ischaemic heart disease, heart failure, diabetes, anxiety, depression) were randomised to a patient-tailored self-management intervention (n=102) or UC (n=99). Daily symptom diaries were completed for 12 months. The primary outcome "COPD exacerbation days per patient per year" was assessed using intention-to-treat analyses.No significant difference was observed in the number of COPD exacerbation days per patient per year (self-management: median 9.6 (interquartile range (IQR) 0.7-31.1); UC: median 15.6 (IQR 3.0-40.3); incidence rate ratio (IRR) 0.87 (95% CI 0.54; 1.39); p=0.546). There was a significantly shorter duration per COPD exacerbation for self-management (self-management: median 8.1 (IQR 4.8-10.1) days; UC: median 9.5 (IQR 7.0-15.1) days; p=0.021), with no between-group differences in the total number of respiratory hospitalisations (IRR 0.76 (95% CI 0.42; 1.35); p=0.348), but a lower probability of ≥1 for respiratory-related hospitalisation compared to UC (relative risk 0.55 (95% CI 0.35; 0.87); p=0.008). No between-group differences were observed in all-cause hospitalisations (IRR 1.07 (95% CI 0.66; 1.72)) or mortality (self-management: n=4 (3.9%); UC: n=7 (7.1%); relative risk 0.55 (95% CI 0.17; 1.84)).Patient-tailored exacerbation action plans for COPD patients with comorbidities did not significantly reduce exacerbation days, but reduced the duration per COPD exacerbation and the risk of having at least one respiratory-related hospitalisation during follow-up, without excess all-cause mortality.

Quantitative STIR of muscle for monitoring nerve regeneration.

PURPOSE: To assess whether short tau inversion recovery (STIR) MRI sequences can provide a tool for monitoring peripheral nerve regeneration, by comparing signal intensity changes in reinnervated muscle over time, and to determine potential clinical time points for monitoring. MATERIALS AND METHODS: For this prospective study, 29 patients with complete traumatic transection of the ulnar or median nerves in the forearm were followed up to 45 months postsurgery. Standardized 1.5 Tesla STIR-MRI scans of hand muscles were obtained at fixed time intervals. Muscle signal intensities were measured semi-quantitatively and correlated to functional outcome. RESULTS: For the patients with good function recovery, mean signal intensity ratios of 1.179 ± 0.039, 1.304 ± 0.180, 1.154 ± 0.121, 1.105 ± 0.046 and 1.038 ± 0.047 were found at 1-, 3-, 6-, 9-, and 12-month follow-up, respectively. In the group with poor function recovery, ratios of 1.240 ± 0.069, 1.374 ± 0.144, 1.407 ± 0.127, 1.386 ± 0.128 and 1.316 ± 0.116 were found. Comparing the groups showed significant differences from 6 months onward (P < 0.001), with normalizing signal intensities in the group with good function recovery and sustained elevated signal intensity in the group with poor function recovery. CONCLUSION: MRI of muscle can be used as a tool for monitoring motor nerve regeneration, by comparing STIR muscle signal intensities over time. A decrease in signal intensity ratio of 50% (as compared to the initial increase) seems to predict good function recovery. Long-term follow-up shows that STIR MRI can be used for at least 15 months after nerve transection to differentiate between denervated and (re)innervated muscles. J. Magn. Reson. Imaging 2016;44:401-410.

Self management for patients with chronic obstructive pulmonary disease.

BACKGROUND: Self management interventions help patients with chronic obstructive pulmonary disease (COPD) acquire and practise the skills they need to carry out disease-specific medical regimens, guide changes in health behaviour and provide emotional support to enable patients to control their disease. Since the first update of this review in 2007, several studies have been published. The results of the second update are reported here. OBJECTIVES: 1. To evaluate whether self management interventions in COPD lead to improved health outcomes.2. To evaluate whether self management interventions in COPD lead to reduced healthcare utilisation. SEARCH METHODS: We searched the Cochrane Airways Group Specialised Register of trials (current to August 2011). SELECTION CRITERIA: Controlled trials (randomised and non-randomised) published after 1994, assessing the efficacy of self management interventions for individuals with COPD, were included. Interventions with fewer than two contact moments between study participants and healthcare providers were excluded. DATA COLLECTION AND ANALYSIS: Two review authors independently assessed trial quality and extracted data. Investigators were contacted to ask for additional information. When appropriate, study results were pooled using a random-effects model. The primary outcomes of the review were health-related quality of life (HRQoL) and number of hospital admissions. MAIN RESULTS: Twenty-nine studies were included. Twenty-three studies on 3189 participants compared self management versus usual care; six studies on 499 participants compared different components of self management on a head-to-head basis. Although we included non-randomised controlled clinical trials as well as RCTs in this review, we restricted the primary analysis to RCTs only and reported these trials in the abstract.In the 23 studies with a usual care control group, follow-up time ranged from two to 24 months. The content of the interventions was diverse. A statistically relevant effect of self management on HRQoL was found (St George's Respiratory Questionnaire (SGRQ) total score, mean difference (MD) -3.51, 95% confidence interval (CI) -5.37 to -1.65, 10 studies, 1413 participants, moderate-quality evidence). Self management also led to a lower probability of respiratory-related hospitalisation (odds ratio (OR) 0.57, 95% CI 0.43 to 0.75, nine studies, 1749 participants, moderate-quality evidence). Over one year of follow-up, eight (95% CI 5 to 14) participants with a high baseline risk of respiratory-related hospital admission needed to be treated to prevent one participant with at least one hospital admission, and 20 (95% CI 15 to 35) participants with a low baseline risk of hospitalisation needed to be treated to prevent one participant with at least one respiratory-related hospital admission.No statistically significant effect of self management on all-cause hospitalisation (OR 0.77, 95% CI 0.45 to 1.30, 6 studies, 1365 participants, low-quality evidence) or mortality (OR 0.79, 95% CI 0.58 to 1.07, 8 studies, 2134 participants, very low-quality evidence) was detected. Also, dyspnoea measured by the (modified) Medical Research Council Scale ((m)MRC) was reduced in individuals who participated in self management (MD -0.83, 95% CI -1.36 to -0.30, 3 studies, 119 participants, low-quality evidence). The difference in exercise capacity as measured by the six-minute walking test was not statistically significant (MD 33.69 m, 95% CI -9.12 to 76.50, 6 studies, 570 participants, very low-quality evidence). Subgroup analyses depending on the use of an exercise programme as part of the intervention revealed no statistically significant differences between studies with and without exercise programmes in our primary outcomes of HRQoL and respiratory-related hospital admissions.We were unable to pool head-to-head trials because of heterogeneity among interventions and controls; thus results are presented narratively within the review. AUTHORS' CONCLUSIONS: Self management interventions in patients with COPD are associated with improved health-related quality of life as measured by the SGRQ, a reduction in respiratory-related hospital admissions, and improvement in dyspnoea as measured by the (m)MRC. No statistically significant differences were found in other outcome parameters. However, heterogeneity among interventions, study populations, follow-up time and outcome measures makes it difficult to formulate clear recommendations regarding the most effective form and content of self management in COPD.

A case of pityriasis rubra pilaris associated with membranous nephropathy.

Pityriasis rubra pilaris (PRP) is a rare idiopathic dermatosis which may be associated with autoimmune diseases, HIV infection, and internal malignancies. Its association with renal diseases is, however, much less recognized. We report a case of PRP with associated membranous nephropathy (MN), which resolved spontaneously with resolution of the dermatosis. This is only the second reported association between PRP and MN of which we are aware. Further reports of such an association will strengthen the evidence for the two conditions being linked and may thereby shed light on the pathogenesis of both PRP and MN.

Rheological Behavior and Printability Study of Tri-Calcium Phosphate Ceramic Inks for Direct Ink Writing Method.

In the biomedical industry, tricalcium phosphate is a bioceramic substance that is frequently employed in the fabrication of scaffolds and bone structures. Fabrication of porous ceramic structures using conventional manufacturing techniques is very challenging because of the brittle nature of the ceramics, which has led to a newly adapted direct ink writing additive manufacturing method. This work investigates the rheology and extrudability of TCP inks to produce near-net-shape structures. Viscosity and extrudability tests found that stable TCP: Pluronic ink of 50 vol.% was more reliable compared to other tested inks prepared from a functional polymer group polyvinyl alcohol. A line study was carried out to identify the printing parameters suitable for printing structures from the selected ink with lesser dimensional error. Printing speed 5 mm/s and extrusion pressure 3 bar was found suitable to print a scaffold through a nozzle of 0.6 mm, keeping the stand-off distance equal to the nozzle diameter. The printed scaffold was further investigated for its physical and morphological structure of the green body. A suitable drying behavior was studied to remove the green body without cracking and wrapping before the sintering of the scaffold.

Exploring Patterns of COPD Exacerbations and Comorbid Flare-Ups.

Background: Comorbidities are known to complicate disease management in patients with Chronic Obstructive Pulmonary Disease (COPD). This is partly due to lack of insight into the interplay of acute exacerbations of COPD (AECOPD) and comorbid flare-ups. This study aimed to explore patterns of AECOPDs and comorbid flare-ups. Methods: Data of increased symptoms were extracted from a 12-month daily symptom follow-up database including patients with COPD and comorbidities (chronic heart failure (CHF), anxiety, depression) and transformed to visualizations of AECOPDs and comorbid flare-up patterns over time. Patterns were subsequently categorized using an inductive approach, based on both predominance (ie, which occurs most often) of AECOPDs or comorbid flare-ups, and their simultaneous (ie, simultaneous start in ≥ 50%) occurrence. Results: We included 48 COPD patients (68 ± 9 years; comorbid CHF: 52%, anxiety: 40%, depression: 38%). In 25 patients with AECOPDs and CHF flare-ups, the following patterns were identified: AECOPDs predominant (n = 14), CHF flare-ups predominant (n = 5), AECOPDs nor CHF flare-ups predominant (n = 6). Of the 24 patients with AECOPDs and anxiety and/or depression flare-ups, anxiety and depression flare-ups occurred simultaneously in 15 patients. In 9 of these 24 patients, anxiety or depression flare-ups were observed independently from each other. In 31 of the included 48 patients, AECOPDs and comorbid flare-ups occurred mostly simultaneously. Conclusion: Patients with COPD and common comorbidities show a variety of patterns of AECOPDs and comorbid flare-ups. Some patients, however, show repetitive patterns that could potentially be used to improve personalized disease management, if recognized.

Sequential MR imaging of denervated and reinnervated skeletal muscle as correlated to functional outcome.

PURPOSE: To prospectively assess the short inversion time inversion-recovery (STIR) magnetic resonance (MR) signal intensity changes of denervated and reinnervated skeletal muscle over time in clinical patients. MATERIALS AND METHODS: This study was approved by the institutional review board, and informed consent was obtained from all patients. Twenty-three patients with complete traumatic transection of the median or ulnar nerve in the forearm were prospectively followed for 12 months after surgical nerve repair. STIR MR images of selected intrinsic hand muscles were obtained 1, 3, 6, 9, and 12 months after nerve repair, and signal intensities of denervated and reinnervated muscles were measured semiquantitatively. After 12 months, hand function was assessed. Signal intensity ratios were correlated to functional outcome with analysis of variance. RESULTS: Of the 23 patients, 10 had good function recovery, while 13 had poor recovery. For the group with good function recovery, mean signal intensity ratios of 1.267 ± 0.060 (standard deviation), 1.357 ± 0.116, 1.297 ± 0.111, 1.205 ± 0.096, and 1.086 ± 0.104 were found at 1-, 3-, 6-, 9-, and 12-month follow-up, respectively. In the group with poor recovery, mean signal intensity ratios of 1.299 ± 0.056, 1.377 ± 0.094, 1.419 ± 0.117, 1.398 ± 0.111, and 1.342 ± 0.095 were found at 1-, 3-, 6-, 9-, and 12-month follow-up, respectively. Comparison of the group with poor function recovery and the group with good function recovery showed significant differences at 6-, 9-, and 12-month follow-up (P = .035, P = .001, and P < .001, respectively), with normalizing signal intensities in the group with good function recovery and sustained high signal intensity in the group with poor function recovery. CONCLUSION: STIR MR imaging can be used to differentiate between denervated and reinnervated muscles for at least 12 months after nerve transection.

New functions for gap junctions.

The most significant finding of the past year in gap junction research has been the association of connexin defects with human diseases. Connexin32 mutations cause X-linked Charcot-Marie-Tooth disease, a demyelinating peripheral neuropathy. Mutations in connexin43 may underlie cardiac malformations in visceroatrial heterotaxia syndromes. Genetic approaches and gene targeting have provided new insights, but also raise new questions concerning connexin function, the significance of connexin diversity and the regulation of intercellular communication.

Molecular cloning of cDNA for rat liver gap junction protein.

An affinity-purified antibody directed against the 27-kD protein associated with isolated rat liver gap junctions was produced. Light and electron microscopic immunocytochemistry showed that this antigen was localized specifically to the cytoplasmic surfaces of gap junctions. The antibody was used to select cDNA from a rat liver library in the expression vector lambda gt11. The largest cDNA selected contained 1,494 bp and coded for a protein with a calculated molecular mass of 32,007 daltons. Northern blot analysis indicated that brain, kidney, and stomach express an mRNA with similar size and homology to that expressed in liver, but that heart and lens express differently sized, less homologous mRNA.

Preparation, characterization, and localization of antisera against bovine MP26, an integral protein from lens fiber plasma membrane.

Polyclonal antisera were prepared in rabbits using both native and chymotrypsin-digested bovine lens fiber plasma membranes. MP26, the principal protein of lens fiber plasma membranes, and CT20, a chymotryptic fragment of MP26, were isolated electrophoretically and used to purify anti-MP26 and anti-CT20 activity from the respective antisera by affinity chromatography. These affinity-purified antisera were characterized by immunoreplica. Immunofluorescence microscopy localized MP26 on sections of methacrylate-embedded lenses in the lens fiber plasma membranes, but not the lens epithelium. Immunocytochemistry of isolated native or chymotrypsin-digested lens fiber plasma membranes localized both the MP26 and the CT20 only in the nonjunctional plasma membranes, with no detectable activity in the lens fiber junctions themselves. Electron microscopy revealed a second set of pentalaminar profiles, thinner by 4 nm than the lens fiber junctions, which contained demonstrable anti-MP26 and anti-CT20 activity following immunocytochemistry. These results indicate either that MP26 is not a component of the lens fiber junctions, or that significant conformational changes accompany assembly of MP26 into lens fiber junctions, resulting in the masking of MP26 antigenic determinants.

Topological distribution of two connexin32 antigenic sites in intact and split rodent hepatocyte gap junctions.

The membrane topology of connexin32, a principal polypeptide of gap junctions in diverse cell types, has been studied in rat and mouse hepatocyte gap junctions using site-specific antisera raised against synthetic oligopeptides corresponding to amino acid sequences deduced from cDNA clones. Based on published hydropathicity maps and identified protease-sensitive cleavage sites, oligopeptides were synthesized corresponding to two hydrophilic domains of connexin32, one predicted to face the cytoplasm, the other predicted to be directed extracellularly. Antisera were raised to keyhole limpet hemocyanin conjugates of the oligopeptides and used to map the distribution of their antigens using indirect immunocytochemistry on isolated gap junctions. The results directly demonstrated the cytoplasmic orientation of an antigen contained within amino acids 98-124 of the connexin32 sequence. The extracellular space in intact, isolated gap junctions is too small to permit binding of antibody molecules, necessitating the experimental separation of the junctional membranes to expose their extracellular surfaces using a urea/alkali procedure. While an antigen contained within amino acids 164-189 was visualized on the extracellular surfaces of some of the separated junctional membranes, variability in the observations and in the splitting procedure left ambiguities concerning the biological relevance of the observations after the denaturing conditions necessary to separate the junctional membranes. Using a different approach, however, the antigen could be exposed in intact liver using a hypertonic disaccharide junction-splitting procedure. The period of time of antigen exposure at the cell surface appears to peak at 30 s and disappear by 2-4 min. Taken together, these data demonstrate the extracellular orientation of an antigen contained within amino acids 164-189, which may be involved in cell-cell interaction within the gap junction.

Gap junctional communication in the early Xenopus embryo.

In the Xenopus embryo, blastomeres are joined by gap junctions that allow the movement of small molecules between neighboring cells. Previous studies using Lucifer yellow (LY) have reported asymmetries in the patterns of junctional communication suggesting involvement in dorso-ventral patterning. To explore that relationship, we systematically compared the transfer of LY and neurobiotin in embryos containing 16-128 cells. In all cases, the junction-permeable tracer was coinjected with a fluorescent dextran that cannot pass through gap junctions. Surprisingly, while LY appeared to transfer in whole-mount embryos, in no case did we observe junctional transfer of LY in fixed and sectioned embryos. The lack of correspondence between data obtained from whole-mounts and from sections results from two synergistic effects. First, uninjected blastomeres in whole-mounts reflect and scatter light originating from the intensely fluorescent injected cell, creating a diffuse background interpretable as dye transfer. Second, the heavier pigmentation in ventral blastomeres masks this scattered signal, giving the impression of an asymmetry in communication. Thus, inspection of whole-mount embryos is an unreliable method for the assessment of dye transfer between embryonic blastomeres. A rigorous and unambiguous demonstration of gap junctional intercellular communication demands both the coinjection of permeant and impermeant tracers followed by the examination of sectioned specimens. Whereas LY transfer was never observed, neurobiotin was consistently transferred in both ventral and dorsal aspects of the embryo, with no apparent asymmetry. Ventralization of embryos by UV irradiation and dorsalization by Xwnt-8 did not alter the patterns of communication. Thus, our results are not compatible with current models for a role of gap junctional communication in dorso-ventral patterning.

In vitro synthesis and membrane insertion of bovine MP26, an integral protein from lens fiber plasma membrane.

Synthesis of MP26, the principal protein of lens fiber plasma membranes, was directed in the reticulocyte lysate system by poly A mRNA enriched from whole bovine lens RNA using oligo (dt)-cellulose chromatography. Synthesized MP26 was enriched by immune precipitation. The in vitro-synthesized MP26 had an electrophoretic mobility indistinguishable from that of the native molecule. MP26 showed a cotranslational requirement for dog pancreas microsomes in order for membrane association to occur. Microsome-associated in vitro-synthesized MP26 showed a sensitivity to digestion with chymotrypsin which was similar to the sensitivity of native MP26 in isolated lens fiber plasma membranes, indicating correct insertion of the MP26 into the microsome. Synthesis and membrane insertion of MP26 using N-formyl-[35S]methionyl tRNA as label demonstrated that no proteolytic processing or significant glycosylation accompanied membrane insertion. Chymotryptic cleavage of membrane-inserted, N-formyl-[35S]methionine-labeled MP26 resulted in loss of label, suggesting that the N-terminal of the in vitro-synthesized MP26 faces the cytoplasm.

Connexin43: a protein from rat heart homologous to a gap junction protein from liver.

Northern blot analysis of rat heart mRNA probed with a cDNA coding for the principal polypeptide of rat liver gap junctions demonstrated a 3.0-kb band. This band was observed only after hybridization and washing using low stringency conditions; high stringency conditions abolished the hybridization. A rat heart cDNA library was screened with the same cDNA probe under the permissive hybridization conditions, and a single positive clone identified and purified. The clone contained a 220-bp insert, which showed 55% homology to the original cDNA probe near the 5' end. The 220-bp cDNA was used to rescreen a heart cDNA library under high stringency conditions, and three additional cDNAs that together spanned 2,768 bp were isolated. This composite cDNA contained a single 1,146-bp open reading frame coding for a predicted polypeptide of 382 amino acids with a molecular mass of 43,036 D. Northern analysis of various rat tissues using this heart cDNA as probe showed hybridization to 3.0-kb bands in RNA isolated from heart, ovary, uterus, kidney, and lens epithelium. Comparisons of the predicted amino acid sequences for the two gap junction proteins isolated from heart and liver showed two regions of high homology (58 and 42%), and other regions of little or no homology. A model is presented which indicates that the conserved sequences correspond to transmembrane and extracellular regions of the junctional molecules, while the nonconserved sequences correspond to cytoplasmic regions. Since it has been shown previously that the original cDNA isolated from liver recognizes mRNAs in stomach, kidney, and brain, and it is shown here that the cDNA isolated from heart recognizes mRNAs in ovary, uterus, lens epithelium, and kidney, a nomenclature is proposed which avoids categorization by organ of origin. In this nomenclature, the homologous proteins in gap junctions would be called connexins, each distinguished by its predicted molecular mass in kilodaltons. The gap junction protein isolated from liver would then be called connexin32; from heart, connexin43.

Targeted ablation of connexin50 in mice results in microphthalmia and zonular pulverulent cataracts.

In the ocular lens, gap junctional communication is a key component of homeostatic mechanisms preventing cataract formation. Gap junctions in rodent lens fibers contain two known intercellular channel-forming proteins, connexin50 (Cx50) and Cx46. Since targeted ablation of Cx46 has been shown to cause senile-type nuclear opacities, it appears that Cx50 alone cannot meet homeostatic requirements. To determine if lens pathology arises from a reduction in levels of communication or the loss of a connexin-specific function, we have generated mice with a targeted deletion of the Cx50 gene. Cx50-null mice exhibited microphthalmia and nuclear cataracts. At postnatal day 14 (P14), Cx50-knockout eyes weighed 32% less than controls, whereas lens mass was reduced by 46%. Cx50-knockout lenses also developed zonular pulverulent cataracts, and lens abnormalities were detected by P7. Deletion of Cx50 did not alter the amounts or distributions of Cx46 or Cx43, a component of lens epithelial junctions. In addition, intercellular passage of tracers revealed the persistence of communication between all cell types in the Cx50-knockout lens. These results demonstrate that Cx50 is required not only for maintenance of lens transparency but also for normal eye growth. Furthermore, these data indicate that unique functional properties of both Cx46 and Cx50 are required for proper lens development.

COOH terminus of occludin is required for tight junction barrier function in early Xenopus embryos.

Occludin is the only known integral membrane protein localized at the points of membrane- membrane interaction of the tight junction. We have used the Xenopus embryo as an assay system to examine: (a) whether the expression of mutant occludin in embryos will disrupt the barrier function of tight junctions, and (b) whether there are signals within the occludin structure that are required for targeting to the sites of junctional interaction. mRNAs transcribed from a series of COOH-terminally truncated occludin mutants were microinjected into the antero-dorsal blastomere of eight-cell embryos. 8 h after injection, the full-length and the five COOH-terminally truncated proteins were all detected at tight junctions as defined by colocalization with both endogenous occludin and zonula occludens-1 demonstrating that exogenous occludin correctly targeted to the tight junction. Importantly, our data show that tight junctions containing four of the COOH-terminally truncated occludin proteins were leaky; the intercellular spaces between the apical cells were penetrated by sulfosuccinimidyl-6-(biotinamido) Hexanoate (NHS-LC-biotin). In contrast, embryos injected with mRNAs coding for the full-length, the least truncated, or the soluble COOH terminus remained impermeable to the NHS-LC-biotin tracer. The leakage induced by the mutant occludins could be rescued by coinjection with full-length occludin mRNA. Immunoprecipitation analysis of detergent-solubilized embryo membranes revealed that the exogenous occludin was bound to endogenous Xenopus occludin in vivo, indicating that occludin oligomerized during tight junction assembly. Our data demonstrate that the COOH terminus of occludin is required for the correct assembly of tight junction barrier function. We also provide evidence for the first time that occludin forms oligomers during the normal process of tight junction assembly. Our data suggest that mutant occludins target to the tight junction by virtue of their ability to oligomerize with full-length endogenous molecules.

Selective interactions among the multiple connexin proteins expressed in the vertebrate lens: the second extracellular domain is a determinant of compatibility between connexins.

Gap junctions are collections of intercellular channels composed of structural proteins called connexins (Cx). We have examined the functional interactions of the three rodent connexins present in the lens, Cx43, Cx46, and Cx50, by expressing them in paired Xenopus oocytes. Homotypic channels containing Cx43, Cx46, or Cx50 all developed high conductance. heterotypic channels composed of Cx46 paired with either Cx43 or Cx50 were also well coupled, whereas Cx50 did not form functional channels with Cx43. We also examined the functional response of homotypic and heterotypic channels to transjunctional voltage and cytoplasmic acidification. We show that all lens connexins exhibited sensitivity to cytoplasmic acidification as well as to voltage, and that voltage-dependent closure of heterotypic channels for a given connexin was dramatically influenced by its partner connexins in the adjacent cell. Based on the observation that Cx43 can discriminate between Cx46 and Cx50, we investigated the molecular determinants that specify compatibility by constructing chimeric connexins from portions of Cx46 and Cx50 and testing them for their ability to form channels with Cx43. When the second extracellular (E2) domain in Cx46 was replaced with the E2 of Cx50, the resulting chimera could no longer form heterotypic channels with Cx43. A reciprocal chimera, where the E2 of Cx46 was inserted into Cx50, acquired the ability to functionally interact with Cx43. Together, these results demonstrate that formation of intercellular channels is a selective process dependent on the identity of the connexins expressed in adjacent cells, and that the second extracellular domain is a determinant of heterotypic compatibility between connexins.