Sleep-wakefulness cycle and behavior in pannexin1 knockout mice.

Pannexins are membrane channel proteins that play a role in a number of critical biological processes (Panchin et al., 2000; Shestopalov, Panchin, 2008). Among other cellular functions, pannexin hemichannels serve as purine nucleoside conduits providing ATP efflux into the extracellular space (Dahl, 2015), where it is rapidly degraded to adenosine. Pannexin1 (Panx1) is abundantly expressed in the brain and has been shown to contribute to adenosine signaling in nervous system tissues (Prochnow et al., 2012). We hypothesized that pannexin1 may contribute to sleep-wake cycle regulation through extracellular adenosine, a well-established paracrine factor in slow wave sleep. To investigate this link, EEG and movement activity throughout the light/dark cycle were compared in Panx1-/- and Panx1+/+ mice. We found a significant increase in waking and a correspondent decrease in slow wave sleep percentages in the Panx1-/- animals. These changes were especially pronounced during the dark period. Furthermore, we found a significant increase in movement activity of Panx1-/- mice. These findings are consistent with the hypothesis that extracellular adenosine is relatively depleted in Panx1-/- animals due to the absence of the ATP-permeable hemichannels. At the same time, sleep rebound after a 6-h sleep deprivation remained unchanged in Panx1-/- mice as compared to the control animals. Behavioral tests revealed that Panx1-/- mice were significantly faster during their descent along the vertical pole but more sluggish during their run through the horizontal pole as compared to the control mice.

Exogenous gene expression and protein targeting in lens fiber cells.

PURPOSE: To test the ability of lens fiber cells at various stages of differentiation to transcribe and translate microinjected DNA templates. METHODS: Expression plasmids encoding green fluorescent protein (GFP) or a GFP-tagged membrane protein (human CD46) were microinjected into organ-cultured embryonic chicken lenses. Protein expression was visualized by confocal microscopy. RESULTS: GFP expression was detected within 12 hours of microinjection, evenly distributed throughout the cytoplasm of the injected cell. All nucleated fiber cells were competent to express GFP, whereas the anucleated central fiber cells were not. When GFP was fused to the C-terminal of CD46, the fusion protein was synthesized intact and properly inserted in the fiber cell plasma membrane. In contrast, N-terminal fusions were cleaved during synthesis, resulting in retention of the GFP tag in the endoplasmic reticulum. CONCLUSIONS: Microinjection of expression plasmids is an effective technique for introducing exogenous genes into individual fiber cells. With this approach, the results show that fiber cells are transcriptionally and translationally competent until the time of organelle loss, and that fiber cells deep within the lens are capable of synthesizing new plasma membrane proteins. The techniques described here should have broad application in studies of fiber cell differentiation and provide a useful complement to conventional transgenic approaches.

Three-dimensional organization of primary lens fiber cells.

PURPOSE: To visualize the three-dimensional organization of primary lens fiber cells. METHODS: The gene for Green Fluorescent Protein (GFP) was introduced into the lens vesicle using two different vector systems: a replication deficient adenovirus or an expression plasmid. Injected embryos were allowed to develop for several days and then were examined by confocal microscopy. RESULTS: Injection of either vector resulted in GFP expression in primary fiber cells. GFP-expressing cells were heterogeneous in shape and length. Some regions of the fibers were varicose, with diameters >10 microm; regions between the varicosities were often extremely thin, with diameters of <2 microm. No differences in the morphologies of GFP-expressing cells were noted between adenovirus and plasmid-injected lenses, suggesting that the irregular, undulating, appearance of the primary fibers was not the result of viral infection. Three-dimensional reconstruction of primary fiber cells revealed that, by E6, the posterior tips of the fibers had detached from the lens capsule. The anterior fiber tips remained in contact with the overlying epithelium for 1 to 2 additional days, demonstrating that the formation of the anterior and posterior sutures was asynchronous. CONCLUSIONS: The three-dimensional cellular organization of GFP-expressing cells is consistent with previous analyses of fiber cell morphology in the embryonic nucleus of adult human and bovine lenses. The present data confirm that the disorganized appearance of primary fiber cells observed in adult lenses is largely a reflection of developmental processes rather than a consequence of aging.

[Construction of the genomic library for cyanobacteria Synechocystis sp. PCC 6803 using a cosmid]. / Konstruirovanie kosmidnoi biblioteki genov tsianobakterii Synechocystis sp. PCC 6803.

The gene library of the phototrophic cyanobacterium Synechocystis PCC6803 was constructed in the cosmid vector Lorist6. About -3 x 10(3) recombinant clones were obtained, of which 760 were selected for further work. Each clone was kept in an individual well of a microtiter plate. The clone grids obtained were hybridized with two DNA probes, which contained the ndhE and dfr genes of Synechocystis PCC6803. Two groups of probe-overlapping cosmids (11 and 8 clones respectively), containing insertions averaging of 39 kb, were isolated. The capacity of the gene library is nine equivalents of the Synechocystis PCC6803 genome and its representativity exceeds 99%.

Pannexins and gap junction protein diversity.

Gap junctions (GJs) are composed of proteins that form a channel connecting the cytoplasm of adjacent cells. Connexins were initially considered to be the only proteins capable of GJ formation. Another family of GJ proteins (innexins) were first found in invertebrates and were proposed to be renamed pannexins after their orthologs were discovered in vertebrates. The lack of both connexins and pannexins in the genomes of some metazoans suggests that other, still undiscovered GJ proteins exist. In vertebrates, connexins and pannexins co-exist. Here we discuss whether vertebrate pannexins have a nonredundant role in animal physiology. Pannexin channels appear to be suited for ATP and calcium signaling and play a role in the maintenance of calcium homeostasis by mechanisms implicating both GJ and nonjunctional function. Suggested roles in the ischemic death of neurons, schizophrenia, inflammation and tumor suppression have drawn much attention to exploring the molecular properties and cellular functions of pannexins.

Expression of autofluorescent proteins reveals a novel protein permeable pathway between cells in the lens core.

The lens of the eye is composed of concentric layers of tightly packed fiber cells. The oldest fibers, those in the lens core, lose their nuclei and other organelles during terminal differentiation. This is thought to ensure the clarity of the lens. The anucleated core fibers are sustained by gap junction-mediated communication with metabolically active cells near the lens surface. In this study, we expressed autofluorescent proteins and microinjected fluorescent markers to probe cell-to-cell communication in different regions of the developing lens. Our data indicate that a novel cell-cell diffusion pathway becomes patent in the lens core during development. This pathway is remarkable in that it is permeable to proteins and other large molecules and is thus distinct from gap junctions. Diffusion of large molecules probably occurs through regions of membrane fusion observed between neighboring cells in the lens core. Further direct evidence for a continuous plasma membrane system was provided by the observation that exogenous membrane proteins expressed in one core fiber cell were able to diffuse laterally into the membranes of adjacent fibers. Thus, the lens core appears to represent a true syncytium within which both membrane proteins and cytoplasmic proteins freely diffuse. Significantly, the outermost edge of the core syncytium encompasses a shell of nucleated, transcriptionally-competent, fiber cells. This arrangement could facilitate the delivery of newly synthesized protein components to the aged and metabolically quiescent cells in the center of the lens.

[Analysis of amino acid sequences of Rhodobacter sphaeroides glutamine synthetase]. / Analiz aminokislotnoi posledovatel'nosti glutaminsintetazy Rhodobacter sphaeroides.

A comparative analysis of the amino acid sequence of glutamine synthetase (GS) of the photosynthetic purple bacterium Rhodobacter sphaeroides revealed that the enzyme is typical for first type procaryotic GSs and structurally resembles GSs of enteric bacteria. The data obtained indicate that the complex phenotype of purple bacterial mutants at the glnA gene coding for GS may be conditioned by specific regulation of nitrogen metabolism in bacterial cells rather than by structural-and-functional peculiarities of GS.