Connexin 43 maintains tissue polarity and regulates mitotic spindle orientation in the breast epithelium.

Cell-cell communication is essential for tissue homeostasis, but its contribution to disease prevention remains to be understood. We demonstrate the involvement of connexin 43 (Cx43, also known as GJA1) and related gap junction in epithelial homeostasis, illustrated by polarity-mediated cell cycle entry and mitotic spindle orientation (MSO). Cx43 localization is restricted to the apicolateral membrane of phenotypically normal breast luminal epithelial cells in 3D culture and in vivo Chemically induced blockade of gap junction intercellular communication (GJIC), as well as the absence of Cx43, disrupt the apicolateral distribution of polarity determinant tight junction marker ZO-1 (also known as TJP1) and lead to random MSO and cell multilayering. Induced expression of Cx43 in cells that normally lack this protein reestablishes polarity and proper MSO in 3D culture. Cx43-directed MSO implicates PI3K-aPKC signaling, and Cx43 co-precipitates with signaling node proteins ß-catenin (CTNNB1) and ZO-2 (also known as TJP2) in the polarized epithelium. The distribution of Cx43 is altered by pro-inflammatory breast cancer risk factors such as leptin and high-fat diet, as shown in cell culture and on tissue biopsy sections. The control of polarity-mediated quiescence and MSO may contribute to the tumor-suppressive role of Cx43.

Draft genome sequence of the purple photosynthetic bacterium Phaeospirillum molischianum DSM120, a particularly versatile bacterium.

Here we present the draft genome sequence of the versatile and adaptable purple photosynthetic bacterium Phaeospirillum molischianum DSM120. This study advances the understanding of the adaptability of this bacterium, as well as the differences between the Phaeospirillum and Rhodospirillum genera.

Thermodynamics of membrane polypeptide oligomerization in light-harvesting complexes and associated structural changes.

Investigation of the equilibrium between the dissociated B777 form of the light-harvesting complex of Rhodospirillum rubrum and the oligomeric B820 form demonstrates that the B777 form consists of bacteriochlorophyll a (BChl) bound to the alpha or beta polypeptide chains; this binding appears to be reasonably stable at room temperature with little dissociation to free BChl and polypeptides. Analysis of the reaction order for the B777 association reaction to form B820 shows that this reaction requires four components, presumably two alpha-B777 units and two beta-B777 units, implying that the B820 subunit contains four BChl molecules. Estimations of the enthalpy and entropy changes associated with the tetramerization give values of, respectively, -175 kJ mol-1 and -0.46 kJ and mol-1 K-1. Soret resonance Raman and Fourier transform preresonance Raman spectra of BChl in detergent together with those of the B777, B820 and native B873 forms of the light harvesting complex illustrate significant changes occurring to the environments of the C-2 acetyl groups (Fischer numbering system) during dissociation to form B820 and a loss of order in the C-9 keto environments on formation of B777. Attenuated total reflectance Fourier transform infrared absorption spectra of the three antennae forms demonstrate little perturbation of the approximately 50% alpha-helical secondary structure during dissociation. These observations are discussed in terms of the energetics of membrane protein folding and the structure of the light harvesting complex.

Detergents modulate dimerization, but not helicity, of the glycophorin A transmembrane domain.

Understanding how the lipid environment influences transmembrane helix association requires thermodynamic measurements that can be interpreted in terms of specific chemical interactions. We have used Förster resonance energy transfer to measure dimerization of the glycophorin A transmembrane helix in detergent micelles. The observed Kd is at least two orders of magnitude weaker in sodium dodecyl sulfate than it is in zwitterionic detergents. In contrast, neither dimerization nor the detergent affects the secondary structure of the glycophorin A helix as measured by far-UV circular dichroism. These measurements support a long standing assumption about the glycophorin A transmembrane domain, that detergents uncouple helix formation from helix dimerization. The approach is applicable to a variety of systems in diverse environments, extending our ability to measure how interactions with complex solvents affect the thermodynamics of oligomerization.

Conformational flexibility and polymerization of vesicular stomatitis virus matrix protein.

The matrix protein of vesicular stomatitis virus (VSV) plays a pivotal role in viral assembly. We previously demonstrated the ability of M protein to self-associate at low salt concentrations. Now, we show the ability of M protein to polymerize in the presence of ZnCl2 in a nucleation-dependent manner. Analysis of kinetics revealed that the nuclei are probably made of three or four molecules of M. These results are consistent with the idea that in vitro self association of M protein is not due to amorphous aggregation but rather reflects an intrinsic ability of M to polymerize. Using attenuated total reflectance Fourier transform infrared spectroscopy, we showed that M polymerization is associated with an increase in the beta-sheet content of the protein. We propose a model explaining both the apparent M protein solubility in infected cells and how M polymerization could promote viral assembly. Data available for other negative strand viruses suggest that M polymerization may be the general basis of viral assembly.

Central lactogenic regulation of maternal behavior in rats: steroid dependence, hormone specificity, and behavioral potencies of rat prolactin and rat placental lactogen I.

Adult virgin female rats display maternal behavior when continuously exposed to foster young for 5-6 days. Central infusions of PRL or placental lactogens (PLs) together with systemic treatment of progesterone (P) and estradiol (E2) stimulate maternal behavior in 1-2 days. In the present set of studies, it was asked whether the actions of lactogenic hormones are dependent upon both E2 and P and specific to lactogenic molecules. Moreover, we wanted to know whether central infusions of rat (r) PRL and PLs were equally effective in inducing maternal behavior. In the first study, adult virgin rats were ovariectomized (ovx) and stereotaxically fitted with bilateral cannulas directed at the medial preoptic area (MPOA). Rats were then assigned to one of four groups: P plus E2, blank (B) plus E2, P plus B, and B plus B. P-filled or B capsules were implanted sc on treatment day 1 and removed on day 11, whereas E2 or B capsules were implanted on day 11. All groups were infused with rPRL (40 ng/side) five times from days 11-13 and injected with bromocriptine (CB-154) sc (days 11-17) to suppress endogenous PRL release. Behavioral testing was conducted daily from days 12-17. It was found that exposure to both P and E2 was necessary to induce a fast onset of maternal behavior in PRL-infused females; priming with P or E2 alone in PRL-treated rats failed to stimulate a fast onset of behavior relative to that in nonsteroid-treated controls. In the second experiment to determine the biochemical specificity of PRL's action, adult nulliparous rats were ovx, implanted with bilateral cannulas directed at the MPOA, treated with both P and E2, injected with CB-154, and infused centrally (five times) with 40 ng (per side) of bovine GH, ovine LH, or vehicle. Central infusions of either bovine GH or ovine LH failed to stimulate maternal behavior, suggesting that the stimulatory actions of PRL are related to its lactogenic properties. In the final study, rats were ovx, fitted with bilateral cannulas directed at the MPOA; treated with P, E2, and CB-154; and given a single set of bilateral infusions of rPL-I or rPRL (40 ng/side.infusion) on day 11, three sets of infusions of rPL-I or rPRL (days 11 and 12), or vehicle infusions. Rats given three infusions of rPL-I and rPRL responded faster than controls, although the effect was not as robust as that in animals given five infusions in the initial study. rPL-I and rPRL groups did not differ from one another. Together these studies indicate that 1) both P and E2 are required for lactogenic stimulation of maternal behavior; 2) the stimulatory actions of PRL and rPLs on maternal behavior are related to their lactogenic properties; 3) extended treatment of females with lactogenic hormones is more effective in stimulating the onset of maternal behavior; and 4) the neural potencies of rPRL and rPL-I are similar. These findings provide support for the idea that the induction of maternal behavior is stimulated by the central actions of lactogenic hormones.

N-methyl-DL-aspartic acid lesions of the medial preoptic area disrupt ongoing parental behavior in male rats.

The effects of axon-sparing, neurotoxic lesions of the medial preoptic area (MPOA) with N-methyl-DL-aspartic acid (NMA) on previously established parental behavior in male rats were investigated. Adult, sexually-inexperienced male rats were gonadectomized and seven days later implanted sc with a single estradiol (E2)-filled Silastic capsule on treatment Day 1. Three progesterone (P4) capsules were implanted sc on treatment Day 3 and removed on Day 21, one day prior to the start of behavioral testing. Males were tested daily with foster pups in order to induce parental behavior, i.e., contacting the test pups, pup retrieval, grouping, and crouching over three foster pups. Full parental behavior appeared in these males after an average of 3 days. After testing on the third consecutive day of parental behavior, parental males were infused bilaterally with either NMA or vehicle into the MPOA. NMA infusions resulted in a significant decline in all components of parental behavior by the next test session, a deficit which persisted throughout the 5 days of post-infusion testing. In contrast, parental care continued to be displayed in animals given vehicle infusions. These findings demonstrate that the cells in the MPOA play an important role in regulating ongoing parental care in male rats and indicate that the neural substrates controlling parental behavior in male and female rats are similar.

Review of the carotid artery loop procedure in sheep.

Carotid loop (CL) surgery involves the permanent externalization of a common carotid artery in a skin tube. The CL facilitates repeated access to the systemic arterial system for blood sampling and blood pressure measurement in laboratory sheep. It eliminates the need for arterial cut-downs and chronic indwelling catheters, reduces the risk of sepsis and infection, and adds flexibility to research protocols. The surgical procedure is aseptically performed under general anesthesia and involves isolation of the common carotid artery, creation of a bipedicled skin tube, and permanent envelopment of the artery in the skin tube. The primary complication is ischemic necrosis with sloughing of the middle of the loop and is usually due to failure to adhere to the critical length-to-width ratio (2.5:1). We have performed 150 CL procedures with an overall success rate of 94%. Nine CL ablations were required, due to necrosis with exposure of the artery (7/9) or stricture formation with loss of patency (2/9). Twenty-two CLs developed complications secondary to partial necrosis, but did not require ablation. Results indicate that the CL is a reliable method to ensure repeated access to the systemic arterial system in sheep. A modification of the standard CL procedure in which the artery is surrounded by a skin tunnel rather than enclosed in a skin loop was performed in 10 sheep. Preliminary results indicate significant reduction in the incidence of complications associated with the standard CL.

Heterologous expression of genes encoding bacterial light-harvesting complex II in Rhodobacter capsulatus and Rhodovulum sulfidophilum.

In the present work we report the high-level expression of foreign genes encoding the light-harvesting (LHII) membrane-spanning polypeptides in photosynthetic bacteria. To do this we first constructed three deletion strains of Rhodovulum (Rhv.) sulfidophilum in which all or part of the puc operon, encoding the peripheral light-harvesting proteins, is missing. To investigate the heterologous expression of the light-harvesting polypeptides from Rb. capsulatus in Rhv. sulfidophilum and vice versa we have reintroduced functional foreign LH genes into these and equivalent strains of Rhodobacter (Rb.) capsulatus. In some cases very high levels of expression were obtained (85%) of those observed in the wild type), while in other cases much lower expression was observed; possible reasons for these differences are discussed. The heterologously expressed proteins were shown to contain normal pigment-binding sites and to be normally and functionally integrated within the host photosynthetic apparatus. The results indicate that heterologous proteins are able to assemble properly and enter into the same protein-protein interactions as their analogs originally present in the host strain.

Structural properties of the tubular appendage spinae from marine bacterium Roseobacter sp. strain YSCB.

Spinae are tubular surface appendages broadly found in Gram-negative bacteria. Little is known about their architecture, function or origin. Here, we report structural characterization of the spinae from marine bacteria Roseobacter sp. YSCB. Electron cryo-tomography revealed that a single filament winds into a hollow flared base with progressive change to a cylinder. Proteinase K unwound the spinae into proteolysis-resistant filaments. Thermal treatment ripped the spinae into ribbons that were melted with prolonged heating. Circular dichroism spectroscopy revealed a dominant beta-structure of the spinae. Differential scanning calorimetry analyses showed three endothermic transformations at 50-85°C, 98°C and 123°C, respectively. The heating almost completely disintegrated the spinae, abolished the 98°C transition and destroyed the beta-structure. Infrared spectroscopy identified the amide I spectrum maximum at a position similar to that of amyloid fibrils. Therefore, the spinae distinguish from other bacterial appendages, e.g. flagella and stalks, in both the structure and mechanism of assembly.

Organisation and evolution of the tol-pal gene cluster.

The genomic context and phylogenetic distribution of the tol-pal gene cluster and homologues to its various components have been investigated. The structure of this operon is well conserved across the gram negative bacteria, and the machine encoded by these genes probably evolved with the appearance of gram negative bacteria. Since the evolutionary appearance of the operon some species appear to have lost the genes. These bacteria seem to fall into two classes, namely obligate intracellular parasites and bacteria that produce large numbers of outer membrane vesicles. The evolution of the alphabeta and gamma proteobacteria was accompanied by the association of an additional gene (ybgC) with the operon. Several coincidences of genomic context argue for an important role of the tol-pal operon in cell envelope maintenance. Genes homologous to tolQ and tolR proved to be very widespread being found throughout the eubacteria, and one example in the archea, this distribution argues for an ancient origin of these genes. The genomic context of these genes often suggests a role in micronutrient uptake. Interestingly in all the cases examined the tolQ and tolR genes or their homologues appear to be present as a pair, with a potential for a tight translational regulation.

Acid denaturation of the B875 light-harvesting complex in membranes of Rhodobacter sphaeroides.

The structural basis for the spectral red shift in the near-IR absorption band of the B875 light-harvesting complex was examined by treatment of membranes from Rhodobacter sphaeroides M21 with acid. This mutant strain lacks the overlapping spectral bands of the B800-850 light-harvesting antenna and gives rise to membrane fragments with both surfaces accessible to protons. At pH 2.2, about half the absorption at 876 nm was converted within 10 min to a 'free' pigment band; the remaining absorption appeared at 880 nm and shifted to ∼845 nm over the next three hours. These spectral shifts could not be reversed by alkali. Approximately one-third of the characteristic near-IR CD signal of B875 was also lost initially and replaced by a broad trough centered near 854 nm. Thereafter, the CD spectrum was dominated by the strong conservative signal of the 845 nm absorbing component which was attributed to an oligomeric bacteriopheophytin a species, probably a dimer. A kinetic analysis of the acid-induced absorption changes suggested a multi-step model with rate constants of 0.37 min(-1) for the initial rapid change and 0.05 and 0.11 min(-1) for the respective subsequent steps. The non-conservative nature of the near-IR CD spectrum of the intact complex, together with the spectral changes observed after the initial loss of near-IR absorption and CD, suggest that pigment-pigment interactions are not solely responsible for the red shift in this complex.

The role of chromophore coupling in tuning the spectral properties of peripheral light-harvesting protein of purple bacteria.

The publication of a structure for the peripheral light-harvesting complex of a purple photosynthetic bacterium (McDermott et al. (1995), Nature 374: 517-521) provides a framework within which we can begin to understand various functional aspects of these complexes, in particular the relationship between the structure and the red-shift of the bacteriochlorophyll Qy transition. In this article we describe calculations of some of the spectral properties expected for an array of chromophores with the observed geometry. We report the stability of the calculated absorption spectrum to minor structural alterations, and deduce that the observed red shift of the 850 nm Qy transition in the B800-850 antenna complexes is about equally attributable to chromophore-chromophore and chromophore-protein interactions, while chromophore-chromophore interactions predominate in generating the red-shift of the 820 nm Qy transition in B800-820 type peripheral liggt-harvesting complexes. Finally we suggest that the red shift in the absorbance of the monomeric Bchl a found in antenna complexes to 800 nm, from 770 nm as observed in most solvents, is largely attributable to a hydrogen bond with the 2-acetyl group of this chromophore.

Neurosteroids affect spatial/reference, working, and long-term memory of female rats.

Female rats take longer to acquire a spatial task during behavioral estrus, when GABA-active progesterone and metabolites are elevated. Whether neurosteroids and neuroactive steroids (neuro(active) steroids), which can act at GABA receptor complexes (GBRs), have activational effects on spatial/reference, working, and long-term memory was investigated. In Experiment 1, ovariectomized Long-Evans rats (N = 107) received oil vehicle or one of six neuro(active) steroids, with varying GBR efficacy (greatest to least efficacious: 5 alpha-pregnan-3 alpha-ol-20-one (THP), 5 alpha-pregnan-3 alpha-ol-11,20-dione, 4-pregnen-3,20-dione 17 alpha-hydroxyprogesterone, 5-pregnen-3 beta-ol-20-one sulfate, and 5-androstan-3 beta-ol-17-one sulfate (DHEAS). Following neuro(active) steroid (3.2 or 6.4 mg/kg) or vehicle sc, rats were tested in a Morris water maze, the following week in a Y maze, and then in an open field. Neuro(active) steroid, but not vehicle, animals had decreased distances to the hidden water maze platform. THP (3.2 and 6.4 mg/kg) animals were faster to find this platform than vehicle animals. In the Y maze, 3.2 mg/kg THP increased percentage correct, but 6.4 mg/kg THP increased latencies to the goal box. DHEAS had the opposite effect, with 3.2 mg/kg increasing latencies to the goal box, while 6.4 mg/kg increased percentage correct. In Experiment 2, N = 75 ovariectomized rats were icv implanted with one of the neuro(active)steroids or cholesterol vehicle and then tested for spatial/reference memory, working and long-term memory, and motoricity/anxiolysis as in Experiment 1. DHEAS implants decreased, while THP increased, latencies and distances to the hidden platform in the Morris water maze. In the Y maze, THP increased latencies and decreased percentage correct, but DHEAS increased the likelihood of correct choice. Open field behavior of animals administered the various neuro(active) steroids (sc or icv) was not different. Thus, of the neuro(active) steroids examined, the neurosteroids THP and DHEAS had the most pronounced activational affects on spatial/reference, working, and long-term memory, independent of motoricity.

Transmembrane helix stability: the effect of helix-helix interactions studied by Fourier transform infrared spectroscopy.

We have measured, using infrared spectroscopy, the hydrogen/deuterium exchange rates of the amide protons in the photosynthetic antenna of Rhodospirillum rubrum. These measurements were made not only on the intact protein in detergent solution but also on two dissociated forms (B820 and B777). We have, on the basis of our knowledge of the structure of this protein, been able to assign the various groups of amide protons that exchange with different time constants to distinct regions of the protein. The most protected group of protons that we observe exchanging with time constants near 6000 min we assign to the transmembrane helices. The slow exchange rates measured for the amide protons of the transmembrane helices of this protein in detergent solution may indicate a destabilization of the helices in detergent solution compared with the membrane. This group of protons is progressively destabilized by stepwise dissociation of the antenna protein, and this destabilization is greater than we can account for by increases in solvent accessibility. We suggest that the observed loss of amide proton protection in the transmembrane helices as they are dissociated might be due to an increase in the helix flexibility and breathing motions as interactions between helices are reduced.

The TolQ-TolR proteins energize TolA and share homologies with the flagellar motor proteins MotA-MotB.

The Tol-Pal system of Escherichia coli is required for the maintenance of outer membrane stability. Recently, proton motive force (pmf) has been found to be necessary for the co-precipitation of the outer membrane lipoprotein Pal with the inner membrane TolA protein, indicating that the Tol-Pal system forms a transmembrane link in which TolA is energized. In this study, we show that both TolQ and TolR proteins are essential for the TolA-Pal interaction. A point mutation within the third transmembrane (TM) segment of TolQ was found to affect the TolA-Pal interaction strongly, whereas suppressor mutations within the TM segment of TolR restored this interaction. Modifying the Asp residue within the TM region of TolR indicated that an acidic residue was important for the pmf-dependent interaction of TolA with Pal and outer membrane stabilization. Analysis of sequence alignments of TolQ and TolR homologues from numerous Gram-negative bacterial genomes, together with analyses of the different tolQ-tolR mutants, revealed that the TM domains of TolQ and TolR present structural and functional homologies not only to ExbB and ExbD of the TonB system but also with MotA and MotB of the flagellar motor. The function of these three systems, as ion potential-driven molecular motors, is discussed

Proton motive force drives the interaction of the inner membrane TolA and outer membrane pal proteins in Escherichia coli.

The Tol-Pal system of the Escherichia coli envelope is formed from the inner membrane TolQ, TolR and TolA proteins, the periplasmic TolB protein and the outer membrane Pal lipoprotein. Any defect in the Tol-Pal proteins or in the major lipoprotein (Lpp) results in the loss of outer membrane integrity giving hypersensitivity to drugs and detergents, periplasmic leakage and outer membrane vesicle formation. We found that multicopy plasmid overproduction of TolA was able to complement the membrane defects of an lpp strain but not those of a pal strain. This result indicated that overproduced TolA has an envelope-stabilizing effect when Pal is present. We demonstrate that Pal and TolA formed a complex using in vivo cross-linking and immunoprecipitation experiments. These results, together with in vitro experiments with purified Pal and TolA derivatives, allowed us to show that Pal interacts with the TolA C-terminal domain. We also demonstrate using protonophore, K+ carrier valinomycin, nigericin, arsenate and fermentative conditions that the proton motive force was coupled to this interaction.

Reevaluation of the electrophoretic migration behavior of soluble globular proteins in the native and detergent-denatured states in polyacrylamide gels.

Although sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis is widely used for estimating molecular masses of proteins, considerable uncertainty still exists both about the structure of SDS-protein complexes and about their mechanism of electrophoretic migration. In this study, soluble globular proteins, with masses of 14-200 kDa, were heat-denatured in the presence of SDS and their relative total molecular volume and net charge were estimated from Ferguson plots of electrophoretic mobility vs acrylamide concentration. Native globular protein served as standards for overall molecular size and effective radii. Results revealed at least two independent electrophoretic migration mechanisms for the SDS-protein complexes: (i) for proteins in the 14-65 kDa range at <15% acrylamide, linear Ferguson plots suggested that they migrated ideally and that their effective radii could be estimated in this manner: (ii) concave plots at higher gel concentrations, and for complexes derived from larger proteins, indicated that migration in these cases could be described by reptation theory. Migration of the large proteins at lower gel concentrations and small proteins at higher gel concentrations was not well described by either theory, representing intermediate behavior not described by these mechanisms. These data support models in which all but the smallest SDS-protein complexes adopt a necklace-like structure in which spherical micelles are distributed along the unfolded polypeptide chain. Possible relations to recent alternative models of gel electrophoresis are also discussed.

Exchanging cofactors in the core antennae from purple bacteria: structure and properties of Zn-bacteriopheophytin-containing LH1.

The core light-harvesting LH1 complex of Rhodospirillum rubrum consists of an assembly of membrane-spanning alpha and beta polypeptides, each of which binds one bacteriochlorophyll (BChl) a molecule. In this work, we describe a technique that allows the replacement of the natural, Mg BChl a cofactors present in this protein by Zn-bacteriopheophytin (Zn-Bpheo). This technique makes use of the well-characterized, reversible dissociation of LH1 induced by the detergent beta-octylglucoside. Incubation of partially dissociated LH1 with exogeneous pigments induces an equilibrium between the protein-bound BChl and the exogeneous pigment. This results in the binding of chemically modified pigments to LH1, in amounts which depend on the pigment composition and concentration of the exchange buffer. This method can yield information on the relative affinities of the LH1 protein-binding sites for the different pigments BChl and Zn-Bpheo and can also be used to obtain fully reassociated LH1 proteins, with a variable content of modified pigment, which may be precisely monitored. Absorption and FT-Raman spectroscopy indicate that this exchange procedure leads to LH1 proteins containing modified pigments, but retaining a binding site structure identical to that of native LH1. Furthermore, examination of the binding curves suggests that there are two distinguishable binding sites, probably corresponding to the two polypeptides, with very different properties. One of these two binding sites shows a marked preference for Zn-Bpheo over BChl, while the other binding site appears to prefer BChl.

Membrane-associated c-type cytochromes from the green sulfur bacterium Chlorobium limicola forma thiosulfatophilum: purification and characterization of cytochrome c553.

Tetraheme cytochromes involved in photosynthetic electron transport have previously been described associated with the reaction centers of purple photosynthetic bacteria; however, similar heme proteins have not until now been characterized in the phylogenetically distinct green sulfur bacteria. In this paper we describe the first isolation and characterization of a multitheme, membrane-associated cytochrome from a green sulfur bacterium, Chlorobium limicola forma thiosulfatophilum. We show that this cytochrome contains a single polypeptide of 32 kDa apparent molecular mass on SDS-PAGE and has a characteristic broad alpha-band absorption at 553 nm. By both low-temperature absorption and electron paramagnetic resonance spectroscopy, we demonstrate that there are at least four distinct heme groups.