5†µm CW Ce3+-doped chalcogenide glass fiber laser with 17% slope efficiency.

Efficient room temperature mid-infrared laser action in a Ce3+-doped chalcogenide fiber was demonstrated. The fiber had a doped selenide glass core in an undoped sulfide glass cladding. The pump source was a CW Fe2+:ZnSe laser emitting at 4.14 µm. The optimized fiber length allowed obtaining up to 7 mW of 5.06 µm output with 17% slope efficiency at room temperature.

Passively Q-switched 5-µm Ce3+-doped selenide glass laser using Fe:CdTe and Fe:CdSe as saturable absorbers.

The first, to the best of our knowledge, mid-infrared Q-switched Ce3+-doped glass laser is demonstrated. As saturable absorbers, Fe2+:CdSe and Fe2+:CdTe are used for the first time. When Q-switched by Fe:CdSe, the laser operates in a multi-pulse regime with an individual pulse width of 110 ns, centered at λ = 5.20 µm. With Fe:CdTe as saturable absorber, 1-3 giant pulses of 30 ns pulse width are generated at λ = 5.13 µm.

Mid-infrared laser performance of Ce3+-doped selenide glass.

An extensive study of a novel room-temperature mid-infrared Ce3+-doped Ge20Sb10Ga5Se65 glass laser is reported. An influence of output-coupler transmission on laser efficiency and emission spectra is investigated. Pumped by a pulsed Fe:ZnSe laser at 4.1 µm, a maximum output energy of 35 mJ is demonstrated at 5.2 µm, with a laser threshold of about 60 mJ and a slope efficiency of 21%. The tuning range of a mid-infrared Ce:glass laser is reported for the first time: with an intracavity prism, the laser is continuously tunable in the spectral range of 4.5-5.6 µm. The internal losses are determined to be below 9% per roundtrip.

Resonantly pumped Ce3+ mid-infrared lasing in selenide glass.

In high purity Ce3+-doped selenide glass pumped by a 4.08 µm Fe:ZnSe laser, 5.1-5.5 µm laser oscillations were observed. This is the first evidence of laser action corresponding to the 2F7/2→2F5/2 transition of Ce3+ ions.

Distributed Bragg reflector fiber laser directly written in a composite fiber manufactured by melting phosphate glass in a silica tube.

We demonstrate a single-frequency distributed Bragg reflector (DBR) fiber laser based on the novel erbium-doped composite fiber fabricated by melting phosphate glass in a silica tube. The fabricated composite fiber was single-mode at the wavelength of 1.55 µm; the measured cutoff wavelength was 1.4 µm. The composite fiber was photosensitive to irradiation at the wavelength of 193 nm. Using the phase mask method, the DBR fiber laser cavity with the total length of 21 mm was inscribed directly into the erbium-doped composite fiber. A stable single-frequency regime of the fabricated DBR laser at the wavelength of 1565 nm is demonstrated.

High-beam quality, high-efficiency laser based on fiber with heavily Yb(3+)-doped phosphate core and silica cladding.

We have fabricated and tested a composite fiber with an Yb(3+)-doped phosphate glass core and silica cladding. Oscillation with a slope efficiency of 74% was achieved using core pumping at 976 nm with fiber lengths of 48-90 mm in a simple laser configuration, where the cavity was formed by a high-reflectivity Bragg grating and the cleaved fiber end. The measured M(2) factors were as low as 1.05-1.22 even though the fiber was multimode at the lasing wavelength.

Phosphate-core silica-clad Er/Yb-doped optical fiber and cladding pumped laser.

We present a composite optical fiber with a Er/Yb co-doped phosphate-glass core in a silica glass cladding as well as cladding pumped laser. The fabrication process, optical properties, and lasing parameters are described. The slope efficiency under 980 nm cladding pumping reached 39% with respect to the absorbed pump power and 28% with respect to the coupled pump power. Due to high doping level of the phosphate core optimal length was several times shorter than that of silica core fibers.

Expanding role of G proteins in tight junction regulation: Galpha(s) stimulates TJ assembly.

Multiple signaling mechanisms regulate epithelial cell tight junction (TJ) assembly and maintenance. Several G proteins are likely to regulate these processes, but only G(i/o) have been specifically tested. Treatment of MDCK cells with cholera toxin, a Galpha(s) activator, accelerated TJ development in the calcium switch as measured by the time to half-maximal [T(50) (H)] transepithelial resistance (TER). Galpha(s) was predominantly localized in the lateral membrane, but a fraction colocalizes with ZO-1 in the TJ. MDCK cell lines expressing epitope-tagged Galpha(s) and constitutively active (R201Calpha(s)) showed a similar localization. TJ assembly was significantly faster in R201Calpha(s)-MDCK cell lines (T(50) (H) of 1.7 versus 3.3 h for controls) without detectable differences in cAMP levels. Confocal studies showed R201Calpha(s)-MDCK cells more rapidly localized ZO-1 and occludin into the developing TJ without affecting E-cadherin or Na(+)/K(+) ATPase localization. Endogenous Galpha(s) and R201Calpha(s) were immunoprecipitated with ZO-1 at baseline and during TJ assembly. The data supports a model of multiple Galpha subunits interacting with TJ proteins to regulate the assembly and maintenance of the TJ.

Reassembly of the tight junction after oxidative stress depends on tyrosine kinase activity.

Oxidative stress compromises the tight junction, but the mechanisms underlying its recovery remain unclear. We developed a model in which oxidative stress reversibly disrupts the tight junction. Exposure of Madin-Darby canine kidney cells to hydrogen peroxide markedly reduced transepithelial resistance and disrupted the staining patterns of the tight junction proteins ZO-1 and occludin. These changes were reversed by catalase. The short-term reassembly of tight junctions was not dependent on new protein synthesis, suggesting that recovery occurs through re-utilization of existing proteins. Although ATP levels were reduced, the reduction was insufficient to explain the observed changes, since a comparable reduction of ATP levels (with 2-deoxy-D-glucose) did not induce these changes. The intracellular hydrogen peroxide scavenger pyruvate protected Madin-Darby canine kidney cells from loss of transepithelial resistance as did the heavy metal scavenger N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine. Of a wide variety of agents examined, only tyrosine kinase inhibitors and protein kinase C inhibitors markedly inhibited tight junction reassembly. During reassembly, tyrosine phosphorylation in or near the lateral membrane, was detected by immunofluorescence. The tyrosine kinase inhibitors genistein and PP-2 inhibited the recovery of transepithelial resistance and perturbed the relocalization of ZO-1 and occludin to the tight junction, indicating that tyrosine kinases, possibly members of the Src family, are critical for reassembly after oxidative stress.

Degradation of heterotrimeric Galpha(o) subunits via the proteosome pathway is induced by the hsp90-specific compound geldanamycin.

One mechanism utilized by cells to maintain signaling pathways is to regulate the levels of specific signal transduction proteins. The compound geldanamycin (GA) specifically interacts with heat shock protein 90 (hsp90) complexes and has been widely utilized to study the role of hsp90 in modulating the function of signaling proteins. In this study, we used GA to demonstrate that levels of heterotrimeric Galpha subunits can be regulated through interactions with hsp90. In a dose-dependent manner, GA significantly reduced the steady state levels of endogenous Galpha(o) expression in two cell lines (PC12 and GH3) and had a similar effect on Galpha(o) transiently expressed in COS cells. Galpha(o) synthesis and degradation was studied in PC12 cells and in transiently transfected COS cells. (35)S labeling followed by immunoprecipitation demonstrated no effect of GA on the rate of Galpha(o) synthesis, but GA accelerated degradation of Galpha(o) in both PC12 cells and COS cells. The use of inhibitors, including lactacystin (a proteosome-specific inhibitor), suggests that Galpha(o) is predominantly degraded through the proteosome pathway. In vitro translated (35)S-labeled Galpha(o) could be detected in hsp90 immunoprecipitates, and this interaction did not require N-terminal myristoylation. Taken together, these results suggest that heterotrimeric Galpha(o) subunits are protected from degradation by interaction with hsp90 and that the interaction of Galpha subunits with heat shock proteins may be a general mechanism for regulating Galpha levels in the cell.

Diode-Pumped Er-Yb:Glass Laser Passively Q Switched by Use of Co(2+):MgAl(2)O(4) as a Saturable Absorber.

We report on passively Q-switched operation of a diode-pumped Er-Yb:glass laser with a Co(2+):MgAl(2)O(4) plate as a saturable absorber. Optical pulses with peak power exceeding 2 kW and a pulse length of 2.3 ns have been generated. Single-longitudinal-mode Q-switched operation at 1.53 mum has been obtained by use of the Co(2+):MgAl(2)O(4) plate as an intracavity etalon. A discussion of the optimal Er(3+) concentration as well as optimization of the cavity design is included.

Interaction of heterotrimeric G protein Galphao with Purkinje cell protein-2. Evidence for a novel nucleotide exchange factor.

The heterotrimeric G protein Galphao is ubiquitously expressed throughout the central nervous system, but many of its functions remain to be defined. To search for novel proteins that interact with Galphao, a mouse brain library was screened using the yeast two-hybrid interaction system. Pcp2 (Purkinje cell protein-2) was identified as a partner for Galphao in this system. Pcp2 is expressed in cerebellar Purkinje cells and retinal bipolar neurons, two locations where Galphao is also expressed. Pcp2 was first identified as a candidate gene to explain Purkinje cell degeneration in pcd mice (Nordquist, D. T., Kozak, C. A., and Orr, H. T. (1988) J. Neurosci. 8, 4780-4789), but its function remains unknown as Pcp2 knockout mice are normal (Mohn, A. R., Feddersen, R. M., Nguyen, M. S., and Koller, B. H. (1997) Mol. Cell. Neurosci. 9, 63-76). Galphao and Pcp2 binding was confirmed in vitro using glutathione S-transferase-Pcp2 fusion proteins and in vitro translated [35S]methionine-labeled Galphao. In addition, when Galphao and Pcp2 were cotransfected into COS cells, Galphao was detected in immunoprecipitates of Pcp2. To determine whether Pcp2 could modulate Galphao function, kinetic constants kcat and koff of bovine brain Galphao were determined in the presence and absence of Pcp2. Pcp2 stimulates GDP release from Galphao more than 5-fold without affecting kcat. These findings define a novel nucleotide exchange function for Pcp2 and suggest that the interaction between Pcp2 and Galphao is important to Purkinje cell function.

Involvement of Galphai2 in the maintenance and biogenesis of epithelial cell tight junctions.

Polarized epithelial cells have highly developed tight junctions (TJ) to maintain an impermeant barrier and segregate plasma membrane functions, but the mechanisms that promote TJ formation and maintain its integrity are only partially defined. Treatment of confluent monolayers of Madin-Darby canine kidney (MDCK) cells with AlF4- (activator of heterotrimeric G protein alpha subunits) results in a 3-4-fold increase in transepithelial resistances (TER), a reliable indicator of TJ integrity. MOCK cells transfected with activated Galpha0 (Q205L) have acclerated TJ formation (Denker, B. M., Saha, C. , Khawaja, S., and Nigam, S. J. (1996) J. Biol. Chem. 271, 25750-25753). Galphai2 has been localized within the tight junction, and a role for Galphai2 in the formation and/or maintenance of the tight junction was studied by transfection of MDCK cells with vector without insert (PC), wild type Galphai2, or a GTPase-deficient mutant (constitutively activated), Q205Lalphai2. Tryptic conformational analysis confirmed expression of a constitutively active Galphai2 in Q205Lalphai2-MDCK cells, and confocal microscopy showed a similar pattern of Galphai2 localization in the three cell lines. Q205Lalphai2-MDCK cells had significantly higher base-line TER values than wild type Galphai2- or PC-MDCK cells (1187 +/- 150 versus 576 +/- 89 (Galphai2); 377 +/- 52 Omega.cm2 (PC)), and both Galphai2- and Q205Lalphai2-transfected cell lines more rapidly develop TER in the Ca2+ switch, a model widely used to study the mechanisms of junctional assembly. Treatment of cells with AlF4- during the Ca2+ switch had little effect on the kinetics of TER development in Galphai2- or Q205Lalphai2-MDCK cells, but PC cells reached half-maximal TER significantly sooner in the presence of AlF4- (similar times to Galphai2-transfected cells). Base-line TER values obtained after the switch were significantly higher for all three cell lines in the presence of AlF4-. These findings indicate that Galphai2 is important for both the maintenance and development of the TJ, although additional Galpha subunits are likely to play a role.

Molecular structure and assembly of the tight junction.

Polarized epithelial cells separate two extremely different cellular milieus. The tight junction (TJ) is the most apical component of the junctional complex and serves as the permeability barrier between these environments. The tight junctional complex appears to be a dynamic and regulated structure. Some of its protein components have been identified and include the transmembrane protein occludin. Nontransmembrane proteins on the cytosolic leaflet including ZO-1, ZO-2, cingulin, 7H6, and several unidentified phosphoproteins are also believed to be part of the TJ. Interactions of some of these proteins with the actin cytoskeleton are a major determinant of TJ structure and may also play a role in the regulation of TJ assembly. Recent progress using the "calcium switch" and the "ATP depletion-repletion" model of TJ formation offers new insight regarding how these structures form. TJ biogenesis appears to be regulated, in part, by classic signal transduction pathways involving heterotrimeric G proteins, release of intracellular Ca2+, and activation of protein kinase C. Although many of the details of the signaling pathways have yet to be defined, these observations may provide insight into how TJs form during tubular development. Furthermore, it may be possible to suggest potential therapeutic targets for intervention in a variety of diseases (e.g., ischemia, toxic injury to the kidney and other epithelial tissue) where TJ integrity has been compromised and reassembly is required.

N-terminal binding domain of Galpha subunits: involvement of amino acids 11-14 of Galphao in membrane attachment.

Heterotrimeric guanine nucleotide binding proteins (G-proteins) transmit signals from membrane receptors to a variety of intracellular effectors. G-proteins reversibly associate with components of the signal transduction system, yet remain membrane attached throughout the cycle of activation. The Galpha subunits remain attached to the plasma membrane through a combination of factors that are only partially defined. We now demonstrate that amino acids within the N-terminal domain of Galpha subunits are involved in membrane binding. We used in vitro translation, a technique widely utilized to characterize functional aspects of G-proteins, and interactions with donor-acceptor membranes to demonstrate that amino acids 11-14 of Galphao contribute to membrane binding. The membrane binding of Galphao lacking amino acids 11-14 (D[11-14]) was significantly reduced at all membrane concentrations in comparison with wild-type Galphao. Several other N-terminal mutants of Galphao were characterized as controls, and these results indicate that differences in myristoylation, palmitoylation and betagamma interactions do not account for the reduced membrane binding of D[11-14]. Furthermore, when membrane attachment of Galphao and mutants was characterized in transiently transfected 35S-labelled and [3H]myristate-labelled COS cells, amino acids 11-14 contributed to membrane binding. These studies reveal that membrane binding of Galpha subunits occurs by a combination of factors that include lipids and amino acid sequences. These regions may provide novel sites for interaction with membrane components and allow additional modulation of signal transduction.

Characterization of clonally derived, spontaneously transformed bone marrow macrophage cell lines from lipopolysaccharide hyporesponsive LPS(d) and normal LPS(n) mice.

Six macrophage cell lines, each derived from a bone marrow macrophage colony grown in soft agar, were established by expansion of the macrophage clones in liquid culture until spontaneous transformation occurred. Four lines originated from the LPS(d) nonresponder mouse strain C3H/HeJ and two from the LPS(n) responder strain CBA/J. The cell lines adhered to plastic and glass surfaces and displayed typical macrophage functions such as phagocytosis and nonspecific esterase activity. Flow cytometry analyses showed that the lines expressed the macrophage surface markers CD11b, CD13, CD32/16, F4/80, and BM8 constitutively. A moderate expression of the adhesion receptor CD11a, but only a very low expression of its ligand CD54, was observed. A minor fraction of the cells in each line constitutively expressed MHC class II antigen, and its expression could be up-regulated in each cell line by treatment with interferon-gamma (IFN-gamma). Secretion of the inflammatory mediators nitric oxide (NO), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-alpha) after induction by three bacterial derivatives, heat-killed Salmonella typhimurium (HKS), lipopolysaccharide (LPS), and the Mycoplasma fermentans-derived amphiphilic lipid MDHM, were examined in detail. Not only did the lines differ in the amounts of mediators secreted in response to any one stimulus, but the doses of MDHM or LPS required for 50% maximal induction of NO varied up to 10-fold among the four LPS(d) cell lines, suggesting considerable functional heterogeneity between the clones. Secretion of large amounts of TNF-alpha was induced in all the cell lines by HKS. Although it could be shown that exogenously added TNF-alpha acted synergistically with IFN-gamma to induce NO release from the cell lines, an autocrine role for TNF-alpha during HKS-IFN-gamma induction of NO synthesis could not be substantiated. Neutralization of TNF-alpha with a specific antibody completely blocked NO induction by exogenous TNF-alpha but did not abrogate NO release either by HKS-IFN-gamma-induced cells or by macrophages treated with supernatant from an HKS-IFN-gamma-activated cell line. These results indicate that the clones are arrested in distinct stages of differentiation and retain some properties of normal untransformed macrophages. They should be helpful tools for investigations into macrophage function.

Analysis of the N-terminal binding domain of Go alpha.

Signalling from membrane receptors through heterotrimeric G-proteins (G alpha and G beta gamma) to intracellular effectors is a highly regulated process. Receptor activation causes exchange of GTP for GDP on G alpha and dissociation of G alpha from G beta gamma. Both subunits remain membrane-associated and interact with a series of other molecules throughout the cycle of activation. The N-terminal binding domain of G alpha subunits interacts with the membrane by several partially defined mechanisms: the anchoring of G alpha to the more hydrophobic G beta gamma subunits, the interaction of N-terminal lipids (palmitate and/or myristate) with the membrane, and attachment of amino acid regions to the membrane {amino acids 11-14 of Go alpha (D[11-14]); Busconi, Boutin and Denker (1997) Biochem. J. 323, 239-244}. We characterized N-terminal mutants of Go alpha with known G beta gamma-binding properties for the ability to interact with phospholipid vesicles and membranes prepared from cultured cells (acceptor membranes). In vitro analysis allows membrane interactions that are important to the activated and depalmitoylated state of G alpha to be characterized. Subcellular localization was also determined in transiently transfected COS cells. All of the mutant proteins are myristoylated, and differences in myristoylation do not account for changes in membrane binding. Disrupting the N-terminal alpha-helix of Go alpha with a proline point mutation at Arg-9 (R9P) does not affect interactions with G beta gamma on sucrose-density gradients but significantly reduces acceptor membrane binding. Deletion of amino acids 6-15 (D[6-15]; reduced G beta gamma binding) or deletion of amino acids 3-21 (D[3-21]); no detectable G beta gamma binding) further reduces acceptor membrane binding. When expressed in COS cells, R9P and D[6-15] are localized in the membrane similar to wild-type Go alpha as a result of the contribution from palmitoylation. In contrast, D[3-21] is completely soluble in COS cells, and no palmitoylation is detected. The binding of Go alpha and mutants translated in vitro to liposomes indicates that Go alpha preferentially binds to neutral phospholipids (phosphatidylcholine). R9P and D[11-14] bind to phosphatidylcholine liposomes like Go alpha, but D[6-15] exhibits no detectable binding. Taken together, these studies suggest that interactions of the N-terminus of G alpha subunits with the membrane may be affected by both membrane proteins and lipids. A detailed understanding of G alpha-membrane interactions may reveal unique mechanisms for regulating signal transduction.

Involvement of a heterotrimeric G protein alpha subunit in tight junction biogenesis.

The tight junction (TJ) of polarized epithelial cells is critical for maintaining an impermeant barrier and epithelial cell polarity. The signaling events important for TJ assembly require regulated calcium stores and protein kinase C (PKC), but the earliest signaling events in the cascade have not been well defined. We now show that Galphai2 in Madin Darby canine kidney (MDCK) cells localizes to a region overlapping with the TJ. To further analyze the localization of Galpha subunits in epithelial cells, rat Galphao, Q205Lalphao (Galphao "activated" by point mutation) and plasmid without insert (PC) were transfected into MDCK cells and localized by immunofluorescence and confocal microscopy. Similar to endogenous Galphai2, Galphao-MDCK cells localize Galphao, (84% similar to Galphai2) in the subapical region overlapping with ZO-1 (zona occludens-I), a key component of the TJ. PC-MDCK cells have no detectable Galphao. In Galphao-MDCK cells, a physical association of Galphao with components of the TJ was detectable by immunoprecipitation of ZO-1. Immunoprecipitates of ZO-1 from Galphao-MDCK cells consistently coprecipitated Galphao. Constitutively active Q205LGalphao localized to the subapical lateral membrane similar to wild-type Galphao. To determine if constitutively activated Galpha subunits can affect TJ biogenesis, the formation of tight junctions in PC, Galphao, and Q205Lalphao-MDCK cells was followed by measurement of transepithelial resistance (TER) during the Ca2+ switch, a model widely used to study mechanisms of junctional assembly. Baseline and post Ca2+ switch TER values did not differ among the cell lines. However, constitutively activated Q205Lalphao-MDCK cells developed TER significantly faster than PC and Galphao cells in the early phase (0-4 h) (54 +/- 4 versus 23 +/- 3 (PC); 12 +/- 1 (Galphao) Omega.m2/h) and late phase (4-h peak) (117 +/- 10 versus 45 +/- 5 (PC); 66 +/- 7 (Galphao) Omega.m2/h) after Ca2+ switch. Peak TER values were significantly higher in Q205Lalphao-MDCK cells (1168 +/- 107 versus 437 +/- 37 (PC); 548 +/- 54 (Galphao) Omega.cm2). These results indicate that Galphao and Q205Lalphao expressed in MDCK cells are localized near the junctional complex, associate with at least one TJ protein, and that activated Galphao accelerates TJ biogenesis without significantly affecting the maintenance of the TJ. Together, these results suggest an important role for heterotrimeric G proteins in TJ assembly.

Interactions between the amino- and carboxyl-terminal regions of G alpha subunits: analysis of mutated G alpha o/G alpha i2 chimeras.

Receptors activate the G alpha subunits of heterotrimeric G proteins by binding to the C-terminus and reducing their affinity for bound GDP, therefore promoting exchange of GDP for GTP. Although this general mechanism is the same for all G alpha subunits, different G alpha subunits vary in nucleotide binding and hydrolysis even though the residues that make up the guanine nucleotide binding site are virtually identical. We have shown previously that truncation of 14 amino acids from the C-terminus of G alpha o decreased the apparent affinity for GDP and permitted us to see an activated conformation with GTP [Denker, B. M., et al. (1992) J. Biol. Chem. 267, 9998-10002]. To test whether mutations in the receptor binding region lead to different phenotypes in closely related G alpha subunits, we made the equivalent deletions in G alpha i2, synthesized the proteins in vitro in a rabbit reticulocyte lysate and used the pattern of native tryptic proteolysis as an index of conformation. The phenotype of truncated G alpha i2 was different from that of truncated G alpha o: GDP affinity was reduced, but we could not detect an activated conformation with GTP (although GTP gamma S activated normally). Analysis of shorter deletions showed that loss of three hydrophobic residues (between 11 and 13 residues from the C-terminus) was responsible for the phenotypes. To define the regions of G alpha o and G alpha i2 that were responsible for their different phenotypes, we used a conserved BamHI site (codon 212) to make chimeras. Each chimera truncated at the C-terminus had the phenotype of the donor of the amino-terminal portion. Both truncated chimeras were activated by GTP gamma S-like wild-type proteins, and both had decreased apparent affinity for GDP. Full-length chimeric subunits behaved like wild-type proteins. The crystal structure of G alpha t and G alpha i1 shows that the three hydrophobic amino acids we have identified make contact with residues in the N- and C-terminal portions of the protein. Our studies point to the importance of the contacts in the N-terminal region (start of beta strands 1 and 3) that may stabilize the C-terminal alpha helix, affect nucleotide binding, and determine the characteristic features of different G alpha subunits.