Genetic risk for schizophrenia is associated with altered visually-induced gamma band activity: evidence from a population sample stratified polygenic risk.

Gamma oscillations (30-90 Hz) have been proposed as a signature of cortical visual information processing, particularly the balance between excitation and inhibition, and as a biomarker of neuropsychiatric diseases. Magnetoencephalography (MEG) provides highly reliable visual-induced gamma oscillation estimates, both at sensor and source level. Recent studies have reported a deficit of visual gamma activity in schizophrenia patients, in medication naive subjects, and high-risk clinical participants, but the genetic contribution to such a deficit has remained unresolved. Here, for the first time, we use a genetic risk score approach to assess the relationship between genetic risk for schizophrenia and visual gamma activity in a population-based sample drawn from a birth cohort. We compared visual gamma activity in a group (N = 104) with a high genetic risk profile score for schizophrenia (SCZ-PRS) to a group with low SCZ-PRS (N = 99). Source-reconstructed V1 activity was extracted using beamformer analysis applied to MEG recordings using individual MRI scans. No group differences were found in the induced gamma peak amplitude or peak frequency. However, a non-parametric statistical contrast of the response spectrum revealed more robust group differences in the amplitude of high-beta/gamma power across the frequency range, suggesting that overall spectral shape carries important biological information beyond the individual frequency peak. Our findings show that changes in gamma band activity correlate with liability to schizophrenia and suggest that the index changes to synaptic function and neuronal firing patterns that are of pathophysiological relevance rather than consequences of the disorder.

Spectrin-dependent and -independent association of F-actin with the erythrocyte membrane.

Binding of F-actin to spectrin-actin-depleted erythrocyte membrane inside-out vesicles was measured using [3H]F-actin. F-actin binding to vesicles at 25 degrees C was stimulated 5-10 fold by addition of spectrin dimers or tetramers to vesicles. Spectrin tetramer was twice as effective as dimer in stimulating actin binding, but neither tetramer nor dimer stimulated binding at 4 degrees C. The addition of purified erythrocyte membrane protein band 4.1 to spectrin-reconstituted vesicles doubled their actin-binding capacity. Trypsinization of unreconstituted vesicles that contain < 10% of the spectrin but nearly all of the band 4.1, relative to ghosts, decreased their F-actin-binding capacity by 70%. Whereas little or none of the residual spectrin was affected by trypsinization, band 4.1 was significantly degraded. Our results show that spectrin can anchor actin filaments to the cytoplasmic surface of erythrocyte membranes and suggest that band 4.1 may be importantly involved in the association.

The role of band 4.1 in the association of actin with erythrocyte membranes.

Spectrin stimulates the association of F-actin with erythrocyte inside-out vesicles. Although inside-out vesicles are nearly devoid of two of the three major cytoskeletal proteins, spectrin and actin, they retain nearly all of the cytoskeletal protein designated band 4.1. Inside-out vesicles which have been substantially depleted of band 4.1 by extraction in 1 M KCl, 0.4 M urea and then reconstituted with spectrin show a markedly diminished ability to bind actin by comparison with vesicles containing normal amounts of band 4.1. This diminution is not due to an impaired ability of the vesicles to bind spectrin. Addition of purified band 4.1 to vesicles either before or after they have been reconstituted with spectrin restores their actin binding capacity to near normal levels as does addition of a spectrin-band 4.1 complex prepared by sucrose gradient centrifugation. Band 4.1 bound to vesicles in the absence of added spectrin has no effect on actin binding. Our results suggest that a spectrin band 4.1 complex is responsible for binding actin to erythrocyte membranes.

A 20-kDa domain is required for phosphatidic acid-induced allosteric activation of phospholipase D from Streptomyces chromofuscus.

Two phospholipase D (PLD) enzymes with both hydrolase and transferase activities were isolated from Streptomyces chromofuscus. There were substantial differences in the kinetic properties of the two PLD enzymes towards monomeric, micellar, and vesicle substrates. The most striking difference was that the higher molecular weight enzyme (PLD57 approximately 57 kDa) could be activated allosterically with a low mole fraction of phosphatidic acid (PA) incorporated into a PC bilayer (Geng et al., J. Biol. Chem. 273 (1998) 12195-12202). PLD42/20, a tightly associated complex of two peptides, one of 42 kDa and the other 20 kDa, had a 4-6-fold higher Vmax toward PC substrates than PLD57 and was not activated by PA. N-Terminal sequencing of both enzymes indicated that both components of PLD42/20 were cleavage products of PLD57. The larger component included the N-terminal segment of PLD57 and contained the active site. The N-terminus of the smaller peptide corresponded to the C-terminal region of PLD57; this peptide had no PLD activity by itself. Increasing the pH of PLD42/20 to 8.9, followed by chromatography of PLD42/20 on a HiTrap Q column at pH 8.5 separated the 42- and 20-kDa proteins. The 42-kDa complex had about the same specific activity with or without the 20-kDa fragment. The lack of PA activation for the 42-kDa protein and for PLD42/20 indicates that an intact C-terminal region of PLD57 is necessary for activation by PA. Furthermore, the mechanism for transmission of the allosteric signal requires an intact PLD57.

Characterization of a soluble ternary complex formed between human interferon-beta-1a and its receptor chains.

The extracellular portions of the chains that comprise the human type I interferon receptor, IFNAR1 and IFNAR2, have been expressed and purified as recombinant soluble His-tagged proteins, and their interactions with each other and with human interferon-beta-1a (IFN-beta-1a) were studied by gel filtration and by cross-linking. By gel filtration, no stable binary complexes between IFN-beta-1a and IFNAR1, or between IFNAR1 and IFNAR2 were detected. However, a stable binary complex formed between IFN-beta-1a and IFNAR2. Analysis of binary complex formation using various molar excesses of IFN-beta-1a and IFNAR2 indicated that the complex had a 1:1 stoichiometry, and reducing SDS-PAGE of the binary complex treated with the cross-linking reagent dissucinimidyl glutarate (DSG) indicated that the major cross-linked species had an apparent Mr consistent with the sum of its two individual components. Gel filtration of a mixture of IFNAR1 and the IFN-beta-1a/IFNAR2 complex indicated that the three proteins formed a stable ternary complex. Analysis of ternary complex formation using various molar excesses of IFNAR1 and the IFN-beta-1a/IFNAR2 complex indicated that the ternary complex had a 1:1:1 stoichiometry, and reducing SDS-PAGE of the ternary complex treated with DSG indicated that the major cross-linked species had an apparent Mr consistent with the sum of its three individual components. We conclude that the ternary complex forms by the sequential association of IFN-beta-1a with IFNAR2, followed by the association of IFNAR1 with the preformed binary complex. The ability to produce the IFN-beta-1a/IFNAR2 and IFN-beta-1a/IFNAR1/IFNAR2 complexes make them attractive candidates for X-ray crystallography studies aimed at determining the molecular interactions between IFN-beta-1a and its receptor.

Biochemical analysis of retroviral structural proteins to identify and quantify retrovirus expressed by an NS0 murine myeloma cell line.

A subclone of the NS0 murine myeloma cell line, frequently used to produce recombinant monoclonal antibodies, was found by a transmission electron microscopy method to express a surprisingly high titer of 10(11) retroviral particles per ml of culture supernatant. Infectivity assays showed a very low infectious titer with the restricted host range expected for a murine amphotropic retrovirus. A Western blot assay for the viral capsid protein was developed to confirm the high titer values and provide a means for monitoring batch consistency and virus removal during the purification process. Mass spectrometry of several of the viral Gag proteins demonstrated that the cell line appeared to produce at least two closely related retroviruses. N-terminal sequencing of three of the Gag proteins demonstrated that these retroviruses were members of the murine leukemia retroviral family. Western blot detection with an antibody for the capsid protein gave a linear standard curve over the range of 0.1-3 ng per lane. This allows the detection of viral titers as low as 6x10(7) virions per ml without the need to concentrate the sample. The Western blot method has higher throughput and less variability than transmission electron microscopy methods and has potential for monitoring viral titer and clearance during development of manufacturing processes.

Expression of genetically engineered immunoconjugates of lymphotoxin and a chimeric anti-ganglioside GD2 antibody.

Human lymphotoxin was genetically conjugated to the constant region of a human gamma 1 immunoglobulin gene at the end of either the second (CH2-LT) or third (CH3-LT) constant region domain. The altered heavy chain constant regions were combined in a plasmid vector together with the variable regions of a mouse anti-ganglioside GD2 antibody 14.18 and the human kappa constant region. The resulting immunoconjugate constructs were expressed in transfected hybridoma cells and tested for both their antibody and lymphotoxin activities. The two constructs were assembled to varying degrees depending on whether the third heavy chain constant region was present. Both forms retained their ability to bind antigen and mediate ADCC but only CH3-LT was able to mediate the lysis of melanoma target cells in the presence of human complement. Lymphotoxin activity, as defined in a cytolytic assay with mouse fibroblasts, was found to increase significantly as a function of heavy chain assembly and to be equivalent to unconjugated lymphotoxin. Neither of the constructs were cytotoxic for antigen-bearing melanoma cells that are normally resistant to lymphotoxin and tumor necrosis factor alpha. Such immunoconjugates may prove useful in targeting cytokines to the site of antigen-bearing cells in vivo. In this case, as a means of eliciting an inflammatory response at the site of a solid tumor.

Preventive gynecologic nursing in an inpatient setting.

A nurse-managed screening program that offers gynecologic examinations and health teaching/counseling to female patients is described. The philosophy of the program includes emphasis on individualized care, health promotion, illness prevention, and fostering of self-care practices. Each physical examination includes a breast and pelvic examination, pap test, and health teaching/counseling in areas of women's health.

Phorbol ester- and Ca2+-dependent phosphorylation of human red cell membrane skeletal proteins.

The addition of the tumor promoting phorbol ester 12-O-tetradecanoyl phorbol 13-acetate to intact human red blood cells activates protein kinase C and stimulates the phosphorylation of the membrane skeletal proteins band 4.1 and band 4.9 as well as two proteins of molecular mass 115 and 110 kDa. We show that 12-O-tetradecanoyl phorbol 13-acetate promotes the association of cytosolic protein kinase C with the red cell membrane and that the enzyme is present on ghost membranes but is largely absent from inside-out vesicles. We show that micromolar Ca2+ added to ghosts also promotes the phosphorylation of band 4.1 and the approximately 100-kDa proteins, a reaction which has not been described previously. Digestion and extraction studies show that the 100-kDa proteins are unrelated to band 3 since they are absent from NaOH stripped membranes, but are found in Triton-prepared cytoskeletons. Digestion of intact red cells with chymotrypsin or neuraminidase, which attack principally band 3 and glycophorin, respectively, markedly inhibits protein kinase C phosphorylation of band 4.1 in red cells and ghosts and of the 100-kDa proteins in ghosts. These enzymes have no effect upon the activity of the Ca2+-activated phosphorylation reaction, suggesting that it does not involve protein kinase C. These results shed light on two phosphorylation reactions which act exclusively on red cell membrane skeletal proteins. Our findings suggest that digestion of the integral membrane proteins band 3 and glycophorin, the principal targets of external protease digestion, affects the activity or specificity of protein kinase C. Finally we have described two apparently novel approximately 100-kDa phosphorylated proteins which are components of Triton-prepared red cell membrane skeletons.

Biochemical characterization of complex formation by human erythrocyte spectrin, protein 4.1, and actin.

Ternary complex formation between the major human erythrocyte membrane skeletal proteins spectrin, protein 4.1, and actin was quantified by measuring cosedimentation of spectrin and band 4.1 with F-actin. Complex formation was dependent upon the concentration of spectrin and band 4.1, each of which promoted the binding of the other to F-actin. Simultaneous measurement of the concentrations of spectrin and band 4.1 in the sedimentable complex showed that a single molecule of band 4.1 was sufficient to promote the binding of a spectrin dimer to F-actin. However, the molar ratio of band 4.1/spectrin in the complex was not fixed, ranging from approximately 0.6 to 2.2 as the relative concentration of added spectrin to band 4.1 was decreased. A mole ratio of 0.6 band 4.1/spectrin suggests that a single molecule of band 4.1 can promote the binding of more than one spectrin dimer to an actin filament. Saturation binding studies showed that in the presence of band 4.1 every actin monomer in a filament could bind at least one molecule of spectrin, yielding ternary complexes with spectrin/actin mole ratios as high as 1.4. Electron microscopy of such complexes showed them to consist of actin filaments heavily decorated with spectrin dimers. Ternary complex formation was not affected by alteration in Mg2+ or Ca2+ concentration but was markedly inhibited by KCl above 100 mM and nearly abolished by 10 mM 2,3-diphosphoglycerate or 10 mM adenosine 5'-triphosphate. Our data are used to refine the molecular model of the red cell membrane skeleton.

Expression and secretion of an assembled tetrameric CH2-deleted antibody in E. coli.

We have expressed in E. coli a functional assembled antibody variant that is secreted into the media. The antibody variant is a CH2-deleted chimeric antibody 14.18, which was previously shown to be a potentially useful reagent for radioimmunodetection of human tumors. The bacterial expression vector contains a dicistronic unit comprised of a L-chain cDNA and a CH2-deleted H-chain cDNA. For translocation across the bacterial membranes, we have replaced the natural signal peptides of the H and L chains with the signal peptide of the bacterial protein pectate lyase B. When expressed in the JM105 host under the control of the trp-lac promoter, the products were secreted into the M9 growth media to about 350 micrograms/L. The secreted antibody, which can be readily purified from the media without any denaturation or renaturation steps, retains antigen-binding activity. SDS-PAGE and nondenaturing size exclusion high-pressure liquid chromatography showed that it is a mixture of assembled HL and fully assembled H2L2. In H2L2, inter-H chain disulfide bonds are not formed, and the two HL half-molecules are likely held together by the trans interaction between the two CH3 domains.

Lymphotoxin beta, a novel member of the TNF family that forms a heteromeric complex with lymphotoxin on the cell surface.

The lymphokine tumor necrosis factor (TNF) has a well-defined role as an inducer of inflammatory responses; however, the function of the structurally related molecule lymphotoxin (LT alpha) is unknown. LT alpha is present on the surface of activated T, B, and LAK cells as a complex with a 33 kd glycoprotein, and cloning of the cDNA encoding the associated protein, called lymphotoxin beta (LT beta), revealed it to be a type II membrane protein with significant homology to TNF, LT alpha, and the ligand for the CD40 receptor. The gene for LT beta was found next to the TNF-LT locus in the major histocompatibility complex (MHC), a region of the MHC with possible linkage to autoimmune disease. These observations raise the possibility that a surface LT alpha-LT beta complex may have a specific role in immune regulation distinct from the functions ascribed to TNF.

The murine anti-human common gamma chain monoclonal antibody CP.B8 blocks the second step in the formation of the intermediate affinity IL-2 receptor.

A murine monoclonal antibody, CP.B8, specific for the extracellular portion of the human common gamma (gammac) chain, and its Fab fragment are shown to block the binding of IL-2 to COS-7 cells transfected with the cDNA for the full-length IL-2 receptor beta (IL-2Rbeta) and gammac chains, components which together comprise the intermediate affinity IL-2 receptor (IL-2R) expressed on the surface of resting T cells, NK cells, and on certain intestinal epithelial cells. To investigate the mechanism of this inhibition, the extracellular portions of the IL-2Rbeta and gammac chains were expressed and purified, and their interactions with each other and with IL-2 were studied by gel filtration and by surface plasmon resonance (SPR). By gel filtration, a stable ternary complex was formed by association of the three proteins, while no stable binary complexes were detected between any two of the three proteins. By SPR analysis, IL-2 was shown to associate rapidly with IL-2Rbeta, forming a binary complex with an equilibrium dissociation constant (Kd) of 800 nM, which permitted subsequent association of the gammac chain. Dissociation of the IL-2/IL-2Rbeta/gammac chain complex was significantly slower than dissociation of the IL-2/IL-2Rbeta complex. Using these model systems, we tested the ability of mAb CP.B8 to inhibit the association of the gammac chain with IL-2 and IL-2Rbeta. By gel filtration, mAb CP.B8 formed a stable complex with the gammac chain, preventing its association with IL-2 and IL-2Rbeta. MAb CP.B8 was also capable of dissociating the gammac chain already complexed with IL-2 and IL-2Rbeta. SPR analysis confirmed these findings and showed, in addition, that the Fab fragment of CP.B8 was also capable of inhibiting the association of the gammac chain with the IL-2/IL-2Rbeta complex. We conclude that mAb CP.B8 blocks the second step in the formation of the intermediate affinity IL-2R on the surface of transfected COS-7 cells by binding at or close to a region on the gammac chain that is involved in contact with IL-2 and/or IL-2Rbeta.