Modulation of connexon densities in gap junctions of horizontal cell perikarya and axon terminals in fish retina: effects of light/dark cycles,interruption of the optic nerve and application of dopamine

Summary In the fish retina, connexon densities of gap junctions in the outer horizontal cells are modulated in response to different light or dark adaptation times and wavelengths. We have examined whether the connexon density is a suitable parameter of gap junction coupling under in situ conditions. Short-term light adaptation evoked low connexon densities, regardless of whether white or red light was used. Short-term dark adaptation evoked high connexon densities; this was more pronounced in the axon terminal than in perikaryal gap junctions. Under a 12 h red light/12 h dark cycle, a significant difference in connexon densities between the light and the dark period could be established in the gap junctions of the perikarya and axon terminals. Under a white light/dark cycle, only the gap junctions of axon terminals showed a significant difference. Crushing of the optic nerve resulted in an increase in connexon densities; this was more pronounced in axon terminals than in perikarya. Dopamine injected into the right eye of white-light-adapted animals had no effect. However, dopamine prevented the effect of optic-nerve crushing on connexon density. The reaction of axon-terminal gap junctions to different conditions thus resembles that of perikaryal gap junctions, but is more intense. Axon terminals are therefore thought to play an important role in the adaptation process.     

Identification of specific proteins and glycoproteins associated with membrane fractions isolated from Zea mays L. coleoptiles

The aim of this work was to identify proteins specific for plant cell membranes which could then be used as unique markers. A crude membrane fraction was isolated from corn coleoptiles and separated on non-linear sucrose density gradients. Separation of endoplasmic reticulum (NADH-cytochrome c reductase), mitochondria (cytochrome c oxidase), golgi (inosine diphosphatase), and plasma membranes (N-1-naphthylphthalamic acid-binding) was achieved. The membrane proteins from the gradient fractions were separated using sodium dodecyl sulphate-poly-acrylamide gel electrophoresis and the gels stained with coomassie blue or with concanavalin A/peroxidase to detect glycoproteins. Proteins specific for the various membranes were identified. Five proteins including two glycoproteins were plasma membrane markers. Protoplasts were isolated and iodinated using lactoperoxidase/glucose oxidase covalently attached to beads. Eleven iodinated proteins were found and three of these corresponded to proteins specifically associated with plasma membranes in the density gradients. Two methods for detecting Ca²⁺-binding proteins following sodium dodecylsulphate polyacrylamide gel electrophoresis were employed. The majority of such proteins were found in the endoplasmatic reticulum and one was specific for plasma membranes. In vitro and in vivo phosphorylation of membrane proteins was examined and the majority of proteins phosphorylated were glycoproteins. Two of the phosphorylated proteins (M_r=110,000 and 20,000) were also iodinated on protoplasts and may be part of the plasma membrane ATPases.Abbreviations ER endoplasmic reticulum - IDP inosine diphosphate - NPA N-1-naphthylphthalamic acid     

Synergy of human neutrophils with fluconazole in killing Candida species

The killing of Candida species by human neutrophils in a long-term 24-h assay and possible synergy with fluconazole (FCZ) for killing was investigated. The test medium (TM) consisted of RPMI-1640, penicillin and streptomycin (P/S), and 10% fresh autologous serum. TM alone was highly fungistatic for Candida species compared to TM without serum. When neutrophils were cocultured in TM with Candida species for 24 h the inoculum colony-forming units (CFU) were always significantly reduced (killing) by 58 to 99%. FCZ was tested over a range of 1–500 g/ml, and though almost always fungistatic itself, it synergized with neutrophils for significantly increased killing of C. albicans (isolate Sh27) (P<0.01) and C. albicans (isolate 94-20) (P<0.05). Killing of non-albicans Candida species was so efficient in the absence of FCZ that demonstration of synergy with FCZ was difficult.     

Study of the role of iron in the anticryptococcal activity of human serum and fluconazole

Anticryptococcal activity of human serum and apotransferrin in RPMI 1640 was studied in vitro. The effects of varying concentrations of FeCl₃ on this activity was investigated. Possible synergy of serum and apotransferrin with fluconazole was also measured. The fungistatic activity of human serum, whether lyophilized, stored at 4 °C, fresh frozen or purchased from commercial sources vs. Cryptococcus neoformans was comparable. There was no significant loss of fungistatic activity after freezing and thawing the serum up to 10 times. The fungistatic activity of human serum was similar when tested in different tissue culture media with the exception of Medium 199. The addition of apotransferrin (2.0 or 0.2 mg/ml) to RPMI 1640 had an inhibitory effect on cryptococcal growth. This effect was reversed by 20 M of FeCl₃ at both apotransferrin concentrations. By contrast, addition of FeCl₃ to human serum and RPMI 1640 did not reverse inhibition of growth. Fluconazole synergized with the human serum preparations described, but not with pooled commercial serum, for fungicidal activity. Synergistic activity of fluconazole and human serum was not affected by the addition of FeCl₃. Apotransferrin did not show any synergistic fungicidal activity with fluconazole.     

Mechanisms responsible for the positive diversity–productivity relationship in Minnesota grasslands

Species’ extinctions have spurred debate on whether interactions among few or among many species cause a positive diversity–productivity relationship in experimentally assembled grasslands. We addressed this question by quantifying the productivity of 14 species across an experimental diversity gradient in Minnesota. We found that interspecific interactions leading to coexistence and competitive displacement both determine which species overyield; i.e. are more productive at high diversity. Overyielding species were either superior N competitors (C4 grasses) or N fixers (legumes). Surprisingly, these species were not most productive in monoculture, thus, the ‘selection’ of productive species in diverse plots did not cause the positive diversity–productivity relationship. Both positive (with legumes) and negative interspecific interactions (with C4 grasses) determined whether individual species overyielded. Foliar pathogens did not cause overyielding, although other natural enemies may be responsible. Overyielding species are not displacing underyielding species over time, implying that other diversity‐promoting interactions also operate in this experiment.     

Kinetics and requirements for activation of macrophages for fungicidal activity: effect of protein synthesis inhibitors and immunosuppressants on activation and fungicidal mechanism.

Peritoneal-and pulmonary macrophages can be activated in vitro with lymphokines (LK) or IFN-gamma, without exogenous lipopolysaccharide, for fungicidal activity against several pathogenic fungi. However, neither the biochemical nor metabolic events of the activation process or of the effector phase have been defined. In the present work we sought to elucidate these events with time-course studies using inhibitors of protein synthesis as well as immunosuppressive agents. We found that protein synthesis inhibitors abrogated the activation process, because cycloheximide (CHX) (1-2 micrograms/ml) prevented activation of macrophages for fungicidal activity against Candida albicans, Blastomyces dermatitidis, and Paracoccidioides brasiliensis. Blocking of the activation process by CHX was not due to macrophage cytotoxicity, and CHX did not impair the ability of nonactivated macrophages to kill Candida parapsilosis. In kinetic studies we showed that activation of macrophages was induced in 4 hr of LK treatment and that CHX had no effect if added after this time. In contrast to CHX, therapeutic concentrations of hydrocortisone (HC), such as less than or equal to 5 micrograms/ml, or cyclosporin A (CsA), 5 micrograms/ml, did not significantly inhibit LK activation of macrophages for killing of fungi. In the effector phase, the fungicidal capacity of activated macrophages in short-term (less than or equal to 4 hr) killing assays could not be abrogated by CHX (5 micrograms/ml), HC (100 micrograms/ml), or CsA (10 micrograms/ml). These results demonstrate that the activation but not the effector mechanism of macrophages for fungicidal activity is blocked by inhibition of protein synthesis. In contrast, therapeutic concentrations of HC or CsA may not interfere with activation of macrophages or their killing mechanisms, thus providing a rationale for antifungal immunotherapy in certain clinical situations (e.g., infection in the immunosuppressed patient).     

PKD controls mitotic Golgi complex fragmentation through a Raf–MEK1 pathway

Before entering mitosis, the stacks of the Golgi cisternae are separated from each other, and inhibiting this process delays entry of mammalian cells into mitosis. Protein kinase D (PKD) is known to be involved in Golgi-to–cell surface transport by controlling the biogenesis of specific transport carriers. Here we show that depletion of PKD1 and PKD2 proteins from HeLa cells by small interfering RNA leads to the accumulation of cells in the G2 phase of the cell cycle and prevents cells from entering mitosis. We further provide evidence that inhibition of PKD blocks mitotic Raf-1 and mitogen-activated protein kinase kinase (MEK) activation, and, as a consequence, mitotic Golgi fragmentation, which could be rescued by expression of active MEK1. Finally, Golgi fluorescence recovery after photobleaching analyses demonstrate that PKD is crucial for the cleavage of the noncompact zones of Golgi membranes in G2 phase. Our findings suggest that PKD controls interstack Golgi connections in a Raf-1/MEK1–dependent manner, a process required for entry of the cells into mitosis.     

PRFS-Based MR Thermometry Versus an Alternative T1 Magnitude Method – Comparative Performance Predicting Thermally Induced Necrosis in Hepatic Tumor Ablation

Objective

To compare the accuracy of a semi-quantitative proton resonance frequency shift (PRFS) thermal mapping interface and an alternative qualitative T1 thermometry model in predicting tissue necrosis in an established routine setting of MRI-guided laser ablation in the human liver.

Materials and Methods

34 cases of PRFS-guided (GRE) laser ablation were retrospectively matched with 34 cases from an earlier patient population of 73 individuals being monitored through T1 magnitude image evaluation (FLASH 2D). The model-specific real-time estimation of necrotizing thermal impact (above 54 °C zone and T1 signal loss, respectively) was correlated in size with the resulting necrosis as shown by lack of enhancement on the first-day contrast exam (T1). Matched groups were compared using the Mann-Whitney test.

Results

Online PRFS guidance was available in 33 of 34 cases. Positive size correlation between calculated impact zone and contrast defect at first day was evident in both groups (p < 0.0004). The predictive error estimating necrosis was median 21 % (range 1 % - 52 %) in the PRFS group and 61 % (range 22 - 84 %) in the T1 magnitude group. Differences in estimating lethal impact were significant (p = 0.004), whereas the real extent of therapy-induced necrosis showed no significant difference (p > 0.28) between the two groups.

Conclusion

PRFS thermometry is feasible in a clinical setting of thermal hepatic tumor ablation. As an interference-free MR-tool for online therapy monitoring its accuracy to predict tissue necrosis is superior to a competing model of thermally induced alteration of the T1 magnitude signal.     

Analytical FcRn affinity chromatography for functional characterization of monoclonal antibodies

The neonatal Fc receptor (FcRn) is important for the metabolic fate of IgG antibodies in vivo. Analysis of the interaction between FcRn and IgG in vitro might provide insight into the structural and functional integrity of therapeutic IgG that may affect pharmacokinetics (PK) in vivo. We developed a standardized pH gradient FcRn affinity liquid chromatography method with conditions closely resembling the physiological mechanism of interaction between IgG and FcRn. This method allows the separation of molecular IgG isoforms, degradation products and engineered molecules based on their affinity to FcRn. Human FcRn was immobilized on the column and a linear pH gradient from pH 5.5 to 8.8 was applied. FcRn chromatography was used in comparison to surface plasmon resonance to characterize different monoclonal IgG preparations, e.g., oxidized or aggregated species. Wild-type and engineered IgGs were compared in vitro by FcRn chromatography and in vivo by PK studies in huFcRn transgenic mice. Analytical FcRn chromatography allows differentiation of IgG samples and variants by peak pattern and retention time profile. The method can distinguish: 1) IgGs with different Fabs, 2) oxidized from native IgG, 3) aggregates from monomer and 4) antibodies with mutations in the Fc part from wild-type IgGs. Changes in the FcRn chromatographic behavior of mutant IgGs relative to the wild-type IgG correlate to changes in the PK profile in the FcRn transgenic mice. These results demonstrate that FcRn affinity chromatography is a useful new method for the assessment of IgG integrity.