Light-induced fluorescence quenching in the light-harvesting chlorophyll a/b protein complex

Summary Irradiation of the principal photosystem II light-harvesting chlorophyll-protein antenna complex, LHC II, with high light intensities brings about a pronounced quenching of the chlorophyll fluorescence. Illumination of isolated thylakoids with high light intensities generates the formation of quenching centres within LHC II in vivo, as demonstrated by fluorescence excitation spectroscopy. In the isolated complex it is demonstrated that the light-induced fluorescence quenching: a) shows a partial, biphasic reversibility in the dark; b) is approximately proportional to the light intensity; c) is almost independent of temperature in the range 0–30°C; d) is substantially insensitive to protein modifying reagents and treatments; e) occurs in the absence of oxygen. A possible physiological importance of the phenomenon is discussed in terms of a mechanism capable of dissipating excess excitation energy within the photosystem II antenna.Abbreviations chla chlorophyll a - chlb chlorophyll b - F₀ fluorescence yield with reaction centers open - F_m fluorescence yield with reaction centres closed - F_i fluorescence at the plateau level of the fast induction phase - LHC II light-harvesting chlorophyll a/b protein complex II - PS II photosystem II - PSI photosystem I - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine     

Centrin- and α-actinin-like immunoreactivity in the ciliary rootlets of insect sensilla

Summary Long ciliary rootlets are a characteristic feature of the dendritic inner segments of the sensory cells in insect sensilla. These rootlets are composed of highly ordered filaments and are regularly cross-striated. Collagenase digestion and immunohistochemistry reveal that the rootlets are probably not composed of collagen fibers. However, double-labeling experiments with phalloidin and anti--actinins show that antibodies to -actinin react with the ciliary rootlets of the sensilla, but do not stain the scolopale, which is composed of actin filaments as visualized by phalloidin. Antibodies to centrin, a contractile protein isolated from flagellar rootlets of green algae, also stain the ciliary rootlets. Within the ciliary rootlets of insect sensilla, -actinin may be associated with filaments other than actin filaments. The immunohistochemical localization of a centrin-like protein suggests that contractions probably occur within the rootlets. The centrin-like protein may play a role during the mechanical transduction or adaptation of the sensilla.     

Presynaptic Mechanism of Action of 4-Aminopyridine: Changes in Intracellular Free Ca2+ Concentration and Its Relationship to B-50 (GAP-43) Phosphorylation

Abstract: Recently we have shown that 4-aminopyridine (4-AP), a drug known to enhance transmitter release, stimulates the phosphorylation of the protein kinase C substrate B-50 (GAP-43) in rat brain synaptosomes and that this effect is dependent on the presence of extracellular Ca²⁺. Hence, we were interested in the relationship between changes induced by 4-AP in the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and B-50 phosphorylation in synaptosomes. 4-AP (100 μ M ) elevates the [Ca²⁺]_i (as determined with fura-2) to approximately the same extent as depolarization with 30 m M K⁺ (from an initial resting level of 240 n M to ∼480 n M after treatment). However, the underlying mechanisms appear to be different: In the presence of 4-AP, depolarization with K⁺ still evoked an increase in [Ca²⁺]_i, which was additive to the elevation caused by 4-AP. Several Ca²⁺ channel antagonists (CdCl₂, LaCl₃, and diphenylhydantoin) inhibited the increase in B-50 phosphorylation by 4-AP. It is interesting that the increase in [Ca²⁺]_i and the increase in B-50 phosphorylation by 4-AP were attenuated by tetrodotoxin, a finding pointing to a possible involvement of Na⁺ channels in this action. These results suggest that 4-AP (indirectly) stimulates both Ca²⁺ influx and B-50 phosphorylation through voltage-dependent channels by a mechanism dependent on Na⁺ channel activity.     

MET inhibition enhances PARP inhibitor efficacy in castration-resistant prostate cancer by suppressing the ATM/ATR and PI3K/AKT pathways

Up to 30% of patients with metastatic castration-resistant prostate cancer (CRPC) patients carry altered DNA damage response genes, enabling the use of poly adenosine diphosphate–ribose polymerase (PARP) inhibitors in advanced CRPC. The proto-oncogene mesenchymal–epithelial transition (MET) is crucial in the migration, proliferation, and invasion of tumour cells. Aberrant expression of MET and its ligand hepatocyte growth factor is associated with drug resistance in cancer therapy. Here, we found that MET was highly expressed in human CRPC tissues and overexpressed in DU145 and PC3 cells in a drug concentration-dependent manner and is closely related to sensitivity to PARP inhibitors. Combining the PARP inhibitor olaparib with the MET inhibitor crizotinib synergistically inhibited CRPC cell growth both in vivo and in vitro. Further analysis of the underlying molecular mechanism underlying the MET suppression-induced drug sensitivity revealed that olaparib and crizotinib could together downregulate the ATM/ATR signaling pathway, inducing apoptosis by inhibiting the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathway, enhancing the olaparib-induced antitumour effect in DU145 and PC3 cells. In conclusion, we demonstrated that MET inhibition enhances sensitivity of CRPC to PARP inhibitors by suppressing the ATM/ATR and PI3K/AKT pathways and provides a novel, targeted therapy regimen for CRPC.     

GoNe encoding a class VIIIb AP2/ERF is required for both extrafloral and floral nectary development in Gossypium

The floral nectary, first recognized and described by Carl Linnaeus, is a remarkable organ that serves to provide carbohydrate-rich nectar to visiting pollinators in return for gamete transfer between flowers. Therefore, the nectary has indispensable biological significance in plant reproduction and even in evolution. Only two genes, CRC and STY, have been reported to regulate floral nectary development. However, it is still unknown what genes contribute to extrafloral nectary development. Here, we report that a nectary development gene in Gossypium (GoNe), annotated as an APETALA 2/ethylene-responsive factor (AP2/ERF), is responsible for the formation of both floral and extrafloral nectaries. GoNe plants that are silenced via virus-induced gene silencing technology and/or knocked out by Cas9 produce a nectariless phenotype. Point mutation and gene truncation simultaneously in duplicated genes Ne₁Ne₂ lead to impaired nectary development in tetraploid cotton. There is no difference in the expression of the CRC and STY genes between the nectary TM-1 and the nectariless MD90ne in cotton. Therefore, the GoNe gene responsible for the formation of floral and extrafloral nectaries may be independent of CRC and STY. A complex mechanism might exist that restricts the nectary to a specific position with different genetic factors. Characterization of these target genes regulating nectary production has provided insights into the development, evolution, and function of nectaries and insect-resistant breeding.     

Erianin induces triple-negative breast cancer cells apoptosis by activating PI3K/Akt pathway

Background: Triple-negative breast cancer (TNBC) is a refractory subtype of breast cancer, 25–30% of which have dysregulation in the PI3K/AKT pathway. The present study investigated the anticancer effect of erianin on TNBC cell line and its underlying mechanism.Methods: After treatment with erianin, MTT assay was employed to determine the MDA-MB-231 and EFM-192A cell proliferation, the nucleus morphological changes were observed by DAPI staining. The cell cycle and apoptotic proportion were detected by flow cytometry. Western blot was performed to determine the cell cycle and apoptosis-related protein expression and PI3K pathways. Finally, the antiproliferative activity of erianin was further confirmed by adding or not adding PI3K agonists SC79.Results: Erianin inhibited the proliferation of MDA-MB-231 and EFM-192A cells in a dose-dependent manner, the IC₅₀ were 70.96 and 78.58 nM, respectively. Erianin could cause cell cycle arrest at the G₂/M phase, and the expressions of p21 and p27 were up-regulated, while the expressions of CDK1 and Cyclin B1 were down-regulated. Erianin also induced apoptosis via the mitochondrial pathway, with the up-regulation of the expression of Cyto C, PARP, Bax, active form of Caspase-3, and Caspase-9. Furthermore, p-PI3K and p-Akt expression were down-regulated by erianin. After co-incubation with SC79, the cell inhibition rate of erianin was decreased, which further confirmed that the attenuated PI3K/Akt pathway was relevant to the pro-apoptotic effect of erianin.Conclusions: Erianin can inhibit the proliferation of TNBC cells and induce cell cycle arrest and apoptosis, which may ascribe to the abolish the activation of the PI3K/Akt pathway.     

Calcium and temperature regulation of the stability of the human platelet integrin GPIIb/IIIa in solution: an analytical ultracentrifugation study

The human platelet integrin GPIIb/IIIa (228 kDa), a Ca-dependent heterodimer formed by the _IIb subunit (GPIIb, 136 kDa) and the ₃ subunit (GPIIIa, 92 kDa), serves as the fibrinogen receptor at the surface of activated platelets. The degree of dissociation of the GPIIb/IIIa heterodimer (s°₂₀ ^*, 8.9 S) into its constituent glycoproteins (GPIIb, 5.8 S; and GPIIIa, 3.9 S) has been assessed by analytical ultracentrifugation in Triton X100 buffers, and its Ca²⁺- and temperature-dependence correlated with Ca²⁺-binding to GPIIb/IIIa and its temperature dependence. At 21°C half-maximal dissociation of GPIIb/IIIa occurs at 5.5 ± 2.5 × 10^–8 M Ca²⁺, very close to the dissociation constant of the high affinity Ca-binding site of GPIIb/IIIa (Kd₁ 8 ± 3 × 10^–8 M) (Rivas and González-Rodríguez, 1991) and much lower than the Kd of the 3.4 medium affinity Ca-binding sites (Kd₂ 4 ± 1.5 × 10^–5 M), which seems to demonstrate that the stability of the heterodimer in solution at room temperature is regulated by the degree of saturation of the high-affinity Ca-binding site. At 4°C, the stability of the heterodimer is apparently Ca²⁺-independent, while at room and physiological temperatures (15–37°C) the degree of dissociation of the heterodimer is regulated by the degree of dissociation of the high- and medium-affinity Ca-binding sites, respectively. On increasing the Ca²⁺ concentration up to 1 × 10^–4 M after dissociation in Triton X100 solutions, the reconstitution of the GPIIb/IIIa heterodimer depends on the time and temperature at which the dissociated heterodimer was maintained, being almost complete within the first 5–10 min at 37°C and within the first 1–2 h at 21°C. After this time, a time- and temperature-dependent irreversible autoassociation of GPIIb (covalent) and GPIIIa (non-covalent) occurs, which hinders both the isolation of permanently stable monoamers of GPIIb and GPIIIa and the reconstitution of the GPIIb/IIIa heterodimer in Triton X100 solutions. Abbreviations: GPIIb, GPIIIa, and GPIIb/IIIa, glycoprotein IIb, IIIa, and the heterodimer formed by them, respectively; s°₂₀ ^*, the sedimentation coefficient of the glycoprotein-detergent complexes determined at 20°C, after extrapolation to zero-glycoprotein concentration Offprint requests to: J. González-Rodríguez     

Different oligosaccharides accumulate in the brain and urine of a cat with α-mannosidosis: structure determination of five brain-derived and seventeen urinary oligosaccharides

Five brain-derived and 17 urinary oligomannose-type oligosaccharides were isolated by ion-exchange chromatography on Mono Q or Dowex, followed by HPLC on Lichrosorb-NH₂ from a Persian cat suffering from -mannosidosis. The structures ofthe carbohydrate chains were determined by 500- or 600-MHz¹H-NMR spectroscopy. Different oligosaccharide patterns were found in brain and urine. 99% of the urinary oligosaccharides possess an (1-6)-linked mannose residue attached to -mannose, whereas only 5% of the brain-derived oligosaccharides contain such a residue. Furthermore, of the urinary carbohydrate chains 71% end with Man1-4GlcNAc1-4GlcNAc and 29% end with Man1-4GlcNAc, whereas the corresponding amounts are 23% and 77%, respectively, for the brain-derived oligosaccharides.Abbreviations MLEV-17 composite pulse devised by M. Levitt - HOHAHA homonuclear Hartman-Hahn spectroscopy - TPPI time-proportional phase incrementation - 2D two dimensional - GlcNAc N-acetylglucosamine - Man mannose - Fuc fucose     

Immunocytochemical localization of conglutin γ and legumin-like globulin in developing and mature seeds ofLupinus albus L.

Summary The distribution of two seed proteins, namely conglutin and a legumin-like globulin, in developing and mature seeds ofLupinus albus L. has been examined by immunocytochemistry and the concomitant modifications of their constituent polypeptides followed by SDS-PAGE. Both proteins were found within vacuolar protein bodies in various tissues of the cotyledons, although with some differences in the distribution patterns. The legumin-like protein was found to be deposited within the large storage parenchyma cells of the cotyledons in a manner similar to that reported for other storage proteins; little or no immunolabelling was associated with the cotyledonary epidermal and vascular parenchyma cells. In contrast conglutin was present in all cell types.A precursor of the legumin-like protein accumulated transiently in the developing cotyledon, but was subsequently modified by proteolytic cleavage. The onset of such modification was concomitant with a transition in the predominant vacuolar forms within the storage parenchyma cells. No precursor molecules of conglutin have been detected in this study, thus indicating that this protein is deposited in the protein bodies in its mature form.Abbreviations LM light microscopy - EM electron microscopy - DAF days after flowering - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - GAR goat antirabbit antiserum