Fluorescence induction in the phycobilisome-containing cyanobacterium Synechococcus sp PCC 7942: Analysis of the slow fluorescence transient

At room temperature, the chlorophyll (Chl) a fluorescence induction (FI) kinetics of plants, algae and cyanobacteria go through two maxima, P at ∼ 0.2-1 and M at ∼ 100-500 s, with a minimum S at ∼ 2-10 s in between. Thus, the whole FI kinetic pattern comprises a fast OPS transient (with O denoting origin) and a slower SMT transient (with T denoting terminal state). Here, we examined the phenomenology and the etiology of the SMT transient of the phycobilisome (PBS)-containing cyanobacterium Synechococcus sp PCC 7942 by modifying PBS → Photosystem (PS) II excitation transfer indirectly, either by blocking or by maximizing the PBS → PS I excitation transfer. Blocking the PBS → PS I excitation transfer route with N-ethyl-maleimide [NEM; A. N. Glazer, Y. Gindt, C. F. Chan, and K.Sauer, Photosynth. Research 40 (1994) 167-173] increases both the PBS excitation share of PS II and Chl a fluorescence. Maximizing it, on the other hand, by suspending cyanobactrial cells in hyper-osmotic media [G. C. Papageorgiou, A. Alygizaki-Zorba, Biochim. Biophys. Acta 1335 (1997) 1-4] diminishes both the PBS excitation share of PS II and Chl a fluorescence. Here, we show for the first time that, in either case, the slow SMT transient of FI disappears and is replaced by continuous P → T fluorescence decay, reminiscent of the typical P → T fluorescence decay of higher plants and algae. A similar P → T decay was also displayed by DCMU-treated Synechococcus cells at 2 °C. To interpret this phenomenology, we assume that after dark adaptation cyanobacteria exist in a low fluorescence state (state 2) and transit to a high fluorescence state (state 1) when, upon light acclimation, PS I is forced to run faster than PS II. In these organisms, a state 2 → 1 fluorescence increase plus electron transport-dependent dequenching processes dominate the SM rise and maximal fluorescence output is at M which lies above the P maximum of the fast FI transient. In contrast, dark-adapted plants and algae exist in state 1 and upon illumination they display an extended P → T decay that sometimes is interrupted by a shallow SMT transient, with M below P. This decay is dominated by a state 1 → 2 fluorescence lowering, as well as by electron transport-dependent quenching processes. When the regulation of the PBS → PS I electronic excitation transfer is eliminated (as for example in hyper-osmotic suspensions, after NEM treatment and at low temperature), the FI pattern of Synechococcus becomes plant-like.     

Synthesis of the β-1,3-glucan, laminarahexaose: NMR and conformational studies

The synthesis of laminarahexaose is described. NMR studies of several of the intermediates leading to the β-1,3-glucan show anomalously small coupling constants for some of the C-1 hydrogens. An X-ray structure for the protected hexasaccharide shows that the small coupling constants are due to some of the glucopyranose rings adopting a twist-boat conformation. The X-ray studies also explain other unexpected NMR observations.     

Ghrelin O-acyltransferase (GOAT) has a preference for n-hexanoyl-CoA over n-octanoyl-CoA as an acyl donor

Ghrelin is a peptide hormone in which serine 3 is modified by n-octanoic acid through GOAT (ghrelin O-acyltransferase). However, the enzymological properties of GOAT remain to be elucidated. We analyzed the in vitro activity of GOAT using the recombinant enzyme. Unexpectedly, although the main active form of ghrelin is modified by n-octanoic acid, GOAT had a strong preference for n-hexanoyl-CoA over n-octanoyl-CoA as an acyl donor. Moreover, a four-amino acid peptide derived from the N-terminal sequence of ghrelin can be modified by GOAT, indicating that these four amino acids constitute the core motif for substrate recognition by the enzyme.     

Discovery of new low-molecular-weight p53–Mdmx disruptors and their anti-cancer activities

Although several p53–Mdm2-binding disruptors have been identified to date, few studies have been published on p53–Mdmx-interaction inhibitors. In the present study, we demonstrated that o-aminothiophenol derivatives with molecular weights of 200–300 selectively inhibited the p53–Mdmx interaction. S-2-Isobutyramidophenyl 2-methylpropanethioate (K-178) (1c) activated p53, up-regulated the expression of its downstream genes such as p21 and Mdm2, and preferentially inhibited the growth of cancer cells with wild-type p53 over those with mutant p53. Furthermore, we found that the S-isobutyryl-deprotected forms 1b and 3b of 1c and S-2-benzamidophenyl 2-methylpropanethioate (K-181) (3c) preferentially inhibited the p53–Mdmx interaction over the p53–Mdm2 interaction, respectively, by using a Flag-p53 and glutathione S-transferase (GST)-fused protein complex (Mdm2, Mdmx, DAPK1, or PPID). In addition, the interaction of p53 with Mdmx was lost by replacing a sulfur atom with an oxygen atom in 1b and 1c. These results suggest that sulfides such as 1b, 3b, 4b, and 5b interfere with the binding of p53–Mdmx, resulting in the dissociation of the two proteins. Furthermore, the results of oral administration experiments using xenografts in nude mice indicated that 1c reduced the volume of tumor masses to 49.0% and 36.6% that of the control at 100 mg/kg and 150 mg/kg, respectively, in 40 days.     

Structural-Thermodynamic Relationships of Interactions in the N-Terminal ATP-Binding Domain of Hsp90

Despite its importance as a target in anti-cancer therapeutics and the numerous rational-based inhibitor design efforts aimed at it, there are only limited data available on structural-thermodynamic relationships of interactions of the N-terminal ATP-binding domain of Hsp90 (N-Hsp90). Here, we redress this by presenting an investigation of binding of nucleotides and ansamycin compounds to this domain. Interactions of nucleotides with N-Hsp90 are relatively weak (> 10 μM) and are strongly enthalpy driven over the temperature range 10-25 °C. Geldanamycin (GA) and its analogues 17-AAG [17-(allylamino)-17-demethoxy-GA] and 17-DMAG (17-N,N-dimethylaminoethylamino-17-demethoxy-GA) bind more strongly and have a dominant favourable enthalpic contribution over the temperature range investigated. We investigated the temperature dependence of the enthalpic contribution to binding. We found that while the ansamycin compounds have the commonly observed negative value, the nucleotides show a negligible or even a positive ΔC_p of binding. These data represent the first observation of a single binding site for which interactions with different ligands result in both negative and positive ΔC_p values. By addressing the likely impact of the potential contributions from protein-ligand interactions, we are able to attribute the anomalous ΔC_p for the nucleotides largely to a change in the conformation of the domain structure and local motion in the lid region of N-Hsp90 with the concomitant exposure of hydrophobic amino acid side chains.     

Phosphorylase activity in relation to starch synthesis in developing Hordeum distichum grain

Activity, control and primer requirements of starch phosphorylase in developing barley endosperm were investigated. Phosphorylase was detected in endosperm extracts from 3 days after anthesis. Unprimed activity was predominant between 2 and 10 days after anthesis, when it constituted 70–80% of total activity, but this proportion declined rapidly as the grain developed. The existence of at least 2 isoenzymes was indicated by studies of pH dependence and phosphate inhibition, and was further supported by acrylamide gel electrophoresis and column chromatography using DEAE-cellulose. The two isoenzymes which ere possibly both glyco proteins, appear in barley endosperm soon after anthesis. One appears capable of unprimed activity, and may be associated with the initiation of a-1,2 glucans, which then serve as primers for starch synthetase. This disappears by 13–15 days after anthesis. The other isoenzyme is capable of some unprimed activity but undergoes modification between 15 and 20 days after anthesis, resulting in the loss of unprimed activity. The relevance of the results to initiation of starch synthesis and to starch synthetase in amyloplasts is discussed.     

Heliothrix oregonensis,gen. nov., sp. nov., a phototrophic filamentous gliding bacterium containing bacteriochlorophyll a

An unusual filamentous, gliding bacterium was found in a few hot springs in Oregon where it formed a nearly unispecific top layer of microbial mats. It contained a bacteriochlorophyll a-like pigment and an abundance of carotenoids. There were no chlorosomes or additional chlorophylls. The organism was aerotolerant and appeared to be photoheterotrophic. It was successfully co-cultured with an aerobic chemoheterotroph in a medium containing glucose and casamino acids. Although it has many characteristics in common with the genus Chloroflexus, the lack of chlorosomes and bacteriochlorophyll c and the aerobic nature of this organism indicate that it should be placed in a new genus. This conclusion is supported by 5S rRNA nucleotide sequence data.     

Androgen receptor suppresses vasculogenic mimicry in hepatocellular carcinoma via circRNA7/miRNA7‐5p/VE‐cadherin/Notch4 signalling

Androgen receptor (AR) can suppress hepatocellular carcinoma (HCC) invasion and metastasis at an advanced stage. Vasculogenic mimicry (VM), a new vascularization pattern by which tumour tissues nourish themselves, is correlated with tumour progression and metastasis. Here, we investigated the effect of AR on the formation of VM and its mechanism in HCC. The results suggested that AR could down‐regulate circular RNA (circRNA) 7, up‐regulate micro RNA (miRNA) 7‐5p, and suppress the formation of VM in HCC Small hairpin circR7 (ShcircR7) could reverse the impact on VM and expression of VE‐cadherin and Notch4 increased by small interfering AR (shAR) in HCC, while inhibition of miR‐7‐5p blocked the formation of VM and expression of VE‐cadherin and Notch4 decreased by AR overexpression (oeAR) in HCC. Mechanism dissection demonstrated that AR could directly target the circR7 host gene promoter to suppress circR7, and miR‐7‐5p might directly target the VE‐cadherin and Notch4 3′UTR to suppress their expression in HCC. In addition, knockdown of Notch4 and/or VE‐cadherin revealed that shVE‐cadherin or shNotch4 alone could partially reverse the formation of HCC VM, while shVE‐cadherin and shNotch4 together could completely suppress the formation of HCC VM. Those results indicate that AR could suppress the formation of HCC VM by down‐regulating circRNA7/miRNA7‐5p/VE‐Cadherin/Notch4 signals in HCC, which will help in the design of novel therapies against HCC.     

The protective effects of MSC‐EXO against pulmonary hypertension through regulating Wnt5a/BMP signalling pathway

The aim of the study was to explore the mechanism of mesenchymal stem cell‐derived exosomes (MSC‐EXO) to protect against experimentally induced pulmonary hypertension (PH). Monocrotaline (MCT)‐induced rat model of PH was successfully established by a single intraperitoneal injection of 50 mg/kg MCT, 3 weeks later the animals were treated with MSC‐EXO via tail vein injection. Post‐operation, our results showed that MSC‐EXO could significantly reduce right ventricular systolic pressure (RVSP) and the right ventricular hypertrophy index, attenuate pulmonary vascular remodelling and lung fibrosis in vivo. In vitro experiment, the hypoxia models of pulmonary artery endothelial cell (PAEC) and pulmonary vascular smooth muscle cell (PASMC) were used. We found that the expression levels of Wnt5a, Wnt11, BMPR2, BMP4 and BMP9 were increased, but β‐catenin, cyclin D1 and TGF‐β1 were decreased in MSC‐EXO group as compared with MCT or hypoxia group in vivo or vitro. However, these increased could be blocked when cells were transfected with Wnt5a siRNA in vitro. Taken together, these results suggested that the mechanism of MSC‐EXO to prevent PH vascular remodelling may be via regulation of Wnt5a/BMP signalling pathway.