Mapping putative contact sites between subunits in a bacterial ATP-binding cassette (ABC) transporter by synthetic peptide libraries

The maltose ATP-binding cassette transporter of Salmonella typhimurium is composed of the soluble periplasmic receptor, MalE, and a membrane-associated complex comprising one copy each of the pore-forming hydrophobic subunits, MalF and MalG, and of a homodimer of the ATP-hydrolyzing subunit, MalK. During the transport process the subunits are thought to undergo conformational changes that might transiently alter molecular contacts between MalFG and MalK(2). In order to map sites of subunit-subunit interactions we have used a comprehensive peptide mapping approach comprising large-scale microsynthesis of labelled probes and array techniques. In particular, we screened the binding of (i) MalFG-derived soluble biotinylated peptides to immobilized MalK, and (ii) radiolabelled MalK to MalFG-derived cellulose membrane-bound peptides. The first approach identified seven peptides (10mers) each of MalF and MalG that specifically bound to MalK. The peptides were localized to TMDs 3 and 6, periplasmic loop P4 and cytoplasmic loops C2 and C3 of MalF, while MalG-derived peptides localized to the N terminus, TMDs 4-6, periplasmic loop P1 and cytoplasmic loop C2. Peptides from C3 and C2, respectively, of MalF and MalG partially encompass the conserved EAA-motif, known to be crucial for interaction with MalK. These results were basically confirmed by screening MalFG-derived peptide arrays consisting of 16mers or 31mers with radiolabelled MalK. This approach also allowed us to perform complete substitutional analyses of peptides in question. The results led to the construction of MalFG variants that were subsequently analyzed for functional consequences in vivo. Growth experiments revealed that most of the mutations had no phenotype, suggesting that the mutated residues themselves are not critical but part of a discontinuous binding site. However, two novel mutations affecting residues from the EAA motifs of MalF (Ile417Glu) and MalG (Phe203Gln/Asn), respectively, displayed severe growth defects, indicating their functional importance. Together, these experimental outcomes identify specific molecular contacts made between MalK and MalFG that extend beyond the well-characterized EAA motif.     

Synthesis and antiproliferative activity of two diastereomeric lignan amides serving as dimeric caffeic acid-l-DOPA hybrids

Two new diastereomeric lignan amides (4 and 5) serving as dimeric caffeic acid-l-DOPA hybrids were synthesized. The synthesis involved the FeCl₃-mediated phenol oxidative coupling of methyl caffeate to afford trans-diester 1a as a mixture of enantiomers, protection of the catechol units, regioselective saponification, coupling with a suitably protected l-DOPA derivative, separation of the two diastereomers thus obtained by flash column chromatography and finally global chemoselective deprotection of the catechol units. The effect of hybrids 4 and 5 and related compounds on the proliferation of two breast cancer cell lines with different metastatic potential and estrogen receptor status (MDA-MB-231 and MCF-7) and of one epithelial lung cancer cell line, namely A-549, was evaluated for concentrations ranging from 1 to 256 μM and periods of treatment of 24, 48 and 72 h. Both hybrids showed interesting and almost equipotent antiproliferative activities (IC₅₀ 64–70 μM) for the MDA-MB-231 cell line after 24–48 h of treatment, but they were more selective and much more potent (IC₅₀ 4–16 μM) for the MCF-7 cells after 48 h of treatment. The highest activity for both hybrids and both breast cancer lines was observed after 72 h of treatment (IC₅₀ 1–2 μM), probably as the result of slow hydrolysis of their methyl ester functions.     

The effects of the caffeic acid phenethyl ester (CAPE) on erythrocyte membrane damage after hind limb ischaemia-reperfusion

Reactive oxygen species have been implicated in pathogenesis injury after ischaemia-reperfusion (I/R). Caffeic Acid Phenethyl Ester (CAPE), an active component of honeybee propolis extract, exhibits antioxidant and anti-inflammatory properties. The aim of this study was to investigate the effects of CAPE on erythrocyte membrane damage after hind limb I/R. Rats were divided into two groups: I/R and I/R with CAPE pre-treatment. They were anaesthetized with intramuscular ketamine 100 mg kg(-1). A 4-h I/R period was performed on the right hind limb of all animals. In the CAPE-treated group, animals received CAPE 10 microm by intraperitoneal injection 1 h before the reperfusion. At the end of the reperfusion period, a midsternotomy was performed. A 5-ml blood sample was withdrawn from the ascending aorta for biochemical assays. Serum and erythrocyte membrane MDA levels were significantly lower in the CAPE-treated group when compared to the I/R group ( p = 0.001 and p<0.001, respectively). Erythrocyte membrane Na(+)-K(+) ATPases activity in the CAPE-treated group was significantly higher than the I/R group ( p<0.001). In conclusion, CAPE seems to be effective in protecting against oxidative stress. Therefore, we suggest that in order to decrease I/R injury, pre-administration of CAPE may be a promising agent for a variety of conditions associated with I/R.     

Acaulonopsis,a new moss genus of the Pottiaceae from Western Cape Province of South Africa,and comments on Vrolijkheidia

Abstract

A new genus, Acaulonopsis (Pottiaceae, Bryophyta), including two new species, A. fynbosensis and A. eureka, is described from the Western Cape Province of South Africa. Together with the similarly much reduced pottiaceous genus Acaulon, Acaulonopsis is unique in the family with a very short seta and spherical capsule, which lacks the apiculus found in other genera. The new genus is also distinguished by short plant stature, and a total of about five ovate leaves that clasp closely the capsules in nearly spherical leaf bases. Continuing bryological study of the fynbos region has recently resulted in a number of startling discoveries of bryophytes new to science and distinct at the genus level. A new combination is made for the South African endemic genus Vrolijkheidia (Pottiaceae) with discussion of its dimorphic habitus.     

Involvement of nitric oxide in the promotion of cell survival by ceramide 1-phosphate

Macrophages play vital roles in inflammatory responses, and their number at sites of inflammation is strictly regulated by cell death and division. Here, we demonstrate that production of nitric oxide (NO) is a major mechanism whereby ceramide-1-phosphate (C1P) blocks apoptosis in macrophages. However, NO failed to stimulate macrophage proliferation. The prosurvival effect of C1P was blocked by inhibitors of inducible NO synthase. The antiapoptotic effect of C1P was also blocked by phosphatidylinositol 3-kinase or nuclear factor-kappa B inhibitors. Moreover, NO reversed the inhibitory effect of C1P on acid sphingomyelinase, but the prosurvival effect of C1P was independent of this action.     

Caffeic acid phenethyl ester induces mitochondria-mediated apoptosis in human myeloid leukemia U937 cells

Caffeic acid phenyl ester (CAPE), a biologically active ingredient of propolis, has several interesting biological properties including antioxidant, anti-inflammatory, antiviral, immunostimulatory, anti-angiogenic, anti-invasive, anti-metastatic and carcinostatic activities. Recently, several groups have reported that CAPE is cytotoxic to tumor cells but not to normal cells. In this study, we investigated the mechanism of CAPE-induced apoptosis in human myeloid leukemia U937 cells. Treatment of U937 cells with CAPE decreased cell viability in a dose-dependent and time-dependent manner. DNA fragmentation assay revealed the typical ladder profile of oligonucleosomal fragments in CAPE-treated U937 cells. In addition, as evidenced by the nuclear DAPI staining experiment, we observed that the nuclear condensation, a typical phenotype of apoptosis, was found in U937 cells treated with 5 μg/ml of CAPE. Therefore, it was suggested that CAPE is a potent agent inducing apoptosis in U937 cells. Apoptotic action of the CAPE was accompanied by release of cytochrome C, reduction of Bcl-2 expression, increase of Bax expression, activation/cleavage of caspase-3 and activation/cleavage of PARP in U937 cells, but not by Fas protein, an initial mediator in the death signaling, or by phospho-eIF2α and CHOP, crucial mediators in ER-mediated apoptosis. From the results, it was concluded that CAPE induces the mitochondria-mediated apoptosis but not death receptors- or ER-mediated apoptosis in U937 cells. Jin and Song contributed equally to this article.     

Does reservoir trophic status influence the feeding and growth of the sharptooth catfish,Clarias gariepinus (Teleostei: Clariidae)?

The diet and growth of sharptooth catfish, Clarias gariepinus, in an oligotrophic system (Kat River Reservoir, Eastern Cape, South Africa) were compared to those in a eutrophic system (Laing Reservoir, Eastern Cape) to determine if the trophic status of a waterbody had an effect on the growth rate of the species. In order of importance, the diet of catfish in Kat River Reservoir consisted of fish, insects, zooplankton, plant material and other items, while the diet of catfish in Laing Reservoir consisted of fish, plant material, zooplankton, other vertebrates and insects. The diets of catfish in the two reservoirs had a similarity index of 68.1% and there was no significant difference in their nutritional value. Fish prey was the most important dietary component in both reservoirs. Temperature regime and zooplankton and zoobenthos density were similar in both systems. However, fish prey density was significantly higher in the eutrophic Laing Reservoir and catfish grew significantly faster in that system. The slower growth rate in Kat River Reservoir was attributed to the higher energy costs associated with the capture of fish prey, which was less abundant than in Laing Reservoir. Trophic status therefore had an indirect effect on catfish growth by influencing the availability of fish prey.     

microRNA (miRNA) speciation in Alzheimer’s disease (AD) cerebrospinal fluid (CSF) and extracellular fluid (ECF)

Human cerebrospinal fluid (CSF), produced by the choroid plexus and secreted into the brain ventricles and subarachnoid space, plays critical roles in intra-cerebral transport and the biophysical and immune protection of the brain. CSF composition provides valuable insight into soluble pathogenic bio-markers that may be diagnostic for brain disease. In these experiments we analyzed amyloid beta (Aβ) peptide and micro RNA (miRNA) abundance in CSF and in short post-mortem interval (PMI <2.1 hr) brain tissue-derived extracellular fluid (ECF) from Alzheimer’s disease (AD) and age-matched control neocortex. There was a trend for decreased abundance of Aβ42 in the CSF and ECF in AD but it did not reach statistical significance (mean age ~72 yr; N=12; p~0.06, ANOVA). The most abundant nucleic acids in AD CSF and ECF were miRNAs, and their speciation and inducibility were studied further. Fluorescent miRNA-array-based analysis indicated significant increases in miRNA-9, miRNA-125b, miRNA-146a, miRNA-155 in AD CSF and ECF (N=12; p<0.01, ANOVA). Primary human neuronal-glial (HNG) cell co-cultures stressed with AD-derived ECF also displayed an up-regulation of these miRNAs, an effect that was quenched using the anti-NF-кB agents caffeic acid phenethyl ester (CAPE) or 1-fluoro-2-[2-(4-methoxy-phenyl)-ethenyl]-benzene (CAY10512). Increases in miRNAs were confirmed independently using a highly sensitive LED-Northern dot-blot assay. Several of these NF-кB-sensitive miRNAs are known to be up-regulated in AD brain, and associate with the progressive spreading of inflammatory neurodegeneration. The results indicate that miRNA-9, miRNA-125b, miRNA-146a and miRNA-155 are CSF- and ECF-abundant, NF-кB-sensitive pro-inflammatory miRNAs, and their enrichment in circulating CSF and ECF suggest that they may be involved in the modulation or proliferation of miRNA-triggered pathogenic signaling throughout the brain and central nervous system (CNS).     

CAPE (caffeic acid phenethyl ester) stimulates glucose uptake through AMPK (AMP-activated protein kinase) activation in skeletal muscle cells

Caffeic acid phenethyl ester (CAPE), a flavonoid-like compound, is one of the major components of honeybee propolis. In the present study, we investigated the metabolic effects of CAPE in skeletal muscle cells and found that CAPE stimulated glucose uptake in differentiated L6 rat myoblast cells and also activated AMPK (AMP-activated protein kinase). In addition, the inhibition of AMPK blocked CAPE-induced glucose uptake, and CAPE activated the Akt pathway in a PI3K (phosphoinositide 3-kinase)-dependent manner. Furthermore, CAPE enhanced both insulin-mediated Akt activation and glucose uptake. In summary, our results suggest that CAPE may have beneficial roles in glucose metabolism via stimulation of the AMPK pathway.