Post-transcriptional regulation of vascular endothelial growth factor mRNA by the product of the VHL tumor suppressor gene.

The VHL tumor suppressor gene is inactivated in patients with von Hippel-Lindau disease and in most sporadic clear cell renal carcinomas. Although VHL protein function remains unclear, VHL does interact with the elongin BC subunits in vivo and regulates RNA polymerase II elongation activity in vitro by inhibiting formation of the elongin ABC complex. Expression of wild-type VHL in renal carcinoma cells with inactivated endogenous VHL resulted in unaltered in vitro cell growth and decreased vascular endothelial growth factor (VEGF) mRNA expression and responsiveness to serum deprivation. VEGF is highly expressed in many tumors, including VHL-associated and sporadic renal carcinomas, and it stimulates neoangiogenesis in growing solid tumors. Despite 5-fold differences in VEGF mRNA levels, VHL overexpression did not affect VEGF transcription initiation or elongation as would have been suggested by VHL-elongin association. These results suggest that VHL regulates VEGF expression at a post-transcriptional level and that VHL inactivation in target cells causes a loss of VEGF suppression, leading to formation of a vascular stroma.

Formation of junctions involved in excitation-contraction coupling in skeletal and cardiac muscle.

During excitation-contraction (e-c) coupling of striated muscle, depolarization of the surface membrane is converted into Ca2+ release from internal stores. This process occurs at intracellular junctions characterized by a specialized composition and structural organization of membrane proteins. The coordinated arrangement of the two key junctional components--the dihydropyridine receptor (DHPR) in the surface membrane and the ryanodine receptor (RyR) in the sarcoplasmic reticulum--is essential for their normal, tissue-specific function in e-c coupling. The mechanisms involved in the formation of the junctions and a potential participation of DHPRs and RyRs in this process have been subject of intensive studies over the past 5 years. In this review we discuss recent advances in understanding the organization of these molecules in skeletal and cardiac muscle, as well as their concurrent and independent assembly during development of normal and mutant muscle. From this information we derive a model for the assembly of the junctions and the establishment of the precise structural relationship between DHPRs and RyRs that underlies their interaction in e-c coupling.

Changes in voltage activation, Cs+ sensitivity, and ion permeability in H5 mutants of the plant K+ channel KAT1.

KAT1 is a voltage-dependent inward rectifying K+ channel cloned from the higher plant Arabidopsis thaliana [Anderson, J. A., Huprikar, S. S., Kochian, L. V., Lucas, W. J. & Gaber, R. F. (1992) Proc. Natl. Acad. Sci. USA 89, 3736-3740]. It is related to the Shaker superfamily of K+ channels characterized by six transmembrane spanning domains (S1-S6) and a putative pore-forming region between S5 and S6 (H5). The 115 region between Pro-247 and Pro-271 in KAT1 contains 14 additional amino acids when compared with Shaker [Aldrich, R. W. (1993) Nature (London) 362, 107-108]. We studied various point mutations introduced into H5 to determine whether voltage-dependent plant and animal K+ channels share similar pore structures. Through heterologous expression in Xenopus oocytes and voltage-clamp analysis combined with phenotypic analysis involving a potassium transport-defective Saccharomyces cerevisiae strain, we investigated the selectivity filter of the mutants and their susceptibility toward inhibition by cesium and calcium ions. With respect to electrophysiological properties, KAT1 mutants segregated into three groups: (i) wild-type-like channels, (ii) channels modified in selectivity and Cs+ or Ca2+ sensitivity, and (iii) a group that was additionally affected in its voltage dependence. Despite the additional 14 amino acids in H5, this motif in KAT1 is also involved in the formation of the ion-conducting pore because amino acid substitutions at Leu-251, Thr-256, Thr-259, and Thr-260 resulted in functional channels with modified ionic selectivity and inhibition. Creation of Ca2+ sensitivity and an increased susceptibility to Cs+ block through mutations within the narrow pore might indicate that both blockers move deeply into the channel. Furthermore, mutations close to the rim of the pore affecting the half-activation potential (U1/2) indicate that amino acids within the pore either interact with the voltage sensor or ion permeation feeds back on gating.

A loop-loop "kissing" complex is the essential part of the dimer linkage of genomic HIV-1 RNA.

RNA-RNA interactions govern a number of biological processes. Several RNAs, including natural sense and antisense RNAs, interact by means of a two-step mechanism: recognition is mediated by a loop-loop complex, which is then stabilized by formation of an extended intermolecular duplex. It was proposed that the same mechanism holds for dimerization of the genomic RNA of human immunodeficiency virus type 1 (HIV-1), an event thought to control crucial steps of HIV-1 replication. However, whereas interaction between the partially self-complementary loop of the dimerization initiation site (DIS) of each monomer is well established, formation of the extended duplex remained speculative. Here we first show that in vitro dimerization of HIV-1 RNA is a specific process, not resulting from simple annealing of denatured molecules. Next we used mutants of the DIS to test the formation of the extended duplex. Four pairs of transcomplementary mutants were designed in such a way that all pairs can form the loop-loop "kissing" complex, but only two of them can potentially form the extended duplex. All pairs of mutants form heterodimers whose thermal stability, dissociation constant, and dynamics were analyzed. Taken together, our results indicate that, in contrast with the interactions between natural sense and antisense RNAs, no extended duplex is formed during dimerization of HIV-1 RNA. We also showed that 55-mer sense RNAs containing the DIS are able to interfere with the preformed HIV-1 RNA dimer.

Truncated elongation factor G lacking the G domain promotes translocation of the 3' end but not of the anticodon domain of peptidyl-tRNA.

The mechanism by which elongation factor G (EF-G) catalyzes the translocation of tRNAs and mRNA on the ribosome is not known. The reaction requires GTP, which is hydrolyzed to GDP. Here we show that EF-G from Escherichia coli lacking the G domain still catalyzed partial translocation in that it promoted the transfer of the 3' end of peptidyl-tRNA to the P site on the 50S ribosomal subunit into a puromycin-reactive state in a slow-turnover reaction. In contrast, it did not bring about translocation on the 30S subunit, since (i) deacylated tRNA was not released from the P site and (ii) the A site remained blocked for aminoacyl-tRNA binding during and after partial translocation. The reaction probably represents the first EF-G-dependent step of translocation that follows the spontaneous formation of the A/P state that is not puromycin-reactive [Moazed, D. & Noller, H. F. (1989) Nature (London) 342, 142-148]. In the complete system--i.e., with intact EF-G and GTP--the 50S phase of translocation is rapidly followed by the 30S phase during which the tRNAs together with the mRNA are shifted on the small ribosomal subunit, and GTP is hydrolyzed. As to the mechanism of EF-G function, the results show that the G domain has an important role, presumably exerted through interactions with other domains of EF-G, in the promotion of translocation on the small ribosomal subunit. The G domain's intramolecular interactions are likely to be modulated by GTP binding and hydrolysis.

Barriers to protein folding: formation of buried polar interactions is a slow step in acquisition of structure.

In the MYL mutant of the Arc repressor dimer, sets of partially buried salt-bridge and hydrogen-bond interactions mediated by Arg-31, Glu-36, and Arg-40 in each subunit are replaced by hydrophobic interactions between Met-31, Tyr-36, and Leu-40. The MYL refolding/dimerization reaction differs from that of wild type in being 10- to 1250-fold faster, having an earlier transition state, and depending upon viscosity but not ionic strength. Formation of the wild-type salt bridges in a hydrophobic environment clearly imposes a kinetic barrier to folding, which can be lowered by high salt concentrations. The changes in the position of the transition state and viscosity dependence can be explained if denatured monomers interact to form a partially folded dimeric intermediate, which then continues folding to form the native dimer. The second step is postulated to be rate limiting for wild type. Replacing the salt bridge with hydrophobic interactions lowers this barrier for MYL. This makes the first kinetic barrier rate limiting for MYL refolding and creates a downhill free-energy landscape in which most molecules which reach the intermediate state continue to form native dimers.

Potentiation of proton transfer function by electrostatic interactions in photosynthetic reaction centers from Rhodobacter sphaeroides: First results from site-directed mutation of the H subunit.

The x-ray crystallographic structure of the photosynthetic reaction center (RC) has proven critical in understanding biological electron transfer processes. By contrast, understanding of intraprotein proton transfer is easily lost in the immense richness of the details. In the RC of Rhodobacter (Rb.) sphaeroides, the secondary quinone (QB) is surrounded by amino acid residues of the L subunit and some buried water molecules, with M- and H-subunit residues also close by. The effects of site-directed mutagenesis upon RC turnover and quinone function have implicated several L-subunit residues in proton delivery to QB, although some species differences exist. In wild-type Rb. sphaeroides, Glu L212 and Asp L213 represent an inner shell of residues of particular importance in proton transfer to QB. Asp L213 is crucial for delivery of the first proton, coupled to transfer of the second electron, while Glu L212, possibly together with Asp L213, is necessary for delivery of the second proton, after the second electron transfer. We report here the first study, by site-directed mutagenesis, of the role of the H subunit in QB function. Glu H173, one of a cluster of strongly interacting residues near QB, including Asp L213, was altered to Gln. In isolated mutant RCs, the kinetics of the first electron transfer, leading to formation of the semiquinone, QB-, and the proton-linked second electron transfer, leading to the formation of fully reduced quinol, were both greatly retarded, as observed previously in the Asp L213 --> Asn mutant. However, the first electron transfer equilibrium, QA-QB <==> QAQB-, was decreased, which is opposite to the effect of the Asp L213 --> Asn mutation. These major disruptions of events coupled to proton delivery to QB were largely reversed by the addition of azide (N3-). The results support a major role for electrostatic interactions between charged groups in determining the protonation state of certain entities, thereby controlling the rate of the second electron transfer. It is suggested that the essential electrostatic effect may be to "potentiate" proton transfer activity by raising the pK of functional entities that actually transfer protons in a coupled fashion with the second electron transfer. Candidates include buried water (H3O+) and Ser L223 (serine-OH2+), which is very close to the O5 carbonyl of the quinone.

The three-dimensional structure of NAD(P)H:quinone reductase, a flavoprotein involved in cancer chemoprotection and chemotherapy: mechanism of the two-electron reduction.

Quinone reductase [NAD(P)H:(quinone acceptor) oxidoreductase, EC 1.6.99.2], also called DT diaphorase, is a homodimeric FAD-containing enzyme that catalyzes obligatory NAD(P)H-dependent two-electron reductions of quinones and protects cells against the toxic and neoplastic effects of free radicals and reactive oxygen species arising from one-electron reductions. These two-electron reductions participate in the reductive bioactivation of cancer chemotherapeutic agents such as mitomycin C in tumor cells. Thus, surprisingly, the same enzymatic reaction that protects normal cells activates cytotoxic drugs used in cancer chemotherapy. The 2.1-A crystal structure of rat liver quinone reductase reveals that the folding of a portion of each monomer is similar to that of flavodoxin, a bacterial FMN-containing protein. Two additional portions of the polypeptide chains are involved in dimerization and in formation of the two identical catalytic sites to which both monomers contribute. The crystallographic structures of two FAD-containing enzyme complexes (one containing NADP+, the other containing duroquinone) suggest that direct hydride transfers from NAD(P)H to FAD and from FADH2 to the quinone [which occupies the site vacated by NAD(P)H] provide a simple rationale for the obligatory two-electron reductions involving a ping-pong mechanism.

An antiestrogen: a phosphotyrosyl peptide that blocks dimerization of the human estrogen receptor.

We have previously identified tyrosine-537 as a constitutively phosphorylated site on the human estrogen receptor (hER). A 12-amino acid phosphotyrosyl peptide containing a selected sequence surrounding tyrosine-537 was used to investigate the function of phosphotyrosine-537. The phosphotyrosyl peptide completely blocked the binding of the hER to an estrogen response element (ERE) in a gel mobility shift assay. Neither the nonphosphorylated tyrosyl peptide nor an unrelated phosphotyrosyl peptide previously shown to inhibit the signal transducers and activators of transcription factor (STAT) blocked binding of the hER to the ERE. The hER phosphotyrosyl peptide was shown by molecular sizing chromatography to dissociate the hER dimer into monomers. The hER specifically bound the 32P-labeled phosphotyrosyl peptide, indicating that the inhibition of ERE binding was caused by the phosphotyrosyl peptide binding directly to the hER and blocking dimerization. These data suggest that the phosphorylation of tyrosine-537 is a necessary step for the formation of the hER dimer. In addition, we propose that the dimerization of the hER occurs by a previously unrecognized Src homology 2 domain (SH2)-like phosphotyrosyl coupling mechanism. Consequently, the phosphotyrosyl peptide represents a class of antagonists that inhibits estrogen action by a mechanism other than interacting with the receptor's hormone binding site.

Mos overexpression in Swiss 3T3 cells induces meiotic-like alterations of the mitotic spindle.

High levels of mos protooncogene product are expressed during oocyte meiotic maturation and Mos has been implicated in formation of the spindle and spindle pole. Here, we show that in Swiss 3T3 cells with 4N DNA content, high levels of Mos lead to the production of binucleated cells. The Swiss 3T3 cells in mitosis, before binucleation occurs, are anastral and the spindle poles are juxtaposed to the cell membrane. These phenotypes may be related to the meiotic process of attachment of the spindle pole to the oocyte membrane during polar body formation. The production of binucleated somatic cells could result from attachment of the altered mitotic spindle pole to the cell membrane that interferes with cytokinesis but not karyokinesis. This can explain at least one form of genetic instability that leads to altered DNA content in tumor cells.

Synthesis of Chromen[4,3-b]pyrrolidines by Intramolecular 1,3-Dipolar Cycloadditions of Azomethine Ylides: An Experimental and Computational Assessment of the Origin of Stereocontrol

Azomethine ylides, generated from imine-derived O-cinnamyl or O-crotonyl salicylaldeyde and α-amino acids, undergo intramolecular 1,3-dipolar cycloaddition, leading to chromene[4,3-b]pyrrolidines. Two reaction conditions are used: (a) microwave-assisted heating (200 W, 185 °C) of a neat mixture of reagents, and (b) conventional heating (170 °C) in PEG-400 as solvent. In both cases, a mixture of two epimers at the α-position of the nitrogen atom in the pyrrolidine nucleus was formed through the less energetic endo-approach (B/C ring fusion). In many cases, the formation of the stereoisomer bearing a trans-arrangement into the B/C ring fusion was observed in high proportions. Comprehensive computational and kinetic simulation studies are detailed. An analysis of the stability of transient 1,3-dipoles, followed by an assessment of the intramolecular pathways and kinetics are also reported.

The Common Agricultural Policy: The Future of the CAP and why real reform will never happen. EUMA Paper Vol. 6 No. 7, May 2010

This paper sets out to examine the Common Agricultural Policy (CAP) of the European Union from its inception to present day 1. Specifically, this paper seeks to answer the following questions: (1) What long-term effects, if any, did the circumstances surrounding, and leading up to the formation of the CAP have; (2) What have internal and external responses been to the CAP; (3) How has the CAP responded to major events both internally (within the European Union), and externally (internationally); (4) What affect does the recently implemented Lisbon Treaty2 have on the CAP, and (5) What is the future of the CAP and CAP reform? In order to answer these questions this paper begins with the contention that the CAP is in fact the largest and strongest driving force of EU expansion. In support of this proposition, this paper first examines the circumstances and events leading to the creation of the CAP in the European Community. Second, this paper examines what long-term effects the circumstances surrounding the CAP’s inception have had on the policy, particularly calling attention to the disproportionate Franco-German CAP benefits. Third, the paper then examines how the CAP has responded to historical events that have had significant effects on the European community, particularly EU expansion, the implementation of the Lisbon Treaty, and the recent worldwide economic crisis. Finally, this paper examines common criticisms of and conflicts surrounding the CAP, both internally and externally, and argues that CAP reform, at least within the current institutional framework of the European Union, can never truly occur.

Eurasian integration. Russia's attempt at the economic unification of the post-Soviet area. OSW Study 44/2013

In 2009, Vladimir Putin, the then Russian prime minister, gave impetus to the establishment of closer relations within what was then a still narrow group of three countries: Russia, Kazakhstan and Belarus. Russia was determined in embarking on the implementation of the principles of the Customs Union among these three states and, since 2012, within the Common Economic Space as well. This process of integration is intended to bring about the introduction of ‘four freedoms’ in this area: the free movement of goods, services, capital and labour. From Moscow’s point of view, building up such integration structures is especially necessary in order to counteract the economic expansion of the European Union and China. It also feels it is important to take measures against the loosening of the bonds between the CIS countries and Russia. At the same time, close co-operation is expected to guarantee for Russia that the strong politico-economic influences in this area will be maintained. Despite the numerous limitations of the integration process, such as the small number of the participating states or limited progress in implementing the CES, this is still the most advanced integration programme in the region seen since the collapse of the USSR. Progress in putting the rules of the Customs Union into practice can be seen as a success for Moscow. In turn, the formation of the CES is still at an early stage, and it is difficult to determine at this point to what extent the three countries will harmonise their markets.

Tectonic evolution of the Proterozoic Itremo Group metasediments in central Madagascar

The major geologic units of the Itremo region in central Madagascar include: (1) upper amphibolite to granulite facies (higher grade) Precambrian rocks, mainly para- and orthogneisses, and migmatites; (2) the newly defined Itremo Nappes, a fold-and-thrust belt containing the Proterozoic Itremo Group sediments, metamorphosed at greenschist to lower amphibolite facies (lower grade) conditions: (3) Middle Neoproterozoic and Late Neoproterozoic-Cambrian intrusives. The stratigraphic succession of the Itremo Group in the eastern part of the Itremo region is, from bottom to top: quartzites, metapelites, metacarbonates and metapelites overlain by metacarbonates. During D1 the Itremo Group sediments were detached from their continental substratum, deformed into a fold-and-thrust nappe (Itremo Nappes), and transported on top of higher grade rocks that are intruded by Middle Neoproterozoic (c. 797–780 Ma) granites and gabbros. A second phase of deformation shortening (D2) affected both the Itremo Sedimentary Nappes and structurally underlying higher-grade rocksunits, and formed large-scale N-S-trending F2 folds. S1 axial plane foliations in Itremo Group sediments are truncated by Late Neoproterozoic-Cambrian granites (c. 570–540 Ma). The age of the formation of the Itremo Nappes is not well constrained: they formed in Neoproterozoic times between 780 and 570 Ma.

Sedimentology at the continental margin of the southern Weddell Sea

During four expeditions with RV "Polarstern" at the continental margin of the southern Weddell Sea, profiling and geological sampling were carried out. A detailed bathymetric map was constructed from echo-sounding data. Sub-bottom profiles, classified into nine echotypes, have been mapped and interpreted. Sedimentological analyses were carried out on 32 undisturbed box grab surface samples, as well as on sediment cores from 9 sites. Apart from the description of the sediments and the investigation of sedimentary structures on X-radiographs the following characteristics were determined: grain-size distributions; carbonate and Corg content; component distibutions in different grain-size fractions; stable oxygen and carbon isotopes in planktic and, partly, in benthic foraminifers; and physical properties. The stratigraphy is based On 14C-dating, oxygen isotope Stages and, at one site, On paleomagnetic measurements and 230Th-analyses The sediments represent the period of deposition from the last glacial maximum until recent time. They are composed predominantly of terrigenous components. The formation of the sediments was controlled by glaciological, hydrographical and gravitational processes. Variations in the sea-ice coverage influenced biogenic production. The ice sheet and icebergs were important media for sediment transport; their grounding caused compaction and erosion of glacial marine sediments on the outer continental shelf. The circulation and the physical and chemical properties of the water masses controlled the transport of fine-grained material, biogenic production and its preservation. Gravitational transport processes were the inain mode of sediment movements on the continental slope. The continental ice sheet advanced to the shelf edge and grounded On the sea-floor, presumably later than 31,000 y.B.P. This ice movement was linked with erosion of shelf sediments and a very high sediment supply to the upper continental slope from the adiacent southern shelf. The erosional surface On the shelf is documented in the sub-bottom profiles as a regular, acoustically hard reflector. Dense sea-ice coverage above the lower and middle continental slope resulted in the almost total breakdown of biogenic production. Immediately in front of the ice sheet, above the upper continental slope, a <50 km broad coastal polynya existed at least periodically. Biogenic production was much higher in this polynya than elsewhere. Intense sea-ice formation in the polynya probably led to the development of a high salinity and, consequently, dense water mass, which flowed as a stream near bottom across the continental slope into the deep sea, possibly contributing to bottom water formation. The current velocities of this water mass presumably had seasonal variations. The near-bottom flow of the dense water mass, in combination with the gravity transport processes that arose from the high rates of sediment accumulation, probably led to erosion that progressed laterally from east to West along a SW to NE-trending, 200 to 400 m high morphological step at the continental slope. During the period 14,000 to 13,000 y.B.P., during the postglacial temperature and sea-level rise, intense changes in the environmental conditions occured. Primarily, the ice masses on the outer continental shelf started to float. Intense calving processes resulted in a rapid retreat of the ice edge to the south. A consequence of this retreat was, that the source area of the ice-rafted debris changed from the adjacent southern shelf to the eastern Weddell Sea. As the ice retreated, the gravitational transport processes On the continental slope ceased. Soon after the beginning of the ice retreat, the sea-ice coverage in the whole research area decreased. Simultaneously, the formation of the high salinity dense bottom water ceased, and the sediment composition at the continental slope then became influenced by the water masses of the Weddell Gyre. The formation of very cold Ice Shelf Water (ISW) started beneath the southward retreating Filchner-Ronne Ice Shelf somewhat later than 12,000 y.B.P. The ISW streamed primarily with lower velocities than those of today across the continental slope, and was conducted along the erosional step on the slope into the deep sea. At 7,500 y.B.P., the grounding line of the ice masses had retreated > 400 km to the south. A progressive retreat by additional 200 to 300 km probably led to the development of an Open water column beneath the ice south of Berkner Island at about 4,000 y.B.P. This in turn may have led to an additional ISW, which had formed beneath the Ronne Ice Shelf, to flow towards the Filcher Ice Shelf. As a result, increased flow of ISW took place over the continental margin, possibly enabling the ISW to spill over the erosional step On the upper continental slope towards the West. Since that time, there is no longer any documentation of the ISW in the sedimentary Parameters on the lower continental slope. There, recent sediments reflect the lower water masses of the Weddell Gyre. The sea-ice coverage in early Holocene time was again so dense that biogenic production was significantly restricted.