Mutation of Pro-258 in transmembrane domain 6 constitutively activates the G protein-coupled alpha-factor receptor.

The alpha-factor pheromone receptor stimulates MATa yeast cells to undergo conjugation. The receptor contains seven transmembrane domains that function in ligand binding and in transducing a signal to the cytoplasmic receptor sequences to mediate G protein activation. A genetic screen was used to isolate receptor mutations that constitutively signal in the absence of alpha-factor. The Pro-258-->Leu (P258L) mutation caused constitutive receptor signaling that was equivalent to about 45% of the maximum level observed in wild-type cells stimulated with alpha-factor. Mutations of both Pro-258 and the adjacent Ser-259 to Leu increased constitutive signaling to > or = 90% of the maximum level. Since Pro-258 occurs in the central portion of transmembrane domain 6, and since proline residues are expected to cause a kink in alpha-helical domains, the P258L mutation is predicted to alter the structure of transmembrane domain 6. The P258L mutation did not result in a global distortion of receptor structure because alpha-factor bound to the mutant receptors with high affinity and induced even higher levels of signaling. These results suggest that sequences surrounding Pro-258 may be involved in ligand activation of the receptor. Conformational changes in transmembrane domain 6 may effect a change in the adjacent sequences in the third intracellular loop that are thought to function in G protein activation. Greater than 90% of all G protein-coupled receptors contain a proline residue at a similar position in transmembrane domain 6, suggesting that this aspect of receptor activation may be conserved in other receptors.

Subunit cleavage of mosquito pro-vitellogenin by a subtilisin-like convertase.

The eukaryotic convertase family plays an important role in posttranslational proteolytic processing and activation of many pro- and polypeptides that have at their cleavage sites the paired basic motif, RX(K/R)R. Recent studies have revealed that the cleavage site of insect pro-vitellogenins (pro-Vg) also contains this motif. To identify and characterize the insect pro-Vg processing enzyme, Vg convertase (VC), its cDNA was cloned from a vitellogenic female fat body cDNA library of the mosquito, Aedes aegypti. The 3735-bp-long VC cDNA has an open reading frame encoding a 115-kDa protein. In vitro transcription/translation of VC cDNA revealed that this 115-kDa protein becomes 140 kDa after co- and posttranslational modifications. The VC deduced amino acid sequence has high similarity to and a domain structure characteristic of furin-like convertases. Northern blot analysis showed that a single 4.2-kb transcript was expressed in the fat body during the first 18 hr of the Vg synthetic period. Coexpression of VC cDNA with mosquito Vg cDNA resulted in correct cleavage of pro-Vg. Thus, this newly identified convertase is, indeed, a functional fat body-specific enzyme for pro-Vg cleavage.

Identification of a minimal sequence of the mouse pro-alpha 1(I) collagen promoter that confers high-level osteoblast expression in transgenic mice and that binds a protein selectively present in osteoblasts.

Based on our previous transgenic mice results, which strongly suggested that separate cell-specific cis-acting elements of the mouse pro-alpha 1(I) collagen promoter control the activity of the gene in different type I collagen-producing cells, we attempted to delineate a short segment in this promoter that could direct high-level expression selectively in osteoblasts. By generating transgenic mice harboring various fragments of the promoter, we identified a 117-bp segment (-1656 to -1540) that is a minimal sequence able to confer high-level expression of a lacZ reporter gene selectively in osteoblasts when cloned upstream of the proximal 220-bp pro-alpha 1(I) promoter. This 220-bp promoter by itself was inactive in transgenic mice and unable to direct osteoblast-specific expression. The 117-bp enhancer segment contained two sequences that appeared to have different functions. The A sequence (-1656 to -1628) was required to obtain expression of the lacZ gene in osteoblasts, whereas the C sequence (-1575 to -1540) was essential to obtain consistent and high-level expression of the lacZ gene in osteoblasts. Gel shift assays showed that the A sequence bound a nuclear protein present only in osteoblastic cells. A mutation in the A segment that abolished the binding of this osteoblast-specific protein also abolished lacZ expression in osteoblasts of transgenic mice.

Differential sensitivity of stilbenedisulfonates in their reaction with band 3 HT (Pro-868-->Leu).

Band 3 HT (Pro-868-->Leu) is a mutant anion exchange protein which has several phenotypic characteristics, including a 2- to 3-fold larger Vmax, and reduced covalent binding of the anion transport inhibitor 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate (H2DIDS). We have used fluorescence kinetic methods to study inhibitor binding to band 3 to determine if the point mutation in band 3 HT produces localized or wide-spread conformational changes within the membrane-bound domain of this transporter. Our results show that covalent binding of H2DIDS by band 3 HT is slower by a factor of 10 to 20 compared with the wild-type protein. In contrast, no such difference in the kinetics was observed for covalent binding of 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS). In addition, the kinetics of H2DIDS release from band 3 HT was abnormal, while the kinetics of 4,4'-dibenzamidostilbene-2,2'-disulfonate (DBDS) release showed no difference when compared with the wild-type protein. We conclude that substitution of leucine for proline at position 868 does not perturb the structure of "lysine A" in the membrane-bound domain of band 3 but rather produces an apparently localized conformational change in the C-terminal subdomain of the protein which alters H2DIDS affinity. When combined with the observation of an increased Vmax, these results suggest that protein structural changes at position 868 influence a turnover step in the transport cycle.

Role of the integument in insect defense: pro-phenol oxidase cascade in the cuticular matrix.

The cuticle of the silkworm Bombyx mori was demonstrated to contain pro-phenol oxidase [zymogen of phenol oxidase (monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1)] and its activating cascade. The activating cascade contained at least one serine proteinase zymogen (latent form of pro-phenol oxidase activating enzyme). When the extracted cascade components were incubated with Ca2+, the latent form of pro-phenol oxidase activating enzyme was itself activated and, in turn, converted through a limited proteolysis of pro-phenol oxidase to phenol oxidase. Immuno-gold localization of prophenol oxidase in the cuticle using a cross-reactive hemolymph anti-pro-phenol oxidase antibody revealed a random distribution of this enzyme in the nonlamellate endocuticle and a specific orderly arrayed pattern along the basal border of the laminae in the lamellate endocuticle of the body wall. Furthermore, prophenol oxidase was randomly distributed in the taenidial cushion of the tracheal cuticle. At the time of pro-phenol oxidase accumulation in the body wall cuticle, no pro-phenol oxidase mRNA could be detected in the epidermal tissue, whereas free-circulating hemocytes contained numerous transcripts of pro-phenol oxidase. Our results suggest that the pro-phenol oxidase is synthesized in the hemocytes and actively transported into the cuticle via the epidermis.

Molecular cloning of insect pro-phenol oxidase: a copper-containing protein homologous to arthropod hemocyanin.

Pro-phenol oxidase [pro-PO; zymogen of phenol oxidase (monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1)] is present in the hemolymph plasma of the silkworm Bombyx mori. Pro-PO is a heterodimeric protein synthesized by hemocytes. A specific serine proteinase activates both subunits through a limited proteolysis. The amino acid sequences of both subunits were deduced from their respective cDNAs; amino acid sequence homology between the subunits was 51%. The deduced amino acid sequences revealed domains highly homologous to the copper-binding site sequences (copper-binding sites A and B) of arthropod hemocyanins. The overall sequence homology between silkworm pro-PO and arthropod hemocyanins ranged from 29 to 39%. Phenol oxidases from prokaryotes, fungi, and vertebrates have sequences homologous to only the copper-binding site B of arthropod hemocyanins. Thus, silkworm pro-PO DNA described here appears distinctive and more closely related to arthropod hemocyanins. The pro-PO-activating serine proteinase was shown to hydrolyze peptide bonds at the carboxyl side of arginine in the sequence-Asn-49-Arg-50-Phe-51-Gly-52- of both subunits. Amino groups of N termini of both subunits were indicated to be N-acetylated. The cDNAs of both pro-PO subunits lacked signal peptide sequences. This result supports our contention that mature pro-PO accumulates in the cytoplasm of hemocytes and is released by cell rupture, as for arthropod hemocyanins.

Meddelanden af Societas pro Fauna et Flora Fennica.

Haft. 47 (1920-1921)

Acta Societatis pro Fauna et Flora Fennica.

v. 7 (1890)

Questiones propositae pro modulo discutiendae Comitiis publicis Harvardini Collegii quod est divin. provid. Cantabrigiae Nov-Anglorum a Laureae Magistralis candidatis, 1660-1753

The bound volume holds handwritten transcriptions of selected Harvard Commencement Quaestiones copied by Isaac Mansfield (Harvard AB 1742). The manuscript volume includes from the 1708 Quaestiones onward, the notation "N.B." next to questions performed by the candidate during the Commencement exercises; the original printed Quaestiones sheets do not note this information. The volume includes Quaestiones transcriptions for which no original broadsides are known to still exists.

Theses, in Comitiis publicis Harvardini Collegii quod est Cantabrigiae Nov-Anglorum, defensae & pro viribus propugnatae a Juvenibus in Artibus initiatis, [1660-1753]

The bound volume holds handwritten transcriptions of selected Harvard Commencement Theses copied by Isaac Mansfield (Harvard AB 1742). The manuscript volume holds only the Theses chosen for public disputation. The volume includes Theses transcriptions for which no original broadsides are known to still exists.

The Pro-Western Turn in Russia's Foreign Policy: Causes, Consequences and Prospects. OSW Point of View, September 2002

From Europe and Poland's point of view, one of the most important recent developments in international politics was the re-orientation of Russia's foreign policy. This paper aims to answer three important questions relating to this issue: 4. When and why did the "pro-Western turn" in the Russian Federation's policy take place? 5. Has it been profitable for Russia? 6. What goals will the Russian policy pursue in the future? An analysis of the last two years in Russia's foreign policy leads to the several conclusions, including those: 5. Clearly, the Russian leaders realise that in the longer term, Russia - in its desire for more influence in the world - will not be able to survive as an independent pole of power in international politics and it will have to join forces with the West (most likely, the European Union). 6. September 11 was not the cause of Russia's pro-Western turn, but rather a catalyst that put the process which started when Vladimir Putin took his office in sharp focus. 7. In the nearest future, this new direction of Russia's foreign policy seems not be challenged by internal opposition in Russia. 8. The "pro-Western turn" proved to be beneficial for Russia, although: d. Russia has not become a strategic ally of the US e. There has been no breakthrough in the relations between Russia and the European Union, and Moscow has not gained any real influence on NATO's important decisions. f. Russia has not become a major decision-maker of international politics. 5. Russia's closing to the West is in Poland's and Europe's interest.

The "Pro-Western Turn" in Russia's Foreign Policy: Causes, Consequences and Prospects. OSW Point of View (unspecified), September 2002

From Europe and Poland's point of view, one of the most important recent developments in international politics was the re-orientation of Russia's foreign policy. This paper aims to answer three important questions relating to this issue: 1. When and why did the "pro-Western turn" in the Russian Federation's policy take place? 2. Has it been profitable for Russia? 3. What goals will the Russian policy pursue in the future? An analysis of the last two years in Russia's foreign policy leads to the several conclusions, including those: a. Clearly, the Russian leaders realise that in the longer term, Russia - in its desire for more influence in the world - will not be able to survive as an independent pole of power in international politics and it will have to join forces with the West (most likely, the European Union). b. September 11 was not the cause of Russia's pro-Western turn, but rather a catalyst that put the process which started when Vladimir Putin took his office in sharp focus. 7. In the nearest future, this new direction of Russia's foreign policy seems not be challenged by internal opposition in Russia. c. The "pro-Western turn" proved to be beneficial for Russia, although: d. Russia has not become a strategic ally of the US e. There has been no breakthrough in the relations between Russia and the European Union, and Moscow has not gained any real influence on NATO's important decisions. f. Russia has not become a major decision-maker of international politics. g. Russia's closing to the West is in Poland's and Europe's interest.

Pro-American non-alignment. Sweden and Finland develop closer military co-operation with the United States. OSW COMMENTARY NUMBER 205/31.03.2016

Russia’s increasingly aggressive policy and its enhanced military activity in the Nordic-Baltic region has led to revaluations in Sweden’s and Finland’s security and defence policies and a rethinking of the formats of their military co-operation. While remaining outside NATO, the two states have been developing closer bilateral defence co-operation and working more closely with the United States, while at the same time developing co-operation with NATO. Sweden and Finland perceive the United States as the guarantor of regional and European security. From their point of view, the United States is currently the country that has both the necessary military capabilities and the political will to react in the event of a conflict between Russia and NATO in the Nordic-Baltic region, in which both countries would inevitably become involved despite their non-aligned status. For Sweden and Finland, intensified co-operation with the United States offers an alternative to NATO membership, which is currently out of the question for domestic political reasons. Meanwhile, the US has also become increasingly aware of the strategic importance of the two states, which, for the purposes of contingency planning, are in fact an extension of NATO’s north-eastern flank.