Podcasts about nh2

Lost Piece Exercise: The audio will tell you the position of most of the pieces on the board. Can you work out where the missing piece is meant to be? To learn more about Don't Move Until You See It and get the free 5-day Conceptualizing Chess Series, head over to https://dontmoveuntilyousee.it/conceptualization FEN for today's exercise: r2qnrk1/pp4b1/2ppb2p/P2Ppp2/NPP1P1pP/R1N1B1P1/2K2PBn/3Q3R b - - 0 1 And the answer is... Nh2

Game Exercise: Close your eyes and follow along with an entire Chess game using the audio below. On each move, try to conceptualize the position clearly and understand how it has changed. Try to follow the game until the end to stretch the amount of moves you can see ahead. To learn more about Don't Move Until You See It and get the free 5-day Conceptualizing Chess Series, head over to https://dontmoveuntilyousee.it/conceptualization PGN for today's exercise: Tartakower vs Euwe (Venice, 1948) 2. 1. e4 e5 2. Nf3 Nc6 3. Bc4 Bc5 4. c3 Bb6 5. d4 Qe7 6. O-O d6 7. h3 Nf6 8. Re1 O-O 9. Na3 Nd8 10. Bf1 Ne8 11. Nc4 f6 12. a4 c6 13. Nxb6 axb6 14. Qb3+ Ne6 15. Qxb6 g5 16. Bc4 h6 17. h4 Kh7 18. hxg5 hxg5 19. dxe5 dxe5 20. Be3 Rh8 21. g3 Kg6 22. Kg2 Nf4+ 23. gxf4 Bh3+ 24. Kg3 exf4+ 25. Bxf4 Qd7 26. Nh2 gxf4+ 27. Kxf4 Rh4+ 28. Ke3 Bg2 29. Nf3 Rxe4+ 30. Kxe4 Nd6+ 31. Kd3 Qf5+ 32. Kd4 Qf4+ 33. Kd3 Qxc4+ 34. Kc2 Bxf3 35. b3 Be4+ 36. Kb2 Qd3 37. Rg1+ Kf7 38. Rac1 Qd2+ 39. Ka3 Nc4+ 40. bxc4 Rxa4+ 41. Kxa4 Qa2+ 42. Kb4 Qb2+ { White resigned. } *

NTD News Today—1/23/20241. Trump to NTD: Will ‘Win by Big Margins' in NH2. Midnight New Hampshire Voters Choose Haley3. NH Residents Want Better Answers to Opioid Crisis4. New Hampshire Farmer Discusses Election5. Trump Urges Supporters Not to Be Complacent6. What a Big NH Win Could Mean for Trump Campaign7. Will Lack of Dem Delegates Benefit Haley?8. The Nation Decides 2024: Live on NTD @ 8PM Et9. DeSantis Unlikely to Be in Administration: Trump10. Kari Lake Focused on Senate Seat, Not Being VP11. Stefanik Would ‘Be Honored' to Be Trump's Running Mate12. Idaho Congress Members Endorse Trump13. Poll: Trump With Wide Margin Lead in NH Primary14. DeSantis Supporters Chose Trump Over Haley 2–1: Polls15. New Hampshire's Largest Paper Endorses Dean Phillips16. Biden Can't Defeat Trump in November: Andrew Yang17. North Dakota Governor Won't Seek 3rd Term18. Fani Willis Faces Setbacks in Corruption Allegations19. Ga: Judge Pauses Testimony of Trump Prosecutor20. Scotus Lets Biden Admin. Remove Texas Razor-Wire21. RPT: Biden Admin. Spent $10B on Illegal Immigrants22. Mexico Wants Probe Into Cartels' US Military Weapons23. Hunter Biden Associates to Testify Behind Closed Doors24. Torrential Rain and Flooding Hit San Diego25. Investors Give Up Futile Wait for China to Fix Economy26. Netflix to Stream WWE Raw27. Scarecrow Protest Marks Farmers' Income Fears28. UK Will Continue Campaign Against Houthis: Cameron29. CIA Video Tries to Recruit Russian Double Agents30. Russian Attacks Kill 5, Wound Dozens: Ukraine31. NATO Signs $1.2B Contract to Replenish Supplies, Aid Ukraine32. Turkey Set to Approve Sweden's NATO Membership Bid33. Robots Help Senior Citizens Fight Loneliness34. Study: Treating Loneliness Helps People With Obesity35. Sydney's Tiny Parks Provide Locals Sanctuary Amid City Life36. WWII TV Series ‘Masters of the Air' Premieres37. Midnight New Hampshire Voters Choose Haley38. NH Residents Want Better Answers to Opioid Crisis39. New Hampshire Farmer Discusses Election40. DeSantis Unlikely to Be in Administration: Trump41. Kari Lake Focused on Senate Seat, Not Being VP42. Analyzing Early Results in NH Primary43. Challenges and Opportunities for Trump, Haley44. Looking Ahead to November's General Election45. Idaho Congress Members Endorse Trump46. Trump and Haley Go Head-to-Head in New Hampshire47. New Hampshire Primary Voters Share Why They Voted48. New Hampshire's Largest Paper Endorses Dean Phillips49. Biden Can't Defeat Trump in November: Andrew Yang50. North Dakota Governor Won't Seek 3rd Term51. Lloyd Austin's 1st Appearance Since Hospitalization52. Scotus Lets Biden Admin. Remove Texas Razor-Wire53. RPT: Biden Admin. Spent $10B on Illegal Immigrants54. Mexico Wants Probe Into Cartels' US Military Weapons55. Mexico Lawsuit Against U.S. Gun Companies Will Proceed56. Hunter Biden Associates to Testify Behind Closed Doors57. Fani Willis Faces Setbacks in Corruption Allegations58. Ga: Judge Pauses Testimony of Trump Prosecutor59. Torrential Rain and Flooding Hit San Diego60. FTC Bans Turbotax Ads for ‘Free' Services61. Apple Launches Security Feature to Combat Thieves62. Former NRA Heads Arrive at NY Court for Trial63. U.S. Business Leaders Visits Taiwan to Expand Cooperation64. 7.1 Magnitude Earthquake Rattles Western China65. Death Toll in China Landslide Rises to 2566. China Lobbies Countries to Praise Its Rights Record67. Buses in Queensland to Install Chinese CCTV Cameras68. UK Will Continue Campaign Against Houthis: Cameron69. CIA Video Tries...

NTD Good Morning—1/23/20241. Trump to NTD: Will 'Win by Big Margins' in NH2. Primary Voting Begins in New Hampshire3. Preview: NH Primary, Biden-Trump Rematch4. NH GOP Voters Weigh Up Final Two Primary Choices5. Trump and Haley Go Head-to-Head in NH6. NH Investigating AI Robocall Impersonating Biden7. GA: Judge Pauses Testimony of Trump Prosecutor8. SCOTUS Lets Biden Admin. Remove Texas Razor-Wire9. US, UK Strike More Houthi Targets in Yemen10. Analysis on New Strikes in Yemen11. 21 Soldiers Killed, Biggest Single Israeli Loss Since Oct. 712. Gaza Hostage Relatives Burst Into Israeli Parliament13. Ukraine: Russian Strikes Hit Gas Pipeline, School14. China Defies Sanctions, Makes Russia its No.1 Oil Supplier15. Cal State Faculty End Strike After Tentative Deal16. Baker Who Refused Gender Transition Cake in Court17. Coast Guard Rescues 9 People From Lake Erie Ice Floe18. How to Prevent and Treat Frostbite19. Trump Tells NTD: Will 'Win by Big Margins' in NH20. Haley's Statements Before Voting21. Preview: NH Primary, Biden-Trump Rematch22. NH GOP Voters Weigh Up Final 2 Primary Choices23. New Hampshire Primary: Trump-Haley Showdown24. Poll: Trump With Wide Margin Lead in NH Primary25. Fani Willis Faces Setbacks in Corruption Allegations26. US, UK Hit More Houthi Targets in Yemen27. IDF Says 21 Soldiers Killed in Southern Gaza28. United Airlines Predicts First Quarter Loss29. SCOTUS Lets Biden Admin. Remove TX Razor-Wire30. Cal State University Strike Ends31. Treasure Hunting on the Rise in UK

To start using Tab for a Cause, go to: http://tabforacause.org/minuteearth2 You might already know that proteins are a fundamental part of your diet, but they're much more than that. LEARN MORE ************** To learn more about this topic, start your googling with these keywords: - Amino acids: are organic compounds that contain amino (–NH2) and carboxyl (–COOH) functional groups, along with a side chain specific to each amino acid. - Proteins: are macromolecules composed of one or more long chains of amino acid residues. Most proteins fold into unique 3D structures. The shape into which a protein naturally folds is known as its native conformation. - Alpha helix (α-helix): is a common motif in the secondary structure of proteins and is a right hand-helix conformation in which every backbone N−H group hydrogen bonds to the backbone C=O group of the amino acid located four residues earlier along the protein sequence. - Beta sheet (β-sheet): is a common motif of the regular protein secondary structure and consists of beta strands (β-strands) connected laterally by at least two or three backbone hydrogen bonds, forming a generally twisted, pleated sheet. - Ribbon diagrams: are 3D schematic representations of protein structure that shows the overall path and organization of the protein backbone in 3D. Ribbon diagrams are generated by interpolating a smooth curve through the polypeptide backbone. α-helices are shown as coiled ribbons or thick tubes, β-strands as arrows, and non-repetitive coils or loops as lines or thin tubes. CREDITS ********* Ever Salazar | Co-Writer, Narrator, Illustrator and Director David Wych | Co-writer and Consultant Aldo de Vos, Know Art | Music MinuteEarth is produced by Neptune Studios LLC https://neptunestudios.info OUR STAFF ************ Sarah Berman • Arcadi Garcia Rius • David Goldenberg Julián Gustavo Gómez • Melissa Hayes • Alex Reich Henry Reich • Peter Reich • Leonardo Souza Ever Salazar • Kate Yoshida OUR LINKS ************ Youtube | https://youtube.com/MinuteEarth TikTok | https://tiktok.com/@minuteearth Twitter | https://twitter.com/MinuteEarth Instagram | https://instagram.com/minute_earth Facebook | https://facebook.com/Minuteearth Website | https://minuteearth.com Apple Podcasts| https://podcasts.apple.com/us/podcast/minuteearth/id649211176 OTHER CREDITS & REFERENCES ********************************** Goodsell, David (2006). Visual Methods from Atoms to Cells. Structure 13, Issue 3:347-354. doi:10.1016/j.str.2005.01.012 Protein 3D images created using Mol* (https://molstar.org/) and structure data from RCSB PDB (https://www.rcsb.org/) Mol* (D. Sehnal, A.S. Rose, J. Kovca, S.K. Burley, S. Velankar (2018) Mol*: Towards a common library and tools for web molecular graphics MolVA/EuroVis Proceedings. doi:10.2312/molva.20181103) Villin folding trajectory by Stefan Doerr - https://figshare.com/authors/Stefan_Doerr/748688 Clathrin Structure (PDB ID: 3IYV) Fotin, A., et al (2004). Molecular model for a complete clathrin lattice from electron cryomicroscopy. Nature 432: 573-579. doi:10.1038/nature03079 Immunoglobulin Structure (PDB IDs: 1IGT & 1IGY) Harris, L.J., et al (1998). Crystallographic structure of an intact IgG1 monoclonal antibody. J Mol Biol 275: 861-872. doi:10.1006/jmbi.1997.1508 ATP Synthase Structure (PDB IDs: 5ARE, 5ARI & 5FIL) Zhou, A., et al (2015). Structure and conformational states of the bovine mitochondrial ATP synthase by cryo-EM. ELife, 4. doi:10.7554/eLife.10180 RCSB PDB Molecule of the Month by David S. Goodsell (The Scripps Research Institute and the RCSB PDB) - https://pdb101.rcsb.org/motm/72 Photosystem II (PDB ID: 5XNL) Su, X., et al (2017). Structure and assembly mechanism of plant C2S2M2-type PSII-LHCII supercomplex. Science 357: 815-820. doi:10.1126/science.aan0327 Ribonuclease (PDB ID: 2AAS) Santoro, J., et al (1993). High-resolution three-dimensional structure of ribonuclease A in solution by nuclear magnetic resonance spectroscopy. J Mol Biol 229: 722-734. doi:10.1006/jmbi.1993.1075 Myosin (PDB ID: 1B7T) Houdusse, A., et al (1999). Atomic structure of scallop myosin subfragment S1 complexed with MgADP: a novel conformation of the myosin head. Cell 97: 459-470. doi:10.1016/s0092-8674(00)80756-4

My AP Biology ThoughtsUnit 1 Episode #46Welcome to My AP Biology Thoughts podcast, my name is Nidhi and I am your host for episode 46 called Unit 1 The Impact of Temperature and pH on enzymes. Today we will be discussing what an enzyme is and how the pH and temperature of the environment affects the enzyme and its substrate. Segment 1: Introduction to The Impact of Temperature and pH on enzymesAn enzyme is a protein or a RNA molecule that acts as a catalyst in chemical reactions, helping to reduce the activation energy needed for the reaction to occur. Often, this speeds up the rate of reaction. Enzymes are not changed or consumed by the reactions they catalyse and as a result can be reused. Enzymes are typically named after the molecules they react with, which is called the substrate, and they end with the suffix ‘-ase'. The active site is the region on the surface of the enzyme which binds to the substrate molecule. The active site and the substrate complement each other in terms of both shape and chemical properties. Enzymes are selective and each enzyme only speeds up a specific reaction. pH is a scale from 1-14 used to specify how acidic or how basic a solution is. A number lower than the neutral 7 is considered an acid while a number higher than 7 is a base. With enzymes, changes in pH can affect active sites by changing its shape or charge and making it harder for substrates to bind. Small changes in pH above or below the Optimum for the enzyme do not cause a permanent change to the enzyme, since the bonds can be reformed. However, extreme changes in pH can cause enzymes to Denature and permanently lose their function. The optimum pH, or the pH where the enzyme is most active, depends on where it normally works. For example, enzymes in the small intestine have an optimum pH of about 7.5, but stomach enzymes have an optimum pH of about 2. Low temperatures result in insufficient thermal energy for the activation of a reaction to proceed. Increasing the temperature will increase the speed and motion of both enzyme and substrate, resulting in higher enzyme activity since a higher kinetic energy will result in more frequent collisions between the enzymes and substrates. At an optimal temperature for the enzyme, the rate of activity will be at its peak. Higher temperatures will cause enzyme stability to decrease, because the thermal energy disrupts the enzyme's hydrogen bonds. This causes the enzyme's active site to lose its shape, resulting in denaturation.  Segment 2: Example of The Impact of Temperature and pH on enzymesTrypsin and pepsin are both enzymes in the digestive system which break protein chains in food into smaller peptide chains or into individual amino acids. Pepsin works in the highly acidic conditions of the stomach. It has an optimum pH of about 1.5. On the other hand, trypsin works in the small intestine, parts of which have a pH of around 7.5. If at a pH of around 7, a substrate attaches itself to the enzyme via two ionic bonds, then a change in pH can definitely make it difficult for the substrate to bond to the enzyme. In an example enzyme, the groups allowing ionic bonding are caused by the transfer of a hydrogen ion from a COOH group in the side chain of one amino acid to an -NH2 group in the side chain of another. At a lower pH, the -COO- will pick up a hydrogen ion and with this an ionic bond can no longer form between the substrate and the enzyme. If those bonds were necessary to attach the substrate and activate it, then at this lower pH, the enzyme won't work. With a pH higher than 7, the NH3+ will lose a hydrogen ion and again an ionic bond can't form. The tertiary structure of the protein is also in part held together by ionic bonds. At very high or very low pH's, these bonds within the enzyme can be disrupted, and it can lose its shape and if it loses its shape, the active site can be lost completely. In the human body the optimum temperature for an enzyme is...

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.04.325118v1?rss=1 Authors: Madasu, M. K., Thang, L. V., Chilukuri, P., Palanisamy, S., Arackal, J. S., Sheahan, T. D., Foshage, A. M., Houghten, R. A., McLaughlin, J. P., McCall, J. G., Al-Hasani, R. Abstract: Noxious cold sensation is commonly associated with peripheral neuropathies, however, there has been limited progress in understanding the mechanism of cold pain. Transient receptor potential (TRP) A1 channels facilitate the perception of noxious cold at the level of dorsal root ganglia (DRG), where kappa opioid receptors (KOR) are also expressed but have not previously been implicated in cold sensation. Here we identify a new role for KOR in enhancing cold hypersensitivity. First, we show that systemic KOR agonism (U50,488, KOR agonist), significantly potentiates the latency to jump and the number of jumps on the cold plate compared controls at 3oC. Importantly, NorBNI (KOR antagonist) attenuates U50,488-induced cold hypersensitivity. However, the central administration of NorBNI does not block U50,488-induced cold hypersensitivity suggesting that peripheral KOR likely modulate this effect. Furthermore, the peripherally-restricted KOR agonist, ff(nle)r-NH2 also induces cold hypersensitivity. Using fluorescent in situ hybridization, we show that KOR mRNA colocalizes with the transcripts for the cold-activated TRPA1 and TRPM8 channels in DRG. Finally, using calcium imaging in DRG, we show that intracellular calcium release is potentiated during the simultaneous application of a TRPA1 agonist, mustard oil (MO), and a KOR agonist (U50,488), when compared to MO alone. This potentiated calcium response is absent in TRPA1 KO mice. Together our data suggest that KOR-induces cold hypersensitivity through modulation of peripheral TRPA1 channels. These findings indicate that whether activation of peripheral KOR is protective or not may be dependent on the pain modality. Copy rights belong to original authors. Visit the link for more info

Quorum Sensing (QS) is a process that enables bacteria to communicate via chemical signalling molecules, which are called autoinducers (AI). When a threshold concentration of QS molecules is reached, the bacteria start their QS regulated gene expression e.g. bioluminescence (Vibrio fischeri), virulence factor secretion (Pseudomonas aeruginosa), biofilm formation (Burkholderia cepacia), sporulation, and mating. It was found that many Gram-negative bacteria use acylated homoserine lactones (AHLs or HSLs) as autoinducers. Due to the broad biological functions of HSLs, the interest in detection and analysis of HSLs is increasing for medical, biotechnological and agricultural applications. In the past years, numerous analytical methods have been developed for HSLs. Conventional analysis, which usually combines chromatography, mass spectrometry (MS), and nuclear magnetic resonance (NMR) has been very successfully applied for identification and quantification of HSLs. But normally, conventional analysis requires many steps of sample preparation, e.g. extraction, pre-concentration and optimisation of conditions to separate individual HSL molecules. In addition, many sensitive bioreporter assays have been developed using different LuxR responsive promoters, which contain LuxR family functional proteins but lack the HSL synthase. A combination of different bioassays is strongly recommended, since no bioreporter is sensitive to all HSLs. Alternatively, in this study, an anti-HSL antibody based immunochemical detection method has been successfully developed and established. HSL molecules consist of a homoserine lactone ring and an acyl side chain (4-18 carbon atoms), and they differ only from side chain length and substitution at C3 atom. Regarding the variation of the molecule structures, four HSL haptens, named HSL1, HSL2, HSL3 and HSL4, were designed for antibody and assay development. HSL1 and HSL3 have a long chain (C11-COOH), but HSL1 has an -oxo and HSL3 has an –OH functional group at the C3 position. In comparison, HSL2 (C5-COOH) and HSL4 (C9-COOH) have shorter side chains and no substitution on the C3 atom. The haptens were synthesised and were covalently coupled to the C-terminal COOH-group of the NH2-residues (lysines) of the carrier proteins (BSA/OVA). Using these HSL hapten-conjugates, rat and mouse anti-HSL monoclonal antibodies (mAbs) were produced, screened and further characterised with enzyme-linked immunosorbent assays (ELISAs). Corresponding to hapten structures, the antibodies showed different selectivities to HSLs with different substitution on C3 position and chain length. Eight mAbs (HSL1-1A5, HSL1-8E1, HSL1/2-2C10, HSL1/2-4H5, HSL4-4C9, HSL4-5H3, HSL4-5E12 and HSL4-6D3) were selected from about 200 mAbs and characterised in detail using coating antigen and enzyme tracer formats. It was demonstrated that the new assays have HSL detection ranges from nM to low µM, which is sensitive enough for detection of HSLs in natural samples according to literature. Interestingly but not surprisingly, AHLs mAbs have at least 20 times higher sensitivity against hydrolysed HSLs (named HSs) than original HSLs, because the conjugation and immunisation conditions, e.g. pH and temperature, for mAb development resulted in HSL hydrolysis. This property of antibodies additionally offers a new sensitive method to detect quorum quenching (QQ) relevant homoserines (HSs), which are important degradation products of HSLs. Comparable results have been obtained by Biacore and Aqua-Optosensor biosensors for HS (L) characterisation. Based on these properties of the mAbs, a detection method of HSLs and HSs in biological samples has been developed and optimised. With the comparison of the real samples before and after hydrolysis treatment, the assays could simply present the relative HSL- and HS- contents in the samples. Similar to bio-reporters, the identification or quantitation of single HSL molecules is not possible only using immunoassay due to the broad recognition of HSLs and HSs. For this purpose, a combination with conventional chemical analysis is a must. However, as a novel sensitive HSL and HS detection method, the developed immunoassays have the advantages of being fast, cost effective and having low sample volume requirement. Using the direct or indirect fluorescence signals of fluorophore labelled anti-HSL mAbs, the in situ experiments with modified Burkholderia cepacia biofilm on ibidi slide (plastic flow chamber from ibidi GmbH) and Pseudomonas putida inoculated barley root have been carried out. Unfortunately, the in situ tests were not successful, mainly due to remaining unspecific background signals. Nevertheless, a few steps, e.g. fluorophore labelling, biofilm formation, and surface blocking have been optimised. The door of HSL in situ tests with the antibodies is still open, if a suitable specific visualising detection method could be found in the future. Certainly, the antibodies can also be broadly applied for many other immunochemical techniques, such as immunosensors or immunoaffinity columns for characterisation or pre-screening of HSLs/HSs, as have been demonstrated successfully.

RNA polymerase II (RNAP II) transiently associates with many different proteins and multiprotein complexes during the mRNA transcription cycle, which includes three phases, initiation, elongation, and termination. This thesis describes structural studies of two factors that facilitate transcription through chromatin. The heterodimeric Saccharomyces cerevisiae elongation factor Spt4-Spt5 (human DSIF) has been identified by biochemical and genetic approaches to help RNAP II transcribe through chromatin. It is assumed that Spt4-Spt5 pauses RNAP II to open a time window for capping enzyme recruitment and addition of a cap to the 5'-end of the nascent RNA. The preparation of milligram quantities of soluble Spt4-Spt5 variants that are suited for structural studies has been achieved. Several strategies to resolve the structure of the RNAP II–Spt4-Spt5 complex were unsuccessful, possibly indicating an intrinsic flexibility of the complex. In addition, there is now evidence for direct links between chromatin modification and transcription elongation. A major player in this process is the histone lysine methyltransferase Set2 which has a modular structure. The catalytic activity of Set2 is mediated by the SET [Su(var)3-9, Enhancer of Zeste, Trithorax] domain. During mRNA elongation, the SRI (Set2 Rpb1-interacting) domain of Set2 binds to the phosphorylated CTD (carboxyl-terminal domain) of RNAP II. The NMR solution structure of yeast Set2 SRI domain has been determined. The structure reveals a novel CTD-binding fold consisting of a left-handed three-helix bundle. Unexpectedly, the SRI domain fold resembles the structure of an RNA polymerase-interacting domain in sigma factors that mediate transcription initiation in bacteria (domain sigma2 in sigma70). NMR titration experiments show that the SRI domain binds a Ser2/Ser5-phosphorylated CTD peptide comprising two heptapeptide repeats and three flanking NH2-terminal residues. Amino acid residues that show strong chemical shift perturbations upon CTD binding cluster in two regions on the SRI surface. The results will enable a detailed analysis of the specific CTD interactions underlying the coupling of transcription and chromatin modification by Set2.

Background: Exogenous use of the intestinal hormone glucagon-like peptide 1 (GLP-1) lowers glycaemia by stimulation of insulin, inhibition of glucagon, and delay of gastric emptying.Aims: To assess the effects of endogenous GLP-1 on endocrine pancreatic secretion and antro-pyloro-duodenal motility by utilising the GLP-1 receptor antagonist exendin(9-39)amide (ex(9-39)NH2).Methods: Nine healthy volunteers underwent four experiments each. In two experiments with and without intravenous infusion of ex(9-39)NH2 300 pmol/kg/min, a fasting period was followed by intraduodenal glucose perfusion at 1 and 2.5 kcal/min, with the higher dose stimulating GLP-1 release. Antro-pyloro-duodenal motility was measured by perfusion manometry. To calculate the incretin effect (that is, the proportion of plasma insulin stimulated by intestinal hormones) the glycaemia observed during the luminal glucose experiments was mimicked using intravenous glucose in two further experiments.Results: Ex(9-39)NH2 significantly increased glycaemia during fasting and duodenal glucose. It diminished plasma insulin during duodenal glucose and significantly reduced the incretin effect by approximately 50%. Ex(9-39)NH2 raised plasma glucagon during fasting and abolished the decrease in glucagon at the high duodenal glucose load. Ex(9-39)NH2 markedly stimulated antroduodenal contractility. At low duodenal glucose it reduced the stimulation of tonic and phasic pyloric motility. At the high duodenal glucose load it abolished pyloric stimulation.Conclusions: Endogenous GLP-1 stimulates postprandial insulin release. The pancreatic textgreeka cell is under the tonic inhibitory control of GLP-1 thereby suppressing postprandial glucagon. GLP-1 tonically inhibits antroduodenal motility and mediates the postprandial inhibition of antral and stimulation of pyloric motility. We therefore suggest GLP-1 as a true incretin hormone and enterogastrone in humans.

Peroxisomales Targeting und Import von Proteinen in Peroxisomen sind für die Entstehung, Wachstum und Funktion von Peroxisomen entscheidend. Ziel dieser Dissertation war, die Region und das Aminosäure-Motiv innerhalb des Adrenoleukodystrophie-Proteins (ALDP) zu identifizieren, welches für das peroxisomale Targeting erforderlich ist. Dieses peroxisomale Membran-Protein ist defekt oder fehlend bei der X-gebundenen Adrenoleukodystrophie, einer überwiegend in der Kindheit auftretenden letalen neurodegenerativen Erkrankung. Unter Verwendung von Deletions- und GFP-Fusionsprotein-Konstrukten konnte die für das peroxisomale Targeting notwendige Region des humanen ALDP auf die Aminosäuren 67-110 eingegrenzt werden. Dabei sind die NH2-terminalen 66 Aminosäuren des ALDP für das peroxisomale Targeting zwar nicht notwendig, sie erhöhen jedoch die Targeting-Effektivität insgesamt. Die für das Targeting notwendige Region ist allerdings alleine nicht auseichend, um ein Fusionsprotein an das Peroxisom zu leiten. Zusätzliche Aminosäuren scheinen für die Stabilisierung und Insertion in die peroxisomale Membran notwendig zu sein, da die Aminosäure-Regionen 1-110 und 67-164 ein Fusionsprotein an das Peroxisom dirigieren können. GFP-Fusionsproteine der dem 67-164 ALDP entsprechenden Regionen des humanen peroxisomalen Membran-Proteins 69 und des Pxa1 der Hefe wurden ebenfalls an das Peroxisom geführt. Damit konnte eine partielle Konservierung der Targeting-Region innerhalb der humanen peroxisomalen ABC-Transporter und zwischen Hefe und Mensch gezeigt werden. Die Targeting-Region beinhaltet ein 14 Aminosäuren (71-84) umfassendes konserviertes Motiv. Eine Deletion des gesamten Motivs oder von Teilen dieses Motivs führt zu einem Verlust des peroxisomalen Targetings des ALDP. Von den getesteten Mutationen einzelner Aminosäuren bewirkt lediglich die Substitution L78F eine signifikante Verminderung der Targeting-Effektivität. Dagegen führt die bei zwei X-ALD Patienten beobachtete Deletion von drei Aminosäuren innerhalb des Motivs zu einem Verlust des peroxisomalen Targetings mit einer partiellen Anreicherung des GFP-Fusionsproteins in Mitochondrien und gibt somit Aufschluss über die molekulare Ätiologie ihrer Erkrankung.

Glycosyl-phosphatidylinositol-anchored membrane proteins (GPI-proteins) are normally identified either by cleavage of the lipid anchor using (glycosyl)phosphatidylinositol-specific phospholipases C or D (GPI-PLs) or by metabolic labeling of the lipid moiety with specific building blocks. Therefore, methods for discrimination between transmembrane proteins and GPI-proteins on the basis of their physicochemical properties are desirable. Here we are presenting a selective extraction method for typical well-characterized mammalian GPI-proteins, e.g., acetylcholine esterase, alkaline phosphatase, 5′-nucleotidase, and lipoprotein lipase, using a derivative of taurocholate. The results were compared to those obtained with well-characterized transmembrane proteins, e.g., insulin receptor and hydroxymethyl glutaryl coenzyme A-reductase, glucose transporters, or aminopeptidase M and several commercially available detergents. With regard to total membrane proteins, it was possible to selectively enrich GPI-proteins up to 8- to 14-fold by using concentrations between 0.1 and 0.3% of 4′-NH2-amino-7β-benzamido-taurocholic acid (BATC). In addition, the cleavage specificity and efficiency of (G)PI-PLs were increased in the presence of identical concentrations of BATC compared to commonly used detergents, e.g., Nonidet P-40. Therefore, the present study shows that the use of BATC facilitates the identification of glycosyl-phosphatidylinositol-anchored membrane proteins.

The mitochondrial processing peptidase (MPP) of Neurospora crassa is constituted by an alpha- and a beta-subunit. We have purified alpha-MPP after expression in Escherichia coli while beta-MPP was purified from mitochondria. A fusion protein between precytochrome b2 and mouse dihydrofolate reductase was expressed in E. coli, and the purified protein was used as substrate for MPP. Both subunits of MPP are required for processing. MPP removes the matrix targeting signal of cytochrome b2 by a single cut, and the resulting presequence peptide is 31 amino acid residues in length. It acts as a competitive inhibitor of processing but has a approximately 30-fold lower affinity for MPP than the preprotein. Competition assays show that MPP recognizes the COOH- terminal portion of the presequence of cytochrome b2 rather than the NH2-terminal part which has the potential to form an amphiphilic helix. Substitution of arginine in position -2 of the matrix targeting sequence of cytochrome b2 prevents processing but not import of a chimeric precursor. Substitution of the tyrosyl residue in position +1 also prevents processing, indicating that MPP interacts with sequences COOH-terminal to the cleavage site. Non-cleavable preprotein is still recognized by MPP. Our data suggest that processing peptidase and import machinery recognize distinct structural elements in preproteins which, however, can be overlapping.

The targeting of proteins to mitochondria involves the recognition of the precursor proteins by receptors on the mitochondrial surface followed by insertion of the precursors into the outer membrane at the general insertion site GIP. Most mitochondrial proteins analyzed so far use a mitochondrial outer membrane protein of 19 kilodaltons (MOM19) as an import receptor. The gene encoding MOM19 has now been isolated. The deduced amino acid sequence predicts that MOM19 is anchored in the outer membrane by an NH2-terminal hydrophobic sequence, while the rest of the protein forms a hydrophilic domain exposed to the cytosol. MOM19 was targeted to the mitochondria via a pathway that is independent of protease-accessible surface receptors and controlled by direct assembly of the MOM19 precursor with GIP.

Binding sites for atrial natriuretic factor (ANF) were determined on isolated rat glomeruli as well as on glomerular membranes. To define optimal conditions, binding of ANF was investigated varying incubation time, temperature and protein concentration. Binding conditions were found to be best at 4°C for 5 hours with 15 μg of glomerular protein. Saturation and affinity cross-linking experiments confirmed the presence of two distinct receptor subtypes - the B-receptor (130 kDa) and the C-receptor (65 kDa). Quantitative differentiation of both ANF binding sites was achieved by competitive displacement with two different unlabeled ANF ligands: a) rANF(99-126) (homologous displacement), b) des(18-22)rANF(4-23)NH2(heterologous displacement). Intact glomeruli and glomerular membranes did not differ significantly in receptor density for the B-receptor (71 ± 37 vs. 94 ± 53 fmol/mg protein) or the C-receptor (976 ± 282 vs. 966 ± 167 fmol/mg protein) or in affinity constants for the B-receptor (43 ± 36 vs. 52 ± 44 pM) or the C-receptor (876 ± 377 vs. 307 ± 36 pM). Glomerular membranes compared to glomeruli showed less nonspecific binding and less intra-assay variation of measuring points done in triplicates. This method of selective displacement should allow to study the influence of various physiological and pathophysiological conditions on the binding properties of B-and C-receptors for ANF.

We have isolated and characterized four collagen-related c-DNA clones (N-COL 1, N-COL 2, N-COL 3, N-COL 4) that are highly expressed in developing nematocytes in hydra. All four c-DNAs as well as their corresponding transcripts are small in size (600-1,000 bp). The deduced amino acid sequences show that they contain a central region consisting of 14 to 16 Gly-X-Y triplets. This region is flanked amino-terminal by a stretch of 14-23 proline residues and carboxy-terminal by a stretch of 6-9 prolines. At the NH2- and COOH-termini are repeated patterns of cysteine residues that are highly conserved between the molecules. A model is proposed which consists of a central stable collagen triple helix of 12-14 nm length from which three 9-22 nm long polyproline II type helices emerge at both ends. Disulfide linkage between cysteine- rich segments in these helices could lead to the formation of oligomeric network structures. Electrophoretic characterization of nematocyst extracts allows resolution of small proline-rich polypeptides that correspond in size to the cloned sequences.

Passage of precursor proteins through translocation contact sites of mitochondria was investigated by studying the import of a fusion protein consisting of the NH2-terminal 167 amino acids of yeast cytochrome b2 precursor and the complete mouse dihydrofolate reductase. Isolated mitochondria of Neurospora crassa readily imported the fusion protein. In the presence of methotrexate import was halted and a stable intermediate spanning both mitochondrial membranes at translocation contact sites accumulated. The complete dihydrofolate reductase moiety in this intermediate was external to the outer membrane, and the 136 amino acid residues of the cytochrome b2 moiety remaining after cleavage by the matrix processing peptidase spanned both outer and inner membranes. Removal of methotrexate led to import of the intermediate retained at the contact site into the matrix. Thus unfolding at the surface of the outer mitochondrial membrane is a prerequisite for passage through translocation contact sites. The membrane-spanning intermediate was used to estimate the number of translocation sites. Saturation was reached at 70 pmol intermediate per milligram of mitochondrial protein. This amount of translocation intermediates was calculated to occupy approximately 1% of the total surface of the outer membrane. The morphometrically determined area of close contact between outer and inner membranes corresponded to approximately 7% of the total outer membrane surface. Accumulation of the intermediate inhibited the import of other precursor proteins suggesting that different precursor proteins are using common translocation contact sites. We conclude that the machinery for protein translocation into mitochondria is present at contact sites in limited number.

Aus dem in situ erzeugten (C5Cl4Li)Mn(CO)3 lassen sich die funktionalisierten Derivate (C5Cl4R)Mn(CO)3 mit R = SnMe3, PPh2, SePh, (SC5Cl4)Mn(CO)3, CHO, COCl, CONH2, CN, NCO und NH2 gewinnen. Auch aus (C5Cl5Rh(1,5-COD) wird über das in situ erzeugte Lithioderivat eine Reihe von Verbindungen (C5Cl4R)Rh(COD) mit R = H, Me, SiMe3, SiMe2H, und SnMe3 erhalten. Die Struktur von (C5Cl4CONH2)Mn(CO)3 wird kristallographisch bestimmt.

Subunit 9 (dicyclohexylcarbodiimide binding protein, 'proteolipid') of the mitochondrial F1F0-ATPase is a nuclearly coded protein in Neurospora crassa. It is synthesized on free cytoplasmic ribosomes as a larger precursor with an NH2-terminal peptide extension. The peptide extension is cleaved off after transport of the protein into the mitochondria. A processing activity referred to as processing peptidase that cleaves the precursor to subunit 9 and other mitochondrial proteins is described and characterized using a cell-free system. Precursor synthesized in vitro was incubated with extracts of mitochondria. Processing peptidase required Mn2+ for its activity. Localization studies suggested that it is a soluble component of the mitochondrial matrix. The precursor was cleaved in two sequential steps via an intermediate-sized polypeptide. The intermediate form in the processing of subunit 9 was also seen in vivo and upon import of the precursor into isolated mitochondria in vitro. The two cleavage sites in the precursor molecule were determined. The data indicate that: (a) the correct NH2-terminus of the mature protein was generated, (b) the NH2-terminal amino acid of the intermediate-sized polypeptide is isoleucine in position-31. The cleavage sites show similarity of primary structure. It is concluded that processing peptidase removes the peptide extension from the precursor to subunit 9 (and probably other precursors) after translocation of these polypeptides (or the NH2-terminal part of these polypeptides) into the matrix space of mitochondria.

Isolated yeast mitochondria were able to take up Neurospora ATPase subunit 9 in vitro although the homologous yeast protein is synthesized within the mitochondria and inserted into the membrane from the matrix side (Tzagoloff, A., and Meagher, P. (1972) J. Biol. Chem. 247, 594- 603). The transfer of the protein was dependent on an energized mitochondrial inner membrane. It was accompanied by proteolytic processing of the precursor to the mature protein with the correct NH2 terminus as determined by Edman degradation of the transferred protein. The possibility is discussed that there are common features in the uptake machinery neither specific for one species nor specific for individual precursor proteins in the same species.