Studies on intramolecular hydrogen bonding between the pyridine nitrogen and the amide hydrogen of the peptide: synthesis and conformational analysis of tripeptides containing novel amino acids with a pyridine ring.

For the first time tripeptides, Z-AA(1)-Xaa-AA(3)-OMe (AA(1) and AA(3) = Gly or Aib, Xaa = 2Pmg and 2Pyg) were prepared containing alpha-methyl-alpha-(2-pyridyl)glycine (2Pmg) and alpha-(2-pyridyl)glycine (2Pyg) by solid-phase Ugi reaction. These results clearly indicate that for the preparation of tripeptides containing an amino acid with a pyridine ring, the solid-phase Ugi reaction is very useful.NMR analysis clarified that 2Pmg-containing tripeptides adopt a unique conformation with an intramolecular hydrogen bond between 2Pmg-NH and the pyridine nitrogen. However, in the case of Z-Gly-2Pyg-Gly-OMe, the intramolecular hydrogen bonding between 2Pyg-NH and the pyridine nitrogen was not observed, whereas Z-Aib-2Pyg-Aib-OMe adopts a unique conformation with an intramolecular hydrogen bond between 2Pyg-NH and a pyridine nitrogen. Conformational analysis of the tripeptides, Z-AA(1)-Xaa-AA(3)-OMe (AA(1), AA(3) = Gly or Aib, Xaa = alpha,alpha-di(2-pyridyl)glycine (2Dpy), alpha-phenyl-alpha-(2-pyridyl)glycine (2Ppg), 2Pmg and 2Pyg), clarified that when an alpha,alpha-disubstituted glycine with a 2-pyridyl group at an alpha-carbon atom is introduced into any peptide, an intramolecular hydrogen bond between a pyridine nitrogen and an amide proton is formed and conformational mobility of the peptide backbone is restricted.     

Phosphate transport in the duodenum and jejunum of goats and its adaptation by dietary phosphate and calcium

Endogenous P(i) recycling is a characteristic feature of the P homeostasis in ruminants. A pronounced salivary P(i) secretion into the rumen is balanced by a high intestinal P(i) absorption and an almost complete renal P(i) reabsorption. In monogastric animals, the major P(i) transport mechanism across the apical membrane of the enterocyte is an Na(+)-dependent transport mediated by NaPi cotransporter type IIb. In ruminants, an Na(+)-, as well as an H(+)-dependent, P(i) transport system seems to exist in the small intestines. Therefore, morphological localization, type of ionic dependence, and ability to adapt to dietary P or Ca restriction of duodenal and jejunal P(i) transport were characterized in goats. In the duodenum, there was an H(+)-dependent, Na(+)-sensitive P(i) transport system that did not belong to the NaPi type II family and was not influenced by dietary P or Ca restriction. In contrast, in the jejunum, there was an Na(+)-dependent, H(+)-sensitive P(i) transport mainly mediated by NaPi IIb. P restriction stimulated the NaPi IIb protein expression, resulting in higher P(i) transport capacity.     

Cleavage of the C-C linkage between the sugar and the aglycon in C-glycosylphloroacetophenone, and the NMR spectral characteristics of the resulting di-C-glycosyl compound

The treatment of unprotected mono-C-beta-D-glucopyranosylphloroacetophenone with a cation-exchange resin in anhydrous acetonitrile afforded both a phloroacetophenone and a di-C-beta-D-glucopyranosylphloroacetophenone. Treatment of an unprotected mono-C-(2-deoxy-beta-D-arabino-hexopyranosyl)phloroacetophenone (mono-C-2-deoxy-beta-D-glucopyranosylphloroacetophenone) also afforded both the aglycon and di-C-(2-deoxy-beta-D-arabino-hexopyranosyl)phloroacetophenone. The reaction mixtures were acetylated, and the structures of the isolated products were determined by NMR spectroscopy. This is the first demonstration of the formation of a di-C-glycosyl compound during the chemical cleavage of the C-C linkage between the sugar and the aglycon in an aryl C-glycosyl derivative.     

Synthesis and characterization of methotrexate-dimyristoylphosphatidylethanolamine derivatives and the glycerophosphorylethanolamine analogs

Research in this laboratory is currently focused on the biochemical and pharmacological properties of liposomes in which an otherwise water-soluble drug is anchored to the lipid bilayers via an appropriate non-polar residue. To this end, we have synthesized three (I-III) methotrexate (MTX) derivatives of dimyristoylphosphatidylethanolamine (DMPE) by conjugation of the alpha and/or gamma glutamyl carboxyl groups of the drug with the amino function of the phospholipid. These derivatives have been characterized analytically and chromatographically as MTX-gamma-DMPE (I), MTX-alpha-DMPE (II), and MTX-alpha, gamma-diDMPE (III). The corresponding glycerophosphorylethanolamine analogs have also been prepared and identified. The biological activity of these compounds (as inhibitors of in vitro cell proliferation and dihydrofolate reductase) is described in the following paper.     

Efficient copulation and the evolutionary loss of the avian intromittent organ

The absence of an intromittent organ (IO) in most species (>97%) of birds is an enigma: birds are the only terrestrial vertebrates in which insemination typically occurs without the use of an IO. In an earlier review, we evaluated six hypotheses to explain the loss of IOs among birds. We found some support for the influence of sperm competition and female choice, but other hypotheses (e.g. avoidance of sexually transmitted disease) could not be tested because of a lack of empirical data. Wesołowski (1999, J. Avian Biol. 30: 483–485) has criticised our female choice and sexually transmitted disease hypotheses as implausible and insufficient to explain the loss of the IO in birds. Instead, he proposes that IOs were lost to minimise the risk of predation (by shortening duration of copulation) and/or to increase the efficiency of sperm transfer. Here we respond to Wesołowski's (1999) criticisms and test his efficient copulation (EC) hypothesis with data on the duration of copulation in 243 species. Contrary to expectations from the EC hypothesis, copulation durations did not differ significantly between species with and without an IO. Thus we can reject the EC hypothesis as an explanation for the loss of IOs in birds.     

Phenol oxidative coupling in the biogenesis of the macrocyclic spermine alkaloids aphelandrine and orantine in Aphelandra sp

A crucial step in the biosynthesis of the spermine alkaloid aphelandrine and its diastereoisomer orantine is an intramolecular cyclization of the intermediate (S)-dihydroxyverbacine. In order to elucidate this step of the biosynthetic pathway, microsomes from the roots of Aphelandra squarrosa Nees were incubated with unlabeled and (D8)-labeled (S)-dihydroxyverbacine. It was shown that the microsomal fraction catalyzes the intramolecular coupling of (S)-dihydroxyverbacine to aphelandrine. This was proven by microsomal transformation of (D8)-labeled (S)-dihydroxyverbacine to (D8)-labeled aphelandrine. The reaction absolutely requires NAPDH and O2. The underlying reaction mechanism is probably an oxidative phenol coupling catalyzed by an aphelandrine synthase. This enzyme is proposed to be a cytochrome P-450 oxidase. The intramolecular cyclization of (S)-dihydroxyverbacine represents an important point in the biogenesis of the aphelandrine-type alkaloids.     

Effective and robust plasmid topology analysis and the subsequent characterization of the plasmid isoforms thereby observed

Within the biopharmaceutical industry, recombinant plasmid DNA is used both as a raw material (e.g. in lentiviral and AAV vector production) as well as an active ingredient (e.g. in DNA vaccines). Consequently, many analytical laboratories are routinely involved with plasmid DNA topoisoform qualitative analysis and quantification. In order to reliably determine plasmid topology, one must ensure that the methodology employed can reliably, precisely and accurately measure qualitatively and quantitatively all topological isoforms. Presented here are an anion-exchange high-performance liquid chromatography (AEC) and an agarose gel electrophoresis (AGE)-based method developed for this purpose. The strategies undertaken to overcome the respective typical problems of limited linear range of quantitation (for AGE) and isoform resolution (for AEC) are described. Also presented is a subsequent direct comparison (for assay precision/accuracy) of these two methods, as well as a package of species characterization [by chloroquine-AGE, enzymatic digestion, multi-angle laser light-scattering (MALLS) and electron microscopy] undertaken to confirm the identity of a minor supercoiled dimeric concatamer observed by both approaches.     

The “balance” argument and the evolution of sex

The “balance” argument suggests that if sex were maladaptive at the individual level, it would be quickly lost from species with asexual/sexual alternation. Williams has suggested an “Aphid-Rotifer” model for such species. It has the following characteristics (a) parthenogenesis is favoured when the environment is stable (b) when the environment changes qualitatively, sex generates offspring with an increased fitness variance compared to parthenogenetic progeny (c) if selection is intense and sib competition occurs then sex is favoured. It is argued here that condition (b) is not essential to the model. An increased fitness variance may be generated by the recombination of unfavourable mutations when one or more of the following occur; (1) founder effects (2) inbreeding (3) haplo-diploidy (4) intra-male competition. Parthenogenesis sustains an advantage when selection is relaxed and the possession of mutations is not reflected in differences in fitness. Sex is favoured when selection is intense and fitness reflects the possession of unfavourable mutations.     

Population growth enhances the mean fixation time of neutral mutations and the persistence of neutral variation

A fundamental result of population genetics states that a new mutation, at an unlinked neutral locus in a randomly mating diploid population, has a mean time of fixation of ～4N(e) generations, where N(e) is the effective population size. This result is based on an assumption of fixed population size, which does not universally hold in natural populations. Here, we analyze such neutral fixations in populations of changing size within the framework of the diffusion approximation. General expressions are derived for the mean and variance of the fixation time in changing populations. Some explicit results are given for two cases: (i) the effective population size undergoes a sudden change, representing a sudden population expansion or a sudden bottleneck; (ii) the effective population changes linearly for a limited period of time and then remains constant. Additionally, a lower bound for the mean time of fixation is obtained for an effective population size that increases with time, and this is applied to exponentially growing populations. The results obtained in this work show, among other things, that for populations that increase in size, the mean time of fixation can be enhanced, sometimes substantially so, over 4N(e,0) generations, where N(e,0) is the effective population size at the time the mutation arises. Such an enhancement is associated with (i) an increased probability of neutral polymorphism in a population and (ii) an enhanced persistence of high-frequency neutral variation, which is the variation most likely to be observed.     

Gynoecium diversity and systematics of the Magnoliales and winteroids

Carpel and ovule structure was compared in representatives of all 11 families of the Magnoliales (Annonaceae, Canellaceae, Degeneriaceae, Eupomatiaccae, Himantandraceae, Magnoliaceae, Myristicaceae) and winteroids (Austrobaileyaceae, Illiciaceae, Sehisandraceae, Winteraceae). Special attention was paid to features that are constant at family level. Bisexual flowers are always protogynous. In all representatives studied the carpels are closed at anthesis. Caipel closure is attained in three different ways: (1) postgenital fusion of inner surfaces (Degeneriaceae, Eupomatiaccae. Winteraceae), or (2) occlusion by secretion (Austrobaileyaceae, Sehisandraceae), or (3) a combination of (1) and (2): in Annonaceae, Canellaceae, Myristicaceae there is a conspicuous secretory canal in the innermost part of the ventral slit; in Illiciaceae and Magnoliaceae there is a narrow canal in the innermost part of the ventral slit; and in Himantandraceae the ventral slit is postgenilally fused in the style but completely open in the ovary. In most families the carpels have a double stigmalic crest or they have two tips in the transversal symmetry plane (i.e. at right angles to the median plane). Stigmas are unicellular papillate in most families but the papillae are bi-to multicellular (uniseriate) in Degeneriaceae and Eupomatiaceae. An unusual cryptic exlracarpellary compitum was found in Himantandraceae and Sehisandraceae. Intrusive oil cells were found in the carpel epidermis of Illiciaceae and Sehisandraceae. Mature ovules vary in length between 0.15 and 1.1 mm. The outer integument is fully annular (not semiannular) in Degeneriaceae, Himantandraceae, Canellaceae, Myristicaceae, and Illiciaceae. A rudimentary aril occurs in Canellaceae, and originates at the same site as in arillate Annonaceae and Myristicaceae. The results most strongly support an Annonaceae-Myristicaceae-Canellaceae alliance, to some degree also an Eupomatiaccac-Degeneriaceae-Himantandraceae-Magnoliaceae alliance, and an Illiciaceae-Schisandraceae-Winteraccae-Austrobaileyaceae alliance.     

Analysis of the conformation and function of the Plasmodium falciparum merozoite proteins MTRAP and PTRAMP

Thrombospondin repeat (TSR)-like domains are structures involved with cell adhesion. Plasmodium falciparum proteins containing TSR domains play crucial roles in parasite development. In particular, the preerythrocytic P. falciparum circumsporozoite protein is involved in hepatocyte invasion. The importance of these domains in two other malaria proteins, the merozoite-specific thrombospondin-related anonymous protein (MTRAP) and the thrombospondin-related apical membrane protein (PTRAMP), were assessed using near-full-length recombinant proteins composed of the extracellular domains produced in Escherichia coli. MTRAP is thought to be released from invasive organelles identified as micronemes during merozoite invasion to mediate motility and host cell invasion through an interaction with aldolase, an actin binding protein involved in the moving junction. PTRAMP function remains unknown. In this study, the conformation of recombinant MTRAP (rMTRAP) appeared to be a highly extended protein (2 nm by 33 nm, width by length, respectively), whereas rPTRAMP had a less extended structure. Using an erythrocyte binding assay, rMTRAP but not rPTRAMP bound human erythrocytes; rMTRAP binding was mediated through the TSR domain. MTRAP- and in general PTRAMP-specific antibodies failed to inhibit P. falciparum development in vitro. Altogether, MTRAP is a highly extended bifunctional protein that binds to an erythrocyte receptor and the merozoite motor.     

肥胖与神经调节

机体的能量获取和能量消耗,在一定时期内,是处于一种相对平衡的状态;获取的能量等于消耗的能量,在这一调节中,神经系统起有重要的作用,如果获取的能量（进食）大于消耗的能量,将产生肥胖,由于很多疾病与肥胖的产生有密切的关系,因此,对能量平衡调节的研究越来越受到重视。本文简要总结了近年来这方面的研究进展,内容包括：（１）饱感的产生与进食的终止;（２）机体脂肪储存信号与进食的调节;（３）与进食有关的中枢;（４）下丘脑中传递与进食有关信息的一级和二级神经元;（５）与临床的关系。     

Decoding errors and the involvement of the E-site

Life depends on the faithful translation of the genetic information into proteins. Ribosomes have developed remarkable mechanisms to ensure the accurate synthesis of proteins. In the first part of this review various types of ribosomal errors and their importance for cell-life are surveyed, while in the second part two important aspects of the ribosomal E-site for the accuracy of translation are considered: (i) The fact that usually misincorporations are not harmful for the cell, since only one in about 400 misincorporations will affect the structure and/or function of a protein, is a function of the E-site. (ii) In contrast, an extremely harmful translational error is a loss of the reading frame, resulting in an immediate loss of the genetic information. Maintenance of the reading frame is one of the most remarkable achievements of the ribosome; only once in about 30,000 elongation cycles is the reading frame lost. A cognate tRNA at the E-site is an essential prerequisite for this high precision.     

Role of magnesium chloride on the purity and activity of ovomucin during the isolation process

Purification of ovomucin is still an empirical technique and sometimes insufficient quantities of ovomucin are purified to allow characterization. Here we aimed to investigate the effect of MgCl(2) on the purity and bioactivity of ovomucin during isoelectric precipitation process and to develop an effective protocol to prepare pure ovomucin with high bioactivity. It was found that addition of MgCl(2) is an alternative approach to remove lysozyme from ovomucin, and that the hemagglutination inhibition (HI) activity of ovomucin with MgCl(2) against New Disease Virus (NDV) was about two times higher than the protein without salts. Thus, an improved procedure comprises a precipitation with 0.05 mol/L CaCl(2) followed by precipitation with 0.05 mol/L MgCl(2) was developed for the isolation of ovomucin. Better adhesion property of ovomucin was observed when low concentration of MgCl(2) was added in the designed ELASA test, whereas the adhesion property of the pure ovomucin without salts to NDV was lower. Thus, magnesium (II) plays an important role in the activity of ovomucin, and the alternative method developed in this study may significantly facilitate the further research on the mechanism of ovomucin activity.     

Reproduction in the red deer female and the effect of oestrogens on the antler cycle and behaviour

Red deer (Cervus elaphus) is a seasonally breeding mammal. The season of reproduction is probably regulated by photoperiod. The rut and the majority of conceptions occur in October. If hind is not allowed to mate the oestrus is repeated at an interval of about 13.5 days. In captivity the mating season is more prolonged and the calving season is extended. The length of gestation is about 235 days. The fat % of milk varies between 6.6 and 17.4. A hind in good condition may attain puberty as 16.5 month old. The smaller doses of stilboestrolum dipropionicum (S, 150-250 mg) induced mainly a female type of sexual behaviour or oestrus. The bigger and more prolonged administration of S (250-300 mg, given twice with a monthly interval) induced a strong male sexual behaviour e.g. roaring, chasing a hind in oestrus, mouting a hind in oestrus or a dummy sprinkled with pheromones and performance of an ejaculatory peak (M). During M the hind was in an almost vertical position and a single ejaculation of saliva was always observed. The artificially induced antlers of hinds without hormonal treatment became broken and their regrowth was very small. After S the velvet was always shed. After larger doses of S the casting was more delayed and the next antler growth was bigger.     

DNA primase and the replication of the telomeres in Oxytricha nova.

An enzymatic activity in crude extracts of macronuclei from the hypotrichous ciliate Oxytricha nova catalyzes the synthesis of RNA consisting of (C4A4)n using an oligodeoxynucleotide template of the telomeric sequence (dG4T4)n. Single-stranded (dG4T4)n is an effective template if it has a random sequence at its 5' end. The enzyme will not use a (dG4T4)n template of any length (up to 64 bases) if it lacks a random sequence at the 5' end. With a random, single-stranded sequence at the 5' end, the (dG4T4)n oligodeoxynucleotide must be at least 36 bases long to work as a template. A 16-base, single-stranded region of (dG4T4)2 is an effective template when joined to a 20-base double-stranded region of (dG4T4)n/(dA4dC4)n, a structural arrangement that is the same as the native telomere of Oxytricha macronuclear DNA. The RNA-synthesizing activity is unaffected by 1.0 mg/ml of alpha-amanitin. Macronuclear extracts have an alpha-amanitin-insensitive, RNA-polymerizing activity that can use a random 55mer oligodeoxynucleotide as a template. This enzyme activity may be the same one that uses (dG4T4)n templates to make (C4A4)n RNA. The (C4A4)n RNA made in the reaction can prime DNA synthesis by the E. coli DNA polymerase I Klenow fragment. Therefore, the RNA polymerase activity fulfills the requirements of the telomere DNA primase that we postulated for replication of telomeres in hypotrichs (Zahler and Prescott, 1988, Nucleic Acids Research 16, 6953-6972).     

Various forms of the celiac trunk and its anastomoses with the superior mesenteric artery]

Three rare varieties of upper abdominal arteries were compared with similar cases in the anatomical literature. An attempt was made to obtain a classification of the supernumerary branches of the celiac trunk and of the anastomoses between the celiac trunk and the superior mesenteric artery. One or more supernumerary branches of the celiac artery can be observed: (1) the superior mesenteric artery; (2) an accessory hepatic artery; (3) a posterior pancreatic artery; (4) a colic artery; (5) an accessory splenic artery; (6) a connecting branch to the superior mesenteric artery, and (7) an inferior phrenic artery. The following types of anastomoses between the celiac artery and the superior mesenteric artery can be distinguished: (1) direct connection; (2) anastomoses within the hepatic artery; (3) anatomoses following pre- or postnatal stenosis, and (4) the pancreatic arcades. For the first type the theory of TANDLER (longitudinal anastomosis) is abandoned. The development of the second type is as yet unresolved. In the case of the last two types a postembryonal formation is possible.     

Structural and thermodynamic analyses of the interaction between melittin and lipopolysaccharide

Lipopolysaccharide (LPS), the major constituent of the outer membrane of Gram-negative bacteria, is the very first site of interactions with the antimicrobial peptides. In this work, we have determined a solution conformation of melittin, a well-known membrane active amphiphilic peptide from honey bee venom, by transferred nuclear Overhauser effect (Tr-NOE) spectroscopy in its bound state with lipopolysaccharide. The LPS bound conformation of melittin is characterized by a helical structure restricted only to the C-terminus region (residues A15-R24) of the molecule. Saturation transfer difference (STD) NMR studies reveal that several C-terminal residues of melittin including Trp19 are in close proximity with LPS. Isothermal titration calorimetry (ITC) data demonstrates that melittin binding to LPS or lipid A is an endothermic process. The interaction between melittin and lipid A is further characterized by an equilibrium association constant (Ka) of 2.85 x 10(6) M(-1) and a stoichiometry of 0.80, melittin/lipid A. The estimated free energy of binding (delta G0), -8.8 kcal mol(-1), obtained from ITC experiments correlates well with a partial helical structure of melittin in complex with LPS. Moreover, a synthetic peptide fragment, residues L13-Q26 or mel-C, derived from the C-terminus of melittin has been found to contain comparable outer membrane permeabilizing activity against Escherichia coli cells. Intrinsic tryptophan fluorescence experiments of melittin and mel-C demonstrate very similar emission maxima and quenching in presence of LPS micelles. The Red Edge Excitation Shift (REES) studies of tryptophan residue indicate that both peptides are located in very similar environment in complex with LPS. Collectively, these results suggest that a helical conformation of melittin, at its C-terminus, could be an important element in recognition of LPS in the outer membrane.     

Main chain and side chain dynamics of the ubiquitin conjugating enzyme variant human Mms2 in the free and ubiquitin-bound States

Protein ubiquitination involves a cascade of enzymatic steps where ubiquitin (Ub) is sequentially transferred as a thiolester intermediate from an E1 enzyme to an E2 enzyme and finally to the protein target with the help of a Ub-protein ligase. Protein ubiquitination brought about by the Ubc13-Mms2 (E2-E2) complex has a unique role in the cell, unrelated to protein degradation. The Mms2-Ubc13 heterodimer links Ub molecules to one another through an isopeptide bond between its own C-terminus and Lys-63 on another Ub. The role of Mms2 is to orient a target-bound Ub molecule such that its Lys-63 is proximal to the C-terminus of the Ub molecule that is covalently linked to the active site of Ubc13. To gain insight into the influence of protein dynamics on the affinity of Ub for Mms2, we have determined pico- to nanosecond time scale fluctuations of the main chain and methyl side chains of human Mms2 in the free and Ub-bound states using solution state (15)N and (2)H nuclear magnetic resonance relaxation measurements. Analysis of the relaxation data allows for a semiquantitative estimation of the conformational entropy change (TDeltaS(NMR)) for the main chain and side chain methyl groups of Mms2 upon binding Ub. The value of TDeltaS(NMR) for the main chain and side chain methyl groups of Mms2 is -8 +/- 2 and -2 +/- 2 kcal mol(-)(1), respectively. The experimental DeltaG(binding) for the Mms2.Ub complex is -6 kcal mol(-)(1). Estimation of DeltaG(binding) using an empirical structure-based approach that does not account for changes in main chain entropy yields a value of -17 +/- 2 kcal mol(-)(1). However, inclusion of TDeltaS(NMR) for the main chain of Mms2 increases the estimated DeltaG(binding) to -9 +/- 3 kcal mol(-)(1). Assuming that changes in Ub main chain dynamics contribute to TDeltaS(NMR) to the same extent as Mms2, the estimated DeltaG(binding) is further reduced to -1 +/- 4 kcal mol(-)(1), a value close to the experimental DeltaG(binding).