Morpholinone mediated oxazolone‐free C‐terminus amide coupling permitting a convergent strategy for peptide synthesis

3‐Substituted‐5‐phenylmorpholinones have been demonstrated to act as N‐protected C‐terminus activated α‐amino acids capable of undergoing solution phase N‐terminus peptide extension following standard coupling procedures. The N‐acylated morpholinones do not undergo epimerisation of the stereocentre of the C‐terminus amino acid residue as oxazolone formation is sterically prevented, although C‐terminus peptide coupling is still possible. This convergent approach to peptide synthesis is exemplified by the preparation of L‐ala‐L‐ala‐L‐ala and L‐ala‐D‐ala‐L‐ala. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Allosteric interaction of nucleotides and tRNA(ala) with E. coli alanyl-tRNA synthetase

Alanyl-tRNA synthetase, a dimeric class 2 aminoacyl-tRNA synthetase, activates glycine and serine at significant rates. An editing activity hydrolyzes Gly-tRNA(ala) and Ser-tRNA(ala) to ensure fidelity of aminoacylation. Analytical ultracentrifugation demonstrates that the enzyme is predominately a dimer in solution. ATP binding to full length enzyme (ARS875) and to an N-terminal construct (ARS461) is endothermic (ΔH = 3-4 kcal mol(-1)) with stoichiometries of 1:1 for ARS461 and 2:1 for full-length dimer. Binding of aminoacyl-adenylate analogues, 5'-O-[N-(L-alanyl)sulfamoyl]adenosine (ASAd) and 5'-O-[N-(L-glycinyl)sulfamoyl]adenosine (GSAd), are exothermic; ASAd exhibits a large negative heat capacity change (ΔC(p) = 0.48 kcal mol(-1) K(-1)). Modification of alanyl-tRNA synthetase with periodate-oxidized tRNA(ala) (otRNA(ala)) generates multiple, covalent, enzyme-tRNA(ala) products. The distribution of these products is altered by ATP, ATP and alanine, and aminoacyl-adenylate analogues (ASAd and GSAd). Alanyl-tRNA synthetase was modified with otRNA(ala), and tRNA-peptides from tryptic digests were purified by ion exchange chromatography. Six peptides linked through a cyclic dehydromoropholino structure at the 3'-end of tRNA(ala) were sequenced by mass spectrometry. One site lies in the N-terminal adenylate synthesis domain (residue 74), two lie in the opening to the editing site (residues 526 and 585), and three (residues 637, 639, and 648) lie on the back side of the editing domain. At least one additional modification site was inferred from analysis of modification of ARS461. The location of the sites modified by otRNA(ala) suggests that there are multiple modes of interaction of tRNA(ala) with the enzyme, whose distribution is influenced by occupation of the ATP binding site.     

Silica, Alumina, and Clay-Catalyzed Alanine Peptide Bond Formation

The peptide bond formation of alanine (ala), ala + glycine (gly), ala + diglycine (gly₂), and ala + gly cyclic anhydride (cyc-gly₂) in drying/wetting cycles at 80°C was studied. Silica, alumina, and representative smectites—montmorillonite and hectorite—were used as catalysts, and the dependence of reaction yields on the available amount of water in the reaction systems was evaluated. Silica and alumina catalyze the formation of oligopeptide mainly in temperature fluctuation experiments, whereas higher amounts of water in the reaction system support clay-catalyzed reactions. Silica and alumina are much more efficient for amino acid dimerization than clays. Whereas only 0.1% of ala oligomerized on hectorite and no reaction proceeded on montmorillonite, about 0.9 and 3.8% alanine converted into its dimer and cyclic anhydride on silica and alumina, respectively. Clay minerals, on the other hand, seem to more efficiently catalyze peptide chain elongation than amino acid dimerization. The reaction yields of ala-gly-gly and gly-gly-ala from ala + gly₂ and ala + cyc-gly₂ reached about 0.3% on montmorillonite and 1.0% on hectorite. The possible mechanisms of these reactions and the relevance of the results for prebiotic chemistry are discussed. Received: 15 December 1996 / Accepted: 1 May 1997     

Alar rim deformities

Alar disharmony is one of the most common abnormalities observed after a rhinoplasty. This article describes three classes in addition to Gunter's classifications of alar/columella deformities, which include concave ala, convex ala caused by convex lateral crus, and convex ala caused by thick alar tissues. These deformities are best visualized from the basilar view. The different surgical techniques for correction of true alar abnormalities are presented. The alar convexity, when it is the result of a misshapen cartilage, is corrected using a lateral crura spanning suture, posterior transection of the lateral crura, or transdomal suture. A thick ala, resulting in convexity, can be thinned through either a direct incision on the ala or an incision in the alar base. A lateral crura strut, an onlay graft, or a rim graft eliminates the concavity. For a slight retraction, an alar rim cartilage graft is an optimal choice. For significant alar retractions, the author's preferred technique is an internal V-to-Y advancement, which is described in detail. An elliptical excision of the alar lining will effectively correct the hanging ala. These techniques have been used to correct alar disharmonies on 58 patients. One patient from the V-Y advancement group exhibited a small area of alar necrosis, and two early patients demonstrated an overcorrection; all were easily resolved with revision surgery. By carefully identifying nasal base and alar abnormalities, harmony can be established to correct an undesirable appearance.     

The effects of a low therapeutic dose of βCG fragment-lytic peptide conjugates on ovarian function and gonadotropin secretion in ewes: a randomized controlled trial

The main objective of this experiment was to determine and compare the effects of two lytic peptide conjugates, Phor21-?CG(ala) and ?CG(ala)-Phor21, at a low therapeutic dose (0.2 mg/kg body weight i.v.), on periovulatory ovarian and endocrine activity, and ensuing luteal function in an ovine experimental model. We hypothesized that the dense expression of LH/hCG receptors on the preovulatory follicle would present an appropriate target for the drugs and disrupt normal ovarian dynamics in sheep. Serum levels of reproductive hormones and ultrasonographic images were used for the assessment of periovulatory events following drug administration in 14 Rideau Arcott ewes; seven animals served as controls. Ovulations were synchronized with intravaginal progestogen-releasing sponges (medroxyprogesterone acetate, 60 mg) that were left in place for 12 days and a single i.m. injection of 750 IU of equine chorionic gonadotropin (eCG) given at sponge withdrawal. Both drugs were administered by i.v. injection 36 h post sponge removal/eCG injection, during the period of increasing LH responsiveness of potential ovulatory follicles and around the expected onset of the preovulatory surge of gonadotropins. No difference (p>0.05) was detected in the number of luteal structures per ewe in control versus treated animals during early luteogenesis. After drug administration, peak FSH concentrations were higher (p<0.05) in Phor21-?CG(ala)-treated compared to control ewes and circulating estradiol concentrations were lower (p<0.05) in ?CG(ala)-Phor21-treated animals. Mean serum progesterone concentrations were lower (p<0.05) in ?CG(ala)-Phor21-treated than control ewes during the luteal phase post-treatment. There were no differences (p>0.05) in the percentage of ewes that lambed or lamb characteristics between the three groups at lambing 9 months post-treatment. In summary, neither Phor21-?CG(ala) nor ?CG(ala)-Phor21 demonstrated adverse effects on the ovulatory process but the treatment with ?CG(ala)-Phor21 significantly depressed follicular and luteal steroidogenesis. With a lack of evidence for disruptive effects on endocrine function and fertility, these obsevations support the use of Phor21-?GG(ala) as a cancer pharmaceutical.     

A new twist to the nasolabial flap for reconstruction of lateral alar defects

The nasolabial flap remains the favored technique for alar and lateral nasal reconstruction. Results with currently popular techniques tend to be inartistic and aesthetically disappointing. Improved results can be achieved, however, by a technique using a medially based nasolabial turnover flap for lining with a distal extension providing the cover. Reconstruction of the ala begins by designing a nasolabial flap with its base as close as possible to the site of the proposed ala. The flap is incised to the required margins, carrying 2 to 3 mm of underlying fat; then, hinged on its base, the flap is flipped over medially like the page of a book. As the proximal flap is sutured to the lining side of the defect, the distal flap gracefully twists 90 degrees and is then folded on itself to form the external surface of the ala. The donor site is closed primarily. With this procedure, a natural-appearing and appropriately positioned ala may be reconstructed in one step, although a second procedure may be helpful to sculpture the margin or precisely position the alar base.     

调内消癖丸对小鼠耳廓微循环的影响

目的观察词内消癖丸活血化瘀、促进小鼠耳廓毛细血管开放,改善微循环的功能,探讨调内消癖丸对小鼠耳廓微循环的影响。方法采用直接给药的试验方法。结果调内消癖丸能促进小鼠耳廓毛细血管开放,改善微循环（P＜0．05,P＜0．01,P＜0．001）。结论调内消癖丸能活血化瘀、改善微循环为临床用药提供依据。     

Ethanolamine kinase controls neuroblast divisions in Drosophila mushroom bodies

The Drosophila mushroom bodies (MBs), paired brain structures composed of vertical and medial lobes, achieve their final organization at metamorphosis. The alpha lobe absent (ala) mutant randomly lacks either the vertical lobes or two of the median lobes. We characterize the ala axonal phenotype at the single-cell level, and show that the ala mutation affects Drosophila ethanolamine (Etn) kinase activity and induces Etn accumulation. Etn kinase is overexpressed in almost all cancer cells. We demonstrate that this enzymatic activity is required in MB neuroblasts to allow a rapid rate of cell division at metamorphosis, linking Etn kinase activity with mitotic progression. Tight control of the pace of neuroblast division is therefore crucial for completion of the developmental program in the adult brain.     

The hanging ala

The relationship of the alar rim to the columella can be altered during rhinoplasty. This may be especially true when a long nose has been shortened. In thin-skinned individuals, elevation of the ala may be accomplished by resection of the caudal aspect of the lateral crus without trimming the lining. In thick-skinned individuals, elevation of the ala may be accomplished by resection of the nasal lining at the cephalad border of the alar cartilage.     

Substrate specificity of a mutant alanyl-transfer ribonucleic acid synthetase of Escherichia coli

The correlation between the in vivo functioning and the in vitro behavior of the thermolabile alanyl-transfer ribonucleic acid (tRNA) synthetase (ARS) of Escherichia coli strain BM113 is presented. As a measure for the ARS activity inside the cell, the amount of acylated tRNA(ala) in vivo was determined. The rapid drop of the per cent tRNA(ala) charged which was observed upon shifting a culture of BM113 to the nonpermissive temperature indicates that in vivo acylation of tRNA(ala) might be the growth-limiting step at high temperature. Since neither growth nor the in vivo charging level of tRNA(ala) was affected by the addition of high l-alanine concentrations to the medium, one may infer that impaired functioning of the mutant enzyme at 40 C seems not to be due to reduced affinity of the enzyme for the amino acid. Separation of bulk tRNA of E. coli and of yeast on benzoylated diethylaminoethyl cellulose and charging of the fractions of the column by wild-type and mutant ARS reveal that only those tRNA species aminoacylated by the wild-type enzyme are also charged by the mutant ARS. Determination of the K(m) values of wild-type and mutant ARS for the three isoaccepting tRNA(ala) species of E. coli shows a ca. 10-fold increase of the apparent K(m) values of the mutant enzyme for all three species. Thus, the mutation proportionally reduces the apparent affinity for tRNA(ala) without causing any detectable recognition errors. Investigation of heat inactivation kinetics of wild-type and mutant ARS without and in the presence of substrates provides further evidence that only the transfer site of the ARS is altered by the mutation. Moreover, whereas both enzymes possess the same pH optimum of the relative maximal velocity, their pH dependence of the K(m) values for tRNA is different. The K(m) of the wild-type enzyme decreases at pH values below 7.0 and that of the mutant enzyme shows the inverse tendency; this again indicates an alteration of the tRNA binding site.     

Mucormycosis of the nasal ala in a leukemic (M7 AML) child. Is surgery of the nasal defect indicated?

Anterior tamponade with Surgicel (oxidized cellulose) was performed on a 5-year-old girl with megakaryoblastic leukemia (M7 AML) and epistaxis. Several days later necrosis of the nasal ala occurred. Debridement was performed and mucormycosis caused by Rhizopus was found in the material. Having cured mucormycosis, a defect of the complete nasal ala remained. The question of a surgical resolution of the disfiguring nasal defect arises.     

A detailed redescription of the mesoderm/neural crest cell boundary in the murine orbitotemporal region integrates the mammalian cranium into a pan-amniote cranial configuration

The morphology of the mammalian chondrocranium appears to differ significantly from those of other amniotes, since the former possesses uniquely developed brain and cranial sensory organs. In particular, a question has long remained unanswered as to the developmental and evolutionary origins of a cartilaginous nodule called the ala hypochiasmatica. In this study, we investigated the embryonic origin of skeletal elements in the murine orbitotemporal region by combining genetic cell lineage analysis with detailed morphological observation. Our results showed that the mesodermal embryonic environment including the ala hypochiasmatica, which appeared as an isolated mesodermal distribution in the neural crest-derived prechordal region, is formed as a part of the mesoderm that continued from the chordal region during early chondrocranial development. The mesoderm/neural crest cell boundary in the head mesenchyme is modified through development, resulting in the secondary mesodermal expansion to invade into the prechordal region. We thus revealed that the ala hypochiasmatica develops as the frontier of the mesodermal sheet stretched along the cephalic flexure. These results suggest that the mammalian ala hypochiasmatica has evolved from a part of the mesodermal primary cranial wall in ancestral amniotes. In addition, the endoskeletal elements in the orbitotemporal region, such as the orbital cartilage, suprapterygoid articulation of the palatoquadrate, and trabecula, some of which were once believed to represent primitive traits of amniotes and to be lost in the mammalian lineage, have been confirmed to exist in the mammalian cranium. Consequently, the mammalian chondrocranium can now be explained in relation to the pan-amniote cranial configuration.     

Immune response to lytic peptides conjugated to a betaCG fragment in treated BALB/C mice

Hecate-betaCG and Phor14-betaCG(ala) are relatively short, amphipathic alpha-helical cationic peptides with the ability to destroy selectively breast, prostate and ovarian cancer cells. Treatment with proteins and peptides frequently initiated antibody formation. Short peptides may minimize the risk of the immune system mobilization after treatment but it is necessary to investigate whether Hecate-betaCG and Phor14-betaCG(ala) induce the immune system to produce antibody and whether they affect the reproductive organs in normal wild-type mice. The results of our experiments showed that specific antibodies, tested by the enzyme-immunoassay, were not detected in the group treated with Hecate-betaCG and Phor14-betaCG(ala). The blood concentrations of both peptides begun to decrease from 60 minutes after injection and after 240 minutes its levels were undetectable. Histopatho-logical examination exhibited degenerative changes in the prostate glands and testes in males and in the ovaries and uteri of females treated with both peptides. In conclusion, our results indicate that both relatively small and rapidly metabolized peptides are not immunogenic and can be used for further investigation as a potential cancer treatment.     

Canavan disease: mutations among Jewish and non-jewish patients.

Canavan disease is an autosomal recessive leukodystrophy caused by the deficiency of aspartoacylase (ASPA). Sixty-four probands were analyzed for mutations in the ASPA gene. Three point mutations--693C-->A, 854A-->C, and 914C-->A--were identified in the coding sequence. The 693C-->A and 914C-->A base changes, resulting in nonsense tyr231-->ter and missense ala305-->glu mutations, respectively, lead to complete loss of ASPA activity in in vitro expression studies. The 854A-->C transversion converted glu to ala in codon 285. The glu285-->ala mutant ASPA has 2.5% of the activity expressed by the wild-type enzyme. A fourth mutation, 433 --2(A-->G) transition, was identified at the splice-acceptor site in intron 2. The splice-site mutation would lead to skipping of exon 3, accompanied by a frameshift, and thus would produce aberrant ASPA. Of the 128 unrelated Canavan chromosomes analyzed, 88 were from probands of Ashkenazi Jewish descent. The glu285-->ala mutation was predominant (82.9%) in this population, followed by the tyr231-->ter (14.8%) and 433 --2(A-->G) (1.1%) mutations. The three mutations account for 98.8% of the Canavan chromosomes of Ashkenazi Jewish origin. The ala305-->glu mutation was found exclusively in non-Jewish probands of European descent and constituted 60% of the 40 mutant chromosomes. Predominant occurrence of certain mutations among Ashkenazi Jewish and non-Jewish patients with Canavan disease would suggest a founding-father effect in propagation of these mutant chromosomes.     

The role of respiration, reactive oxygen species and oxidative stress in mother cell-specific ageing of yeast strains defective in the RAS signalling pathway

We show that the dominant activated allele of the yeast RAS gene, RAS2(ala18,val19), led to redox imbalance in exponential-phase cells and to excretion of almost all of the cellular glutathione into the medium when the cells reached early-stationary phase. The mitochondria of the mutant stained strongly with dihydrorhodamine 123 (DHR) and the cells displayed a very short mother cell-specific lifespan. Adding 1 mM reduced glutathione (GSH) to the medium partly restored the lifespan. The corresponding RAS2(+) rho-zero strain also displayed a short lifespan, excreted nearly all of its GSH, and stained positively with DHR. Adding 1 mM GSH completely restored the lifespan of the RAS2(+) rho-zero strain to that of the wild-type cells. The double mutant RAS2(ala18,val19) rho-zero cells showed the same lifespan as the RAS2(ala18,val19) cells, and the effect of glutathione in restoring the lifespan was the same, indicating that both mutations shorten lifespan through a similar mechanism. In the RAS2(ala18,val19) mutant strain and its rho-zero derivative we observed for the first time a strong electron spin resonance (ESR) signal characteristic of the superoxide radical anion. The mutant cells were, therefore, producing superoxide in the absence of a complete mitochondrial electron transport chain, pointing to the existence of a possible non-mitochondrial source for ROS generation. Our results indicate that oxidative stress resulting from a disturbance of redox balance can play a major role in mother cell-specific lifespan determination of yeast cells.     

Schistosoma mansoni: incorporation and metabolism of protein amino acids in vitro

1. The amino acid composition of total proteins in six stages of the life cycle of Schistosoma mansoni was determined by routine autoanalysis of acid hydrolysates. Aspartate, glutamate and glycine were consistently the most abundant protein amino acids in all stages. 2. Incorporation of each of the protein amino acids into adult and egg proteins was determined using 72 hr cultures in complex media. Incorporation rates varied widely and there was no correlation between abundance in protein and the rate of incorporation. 3. Only five amino acids were interconverted to other amino acids which were themselves incorporated into worm and egg proteins (ala, arg, asp, gly, ser); of these only two (glu from ala and pro from arg) appeared to be of quantitative significance. Exogenous glucose yielded only three protein amino acids (ala, asp, glu). 4. The data are considered in the light of differences in egg and adult protein synthesis and with particular regard to potential chemotherapy at this level.     

Relationship between bound and free amino acids in the brain of growing rats]

Total pool of glutamate, glutamine and GABA in the hemispheres increases during postnatal life of rats, the increase being due to that in free and bound forms of amino acids. In the cerebellum of 1-day rats, the content of free and bound glu, gln asp, GABA, bound ala and free gly is lower, whereas the level of free glu and ala, bound gly is higher than in mature animals. To the end of the 1st week, total amino acid content decreases, except GABA, which is increased. Aminon acid content begins to increase at the 21th and 28th days of postnatal life.